Search

The microbiota—the diverse set of commensal microbes that normally colonize humans—represents the first line of defense against infectious diseases. In this review, we summarize the direct and indirect mechanisms by which the microbiota modulates susceptibility to, and severity of, infections, with a focus on immunological mechanisms. Moreover, we highlight some of the ways that modern-world lifestyles have influenced the structure-function relationship between the microbiota and infectious diseases. Ultimately, understanding how the microbiota influences infectious risks will facilitate development of microbiota-derived therapeutics that bolster host defenses.

An experiment was conducted during Kahrif 2017 and Kharif 2018 at department of plant pathology, Rajasthan collage of Agriculture, Udaipur, Raj. to find out the average yield loss assessment caused due to Curvularia leaf spot of maize. During Kharif 2017 estimated average yield loss was 21.69% with 18.56 % PDI and 1032.33 kg/plot yield in protected plot similarly in Kharif 2018 yield loss was 22.49% and 992.73 kg /plot yield with 18.07% PDI. On average yield loss caused due to CLS in maize in both years was estimated 22.06%.

The aim of the present study is to evaluate the neuroprotective effect of methanol extract of Garcinia morella Desr leaves in monosodium glutamate (MSG) induced neurotoxicity in rats. The MSG 2 g/kg, i.p. was used to induce the neurotoxicity. The rats were administrated with methanol extract of Garcinia morella Desr leaves (MEGM) 200 and 400 mg/kg, p.o. and Dextromethorphan 30 mg/kg, p.o. after 1 h injection of MSG. The animals were evaluated for its behavioural factors such as muscle grip and locomotor activity. At the end of the study, the antioxidant enzymes levels, neurotransmitter levels, TNFα, ß-amyloid and mineral levels were estimated in brain homogenate. The MEGM treated group shows significant improvement of behavioural and locomotor activity and muscle strength against MSG induced neurotoxicity. The glutathione, SOD, catalase and total protein levels were significantly increased in MEGM treated group. However, significant reduction within the level of varied neurotransmitters like AChE, Dopamine, TNFα, ß-amyloid were observed on treatment with MEGM extract. Further, MEGM also significantly decrease the MSG induced toxicity through declined levels of Ca+2 and Na+ with increased levels of K+. In conclusion, the present study suggests that the MEGM has significant neuroprotective activity against MSG induced neurotoxic rats.

Activation of invariant natural killer T (iNKT) cells by α-galactosylceramides (α-GalCers) stimulates strong immune responses and potent anti-tumor immunity. Numerous modifications of the glycolipid structure have been assessed to derive activating ligands for these T cells with altered and potentially advantageous properties in the induction of immune responses. Here, we synthesized variants of the prototypical α-GalCer, KRN7000, with amide-linked phenyl alkane substitutions on the C4″-position of the galactose ring. We show that these variants have weak iNKT cell stimulating activity in mouse models but substantially greater activity for human iNKT cells. The most active of the C4″-amides in our study showed strong anti-tumor effects in a partially humanized mouse model for iNKT cell responses. In silico analysis suggested that the tether length and degree of flexibility of the amide substituent affected the recognition by iNKT cell antigen receptors of the C4″-amide substituted glycolipids in complex with their antigen presenting molecule CD1d. Our findings establish the use of stable C4″-amide linked additions to the sugar moiety for further exploration of the immunological effects of structural modifications of iNKT cell activating glycolipids and highlight the critical need for more accurate animal models to assess these compounds for immunotherapeutic potential in humans.

Decline in insulin sensitivity due to dysfunction of adipose tissue (AT) is one of the earliest pathogenic events in type 2 diabetes. We hypothesize that differential DNA methylation (DNAm) controls insulin sensitivity and obesity by modulating transcript expression in AT. Integrating AT DNAm profiles with transcript profile data measured in a cohort of 230 African Americans (AAs) from the African American Genetics of Metabolism and Expression cohort, we performed cis-expression quantitative trait methylation (cis-eQTM) analysis to identify epigenetic regulatory loci for glucometabolic trait–associated transcripts. We identified significantly associated cytosine-guanine dinucleotide regions for 82 transcripts (false discovery rate [FDR]-P < 0.05). The strongest eQTM locus was observed for the proopiomelanocortin (POMC; ρ = −0.632, P = 4.70 × 10−27) gene. Epigenome-wide association studies (EWAS) further identified 155, 46, and 168 cytosine-guanine dinucleotide regions associated (FDR-P < 0.05) with the Matsuda index, SI, and BMI, respectively. Intersection of EWAS, transcript level to trait association, and eQTM results, followed by causal inference test identified significant eQTM loci for 23 genes that were also associated with Matsuda index, SI, and/or BMI in EWAS. These associated genes include FERMT3, ITGAM, ITGAX, and POMC. In summary, applying an integrative multiomics approach, our study provides evidence for DNAm-mediated regulation of gene expression at both previously identified and novel loci for many key AT transcripts influencing insulin resistance and obesity.

Mycobacterium tuberculosis, responsible for Tuberculosis (TB), remains the leading cause of mortality among infectious diseases worldwide from a single infectious agent, with an estimated 1.7 million deaths in 2016. Biotin is an essential cofactor in M. tuberculosis that is required for lipid biosynthesis and gluconeogenesis. M. tuberculosis relies on de novo biotin biosynthesis to obtain this vital cofactor since it cannot scavenge sufficient biotin from a mammalian host. The biotin biosynthetic pathway in M. tuberculosis has been well studied and rigorously genetically validated providing a solid foundation for medicinal chemistry efforts. This review examines the mechanism and structure of the enzymes involved in biotin biosynthesis and ligation, summarizes the reported genetic validation studies of the pathway, and then analyzes the most promising inhibitors and natural products obtained from structure-based drug design and phenotypic screening.

Radial neuron migration in the developing cerebral cortex is a complex journey, starting in the germinal zones and ending in the cortical plate. In mice, migratory distances can reach several hundreds of microns, or millimeters in humans. Along the migratory path, radially migrating neurons slither through cellularly dense and complex territories before they reach their final destination in the cortical plate. This task is facilitated by radial glia, the neural stem cells of the developing cortex. Indeed, radial glia have a unique bipolar morphology, enabling them to serve as guides for neuronal migration. The key guiding structure of radial glia is the basal process, which traverses the entire thickness of the developing cortex. Neurons recognize the basal process as their guide and maintain physical interactions with this structure until the end of migration. Thus, the radial glia basal process plays a key role during radial migration. In this review, we highlight the pathways enabling neuron-basal process interactions during migration, as well as the known mechanisms regulating the morphology of the radial glia basal process. Throughout, we describe how dysregulation of these interactions and of basal process morphology can have profound effects on cortical development, and therefore lead to neurodevelopmental diseases.

Nucleotide-binding leucine-rich repeat (NLR) immune receptors play a critical role in defence against pathogens in plants and animals. However, we know very little about NLR-interacting proteins and the mechanisms that regulate NLR levels. Here, we used proximity labeling (PL) to identify the proteome proximal to N, which is an NLR that confers resistance to Tobacco mosaic virus (TMV). Evaluation of different PL methods indicated that TurboID-based PL provides more efficient levels of biotinylation than BioID and BioID2 in plants. TurboID-based PL of N followed by quantitative proteomic analysis and genetic screening revealed multiple regulators of N-mediated immunity. Interestingly, a putative E3 ubiquitin ligase, UBR7, directly interacts with the TIR domain of N. UBR7 downregulation leads to an increased amount of N protein and enhanced TMV resistance. TMV-p50 effector disrupts the N-UBR7 interaction and relieves negative regulation of N. These findings demonstrate the utility of TurboID-based PL in plants and the N-interacting proteins we identified enhance our understanding of the mechanisms underlying NLR regulation.

Ayurveda and contemporary science comprehends human body as model to understand disease state from very different perspective hence their methodology to describe disease manifestation also seems very different as they observe same problem from different viewpoints. For more precision in healthcare system it is essential that best of both systems need to be integrated, In Ayurvedic texts, the description of concept of basic constitution/Prakriti in health and disease states is well described. The scripts are not clearly understandable and hence its importance has not been properly understood by contemporary biological researchers and thus not utilized. According to Ayurveda individual’s basic constitution (Prakriti) influences and directs one’s disease status, its treatment & lifestyle regimen. In genomics, individual’s basic constitution is decided by its genetic makeup. The current trend in Ayurveda-related biomolecular studies is establishment of high correlation between Prakriti and Genomics. This approach of Ayurgenomics would facilitate the development of alternative methods for cost effective screening of predisposed individuals in the population. This would result in development of an integrated approach to systems biology for disease and health state. For the establishment and success of this concept the prerequisite is integration of Ayurveda into mainstream contemporary biology to achieve global acceptability for the concepts and science of Ayurveda, and for this Ayurvedic concept based phenotypic (Prakriti) assessment needs to be made so that difference among individuals from large population based on their physical, physiological and psychological status can be observed and they can be categorized by their specific predominant Prakriti i.e. Vata, Pitta, Kapha. Information about individual predominant Prakriti can be further analyzed on certain genomic parameters related to gene expression, genetic, epigenetic and biochemical factors, which can be further utilized for Integration of Ayurveda with Genomics for systems biology approach in predictive and personalized medicine.

