Miltefosine inhibits Chikungunya virus replication in human primary dermal fibroblasts.

Background: Chikungunya virus (CHIKV) is a re-emerging pathogen that has caused widespread outbreaks affecting millions of people around the globe. Currently, there is no specific therapeutic drug against CHIKV, with symptomatic treatment only to manage the disease. Pi3-akt signaling has been implicated in infection of several viruses including that of CHIKV. Effect of Pi3-akt signaling inhibitors on CHIKV replication was evaluated in this study. Methods: Human primary dermal fibroblast cells were treated with inhibitors of the Pi3-akt signaling pathway. Suppression of CHIKV replication was evaluated as reduction in virus titer in cell supernatants. Effect of miltefosine (MF) on CHIKV replication was evaluated in pre and post treatment regimen. Inhibition of virus replication was determined by cell growth, virus titer and western blot. Results: Inhibition of Akt-phosphorylation significantly inhibited CHIKV replication. No effect on CHIKV replication was observed after treatment with Pi3-kinase and mTOR activation inhibitors. Further, MF, an FDA-approved Akt-inhibitor, inhibited CHIKV replication in pre- and post-infection treatment regimens. Conclusion: Data suggests that Akt-phosphorylation can be an amenable target of therapy against CHIKV infection. This is the first study to show inhibition of CHIKV replication by MF, and presents a case for further development of MF as an anti-CHIKV drug.

Hemorrhage enhances cytokine, complement component 3, and caspase-3, and regulates microRNAs associated with intestinal damage after whole-body gamma-irradiation in combined injury.

Hemorrhage following whole-body γ-irradiation in a combined injury (CI) model increases mortality compared to whole-body γ-irradiation alone (RI). The decreased survival in CI is accompanied by increased bone marrow injury, decreased hematocrit, and alterations of miRNA in the kidney. In this study, our aim was to examine cytokine homeostasis, susceptibility to systemic bacterial infection, and intestinal injury. More specifically, we evaluated the interleukin-6 (IL-6)-induced stress proteins including C-reactive protein (CRP), complement 3 (C3), Flt-3 ligand, and corticosterone. CD2F1 male mice received 8.75 Gy 60Co gamma photons (0.6 Gy/min, bilateral) which was followed by a hemorrhage of 20% of the blood volume. In serum, RI caused an increase of IL-1, IL-2, IL-3, IL-5, IL-6, IL-12, IL-13, IL-15, IL-17A, IL-18, G-CSF, CM-CSF, eotaxin, IFN-γ, MCP-1, MIP, RANTES, and TNF-α, which were all increased by hemorrhage alone, except IL-9, IL-17A, and MCP-1. Nevertheless, CI further elevated RI-induced increases of these cytokines except for G-CSF, IFN- γ and RANTES in serum. In the ileum, hemorrhage in the CI model significantly enhanced RI-induced IL-1ß, IL-3, IL-6, IL-10, IL-12p70, IL-13, IL-18, and TNF-α concentrations. In addition, Proteus mirabilis Gram(-) was found in only 1 of 6 surviving RI mice on Day 15, whereas Streptococcus sanguinis Gram(+) and Sphingomonas paucimobilis Gram(-) were detected in 2 of 3 surviving CI mice (with 3 CI mice diseased due to inflammation and infection before day 15) at the same time point. Hemorrhage in the CI model enhanced the RI-induced increases in C3 and decreases in CRP concentrations. However, hemorrhage alone did not alter the basal levels, but hemorrhage in the CI model displayed similar increases in Flt-3 ligand levels as RI did. Hemorrhage alone altered the basal levels of corticosterone early after injury, which then returned to the baseline, but in RI mice and CI mice the increased corticosterone concentration remained elevated throughout the 15 day study. CI increased 8 miRNAs and decreased 10 miRNAs in serum, and increased 16 miRNA and decreased 6 miRNAs in ileum tissue. Among the altered miRNAs, CI increased miR-34 in the serum and ileum which targeted an increased phosphorylation of ERK, p38, and increased NF-κB, thereby leading to increased iNOS expression and activation of caspase-3 in the ileum. Further, let-7g/miR-98 targeted the increased phosphorylation of STAT3 in the ileum, which is known to bind to the iNOS gene. These changes may correlate with cell death in the ileum of CI mice. The histopathology displayed blunted villi and villus edema in RI and CI mice. Based on the in silico analysis, miR-15, miR-99, and miR-100 were predicted to regulate IL-6 and TNF. These results suggest that CI-induced alterations of cytokines/chemokines, CRP, and C3 cause a homeostatic imbalance and may contribute to the pathophysiology of the gastrointestinal injury. Inhibitory intervention in these responses may prove therapeutic for CI and improve recovery of the ileal morphologic damage.

