Vitamin E alleviates chlorpyrifos induced glutathione depletion, lipid peroxidation and iron accumulation to inhibit ferroptosis in hepatocytes and mitigate toxicity in zebrafish.

Organophosphates, a widely used group of pesticides, can cause severe toxicity in human beings and other non-target organisms. Liver, being the primary site for xenobiotic metabolism, is extremely vulnerable to xenobiotic-induced toxicity. Considering the numerous vital functions performed by the liver, including xenobiotic detoxification, protecting this organ from the ubiquitous pesticides in our food and environment is essential for maintaining homeostasis. In this study, we have investigated the impact of the organophosphate pesticide, Chlorpyrifos (CPF), on zebrafish liver at a concentration (300 µg/L) which is environmentally realistic. We have also demonstrated the role of dietary supplementation of α-tocopherol or Vitamin E (Vit E) (500 mg/kg feed) in mitigating pesticide-induced liver toxicity. Mechanistically, we showed that Vit E resulted in significant elevation of the Nrf2 and its downstream antioxidant enzyme activities and gene expressions, especially that of GST and GPx, resulting in reduction of CPF-induced intracellular lipid ROS and hepatic LPO. Further interrogation, such as analysis of GSH: GSSG ratio, intracellular iron concentration, iron metabolizing genes, mitochondrial dysfunction etc. revealed that CPF induces ferroptosis which can be reversed by Vit E supplementation. Ultimately, reduced concentration of CPF in zebrafish serum and flesh highlighted the role of Vit E in ameliorating CPF toxicity.

Intestinal microbiota signatures of clinical response and immune-related adverse events in melanoma patients treated with anti-PD-1.

Ample evidence indicates that the gut microbiome is a tumor-extrinsic factor associated with antitumor response to anti-programmed cell death protein-1 (PD-1) therapy, but inconsistencies exist between published microbial signatures associated with clinical outcomes. To resolve this, we evaluated a new melanoma cohort, along with four published datasets. Time-to-event analysis showed that baseline microbiota composition was optimally associated with clinical outcome at approximately 1 year after initiation of treatment. Meta-analysis and other bioinformatic analyses of the combined data show that bacteria associated with favorable response are confined within the Actinobacteria phylum and the Lachnospiraceae/Ruminococcaceae families of Firmicutes. Conversely, Gram-negative bacteria were associated with an inflammatory host intestinal gene signature, increased blood neutrophil-to-lymphocyte ratio, and unfavorable outcome. Two microbial signatures, enriched for Lachnospiraceae spp. and Streptococcaceae spp., were associated with favorable and unfavorable clinical response, respectively, and with distinct immune-related adverse effects. Despite between-cohort heterogeneity, optimized all-minus-one supervised learning algorithms trained on batch-corrected microbiome data consistently predicted outcomes to programmed cell death protein-1 therapy in all cohorts. Gut microbial communities (microbiotypes) with nonuniform geographical distribution were associated with favorable and unfavorable outcomes, contributing to discrepancies between cohorts. Our findings shed new light on the complex interaction between the gut microbiome and response to cancer immunotherapy, providing a roadmap for future studies.

Gut Microbiome Directs Hepatocytes to Recruit MDSCs and Promote Cholangiocarcinoma.

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.

Role of a 19S Proteasome Subunit- PSMD10Gankyrin in Neurogenesis of Human Neural Progenitor Cells.

PSMD10Gankyrin, a proteasome assembly chaperone, is a widely known oncoprotein which aspects many hall mark properties of cancer. However, except proteasome assembly chaperon function its role in normal cell function remains unknown. To address this issue, we induced PSMD10Gankyrin overexpression in HEK293 cells and the resultant large-scale changes in gene expression profile were analyzed. We constituted networks from microarray data of these differentially expressed genes and carried out extensive topological analyses. The overrecurring yet consistent theme that appeared throughout analysis using varied network metrics is that all genes and interactions identified as important would be involved in neurogenesis and neuronal development. Intrigued we tested the possibility that PSMD10Gankyrin may be strongly associated with cell fate decisions that commit neural stem cells to differentiate into neurons. Overexpression of PSMD10Gankyrin in human neural progenitor cells facilitated neuronal differentiation via ß-catenin Ngn1 pathway. Here for the first time we provide preliminary and yet compelling experimental evidence for the involvement of a potential oncoprotein - PSMD10Gankyrin, in neuronal differentiation.

Gut microbiome-mediated bile acid metabolism regulates liver cancer via NKT cells.

