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Introduction: Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease ranging from liver steatosis to nonalcoholic steatohepatitis (NASH). Besides lifestyle modifications, Vitamin E (800 IU/day) is generally recommended for NASH. Vitamin E monotherapy is not sufficient for the multifaceted disease like NALFD. The combination of Vitamin E 400IU and Fraxinus excelsior 500 mg twice daily was found to be better than vitamin E 400 IU twice daily in improving the lipid profile and liver function parameters in patients with NAFLD. We conducted a study to assess safety and effectiveness of Vitamin E plus Fraxinus excelsior in Indian patients with NAFLD in real-world settings. Patients and Methods: This was a non-interventional study in NAFLD patients with varying grades of steatosis conducted by 234 physicians across India from January 2018 to August 2018. Patients received combination of Vitamin E (400 IU) and Fraxinus excelsior (500 mg) soft gelatin capsules twice daily after meals for 12 weeks. Effectiveness of the treatment was assessed at visit 2 (6 weeks) and visit 3 (12 weeks, end of study) from baseline. The parameters for assessment included severity of liver steatosis, liver function parameters and the global assessment of safety and effectiveness. Results: A total of 1114 patients were included in the study. At baseline, majority of the patients (71.18%) had Grade II liver steatosis followed by 21.01% and 7.81% patients who had Grade III and Grade I liver steatosis, respectively. After 12 weeks of treatment with vitamin E and Fraxinus excelsior combination, 21% patients had no steatosis, 58.79% patients were in Grade 1 steatosis, 19.57% in grade II steatosis and only 0.63% patients in Grade III steatosis. The mean percentage reduction in aspartate aminotransferase (AST) level at week 6 and week 12 from baseline was 24.92% and 43.79%, respectively. Similarly, the mean percentage reduction in alanine aminotransferase (ALT) level at week 6 and week 12 from baseline was 24.37% and 44.05% respectively. The mean reductions in AST and ALT were significant at week 6 and week 12. More than 50% of the investigators rated treatment as excellent for the safety and effectiveness. Conclusion: Evidence from this Indian real-life study suggests that Vitamin E (400 IU) and Fraxinus excelsior (500 mg) is safe and effective for the treatment of NAFLD in routine clinical practice. Its consumption is associated with improvement in hepatic steatosis and liver function parameters (AST and ALT). Given the limited therapeutic options in NAFLD, this combination has the potential to bridge the therapeutic gap in management of NAFLD.

BACKGROUND: Induction therapy has not been proven to be beneficial for patients with clinical T2N0 esophageal adenocarcinoma. Surgery alone is associated with disappointing survival for patients found to have nodal disease on final pathologic examination. The aim of this study was to identify factors that predict pathologic nodal involvement in patients with endoscopic ultrasound (EUS)–proven T2N0 esophageal adenocarcinoma. METHODS: We retrospectively reviewed patients with EUS-staged T2N0 (uT2N0) esophageal adenocarcinoma treated with surgery alone. Final pathologic staging was compared with clinical staging. Demographic and clinicopathologic variables were evaluated as putative risk factors for nodal metastases. Logistic regression models were used to identify factors associated with nodal involvement. Kaplan-Meier analysis was performed to compare overall and recurrence-free survival between patients with (N+) and without (N−) nodal disease. RESULTS: We identified 80 patients with uT2N0 esophageal adenocarcinoma treated with surgery alone. Clinical staging with EUS was inaccurate for 73 patients (91%). Twenty-eight patients (35%) had pathologic N+ disease at resection. Five-year overall survival was 67% for N− patients and 41% for N+ patients (p=0.006). Recurrence-free survival was 65% for N− patients and 32% for N+ patients (p=0.0043). Univariable analysis identified vascular invasion and neural invasion as risk factors for nodal metastasis. Multivariable analysis identified vascular invasion as an independent predictor of pathologic nodal involvement. CONCLUSIONS: EUS is inaccurate for staging of T2N0 esophageal adenocarcinoma and often fails to identify nodal involvement. Identification of vascular invasion on preoperative biopsy should be explored as a prognostic marker to select patients for induction therapy.

Gut dysbiosis is commonly observed in patients with cirrhosis and chronic gastrointestinal disorders; however, its effect on antitumor immunity in the liver is largely unknown. Here we studied how the gut microbiome affects antitumor immunity in cholangiocarcinoma. Primary sclerosing cholangitis (PSC) or colitis, two known risk factors for cholangiocarcinoma which promote tumor development in mice, caused an accumulation of CXCR2+ polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC). A decrease in gut barrier function observed in mice with PSC and colitis allowed gut-derived bacteria and lipopolysaccharide to appear in the liver and induced CXCL1 expression in hepatocytes through a TLR4-dependent mechanism and an accumulation of CXCR2+ PMN-MDSCs. In contrast, neomycin treatment blocked CXCL1 expression and PMN-MDSC accumulation and inhibited tumor growth even in the absence of liver disease or colitis. Our study demonstrates that the gut microbiome controls hepatocytes to form an immunosuppressive environment by increasing PMN-MDSCs to promote liver cancer. Significance: MDSCs have been shown to be induced by tumors and suppress antitumor immunity. Here we show that the gut microbiome can control accumulation of MDSCs in the liver in the context of a benign liver disease or colitis. See related commentary by Chagani and Kwong, p. 1014. This article is highlighted in the In This Issue feature, p. 995

The Eleutheronema tetradactylum is a protandrous, hermaphrodite, marine perciformes fish. The body length of this fish acts as an important diagnostic marker for male and female discrimination. The present study describes for the first time the ultrastructural characteristics on the medial surface of the sagitta otolith in different body size groups of males of E. tetradactylum (Polynemidae: Perciformes) using scanning electron microscopy. The sagitta is a spindle-shaped structure that includes a well-developed rostrum and a poorly developed antirostrum. The sulcus is ostio-pseudocaudal type, almost straight and devoid of the collum. The ostium is a well-developed, vase-shaped structure. The cauda includes the colliculum and a well-developed caudal bulb with several distinct growth stripes. The length of the caudal bulb is significantly correlated to the growth of the body size of the fish. The excisura major is indistinct and the excisura minor is absent. The cristae are distinct on both sides of the sulcus. The one-way ANOVA test revealed that the development of several sagitta features shows significant differences in various body size groups of E. tetradactylum. The growth of the sagitta length is more closely related to the fork length than the sagitta width. Therefore, the sagitta length and the caudal bulb length can be used as important predictors to evaluate the fish size. The cauda region of the sagitta in E. tetradactylum is unique as well as more decorative than those of another Polynemidae fish and other hermaphrodite, marine perciformes fishes. The sagitta characteristics of E. tetradactylum might be advantageous in the identification of the sex and the taxonomy of the hermaphrodite fish species.

