RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 61/947,420 filed on March 3, 2014, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention is related to the identification of markers of a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury. Inventive markers include DNA, RNA, or microRNA (miRNA) that may play a role in central nervous system function and therapy. The invention also relates to diagnostics, devices and therapeutic materials and methods, including methods for detecting biomarkers, and the diagnostic method for aiding and monitoring the propensity or progression of an injury or condition.

BACKGROUND OF THE INVENTION

[0003] The field of clinical neurology remains frustrated by the recognition that secondary injury to a central nervous system tissue associated with physiologic response to the initial insult could be lessened if only the initial insult could be rapidly diagnosed or in the case of a progressive disorder before stress on central nervous system tissues reached a preselected threshold. Traumatic, ischemic, and neurotoxic chemical insult, along with generic disorders, all present the prospect of brain damage. While the diagnosis of severe forms of each of these causes of brain damage is straightforward through clinical response testing and computed tomography (CT) and magnetic resonance imaging (MRI) testing, these diagnostics have their limitations in that spectroscopic imaging is both costly and time consuming while clinical response testing of incapacitated individuals is of limited value and often precludes a nuanced diagnosis. Additionally, owing to the limitations of existing diagnostics, situations under which a subject experiences a stress to their neurological condition such that the subject often is unaware that damage has occurred or seek treatment as the subtle symptoms often quickly resolve. The lack of treatment of these mild to moderate challenges to neurologic condition of a subject can have a cumulative effect or subsequently result in a severe brain damage event which in either case has a poor clinical prognosis.

[0004] In order to overcome the limitations associated with spectroscopic and clinical response diagnosis of neurological condition, there is increasing attention on the use of biomarkers as internal indicators of change as to molecular or cellular level health condition of a subject. As detection of biomarkers uses a sample obtained from a subject and detects the biomarkers in that sample, typically cerebrospinal fluid, blood, or plasma, biomarker detection holds the prospect of inexpensive, rapid, and objective measurement of neurological condition. With the attainment of rapid and objective indicators of neurological condition allows one to determine severity of a non-normal brain condition on a scale with a degree of objectivity, predict outcome, guide therapy of the condition, as well as monitor subject responsiveness and recovery. Additionally, such information as obtained from numerous subjects allows one to gain a degree of insight into the mechanism of brain injury.

[0005] According to the U.S. GAO (GAO, 2008), traumatic brain injury (TBI) "has emerged as a leading injury among service members" serving in Iraq and Afghanistan. The RAND Corporation estimated that approximately 320,000 soldiers have experienced a TBI during deployment in years 2001-2008. Further, TBI, especially in its mild form (mTBI) is often accompanied by posttraumatic stress syndrome (PTSD). Improvised explosive devices (IEDs), which military members often encounter during combat, are largely to blame for the increase in TBIs.

[0006] A blast generates primary damage at gas-fluid interfaces. As its over-pressurization wave propagates through the body, a blast can produce pulmonary barotraumas, tympanic membrane ruptures with middle ear damage, abdominal hemorrhage and perforation, rupture of the eye balls, and concussions. Especially when repeated, these can lead to sustained neuro- somatic damage and neurodegeneration.

[0007] Often, this damage is not detected as it occurs. Mild or moderate brain injuries (mTBI) are often overlooked as emergency medical staff directs their attention towards more visible injuries. Symptoms of blast brain injury often manifest only sometime after the injury. Further, when later symptoms appear, their true etiology need not be evident, given intervening events in the medical history of the patient.

[0008] Thus, tools to diagnose mTBI are important in order to manage and treat military personnel and veterans. Diagnostic tools would be especially valuable if they could classify the trauma by severity and type. Such tools would have "dual use" in civilian medicine, where mTBI is associated with automobile accidents, sports injuries, and geriatric trauma, inter alia. At least 1.7 million people sustain an mTBI annually in U.S.

[0009] While imaging can diagnose major trauma, it cannot do so for mTBI. Therefore, many laboratories have sought to identify molecular markers and their associated physiological pathways arising from mTBI. Very few of which focus on the identification of nucleic acid markers.

[0010] In principle, a useful molecular marker of mTBI could be any molecule that appears in a traumatized patient in an easily retrieved sample (e.g. blood, urine, saliva) that is absent in an analogous sample obtained from an untraumatized individual. Several mechanisms might generate such makers. For example, the blood-brain barrier (BBB) normally prevents molecules present in the brain from gaining access to the serum (and vice versa). To the extent to which mTBI compromises the BBB, molecules normally concentrated in the brain could appear in the serum, where their presence could be recognized.

[0011] Alternatively, the molecular inventory of the serum could be changed as a consequence of mTBI without compromising the BBB, but rather as a system-wide response to the injury. Here, the concentration of markers could either increase or decrease. Most useful, of course, would be systemic markers specific for brain trauma, rather than trauma generally.

[0012] A variety of studies have identified proteins that can reliably detect TBI in model organisms, using panels of antibodies. Unfortunately, antibody-based approaches target epitopes that are rarely conserved across species. Thus, assays that work in animal models typically do not work in humans, thus frustrating the medical diagnostic community. Furthermore, such assays are limited by the insensitivity inherent in antibody-based assays. For these and other reasons, there remains an unmet need to discover novel non- protein biomarkers to support more fast, sensitive and simple TBI diagnoses and prognoses.

[0013] In 1988, one of the Pi's proposed that extracellular R A might exist in multicellular eukaryotes acting as a communicator between cells (Benner, 1988). This proposal was based on an analysis of extracellular ribonucleases (Nambiar et al., 1984), their evolution (Sassi et al., 2007), and their brain-toxicity (Benner, 2002), together with scattered reports of extracellular RNA and the (then confusing) data emerging from studies with "antisense" nucleotide analogues.

[0014] Extracellular RNA has been proposed as a target to diagnose and prognose various diseases. Intracellular miRNA molecules are released from cells and tissues into body fluids (CSF, plasma, serum, urine or saliva), where they are readily detected (see Figure 1). Indeed, miRNAs in human blood are protected from RNase activity, remaining stable even when subjected to conditions that would normally degrade RNA, such as boiling, low or high pH, extended storage, and freeze-thaw cycles. miRNAs can be recovered from archived 10-year-old human serum samples and in unrefrigerated dried serum blots. [0015] A need persists for the development of improved biomarkers in nearly all neurological disorders, diseases, and injuries, including traumatic brain injury, and more specifically mild and moderate traumatic brain injury. As such, there is an unmet need for new biomarkers that individually, or in combination with other biomarkers or diagnostic modalities, deliver the required sensitivity and specificity for early detection and prognosis of neurological disorders, diseases, and injuries, traumatic brain injury, and mild and moderate traumatic brain injury. In particular, simple tests for these neurological conditions that can be performed on readily-accessible biological fluids are needed. SUMMARY OF INVENTION

[0016] The present invention provides novel miRNA biomarkers which are indicative of Alzheimer's Disease, and which may be used to accurately diagnose A neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in a subject. These biomarkers include miR-34a, miR-31, miR-206, miR-34c, miR-410, miR-137, miR-7a, mir-579, mir-548f, mir-548e, mir-527, mir-153, mir-518a-5p, mir-182, mir-382, mir-495, mir-181b, mir-181c, mir- 590-3p, mir-181d, mir-377, mir-15b, mir-548g, mir-1279, mir-582-5p, mir-103, mir-130a, mir- 301b, mir-96, mir-27b, mir-548c-3p, mir-203, mir-370, mir-141, mir-200a, mir-548p, mir-548a- 3p, mir-19a, mir-454, mir-130b, mir-27a, mir-7, mir-338-5p, mir-148a, mir-148b, mir-152, mir- 561, mir-19b, mir-525-5p, mir-576-5p. In some embodiments, the methods entail detection of extracellular, circulating miRNAs in a suitable sample, preferably whole blood, plasma, serum, CSF, urine, or saliva.

[0017] In at least one embodiment the invention provides a method of diagnosing a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in a subject, which includes determining the level of a panel of miRNA biomarkers in a biological sample containing circulating miRNA from a subject, the panel comprising of miR-34a, miR-31, miR-206, miR-34c, miR-410, miR-137, miR-7a, wherein a difference in the level of the panel of miRNA's versus that in a normal subject as determined relative to a suitable control is indicative of a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in a subject.

[0018] In at least one embodiment, the invention provides a method of diagnosing a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in a subject by determining the level of at least one miRNA in a sample containing circulating miRNA derived from the subject, wherein the at least one miRNA is miR-34a, miR-31, miR- 206, miR-34c, miR-410, miR-137, miR-7a, mir-579, mir-548f, mir-548e, mir-527, mir-153, mir- 518a-5p, mir-182, mir-382, mir-495, mir-181b, mir-181c, mir-590-3p, mir-181d, mir-377, mir- 15b, mir-548g, mir-1279, mir-582-5p, mir-103, mir-130a, mir-301b, mir-96, mir-27b, mir-548c- 3p, mir-203, mir-370, mir-141, mir-200a, mir-548p, mir-548a-3p, mir-19a, mir-454, mir-130b, mir-27a, mir-7, mir-338-5p, mir-148a, mir-148b, mir-152, mir-561, mir-19b, mir-525-5p, mir- 576-5p, or combinations thereof, and wherein a difference in the level of the at least one miRNA versus that in a normal subject as determined relative to a suitable control is indicative of a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in the subject. For example, in one embodiment, the method includes determining the level of at least one miRNA in a sample containing circulating miRNA derived from the subject, wherein the at least one miRNA is miR-34a, miR-31, miR-206, miR-34c, miR-410, miR-137, miR-7a, mir-579, mir-548f, mir-548e, mir-527, mir-153, mir-518a-5p, mir-182, mir-382, mir-495, mir-181b, mir- 181c, mir-590-3p, mir-181d, mir-377, mir-15b, mir-548g, mir-1279, mir-582-5p, mir-103, mir- 130a, mir-301b, mir-96, mir-27b, mir-548c-3p, mir-203, mir-370, mir-141, mir-200a, mir-548p, mir-548a-3p, mir-19a, mir-454, mir-130b, mir-27a, mir-7, mir-338-5p, mir-148a, mir-148b, mir- 152, mir-561, mir-19b, mir-525-5p, mir-576-5p, or combinations thereof, and wherein a decrease in the level of the at least one miRNA relative to a control is indicative of a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in the subject. Optionally, the method may further comprise providing a diagnosis that the subject has or does not have a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury based on the level of at least one miRNA in the sample. In one embodiment, the method may further comprise correlating a difference in the level or levels of at least one miRNA relative to a suitable control with a diagnosis of a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in the subject.

[0019] In one embodiment, the method comprises determining the level of one miRNA, miR-34a, miR-31, miR-206, miR-34c, miR-410, miR-137, miR-7a, mir-579, mir-548f, mir-548e, mir-527, mir-153, mir-518a-5p, mir-182, mir-382, mir-495, mir-181b, mir-181c, mir-590-3p, mir-181d, mir-377, mir-15b, mir-548g, mir-1279, mir-582-5p, mir-103, mir-130a, mir-301b, mir-96, mir-27b, mir-548c-3p, mir-203, mir-370, mir-141, mir-200a, mir-548p, mir-548a-3p, mir-19a, mir-454, mir-130b, mir-27a, mir-7, mir-338-5p, mir-148a, mir-148b, mir-152, mir-561, mir-19b, mir-525-5p, mir-576-5p, in the sample, wherein the difference in the level of the miRNA versus that in a normal subject is an increase or decrease by greater than a 1.5 fold change. [0020] In at least one embodiment the method comprises determining the level of two or more miRNAs in the sample. In at least one embodiment, the method may further comprise determining the level of and additional miRNA, not the same as the first, selected from miR-34a, miR-31, miR-206, miR-34c, miR-410, miR-137, miR-7a, mir-579, mir-548f, mir-548e, mir-527, mir-153, mir-518a-5p, mir-182, mir-382, mir-495, mir-181b, mir-181c, mir-590-3p, mir-181d, mir-377, mir-15b, mir-548g, mir-1279, mir-582-5p, mir-103, mir-130a, mir-301b, mir-96, mir- 27b, mir-548c-3p, mir-203, mir-370, mir-141, mir-200a, mir-548p, mir-548a-3p, mir-19a, mir- 454, mir-130b, mir-27a, mir-7, mir-338-5p, mir-148a, mir-148b, mir-152, mir-561, mir-19b, mir- 525-5p, mir-576-5p, or combinations thereof. In at least one embodiment, the method involves determining the level of one of the following combinations of miRNAs: miR-34a, miR-31, miR- 206, miR-34c, miR-410, miR-137, miR-7a.