Deinococcus Mn2+-peptide complex: A novel approach to alphavirus vaccine development.

Over the last ten years, Chikungunya virus (CHIKV), an Old World alphavirus has caused numerous outbreaks in Asian and European countries and the Americas, making it an emerging pathogen of great global health importance. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, on the other hand, has been developed as a bioweapon in the past due to its ease of preparation, aerosol dispersion and high lethality in aerosolized form. Currently, there are no FDA approved vaccines against these viruses. In this study, we used a novel approach to develop inactivated vaccines for VEEV and CHIKV by applying gamma-radiation together with a synthetic Mn-decapeptide-phosphate complex (MnDpPi), based on manganous-peptide-orthophosphate antioxidants accumulated in the extremely radiation-resistant bacterium Deinococcus radiodurans. Classical gamma-irradiated vaccine development approaches are limited by immunogenicity-loss due to oxidative damage to the surface proteins at the high doses of radiation required for complete virus-inactivation. However, addition of MnDpPi during irradiation process selectively protects proteins, but not the nucleic acids, from the radiation-induced oxidative damage, as required for safe and efficacious vaccine development. Previously, this approach was used to develop a bacterial vaccine. In the present study, we show that this approach can successfully be applied to protecting mice against viral infections. Irradiation of VEEV and CHIKV in the presence of MnDpPi resulted in substantial epitope preservation even at supra-lethal doses of gamma-rays (50,000Gy). Irradiated viruses were found to be completely inactivated and safe in vivo (neonatal mice). Upon immunization, VEEV inactivated in the presence of MnDpPi resulted in drastically improved protective efficacy. Thus, the MnDpPi-based gamma-inactivation approach described here can readily be applied to developing vaccines against any pathogen of interest in a fast and cost-effective manner.

Differential host gene responses from infection with neurovirulent and partially-neurovirulent strains of Venezuelan equine encephalitis virus.

BACKGROUND: Venezuelan equine encephalitis virus (VEEV) is an alphavirus in the family Togaviridae. VEEV causes a bi-phasic illness in mice where primary replication in lymphoid organs is followed by entry into the central nervous system (CNS). The CNS phase of infection is marked by encephalitis and large scale neuronal death ultimately resulting in death. Molecular determinants of VEEV neurovirulence are not well understood. In this study, host gene expression response to highly neurovirulent VEEV (V3000 strain) infection was compared with that of a partially neurovirulent VEEV (V3034 strain) to identify host factors associated with VEEV neurovirulence. METHODS: Whole genome microarrays were performed to identify the significantly modulated genes. Microarray observations were classified into three categories i.e., genes that were similarly modulated against both V3000 and V3034 infections, and genes that were uniquely modulated in infection with V3034 or V3000. Histologic sections of spleen and brain were evaluated by hematoxylin and eosin stains from all the mice. RESULTS: V3000 infection induced a greater degree of pathology in both the spleen and brain tissue of infected mice compared to V3034 infection. Genes commonly modulated in the spleens after V3000 or V3034 infection were associated with innate immune responses, inflammation and antigen presentation, however, V3000 induced a gene response profile that suggests a stronger inflammatory and apoptotic response compared to V3034. In the brain, both the strains of VEEV induced an innate immune response reflected by an upregulation of the genes involved in antigen presentation, interferon response, and inflammation. Similar to the spleen, V3000 was found to induce a stronger inflammatory response than V3034 in terms of induction of pro-inflammatory genes and associated pathways. Ccl2, Ccl5, Ccl6, and Ly6 were uniquely upregulated in V3000 infected mouse brains and correlated with the extensive inflammation observed in the brain. CONCLUSION: The common gene profile identified from V3000 and V3034 exposure can help in understanding a generalized host response to VEEV infection. Inflammatory genes that were uniquely identified in mouse brains with V3000 infection will help in better understanding the lethal neurovirulence of VEEV. Future studies are needed to explore the roles played by the genes identified in VEEV induced encephalitis.