The present research explored the potential of probe ultrasound homogenizer and hydrocolloids in formulating and stabilizing mayonnaise. The effect of ultrasonic (US) treatment (12 min at 20%, 30%, 40% amplitude) and stabilizers xanthan gum (XG; 0.5% w/w), guar gum (GG; 0.5% w/w), and XG/GG (1:1) on emulsion storage stability (ESS), physicochemical and rheological properties of mayonnaise were studied. Rheological data comprising of shear rate ( γ ˙ ) and shear stress ( τ ) was analysed through four different rheological models (Herschel–Bulkley, Casson, Power Law, and Carreau-Yasuda models), and their fitness was evaluated. The samples exhibited non-Newtonian, pseudoplastic behaviour with flow behaviour index (n)

Even in the twenty-first century, rabies remains one of the most dreaded diseases in many parts of the world. An effective chemotherapeutic still remains elusive. The present study was aimed at in vitro evaluation of crude extracts of Allamanda cathartica and Cynodon dactylon for their potential anti-rabies activity based on the principle of immunofluorescence. The extracts were tested for cytotoxicity and screened for the presence of phytochemicals. While A. cathartica extracts were found to be non-toxic, the CC50 of C. dactylon (water and methanol) cold extracts were found to be 8.17 and 9.20 mg/mL respectively on BHK-21 cell line. Rapid Fluorescent Focus Inhibition Test (RFFIT) was used to evaluate anti-rabies activities of these plants against the rabies challenge virus standard strain. We observed 50% inhibition of 10 TCID50 CVS at 5 mg/mL (IC50) whereas florescence (no inhibition) was observed with A. cathartica extracts. The present study highlights the use of modified RFFIT as a method of choice for testing anti-rabies activity over assays based on evaluation of cytopathic effect.

The term “virtuality” has been quoted as “real but not actual, ideal but not abstract” by Marcel Proust. Visual-based reality is gaining popularity in dentistry because of its use in dealing with image-guided and robot-assisted surgery. It has provided a new approach for improving the treatments and delivering education in dentistry. The major throwback was the use of images registration by endoscope camera devices in the form of a video which can be presented on a monitor where the operator is able to observe the site inside the patient. The technologies of modern science have amazed and also fascinate our vision regarding working with patients. This literature review provides only a glance at the future of intelligence science.

Macrochlamys indica is a hermaphrodite terrestrial pulmonate mollusk. Transmission electron microscope studies were done on the ovotestis of young and adult (older) M. indica which are elaborated in this paper.The ovotestis contains numerous lobes each of which contains many ovoid shaped acini which are occupied by stages of spermatogenesis and a single oocyte. In younger snails, the acini contain stages of developing spermatogenesis, whereas each acinus of older snails is composed of single large oocyte and few stags of spermatogenesis. The number of Sertoli cells is high in the acini of younger snails than in older snails. Details of the cellular organization of the Sertoli cell are described. Some long thin threads extend from the acinar boundary to acinar lumen. The anterior end of these threads is either free or directly connected to the developing cells of spermatogenesis. There are two types of cells in the interacinar space of the ovotestis in both younger and older snails. One cell is small oval interstitial cell and other is thin elongated periacinar cell. The acinar boundary contains secretory cells with deeply stained nucleus. In the acinus of older snails, the Sertoli cells do not form any barrier between oocyte and spermatogenic cells.Functions of the periacinar cell and interacinar cell are discussed. It is found that the spermatogenesis is highly active in younger snails with single dormant oocyte while process of oogenesis predominates in the older snails. The reproductive strategy of this pulmonate species thus depends on the individual's body size and their maturity.

Microscopic anatomy of Haller’s organ of snake inhabiting tick, Amblyomma gervaisi and Amblyomma helvolum is described. The Haller’s organ consists of two parts, the anterior pit and the proximal capsule. The surface ultrastructural studies by using scanning electron microscope (SEM) on this olfactory organ of both the species revealed some species specific features like slit pattern. Anterior pit consists of six sensilla in A. gervaisi and four in A. helvolum. These sensilla at their base region are connected to each other by a channel. Transmission electron microscope (TEM) observation of this organ of A. gervaisi shows that the lumen of each sensilla and the channel are associated with muscles and bunches of nerve fibres, respectively. Proximal capsule consists of few pyramid shaped elevated regions, each of which at their ventral surface bears cluster of one bipolar sensory cell guarded by two lateral supportive cells. Cytomorphology of the sensory cell exhibit distinct nucleus, mitochondria, ...

Mycobacteriophages infect mycobacteria, resulting in their death. Therefore, the possibility of using them as therapeutic agents against the deadly mycobacterial disease tuberculosis (TB) is of great interest. To obtain better insight into the dynamics of mycobacterial inactivation by mycobacteriophages, this study was initiated using mycobacteriophage D29 and Mycobacterium smegmatis as the phage-host system. Here, we implemented a goal-oriented iterative cycle of experiments on one hand and mathematical modeling combined with Monte Carlo simulations on the other. This integrative approach lends valuable insight into the detailed kinetics of bacterium-phage interactions. We measured time-dependent changes in host viability during the growth of phage D29 in M. smegmatis at different multiplicities of infection (MOI). The predictions emerging out of theoretical analyses were further examined using biochemical and cell biological assays. In a phage-host interaction system where multiple rounds of infection are allowed to take place, cell counts drop more rapidly than expected if cell lysis is considered the only mechanism for cell death. The phenomenon could be explained by considering a secondary factor for cell death in addition to lysis. Further investigations reveal that phage infection leads to the increased production of superoxide radicals, which appears to be the secondary factor. Therefore, mycobacteriophage D29 can function as an effective antimycobacterial agent, the killing potential of which may be amplified through secondary mechanisms.