[0021] In at least one embodiment, the method may comprise determining the levels of two or more miRNAs in a sample containing circulating miRNA from the subject, comparing the levels of the two or more miRNAs in the sample to a set of data representing levels of the same miRNAs present in normal subjects and subjects having a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury, and diagnosing the subject as having or not having a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury based on the comparison. In some embodiments, the two or more miRNAs may include any number of combinations selected from the following miRNA's: miR-34a, miR-31, miR-206, miR-34c, miR-410, miR-137, miR-7a, mir-579, mir-548f, mir-548e, mir-527, mir-153, mir- 518a-5p, mir-182, mir-382, mir-495, mir-181b, mir-181c, mir-590-3p, mir-181d, mir-377, mir- 15b, mir-548g, mir-1279, mir-582-5p, mir-103, mir-130a, mir-301b, mir-96, mir-27b, mir-548c- 3p, mir-203, mir-370, mir-141, mir-200a, mir-548p, mir-548a-3p, mir-19a, mir-454, mir-130b, mir-27a, mir-7, mir-338-5p, mir-148a, mir-148b, mir-152, mir-561, mir-19b, mir-525-5p, mir- 576-5p.

[0022] In at least one embodiment, the method may optionally include performing at least one additional test to facilitate the diagnosis of a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury such as Glasgow Coma Scale (GCS), CT, or MRI.

[0023] The level of a miRNA may be detected by any suitable method. For example, a miRNA may be detected using an agent which specifically hybridizes to the miRNA. In certain embodiments, the level of a miRNA may be detected using amplification methods, hybridization methods, and/or sequencing methods. In one embodiment, the level of miRNA is detected using quantitative PCR. [0024] In another aspect, the invention provides a method of diagnosing a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in a subject, comprising measuring the level of at least one miRNA in a blood-derived sample from the subject, wherein the at least one miRNA is miR-34a, miR-31, miR-206, miR-34c, miR-410, miR-137, miR-7a, mir-579, mir-548f, mir-548e, mir-527, mir-153, mir-518a-5p, mir-182, mir- 382, mir-495, mir-181b, mir-181c, mir-590-3p, mir-181d, mir-377, mir-15b, mir-548g, mir- 1279, mir-582-5p, mir-103, mir-130a, mir-301b, mir-96, mir-27b, mir-548c-3p, mir-203, mir- 370, mir-141, mir-200a, mir-548p, mir-548a-3p, mir-19a, mir-454, mir-130b, mir-27a, mir-7, mir-338-5p, mir-148a, mir-148b, mir-152, mir-561, mir-19b, mir-525-5p, mir-576-5p, or combinations thereof, and correlating a difference in the level or levels in the subject relative to a suitable control with a diagnosis of a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in the subject.

[0025] In at least one embodiment, the invention provides a method of monitoring the course of a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in a subject, by determining the level of at least one miRNA in a first sample containing circulating miRNA from a subject, determining the level of said at least one miRNA in a second sample containing circulating miRNA from said subject, wherein said second sample is obtained after said first sample, and comparing the levels determined in the first sample with levels determined in the second sample, wherein said levels are indicative of A neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury progression, and wherein the at least one miRNA is miR-34a, miR-31, miR-206, miR-34c, miR-410, miR-137, miR-7a, mir-579, mir-548f, mir-548e, mir-527, mir-153, mir-518a-5p, mir-182, mir-382, mir-495, mir-181b, mir- 181c, mir-590-3p, mir-181d, mir-377, mir-15b, mir-548g, mir-1279, mir-582-5p, mir-103, mir- 130a, mir-301b, mir-96, mir-27b, mir-548c-3p, mir-203, mir-370, mir-141, mir-200a, mir-548p, mir-548a-3p, mir-19a, mir-454, mir-130b, mir-27a, mir-7, mir-338-5p, mir-148a, mir-148b, mir- 152, mir-561, mir-19b, mir-525-5p, mir-576-5p, or combinations thereof.

[0026] In at least one embodiment the invention provides a method of treating a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in a subject, by determining the level of at least one miRNA in a sample containing circulating miRNA from said subject, wherein a difference in the level of the at least one miRNA versus that in a normal subject as determined relative to a suitable control is indicative of A neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in the subject; and, if a difference in the level of at least one miRNA is detected, administering a therapeutically effective amount of a composition comprising an Alzheimer's therapeutic to the subject. In one embodiment, the at least one miRNA is miR-34a, miR-31, miR-206, miR-34c, miR-410, miR-137, miR-7a, mir-579, mir-548f, mir-548e, mir-527, mir-153, mir-518a-5p, mir- 182, mir-382, mir-495, mir-181b, mir-181c, mir-590-3p, mir-181d, mir-377, mir-15b, mir-548g, mir-1279, mir-582-5p, mir-103, mir-130a, mir-301b, mir-96, mir-27b, mir-548c-3p, mir-203, mir-370, mir-141, mir-200a, mir-548p, mir-548a-3p, mir-19a, mir-454, mir-130b, mir-27a, mir- 7, mir-338-5p, mir-148a, mir-148b, mir-152, mir-561, mir-19b, mir-525-5p, mir-576-5p, or combinations thereof.

[0027] In another aspect, the invention provides a method of treating a subject having a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury, by identifying a subject having A neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in which the level of at least one miRNA in a sample containing circulating miRNA from the subject is different (e.g., decreased) relative to a suitable control, and administering a therapeutically effective amount of an Alzheimer's therapeutic to the subject. In one embodiment, the at least one miRNA is miR-34a, miR-31, miR-206, miR-34c, miR-410, miR-137, miR-7a, mir-579, mir-548f, mir-548e, mir-527, mir-153, mir-518a-5p, mir-182, mir- 382, mir-495, mir-181b, mir-181c, mir-590-3p, mir-181d, mir-377, mir-15b, mir-548g, mir- 1279, mir-582-5p, mir-103, mir-130a, mir-301b, mir-96, mir-27b, mir-548c-3p, mir-203, mir- 370, mir-141, mir-200a, mir-548p, mir-548a-3p, mir-19a, mir-454, mir-130b, mir-27a, mir-7, mir-338-5p, mir-148a, mir-148b, mir-152, mir-561, mir-19b, mir-525-5p, mir-576-5p, or combinations thereof.

[0028] In another aspect, the invention provides a kit for diagnosing a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in a subject, containing an agent that selectively detects the presence of at least one miRNA in a sample containing circulating miRNAs from a subject, wherein the at least one miRNA is miR-34a, miR-31, miR-206, miR-34c, miR-410, miR-137, miR-7a, mir-579, mir-548f, mir-548e, mir-527, mir-153, mir-518a-5p, mir-182, mir-382, mir-495, mir-181b, mir-181c, mir-590-3p, mir-181d, mir-377, mir-15b, mir-548g, mir-1279, mir-582-5p, mir-103, mir-130a, mir-301b, mir-96, mir- 27b, mir-548c-3p, mir-203, mir-370, mir-141, mir-200a, mir-548p, mir-548a-3p, mir-19a, mir- 454, mir-130b, mir-27a, mir-7, mir-338-5p, mir-148a, mir-148b, mir-152, mir-561, mir-19b, mir- 525-5p, mir-576-5p, or combinations thereof, and instructions for determining the level of said at least one miRNA, wherein a difference in the level of the at least one miRNA versus that in a normal subject as determined relative to a suitable control is indicative of a neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury in the subject. Such a kit may optionally contain an agent that selectively detects the presence of at least one additional miRNA in the sample. In some embodiments, the agent specifically hybridizes to a miRNA.

[0029] In at least one embodiment, a method for predicting the clinical outcome of a patient suffering from a neurological condition or disorder, traumatic brain injury (TBI) or multiple- organ injury is provided which includes obtaining a biological sample from a subject, then assessing the level of expression or activity of at least one miRNA in the sample, wherein the at least one miRNA is selected from the group consisting of miR-34a, miR-31, miR-206, miR-34c, miR-410, miR-137, miR-7a, mir-579, mir-548f, mir-548e, mir-527, mir-153, mir-518a-5p, mir- 182, mir-382, mir-495, mir-181b, mir-181c, mir-590-3p, mir-181d, mir-377, mir-15b, mir-548g, mir-1279, mir-582-5p, mir-103, mir-130a, mir-301b, mir-96, mir-27b, mir-548c-3p, mir-203, mir-370, mir-141, mir-200a, mir-548p, mir-548a-3p, mir-19a, mir-454, mir-130b, mir-27a, mir- 7, mir-338-5p, mir-148a, mir-148b, mir-152, mir-561, mir-19b, mir-525-5p, mir-576-5p. The level of expression or activity of the at least one miRNA is then assessed in a biological sample from a control subject having a good clinical outcome and compared to the injured patient. In at least one embodiment, a significantly modulated level of expression or activity in the biological sample from the subject as compared to the expression or activity level in the biological sample from the control subject predicts the clinical outcome of the patient.

[0030] In at least one embodiment, the method or kit provided is for the diagnosis or detection of a traumatic brain injury (TBI). In at least one embodiment, the TBI is a mild or moderate TBI (mTBI).

[0031] In at least one embodiment the biological sample of the method or kits provided is whole blood, serum, plasma, CSF, urine, and saliva.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a schematic illustration showing the fate of brain injury biomarkers. The pathway of genesis of biomarkers from the brain to the eventual release of such biomarkers into biofluids, such as CSF, blood, urine, saliva, sweat etc. provide an opportunity for biomarker detection with low invasiveness.

[0033] Figure 2 is a schematic illustration showing the role of miRNA brain injury biomarkers as it relates to cellular repair, differentiation, and necrosis.

[0034] Figure 3 is a schematic illustration showing miRNA target mapping for neuronal injuries and disorders based on mapping process related to certain neural proteins. [0035] Figure 4 provides A) a graphic representation of two blast set-ups, and b) calibration of overpressure on a rat head.

[0036] Figure 5 provides a collection of pathway maps grouped hierarchically into folders according to main biological processes representing miRNA's changing >1.5 fold after blast TBI.

[0037] Figure 6 illustrates the results after blast exposure and measuring existing protein biomarkers of blast induced TBI showing elevated Interleukines (IL-1 and IL-10) and Selectines (E and L) in rat blood after different types of blast exposure.

[0038] Figure 7 illustrates the results after blast exposure and measuring existing protein biomarkers of blast induced TBI showing elevated GFAP and CNPase.

[0039] Figure 8 illustrates the results after blast exposure and measuring existing protein biomarkers of blast induced TBI showing elevated UCH-L1 and NSE.

[0040] Figure 9 provides a schematic for the planned architecture to detect miRNA.

[0041] Figure 10 illustrates that the miRNA can be released by introducing a second C=0 species (acetone).

[0042] Figure 11 illustrates that after being concentrated and secured within a bead, the miRNA can serve as the template for the ligation of two fragments that are AEGIS -standard DNA chimeras

DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention has utility in the diagnosis and management of abnormal neurological condition or disorder, traumatic brain injury (TBI) or multiple-organ injury through detection of cellular material released following injury or disease. Specifically, the invention has utility in the use of a diagnostic to identify or classify disease or injury subtype, specifically a traumatic brain injury (TBI) subtype (mild, moderate or severe), alone or in combination with multiple organ injury as well as for identifying potential therapeutics effective for the particular traumatic brain injury subtype the subject has endured.

[0044] The subject invention also has utility as a means of detecting neurological trauma or condition that is predictive or indicative of future disease or injury. Illustratively, the subject invention has utility as a safety or efficacy screening protocol in vivo or in vitro for drug development. Drug development is not limited to drugs directed to neurological conditions. In a preferred embodiment the inventive biomarkers have utility to detect expected or unexpected neurological side effects in in vitro animal studies as a means of selecting a lead compound for analyses or as a means of assessing safety of a previously identified drug candidate.