Gastrointestinal (GI) syndrome is a serious side effect and dose-limiting toxicity observed in patients undergoing lower-abdominal radiotherapy. Previous mouse studies show that p53 gene dosage determines susceptibility to GI syndrome development. However, the translational relevance of p53 activity has not been addressed. Here, we used a knock-in mouse in which the p53–Mdm2 negative feedback loop is genetically disrupted. These mice retain biallelic p53 and thus, normal basal p53 levels and activity. However, due to the lack of p53-mediated Mdm2 transcription, irradiated Mdm2(P2/P2) mice exhibit enhanced acute p53 activity, which protects them from GI failure. Intestinal crypt cells residing in the +4 and higher positions exhibit decreased apoptosis, increased p21 expression, and hyperproliferation to reinstate intestinal integrity. Correspondingly, pharmacological augmentation of p53 activity in wild-type mice with an Mdm2 inhibitor protects against GI toxicity without affecting therapeutic outcome. Our results suggest that transient disruption of the p53–Mdm2 interaction to enhance p53 activity could be a viable prophylactic strategy for alleviating GI syndrome in patients undergoing radiotherapy.

Nucleotide-binding leucine-rich repeat (NLR) immune receptors play a critical role in defence against pathogens in plants and animals. However, we know very little about NLR-interacting proteins and the mechanisms that regulate NLR levels. Here, we used proximity labeling (PL) to identify the proteome proximal to N, which is an NLR that confers resistance to Tobacco mosaic virus (TMV). Evaluation of different PL methods indicated that TurboID-based PL provides more efficient levels of biotinylation than BioID and BioID2 in plants. TurboID-based PL of N followed by quantitative proteomic analysis and genetic screening revealed multiple regulators of N-mediated immunity. Interestingly, a putative E3 ubiquitin ligase, UBR7, directly interacts with the TIR domain of N. UBR7 downregulation leads to an increased amount of N protein and enhanced TMV resistance. TMV-p50 effector disrupts the N-UBR7 interaction and relieves negative regulation of N. These findings demonstrate the utility of TurboID-based PL in plants and the N-interacting proteins we identified enhance our understanding of the mechanisms underlying NLR regulation., Plant NLR receptors trigger immune signaling following recognition of pathogen effectors. Here, Zhang et al. optimize a TurboID-based proximity labeling approach and show that it can be used to identify interacting partners of N, an NLR that confers resistance to Tobacco mosaic virus.

Studies over the last decade have transformed our previously simplistic view of microbes, having only a pathogenic role in disease to a more robust understanding that they are critical for maintaining human health. Indeed, our microbiota-the collection of commensal organisms that live in and on each of us-contributes to nearly every facet of host physiology, from ontogeny of the immune system to neurological function to metabolism. Although the specific details of these host-microbe interactions are still being elucidated for most diseases, the coupling of clinical samples with animal models of disease have provided key insights. This review provides some general background on the microbiota, highlights a few examples of how the microbiota influences diseases of the central nervous system, and provides a perspective for how these findings may be clinically translatable. .

During Ag priming, naive CD4+ T cells differentiate into subsets with distinct patterns of cytokine expression that dictate to a major extent their functional roles in immune responses. We identified a subset of CD4+ T cells defined by secretion of IL-3 that was induced by Ag stimulation under conditions different from those associated with previously defined functional subsets. Using mouse models of bacterial and viral infections, we showed that IL-3–secreting CD4+ T cells were generated by infection at the skin and mucosa but not by infections introduced directly into the blood. Most IL-3–producing T cells coexpressed GM-CSF and other cytokines that define multifunctionality. Generation of IL-3–secreting T cells in vitro was dependent on IL-1 family cytokines and was inhibited by cytokines that induce canonical Th1 or Th2 cells. Our results identify IL-3–secreting CD4+ T cells as a potential functional subset that arises during priming of naive T cells in specific tissue locations.

Compelling evidence suggest that germs cells are predominantly sensitive to DNA damaging agents in comparison to other cells. High fidelity DNA repair in testicular cells thus becomes indispensable to preserve the genomic integrity for passing on to the progeny. Compromised DNA repair machinery in the testicular cells may result in impaired spermatogenesis and infertility. It remains unclear if the alterations in the expression of DNA repair genes correlate with azoospermia and male infertility. In the present study, 54 non-obstructive azoospermic infertile patients with hypospermatogenesis (HS, n = 26), maturation arrest (MA, n = 15), Sertoli cell only syndrome (SCOS, n = 13) and 14 controls with obstructive azoospermia, but normal spermatogenesis were recruited. Expression profiling of 84 DNA repair genes in testicular biopsy samples was performed using PCR array. Out of 84 genes, 27, 64 and 28 genes showed >5 fold down-regulation in the HS, MA and SCOS groups, respectively. On the basis of differential expression and their functional significance in spermatogenesis, ten genes (MSH2, BRIP1, CCNH, LIG4, MGMT, NTHL1, PMS1, DMC1, POLB and XPA) were selected for validation of transcript levels in a higher number of cases using RT-PCR, which corroborated the findings of array. Four genes (MSH2, LIG4, PMS1 and DMC1) were analyzed for protein levels using immunohistochemistry, which further validated the loss of DNA repair gene expression. Caspase-3 immunostaining showed that the loss of DNA repair correlated with increased testicular apoptosis in patients. Maturation arrest showed the highest apoptotic index with maximum number of downregulated genes. We conclude that the loss of DNA repair genes expression in testis correlates with increased apoptosis, azoospermia and infertility.

The synthesis, absolute stereochemical configuration, complete biological characterization, mechanism of action and resistance, and pharmacokinetic properties of (S)-(−)-acidomycin are described. Acidomycin possesses promising antitubercular activity against a series of contemporary drug susceptible and drug-resistant M. tuberculosis strains (MICs = 0.096–6.2 μM), but is inactive against non-tuberculosis mycobacteria, gram-positive and gram-negative pathogens (MICs > 1000 μM). Complementation studies with biotin biosynthetic pathway intermediates and subsequent biochemical studies confirmed acidomycin inhibits biotin synthesis with a K(i) of approximately 1 μM through the competitive inhibition of biotin synthase (BioB) and also stimulates unproductive cleavage of S-adenosylmethionine (SAM) to generate the toxic metabolite 5′-deoxyadenosine. Cell studies demonstrate acidomycin selectively accumulates in M. tuberculosis providing a mechanistic basis for the observed antibacterial activity. The development of spontaneous resistance by M. tuberculosis to acidomycin was difficult and only low-level resistance to acidomycin was observed by overexpression of BioB. Collectively, the results provide a foundation to advance acidomycin and highlight BioB as a promising target.

Background: Shankhpushpi is an Ayurvedic drug, widely used for its actions on the central nervous system, especially to improve intellect and boost memory. Four botanicals viz. Canscora decussata Schult. (CD), Clitorea ternatea Linn. (CT), Convolvulus pluricaulis Choisy. (CP) and Evolvulus alsinoides Linn. (EA) are considered as sources of Shankhpushpi by Indian practitioners on the basis of their morphological descriptions given in ancient texts. Objective: The present study was undertaken to evaluate the neuropharmacological effect of four herbs commonly identified as source of Shankhpushpi. Materials and methods: Methanol extracts of all four varieties were tested and evaluated in vitro and in vivo for their neuropharmacological effects. Experiments such as protection against β-amyloid induced neurotoxicity on brain cell line (Neuro 2A), antioxidant potential, AchE (acetylcholinesterase enzyme) inhibition, and 5-LOX (lipoxygenase) enzyme inhibition were conducted for in vitro evaluation. For in vivo evaluation, scopolamine (0.3 mg/kg i.p.) induced memory retrieval using pole climbing apparatus and Morris water maze were performed in rat models. Results: It was found that protective effects of EA and CD against β-amyloid induced neurotoxicity in Neuro 2A cells were significantly higher than CT and CP. EA proved to be superior than other varieties on the basis of antioxidant activity, AchE inhibitory and LOX inhibitory activities. The preventive activity of EA on scopolamine induced memory retrieval in pole climbing and Morris water maze task in rats was found to be higher than that of CD, CT and CP. Conclusion: EA has remarkable neuropharmacological effect as compared to other three varieties of Shankhpushpi. This effect may be attributed due to the presence of steroids (stigmasterol and betulinic acid), coumarins (scopoletin) and flavonoids (β-carotene and chlorogenic acid). Hence it can be used as a promising lead in development and management of neuronal disorders including Alzheimer's disease. Keywords: Neuroprotection, Lipoxygenase, Enzyme inhibition, Memory, Shankhpushpi

Interleukin-3 (IL-3) is produced during infections caused by parasites, bacteria and viruses, but its contribution to immunity in this context remains largely unknown. In mouse models of parasitic infections, in which the effects of IL-3 have been most extensively studied, IL-3 has been variously reported as protective, detrimental or inconsequential. Similarly, mixed results have been reported in viral and bacterial infection models. Here, we investigated the effects of IL-3 in mouse models of Mycobacterium tuberculosis and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections by assessing the pathogen burden, disease manifestations and survival following infection. After infection with M. tuberculosis, IL-3 deficient mice showed higher bacillary burden, increased lung pathology and reduced survival compared to wild type mice. After infection with HSV-1 through cutaneous route and HSV-2 through vaginal route, IL-3 deficient mice showed higher viral burden, increased disease manifestations and reduced survival compared to wild type mice. Our results show that IL-3 makes a subtle but significant contribution to protective immunity in these mouse models of bacterial and viral infections.