Objective: The soxhlet and cold extracts of Datura metel Linn. were evaluated for in vitro antirabies activity. Materials and Methods: Soxhlet and cold extraction method were used to extract Datura (fruit and seed) extracts. In vitro cytotoxicity assay was performed by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. Based on the CC50 range, the in vitro antirabies activity of the extracts was screened by rapid fluorescent focus inhibition test and molecular method. Results: The Datura (fruit and seed) extracts were not cytotoxic below 5 mg/ml (CC50). Titer of 10−4 rabies virus challenge virus standard (RV CVS) (1 50% tissue culture infective dose [1 TCID50]) was obtained by RFFT method and the challenge dose of 10 TCID50 was used for antirabies assay. Datura fruit and seed (soxhlet and cold) extracts showed 50% inhibition of RV CVS at 2.5 mg/ml and 1.25 mg/ml (inhibitory concentration 50% [IC50]), respectively. The tested extracts showed selectivity index (CC50/IC50) ranging from 2 to 4. The viral RNA was extracted and real-time reverse transcription-polymerase chain reaction was performed which also revealed a 2-fold reduction of viral load at 1.25 mg/ml of the Datura seed (soxhlet methanolic and cold aqueous) extracts. Conclusion: To the best of our knowledge, this is the first study of in vitro antiviral activity of D. metel Linn. against rabies virus. Datura seed extracts have a potential in vitro antirabies activity and, in future, can be further screened for in vivo activity against rabies virus in murine model. SUMMARY In the present study, Datura metel. Linn showed and in-vitro anti rabies activity in Vero cell line which was determined by RFFIT method and PCR method

Hereditary deafness is one of the most common disabilities affecting newborns. Many forms of hereditary deafness are caused by morphological defects of the stereocilia bundles on the apical surfaces of inner ear hair cells, which are responsible for sound detection. We explored the effectiveness of gene therapy in restoring the hair cell stereocilia architecture in the whirlin mouse model of human deafness, which is deaf due to dysmorphic, short stereocilia. Wild-type whirlin cDNA was delivered via adeno-associated virus (AAV8) by injection through the round window of the cochleas in neonatal whirler mice. Subsequently, whirlin expression was detected in infected hair cells (IHCs), and normal stereocilia length and bundle architecture were restored. Whirlin gene therapy also increased inner hair cell survival in the treated ears compared to the contralateral nontreated ears. These results indicate that a form of inherited deafness due to structural defects in cochlear hair cells is amenable to restoration through gene therapy.

Background: The treatment modalities of resistant hypertension (RH) remain a clinical challenge, often requiring secondary/add-on drugs with first-line therapy to control blood pressure (BP). This study was conducted to explore and understand the preferences and practices of Indian physicians towards the use of vasodilator (especially di-hydralazine) in the management of RH.Methods: This was a cross-sectional, observational, web-based physician survey. The study included cardiologist, nephrologist and consultant physicians from different geographical regions of India. A web-based physician survey questionnaire (PSQ) was created in google forms and the link was circulated to the physicians. Responses obtained were analysed.Results: A total of 457 physicians participated in this survey. In majority of the physicians, vasodilators were the treatment choice as secondary or add-on drugs with first line therapy to control BP in RH; especially hydralazine/di-hydralazine preferred the most. Majority of the physicians preferred to combine vasodilator with beta blocker and diuretic in patients with uncontrolled and RH. Cardiac failure, followed by chronic kidney disease (CKD), diabetes, dyslipidaemia, hypertensive emergency and angina were the common patient profile in RH in which majority physicians prescribed vasodilator (di-hydralazine). Majority of the physicians rated vasodilator di-hydralazine as “good-very good” in terms of efficacy, safety, tolerability, patient compliance and patient satisfaction in RH.Conclusions: Overall, vasodilators (hydrazinophthalazine derivatives) are preferred as add-on drugs along with first-line drugs in RH. Physician’s opinion towards the use of di-hydralazine was positive. Di-hydralazine may be preferred as an add-on therapeutic option to control BP in RH, however randomized clinical trials are needed for recommendation in cardio-renal medicine.

INTRODUCTION Primary liver tumors and liver metastasis currently represent the leading cause of cancer-related deaths. The liver intimately cross-talks with the gut and performs many essential functions related to digestion, metabolism of nutrients, and clearance of bacterial metabolites. Diseased livers are often associated with altered gut bacterial composition, or dysbiosis, and it has been suggested that gut bacterial products contribute to malignant transformation of hepatocytes. The liver is exposed to the gut microbiome through the portal vein and is an immunological organ that is heavily populated by immune cells. Emerging studies have shown that gut commensal bacteria are important regulators of antitumor immunity. Although it has been established that the gut microbiome influences the efficacy of cancer immunotherapy, the role of gut bacteria in antitumor surveillance in the liver is poorly understood. RATIONALE The liver is exposed to gut bacterial metabolites and products by way of blood from the intestine, which comprises 70% of the whole liver blood supply. Changes in the gut microbiome may affect immune cell function in the liver, and commensal bacteria can mediate the metabolism of primary into secondary bile acids, which recirculate back into the liver through the enterohepatic circulation. Given that bile acids are known to be involved in liver cancer development, we focused on the role of bile acids in immunosurveillance of tumors growing in the liver. We altered gut bacteria and examined changes of hepatic immune cells and antitumor immunity directed against liver tumors. Uncovering how the gut microbiome uses bile acids to shape immunity to liver cancer may have future therapeutic applications. RESULTS Using one primary liver model and three liver metastasis models, we found that altering commensal gut bacteria induced a liver-selective antitumor effect. A selective increase of hepatic CXCR6 + natural killer T (NKT) cells was observed, independent of mouse strain, gender, or presence of liver tumors. The accumulated hepatic NKT cells showed an activated phenotype and produced more interferon-γ upon antigen stimulation. In vivo studies using both antibody-mediated cell depletion and NKT-deficient mice confirmed that NKT cells mediated the inhibition of tumor growth in the liver. Further investigation showed that NKT cell accumulation was regulated by the expression of CXCL16, the solo ligand for CXCR6, on liver sinusoidal endothelial cells, which form the lining of liver capillaries and the first barrier for the blood coming from the gut entering the liver. Primary bile acids increased CXCL16 expression, whereas secondary bile acids showed the opposite effect. Removing gram-positive bacteria by antibiotic treatment with vancomycin, which contains the bacteria mediating primary-to-secondary bile acid conversion, was sufficient to induce hepatic NKT cell accumulation and decrease liver tumor growth. Feeding secondary bile acids or colonization of bile acid–metabolizing bacteria, reversed both NKT cell accumulation and inhibition of liver tumor growth in mice with altered gut commensal bacteria. In nontumor liver tissue from human patients with primary liver cancer, primary bile acid chenodeoxycholic acid (CDCA) levels correlated with CXCL16 expression, whereas an inverse correlation was observed with secondary bile acid glycolithocholate (GLCA), suggesting that the finding may apply to humans. CONCLUSION We describe a mechanism by which the gut microbiome uses bile acids as messengers to control a chemokine-dependent accumulation of hepatic NKT cells and antitumor immunity in the liver, against both primary and metastatic liver tumors. These findings not only have possible implications for future cancer therapeutic studies but also provide a link between the gut microbiome, its metabolites, and immune responses in the liver.

