mTOR pathway inhibition prevents neuroinflammation and neuronal death in a mouse model of cerebral palsy.

BACKGROUND AND PURPOSE: Mammalian target of rapamycin (mTOR) pathway signaling governs cellular responses to hypoxia and inflammation including induction of autophagy and cell survival. Cerebral palsy (CP) is a neurodevelopmental disorder linked to hypoxic and inflammatory brain injury however, a role for mTOR modulation in CP has not been investigated. We hypothesized that mTOR pathway inhibition would diminish inflammation and prevent neuronal death in a mouse model of CP. METHODS: Mouse pups (P6) were subjected to hypoxia-ischemia and lipopolysaccharide-induced inflammation (HIL), a model of CP causing neuronal injury within the hippocampus, periventricular white matter, and neocortex. mTOR pathway inhibition was achieved with rapamycin (an mTOR inhibitor; 5mg/kg) or PF-4708671 (an inhibitor of the downstream p70S6kinase, S6K, 75 mg/kg) immediately following HIL, and then for 3 subsequent days. Phospho-activation of the mTOR effectors p70S6kinase and ribosomal S6 protein and expression of hypoxia inducible factor 1 (HIF-1α) were assayed. Neuronal cell death was defined with Fluoro-Jade C (FJC) and autophagy was measured using Beclin-1 and LC3II expression. Iba-1 labeled, activated microglia were quantified. RESULTS: Neuronal death, enhanced HIF-1α expression, and numerous Iba-1 labeled, activated microglia were evident at 24 and 48 h following HIL. Basal mTOR signaling, as evidenced by phosphorylated-S6 and -S6K levels, was unchanged by HIL. Rapamycin or PF-4,708,671 treatment significantly reduced mTOR signaling, neuronal death, HIF-1α expression, and microglial activation, coincident with enhanced expression of Beclin-1 and LC3II, markers of autophagy induction. CONCLUSIONS: mTOR pathway inhibition prevented neuronal death and diminished neuroinflammation in this model of CP. Persistent mTOR signaling following HIL suggests a failure of autophagy induction, which may contribute to neuronal death in CP. These results suggest that mTOR signaling may be a novel therapeutic target to reduce neuronal cell death in CP.

Sequence dependence of isothermal DNA amplification via EXPAR.

Isothermal nucleic acid amplification is becoming increasingly important for molecular diagnostics. Therefore, new computational tools are needed to facilitate assay design. In the isothermal EXPonential Amplification Reaction (EXPAR), template sequences with similar thermodynamic characteristics perform very differently. To understand what causes this variability, we characterized the performance of 384 template sequences, and used this data to develop two computational methods to predict EXPAR template performance based on sequence: a position weight matrix approach with support vector machine classifier, and RELIEF attribute evaluation with Naïve Bayes classification. The methods identified well and poorly performing EXPAR templates with 67-70% sensitivity and 77-80% specificity. We combined these methods into a computational tool that can accelerate new assay design by ruling out likely poor performers. Furthermore, our data suggest that variability in template performance is linked to specific sequence motifs. Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates. Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers. Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions.

Integrated nucleic acid testing system to enable TB diagnosis in peripheral settings.

To facilitate treatment and limit transmission of tuberculosis (TB), new methods are needed to enable rapid and affordable diagnosis of the disease in high-burden low-resource settings. We have developed a prototype integrated nucleic acid testing device to detect Mycobacterium tuberculosis (M.tb) in sputum. The device consists of a disposable cartridge and compact, inexpensive instrument that automates pathogen lysis, nucleic acid extraction, isothermal DNA amplification and lateral flow detection. A liquefied and disinfected sputum sample is manually injected into the cartridge, and all other steps are automated, with a result provided in <1.5 h. Cell disruption and DNA extraction is executed within a four-port active valve containing a miniature bead blender (based on PureLyse® technology, Claremont BioSolutions LLC). The DNA-containing eluate is combined with dry master-mix reagents and target DNA is isothermally amplified. Amplified master-mix is then pumped into a lateral flow strip chamber for detection. The entire process is performed in a single-use closed-system cartridge to prevent amplicon carryover. For testing of M.tb-spiked sputum the system provided a limit of detection of 5 × 103 colony forming units (CFU) per mL. None of the negative sputum-only controls yielded a false-positive result. Testing of 45 clinical sputum specimens from TB cases and controls relative to a validated manual qPCR-based comparator method revealed a preliminary sensitivity of 90% and specificity of 96%. With further development, the herein described integrated nucleic acid testing device can enable TB diagnosis and treatment initiation in the same clinical encounter in near-patient low-resource settings of high TB burden countries.

Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities.

Rapid isothermal nucleic acid amplification technologies can enable diagnosis of human pathogens and genetic variations in a simple, inexpensive, user-friendly format. The isothermal exponential amplification reaction (EXPAR) efficiently amplifies short oligonucleotides called triggers in less than 10 min by means of thermostable polymerase and nicking endonuclease activities. We recently demonstrated that this reaction can be coupled with upstream generation of trigger oligonucleotides from a genomic target sequence, and with downstream visual detection using DNA-functionalized gold nanospheres. The utility of EXPAR in clinical diagnostics is, however, limited by a nonspecific background amplification phenomenon, which is further investigated in this report. We found that nonspecific background amplification includes an early phase and a late phase. Observations related to late phase background amplification are in general agreement with literature reports of ab initio DNA synthesis. Early phase background amplification, which limits the sensitivity of EXPAR, differs however from previous reports of nonspecific DNA synthesis. It is observable in the presence of single-stranded oligonucleotides following the EXPAR template design rules and generates the trigger sequence expected for the EXPAR template present in the reaction. It appears to require interaction between the DNA polymerase and the single-stranded EXPAR template. Early phase background amplification can be suppressed or eliminated by physically separating the template and polymerase until the final reaction temperature has been reached, thereby enabling detection of attomolar starting trigger concentrations.

A Drosophila KCNQ channel essential for early embryonic development.

The mammalian voltage-dependent KCNQ channels are responsible for distinct types of native potassium currents and are associated with several human diseases. We cloned a novel Drosophila KCNQ channel (dKCNQ) based on its sequence homology to the mammalian genes. When expressed in Chinese hamster ovary cells, dKCNQ gives rise to a slowly activating and slowly deactivating current that activates in the subthreshold voltage range. Like the M-current produced by mammalian KCNQ channels, dKCNQ current is sensitive to the KCNQ-specific blocker linopirdine and is suppressed by activation of a muscarinic receptor. dKCNQ is also similar to the mammalian channels in that it binds calmodulin (CaM), and CaM binding is necessary to produce functional currents. In situ hybridization analysis demonstrates that dKCNQ mRNA is present in brain cortical neurons, the cardia (proventriculus), and the nurse cells and oocytes of the ovary. We generated mutant flies with deletions in the genomic sequence of dKCNQ. Embryos produced by homozygous deletion females exhibit disorganized nuclei and fail to hatch, suggesting strongly that a maternal contribution of dKCNQ protein and/or mRNA is essential for early embryonic development.

MONaKA, a novel modulator of the plasma membrane Na,K-ATPase.

We have cloned and characterized mouse and human variants of MONaKA, a novel protein that interacts with and modulates the plasma membrane Na,K-ATPase. MONaKA was cloned based on its sequence homology to the Drosophila Slowpoke channel-binding protein dSlob, but mouse and human MONaKA do not bind to mammalian Slowpoke channels. At least two splice variants of MONaKA exist; the splicing is conserved perfectly between mouse and human, suggesting that it serves some important function. Both splice variants of MONaKA are expressed widely throughout the CNS and peripheral nervous system, with different splice variant expression ratios in neurons and glia. A yeast two-hybrid screen with MONaKA as bait revealed that it binds tightly to the beta1 and beta3 subunits of the Na,K-ATPase. The association between MONaKA and Na,K-ATPase beta subunits was confirmed further by coimmunoprecipitation from transfected cells, mouse brain, and cultured mouse astrocytes. A glutathione S-transferase-MONaKA fusion protein inhibits Na,K-ATPase activity from whole brain or cultured astrocytes. Furthermore, transfection of MONaKA inhibits 86Rb+ uptake via the Na,K-ATPase in intact cells. These results are consistent with the hypothesis that MONaKA modulates brain Na,K-ATPase and may thereby participate in the regulation of electrical excitability and synaptic transmission.

Experimental methods to study the role of the peripheral cannabinoid receptor in immune function.