[0045] Finally, the subject invention also has utility as introducing new devices for detecting and providing ready and usable clinical indication of neurological trauma or condition that is predictive or indicative of future disease or injury or that may be used in determining the appropriate medical intervention, such as predicting outcome of imaging.

[0046] As used herein an injury is an alteration in cellular or molecular integrity, activity, level, robustness, state, or other alteration that is traceable to an event. Injury illustratively includes a physical, mechanical, chemical, biological, functional, infectious, or other modulator of cellular or molecular characteristics. An event is illustratively, a physical trauma such as an impact (percussive) or a biological abnormality such as a stroke resulting from either blockade or leakage of a blood vessel. An event is optionally an infection by an infectious agent. A person of skill in the art recognizes numerous equivalent events that are encompassed by the terms injury or event.

[0047] An injury is optionally a physical event such as a percussive impact. An impact is the like of a percussive injury such as resulting to a blow to the head that either leaves the cranial structure intact or results in breach thereof. Experimentally, several impact methods are used illustratively including controlled cortical impact (CCI) at a 1.6 mm depression depth, equivalent to severe TBI in human. This method is described in detail by Cox, CD, et al, J Neurotrauma, 2008; 25(11): 1355-65. It is appreciated that other experimental methods producing impact trauma are similarly operable.

[0048] TBI may also result from stroke. Ischemic stroke is optionally modeled by middle cerebral artery occlusion (MCAO) in rodents. UCHL1 protein levels, for example, are increased following mild MCAO which is further increased following severe MCAO challenge. Mild MCAO challenge may result in an increase of protein levels within two hours that is transient and returns to control levels within 24 hours. In contrast, severe MCAO challenge results in an increase in protein levels within two hours following injury and may be much more persistent demonstrating statistically significant levels out to 72 hours or more.

[0049] Without being restricted to one theory or model, one proposed delineation between mild-traumatic brain injury (MTBI) and TBI are the recognizable increase or decrease in molecular biomarkers in biological fluids following injury. Illustrative examples of molecular markers include those described by Kobeissy FH, et al, Mol Cell Proteomics, 2006; 5:1887- 1898. A proposed definition of TBI is the presence of at least one recognizable molecular biomarker with at least two-fold increased or decreased levels in cortical tissue 48 h following experimental TBI (rat controlled cortical impact with controlled cortical impact at a 1.6 mm depression depth, equivalent to severe TBI in humans).

[0050] The term "biomarker" as used herein represents antibodies, DNA, R A, miR A, fragments of RNA, fragments of DNA, peptides, proteins, lipids, or other biological material whose presence, absence, level or activity is correlative of or predictive of neurological damage or disease. Inventive protein and miRNA biomarkers are listed in Table 1.

Table 1

miRNA Markers

hsa-mir579 hsa-mir-548f hsa-mir-548e hsa-mir-527 hsa-mir-153 hsa-mir-518a-5p hsa-mir-182 hsa-mir-382 hsa-mir-495 hsa-mir-181b hsa-mir-181c hsa-mir-590-3p hsa-mir-181d hsa-mir-377 hsa-mir-15b hsa-mir-548g hsa-mir-1279 hsa-mir-582-5p hsa-mir-103 hsa-mir-130a hsa-mir-301b hsa-mir-96 hsa-mir-27b hsa-mir-548c-3p hsa-mir-203 hsa-mir-370 hsa-mir-141 hsa-mir-200a hsa-mir-548p hsa-mir-548a-3p hsa-mir-19a hsa-mir-454 hsa-mir-130b hsa-mir-27a hsa-mir-7 hsa-mir-338-5p hsa-mir-148a hsa-mir-148b hsa-mir-152 hsa-mir-561 hsa-mir-19b hsa-mir-525-5p rno-mi -34a rno-miR-31 rno-miR-206 rno-miR-34c rno-miR-410 hsa-mir-576-5p rno-miR-137 rno-miR-7a

PROTEIN MARKERS

PIK3R2 FOX03 PARK7 FOXOl RPS6KB1 TP53

PDK1 TSC2 PIK3CA FRAP1 EIF4EBP1 TSC1

PPP2R4 AKT1 SGK PTEN EIF4E RHEB

[0051] It is appreciated that the miRNA's and proteins are not known in the art. The selection of the particular miRNA's were based on specific mapping of proteins, and selecting miRNA's, from thousands of potential miRNA's. It is further appreciated that the miRNA's selected were related to proteins known for being modulated by a neurological condition or disorders, and certain processes relating to the miRNA's were selected based on their relevance only to neural injuries or disorders. Figures 2 and 3 illustrate the selection process of the inventive miRNA's.

[0052] In a first aspect of the present invention a biomarker is a polynucleic acid such as an oligonucleotide. An oligonucleotide is a DNA or RNA molecule. Preferred examples of RNA molecules are mRNA and miRNA molecules. [0053] RNA molecules were historically believed to have short half-lives in plasma. More recently, studies indicated that RNA molecules may be protected in plasma by protein or lipid vesicles. As such, RNA molecules released following or during TBI, for example, can be detected in blood, plasma, serum, CSF, or other biological material and be associated with the presence of injury in the inventive method. Numerous methods are known in the art for isolating RNA from a biological sample. Illustratively, the methods described by El-Hefnaway, T, et al., Clinical Chem., 2004; 50(3);564-573, the contents of which are incorporated herein by reference, are operable in the present invention.

[0054] Primer and probe designs are within the level of skill in the art. Any suitable primer and probe as well as labels thereon are operable for the detection of mRNA biomarkers in the subject invention. Illustratively, primer and probe design can be performed using automated programs available from commercial sources. Alternatively, numerous commercial suppliers provide primer and probe design services including Applied Biosystems (Foster City, CA).

[0055] An inventive method for RNA illustratively includes obtaining a biological sample from a subject that may be suspected of having a neurological condition; obtaining RNA from said sample; analyzing the RNA for the presence of an RNA biomarker; comparing the level of RNA biomarker detected with the level of RNA biomarker from a subject without a neurological condition; and diagnosing the presence or absence of a neurological condition in the suspect subject.

[0056] Optionally, the inventive method involves analyzing the biological sample for the presence of a plurality of biomarkers. A plurality can be any number greater than one. Preferably, two biomarkers are analyzed. More biomarkers may be simultaneously or sequentially assayed for in the inventive method illustratively including three, four, five, six, seven, eight, nine, 10, 20, 50, 100, 1000, or any number between or greater.

[0057] Preferred methods for the detection and quantitation of biomarkers is real-time PCR (RT-PCR). RT-PCR allows for the simultaneous amplification and quantitation of a plurality of biomarkers simultaneously. Alternatively, mass spectroscopy such as electrospray ionization mass spectroscopy coupled with time of flight detection and high performance liquid chromatography are similarly operable. It is appreciated that other methods are similarly operable for detection as will be appreciated by one of ordinary skill in the art.

[0058] Numerous miRNA molecules are operable as biomarkers in the subject invention. The term "miRNA" is used according to its ordinary and plain meaning and refers to a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation. Examples include miRNA molecules that regulate the expression of one or more proteins listed in Table 1. Several miRNA molecules have been identified and are operable as biomarkers in the inventive methods. Illustratively, miRNA molecules described by Redell, JB, et al, J. Neurosci. Res., 2009; 87: 1435-48; Lei, P., et al, Brain Res., doi: 10.1016/j.brainres.2009.05.074; 5 Lu, N, et al, Exp. Neurology, 2009; doi: 10.1016/j.expneurol.2009.06.015; and Jeyaseelan, K, et al, Stroke, 2008; 39:959-966, the contents of each are incorporated herein by reference for the miRNAs defined therein, but also specifically for methods of isolation and quantitation of miRNA described in each reference. These methods, or modifications thereof, that are recognized by one of ordinary skill in the art are used in the present inventive method.

10 [0059] Other inventive miRNA' s for the diagnosis of a neural injury or neuronal disorder include those marker identified in Table 2, below, represent miRNA changes following mild blast TBI in rat model.

[0060] It is appreciated that the patterns of biomarkers such as RNA, miRNA, DNA, and autoantibodies is operable to locate the site and severity of neuronal abnormality. Illustratively, damage to the brain reveals a different pattern of a plurality of biomarkers than does damage to other regions of the central nervous system. Also, damage to the hippocampus will produce a different pattern of biomarkers than damage to the frontal lobe. As such, localization of injury is achieved by comparative detection of a plurality of biomarkers. For example, miRNA levels within cells are altered in specific patterns in response to TBI. (See Redell, J, et al, J. Neurosci. Res., 2009; 87: 1435-1448.) The present inventors have surprisingly discovered that the levels of miRNA biomarkers that regulate expression of the proteins in Table 1 are similarly altered by either upregulation or downregulation dependent on the severity of injury or the time since onset of injury. The pattern of miRNA and other biomarkers changes as injury or disease progresses. This may be a result of secondary injury events, delayed cell apoptosis, or other mechanism altering the release of RNA, DNA, or protein. Redell, J, incorporated herein by reference above, illustrates alteration of miRNA biomarkers at 3 hours, and 24 hours. Some miRNAs are upregulated at 3 hours whereas others are only upregulated at 24 hours. Similar results are observed for downregulation of miRNA. As such, the regulation of miRNA biomarkers, the method of their detection, and the temporal alteration in expression of Redell, J, et al., J. Neurosci. Res., 2009; 87: 1435-1448 are each incorporated herein by reference as equally applicable to the subject invention. Similarly, the temporal nature of miRNA expression in response to stroke as observed by Jeyaseelan, K, et al, Stroke, 2008; 39:959-966 is also incorporated herein by reference for the particular miRNAs taught therein as well as the methods of isolation, quantification, and detection taught therein. As such, a preferred embodiment screens a biological sample for a first and a second biomarker. Greater numbers are similarly operable.

[0061] The detection of inventive biomarkers is also operable as a means to screen potential drug candidates or analyze safety of previously identified drug candidates. These assays may be either in vitro or in vivo. In vivo screening or assay protocols illustratively include measurement of a biomarker in an animal illustratively including a mouse, rat, or human. Studies to determine or monitor levels of miRNA' s are optionally combined with behavioral analyses or motor deficit analyses such as: motor coordination tests illustratively including Rotarod, beam walk test, gait analysis, grid test, hanging test and string test; sedation tests illustratively including those detecting spontaneous locomotor activity in the open-field test; sensitivity tests for allodynia - cold bath tests, hot plate tests at 38°C and Von Frey tests; sensitivity tests for hyperalgesia - hot plate tests at 52°C and Randall-Sellito tests; and EMG evaluations such as sensory and motor nerve conduction, Compound Muscle Action Potential (CMAP) and h-wave reflex.

[0062] The inventive biomarker analyses are illustratively operable to detect, diagnose, or treat a disease state or screen for chemical or other therapeutics to treat disease. Diseases or conditions illustratively screenable include but are not limited to: myelin involving diseases such as multiple sclerosis, stroke, amyotrophic lateral sclerosis (ALS), chemotherapy, cancer, Parkinson's disease, nerve conduction abnormalities stemming from chemical or physiological abnormalities such as ulnar neuritis and carpel tunnel syndrome, other peripheral neuropathies illustratively including sciatic nerve crush (traumatic neuropathy), streptozotozin (STZ) (diabetic neuropathy), antimitotic-induced neuropathies (chemotherapy-induced neuropathy), experimental autoimmune encephalomyelitis (EAE), delayed-type hypersensitivity (DTH), rheumatoid arthritis, epilepsy, pain, neuropathic pain, and intra-uterine trauma.