The interaction of host cells with mycobacteria is complex and can lead to multiple outcomes ranging from bacterial clearance to progressive or latent infection. Autophagy is recognized as one component of host cell responses that has an essential role in innate and adaptive immunity to intracellular bacteria. Many microbes, including Mycobacterium tuberculosis, have evolved to evade or exploit autophagy, but the precise mechanisms and virulence factors are mostly unknown. Through a loss-of-function screening of an M. tuberculosis transposon mutant library, we identified 16 genes that contribute to autophagy inhibition, six of which encoded the PE/PPE protein family. Their expression in Mycobacterium smegmatis confirmed that these PE/PPE proteins inhibit autophagy and increase intracellular bacterial persistence or replication in infected cells. These effects were associated with increased mammalian target of rapamycin (mTOR) activity and also with decreased production of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). We also confirmed that the targeted deletion of the pe/ppe genes in M. tuberculosis resulted in enhanced autophagy and improved intracellular survival rates compared to those of wild-type bacteria in the infected macrophages. Differential expression of these PE/PPE proteins was observed in response to various stress conditions, suggesting that they may confer advantages to M. tuberculosis by modulating its interactions with host cells under various conditions. Our findings demonstrated that multiple M. tuberculosis PE/PPE proteins are involved in inhibiting autophagy during infection of host phagocytes and may provide strategic targets in developing therapeutics or vaccines against tuberculosis.

Macroautophagy/autophagy plays a dual role in many physiological processes of multicellular eukaryotes. In plants, autophagy can be used by both host and pathogen for a beneficiary infection outcome. Plants employ a two-tier innate immune system to defend against invading pathogens. Cell surface localized pattern recognition receptors recognize conserved pathogen-associated molecular patterns (PAMPs) and launch pattern-triggered immunity (PTI) to provide broad-spectrum resistance. Pathogens inject a battery of effector proteins into their hosts to counter PTI and compromise the primary immune response. Hosts induce a second layer of defense called effector-triggered immunity (ETI) to counter the effects of these effectors. In addition to ETI and PTI, autophagy is emerging as a central cellular process modulated by both host and pathogens toward their respective advantage. Pathogens lacking the ability to inject effectors are compromised in virulence. However, molecular targets and biochemical characterization of most of these effector proteins remain elusive. In a recent paper we presented a systematic analysis of interaction between autophagy proteins of Arabidopsis thaliana with effectors from bacterial, fungal, oomycete and nematode pathogens. Abbreviations: ATG, a u t opha g y related; BiFC, b imolecular f luorescence c omplementation; ETI, effector-triggered immunity; PAMPs, pathogen-associated molecular patterns; PTI, pattern-triggered immunity.

Peroxiredoxins (Prxs) are antioxidant proteins that are involved in cellular defence against reactive oxygen species and reactive nitrogen species. Humans have six peroxiredoxins, hPrxI-VI, out of which hPrxI and hPrxII belongs to the typical 2-Cys class sharing 90% conservation in their amino acid sequence including catalytic residues required to carry out their peroxidase and chaperone activities. Despite the high conservation between hPrxI and hPrxII, hPrxI behaves differently from hPrxII in its peroxidase and chaperone activity. We recently showed in yeast that in the absence of Tsa1 and Tsa2 (orthologs of hPrx) hPrxI protects the cells against different stressors whereas hPrxII does not. To understand this difference, we expressed catalytic mutants of hPrxI in yeast cells lacking the orthologs of hPrxI/II. We found that the catalytic mutants lacking peroxidase function including hPrxIC52S, hPrxIC173S, hPrxIT49A, hPrxIP45A and hPrxIR128A were not able to grow on media with nitrosative stressor (sodium nitroprusside) and unable to withstand heat stress, but surprisingly they were able to grow on an oxidative stressor (H2O2). Interestingly, we found that hPrxI increases the expression of antioxidant genes, GPX1 and SOD1, and this is also seen in the case of a catalytic mutant, indicating hPrxI can indirectly reduce oxidative stress independently of its own peroxidase function and thus suggesting a novel role of hPrxI in altering the expression of other antioxidant genes. Furthermore, hPrxIC83T was resistant to hyperoxidation and formation of stable high molecular weight oligomers, which is suggestive of impaired chaperone activity. Our results suggest that the catalytic residues of hPrxI are essential to counter the nitrosative stress whereas Cys83 in hPrxI plays a critical role in hyperoxidation of hPrxI., Graphical abstract Image 1

Daxx loss causes coordinate dysregulation of ERVs and protein-coding genes after physiologic stress and impairs tissue recovery., Tumor sequencing studies have emphasized the role of epigenetics and altered chromatin homeostasis in cancer. Mutations in DAXX, which encodes a chaperone for the histone 3.3 variant, occur in 25% of pancreatic neuroendocrine tumors (PanNETs). To advance our understanding of physiological functions of Daxx, we developed a conditional Daxx allele in mice. We demonstrate that Daxx loss is well tolerated in the pancreas but creates a permissive transcriptional state that cooperates with environmental stress (inflammation) and other genetic lesions (Men1 loss) to alter gene expression and cell state, impairing pancreas recovery from inflammatory stress in vivo. The transcriptional changes are associated with dysregulation of endogenous retroviral elements (ERVs), and dysregulation of endogenous genes near ERVs is also observed in human PanNETs with DAXX mutations. Our results reveal a physiologic function of DAXX, provide a mechanism associated with impaired tissue regeneration and tumorigenesis, and expand our understanding of ERV regulation in somatic cells.

Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.

Objective: This case report will highlight the importance of histopathologic analysis of the gonads to differentiate ovotesticular disorder of sexual development (OT-DSD) from mixed gonadal dysgenesis. Both disorders may have the same clinical and cytogenetic presentation, but the latter presents high risk for gonadal malignancy, hence there is usually a need for early gonadectomy, while the former has a low risk for malignancy. Therefore OT-DSD patients may have the opportunity for spontaneous pubertal development and fertility with regular tumor surveillance.Methods: We report the case of a 22-year-old patient reared as a male with a 46,XY karyotype diagnosed as OT-DSD by gonadal biopsy. The patient presented with microphallus with chordee, penoscrotal hypospadias, poor virilization, and development of gynecomastia at puberty.Results: Histopathologic examination of the single tubule biopsy of the left gonad showed seminiferous tubules lined by Sertoli cells and Leydig cells in the interstitial space. Biopsy of the right gonad showed an ovary with Graafian follicles and fallopian tubes. There was also a uterus with the endometrium in the proliferative phase.Conclusion: OT-DSD is a rare disorder of sexual development characterized by the presence of both ovarian tissue with mature ovarian follicles as well as testicular tissue with distinct seminiferous tubules. These may present either in the same gonad or separately as opposite-side gonads in the same individual. It is associated with variable genotypes of which the most common karyotype is 46,XX with the 46,XY karyotype found in only 10% of cases.Abbreviations: DSD Disorder of sexual development OT-DSD ovotesticular disorder of sexual development

Lipid metabolism forms the heart and soul of Mycobacterium tuberculosis life cycle. Starting from macrophage invasion at cholesterol rich micro-domains to a sustainable survival for infection by utilizing cholesterol, Mycobacterium displays the nexus of metabolic pathways around host derived lipids. mce4 operon acts as cholesterol import system in M. tuberculosis and here we demonstrate role of mce4A gene of this operon in cholesterol catabolism. Here M. tuberculosis H37Rv overexpressing Rv3499c (mce4A) recombinant was used as a model to decipher the metabolic flux during intake and utilization of host lipids by mycobacteria. We analysed the impact of mce4A expression on carbon shift initiated during cholesterol utilization necessary for long term survival of mycobacterium. Through transcriptional analysis, upregulation in methylcitrate cycle (MCC) and methylmalonyl pathway (MMP) genes was observed in Rv3499c overexpressing recombinants of M. tuberculosis H37Rv. Up-regulation of methylmalonyl pathway associated enzyme encoding genes increased accumulation of virulence associated mycobacterial lipids phthiocerol dimycocerates (PDIM) and sulfolipid (SL1). We demonstrate that MCC and MMP associated enzyme encoding genes are upregulated upon mce4A overexpression and lead to enhanced accumulation of PDIM and SL1 which are responsible for pathogenicity of M. tuberculosis.

