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Background: The commensal microbiota of mosquitoes impacts their development, immunity, and competency, and could provide a target for alternative entomological control approaches. However, despite the importance of the mosquito/microbiota interactions, little is known about the relative contribution of endogenous and exogenous factors in shaping the bacterial communities of mosquitoes. Methods: We used a high-throughput sequencing-based assay to characterize the bacterial composition and diversity of 665 individual field-caught mosquitoes, as well as their species, genotype at an insecticide resistance locus, blood-meal composition, and the eukaryotic parasites and viruses they carry. We then used these data to rigorously estimate the individual effect of each parameter on the bacterial diversity as well as the relative contribution of each parameter to the microbial composition. Results: Overall, multivariate analyses did not reveal any significant contribution of the mosquito species, insecticide resistance, or blood meal to the bacterial composition of the mosquitoes surveyed, and infection with parasites and viruses only contributed very marginally. The main driver of the bacterial diversity was the location at which each mosquito was collected, which explained roughly 20% of the variance observed. Conclusions: This analysis shows that when confounding factors are taken into account, the site at which the mosquitoes are collected is the main driver of the bacterial diversity of wild-caught mosquitoes, although further studies will be needed to determine which specific components of the local environment affect bacterial composition. [ABSTRACT FROM AUTHOR]

Plasmodium vivax and P. falciparum, the parasites responsible for most human malaria worldwide, exhibit striking biological differences, which have important clinical consequences. Unfortunately, P. vivax, unlike P. falciparum, cannot be cultivated continuously in vitro, which limits our understanding of its biology and, consequently, our ability to effectively control vivax malaria. Here, we describe single-cell gene expression profiles of 9,215 P. vivax parasites from bloodstream infections of Aotus and Saimiri monkeys. Our results show that transcription of most P. vivax genes occurs during short periods of the intraerythrocytic cycle and that this pattern of gene expression is conserved in other Plasmodium species. However, we also identify a strikingly high proportion of species-specific transcripts in late schizonts, possibly associated with the specificity of erythrocyte invasion. Our findings provide new and robust markers of blood-stage parasites, including some that are specific to the elusive P. vivax male gametocytes, and will be useful for analyzing gene expression data from laboratory and field samples. Analysis of individual Plasmodium vivax parasites reveals the tight control of the expression of most genes during the intra-erythrocytic cycle and the differentiation of male and female gametocytes, and highlights differences between the development of P. vivax and P. falciparum. [ABSTRACT FROM AUTHOR]

The study of the microbial communities has gained traction in recent years with the advent of next-generation sequencing with, or without, PCR-based amplification of the 16S ribosomal RNA region. Such studies have been applied to topics as diverse as human health and environmental ecology. Fewer studies have investigated taxa outside of bacteria, however. We present here data demonstrating the utility of studying taxa outside of bacteria including algae, diatoms, archaea and fungi. Here, we show how location along the Cuyahoga River as well as a transient rainfall event heavily influence the microbial composition. Our data reveal how individual OTUs vary between samples and how the patterns of OTU abundance can accurately predict sampling location. The clustering of samples reveals that these taxa are all sensitive to water conditions in unique ways and demonstrate that, for our dataset, algae was most distinctive between sample groups, surpassing bacteria. Diversity between sampling sites could allow studies investigating pollution or water quality to identify marker OTUs or patterns of OTU abundance as indicators to assess environmental conditions or the impact of human activity. We also directly compare data derived from primers amplifying distinct taxa and show that taxa besides bacteria are excellent indicators of water condition. [ABSTRACT FROM AUTHOR]