[0063] Analyses of blast injury to a subject produced several inventive correlations between miRNA's and neuronal injury. Neuronal injury is optionally the result of whole body blast, blast force to a particular portion of the body, or the result of other neuronal trauma or disease that produces detectable or differentiatable levels of biomarkers. Thus, identifying pathogenic pathways of primary blast brain injury (BBI) in reproducible experimental models is helpful to the development of diagnostic algorithms for differentiating severe, moderate and mild (mTBI) from posttraumatic stress disorder (PTSD). Accordingly, a number of experimental animal models have been implemented to study mechanisms of blast wave impact and include rodents and larger animals such as sheep. However, because of the rather generic nature of blast generators used in the different studies, the data on brain injury mechanisms and putative biomarkers have been difficult to analyze and compare.

[0064] To provide correlations between neurological condition and measured quantities of miRNA biomarkers and others, samples of CSF or serum are collected from subjects with the samples being subjected to measurement of miRNA biomarkers. The subjects vary in neurological condition. Detected levels of miRNA biomarkers are then correlated with CT scan results as well as GCS scoring. Based on these results, an inventive assay is developed and validated. It is appreciated that miRNA biomarkers, in addition to being obtained from CSF and serum, are also readily obtained from blood, plasma, saliva, urine, as well as solid tissue biopsy. While CSF is a preferred sampling fluid owing to direct contact with the nervous system, it is appreciated that other biological fluids have advantages in being sampled for other purposes and therefore allow for inventive determination of neurological condition as part of a battery of tests performed on a single sample such as blood, plasma, serum, saliva or urine. It should further ne appreciated that use of alternative fluids than CSF is further preferred because of the non- invasive and routine medical nature of taking such samples.

[0065] A biological sample is obtained from a subject by conventional techniques. For example, CSF is obtained by lumbar puncture. Blood is obtained by venipuncture, while plasma and serum are obtained by fractionating whole blood according to known methods. Surgical techniques for obtaining solid tissue samples are well known in the art. For example, methods for obtaining a nervous system tissue sample are described in standard neurosurgery texts such as Atlas of Neurosurgery: Basic Approaches to Cranial and Vascular Procedures, by F. Meyer, Churchill Livingstone, 1999; Stereotactic and Image Directed Surgery of Brain Tumors, 1st ed., by David G. T. Thomas, WB Saunders Co., 1993; and Cranial Microsurgery: Approaches and Techniques, by L. N. Sekhar and E. De Oliveira, 1st ed., Thieme Medical Publishing, 1999. Methods for obtaining and analyzing brain tissue are also described in Belay et al., Arch. Neurol. 58: 1673-1678 (2001); and Seijo et al, J. Clin. Microbiol. 38: 3892-3895 (2000).

[0066] Preferably, a biomarker is selective for detecting or diagnosing neurological conditions such as brain injury and the like. More preferably, a biomarker is both specific and effective for the detection and distinguishing levels of TBI. Such biomarkers are optionally termed neuro active biomarkers.

[0067] Biomarker analyses are preferably performed using biological samples or fluids. Illustrative biological samples operable herein illustratively include, cells, tissues, cerebral spinal fluid (CSF), artificial CSF, whole blood, serum, plasma, cytosolic fluid, urine, feces, stomach fluids, digestive fluids, saliva, nasal or other airway fluid, vaginal fluids, semen, buffered saline, saline, water, or other biological fluid recognized in the art.

[0068] In addition to increased cell expression, biomarkers also appear in biological fluids in communication with injured cells. Obtaining biological fluids such as cerebrospinal fluid (CSF), blood, plasma, serum, saliva and urine, from a subject is typically much less invasive and traumatizing than obtaining a solid tissue biopsy sample. Thus, samples that are biological fluids are preferred for use in the invention. CSF, in particular, is preferred for detecting nerve damage in a subject as it is in immediate contact with the nervous system and is readily obtainable. It is appreciated serum is a preferred biological sample as it is much more easily obtainable and presents much less risk of further injury or side-effect to a donating subject.

[0069] After insult, nerve cells in in vitro culture or in situ in a subject express altered levels or activities of one or more proteins or RNA molecules than do such cells not subjected to the insult. Thus, samples that contain nerve cells, e.g., a biopsy of a central nervous system or peripheral nervous system tissue are suitable biological samples for use in the invention. In addition to nerve cells, however, other cells express illustratively αΙΙ-spectrin including, for example, cardiomyocytes, myocytes in skeletal muscles, hepatocytes, kidney cells and cells in testis. A biological sample including such cells or fluid secreted from these cells might also be used in an adaptation of the inventive methods to determine and/or characterize an injury to such non-nerve cells.

[0070] A subject as used herein illustratively includes a dog, a cat, a horse, a cow, a pig, a sheep, a goat, a chicken, non-human primate, a human, a rat, guinea pig, hamster, and a mouse. Because the present invention preferably relates to human subjects, a preferred subject for the methods of the invention is a human being.

[0071] Subjects who most benefit from the present invention are those suspected of having or at risk for developing abnormal neurological conditions or injury, such as victims of brain injury caused by traumatic insults (e.g., gunshot wounds, automobile accidents, sports accidents, shaken baby syndrome, other percussive injuries), ischemic events (e.g., stroke, cerebral hemorrhage, cardiac arrest), neurodegenerative disorders (such as Alzheimer's, Huntington's, and Parkinson's diseases; prion-related disease; other forms of dementia), epilepsy, substance abuse (e.g., from amphetamines, Ecstasy/MDMA, or ethanol), and peripheral nervous system pathologies such as diabetic neuropathy, chemotherapy-induced neuropathy and neuropathic pain.

[0072] To provide correlations between a neurological condition and measured quantities of biomarkers CSF or serum are preferable biological fluids. Illustratively, samples of CSF or serum are collected from subjects with the samples being subjected to measurement of biomarkers. The subjects vary in neurological condition. Detected levels of one or more biomarkers are then correlated with either recognized or standardized baseline levels or optionally CT scan results as well as GCS scoring. Based on these results, an inventive assay is optionally developed and validated. It is appreciated that neuroactive biomarkers, in addition to being obtained from CSF and serum, are also readily obtained from blood, plasma, saliva, urine, as well as solid tissue biopsy. While CSF is a preferred sampling fluid owing to direct contact with the nervous system, it is appreciated that other biological fluids have advantages in being sampled for other purposes and therefore allow for inventive determination of neurological condition alone or as part of a battery of tests performed on a single sample such as blood, plasma, serum, saliva or urine.

[0073] Baseline levels of biomarkers are those levels obtained in the target biological sample in the species of desired subject in the absence of a known neurological condition. These levels need not be expressed in hard concentrations, but may instead be known from parallel control experiments and expressed in terms of fluorescent units, density units, and the like. Typically, in the absence of a neurological condition some miRNAs are present in biological samples at a negligible amount. However, other miR A's are highly abundant in neural tissue and fluids. Determining the baseline levels of the abundant miRNA's in neurons, plasma, or CSF, for example, of particular species is well within the skill of the art. Similarly, determining the concentration of baseline levels of other biomarkers is well within the skill of the art.

[0074] As used herein the term "diagnosing" means recognizing the presence or absence of a neurological or other condition such as an injury or disease. Diagnosing is optionally referred to as the result of an assay wherein a particular ratio or level of a biomarker is detected or is absent.

[0075] As used herein a "ratio" is either a positive ratio wherein the level of the target is greater than the target in a second sample or relative to a known or recognized baseline level of the same target. A negative ratio describes the level of the target as lower than the target in a second sample or relative to a known or recognized baseline level of the same target. A neutral ratio describes no observed change in target biomarker.

[0076] As used herein the term "administering" is delivery of a therapeutic to a subject. The therapeutic is administered by a route determined to be appropriate for a particular subject by one skilled in the art. For example, the therapeutic is administered orally, parenterally (for example, intravenously, by intramuscular injection, by intraperitoneal injection, intratumorally, by inhalation, or transdermally. The exact amount of therapeutic required will vary from subject to subject, depending on the age, weight and general condition of the subject, the severity of the neurological condition that is being treated, the particular therapeutic used, its mode of administration, and the like. An appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein or by knowledge in the art without undue experimentation.

[0077] An exemplary process for detecting the presence or absence of one or more neuroactive biomarkers in a biological sample involves obtaining a biological sample from a subject, such as a human, contacting the biological sample with a compound or an agent capable of detecting of the biomarker being analyzed, illustratively including a primer, a probe, antigen, or antibody, and analyzing the sample for the presence of the biomarker. It is appreciated that other detection methods are similarly operable illustratively contact with a protein or nucleic acid specific stain.

[0078] An inventive process can be used to detect miRNA biomarkers and one or more other miRNA biomarkers in a biological sample in vitro, as well as in vivo. The quantity of expression of miRNA biomarkers in a sample is compared with appropriate controls such as a first sample known to express detectable levels of the marker being analyzed (positive control) and a second sample known to not express detectable levels of the marker being analyzed (a negative control). For example, in vitro techniques for detection of a marker include enzyme linked immunosorbent assays (ELISAs), radioimmuno assay, radioassay, Western blot, Southern blot, northern blot, immunoprecipitation, immunofluorescence, mass spectrometry, RT-PCR, PCR, liquid chromatography, high performance liquid chromatography, enzyme activity assay, cellular assay, positron emission tomography, mass spectroscopy, combinations thereof, or other technique known in the art. Furthermore, in vivo techniques for detection of a marker include introducing a labeled agent that specifically binds the marker into a biological sample or test subject. For example, the agent can be labeled with a radioactive marker whose presence and location in a biological sample or test subject can be detected by standard imaging techniques.

[0079] RNA and DNA binding antibodies are known in the art. Illustratively, an RNA binding antibody is synthesized from a series of antibody fragments from a phage display library. Illustrative examples of the methods used to synthesize RNA binding antibodies are found in Ye, J, et al, PNAS USA, 2008; 105:82-87 the contents of which are incorporated herein by reference as methods of generating RNA binding antibodies. As such, it is within the skill of the art to generate antibodies to RNA based biomarkers.

[0080] DNA binding antibodies are similarly well known in the art. Illustrative methods of generating DNA binding antibodies are found in Watts, RA, et al, Immunology, 1990; 69(3): 348-354 the contents of which are incorporated herein by reference as an exemplary method of generating anti-DNA antibodies.

[0081] An antibody is optionally labeled. A person of ordinary skill in the art recognizes numerous labels operable herein. Labels and labeling kits are commercially available optionally from Invitrogen Corp, Carlsbad, CA. Labels illustratively include, fluorescent labels, biotin, peroxidase, radionucleotides, or other label known in the art.

[0082] A myriad of detectable labels that are operative in a diagnostic assay for miRNA's are known in the art. Agents used in methods for detecting miRNA's or another biomarkers are conjugated to a detectable label, e.g., an enzyme such as horseradish peroxidase. Agents labeled with horseradish peroxidase can be detected by adding an appropriate substrate that produces a color change in the presence of horseradish peroxidase. Several other detectable labels that may be used are known. Common examples of these include alkaline phosphatase, horseradish peroxidase, fluorescent compounds, luminescent compounds, colloidal gold, magnetic particles, biotin, radioisotopes, and other enzymes. It is appreciated that a primary/secondary antibody system is optionally used to detect one or more biomarkers. A primary antibody that specifically recognizes one or more biomarkers is exposed to a biological sample that may contain the biomarker of interest. A secondary antibody with an appropriate label that recognizes the species or isotype of the primary antibody is then contacted with the sample such that specific detection of the one or more biomarkers in the sample is achieved.

[0083] The present invention employs a step of correlating the presence or amount of at least one miR A or protein biomarker selected from Table 1 or Table 2 in a biological sample with the severity and/or type of nerve cell injury. The amount of miRNA biomarkers in the biological sample is associated with neurological condition for traumatic brain injury as detailed in the examples. The results of an inventive assay to synergistically measure miRNA biomarkers and one or more other biomarkers can help a physician determine the type and severity of injury with implications as to the types of cells that have been compromised. These results are in agreement with CT scan and GCS results, yet are quantitative, obtained more rapidly, and at far lower cost.