Bacteriophages are more abundant than any other organism on our planet. The interaction of bacteriophages and bacteria and their coevolution is well known. In this chapter, we describe various aspects of modeling such systems and their dynamics. We explore their interaction in: (i) liquid media, which leads to well-mixed populations and (ii) solid media, where their interaction is spatially restricted. Such modeling, when used in conjunction with experiments would not only shed deep insight into the underlying dynamics but also provide useful clues toward potential therapeutic applications.

Rabies is an acute viral infection that causes encephalomyelitis in almost all warm blooded animals and is invariably fatal once the clinical signs appear. The present study was carried out to assess the effect of recombinant human interferon alpha (rhIFN α-2A) treatment on the survival of rabies infected mice and its correlation with cytokines expression. The gene expression of TNF-α and IL-6 was measured by SYBR Green Real Time PCR for two groups—“Pre-exposure” (mice were inoculated with rhIFN α-2A prior to rabies infection) and “Post-exposure” (mice were inoculated with rhIFN α-2A post rabies virus infection). Delayed mortality was observed in interferon treated infected groups. In addition, statistically significant decrease (P

Objectives/Hypothesis Gene therapy offers the possibility of delivering corrective genetic materials to the cochlea, potentially improving hearing. In animals, the most commonly used surgical methods for viral gene therapy delivery to the cochlea are the round window and the cochleostomy approaches. However, the patterns of viral infection and the effects on hearing have not been directly compared between these two approaches. In this study, we compare the patterns of cochlear infection and effects on hearing between these two surgical approaches using adeno-associated virus serotype 2/8 (AAV8) as the gene delivery vehicle. Study Design Animal study and basic science research. Methods One- to two-month-old CBA/J mice were used in this study. AAV8-green fluorescent protein (GFP) was delivered to the cochlea by either the round window or the cochleostomy approach (described below). Auditory brainstem response was used to examine hearing thresholds before and after surgery. Animals were examined at 1, 2, 3, and 4 weeks after surgery for the patterns of cochlear infection and hearing loss. Results Cochlear gene transfer was successful through both surgical approaches. In both approaches, AAV8-GFP mostly infected the inner hair cells. There was occasional low-level infection of the outer hair cells and supporting cells. The two surgical approaches resulted in comparable viral infection efficiencies. The round window approach resulted in less surgical trauma, as indicated by hearing loss, than the cochleostomy approach. Conclusions Adeno-associated virus-mediated gene transfer to the cochlea can be accomplished using either the round window or the cochleostomy surgical approach. The round window approach resulted in less hearing loss compared to the cochleostomy approach. Level of Evidence NA Laryngoscope, 125:2557–2564, 2015

Cisplatin (cis-diamminedichloroplatinum) is widely used as a chemotherapeutic drug for genitourinary, breast, lung and head and neck cancers. Though effective in inducing apoptosis in cancer cells, cisplatin treatment causes severe hearing loss among patients. Steroids have been shown to mitigate cisplatin-induced hearing loss. However, steroids may interfere with the anti-cancer properties of cisplatin if administered systemically, or are rapidly cleared from the middle and inner ear and hence lack effectiveness when administered intra-tympanically. In this work, we deliver prednisolone-loaded nanoparticles magnetically to the cochlea of cisplatin-treated mice. This magnetic delivery method substantially reduced hearing loss in treated animals at high frequency compared to control animals or animals that received intra-tympanic methylprednisolone. The method also protected the outer hair cells from cisplatin-mediated ototoxicity.

The microbiota is composed of commensal bacteria and other microorganisms that live on the epithelial barriers of the host. The commensal microbiota is important for the health and survival of the organism. Microbiota influences physiological functions from the maintenance of barrier homeostasis locally to the regulation of metabolism, haematopoiesis, inflammation, immunity and other functions systemically. The microbiota is also involved in the initiation, progression and dissemination of cancer both at epithelial barriers and in sterile tissues. Recently, it has become evident that microbiota, and particularly the gut microbiota, modulates the response to cancer therapy and susceptibility to toxic side effects. In this Review, we discuss the evidence for the ability of the microbiota to modulate chemotherapy, radiotherapy and immunotherapy with a focus on the microbial species involved, their mechanism of action and the possibility of targeting the microbiota to improve anticancer efficacy while preventing toxicity.

Commensal microorganisms (the microbiota) live on all the surface barriers of our body and are particularly abundant and diverse in the distal gut. The microbiota and its larger host represent a metaorganism in which the cross talk between microbes and host cells is necessary for health, survival, and regulation of physiological functions locally, at the barrier level, and systemically. The ancestral molecular and cellular mechanisms stemming from the earliest interactions between prokaryotes and eukaryotes have evolved to mediate microbe-dependent host physiology and tissue homeostasis, including innate and adaptive resistance to infections and tissue repair. Mostly because of its effects on metabolism, cellular proliferation, inflammation, and immunity, the microbiota regulates cancer at the level of predisposing conditions, initiation, genetic instability, susceptibility to host immune response, progression, comorbidity, and response to therapy. Here, we review the mechanisms underlying the interaction of the microbiota with cancer and the evidence suggesting that the microbiota could be targeted to improve therapy while attenuating adverse reactions.