Marijuana components, such as delta-9-tetrahydrocannabinol, and endogenous cannabinoids, such as anandamide and 2-arachydonoylglycerol, alter diverse immune functions. Two cannabinoid receptors have been discovered to date, the central cannabinoid receptor (CB1R) and the peripheral cannabinoid receptor (CB2R). The CB1R is expressed predominantly in the central nervous system. The CB2R is expressed mainly in cells of the immune system, suggesting that the CB2R is involved in immunoregulatory events. Cannabinoids have been shown to modulate diverse immune functions including cytokine production, lymphocyte proliferation, and humoral and cell-mediated immune responses. In addition, cannabinoids have been shown to induce different signal transduction pathways. However, the role of cannabinoids and their receptors in the immune system remains unclear. The objective of the experimental methods described herein is to investigate the role of CB2R activation in specific splenocyte and macrophage functions using a mouse lacking the CB2R. Interestingly, our findings,thus far suggest that basal CB2R activation modulates lymphocyte proliferation and cytokine secretion and macrophage phagocytic activity. Therefore, data obtained using the methodology described in this chapter will help us elucidate the role of cannabinoids and the CB2R in the immune system.

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy.

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Pilot study of a rapid and minimally instrumented sputum sample preparation method for molecular diagnosis of tuberculosis.

Nucleic acid amplification testing (NAAT) enables rapid and sensitive diagnosis of tuberculosis (TB), which facilitates treatment and mitigates transmission. Nucleic acid extraction from sputum constitutes the greatest technical challenge in TB NAAT for near-patient settings. This report presents preliminary data for a semi-automated sample processing method, wherein sputum is disinfected and liquefied, followed by PureLyse(®) mechanical lysis and solid-phase nucleic acid extraction in a miniaturized, battery-operated bead blender. Sputum liquefaction and disinfection enabled a >10(4) fold reduction in viable load of cultured Mycobacterium tuberculosis (M.tb) spiked into human sputum, which mitigates biohazard concerns. Sample preparation via the PureLyse(®) method and a clinically validated manual method enabled positive PCR-based detection for sputum spiked with 10(4) and 10(5) colony forming units (cfu)/mL M.tb. At 10(3) cfu/mL sputum, four of six and two of six samples amplified using the comparator and PureLyse(®) method, respectively. For clinical specimens from TB cases and controls, the two methods provided 100% concordant results for samples with 1 mL input volume (N = 41). The semi-automated PureLyse(®) method therefore performed similarly to a validated manual comparator method, but is faster, minimally instrumented, and can be integrated into TB molecular diagnostic platforms designed for near-patient low-resource settings.

Simple system for isothermal DNA amplification coupled to lateral flow detection.

Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF) detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb) genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP) or the Exponential Amplification Reaction (EXPAR), both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden.

Point-of-care nucleic acid testing for infectious diseases.

Nucleic acid testing for infectious diseases at the point of care is beginning to enter clinical practice in developed and developing countries; especially for applications requiring fast turnaround times, and in settings where a centralized laboratory approach faces limitations. Current systems for clinical diagnostic applications are mainly PCR-based, can only be used in hospitals, and are still relatively complex and expensive. Integrating sample preparation with nucleic acid amplification and detection in a cost-effective, robust, and user-friendly format remains challenging. This review describes recent technical advances that might be able to address these limitations, with a focus on isothermal nucleic acid amplification methods. It briefly discusses selected applications related to the diagnosis and management of tuberculosis, HIV, and perinatal and nosocomial infections.

Creating an anticoagulant patient education class.

A patient education workgroup was developed on a progressive care medical/vascular surgical unit. The workgroup identified patient education needs regarding discharge education for postsurgical patients and those discharging with oral anticoagulants (OAC). Staff surveys aided the workgroup in identifying a need for additional discharge education for patients and families. After various methods of patient education were explored, it was determined the workgroup could best meet the needs of the patient population through a class format providing group discussion and interaction. Logistical details and class formatting were configured to meet both the needs of the patients and the nursing staff. Current institutional patient education pamphlets were used to develop the content for the class. Physician review and input were obtained during the development of the content. A patient education specialist was also consulted to ensure proper literacy levels were used. To meet the Joint Commission National Patient Safety Goal regarding anticoagulant safety, the content focused on home management, which included the following: knowledge of INR goal range, dietary factors, when to call the provider and safety precautions. Other topics to promote self-efficacy in anticoagulation therapy were also included in the content. Postclass evaluations completed by patients and families provided useful feedback for continuous improvement and patient satisfaction. Preliminary survey results indicate high patient satisfaction with the class. Plans include a quality improvement project to evaluate the effectiveness of the patient education class on OAC.