[0084] The present invention optionally includes the presence of one or more therapeutic agents that may alter one or more characteristics of a target biomarker. A therapeutic optionally serves as an agonist or antagonist of a target biomarker or upstream effector of a biomarker. A therapeutic optionally affects a downstream function of a biomarker. For example, Acetylcholine (Ach) plays a role in pathological neuronal excitation and TBI-induced muscarinic cholinergic receptor activation may contribute to excitotoxic processes. As such, biomarkers optionally include levels or activity of Ach or muscarinic receptors. Optionally, an operable biomarker is a molecule, protein, nucleic acid or other that is affected by the activity of muscarinic receptor(s). As such, therapeutics operable in the subject invention illustratively include those that modulate various aspects of muscarinic cholinergic receptor activation.

[0085] Specific mucarinic receptors operable as therapeutic targets or modulators of therapeutic targets include the Mi, M ₂ , M ₃ , M ₄ , and M ₅ muscarinic receptors.

[0086] The suitability of the muscarinic cholinergic receptor pathway in detecting and treating TBI arises from studies that demonstrated elevated ACh in brain cerebrospinal fluid (CSF) following experimental TBI (Gorman et al., 1989; Lyeth et al., 1993a) and ischemia (Kumagae and Matsui, 1991), as well as the injurious nature of high levels of muscarinic cholinergic receptor activation through application of cholinomimetics (Olney et al., 1983; Turski et al., 1983). Furthermore, acute administration of muscarinic antagonists improves behavioral recovery following experimental TBI (Lyeth et al., 1988a; Lyeth et al., 1988b; Lyeth and Hayes, 1992; Lyeth et al, 1993b; Robinson et al, 1990).

[0087] A therapeutic operable in the subject invention is illustratively any molecule, compound, family, extract, solution, drug, pro-drug, or other mechanism that is operable for changing, preferably improving, therapeutic outcome of a subject at risk for or victim of a neuronal injury such as TBI or MTBI. A therapeutic is optionally a muscarinic cholinergic receptor modulator such as an agonist or antagonist. An agonist or antagonist may by direct or indirect. An indirect agonist or antagonist is optionally a molecule that breaks down or synthesizes acetylcholine or other muscarinic receptor related molecule illustratively, molecules currently used for the treatment of Alzheimer's disease. Cholinic mimetics or similar molecules are operable herein. An exemplary list of therapeutics operable herein include: dicyclomine, scoplamine, milameline, N-methyl-4-piperidinylbenzilate NMP, pilocarpine, pirenzepine, acetylcholine, methacholine, carbachol, bethanechol, muscarine, oxotremorine M, oxotremorine, thapsigargin, calcium channel blockers or agonists, nicotine, xanomeline, BuTAC, clozapine, olanzapine, cevimeline, aceclidine, arecoline, tolterodine, rociverine, IQNP, indole alkaloids, himbacine, cyclostellettamines, deriviatives thereof, pro-drugs thereof, and combinations thereof. A therapeutic is optionally a molecule operable to alter the level of or activity of a calpain or caspase. Such molecules and their administration are known in the art.

[0088] An inventive method illustratively includes a process for diagnosing a neurological condition in a subject, treating a subject with a neurological condition, or both. In a preferred embodiment an inventive process illustratively includes obtaining a biological sample from a subject. The biological sample is assayed by mechanisms known in the art for detecting or identifying the presence of one or more biomarkers present in the biological sample. Based on the amount or presence of a target biomarker in a biological sample, a ratio of one or more biomarkers is optionally calculated. The ratio is optionally the level of one or more biomarkers relative to the level of another biomarker in the same or a parallel sample, or the ratio of the quantity of the biomarker to a measured or previously established baseline level of the same biomarker in a subject known to be free of a pathological neurological condition. The ratio allows for the diagnosis of a neurological condition in the subject. An inventive process also administers a therapeutic to the subject that will either directly or indirectly alter the ratio of one or more biomarkers.

[0089] An inventive process is also provided for diagnosing and treating a multiple-organ injury. Multiple organs illustratively include subsets of neurological tissue such as brain, spinal cord and the like, or specific regions of the brain such as cortex, hippocampus and the like. The inventive process illustratively includes assaying for a plurality of biomarkers in a biological sample obtained from a subject wherein the biological was optionally in fluidic contact with an organ suspected of having undergone injury or control organ when the biological sample was obtained from the subject. The inventive process determines a first subtype of organ injury in based on a first ratio of a plurality of biomarkers. The inventive process also determines a second subtype of a second organ injury based on a second ration of the plurality of biomarkers in the biological sample. The ratios are illustratively determined by processes described herein or known in the art.

[0090] Treatment of a multiple organ injury in the inventive process is illustratively achieved by administering to a subject at least one therapeutic antagonist or agonist effective to modulate the activity of a protein whose activity is altered in response to the first organ injury, and administering at least one therapeutic agonist or antagonist effective to modulate the activity of a protein whose activity is altered in response to a second organ injury.

[0091] Many amplification-based methods exist for detecting the level of miRNA nucleic acid sequences, including, but not limited to, PCR, RT-PCR, qPCR, and rolling circle amplification. Other amplification-based techniques include, for example, ligase chain reaction, multiplex ligatable probe amplification, in vitro transcription (IVT), strand displacement amplification, transcription-mediated amplification, RNA (Eberwine) amplification, and other methods that are known to persons skilled in the art.

[0092] A typical PCR reaction includes multiple steps, or cycles, that selectively amplify target nucleic acid species: a denaturing step, in which a target nucleic acid is denatured; an annealing step, in which a set of PCR primers (i.e., forward and reverse primers) anneal to complementary DNA strands, and an elongation step, in which a thermostable DNA polymerase elongates the primers. By repeating these steps multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target sequence. Typical PCR reactions include 20 or more cycles of denaturation, annealing, and elongation. In many cases, the annealing and elongation steps can be performed concurrently, in which case the cycle contains only two steps. A reverse transcription reaction (which produces a cDNA sequence having complementarity to a miRNA) may be performed prior to PCR amplification. Reverse transcription reactions include the use of, e.g., a RNA-based DNA polymerase (reverse transcriptase) and a primer.

[0093] Kits for quantitative real time PCR of miRNA are known, and are commercially available. Examples of suitable kits include, but are not limited to, the TaqMan.RTM. miRNA Assay (Applied Biosystems) and the mirVana.TM. qRT-PCR miRNA detection kit (Ambion). The miRNA can be ligated to a single stranded oligonucleotide containing universal primer sequences, a polyadenylated sequence, or adaptor sequence prior to reverse transcriptase and amplified using a primer complementary to the universal primer sequence, poly(T) primer, or primer comprising a sequence that is complementary to the adaptor sequence.

[0094] In some instances, custom qRT-PCR assays can be developed for determination of miRNA levels. Custom qRT-PCR assays to measure miRNAs in a biological sample, e.g., a body fluid, can be developed using, for example, methods that involve an extended reverse transcription primer and locked nucleic acid modified PCR. Custom miRNA assays can be tested by running the assay on a dilution series of chemically synthesized miRNA corresponding to the target sequence. This permits determination of the limit of detection and linear range of quantitation of each assay. Furthermore, when used as a standard curve, these data permit an estimate of the absolute abundance of miRNAs measured in biological samples.

[0095] Amplification curves may optionally be checked to verify that Ct values are assessed in the linear range of each amplification plot. Typically, the linear range spans several orders of magnitude. For each candidate miRNA assayed, a chemically synthesized version of the miRNA can be obtained and analyzed in a dilution series to determine the limit of sensitivity of the assay, and the linear range of quantitation. Relative expression levels may be determined, for example, according to the 2(-.DELTA.. DELTA. C(T)) Method, as described by Livak et al., Analysis of relative gene expression data using real-time quantitative PCR and the 2(-.DELTA.. DELTA. C(T)) Method. Methods (2001) December; 25(4):402-8.

[0096] In some embodiments, two or more miRNAs are amplified in a single reaction volume. For example, multiplex q-PCR, such as qRT-PCR, enables simultaneous amplification and quantification of at least two miRNAs of interest in one reaction volume by using more than one pair of primers and/or more than one probe. The primer pairs comprise at least one amplification primer that specifically binds each miRNA, and the probes are labeled such that they are distinguishable from one another, thus allowing simultaneous quantification of multiple miRNAs.

[0097] Rolling circle amplification is a DNA-polymerase driven reaction that can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions (see, for example, Lizardi et al, Nat. Gen. (1998) 19(3):225-232; Gusev et al, Am. J. Pathol. (2001) 159(l):63-69; Nallur et al, Nucleic Acids Res. (2001) 29(23):E118). In the presence of two primers, one hybridizing to the (+) strand of DNA, and the other hybridizing to the (-) strand, a complex pattern of strand displacement results in the generation of over 10 9 copies of each DNA molecule in 90 minutes or less. Tandemly linked copies of a closed circle DNA molecule may be formed by using a single primer. The process can also be performed using a matrix-associated DNA. The template used for rolling circle amplification may be reverse transcribed. This method can be used as a highly sensitive indicator of miRNA sequence and expression level at very low miRNA concentrations (see, for example, Cheng et al., Angew Chem. Int. Ed. Engl. (2009) 48(18):3268-72; Neubacher et al, Chembiochem. (2009) 10(8): 1289-91).

[0098] miRNA may also be detected using hybridization-based methods, including but not limited to hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, and in situ hybridization. Microarrays can be used to measure the expression levels of large numbers of miRNAs simultaneously. Microarrays can be fabricated using a variety of technologies, including printing with fine -pointed pins onto glass slides, photolithography using pre -made masks, photolithography using dynamic micromirror devices, ink-jet printing, or electrochemistry on microelectrode arrays. Also useful are microfluidic TaqMan Low-Density Arrays, which are based on an array of microfluidic qRT- PCR reactions, as well as related microfluidic qRT-PCR based methods. In one example of microarray detection, various oligonucleotides (e.g., 200+5 '-amino-modified-C6 oligos) corresponding to human sense miRNA sequences are spotted on three-dimensional CodeLink slides (GE Health/Amersham Biosciences) at a final concentration of about 20 .mu.M and processed according to manufacturer's recommendations. First strand cDNA synthesized from 20 .mu.g TRIzol-purified total RNA is labeled with biotinylated ddUTP using the Enzo Bio Array end labeling kit (Enzo Life Sciences Inc.). Hybridization, staining, and washing can be performed according to a modified Affymetrix Antisense genome array protocol.

[0099] Axon B-4000 scanner and Gene-Pix Pro 4.0 software or other suitable software can be used to scan images. Non-positive spots after background subtraction, and outliers detected by the ESD procedure, are removed. The resulting signal intensity values are normalized to per-chip median values and then used to obtain geometric means and standard errors for each miRNA. Each miRNA signal can be transformed to log base 2, and a one-sample t test can be conducted. Independent hybridizations for each sample can be performed on chips with each miRNA spotted multiple times to increase the robustness of the data.

[00100] Microarrays can be used for the expression profiling of miRNAs in diseases. For example, RNA can be extracted from a sample and, optionally, the miRNAs are size-selected from total RNA. Oligonucleotide linkers can be attached to the 5' and 3' ends of the miRNAs and the resulting ligation products are used as templates for an RT-PCR reaction. The sense strand PCR primer can have a fluorophore attached to its 5' end, thereby labeling the sense strand of the PCR product. The PCR product is denatured and then hybridized to the microarray. A PCR product, referred to as the target nucleic acid that is complementary to the corresponding miRNA capture probe sequence on the array will hybridize, via base pairing, to the spot at which the, capture probes are affixed. The spot will then fluoresce when excited using a microarray laser scanner.

[00101] The fluorescence intensity of each spot is then evaluated in terms of the number of copies of a particular miRNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular miRNA.

[00102] Total RNA containing the miRNA extracted from a body fluid sample can also be used directly without size-selection of the miRNAs. For example, the RNA can be 3' end labeled using T4 RNA ligase and a fluorophore-labeled short RNA linker. Fluorophore-labeled miRNAs complementary to the corresponding miRNA capture probe sequences on the array hybridize, via base pairing, to the spot at which the capture probes are affixed. The fiuorescence intensity of each spot is then evaluated in terms of the number of copies of a particular miRNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular miRNA. Several types of microarrays can be employed including, but not limited to, spotted oligonucleotide microarrays, pre-fabricated oligonucleotide microarrays or spotted long oligonucleotide arrays.