Influenza is a serious respiratory illness which can be debilitating and cause complications that lead to hospitalization and death. Although influenza vaccine can prevent influenza virus infection, the only therapeutic options to treat influenza virus infection are antiviral agents. Given temporal and geographic changes and the shifts in antiviral drug resistance among influenza viruses, it is time to consider natural antiviral agents against influenza virus. Jatropha curcas is known for various medicinal uses. Its antimicrobial, anti-cancer and anti-HIV activity has been well recognized. Because of its broad-spectrum activity, we investigated aqueous and methanol leaf extracts for cytotoxicity and its potential to inhibit hemagglutinin protein of influenza virus. The bioactive compounds from leaf extracts were characterized by high-performance thinlayer chromatography which revealed the presence of major phytochemicals including flavonoids, saponins and tannins. The cytotoxic concentration 50 for aqueous and methanol extracts were determined using trypan blue dye exclusion assay. Inhibition of hemagglutinin protein was assessed using minimal cytotoxic concentrations of the extracts and 10(2.5) TCID50 (64 HA titre) of the Influenza A (H1N1) virus with different exposure studies using hemagglutination assay. Aqueous and methanol extracts were found to be non toxic to Madin darby canine kidney cells below concentration of 15.57 and 33.62 mg/mL for respectively. Inhibition of hemagglutinin was studied using reducing hemagglutination titre which confirmed that the J. curcas extracts have direct effect on the process of virus adsorption leading to its inhibition. Our results provide the information which shows the potential of Jatropha extracts in the treatment of influenza A (H1N1) virus infection. With an established reduced toxicity and prevention of infection by inhibiting hemagglutinin protein, these extracts and its derivatives may be further developed as broad spectrum anti-influenza drugs for prevention and treatment of infections by different types of influenza viruses with further mechanistic studies on anti-influenza.

Mechanosensory hair cells are the receptor cells of hearing and balance. Hair cells are sensitive to death from exposure to therapeutic drugs with ototoxic side effects, including aminoglycoside antibiotics and cisplatin. We recently showed that the induction of heat shock protein 70 (HSP70) inhibits ototoxic drug–induced hair cell death. Here, we examined the mechanisms underlying the protective effect of HSP70. In response to heat shock, HSP70 was induced in glia-like supporting cells but not in hair cells. Adenovirus-mediated infection of supporting cells with Hsp70 inhibited hair cell death. Coculture with heat-shocked utricles protected nonheat-shocked utricles against hair cell death. When heat-shocked utricles from Hsp70–/– mice were used in cocultures, protection was abolished in both the heat-shocked utricles and the nonheat-shocked utricles. HSP70 was detected by ELISA in the media surrounding heat-shocked utricles, and depletion of HSP70 from the media abolished the protective effect of heat shock, suggesting that HSP70 is secreted by supporting cells. Together our data indicate that supporting cells mediate the protective effect of HSP70 against hair cell death, and they suggest a major role for supporting cells in determining the fate of hair cells exposed to stress.

Purpose: Influenza has a major impact on public heath, annually affecting 15-20% of the global population. Information on the activity of influenza virus in Mumbai is limited. The present study was carried out to determine the prevalence of influenza viruses causing acute respiratory infections in children by molecular methods. Objective: To study the prevalence of influenza viruses among the paediatric population in Mumbai by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR). Materials and Methods: From July 2007 to July 2009, 100 respiratory samples (nasal and throat swabs) were collected from paediatric patients with acute respiratory symptoms. attending out patients department, and admitted to the paediatric wards of B. J. Wadia Hospital for Children, Mumbai. The samples were collected and processed as per World Health Organization (WHO) guidelines. Viral RNA was extracted and one-step rRT-PCR was performed to detect influenza type A (H1 and H3) and influenza type B virus. Results: Out of 100 samples processed by rRT-PCR, a total of 11 samples (11%) were positive for influenza virus. The typing for influenza A subtypes showed 1% (1) positivity for H1 and 5% (5) positivity for H3 subtypes and 5% (5) samples tested positive for influenza type B virus. Conclusion: It was observed that both influenza type A and B viruses were prevalent in Mumbai during the study period. Such surveillance data are important in the early detection of any antigenic variants that may be helpful in global influenza vaccine preparation and for any pandemic preparedness activity.

Cell specific targeting is an emerging field in nanomedicine. Homing of the multifunctional nanoparticles (MFNPs) is achieved by the conjugation of targeting moieties on the nanoparticle surface. The inner ear is an attractive target for new drug delivery strategies as it is hard to access and hearing loss is a significant worldwide problem. In this work we investigated the utility of a Nerve Growth Factor-derived peptide (hNgf_EE) functionalized nanoparticles (NPs) to target cells of the inner ear. These functionalized NPs were introduced to organotypic explant cultures of the mouse inner ear and to PC-12 rat pheochromocytoma cells. The NPs did not show any signs of toxicity. Specific targeting and higher binding affinity to spiral ganglion neurons, Schwann cells and nerve fibers of the explant cultures were achieved through ligand mediated multivalent binding to tyrosine kinase receptors and to p75 neurotrophin receptors. Unspecific uptake of NPs was investigated using NPs conjugated with scrambled hNgf_EE peptide. Our results indicate a selective cochlear cell targeting by MFNPs, which may be a potential tool for cell specific drug and gene delivery to the inner ear.

We present a fresh and broad yet simple approach towards information retrieval in general and diagnostics in particular by applying the theory of complex networks on multidimensional, dynamic images. We demonstrate a successful use of our method with the time series generated from high content thermal imaging videos of patients suffering from the aqueous deficient dry eye (ADDE) disease. Remarkably, network analyses of thermal imaging time series of contact lens users and patients upon whom Laser-Assisted in situ Keratomileusis (Lasik) surgery has been conducted, exhibit pronounced similarity with results obtained from ADDE patients. We also propose a general framework for the transformation of multidimensional images to networks for futuristic biometry. Our approach is general and scalable to other fluctuation-based devices where network parameters derived from fluctuations, act as effective discriminators and diagnostic markers., Replaced by published version. Detailed Supplementary Information on journal website

Rabies remains an important public health problem in the world due to uncontrolled enzootic rabies. Although rabies associated fatalities may be prevented with timely immunoprophylaxis, but till date a therapeutic molecule has remained elusive. We investigated the role of rhuIFN α-2a in murine model challenged with rabies virus. Titre of 10 4.25 LD 50 /0.03 ml of 10% w/v RV CVS stock suspension were obtained. Based on 1LD 50 titre, challenge dose of 50 LD 50 was administered along with rhuIFN α-2a with pre-exposure (primed) and post-exposure with the rabies virus. Both showed increased survival time as compared with the virus controls. These fi ndings suggest that the rhuIFN α-2a might have some anti-viral activity, which can be used for the treatment of rabies infection. Further research on the effi cacy of interferon along with anti-viral drugs for the treatment will be helpful in designing combination therapy against the disease.