Differential expression of microRNAs in the brains of mice subjected to increasing grade of mild traumatic brain injury.

OBJECTIVE: To investigate the effect of heterogeneity in mTBI on miRNA expression in mouse brain and to identify molecular pathways targeted by the modulated miRNAs. METHODS: A weight drop device was used to induce four increasing grades of mTBI. MiRNA expression was evaluated using TaqMan rodent miRNA arrays. Bioinformatics analysis was done using the DIANA miRPath tool and Ingenuity Pathway Analysis software. Histology of brain sections was evaluated using H&E staining. RESULTS: No histologic lesions were observed in the brains of injured mice; however, significant modulation in miRNA expression profile was observed. Global miRNA profiling indicated a trend of decrease in the number of modulated miRNAs from 24 hours to day 7 post-injury, except for the most severe grade of mTBI. Canonical pathways like calcium signalling, synaptic pathways and axon guidance pathway were the major targets of the modulated miRNAs. Network correlation analyses indicated an interaction between the modulated miRNAs and putative protein biomarkers of TBI. CONCLUSIONS: The data demonstrated that varying intensities of mTBI induced a differential miRNA expression profile in the brain post-injury. Pathways such as calcium and synaptic signalling were major targets of modulated miRNAs and may play a role in the pathophysiology of mTBI.

MDP: A Deinococcus Mn2+-Decapeptide Complex Protects Mice from Ionizing Radiation.

The radioprotective capacity of a rationally-designed Mn2+-decapeptide complex (MDP), based on Mn antioxidants in the bacterium Deinococcus radiodurans, was investigated in a mouse model of radiation injury. MDP was previously reported to be extraordinarily radioprotective of proteins in the setting of vaccine development. The peptide-component (DEHGTAVMLK) of MDP applied here was selected from a group of synthetic peptides screened in vitro for their ability to protect cultured human cells and purified enzymes from extreme damage caused by ionizing radiation (IR). We show that the peptides accumulated in Jurkat T-cells and protected them from 100 Gy. MDP preserved the activity of T4 DNA ligase exposed to 60,000 Gy. In vivo, MDP was nontoxic and protected B6D2F1/J (female) mice from acute radiation syndrome. All irradiated mice treated with MDP survived exposure to 9.5 Gy (LD70/30) in comparison to the untreated mice, which displayed 63% lethality after 30 days. Our results show that MDP provides early protection of white blood cells, and attenuates IR-induced damage to bone marrow and hematopoietic stem cells via G-CSF and GM-CSF modulation. Moreover, MDP mediated the immunomodulation of several cytokine concentrations in serum including G-CSF, GM-CSF, IL-3 and IL-10 during early recovery. Our results present the necessary prelude for future efforts towards clinical application of MDP as a promising IR countermeasure. Further investigation of MDP as a pre-exposure prophylactic and post-exposure therapeutic in radiotherapy and radiation emergencies is warranted.

A Panel of Serum MiRNA Biomarkers for the Diagnosis of Severe to Mild Traumatic Brain Injury in Humans.

MicroRNAs (MiRNAs) are small endogenous RNA molecules and have emerged as novel serum diagnostic biomarkers for several diseases due to their stability and detection at minute quantities. In this study, we have identified a serum miRNA signature in human serum samples of mild to severe TBI, which can be used for diagnosis of mild and moderate TBI (MMTBI). Human serum samples of MMTBI, severe TBI (STBI), orthopedic injury and healthy controls were used and miRNA profiling was done using taqman real time PCR. The real time PCR data for the MMTBI, STBI and orthopedic injury was normalized to the control samples which showed upregulation of 39, 37 and 33 miRNAs in MMTBI, STBI and orthopedic injury groups respectively. TBI groups were compared to orthopedic injury group and an up-regulation of 18 and 20 miRNAs in MMTBI and STBI groups was observed. Among these, a signature of 10 miRNAs was found to be present in both MMTBI and STBI groups. These 10 miRNAs were validated in cerebrospinal fluid (CSF) from STBI and four miRNAs were found to be upregulated in CSF. In conclusion, we identified a subset of 10 unique miRNAs which can be used for diagnosis of MMTBI and STBI.

Phytochemicals in Wound Healing.