[00103] miRNAs can also be detected without amplification using the nCounter Analysis System (NanoString Technologies, Seattle, Wash.). This technology employs two nucleic acid- based probes that hybridize in solution (e.g., a reporter probe and a capture probe). After hybridization, excess probes are removed, and probe/target complexes are analyzed in accordance with the manufacturer's protocol. nCounter miRNA assay kits are available from NanoString Technologies, which are capable of distinguishing between highly similar miRNAs with great specificity. miRNAs can also be detected using branched DNA (bDNA) signal amplification (see, for example, Urdea, Nature Biotechnology (1994), 12:926-928). miRNA assays based on bDNA signal amplification are commercially available. One such assay is the QuantiGene.RTM. 2.0 miRNA Assay (Affymetrix, Santa Clara, Calif). [00104] Northern Blot and in situ hybridization may also be used to detect miRNAs. Suitable methods for performing Northern Blot and in situ hybridization are known in the art.

[00105] Advanced sequencing methods can likewise be used as available. For example, miRNAs can be detected using Illumina.RTM. Next Generation Sequencing (e.g., Sequencing- By-Synthesis or TruSeq methods, using, for example, the HiSeq, HiScan, GenomeAnalyzer, or MiSeq systems (Illumina, Inc., San Diego, Calif.)). miRNAs can also be detected using Ion Torrent Sequencing (Ion Torrent Systems, Inc., Gulliford, Conn.), or other suitable methods of semiconductor sequencing.

[00106] Mass spectroscopy can be used to quantify miRNA using RNase mapping. Isolated RNAs can be enzymatically digested with RNA endonucleases (RNases) having high specificity (e.g., RNase Tl, which cleaves at the 3'-side of all unmodified guanosine residues) prior to their analysis by MS or tandem MS (MS/MS) approaches. The first approach developed utilized the on-line chromatographic separation of endonuclease digests by reversed phase HPLC coupled directly to ESI-MS. The presence of posttranscriptional modifications can be revealed by mass shifts from those expected based upon the RNA sequence. Ions of anomalous mass/charge values can then be isolated for tandem MS sequencing to locate the sequence placement of the posttranscriptionally modified nucleoside.

[00107] Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has also been used as an analytical approach for obtaining information about posttranscriptionally modified nucleosides. MALDI-based approaches can be differentiated from ESI-based approaches by the separation step. In MALDI-MS, the mass spectrometer is used to separate the miRNA.

[00108] To analyze a limited quantity of intact miRNAs, a system of capillary LC coupled with nanoESI-MS can be employed, by using a linear ion trap-orbitrap hybrid mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific) or a tandem-quadrupole time-of-flight mass spectrometer (QSTAR.RTM. XL, Applied Biosystems) equipped with a custom-made nanospray ion source, a Nanovolume Valve (Valco Instruments), and a splitless nano HPLC system (DiNa, KYA Technologies). Analyte/TEAA is loaded onto a nano-LC trap column, desalted, and then concentrated. Intact miRNAs are eluted from the trap column and directly injected into a CI 8 capillary column, and chromatographed by RP-HPLC using a gradient of solvents of increasing polarity. The chromatographic eluent is sprayed from a sprayer tip attached to the capillary column, using an ionization voltage that allows ions to be scanned in the negative polarity mode. [00109] Additional methods for miRNA detection and measurement include, for example, strand invasion assay (Third Wave Technologies, Inc.), surface plasmon resonance (SPR), cDNA, MTDNA (metallic DNA; Advance Technologies, Saskatoon, SK), and single-molecule methods such as the one developed by US Genomics. Multiple miRNAs can be detected in a microarray format using a novel approach that combines a surface enzyme reaction with nanoparticle-amplified SPR imaging (SPRI). The surface reaction of poly(A) polymerase creates poly(A) tails on miRNAs hybridized onto locked nucleic acid (LNA) microarrays. DNA- modified nanoparticles are then adsorbed onto the poly(A) tails and detected with SPRI. This ultrasensitive nanoparticle-amplified SPRI methodology can be used for miRNA profiling at attamole levels.

[00110] In certain embodiments, labels, dyes, or labeled probes and/or primers are used to detect amplified or unamp lifted miRNAs. The skilled artisan will recognize which detection methods are appropriate based on the sensitivity of the detection method and the abundance of the target. Depending on the sensitivity of the detection method and the abundance of the target, amplification may or may not be required prior to detection. One skilled in the art will recognize the detection methods where miRNA amplification is preferred.

[00111] A probe or primer may include standard (A, T or U, G and C) bases, or modified bases. Modified bases include, but are not limited to, the AEGIS bases (from Eragen Biosciences), which have been described, e.g., in U.S. Pat. Nos. 5,432,272, 5,965,364, and 6,001,983. In certain aspects, bases are joined by a natural phosphodiester bond or a different chemical linkage. Different chemical linkages include, but are not limited to, a peptide bond or a Locked Nucleic Acid (LNA) linkage, which is described, e.g., in U.S. Pat. No. 7,060,809.

[00112] In a further aspect, oligonucleotide probes or primers present in an amplification reaction are suitable for monitoring the amount of amplification product produced as a function of time. In certain aspects, probes having different single stranded versus double stranded character are used to detect the nucleic acid. Probes include, but are not limited to, the 5'- exonuclease assay (e.g., TaqMan.TM.) probes (see U.S. Pat. No. 5,538,848), stem-loop molecular beacons (see, e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517), stemless or linear beacons (see, e.g., WO 9921881, U.S. Pat. Nos. 6,485,901 and 6,649,349), peptide nucleic acid (PNA) Molecular Beacons (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, e.g. U.S. Pat. No. 6,329,144), non-FRET probes (see, e.g., U.S. Pat. No. 6,150,097), Sunrise. TM./AmplifluorB.TM. probes (see, e.g., U.S. Pat. No. 6,548,250), stem-loop and duplex Scorpion.TM. probes (see, e.g., U.S. Pat. No. 6,589,743), bulge loop probes (see, e.g., U.S. Pat. No. 6,590,091), pseudo knot probes (see, e.g., U.S. Pat. No. 6,548,250), cyclicons (see, e.g., U.S. Pat. No. 6,383,752), MGB Eclipse.TM. probe (Epoch Biosciences), hairpin probes (see, e.g., U.S. Pat. No. 6,596,490), PNA light-up probes, antiprimer quench probes (Li et al, Clin. Chem. 53:624-633 (2006)), self-assembled nanoparticle probes, and ferrocene-modified probes described, for example, in U.S. Pat. No. 6,485,901.

[00113] In certain embodiments, one or more of the primers in an amplification reaction can include a label. In yet further embodiments, different probes or primers comprise detectable labels that are distinguishable from one another. In some embodiments a nucleic acid, such as the probe or primer, may be labeled with two or more distinguishable labels.

[00114] In some aspects, a label is attached to one or more probes and has one or more of the following properties: (i) provides a detectable signal; (ii) interacts with a second label to modify the detectable signal provided by the second label, e.g., FRET (Fluorescent Resonance Energy Transfer); (iii) stabilizes hybridization, e.g., duplex formation; and (iv) provides a member of a binding complex or affinity set, e.g., affinity, antibody-antigen, ionic complexes, hapten-ligand (e.g., biotin-avidin). In still other aspects, use of labels can be accomplished using any one of a large number of known techniques employing known labels, linkages, linking groups, reagents, reaction conditions, and analysis and purification methods.

[00115] miRNAs can be detected by direct or indirect methods. In a direct detection method, one or more miRNAs are detected by a detectable label that is linked to a nucleic acid molecule. In such methods, the miRNAs may be labeled prior to binding to the probe. Therefore, binding is detected by screening for the labeled miRNA that is bound to the probe. The probe is optionally linked to a bead in the reaction volume.

[00116] In certain embodiments, nucleic acids are detected by direct binding with a labeled probe, and the probe is subsequently detected. In one embodiment of the invention, the nucleic acids, such as amplified miRNAs, are detected using FlexMAP Microspheres (Luminex) conjugated with probes to capture the desired nucleic acids. Some methods may involve detection with polynucleotide probes modified with fluorescent labels or branched DNA (bDNA) detection, for example.

[00117] In other embodiments, nucleic acids are detected by indirect detection methods. For example, a biotinylated probe may be combined with a streptavidin-conjugated dye to detect the bound nucleic acid. The streptavidin molecule binds a biotin label on amplified miRNA, and the bound miRNA is detected by detecting the dye molecule attached to the streptavidin molecule. In one embodiment, the streptavidin-conjugated dye molecule comprises Phycolink.RTM. Streptavidin R-Phycoerythrin (PROzyme). Other conjugated dye molecules are known to persons skilled in the art.

[00118] Labels include, but are not limited to: light-emitting, light-scattering, and light- absorbing compounds which generate or quench a detectable fluorescent, chemiluminescent, or bioluminescent signal (see, e.g., Kricka, L., Nonisotopic DNA Probe Techniques, Academic Press, San Diego (1992) and Garman A., Non-Radioactive Labeling, Academic Press (1997).). A dual labeled fluorescent probe that includes a reporter fluorophore and a quencher fluorophore is used in some embodiments. It will be appreciated that pairs of fluorophores are chosen that have distinct emission spectra so that they can be easily distinguished.

[00119] In certain embodiments, labels are hybridization-stabilizing moieties which serve to enhance, stabilize, or influence hybridization of duplexes, e.g., intercalators and intercalating dyes (including, but not limited to, ethidium bromide and SYBR-Green), minor-groove binders, and cross-linking functional groups (see, e.g., Blackburn et al, eds. "DNA and RNA Structure" in Nucleic Acids in Chemistry and Biology (1996)).

[00120] In other embodiments, methods relying on hybridization and/or ligation to quantify miRNAs may be used, including oligonucleotide ligation (OLA) methods and methods that allow a distinguishable probe that hybridizes to the target nucleic acid sequence to be separated from an unbound probe. As an example, HARP-like probes, as disclosed in U.S. Publication No. 2006/0078894 may be used to measure the quantity of miRNAs. In such methods, after hybridization between a probe and the targeted nucleic acid, the probe is modified to distinguish the hybridized probe from the unhybridized probe. Thereafter, the probe may be amplified and/or detected. In general, a probe inactivation region comprises a subset of nucleotides within the target hybridization region of the probe. To reduce or prevent amplification or detection of a HARP probe that is not hybridized to its target nucleic acid, and thus allow detection of the target nucleic acid, a post-hybridization probe inactivation step is carried out using an agent which is able to distinguish between a HARP probe that is hybridized to its targeted nucleic acid sequence and the corresponding unhybridized HARP probe. The agent is able to inactivate or modify the unhybridized HARP probe such that it cannot be amplified.

[00121] A probe ligation reaction may also be used to quantify miRNAs. In a Multiplex Ligation-dependent Probe Amplification (MLPA) technique (Schouten et al., Nucleic Acids Research 30:e57 (2002)), pairs of probes which hybridize immediately adjacent to each other on the target nucleic acid are ligated to each other driven by the presence of the target nucleic acid. In some aspects, MLPA probes have flanking PCR primer binding sites. MLPA probes are specifically amplified when ligated, thus allowing for detection and quantification of miRNA biomarkers.

[00122] The subject invention illustratively includes a composition for distinguishing the magnitude of a neurological condition in a subject. An inventive composition is either an agent entity or a mixture of multiple agents. In a preferred embodiment a composition is a mixture. The mixture optionally contains a biological sample derived from a subject. The subject is optionally suspected of having a neurological condition. The biological sample in communication with the nervous system of the subject prior to being isolated from the subject.

[00123] The kit is also provided that encompasses a substrate suitable for associating with the target biomarker in a biological sample. The biological sample is optionally provided with the kit or is obtained by a practitioner for use with an inventive kit. An inventive kit also includes at least two antibodies that specifically and independently bind to at least two biomarkers. The antibodies preferably distinguish between the two biomarkers. Preferably, a first antibody is specific and independent for binding and detecting a first biomarker. A second antibody is specific and independent for binding and detecting a second biomarker. In this way the presence or absence of multiple biomarkers in a single biological sample can be determined or distinguished.