The syndrome of cancer cachexia is currently defined as a state of ill health, malnutrition and physical wasting with marked white adipose tissue (WAT) and skeletal muscle mass wasting, representing the clinical consequence of a chronic and systemic inflammatory response. Over the last decade, WAT has been recognized as an important endocrine organ, and earning a lot of attention during cancer cachexia development. We investigated the role of microbiota along the cachexia associated cancer. We performed experiments with conventional and Germ Free mice (GF) (n=6 in each group) of 8-10 weeks old C57B/6, which were subcutaneously injected with LLC cells [4x106 cells in 0.2 mL; Tumor-bearing, (TB) or PBS control (C)]. We performed Immunohistochemistry, RT- qPCR, and Western Blot. We observed that GF Tumor-bearing mice have increased several symptoms of the cachexia compared to conventional TB mice. The WAT mass was decreased 50% in GF Tumor-bearing mice compared to all groups, which indicates a pathway related to lipolysis, as we found increased level of phosphorylated enzymes in GF Tumor-bearing mice. We also observed that GF Tumor bearing mice decreased skeletal muscle mass and gene expression that are related with atrophy were increased in GF Tumor bearing mice. Our data suggested that homeostasis of microbiota may impair the development of the cachexia syndrome. Citation Format: Rodrigo Xavier das Neves, Soumen Roy, Amiran Dzutsev, April Huang, Loretta Smith, Simone Difilippantonio, Hawes Misty, Marília Seelaender, Giorgio Trinchieri. Germ free mice accelerate cachexia-associated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 451. doi:10.1158/1538-7445.AM2017-451

Chemotherapy induced toxicity severely affect the cancer survivors and lowers the quality of life. By 2020, there will be more than 18 million of cancer survivors all over the world. Majority of them might develop long term nephrotoxicity, ototoxicity and gut toxicity. In addition, chemotherapy may also facilitate the initiation and progression of cachexia. Recent studies have shown that gut microbiota modulates the efficacy of anti-cancer chemotherapy, however very limited knowledge is available regarding the role of gut microbiota in regulating systemic toxicity and cachexia. We hypothesized that gut microbiota modulates cisplatin induced systemic toxicity as well as cachexia. Four groups (n=10 in each group) of 6-8 weeks old C57B/6 mice were treated with cisplatin, cisplatin+antibiotics cocktails (ABX), ABX only and control. ABX cocktail contained primaxin, vancomycin and neomycin. This experiment was validated using C57B/6 germ free mice. We performed anti-p-γ-H2AX based toxicity assay for DNA damage. In addition, we performed immunohistochemistry for studying cachexia. H&E staining and DNA damage were assessed by light and super resolution confocal microscopy (Zeiss 880) and quantified by 3-D reconstruction using IMARIS. We also performed high content imaging to evaluate DNA damage in bone marrow and spleen. Body weight and kidney blood content were also analyzed to determine the degree of toxicity. Depletion of gut microbiota significantly reduced nephrotoxicity and gut toxicity. In addition, we found that depletion of gut microbiota prevents muscle and adipose tissue loss by down-regulating UCP-1and PGC-1α in adipose tissue and MURF-1 and Atrogin-1 in muscle. Experiments are confirmed by using Germ free mice. Our data suggest that modulation of gut microbiota may be utilized to reduce chemotherapy associated cachexia and systemic toxicity. Citation Format: Soumen Roy, Rodrigo Das Neves, Amiran Dzutsev, Carolyne Smith, Bathai Edwards, Miranda Dawson, Simone Difilippantonio, Loretta Smith, April Huang, Young Kim, Giorgio Trinchieri. Gut microbiota regulates cisplatin mediated cachexia and systemic toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4926. doi:10.1158/1538-7445.AM2017-4926

Hearing loss and balance disorders affect millions of people worldwide. Sensory transduction in the inner ear requires both mechanosensory hair cells (HCs) and surrounding glia-like supporting cells (SCs). HCs are susceptible to death from aging, noise overexposure, and treatment with therapeutic drugs that have ototoxic side effects; these ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic drug cisplatin. Although both classes of drugs are known to kill HCs, their effects on SCs are less well understood. Recent data indicate that SCs sense and respond to HC stress, and that their responses can influence HC death, survival, and phagocytosis. These responses to HC stress and death are critical to the health of the inner ear. Here we have used live confocal imaging of the adult mouse utricle, to examine the SC responses to HC death caused by aminoglycosides or cisplatin. Our data indicate that when HCs are killed by aminoglycosides, SCs efficiently remove HC corpses from the sensory epithelium in a process that includes constricting the apical portion of the HC after loss of membrane integrity. SCs then form a phagosome, which can completely engulf the remaining HC body, a phenomenon not previously reported in mammals. In contrast, cisplatin treatment results in accumulation of dead HCs in the sensory epithelium, accompanied by an increase in SC death. The surviving SCs constrict fewer HCs and display impaired phagocytosis. These data are supported by in vivo experiments, in which cochlear SCs show reduced capacity for scar formation in cisplatin-treated mice compared with those treated with aminoglycosides. Together, these data point to a broader defect in the ability of the cisplatin-treated SCs, to preserve tissue health in the mature mammalian inner ear.