Significance: Traditional therapies, including the use of dietary components for wound healing and skin regeneration, are very common in Asian countries such as China and India. The increasing evidence of health-protective benefits of phytochemicals, components derived from plants is generating a lot of interest, warranting further scientific evaluation and mechanistic studies. Recent Advances: Phytochemicals are non-nutritive substances present in plants, and some of them have the potential to provide better tissue remodeling when applied on wounds and to also act as proangiogenic agents during wound healing. Critical Issues: In this review, we briefly discuss the current understanding, important molecular targets, and mechanism of action(s) of some of the phytochemicals such as curcumin, picroliv, and arnebin-1. We also broadly review the multiple pathways that these phytochemicals regulate to enhance wound repair and skin regeneration. Future Directions: Recent experimental data on the effects of phytochemicals on wound healing and skin regeneration establish the potential clinical utility of plant-based compounds. Additional research in order to better understand the exact mechanism and potential targets of phytochemicals in skin regeneration is needed. Human studies a2nd clinical trials are pivotal to fully understand the benefits of phytochemicals in wound healing and skin regeneration.

1,5-Iodonaphthyl azide-inactivated V3526 protects against aerosol challenge with virulent venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is a New World alphavirus. VEEV is highly infectious in aerosolized form and has been identified as a bio-terrorism agent. There is no licensed vaccine for prophylaxis against VEEV. The current IND vaccine is poorly immunogenic and does not protect against an aerosol challenge with virulent VEEV. We have previously shown that VEEV inactivated by 1,5-iodonaphthyl azide (INA) protects against footpad challenge with virulent VEEV. In this study, we inactivated an attenuated strain of VEEV, V3526, with INA and evaluated its protective efficacy against aerosol challenge with wild type VEEV. We demonstrated that among three routes of immunization, intramuscular immunization with INA-inactivate V3526 (INA-iV3526) provided complete protection against aerosol challenge with virulent VEEV. Our data suggests that INA-iV3526 can be explored further for development as an effective vaccine candidate against aerosol challenge of virulent VEEV.

Hemorrhage Exacerbates Radiation Effects on Survival, Leukocytopenia, Thrombopenia, Erythropenia, Bone Marrow Cell Depletion and Hematopoiesis, and Inflammation-Associated microRNAs Expression in Kidney.

Exposure to high-dose radiation results in detrimental effects on survival. The effects of combined trauma, such as radiation in combination with hemorrhage, the typical injury of victims exposed to a radiation blast, on survival and hematopoietic effects have yet to be understood. The purpose of this study was to evaluate the effects of radiation injury (RI) combined with hemorrhage (i.e., combined injury, CI) on survival and hematopoietic effects, and to investigate whether hemorrhage (Hemo) enhanced RI-induced mortality and hematopoietic syndrome. Male CD2F1 mice (10 weeks old) were given one single exposure of γ- radiation (60Co) at various doses (0.6 Gy/min). Within 2 hr after RI, animals under anesthesia were bled 0% (Sham) or 20% (Hemo) of total blood volume via the submandibular vein. In these mice, Hemo reduced the LD50/30 for 30-day survival from 9.1 Gy (RI) to 8.75 Gy (CI) with a DMF of 1.046. RI resulted in leukocytopenia, thrombopenia, erythropenia, and bone marrow cell depletion, but decreased the caspase-3 activation response. RI increased IL-1ß, IL-6, IL-17A, and TNF-α concentrations in serum, bone marrow, ileum, spleen, and kidney. Some of these adverse alterations were magnified by CI. Erythropoietin production was increased in kidney and blood more after CI than RI. Furthermore, CI altered the global miRNAs expression in kidney and the ingenuity pathway analysis showed that miRNAs viz., let-7e, miR-30e and miR-29b that were associated with hematopoiesis and inflammation. This study provides preliminary evidence that non-lethal Hemo exacerbates RI-induced mortality and cell losses associated with high-dose γ-radiation. We identified some of the initial changes occurring due to CI which may have facilitated in worsening the injury and hampering the recovery of animals ultimately resulting in higher mortality.

Eastern equine encephalitis virus in mice I: clinical course and outcome are dependent on route of exposure.