[00124] In the kit, the biological sample can be CSF or blood, and the agent can be an antibody, aptamer, primer, probe, or other molecule that specifically binds at least one biomarker for a neurological condition. Suitable agents are described above. The kit can also include a detectable label such as one conjugated to the agent, or one conjugated to a substance that specifically binds to the agent (e.g., a secondary antibody).

[00125] The invention employs a step of correlating the presence or amount of a biomarker in a biological sample with the severity and/or type of nerve cell (or other biomarker-expressing cell) injury. The amount of biomarker(s) in the biological sample directly relates to severity of nerve tissue injury as a more severe injury damages a greater number of nerve cells which in turn causes a larger amount of biomarker(s) to accumulate in the biological sample (e.g., CSF; serum).

[00126] Also, the level of or kinetic extent of miRNA biomarkers present in a biological sample may optionally distinguish mild injury from a more severe injury. In an illustrative example, severe MCAO (2h) produces modulated levels of miRNA in both CSF and serum relative to mild challenge (30 min) while both produce miRNA levels in excess of uninjured subjects. Moreover, the persistence or kinetic extent of the markers in a biological sample is indicative of the severity of the injury with greater injury indicating increases persistence of miRNA biomarkers in the subject that is measured by an inventive process in biological samples taken at several timepoints following injury.

[00127] The results of such a test can help a physician determine whether the administration a particular therapeutic such as calpain and/or caspase inhibitors or muscarinic cholinergic receptor antagonists might be of benefit to a patient. This application may be especially important in detecting age and gender difference in cell death mechanism.

[00128] Methods involving conventional biological techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed. Ausubel et al, Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Immunological methods (e.g., preparation of antigen-specific antibodies, immunoprecipitation, and immunoblotting) are described, e.g., in Current Protocols in Immunology, ed. Coligan et al, John Wiley & Sons, New York, 1991; and Methods of Immunological Analysis, ed. Masseyeff et al, John Wiley & Sons, New York, 1992.

[00129] Various aspects of the present invention are illustrated by the following non-limiting examples. The examples are for illustrative purposes and are not a limitation on any practice of the present invention. It will be understood that variations and modifications can be made without departing from the spirit and scope of the invention. While the examples are generally directed to mammalian tissue, specifically, analyses of rat tissue, a person having ordinary skill in the art recognizes that similar techniques and other techniques know in the art readily translate the examples to other mammals such as humans. Reagents illustrated herein are commonly cross reactive between mammalian species or alternative reagents with similar properties are commercially available, and a person of ordinary skill in the art readily understands where such reagents may be obtained.

Material and Methods

Animals

[00130] Male Sprague-Dawley rats (200-250 g) are randomly divided into control and TBI groups. After 4 weeks, the animals (n=6/group) are sacrificed and the brain tissues are flash- frozen for gene expression and microRNA analysis or placed in 10% formalin for paraffin embedding.

Microarray Analysis for miRNA Expression [00131] MicroRNAs are extracted from brain tissues using the mirVana miRNA isolation kit (Ambion Inc., Austin, Tex., USA). Briefly, the tissues are homogenized in the Lysis/Binding solution. miRNA additive (1 : 10) and equal volume acid-phenol:chloroform is added to the lysate and incubated for 10 min on ice. Following centrifugation and removal of the aqueous phase, the mixture is incubated in ethanol. The mixture is passed through the filter cartridge and is eluted with elution solution.

[00132] Brain tissue miRNA arrays are analyzed using Agilent miRNA Arrays

(http ://assuragen. com).

[00133] PCR-based miRNA array analyses are carried out to examine alteration of miRNAs expression in human brain tissue that suffered TBI using TaqMan PCR system according to the manufacturer's instructions.

miRNA Analysis by RT-PCR

[00134] miRNAs are isolated using mirVana kit Real-time PCR is used to validate array data using primers. For a final reaction volume of 20 μί, the following reagents are added: 10 TaqMan twice. Universal PCR Master Mix (No AmpErase UNG), 8 Nuclease-free water, 1 μΐ _^ TaqMan microRNA probe and 1 cDNA product. The data is normalized to account for differences in reverse-transcription efficiencies and amount of template in the reaction mixtures. Western Blotting

[00135] TBI tissues are homogenized, centrifuged and resuspended in ice cold RIPA lysis buffer (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM

EDTA). Cell lysates are subsequently sonicated and the protein is quantified by BCA assay (Pierce Endogen, IL, USA). To confirm expression of MAPK (ERK1/2) (New England Biolab), 30 μg of protein was loaded and size-fractionated on a 10%> SDS-PAGE and blotted overnight onto a PVDF membrane (BIO-RAD Hercules, Calif, USA). Nonspecific sites were blocked in a 5% solution of nonfat milk powder in TBS. This was followed by incubation with goat anti- rabbit secondary IgG antibody with horseradish peroxidase conjugate (Santa Cruz Biotechnology Inc., CA, USA) using 1 : 1000 and 1 :5000 dilutions, respectively. Lysates are visualized with enhanced chemiluminescence Advance Western blot detection system (Amersham Biosciences, Piscataway, N.J., USA).

EXAMPLE 1

[00136] In vivo model of TBI injury model: A controlled cortical impact (CCI) device is used to model TBI on rats as previously described (Pike et al, 1998). Adult male (280-300 g) Sprague- Dawley rats (Harlan: Indianapolis, IN) are anesthetized with 4% isoflurane in a carrier gas of 1 : 1 0 ₂ /Ν ₂ 0 (4 min.) and maintained in 2.5% isoflurane in the same carrier gas. Core body temperature is monitored continuously by a rectal thermistor probe and maintained at 37±1°C by placing an adjustable temperature controlled heating pad beneath the rats. Animals are mounted in a stereotactic frame in a prone position and secured by ear and incisor bars. (See Figure 4). Following a midline cranial incision and reflection of the soft tissues, a unilateral (ipsilateral to site of impact) craniotomy (7 mm diameter) is performed adjacent to the central suture, midway between bregma and lambda. The dura mater is kept intact over the cortex. Brain trauma is produced by impacting the right (ipsilateral) cortex with a 5 mm diameter aluminum impactor tip (housed in a pneumatic cylinder) at a velocity of 3.5 m/s with a 1.6 mm compression and 150 ms dwell time. Sham-injured control animals are subjected to identical surgical procedures but do not receive the impact injury. Appropriate pre- and post-injury management is preformed to insure compliance with guidelines set forth by the University of Florida Institutional Animal Care and Use Committee and the National Institutes of Health guidelines detailed in the Guide for the Care and Use of Laboratory Animals. In addition, research is conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to principles stated in the "Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 edition."

EXAMPLE 2

[00137] Middle cerebral artery occlusion (MCAO) injury model: Rats are incubated under isoflurane anesthesia (5% isoflurane via induction chamber followed by 2% isoflurane via nose cone), the right common carotid artery (CCA) of the rat is exposed at the external and internal carotid artery (ECA and ICA) bifurcation level with a midline neck incision. The ICA is followed rostrally to the pterygopalatine branch and the ECA is ligated and cut at its lingual and maxillary branches. A 3-0 nylon suture is then introduced into the ICA via an incision on the ECA stump (the suture's path was visually monitored through the vessel wall) and advanced through the carotid canal approximately 20 mm from the carotid bifurcation until it becomes lodged in the narrowing of the anterior cerebral artery blocking the origin of the middle cerebral artery. The skin incision is then closed and the endo vascular suture left in place for 30 minutes or 2 hours. Afterwards the rat is briefly reanesthetized and the suture filament is retracted to allow reperfusion. For sham MCAO surgeries, the same procedure is followed, but the filament is advanced only 10 mm beyond the internal-external carotid bifurcation and is left in place until the rat is sacrificed. During all surgical procedures, animals are maintained at 37 ± 1°C by a homeothermic heating blanket (Harvard Apparatus, Holliston, MA, U.S.A.). It is important to note that at the conclusion of each experiment, if the rat brains show pathologic evidence of subarachnoid hemorrhage upon necropsy they are excluded from the study. Appropriate pre- and post-injury management is preformed to insure compliance with all animal care and use guidelines.

EXAMPLE 3

[00138] Tissue and Sample Preparation: At the appropriate time points (2, 6, 24 hours and 2, 3, 5 days) after injury, animals are anesthetized and immediately sacrificed by decapitation. Brains are quickly removed, rinsed with ice cold PBS and halved. The right hemisphere (cerebrocortex around the impact area and hippocampus) is rapidly dissected, rinsed in ice cold PBS, snap-frozen in liquid nitrogen, and stored at -80°C until used. For immunohistochemistry, brains are quick frozen in dry ice slurry, sectioned via cryostat (20 μιη) onto SUPERFROST PLUS GOLD® (Fisher Scientific) slides, and then stored at -80°C until used. For the left hemisphere, the same tissue as the right side is collected. For Western blot analysis, the brain samples are pulverized with a small mortar and pestle set over dry ice to a fine powder. The pulverized brain tissue powder is then lysed for 90 min at 4°C in a buffer of 50 mM Tris (pH 7.4), 5 mM EDTA, 1% (v/v) Triton X-100, 1 mM DTT, lx protease inhibitor cocktail (Roche Biochemicals). The brain lysates are then centrifuged at 15,000xg for 5 min at 4°C to clear and remove insoluble debris, snap-frozen, and stored at -80°C until used.

[00139] Candidate miRNA biomarkers identified using the high-throughput Nanostring platform were then validated by using the stem-loop TaqMan RT-qPCR miRNA assay (Life Technologies). Briefly, a RT primer pool was created with specific miRNA RT primers (miR- 34a, miR-31, miR-206, miR-34c, miR-410, miR-137, miR-7a, mir-579, mir-548f, mir-548e, mir- 527, mir-153, mir-518a-5p, mir-182, mir-382, mir-495, mir-181b, mir-181c, mir-590-3p, mir- 181d, mir-377, mir-15b, mir-548g, mir-1279, mir-582-5p, mir-103, mir-130a, mir-301b, mir-96, mir-27b, mir-548c-3p, mir-203, mir-370, mir-141, mir-200a, mir-548p, mir-548a-3p, mir-19a, mir-454, mir-130b, mir-27a, mir-7, mir-338-5p, mir-148a, mir-148b, mir-152, mir-561, mir-19b, mir-525-5p, mir-576-5p) at a final concentration of 0.05.times, in 1. times. TE. miRNA-specific primers and probes were purchased from Life Technologies. A 15 .mu.l RT reaction was set up containing 6 .mu.l of the RT primer pool, 0.3 .mu.L dNTPS (100 mM), 3 .mu.l of Multiscribe RT (50 U/.mu.l), 1.5 .mu.l of the lO.times. Reverse Transcription buffer and 0.2 .mu.l of RNAseIN (20 U/.mu.l) and water. All Reverse Transcription components were contained in the miRNA RT kit (Life Technologies; #4366596). Three .mu.l of total RNA (1 : 10 dilution) was added as template for each sample and the reaction was incubated on ice for 5 min followed by 30 min at 16. degree. C, 30 min at 42. degree. C, and 5 min at 85. degree. C. for enzyme inactivation. The reaction was then stored at 4. degree. C. A second pool of pre-amplification primers was then created with each PCR primer probe (20.times.) for the same assays mixed at a final concentration of 0.2.times. in 1. times. TE. A pre-amplification reaction was set up containing 2.times.. This was followed by a 99.9. degree. C. incubation for 10 min, and then the reaction was diluted by adding 175 .mu.l of 0.1. times. TE (pH 8.0) and mixed by inversion. Two .mu.l of the pre-amplification product was then used for individual standard TaqMan qPCR reactions (in duplicate) following standard protocol (Life Technologies; P/N 4364031 -Rev D) on an ABI7500 instrument. Ct values were calculated by setting a manual threshold of 0.2 and an automatic baseline for all the reactions in a single study (SDS 2.4, Life Technologies) for uniform analysis.