Cisplatin is a highly successful and widely used chemotherapy for the treatment of various solid malignancies in both adult and pediatric patients. Side effects of cisplatin treatment include nephrotoxicity and ototoxicity. Cisplatin ototoxicity results from damage to and death of cells in the inner ear, including sensory hair cells. We showed previously that heat shock inhibits cisplatin-induced hair cell death in whole-organ cultures of utricles from adult mice. Since heat shock protein 70 (HSP70) is the most upregulated HSP in response to heat shock, we investigated the role of HSP70 as a potential protectant against cisplatin-induced hair cell death. Our data using utricles from HSP70 (-/-) mice indicate that HSP70 is necessary for the protective effect of heat shock against cisplatin-induced hair cell death. In addition, constitutive expression of inducible HSP70 offered modest protection against cisplatin-induced hair cell death. We also examined a second heat-inducible protein, heme oxygenase-1 (HO-1, also called HSP32). HO-1 is an enzyme responsible for the catabolism of free heme. We previously showed that induction of HO-1 using cobalt protoporphyrin IX (CoPPIX) inhibits aminoglycoside-induced hair cell death. Here, we show that HO-1 also offers significant protection against cisplatin-induced hair cell death. HO-1 induction occurred primarily in resident macrophages, with no detectable expression in hair cells or supporting cells. Depletion of macrophages from utricles abolished the protective effect of HO-1 induction. Together, our data indicate that HSP induction protects against cisplatin-induced hair cell death, and they suggest that resident macrophages mediate the protective effect of HO-1 induction.

Background Interaction between immune system and Chandipura virus (CHPV) during different stages of its life cycle remain poorly understood. The exact route of virus entry into the blood and CNS invasion has not been clearly defined. The present study was undertaken to assess the population in PBMC that supports the growth of virus and to detect active virus replication in PBMC as well as its subsets. Methods PBMC subsets viz.: CD3+, CD14+, CD19+, CD56+cells were separated and infected with CHPV. The infected cells were then assessed for transcription (N gene primer) and replication (NP gene primer) of CHPV by PCR. The supernatant collected from infected cells were titrated in Baby Hamster Kidney (BHK) cells to assess virus release. The cytokine and chemokine expression was quantified by flow cytometry. Results Amplification of N and NP gene was detected in CD14+ (monocyte) and CD19+ (B cell), significant increase in virus titre was also observed in these subsets. It was observed that, although the levels of IL-6 and IL-10 were elevated in CD14+ cells as compared to CD19+cells, the differences were not significant. However the levels of TNFα and IL-8 were significantly elevated in CD14+ cells than in CD19+cells. The levels of chemokine (CXCL9, CCL5, CCL2, CXCL10) were significantly elevated in CHPV infected PBMC as compared to uninfected cells. CCL2 and CXCL9 were significantly increased in CHPV infected CD14+cells as compared to CD19+ cells. Conclusion CD14+and CD19+cells support active replication of CHPV. High viral load was detected in CD14+ cells infected with CHPV hence it might be the primary target cells for active replication of CHPV. An elevated levels of cytokines and chemokines observed in CD14+ cells may help in predicting the pathogenecity of CHPV and possible entry into the central nervous system.

The present study was carried out to monitor influenza viruses by identifying the virus and studying the seasonal variation during 2007–2009 in Mumbai. A total of 193 clinical respiratory samples (nasal and throat swab) were collected from patients having influenza like illness in Mumbai region. One-step real-time reverse-transcriptase PCR (rRTPCR) was used to detect Influenza type A (H1 and H3) and Influenza type B virus. Isolation of the virus was carried out using in vitro system which was further confirmed and typed by hemagglutination assay and hemagglutination inhibition assay. Out of 193 samples 24 (12.4 3%) samples tested positive for influenza virus, of which 13 (6.73 %) were influenza type A virus and 10 (5.18 %) were influenza type B virus, while 1 sample (0.51 %) was positive for both. By culture methods, 3 (1.55 %) viral isolates were obtained. All the three isolates were found to be Influenza type B/Malaysia (Victoria lineage) by Hemagglutination Inhibition Assay. The data generated from the present study reveals that both Influenza type A and B are prevalent in Mumbai with considerable activity. The peak activity was observed during monsoon season.

A total of 100 blood and 18 urine samples of rodents and suspected dogs were collected from Mumbai, India during 2006-2008. In order to determine the role of animals in transmission of the disease to humans, all the samples were screened retrospectively by real-time polymerase chain reaction for leptospiral DNA and antibodies were detected using microscopic agglutination test. Leptopsiral DNA was detected from two blood and five urine samples from rodents. Of a total of 71 rodent and dog samples investigated for anti-Leptospira antibodies, 14 (19.7%) were positive. Pyrogenes was the predominant serovar found in 100.0% (7/7) and 85.7% (6/7) from suspected canine cases and rodents, respectively; followed by Icterohemorrhagiae, which was found in one rodent sample 14.28% (1/7). The study proves that there is high prevalence of leptospirosis in rodents and dogs in this region, which proves possible role of these animals in transmission of leptospires to humans. Hence it is imperative to necessary control measures to prevent human leptospirosis.

Therapeutic drugs with ototoxic side effects cause significant hearing loss for thousands of patients annually. Two major classes of ototoxic drugs are cisplatin and the aminoglycoside antibiotics, both of which are toxic to mechanosensory hair cells, the receptor cells of the inner ear. A critical need exists for therapies that protect the inner ear without inhibiting the therapeutic efficacy of these drugs. The induction of heat shock proteins (HSPs) inhibits both aminoglycoside- and cisplatin-induced hair cell death and hearing loss. We hypothesized that exposure to sound that is titrated to stress the inner ear without causing permanent damage would induce HSPs in the cochlea and inhibit ototoxic drug–induced hearing loss. We developed a sound exposure protocol that induces HSPs without causing permanent hearing loss. We used this protocol in conjunction with a newly developed mouse model of cisplatin ototoxicity and found that preconditioning mouse inner ears with sound has a robust protective effect against cisplatin-induced hearing loss and hair cell death. Sound therapy also provided protection against aminoglycoside-induced hearing loss. These data indicate that sound preconditioning protects against both classes of ototoxic drugs, and they suggest that sound therapy holds promise for preventing hearing loss in patients receiving these drugs.