BACKGROUND: Eastern equine encephalitis virus (EEEV), an arbovirus, is an important human and veterinary pathogen belonging to one of seven antigenic complexes in the genus Alphavirus, family Togaviridae. EEEV is considered the most deadly of the mosquito-borne alphaviruses due to the high case fatality rate associated with clinical infections, reaching up to 75 % in humans and 90 % in horses. In patients that survive acute infection, neurologic sequelae are often devastating. Although natural infections are acquired by mosquito bite, EEEV is also highly infectious by aerosol. This fact, along with the relative ease of production and stability of this virus, has led it to being identified as a potential agent of bioterrorism. METHODS: To characterize the clinical course and outcome of EEEV strain FL93-939 infection, we compared clinical parameters, cytokine expression, viremia, and viral titers in numerous tissues of mice exposed by various routes. Twelve-week-old female BALB/c mice were infected by the intranasal, aerosol, or subcutaneous route. Mice were monitored for clinical signs of disease and euthanized at specified time points (6 hpi through 8 dpi). Blood and tissues were harvested for cytokine analysis and/or viral titer determination. RESULTS: Although all groups of animals exhibited similar clinical signs after inoculation, the onset and severity differed. The majority of those animals exposed by the aerosol route developed severe clinical signs by 4 dpi. Significant differences were also observed in the viral titers of target tissues, with virus being detected in the brain at 6 hpi in the aerosol study. CONCLUSION: The clinical course and outcome of EEEV infection in mice is dependent on route of exposure. Aerosol exposure to EEEV results in acute onset of clinical signs, rapid neuroinvasion, and 100 % mortality.

Eastern equine encephalitis virus in mice II: pathogenesis is dependent on route of exposure.

BACKGROUND: Eastern equine encephalitis virus (EEEV) is an alphavirus with a case fatality rate estimated to be as high as 75 % in humans and 90 % in horses. Surviving patients often have long-lasting and severe neurological sequelae. At present, there is no licensed vaccine or therapeutic for EEEV infection. This study completes the clinical and pathological analysis of mice infected with a North American strain of EEEV by three different routes: aerosol, intranasal, and subcutaneous. Such an understanding is imperative for use of the mouse model in vaccine and antiviral drug development. METHODS: Twelve-week-old female BALB/c mice were infected with EEEV strain FL93-939 by the intranasal, aerosol, or subcutaneous route. Mice were euthanized 6 hpi through 8 dpi and tissues were harvested for histopathological and immunohistochemical analysis. RESULTS: Viral antigen was detected in the olfactory bulb as early as 1-2 dpi in aerosol and intranasal infected mice. However, histologic lesions in the brain were evident about 24 hours earlier (3 dpi vs 4 dpi), and were more pronounced following aerosol infection relative to intranasal infection. Following subcutaneous infection, viral antigen was also detected in the olfactory bulb, though not as routinely or as early. Significant histologic lesions were not observed until 6 dpi. CONCLUSION: These pathologic studies suggest EEEV enters the brain through the olfactory system when mice are exposed via the intranasal and aerosol routes. In contrast, the histopathologic lesions were delayed in the subcutaneous group and it appears the virus may utilize both the vascular and olfactory routes to enter the brain when mice are exposed to EEEV subcutaneously.

Inactivation of non-enveloped virus by 1,5 iodonaphthyl azide.

BACKGROUND: A photoactive hydrophobic agent 1,5-iodonaphthyl-azide (INA), has been previously shown to completely inactivate the enveloped viruses. INA sequesters into the lipid bilayer of the virus envelope and upon UV-irradiation bind to the hydrophobic domains of the envelope glycoproteins. In our earlier study, we have shown that the Venezuelan equine encephalitis virus (VEEV) genomic RNA was also inactivated during the inactivation of the virus with INA. FINDINGS: In the present study, we evaluated if the RNA inactivation property of INA can be used to inactivate non-enveloped RNA viruses. Encephalomyocarditis virus (EMCV) was used as a model non-enveloped virus. Treatment with INA followed by UV-irradiation resulted in complete inactivation of EMCV. RNA isolated from INA-inactivated EMCV was non-infectious and INA was found to be associated with the viral RNA genome. INA-inactivated EMCV induced robust total antibody response. However binding capacity of INA-inactivated EMCV to neutralizing antibody was inhibited. CONCLUSION: This is the first study to show that INA can completely inactivate non-enveloped virus. Our results suggest that the amino acid composition of the neutralizing epitope may interfere with the protective antibody response generated by the INA-inactivated non-enveloped virus.