[00140] The .DELTA..DELTA.Ct method (Life Technologies, see also Livak K J, Schmittgen T D, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-.DELTA..DELTA. C(T)) Method. Methods. (2001) December; 25(4):402-8) was used for analysis, with the geometric mean of each of the miRNAs, and the average relative Ct values of NC patients being used to calibrate all the individual values. Linear fold changes were then calculated and plotted on scatter plots using Prism (GraphPad Prism Software, San Diego, Calif- USA), (see Figure 5 and Table 2). Protein biomarkers normally associated with TBI were further analyzed to determine well accepted correlations of injury. Interleukins (1, 10), Selectin (E,L), NSE, GFAP, CNPase, and UCH-L1 were all measured and showed correlations of injury to validate the fold change modulations found in miRNAs. (See Figures 6-8)

EXAMPLE 4

Single Step Procedure for the Capture of miRNAs

[00141] As they are presented in biological fluids, miRNAs are scarce and contaminated by large amounts of other nucleic acids (including RNA), other macromolecules (including proteins), and a rich collection of small molecular species. This makes it challenging to capture miRNAs in high yield by enzymatic steps, especially those present in low concentrations. Instead, this is a class of recovery task well suited for "click" chemical reactions where a single reagent transforms the miRNA into a reactive species that does a "click" chemical reaction that binds the miRNA to a solid support.

[00142] A periodate or other hypervalent iodine species is used that specifically converts the 2',3'-diol at the 3'-end of the miRNA to a species that has two carbonyl (C=0) groups. This reagent is used to cleave fluorescent tags from nucleosides at each step in a "sequencing using cyclic reversible termination". At room temperature with lOOmM reagent, the reaction is >99% complete in one minute.

[00143] Many miRNA-detection architectures concentrated miRNA by first resolving a sample using gel electrophoresis. This has two disadvantages: (a) the volume of sample that can be processed is limited by the capacity of the gel and (b) gel electrophoresis is slow and requires talented staff to perform.

[00144] Accordingly, the assay uses reagents that are placed inside of size-exclusion beads, similar to those used for size-exclusion chromatography of proteins. This allows an indefinite volume of serum, urine, or other sample to be passed through a column, by passive gravity driven flow. Only the small miRNAs enter the size-excluded volume; large RNA and protein molecules pass directly through the support. Within the size-excluded volume are bound two reagents. The first is a periodate equivalent that generates carbonyl groups from the 2',3'-diol unit at the 3 '-end of the miRNA. Then, the C=0 group as an imine. Alternatively, solution periodate can be used to generate the C=0 units in the bulk solution, with only small miRNAs entering the capture zone because of size exclusion to encounter the imine forming reagent.

[00145] Figure 9 provides a schematic for the planned architecture to detect miRNA. Separation. Passive flow (driven by gravitation or by capillary flow) occurs through a size exclusion stationary phase will allow short (20-22 nucleotide) miRNAs to enter a capture zone within a "size excluded" volume; larger nucleic acids and most proteins move along the support without entering the capture zone. The sample is first diluted with dimethylsulfoxide as a denaturant and stabilizer. Once inside the capture volume, the miRNA analyte is cleaved by a support-bound periodate equivalent to yield a dialdehyde (shown) or a diketone. In the same capture volume, this dialdehyde is immediately converted to an oxime by reacting with an aminoxy unit that is also immobilized within the capture zone. By trapping scarce miRNA by chemical reactions having half-lives measured in seconds, this architecture does not rely on the success of enzymatic reactions that must recruit multiple substrates (as in polyA tailing). So captured, the beads can be hybridized directly to various miRNAs carrying different fluorescent tags to determine directly whether miRNAs were present in the biological sample. Here, ca. 10 ¹⁴ molecules of miRNA are required for observation to be direct. Thus, this is useful for detecting miRNAs in large amounts (or, conversely, by passing arbitrarily large amounts of fluid through the support). [00146] With the scarce miRNA captured and secured inside of size-exclusion beads, downstream reactions can occur as quickly as desired or (if the sample must be stored) at leisure. Many different downstream architectures are possible. Here, the dimethylsulfoxide is removed, allowing the size exclusion gel to swell, and allowing the entry of T4 RNA ligase (mw 43500 daltons) and an AEGIS tag with a 5 '-end blocked. This creates a chimera that is ready for PCR where one primer is specific for the miRNA and the other is general for the AEGIS tag. A linear amplification is made by run-off reverse transcriptase copying of the bound chimera.

[00147] The carbonyl compound is then immediately captured as an oxime through the reaction of an aminoxy unit that is attached to the 5 '-end of an immobilized DNA molecule that contains AEGIS components. This chemical reaction is likewise quite rapid, and leads to a miRNA-AEGIS chimera and that is immobilized within the size-excluded volume until such time as it is analyzed. Analogous chemistry is used in the SNAP2 architecture reported by the Benner laboratory to generate on-template assembly of primers. This sample capture architecture is expected to be both robust and free of bias. Any microRNA that enters the size excluded volume will encounter between 1000 and 10000 periodate equivalents before exiting, ensuring that it will certainly be converted to an oxime-capturable form. Once formed, the carbonyl-containing product will encounter between 1000 and 10000 aminoxy molecules before it can leave that volume, ensuring essentially quantitative capture. The size exclusion principle protects the capture miRNA from any enzymes that might degrade it. Therefore, we expect that the substrate holding the miRNA will be storeable and stable to storage and transport. Further, we expect that entry of miRNAs into the size-excluding space will be sequence-independent, as all miRNAs have the same size. Unlike with electrophoretic separation, this sample preparation is quick and inexpensive to run.

EXAMPLE 6

Amplification of the Chemically Captured miRNAs

[00148] The simplest architecture, shown in Figure 9, generates a few copies of the target by priming from the bound miRNA target to give an AEGIS -tagged product via run-off reverse transcription. While this permits the detection of abundant miRNAs, especially if a large amount of serum has been passed through the size-exclusion column, positive identification of scarce miRNAs requires amplification. Thus, after the miRNA molecules are captured, the advantages of miRNAs as nucleic acids (they can be PCR amplified) must be exploited. Here, two features of the products of the click chemistry are exploited. First, although the oxime linkage used to capture the miRNAs is quite stable, it can be undone by a reaction that generated another oxime. Thus, the chimera is released from the support by treating it with 100 mM acetone or biacetyl to generate a free AEGIS-miRNA chimera. Here, it is amplified in solution in a standard PCR reaction. Alternatively, it is captured by hybridization to a complementary segment of AEGIS DNA, either free in solution or on a support. Here, labeled probes having a complementary sequence. Second, the dialdehyde itself has unusual "click" reactivity. If treated with carboxymorpholine, it will suffer beta elimination to cleave off the oxidized nucleotide to generate an miRNA sequence that is one nucleotide shorter, entirely natural in structure, and having a 3'-0-phosphate unit (Figure 10). The 3 '-Ophosphate is also cleaved, in one pot in situ, to generate an entirely natural RNA molecule.

[00149] Figure 10. After being concentrated and secured within a bead, the miRNA can be observed or linearly amplified while attached (see Figure 9), or as illustrated in Figure 10 it can be released by introducing a second C=0 species (here, acetone). Treatment with carboymorpholine causes spontaneous fragmentation of the dialdehyde, generating a native miRNA (shortened by one nucleotide) with a 3 '-phosphate group, which can in turn be removed to yield another end that can be ligated using T4 RNA ligase, tailed, or otherwise processed. Because of the "click" capture, none of these reactions need be done in complex biological fluids at high dilution. Further, if an Aegis-tag has been placed at the 5 '-end of the miRNA sequence, the downstream processing can be done on chimeras attached to a support through AEGIS- AEGIS hybridization.

[00150] With these tools added to the click chemistry, size exclusion capture, AEGIS, SAMRS, and SNAP2 technologies mentioned above, the analytical chemist has full control over where the miRNA molecules go once they have been concentrated and captured within the size exclusion volume. With -100% capture of the initial miRNA, the goal is to construct a downstream architecture that does not lose any molecules before they are able to be PCR amplified. While many architectures are conceivable, we see certain advantages in the architecture described in Figure 11, which uses the bound miRNA to serve as a template for ligation.

[00151] Here, while the miRNA is still bound as a double oxime to the support, two sets of chimeric AEGIS -DNA probes are introduced. These are designed to have standard DNA segments at their ends that allows them to bind flush and adjacent to the miRNA; their AEGIS ends are single stranded, and are specific for the miRNA that their standard ends bind to. When ligase is added, a new oligomer, approximately 60 nucleotides long, is created. Because no AEGIS nucleotides are found in any natural sequences, the amplicon is entirely novel in any environment. Further, because the AEGIS tags are designed to be different for different miRNAs, different entirely novel amplicons will be created on the solid support for each miRNA. GACTZP PCR amplification of these will generate amplicons that will be captured with probes attached to Luminex beads or arrays carrying the miRNA sequence itself. Figure 11 shows that after being concentrated and secured within a bead, the miRNA can serve as the template for the ligation of two fragments that are AEGIS -standard DNA chimeras. The standard segments (green) bind to the bound miRNA and are ligated, either chemically or enzymatically. The AEGIS-segments (illustrated at the bottom) have different sequences for different miRNA targets. Thus, the ligation creates an entirely new target for GACTZP PCR, one that cannot be found in any natural biological sample, and one that is specific for the particular miRNA that served as the ligation template.

[00152] For downstream processing, direct PCR is, of course, the simplest approach. However, in the presence of T4 RNA ligase or CircLigase, the product can be cyclized to give a 60mer single stranded product which is a hybrid of DNA and RNA and that contains a single unnatural linker. Here, the strategic use of a support allows the cyclization to occur after all of the AEGIS -DNA chimeras have been washed away. This prevents complication by the ligase- catalyzed formation of concatamers. This cyclized product can then be isothermally amplified using SSII reverse transcriptase to give a concatamer of many identical sequences Here, the orthogonality of AEGIS nucleobases (they do not bind to standard nucleobases) ensures clean amplification. As the ligated AEGIS -DNA chimera is made from DNA, concatenated products are formed in high yields and for substantial lengths.

EXAMPLE 7

Using Toehold Catalysis and Self-Avoiding Molecular Recognition Systems (SAMRS)

[00153] A flexible assay to detect single miRNAs presented in biological fluids at low concentrations is provided for use in a clinical laboratory. First an increasing the level of multiplexing, then decreasing the number of PCR cycles that are required to get detectable signal from scarce miRNA products. Self-avoiding molecular recognition systems (SAMRS) can be used to allow indefinitely large amounts of multiplexing. In the ligase-based architecture, the principle problem in the ligation of libraries is that library components will find other components that have sufficient matching to allow offtarget ligation, which leads to "a mess". Adding SAMRS nucleotides into certain regions of the probes (illustrated in Figures 11 & 12 as the upper most sequences) solves this problem. Figure 12 illustrates non-enzymatic amplification. Non-enzymatic amplification requires that a ligated product dissociate from the bound miRNA to allow it to template a second (and third ...) ligation. A "toehold" (lower left sequence in Figure 12 (left) and splicing the SAMRS (right)) attached to one ligating component allows this by binding to a short part of the single stranded AEGIS region of its partner. This allows the standard magenta region to invade the duplex that holds the ligated product to the covalently bound miRNA. This "peels off the ligated product, releasing it to solution for PCR amplification, and allowing the bound miRNA to join still more fragments. One approach to decrease the amount of downstream PCR is to get the covalently bound miRNA to template the ligation of more than one AEGIS -DNA chimera. Normally, this is not possible without temperature cycling. However, if one attaches to one ligating component a short segment of AEGIS DNA that is complementary to a short segment of the single stranded AEGIS region of its ligation partner, and attach to that a short segment of standard DNA that is complementary to the 3 '-end of the miRNA, the ligated product will have a tail that uses the single stranded region of the AEGIS DNA as a toehold to invade the duplex that holds the ligated product to the miRNA (Figure 12). This allows the lower left illustrated region to "peel off the ligated product, releasing it to solution for PCR amplification, and allowing the bound miRNA to join still more fragments.

Other Embodiments

[00154] The foregoing description is illustrative of particular embodiments of the invention, but is not meant to be a limitation upon the practice thereof. The following claims, including all equivalents thereof, are intended to define the scope of the invention.

[00155] All references mentioned herein are each incorporated herein by reference as if the contents of each reference were fully and explicitly included for the materials for which each reference is cited.