Objectives: The presence of drug-resistant HIV is a major global concern and warrants the development of novel anti-virals as alternative and inexpensive therapy. In the current study, potentially drug-resistant HIV was isolated and previously unreported anti-viral activity of Jatropha curcas Linn. leaf extracts was assessed. Methods: HIV isolation was done using in vitro micro co-culture methods followed by drug susceptibility assays to determine resistance to Zidovudine (AZT), Lamivudine (3TC) and Stavudine (d4T). Soxhlet apparatus was used for extraction of metabolites from leaves of Jatropha curcas Linn. and Methanolic and Aqueous Extracts were chosen for further study. Secondary metabolites were detected by High-Performance Thin Layer Chromatography and in vitro cytotoxicity established by MTT assay. The extracts were then used in post- and pre-infection studies by measuring inhibition of HIV replication to determine anti-viral activity. Results: Seven HIV isolates were obtained (isolation rate: 23.33%) with drug IC50 values ranging from 0.001418- 82.73 μM AZT, 2.645-15.35 μM 3TC and 18.55-66.23 μM d4T. Tannins, Flavonoids, Saponins were detected in Aqueous Extract and Flavonoids, Saponins in Methanolic Extract. The CC50 values were 32.07 mg/mL and 35.5 mg/ mL for Aqueous and Methanolic Extracts respectively. Anti-viral activity was evaluated by inhibition of HIV replication as determined by HIV p24 antigen ELISA. Post-infection (4 isolates) interaction studies showed IC50 values ranging from 0.0255-0.4137 mg/mL and 0.00073-0.1278 mg/mL for Aqueous and Methanolic Extracts respectively and preinfection (1 isolate) interaction studies showed 100% inhibition by Methanolic and 97.19% inhibition by Aqueous Extract at 25 mg/mL each. Conclusions: HIV isolates potentially resistant to AZT/3TC/d4T were obtained and Jatropha curcas Linn. leaf extracts showed effective anti-viral and probable entry inhibition activity against potentially drug-resistant HIV, which has not been reported earlier. The study indicates that Jatropha curcas Linn. is a good candidate for anti-HIV therapy with further research.

Sanjeev Ranjan,1 Rohit Sood,1 Jozsef Dudas,2 Rudolf Glueckert,2 Anneliese Schrott-Fischer,2 Soumen Roy,2 Ilmari Pyykkö,3 Paavo KJ Kinnunen11Helsinki Biophysics and Biomembrane Group, Department of Biomedical Engineering and Computational Science, Aalto University, Espoo, Finland; 2Department of Otorhinolaryngology, Innsbruck Medical University, Innsbruck, Austria; 3Department of Otolaryngology, University of Tampere Medical School, Tampere, FinlandBackground: The neurotrophic receptor tyrosine kinase B (TrkB) has diverse signaling roles in neurons and tumor cells. Accordingly, its suppressive targeting is of interest in neuroblastoma and other tumors, whereas its role in improving survival is focused in neurons. Here we describe targeting of TrkB-binding peptide-conjugated liposomes (PCL) to the TrkB-expressing mouse macrophage-like cell line RAW264, and to all-trans-retinoic acid-treated neuron-like TrkB+ SH-SY5Y human neuroblastoma cells.Methods: Binding and internalization of PCL was monitored by flow cytometry and confocal fluorescence microscopy.Results: Internalization of TrkB-targeted PCL by RAW264 cells was enhanced and faster when compared with PCL having the corresponding scrambled peptide. Likewise, binding and augmented uptake were confirmed for TrkB+ SH-SY5Y cells, with targeted PCL appearing in the cytoplasm after 20 minutes of incubation.Conclusion: We demonstrate here the feasibility of targeting liposomes to TrkB-expressing cells by 18-mer peptides, promoting cellular uptake (at least partly into endosomes) via receptor-mediated pathways.Keywords: liposomes, targeting, tyrosine kinase B, peptide

Hearing loss is a very significant health problem. The methods currently available for inner ear drug delivery are limited and a noninvasive cell-specific drug delivery strategy needs to be found. Aim: In this study we investigated the ability of polymersomes, lipid core nanocapsules and hyperbranched poly-L-lysine to cross the round window membrane. Materials & Methods: Nanoparticles (NPs) used in this study have different size and chemical compositions. Freshly frozen human temporal bones were used for this investigation. Intact human round window membrane within the freshly frozen human temporal bone served as an excellent model to test the membrane permeation and distribution within the tissues. Results: In this investigation we were able to visualize the NPs across the round window membrane. The NPs were subsequently found to be distributed in the sensory hair cells, nerve fibers and to other cells of the cochlea. Conclusion: This finding raises hope in terms of future multifunctional NP-based drug delivery strategy to the human inner ear.

Background: Japanese encephalitis is a leading form of viral encephalitis, prevalent mostly in South Eastern Asia caused by Japanese encephalitis virus (JEV). It is transmitted by the mosquitoes of the Culex sp. The disease affects children and results in 50% result in permanent neuropsychiatric disorder. There arises a need to develop a safe, affordable, and potent anti-viral agent against JEV. This study aimed to assess the antiviral activity of ajwain (Trachyspermum ammi: Umbellifereae) essential oil against JEV. Materials and Methods: Ajwain oil was extracted by distillation method and in vitro cytotoxicity assay was performed in vero cell line by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay method. JEV titer was determined by plaque assay and in vitro antiviral activity of ajwain oil was quantified by the plaque reduction neutralization test (PRNT). Results: Cytotoxic concentration of the oil was found to be 1 mg/ml by MTT assay. The titer of the virus pool was found to be 50× 10 7 PFU/ml. we observed 80% and 40% virus inhibition in 0.5mg/ml of ajwain oil by PRNT method in preexposure treatment and postexposure treatment (antiviral activity), respectively. Conclusion: Our data indicate ajwain oil has potential in vitro antiviral activity against JEV. Further, the active biomolecule will be purified and evaluated for anti-JEV activity and also to scale up for in vivo trial to evaluate the efficacy of ajwain oil in future.

Methods In vitro micro-co-culture was employed for virus isolation followed by drug susceptibility assays to determine resistance to Azidothymidine (AZT) and Lamivudine (3TC). Jatropha curcasleaves were extracted using Soxhlet apparatus. Methanolic (ME) and aqueous (AE) extracts were chosen for further study. Secondary metabolites were detected by High-Performance Thin Laye rChromatography and in vitro cytotoxicity established by MTT assay. Anti-viral activity was evaluated by p24 inhibitionin postand pre-infection interaction studies.