Primary liver tumors and liver metastasis currently represent the leading cause of cancer-related death. Commensal bacteria are important regulators of antitumor immunity, and although the liver is exposed to gut bacteria, their role in antitumor surveillance of liver tumors is poorly understood. We found that altering commensal gut bacteria in mice induced a liver-selective antitumor effect, with an increase of hepatic CXCR6+ natural killer T (NKT) cells and heightened interferon-γ production upon antigen stimulation. In vivo functional studies showed that NKT cells mediated liver-selective tumor inhibition. NKT cell accumulation was regulated by CXCL16 expression of liver sinusoidal endothelial cells, which was controlled by gut microbiome-mediated primary-to-secondary bile acid conversion. Our study suggests a link between gut bacteria-controlled bile acid metabolism and liver antitumor immunosurveillance.

Magnetic Nanoparticle Mediated Steroid Delivery Mitigates Cisplatin Induced Hearing Loss.

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.

Microbiota: a key orchestrator of cancer therapy.

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.

Microbes and Cancer.

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.

Monocytes and B cells support active replication of Chandipura virus.

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.

Gene Therapy Restores Hair Cell Stereocilia Morphology in Inner Ears of Deaf Whirler Mice.

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.

Using complex networks towards information retrieval and diagnostics in multidimensional imaging.

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.

Evaluation of antiviral activity of essential oil of Trachyspermum Ammi against Japanese encephalitis virus.

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.

Mapping networks of light-dark transition in LOV photoreceptors.

MOTIVATION: In optogenetics, designing modules of long or short signaling state lifetime is necessary for control over precise cellular events. A critical parameter for designing artificial or synthetic photoreceptors is the signaling state lifetime of photosensor modules. Design and engineering of biologically relevant artificial photoreceptors is based on signaling mechanisms characteristic of naturally occurring photoreceptors. Therefore identifying residues important for light-dark transition is a definite first step towards rational design of synthetic photoreceptors. A thorough grasp of detailed mechanisms of photo induced signaling process would be immensely helpful in understanding the behaviour of organisms. RESULTS: Herein, we introduce the technique of differential networks. We identify key biological interactions, using light-oxygen-voltage domains of all organisms whose dark and light state crystal structures are simultaneously available. Even though structural differences between dark and light states are subtle (other than the covalent bond formation between flavin chromophore and active site Cysteine), our results successfully capture functionally relevant residues and are in complete agreement with experimental findings from literature. Additionally, using sequence-structure alignments, we predict functional significance of interactions found to be important from network perspective yet awaiting experimental validation. Our approach would not only help in minimizing extensive photo-cycle kinetics procedure but is also helpful in providing first-hand information on the fundamentals of photo-adaptation and rational design of synthetic photoreceptors in optogenetics. CONTACT: devrani.dbs@presiuniv.ac.in or soumen@jcbose.ac.in SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cochlear gene transfer mediated by adeno-associated virus: Comparison of two surgical approaches.

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.

Heat shock protein-mediated protection against Cisplatin-induced hair cell death.

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.

Inner ear supporting cells protect hair cells by secreting HSP70.

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.

Evaluation of Jatropha curcas Linn. leaf extracts for its cytotoxicity and potential to inhibit hemagglutinin protein of influenza virus.

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.

Peptide-mediated targeting of liposomes to TrkB receptor-expressing cells.

BACKGROUND: 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.

Visualization and analysis of superparamagnetic iron oxide nanoparticles in the inner ear by light microscopy and energy filtered TEM.

Nanoparticles as potential carriers for local drug transfer are an alternative to systemic drug delivery into the inner ear. We report on the first in vitro tests of a new ferrogel consisting of superparamagnetic iron oxide nanoparticles (SPIONs) and a Pluronic(®) F127 (PF127) copolymer. Pluronic copolymers possess a unique viscosity-adjustable property that makes PF127 gels easy to handle compared to conventional cross-linked hydrogels. This ferrogel was successfully tested in cadaver human temporal bones as well as in organotypic explant cultures of mouse inner ears. SPIONs were identified by light microscopy and localized with different imaging modes in energy-filtered transmission electron microscopy. Our approach shows a promising possibility to use iron oxide nanoparticles, which are suitable for visualization and characterization at both the light- and electron-microscopic levels. FROM THE CLINICAL EDITOR: The authors report the first in vitro tests of a new ferrogel consisting of superparamagnetic iron oxide nanoparticles (SPIONs) and a Pluronic® F127 (PF127) copolymer for drug delivery in the inner ear, demonstrasting a promising possibility to use iron oxide nanoparticles, which are suitable for visualization and characterization at both the light- and electron-microscopic levels.