OBJECTIVE Tissue-specific gene expression is associated with individual metabolic measures. However, these measures may not reflect the true but latent underlying biological phenotype. This study reports gene expression associations with multidimensional glucometabolic characterizations of obesity, glucose homeostasis, and lipid traits. METHODS Factor analysis was computed by using orthogonal rotation to construct composite phenotypes (CPs) from 23 traits in 256 African Americans without diabetes. Genome-wide transcript expression data from adipose and muscle were tested for association with CPs, and expression quantitative trait loci (eQTLs) were identified by associations between cis-acting single-nucleotide polymorphisms (SNPs) and gene expression. RESULTS The factor analysis identified six CPs. CPs 1 through 6 individually explained 34%, 12%, 9%, 8%, 6%, and 5% of the variation in 23 glucometabolic traits studied. There were 3,994 and 929 CP-associated transcripts identified in adipose and muscle tissue, respectively; CP2 had the largest number of associated transcripts. Pathway analysis identified multiple canonical pathways from the CP-associated transcripts. In adipose and muscle, significant cis-eQTLs were identified for 558 and 164 CP-associated transcripts (q-value

Background and Objectives: Type 2 diabetes is a multifactorial disorder that results from the interaction between genetic predisposition and environmental factors. Different Prakriti (body constitution) individuals have different susceptibility for the diseases, and this Prakriti is determined by both genetic and environmental factor. This study was undertaken to determine the association status of Methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C with type 2 diabetes and Prakriti. Materials and Methods: After informed consent, 54 patients with type 2 diabetes and 56 individuals as normal controls were analyzed. Their constitution and pathological data were collected and MTHFR C677T and A1298C genotypes were determined. Results: Kapha/Kapha-Pittaja Prakriti were associated and found to be strong risk factors (Chi-square test = 39.67, P < 0.00001, odds ratio [OR] = 16.133, 95% confidence interval [CI] = 6.32–41.20) for type 2 diabetes. MTHFR C677T was associated (Chi-square test = 7.743, P = 0.02) with type 2 diabetes where the major CC genotype was found to be a risk for type 2 diabetes (OR = 3.78, 95% CI = 1.14–12.45). A1298C was not associated with type 2 diabetes (Chi-square test = 2.264, P = 0.322). None of the Prakriti was associated with C677T and A1298C variants. Interpretation and Conclusion: In the present study, an extremely strong association between Prakriti (Kaphaja/Kapha-Pittaja) and type 2 diabetes (P < 0.00001) was detected. The present study gives a strong clue for the association of Prakriti (body constitutional) and clinical phenotype.

Analysis of Ag-specific CD4+ T cells in mycobacterial infections at the transcriptome level is informative but technically challenging. Although several methods exist for identifying Ag-specific T cells, including intracellular cytokine staining, cell surface cytokine-capture assays, and staining with peptide:MHC class II multimers, all of these have significant technical constraints that limit their usefulness. Measurement of activation-induced expression of CD154 has been reported to detect live Ag-specific CD4+ T cells, but this approach remains underexplored and, to our knowledge, has not previously been applied in mycobacteria-infected animals. In this article, we show that CD154 expression identifies adoptively transferred or endogenous Ag-specific CD4+ T cells induced by Mycobacterium bovis bacillus Calmette-Guérin vaccination. We confirmed that Ag-specific cytokine production was positively correlated with CD154 expression by CD4+ T cells from bacillus Calmette-Guérin–vaccinated mice and show that high-quality microarrays can be performed from RNA isolated from CD154+ cells purified by cell sorting. Analysis of microarray data demonstrated that the transcriptome of CD4+ CD154+ cells was distinct from that of CD154− cells and showed major enrichment of transcripts encoding multiple cytokines and pathways of cellular activation. One notable finding was the identification of a previously unrecognized subset of mycobacteria-specific CD4+ T cells that is characterized by the production of IL-3. Our results support the use of CD154 expression as a practical and reliable method to isolate live Ag-specific CD4+ T cells for transcriptomic analysis and potentially for a range of other studies in infected or previously immunized hosts.

Increased levels of inhibitors of the p53 tumor suppressor such as Mdm2 and Mdm4 drive tumor development and thus serve as targets for therapeutic intervention. Recently, digestive organ expansion factor (Diexf) has been identified as a novel inhibitor of p53 in zebrafish. Here, we address the potential role of Diexf as a regulator of the p53 pathway in mammals by generating Diexf knockout mice. We demonstrate that, similar to Mdm2 and Mdm4, homozygous deletion of Diexf is embryonic lethal. However, unlike in Mdm2 and Mdm4 mice, loss of p53 does not rescue this phenotype. Moreover, Diexf heterozygous animals are not sensitive to sub-lethal ionizing radiation. Thus, we conclude that Diexf is an essential developmental gene in the mouse, but is not a significant regulator of the p53 pathway during development or in response to ionizing radiation.

ADIPOQ/adiponectin, an adipocytokine secreted by adipocytes in the breast tumor microenvironment, negatively regulates cancer cell growth hence increased levels of ADIPOQ/adiponectin are associated with decreased breast cancer growth. However, its mechanisms of action remain largely elusive. We report that ADIPOQ/adiponectin induces a robust accumulation of autophagosomes, increases MAP1LC3B-II/LC3B-II and decreases SQSTM1/p62 in breast cancer cells. ADIPOQ/adiponectin-treated cells and xenografts exhibit increased expression of autophagy-related proteins. LysoTracker Red-staining and tandem-mCherry-GFP-LC3B assay show that fusion of autophagosomes and lysosomes is augmented upon ADIPOQ/adiponectin treatment. ADIPOQ/adiponectin significantly inhibits breast cancer growth and induces apoptosis both in vitro and in vivo, and these events are preceded by macroautophagy/autophagy, which is integral for ADIPOQ/adiponectin-mediated cell death. Accordingly, blunting autophagosome formation, blocking autophagosome-lysosome fusion or genetic-knockout of BECN1/Beclin1 and ATG7 effectively impedes ADIPOQ/adiponectin induced growth-inhibition and apoptosis-induction. Mechanistic studies show that ADIPOQ/adiponectin reduces intracellular ATP levels and increases PRKAA1 phosphorylation leading to ULK1 activation. AMPK-inhibition abrogates ADIPOQ/adiponectin-induced ULK1-activation, LC3B-turnover and SQSTM1/p62-degradation while AMPK-activation potentiates ADIPOQ/adiponectin's effects. Further, ADIPOQ/adiponectin-mediated AMPK-activation and autophagy-induction are regulated by upstream master-kinase STK11/LKB1, which is a key node in antitumor function of ADIPOQ/adiponectin as STK11/LKB1-knockout abrogates ADIPOQ/adiponectin-mediated inhibition of breast tumorigenesis and molecular analyses of tumors corroborate in vitro mechanistic findings. ADIPOQ/adiponectin increases the efficacy of chemotherapeutic agents. Notably, high expression of ADIPOQ receptor ADIPOR2, ADIPOQ/adiponectin and BECN1 significantly correlates with increased overall survival in chemotherapy-treated breast cancer patients. Collectively, these data uncover that ADIPOQ/adiponectin induces autophagic cell death in breast cancer and provide in vitro and in vivo evidence for the integral role of STK11/LKB1-AMPK-ULK1 axis in ADIPOQ/adiponectin-mediated cytotoxic autophagy.

Tumor suppressor and upstream master kinase Liver kinase B1 (LKB1) plays a significant role in suppressing cancer growth and metastatic progression. We show that low-LKB1 expression significantly correlates with poor survival outcome in breast cancer. In line with this observation, loss-of-LKB1 rendered breast cancer cells highly migratory and invasive, attaining cancer stem cell-like phenotype. Accordingly, LKB1-null breast cancer cells exhibited an increased ability to form mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2), properties also observed in spontaneous tumors in Lkb1−/− mice. Conversely, LKB1-overexpression in LKB1-null cells abrogated invasion, migration and mammosphere-formation. Honokiol (HNK), a bioactive molecule from Magnolia grandiflora increased LKB1 expression, inhibited individual cell-motility and abrogated the stem-like phenotype of breast cancer cells by reducing the formation of mammosphere, expression of pluripotency-factors and aldehyde dehydrogenase activity. LKB1, and its substrate, AMP-dependent protein kinase (AMPK) are important for HNK-mediated inhibition of pluripotency factors since LKB1-silencing and AMPK-inhibition abrogated, while LKB1-overexpression and AMPK-activation potentiated HNK’s effects. Mechanistic studies showed that HNK inhibited Stat3-phosphorylation/activation in an LKB1-dependent manner, preventing its recruitment to canonical binding-sites in the promoters of Nanog, Oct4 and Sox2. Thus, inhibition of the coactivation-function of Stat3 resulted in suppression of expression of pluripotency factors. Further, we showed that HNK inhibited breast tumorigenesis in mice in an LKB1-dependent manner. Molecular analyses of HNK-treated xenografts corroborated our in vitro mechanistic findings. Collectively, these results present the first in vitro and in vivo evidence to support crosstalk between LKB1, Stat3 and pluripotency factors in breast cancer and effective anticancer modulation of this axis with HNK treatment.