Chikungunya Virus Infection Alters Expression of MicroRNAs Involved in Cellular Proliferation, Immune Response and Apoptosis.

OBJECTIVE(S): Chikungunya virus (CHIKV) is a reemerging virus of significant importance that has caused large-scale outbreaks in the countries with a temperate climate. CHIKV causes debilitating arthralgia which can persist for weeks and up to a year. Fibroblast cells are the main target of CHIKV infection. In this study, we analyzed microRNA (miRNA) modulation in the fibroblast cells infected with CHIKV at an early stage of infection. METHODS: 760 miRNAs were analyzed for modulation following infection with CHIKV at 6 h after infection. Bioinformatic analysis was done to identify the signaling pathway that may be targeted by the significantly modulated miRNAs. Validation of the miRNAs was done using a singleplex miRNA assay and protein target validation of modulated miRNAs was done by Western blot analysis. RESULTS: Computational analysis of the significantly modulated miRNAs indicated their involvement in signaling pathways such as Toll-like receptor, mTOR, JAK-STAT and Pi3-Akt pathways, which have been shown to play important roles during CHIKV infection. Topoisomerase IIß, a target of two of the modulated miRNAs, was downregulated upon CHIKV infection. CONCLUSION(S): We identified several miRNAs that may play important roles in early events after CHIKV infection and can be potential therapeutic targets against CHIKV infection.

Identification of serum microRNA signatures for diagnosis of mild traumatic brain injury in a closed head injury model.

Wars in Iraq and Afghanistan have highlighted the problems of diagnosis and treatment of mild traumatic brain injury (mTBI). MTBI is a heterogeneous injury that may lead to the development of neurological and behavioral disorders. In the absence of specific diagnostic markers, mTBI is often unnoticed or misdiagnosed. In this study, mice were induced with increasing levels of mTBI and microRNA (miRNA) changes in the serum were determined. MTBI was induced by varying weight and fall height of the impactor rod resulting in four different severity grades of the mTBI. Injuries were characterized as mild by assessing with the neurobehavioral severity scale-revised (NSS-R) at day 1 post injury. Open field locomotion and acoustic startle response showed behavioral and sensory motor deficits in 3 of the 4 injury groups at day 1 post injury. All of the animals recovered after day 1 with no significant neurobehavioral alteration by day 30 post injury. Serum microRNA (miRNA) profiles clearly differentiated injured from uninjured animals. Overall, the number of miRNAs that were significantly modulated in injured animals over the sham controls increased with the severity of the injury. Thirteen miRNAs were found to identify mTBI regardless of its severity within the mild spectrum of injury. Bioinformatics analyses revealed that the more severe brain injuries were associated with a greater number of miRNAs involved in brain related functions. The evaluation of serum miRNA may help to identify the severity of brain injury and the risk of developing adverse effects after TBI.

Second generation inactivated eastern equine encephalitis virus vaccine candidates protect mice against a lethal aerosol challenge.

Currently, there are no FDA-licensed vaccines or therapeutics for eastern equine encephalitis virus (EEEV) for human use. We recently developed several methods to inactivate CVEV1219, a chimeric live-attenuated eastern equine encephalitis virus (EEEV). Dosage and schedule studies were conducted to evaluate the immunogenicity and protective efficacy of three potential second-generation inactivated EEEV (iEEEV) vaccine candidates in mice: formalin-inactivated CVEV1219 (fCVEV1219), INA-inactivated CVEV1219 (iCVEV1219) and gamma-irradiated CVEV1219 (gCVEV1219). Both fCVEV1219 and gCVEV1219 provided partial to complete protection against an aerosol challenge when administered by different routes and schedules at various doses, while iCVEV1219 was unable to provide substantial protection against an aerosol challenge by any route, dose, or schedule tested. When evaluating antibody responses, neutralizing antibody, not virus specific IgG or IgA, was the best correlate of protection. The results of these studies suggest that both fCVEV1219 and gCVEV1219 should be evaluated further and considered for advancement as potential second-generation inactivated vaccine candidates for EEEV.

Serum and amygdala microRNA signatures of posttraumatic stress: fear correlation and biomarker potential.