AMPA receptor (AMPAR) trafficking has emerged as a fundamental concept for understanding mechanisms of learning and memory as well as many neurological disorders. Classical conditioning is a simple and highly conserved form of associative learning. Our studies use an ex vivo brainstem preparation in which to study cellular mechanisms underlying learning during a neural correlate of eyeblink conditioning. Two stages of AMPAR synaptic delivery underlie conditioning utilizing sequential trafficking of GluA1-containing AMPARs early in conditioning followed by replacement with GluA4 subunits later. Subunit-selective trafficking of AMPARs is poorly understood. Here, we focused on identification of auxiliary chaperone proteins that traffic AMPARs. The results show that auxiliary proteins TARPγ8 and GSG1L are colocalized with AMPARs on abducens motor neurons that generate the conditioning. Significantly, TARPγ8 was observed to chaperone GluA1-containing AMPARs during synaptic delivery early in conditioning while GSG1L chaperones GluA4 subunits later in conditioning. Interestingly, TARPγ8 remains at the membrane surface as GluA1 subunits are withdrawn and associates with GluA4 when they are delivered to synapses. These data indicate that GluA1- and GluA4-containing AMPARs are selectively chaperoned by TARPγ8 and GSG1L, respectively. Therefore, sequential subunit-selective trafficking of AMPARs during conditioning is achieved through the timing of their interactions with specific auxiliary proteins.

M3 muscarinic receptor (M3R) expression is increased in colon cancer; M3R activation stimulates colon cancer cell invasion via cross-talk with epidermal growth factor receptors (EGFR), post-EGFR activation of mitogen-activated protein kinase (MAPK) extracellular signal-related kinase 1/2 (ERK1/2), and induction of matrix metalloproteinase-1 (MMP1) expression. MMP1 expression is strongly associated with tumor metastasis and adverse outcomes. Here, we asked whether other MAPKs regulate M3R agonist-induced MMP1 expression. In addition to activating ERK1/2, we found that treating colon cancer cells with acetylcholine (ACh) stimulated robust time- and dose-dependent phosphorylation of p38 MAPK. Unlike ERK1/2 activation, ACh-induced p38 phosphorylation was EGFR-independent and blocked by inhibiting protein kinase C-α (PKC-α). Inhibiting activation of PKC-α, EGFR, ERK1/2, or p38-α/β alone attenuated, but did not abolish ACh-induced MMP1 expression, a finding that predicted potentiating interactions between these pathways. Indeed, ACh-induced MMP1 expression was abolished by incubating cells with either an EGFR or MEK/ERK1/2 inhibitor combined with a p38-α/β inhibitor. Activating PKC-α and EGFR directly with the combination of phorbol 12-myristate 13-acetate (PMA) and EGF potentiated MMP1 gene and protein expression, and cell invasion. PMA- and ACh-induced MMP1 expression were strongly diminished by inhibiting Src and abolished by concurrently inhibiting both p38-α/β and Src, indicating that Src mediates the cross-talk between PKC-α and EGFR signaling. Using siRNA knockdown, we identified p38-α as the relevant p38 isoform. Collectively, these studies uncover novel functional interactions between post-muscarinic receptor signaling pathways that augment MMP1 expression and drive colon cancer cell invasion; targeting these potentiating interactions has therapeutic potential.

Disruption of the p53 tumor suppressor pathway is a primary cause of tumorigenesis. In addition to mutation of the p53 gene itself, overexpression of major negative regulators of p53, MDM2 and MDM4, also act as drivers for tumor development. Recent studies suggest that expression of splice variants of Mdm2 and Mdm4 may be similarly involved in tumor development. In particular, multiple studies show that expression of a splice variant of MDM4, MDM4-S correlates with tumor aggressiveness and can be used as a prognostic marker in different tumor types. However, in the absence of prospective studies, it is not clear whether expression of MDM4-S in itself is oncogenic or is simply an outcome of tumorigenesis. Here we have examined the role of Mdm4-S in tumor development in a transgenic mouse model. Our results suggest that splicing of Mdm4 does not promote tumor development and does not cooperate with other oncogenic insults to alter tumor latency or aggressiveness. We conclude that Mdm4-S overexpression is a consequence of splicing defects in tumor cells rather than a cause of tumor evolution.

The p53 inhibitor Mdm4 is present at high levels in multiple human cancers. Overexpression of Mdm4 in mice drives the spontaneous development of mostly lymphomas and sarcomas. In this study, we explored the ability of Mdm4 to cooperate with lesions in tumour development. The Mdm4 transgene contributed to mammary tumour development in a BALB/cJ background. High levels of Mdm4 enhanced tumour development in a mutant p53R172H heterozygous background, and reduced the need to lose the wild-type p53 allele, as compared with mice heterozygous only for the p53R172H mutation. Additionally, high levels of Mdm4 cooperated with an oncogenic K-ras mutation to drive lung tumourigenesis in vivo. Finally, we examined p53-independent functions of Mdm4 by studying the contribution of Mdm4 to tumour development in the absence of p53. Whereas the overall survival times of p53-null mice with and without the Mdm4 transgene were similar, male mice with both alterations showed significantly shorter survival than p53-null male mice, and showed differences in tumour spectrum, demonstrating a p53-independent function of Mdm4 in tumourigenesis. Furthermore, p53-null mice with the highest level of Mdm4 tended to have multiple tumours. Thus, a detailed analysis of Mdm4 transgenic mice in various genetic backgrounds shows synergy in tumour development in vivo. Mdm4 may thus serve as a therapeutic target in cancers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Gut-innervating nociceptor sensory neurons respond to noxious stimuli by initiating protective responses including pain and inflammation; however, their role in enteric infections is unclear. Here, we find that nociceptor neurons critically mediate host defense against the bacterial pathogen Salmonella enterica serovar Typhimurium (STm). Dorsal root ganglia nociceptors protect against STm colonization, invasion, and dissemination from the gut. Nociceptors regulate the density of microfold (M) cells in ileum Peyer's patch (PP) follicle-associated epithelia (FAE) to limit entry points for STm invasion. Downstream of M cells, nociceptors maintain levels of segmentous filamentous bacteria (SFB), a gut microbe residing on ileum villi and PP FAE that mediates resistance to STm infection. TRPV1+ nociceptors directly respond to STm by releasing calcitonin gene-related peptide (CGRP), a neuropeptide that modulates M cells and SFB levels to protect against Salmonella infection. These findings reveal a major role for nociceptor neurons in sensing and defending against enteric pathogens.

Mucosa-associated lymphoid tissues contain roughly 80% of all immune cells and produce virtually all of the body’s IgA1–3. Although the majority of IgA-secreting cells educated within a mucosal site home back to the same anatomic region, some cells are also found in distant mucosal tissues2–6. These observations underlie the notion of a common mucosal immune system, which holds that anatomically unrelated mucosal sites are functionally connected by a shared immune system2,3. However, the ontological basis of this separation between site of immune education and functionality has remained elusive. Here we show that mice lacking Peyer’s patches (PPs)—small-intestinal lymphoid tissue covered by antigen-sampling M cells—have no immunologic defect in the small-intestinal lamina propria. Surprisingly, the primary immunological abnormality in PP-deficient mice was a reduction in colonic B cells, including plasmablasts but not plasma cells. Adoptive transfer experiments conclusively demonstrated that PP-derived cells preferentially give rise to colonic—but not small-intestinal—B cells and plasmablasts. Finally, these PP-derived colonic B cells were critical for restraining colonic inflammation. Thus, PPs bridge the small-intestinal and colonic immune systems and provide a clear example of immune education being required in an anatomic compartment distinct from the effector site. Our findings, which highlight that the majority of fecal IgA is produced by colonic plasmablasts that originate from PPs, will help inform design of mucosal vaccines.

Prior to generating a new mouse model, it is important to plan the method that will be used to detect which of the mice generated have the mutation(s) desired. Nearly, all types of mutations may be detected using PCR. However, the choice of primers will differ depending upon the method used to generate the model. Transgenic mice should be genotyped across a unique junction fragment. Targeted ES cells used to generate knock-out or knock-in mice should be genotyped using primers from a unique marker in the construct and a region outside of the construct. Targeting in ES cells can also be detected using a genomic Southern blot. Mice targeted using CRISPR/Cas9 should have the region of interest amplified using PCR, and then be assessed for size changes (for large changes in sequence) by Surveyor Assay (for gene knock-out and point mutations) and/or sequenced to verify the mutation. Each of these models has a unique requirement for genotyping, and failure to understand the requirements can easily lead to loss of the gene in subsequent generations.

Dicer1 functions as a tumor suppressor in mouse models. In humans, somatic mutations are associated with many cancers in adults, and patients with DICER1 syndrome with DICER1 germline mutations are susceptible to childhood cancers. Dicer is phosphorylated by the ERK-MAP kinase pathway and because this pathway is activated in human cancers, we asked whether phosphorylated Dicer1 contributed to tumor development. In human endometrioid cancers, we discovered that phosphorylated DICER1 is significantly associated with invasive disease. To test a direct involvement of Dicer1 phosphorylation in tumor development, we studied mice with phosphomimetic alterations at the two conserved serines phosphorylated by ERK and discovered that a phosphomimetic Dicer1 drives tumor development and dissemination in two independent murine cancer models (KRas+/LA1 and p53+/−). Our findings demonstrate that phosphomimetic Dicer1 promotes tumor development and invasion. Significance: This work highlights the relevance of Dicer1 phosphorylation in mammalian tumor development and dissemination.

SUMMARYGut-innervating nociceptor sensory neurons respond to noxious/tissue-damaging stimuli by initiating protective responses and releasing mediators that regulate tissue inflammation, gastrointestinal secretion, and motility. The role of nociceptors in host defense against enteric pathogens is unclear. Here, we found that gut-extrinsic nociceptor neurons are critical in protecting the host against Salmonella typhimurium (STm) infection. Nociceptors responded to STm by releasing the neuropeptide calcitonin gene-related peptide (CGRP). Targeted depletion of Nav1.8 and TRPV1 neurons from gut-extrinsic dorsal root ganglia and vagal ganglia increased STm colonization, invasion, and dissemination. Nociceptors regulated the gut microbiota at homeostasis, specifically segmented filamentous bacteria (SFB) levels in the ileum, which protected against STm by colonization resistance. Nociceptors also regulated the density of microfold epithelial cells in the Peyer’s patch via CGRP to limit points of entry for STm invasion into host tissues. Understanding how host sensory neurons crosstalk with pathogenic bacteria may impact treatments for enteric infections.HIGHLIGHTSNav1.8 and TRPV1 nociceptors defend against Salmonella typhimurium (STm) infectionNociceptors shape the gut microbiota and SFB levels which resist pathogen colonizationNociceptors suppress Peyer’s patch microfold cell density to limit pathogen invasionNeurons sense STm and release CGRP to modulate microfold cells and host defense

Studies over the last decade have transformed our previously simplistic view of microbes having only a pathogenic role in disease to a more robust understanding that they are critical for maintaining human health. Indeed, our microbiota—the collection of commensal organisms that live in and on each of us—contributes to nearly every facet of host physiology, from metabolic functions to maturation of the immune system and everything in between. Although the specific details of these host–microbe interactions are still being elucidated for most diseases, the marriage of clinical samples with animal models of disease have provided key insights. This chapter provides some general background on the microbiota, highlights a few examples of how the microbiota influences infectious diseases, and provides some context for how these findings may be clinically translatable.

Under a Creative Commons license.-- et al., Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression., Drs. Feitelson and Arzumanyan were supported by NIH (AI076535) and by Temple University. Dr. Rob J. Kulathinal was supported by the National Science Foundation, and by the American Cancer Society. Dr. Marino was supported by grant from University Roma Tre (CLA 2013) and by the Italian Association for Cancer Research (no. IG15221). Dr. Georgakilas was supported by the EU Marie Curie Reintegration Grant (MC-CIG-303514), Greek National funds through the Operational Program ‘Educational and Lifelong Learning of the National Strategic Reference Framework (NSRF)- Research Funding Program: THALES (MIS 379346) and COST Action CM1201 ‘Biomimetic Radical Chemistry.’ Dr. Amedei was supported by the Italian Ministry of University and University of Italy. Dr. Amin was supported by the Terry Fox Foundation (TF-36), UAEU Program for Advanced Research (UPAR25183), Al-Jalila Foundation (AJF201454) and Zayed Center for Health Sciences (ZCHS2014). Dr. Sanchez-Garcia was supported by FEDER, by MICINN (SAF2012-32810), by NIH (R01 CA109335-04A1), by Junta de Castilla y León (BIO/SA06/13), by the ARIMMORA project (FP7-ENV-2011, EU 7th Framework Program) and by the EuroSyStem and the DECIDE Network (EU FP7). Dr. Sharma was funded by NIH grants (R01CA131294, CA155686), the Avon Foundation and a Breast Cancer Research Foundation grant (90047965). Dr. Saxena was supported by a grant from NIH (K01DK077137 and R03DK089130). Dr. Singh was supported by the Fast Track Scheme for Young Scientists, Department of Science and Technology, India (SR/FT/LS-063/2008). Dr. Honoki was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan (no. 24590493). Dr. Ciriolo was supported by the Italian Association for Cancer Research (AIRC – grant #IG10636). Dr. Aquilano was supported by MIUR-PRIN (20125S38FA 002) and Ministero della Salute (GR-2011-02348047). Dr. Chen was funded from the Ovarian and Prostate Cancer Research Trust, UK. Dr. Mohammed is supported by the Purdue University Center for Cancer Research. W. Nicol Keith & Alan Bilsland were supported by the University of Glasgow, Beatson Oncology Centre Fund, and Cancer Research UK (http://www.cancerresearchuk.org) grant C301/A14762.

Receptor-like cytoplasmic kinases (RLCKs) inArabidopsisplay a central role in the integration of signaling input from various growth and immune signaling pathways. BOTRYTIS-INDUCED KINASE 1 (BIK1), belonging to the RLCK family, is an important player in defense against bacterial and fungal pathogens and in ethylene and brassinosteroid hormone signaling. In this study, the purification and crystallization of a first member of the class VI family of RLCK proteins, BIK1, are reported. BIK1 was crystallized using the microbatch-under-oil method. X-ray diffraction data were collected to 2.35 Å resolution. The crystals belonged to the monoclinic space groupC2, with two monomers per asymmetric unit.

The antibacterial properties of twenty-eight novel ricinoleic acid glycosides synthesized by Koenigs–Knorr glycosylation are reported. Seven of them were found to show promising wide spectrum antibacterial activity against Gram positive bacteria of which two compounds, the mannopyranosyl- and the arabinofuranosyl derivatives, were proven effective against various non-clinical/clinical/NorA-overexpressed/resistant strains of Staphylococcus aureus as well as other Gram +ve bacteria such as Bacillus subtilis ATCC 6051 and Micrococcus luteus MTCC 2470. It was found that both the presence of the sugar and its structure are necessary and important for the compounds to be bioactive. The methyl ester protection of the carboxylic acid moiety of the ricinoleic acid unit was also found to be important for imparting good bioactivity to the molecule. Based on the membrane permeability and cell disintegration studies, these compounds are found to increase the bacterial cell membrane permeability, subsequently causing cell death.

Macroautophagy/autophagy plays a dual role in many physiological processes of multicellular eukaryotes. In plants, autophagy can be used by both host and pathogen for a beneficiary infection outcome. Plants employ a two-tier innate immune system to defend against invading pathogens. Cell surface localized pattern recognition receptors recognize conserved pathogen-associated molecular patterns (PAMPs) and launch pattern-triggered immunity (PTI) to provide broad-spectrum resistance. Pathogens inject a battery of effector proteins into their hosts to counter PTI and compromise the primary immune response. Hosts induce a second layer of defense called effector-triggered immunity (ETI) to counter the effects of these effectors. In addition to ETI and PTI, autophagy is emerging as a central cellular process modulated by both host and pathogens toward their respective advantage. Pathogens lacking the ability to inject effectors are compromised in virulence. However, molecular targets and biochemical characterization of most of these effector proteins remain elusive. In a recent paper we presented a systematic analysis of interaction between autophagy proteins of Arabidopsis thaliana with effectors from bacterial, fungal, oomycete and nematode pathogens. Abbreviations: ATG, autophagy related; BiFC, bimolecular fluorescence complementation; ETI, effector-triggered immunity; PAMPs, pathogen-associated molecular patterns; PTI, pattern-triggered immunity