Exposure to acute traumatic stress can cause permanent changes in neurological circuitry and may lead to the development of an anxiety disorder known as posttraumatic stress disorder (PTSD). Current diagnosis of PTSD is based on clinical or behavioral symptom assessment, however, these are not definitive due to overlapping symptoms with other psychiatric disorders or mild traumatic brain injury (mTBI). No FDA approved diagnostic tests or biomarkers are currently available for diagnosis of PTSD. Recently, circulating miRNAs have emerged as novel biomarkers of many diseases. In this study, we have examined the altered expression of serum and amygdala miRNAs in an animal model of PTSD. Differentially expressed and statistically significant miRNAs in serum were validated for their presence in amygdala of corresponding animals. A panel of nine stress-responsive miRNAs viz., miR-142-5p, miR-19b, miR-1928, miR-223-3p, miR-322∗, miR-324, miR-421-3p and miR-463∗ and miR-674∗ were identified, and may have potential as biomarker(s) for PTSD. Further validations by bioinformatics and system biology approaches indicate that five miRNAs such as miR-142-5p, miR-19b, miR-1928, miR-223 and miR-421-3p may play a potential role in the regulation of genes associated with delayed and exaggerated fear. To the best of our knowledge, this is the first report demonstrating the plausibility of using circulating miRNAs as biomarkers of PTSD.

Artificial microRNAs can effectively inhibit replication of Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus is a member of the alphavirus family and genus togaviridae. VEEV is highly infectious in aerosol form and has been weaponized in the past making it a potential biothreat agent. At present, there are no FDA approved antiviral treatments or vaccines for VEEV. Artificial microRNAs are small molecules which are expressed through endogenous microRNA machinery by RNA polymerase II. These artificial microRNAs effectively inhibit gene expression and are non-toxic to the host cell. VEEV RNA dependent RNA polymerase (RdRp) is central to VEEV replication. Therefore, we hypothesize that targeted inhibition of VEEV RdRp using artificial microRNAs may efficiently inhibit VEEV replication. Five artificial microRNAs were tested in vitro in BHK cells. Three of these artificial miRNAs showed significant inhibition of VEEV replication. Further, these microRNAs were cloned into the expression vector in combination to see the synergistic effect on VEEV replication. Combination of more than one miRNA did not result in significant inhibition of virus replication. In conclusion, we have shown that RNAi through artificial microRNAs effectively inhibits VEEV replication and is significantly less toxic in comparison to siRNAs.

Skin regenerative potentials of curcumin.

Curcumin, an active constituent of the spice turmeric, is well known for its chemopreventive properties and is found to be beneficial in treating various disorders including skin diseases. Curcumin protects skin by quenching free radicals and reducing inflammation through the inhibition of nuclear factor-kappa B. Curcumin also affects other signaling pathways including transforming growth factor-ß and mitogen-activated protein kinase pathway. Curcumin also modulates the phase II detoxification enzymes which are crucial in detoxification reactions and for protection against oxidative stress. In the present review, the biological mechanisms of the chemopreventive potential of curcumin in various skin diseases like psoriasis, vitiligo, and melanoma is discussed. The application of curcumin in skin regeneration and wound healing is also elucidated. We also explored the recent innovations and advances involved in the development of transdermal delivery systems to enhance the bioavailability of curcumin, particularly in the skin. Recent clinical trials pertaining to the use of curcumin in skin diseases establishes its benefits and also the need for additional clinical trials in other diseases are discussed.

Inactivation of Chikungunya virus by 1,5 iodonapthyl azide.

BACKGROUND: Chikungunya virus (CHIKV) is an arthropod borne alphavirus of the family Togaviridae. CHIKV is a reemerging virus for which there is no safe prophylactic vaccine. A live attenuated strain of CHIKV, CHIK181/25, was previously demonstrated to be highly immunogenic in humans, however, it showed residual virulence causing transient arthralgia. FINDINGS: In this study, we demonstrate the complete inactivation of CHIKV181/25 by 1,5 iodonapthyl azide (INA). No cytopathic effect and virus replication was observed in cells infected with the INA-inactivated CHIKV. However, a reduction in the INA-inactivated CHIK virus-antibody binding capacity was observed by western blot analysis. CONCLUSION: INA completely inactivated CHIKV and can further be explored for developing an inactivated-CHIKV vaccine.