Under a Creative Commons license.-- Review.-- et al., Targeted therapies and the consequent adoption of >personalized> oncology have achieved notablesuccesses in some cancers; however, significant problems remain with this approach. Many targetedtherapies are highly toxic, costs are extremely high, and most patients experience relapse after a fewdisease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistantimmortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are notreliant upon the same mechanisms as those which have been targeted). To address these limitations, aninternational task force of 180 scientists was assembled to explore the concept of a low-toxicity >broad-spectrum> therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspectsof relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a widerange of high-priority targets (74 in total) that could be modified to improve patient outcomes. For thesetargets, corresponding low-toxicity therapeutic approaches were then suggested, many of which werephytochemicals. Proposed actions on each target and all of the approaches were further reviewed forknown effects on other hallmark areas and the tumor microenvironment. Potential contrary or procar-cinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixedevidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of therelationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. Thisnovel approach has potential to be relatively inexpensive, it should help us address stages and types ofcancer that lack conventional treatment, and it may reduce relapse risks. A proposed agenda for futureresearch is offered., Amr Amin was funded by Terry Fox Foundation Grant # TF-13-20 and UAEU Program for Advanced Research (UPAR) #31S118; Jack Arbiser was funded by NIHAR47901; Alexandra Arreola was funded by NIH NRSA Grant F31CA154080; Alla Arzumanyan was funded by NIH (NIAID) R01: Combination therapies for chronic HBV, liver disease, and cancer (AI076535); Work in the lab of Asfar S. Azmi is supported by NIH R21CA188818 as well as from Sky Foundation Inc. Michigan; Fabian Benencia was supported by NIH Grant R15 CA137499-01; Alan Bilsland was supported by the University of Glasgow, Beatson Oncology Centre Fund, CRUK (www.cancerresearchuk.org) Grant C301/A14762; Amancio Carnero was supported by grants from the Spanish Ministry of Economy and Competitivity, ISCIII (Fis: PI12/00137, RTICC: RD12/0036/0028) co-funded by FEDER from Regional Development European Funds (European Union), Consejeria de Ciencia e Innovacion (CTS-6844 and CTS-1848) and Consejeria de Salud of the Junta de Andalucia (PI-0135-2010 and PI-0306-2012). His work on this project has also been made possible thanks to the Grant PIE13/0004 co-funded by the ISCIII and FEDER funds; Stephanie C. Casey was supported by NIH Grant F32CA177139; Mrinmay Chakrabarti was supported by the United Soybean Board; Rupesh Chaturvedi was supported by an NIH NCCAM Grant (K01AT007324); Georgia Zhuo Chen was supported by an NIH NCI Grant (R33 CA161873-02); Helen Chen acknowledges financial support from the Michael Cuccione Childhood Cancer Foundation Graduate Studentship; Sophie Chen acknowledges financial support from the Ovarian and Prostate Cancer Research Trust, UK; Yi Charlie Chen acknowledges financial support from the West Virginia Higher Education Policy Commission/Division of Science Research, his research was also supported by NIH grants (P20RR016477 and P20GM103434) from the National Institutes of Health awarded to the West Virginia IDeA Network of Biomedical Research Excellence; Maria Rosa Ciriolo was partially supported by the Italian Association for Cancer Research (AIRC) Grants #IG10636 and #15403; Helen M. Coley acknowledges financial support from the GRACE Charity, UK and the Breast Cancer Campaign, UK; Marisa Connell was supported by a Michael Cuccione Childhood Cancer Foundation Postdoctoral Fellowship; Sarah Crawford was supported by a research grant from Connecticut State University; Charlotta Dabrosin acknowledges financial support from the Swedish Research Council and the Swedish Research Society; Giovanna Damia gratefully acknowledges the generous contributions of The Italian Association for Cancer Research (IG14536 to G.D.), Santanu Dasgupta gratefully acknowledges the support of the University of Texas Health Science Centre at Tyler, Elsa U. Pardee Foundation; William K. Decker was supported in part by CPRIT, the Cancer Prevention and Research Institute of Texas; Anna Mae E. Diehl was supported by NIH National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the NIH National Institute on Alcohol Abuse and Alcoholism (NIAAA), Gilead and Shire Pharmaceuticals; Q. Ping Dou was partially supported by NIH/NCI (1R01CA20009, 5R01CA127258-05 and R21CA184788), and NIH P30 CA22453 (to Karmanos Cancer Institute); Janice E. Drew was supported by the Scottish Government's Rural and Environment Science and Analytical Services Division; Eyad Elkord thanks the National Research Foundation, United Arab Emirates University and the Terry Fox Foundation for supporting research projects in his lab; Bassel El-Rayes was supported by Novartis Pharmaceutical, Aveo Pharmaceutical, Roche, Bristol Myers Squibb, Bayer Pharmaceutical, Pfizer, and Kyowa Kirin; Mark A. Feitelson was supported by NIH/NIAID Grant AI076535, Dean W. Felsher was supported by NIH grants (R01CA170378, U54CA149145, and U54CA143907); Lynnette R Ferguson was financially supported by the Auckland Cancer Society and the Cancer Society of New Zealand; Gary L. Firestone was supported by NIH Public Service Grant CA164095 awarded from the National Cancer Institute; Christian Frezza “would like to acknowledge funding from a Medical Research Council CCU-Program Grant on cancer metabolism, and a unique applicant AICR project grant”; Mark M. Fuster was supported by NIH Grant R01-HL107652; Alexandros G. Georgakilas was supported by an EU Marie Curie Reintegration Grant MC-CIG-303514, Greek National funds through the Operational Program ‘Educational and Lifelong Learning of the National Strategic Reference Framework (NSRF)-Research Funding Program THALES (Grant number MIS 379346) and COST Action CM1201 ‘Biomimetic Radical Chemistry’; Michelle F. Green was supported by a Duke University Molecular Cancer Biology T32 Training Grant; Brendan Grue was supported by a National Sciences Engineering and Research Council Undergraduate Student Research Award in Canada; Dorota Halicka was supported by by NIH NCI grant NCI RO1 28704; Petr Heneberg was supported by the Charles University in Prague projects UNCE 204015 and PRVOUK P31/2012, by the Czech Science Foundation projects 15-03834Y and P301/12/1686, by the Czech Health Research Council AZV project 15-32432A, and by the Internal Grant Agency of the Ministry of Health of the Czech Republic project NT13663-3/2012; Matthew D. Hirschey wishes to acknowledge Duke University Institutional Support, the Duke Pepper Older Americans Independence Center (OAIC) Program in Aging Research supported by the National Institute of Aging (P30AG028716-01) and NIH/NCI training grants to Duke University (T32-CA059365-19 and 5T32-CA059365), Lorne J. Hofseth was supported by NIH grants (1R01CA151304, 1R03CA1711326, and 1P01AT003961); Kanya Honoki was supported in part by the grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 24590493); Hsue-Yin Hsu was supported in part by grants from the Ministry of Health and Welfare (CCMP101-RD-031 and CCMP102-RD-112) and Tzu-Chi University (61040055-10) of Taiwan; Lasse D. Jensen was supported by Svenska Sallskapet for Medicinsk Forskning, Gosta Fraenkels Stiftelse, Ak.e Wibergs Stiftelse, Ollie och Elof Ericssons Stiftelse, Linkopings Universitet and the Karolinska Institute, Sweden; Wen G. Jiang wishes to acknowledge the support by Cancer Research Wales, the Albert Hung Foundation, the Fong Family Foundation, and Welsh Government A4B scheme; Lee W. Jones was supported in part by grants from the NIH NCI; W Nicol Keith was supported by the University of Glasgow, Beatson Oncology Centre Fund, CRUK (www.cancerresearchuk.org) Grant C301/A14762; Sid P. Kerkar was supported by the NIH Intramural Research Program; Rob J. Kulathinal was supported by the National Science Foundation, and the American Cancer Society; Byoung S. Kwon was supported in part by National Cancer Center (NCC-1310430-2) and National Research Foundation (NRF-2005-0093837); Anne Le was supported by Sol Goldman Pancreatic Cancer Research Fund Grant 80028595, a Lustgarten Fund Grant 90049125 and Grant NIHR21CA169757 (to Anne Le); Michael A. Lea was funded by the The Alma Toorock Memorial for Cancer Research; Ho-Young Lee., This work was supported by grants from the National Research Foundation of Korea (NRF), the Ministry of Science, ICT & Future Planning (MSIP), Republic of Korea (Nos. 2011-0017639 and 2011-0030001) and by a NIH Grant R01 CA100816; Liang-Tzung Lin was supported in part by a grant from the Ministry of Education of Taiwan (TMUTOP103005-4); Jason W. Locasale acknowledges support from NIH awards (CA168997 and AI110613) and the International Life Sciences Institute; Bal L. Lokeshwar was supported in part by United States’ Public Health Services Grants: NIH R01CA156776 and VA-BLR&D Merit Review Grant No. 5I01-BX001517-02; Valter D. Longo acknowledges support from NIH awards (P01AG034906 and R01AG020642) and from the V Foundation; Costas A. Lyssiotis was funded in part by the Pancreatic Cancer Action Network as a Pathway to Leadership Fellow and through a Dale F. Frey Breakthrough award from the Damon Runyon Cancer Research Foundation; Karen L. MacKenzie wishes to acknowledge the support from the Children's Cancer Institute Australia (affiliated with the University of New South Wales, Australia and the Sydney Children's Hospital Network); Maria Marino was supported by grant from University Roma Tre to M.M. (CLA 2013) and by the Italian Association for Cancer Research (AIRC-Grant #IG15221), Ander Matheu is funded by Carlos III Health Institute (AM: CP10/00539), Basque Foundation for Science (IKERBASQUE) and Marie Curie CIG Grant (AM: 2012/712404); Christopher Maxwell was supported by funding from the Canadian Institutes of Health Research, in partnership with the Avon Foundation for Women (OBC-134038) and the Canadian Institutes of Health Research New Investigator Salary Award (MSH-136647); Eoin McDonnell received Duke University Institutional Support; Kapil Mehta was supported by Bayer Healthcare System G4T (Grants4Targets); Gregory A. Michelotti received support from NIH NIDDK, NIH NIAAA, and Shire Pharmaceuticals; Vinayak Muralidhar was supported by the Harvard-MIT Health Sciences and Technology Research Assistantship Award; Elena Niccolai was supported by the Italian Ministry of University and the University of Italy; Virginia R. Parslow gratefully acknowledges the financial support of the Auckland Cancer Society Research Centre (ACSRC); Graham Pawelec was supported by the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF) Grant number 16SV5536K, and by the European Commission (FP7 259679 “IDEAL”); Peter L. Pedersen was supported by NIH Grant CA-10951; Brad Poore was supported by Sol Goldman Pancreatic Cancer Research Fund Grant 80028595, the Lustgarten Fund Grant 90049125, and Grant NIHR21CA169757 (to Anne Le); Satya Prakash was supported by a Canadian Institutes of Health Research Grant (MOP 64308); Lizzia Raffaghello was supported by an NIH Grant (P01AG034906-01A1) and Cinque per Mille dell’IRPEF–Finanziamento della Ricerca Sanitaria; Jeffrey C. Rathmell was supported by an NIH Grant (R01HL108006); Swapan K. Ray was supported by the United Soybean Board; Domenico Ribatti received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under Grant agreement n°278570; Luigi Ricciardiello was supported by the AIRC Investigator Grants 10216 and 13837, and the European Community's Seventh Framework Program FP7/2007–2013 under Grant agreement 311876; Francis Rodier acknowledges the support of the Canadian Institute for Health Research (FR: MOP114962, MOP125857), Fonds de Recherche Québec Santé (FR: 22624), and the Terry Fox Research Institute (FR: 1030), Gian Luigi Russo contributed to this effort while participating in the Fulbright Research Scholar Program 2013–14; Isidro Sanchez-Garcia is partially supported by FEDER and by MICINN (SAF2012-32810), by NIH Grant (R01 CA109335-04A1), by Junta de Castilla y León (BIO/SA06/13) and by the ARIMMORA project (FP7-ENV-2011, European Union Seventh Framework Program). Isidro Sanchez-Garcia's lab is also a member of the EuroSyStem and the DECIDE Network funded by the European Union under the FP7 program; Andrew J. Sanders wishes to acknowledge the support by Cancer Research Wales, the Albert Hung Foundation, the Fong Family Foundation, and Welsh Government A4B scheme; Neeraj K. Saxena was supported by grant funding from NIH NIDDK (K01DK077137, R03DK089130); Dipali Sharma was partially funded by NIH NCI grants (R01CA131294, R21 CA155686), the Avon Foundation and a Breast Cancer Research Foundation Grant (90047965); Markus David Siegelin received funding from National Institute of Health, NINDS Grant K08NS083732, and the 2013 AACR-National Brain Tumor Society Career Development Award for Translational Brain Tumor Research, Grant Number 13-20-23-SIEG; Neetu Singh was supported by funds from the Department of Science and Technology (SR/FT/LS-063/2008), New Delhi, India; Carl Smythe was supported by Yorkshire Cancer Research and The Wellcome Trust, UK; Carmela Spagnuolo was supported by funding from Project C.I.S.I.A., act n. 191/2009 from the Italian Ministry of Economy and Finance Project CAMPUS-QUARC, within program FESR Campania Region 2007/2013, objectives 2.1, 2.2; Diana M. Stafforini was supported by grants from the National Cancer Institute (5P01CA073992), IDEA Award W81XWH-12-1-0515 from the Department of Defense, and by the Huntsman Cancer Foundation; John Stagg was supported by the Canadian Institutes of Health Research; Pochi R. Subbarayan was supported by the University of Miami Clinical and Translational Science Institute (CTSI) Pilot Research Grant (CTSI-2013-P03) and SEEDS You Choose Awards; Phuoc T. Tran was funded by the DoD (W81XWH-11-1-0272 and W81XWH-13-1-0182), a Kimmel Translational Science Award (SKF-13-021), an ACS Scholar award (122688-RSG-12-196-01-TBG) and the NIH (R01CA166348); Kathryn E. Wellen receives funding from the National Cancer Institute, Pancreatic Cancer Action Network, Pew Charitable Trusts, American Diabetes Association, and Elsa U. Pardee Foundation; Huanjie Yang was partially supported by the Scientific Research Foundation for the Returned Oversea Scholars, State Education Ministry and Scientific and Technological Innovation Project, Harbin (2012RFLXS011), Paul Yaswen was supported by funding from the United States National Institutes of Health (ES019458) and the California Breast Cancer Research Program (17UB-8708); Clement Yedjou was supported by a grant from the National Institutes of Health (Grant # G1200MD007581), through the RCMI-Center for Environmental Health; Xin Yin was supported by NIH/National Heart, Lung, and Blood Institute Training Grant T32HL098062.; Jiyue Zhu was supported by NIH Grant R01GM071725; Massimo Zollo was supported by the European FP7-TuMIC HEALTH-F2-2008-201662, the Italian Association for Cancer research (AIRC) Grant IG # 11963 and the Regione Campania L.R:N.5, the European National Funds PON01-02388/1 2007-2013.

DICER1 gene alterations and decreased expression are associated with developmental disorders and diseases in humans. Oscillation of Dicer1 phosphorylation and dephosphorylation regulates its function during the oocyte-to-embryo transition in Caenorhabditis elegans . Dicer1 is also phosphorylated upon FGF stimulation at conserved serines in mouse embryonic fibroblasts and HEK293 cells. However, whether phosphorylation of Dicer1 has a role in mammalian development remains unknown. To investigate the consequence of constitutive phosphorylation, we generated phosphomimetic knock-in mouse models by replacing conserved serines 1712 and 1836 with aspartic acids individually or together. Dicer1 S1836D/S1836D mice display highly penetrant postnatal lethality, and the few survivors display accelerated aging and infertility. Homozygous dual-phosphomimetic Dicer1 augments these defects, alters metabolism-associated miRNAs, and causes a hypermetabolic phenotype. Thus, constitutive phosphorylation of Dicer1 results in multiple pathologic processes in mice, indicating that phosphorylation tightly regulates Dicer1 function and activity in mammals.

Insulin resistance (IR) is a harbinger of type 2 diabetes (T2D) and partly determined by genetic factors. However, genetically regulated mechanisms of IR remain poorly understood. Using gene expression, genotype, and insulin sensitivity data from the African American Genetics of Metabolism and Expression (AAGMEx) cohort, we performed transcript-wide correlation and expression quantitative trait loci (eQTL) analyses to identify IR-correlated cis-regulated transcripts (cis-eGenes) in adipose tissue. These IR-correlated cis-eGenes were tested in the European ancestry individuals in the Metabolic Syndrome in Men (METSIM) cohort for trans-ethnic replication. Comparison of Matsuda index–correlated transcripts in AAGMEx with the METSIM study identified significant correlation of 3,849 transcripts, with concordant direction of effect for 97.5% of the transcripts. cis-eQTL for 587 Matsuda index–correlated genes were identified in both cohorts. Enoyl-CoA hydratase domain-containing 3 (ECHDC3) was the top-ranked Matsuda index–correlated cis-eGene. Expression levels of ECHDC3 were positively correlated with Matsuda index, and regulated by cis-eQTL, rs34844369 being the top cis-eSNP in AAGMEx. Silencing of ECHDC3 in adipocytes significantly reduced insulin-stimulated glucose uptake and Akt Ser473 phosphorylation. RNA sequencing analysis identified 691 differentially expressed genes in ECHDC3-knockdown adipocytes, which were enriched in γ-linolenate biosynthesis, and known IR genes. Thus, our studies elucidated genetic regulatory mechanisms of IR and identified genes and pathways in adipose tissue that are mechanistically involved in IR.