DNA methylation is traditionally thought to be established during early development and to remain mostly unchanged thereafter in healthy tissues, although recent studies have shown that this epigenetic mark can be more dynamic. Epigenetic changes occur in the liver after birth, but the timing and underlying biological processes leading to DNA methylation changes are not well understood. We hypothesized that this epigenetic reprogramming was the result of terminal differentiation of hepatocyte precursors. Using genomic approaches, we characterized the DNA methylation patterns in mouse liver from E18.5 until adulthood to determine if the timing of the DNA methylation change overlaps with hepatocyte terminal differentiation, and to examine the genomic context of these changes and identify the regulatory elements involved. Out of 271,325 CpGs analyzed throughout the genome, 214,709 CpGs changed DNA methylation by more than 5% (e.g., from 5 to 10% methylation) between E18.5 and 9 wk of age, and 18,863 CpGs changed DNA methylation by more than 30%. Genome-scale data from six time points between E18.5 and P20 show that DNA methylation changes coincided with the terminal differentiation of hepatoblasts into hepatocytes. We also showed that epigenetic reprogramming occurred primarily in intergenic enhancer regions while gene promoters were less affected. Our data suggest that normal postnatal hepatic development andmaturation involves extensive epigenetic remodeling of the genome, and that enhancers play a key role in controlling the transition from hepatoblasts to fully differentiated hepatocytes. Our study provides a solid foundation to support future research aimed at further revealing the role of epigenetics in stem cell biology. [ABSTRACT FROM AUTHOR]

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Aims: Epidemiological and animal studies have shown that maternal diet can influence metabolism in adult offspring. However, the molecular mechanisms underlying these changes remain poorly understood. Here, we characterize the phenotypes induced by maternal obesity in a mouse model and examine gene expression and epigenetic changes induced by maternal diet in adult offspring. Methods: We analyzed genetically identical male mice born from dams fed a high- or low-fat diet throughout pregnancy and until day 21 postpartum. After weaning, half of the males of each group were fed a high-fat diet, the other half a low-fat diet. We first characterized the genome-wide gene expression patterns of six tissues of adult offspring - liver, pancreas, white adipose, brain, muscle and heart. We then measured DNA methylation patterns in liver at selected loci and throughout the genome. Results: Maternal diet had a significant effect on the body weight of the offspring when they were fed an obesogenic diet after weaning. Our analyses showed that maternal diet had a pervasive effect on gene expression, with a pronounced effect in liver where it affected many genes involved in inflammation, cholesterol synthesis and RXR activation. We did not detect any effect of the maternal diet on DNA methylation in the liver. Conclusions: Overall, our findings highlighted the persistent influence of maternal diet on adult tissue regulation and suggested that the transcriptional changes were unlikely to be caused by DNA methylation differences in adult liver. [ABSTRACT FROM AUTHOR]

Abstract: Background: Mutations in mitochondrial DNA (mtDNA) cause a variety of pathologic states in human patients. Development of animal models harboring mtDNA mutations is crucial to elucidating pathways of disease and as models for preclinical assessment of therapeutic interventions. Scope of review: This review covers the knowledge gained through animal models of mtDNA mutations and the strategies used to produce them. Animals derived from spontaneous mtDNA mutations, somatic cell nuclear transfer (SCNT), nuclear translocation of mitochondrial genes followed by mitochondrial protein targeting (allotopic expression), mutations in mitochondrial DNA polymerase gamma, direct microinjection of exogenous mitochondria, and cytoplasmic hybrid (cybrid) embryonic stem cells (ES cells) containing exogenous mitochondria (transmitochondrial cells) are considered. Major conclusions: A wide range of strategies have been developed and utilized in attempts to mimic human mtDNA mutation in animal models. Use of these animals in research studies has shed light on mechanisms of pathogenesis in mitochondrial disorders, yet methods for engineering specific mtDNA sequences are still in development. General significance: Research animals containing mtDNA mutations are important for studies of the mechanisms of mitochondrial disease and are useful for the development of clinical therapies. This article is part of a Special Issue entitled Biochemistry of Mitochondria. [Copyright &y& Elsevier]

The characterization of mitochondrial diseases has proceeded rapidly since the first descriptions of mitochondrial DNA (mtDNA)-linked disease mutations appeared in the late 1980s. To elucidate mechanisms of a variety of mitochondrial disorders and disease, both in vitro and in vivo modeling systems have been exploited. To produce these models, numerous approaches have been undertaken due to the difficulty associated with targeted mutagenesis and directed modification of the mitochondrial genome. Currently available models of mitochondrial disease are discussed in this paper, including our xenomitochondrial mice. In this model, mitochondria from one donor species are transferred to another. By doing so, cells and animals were generated with varying levels of heteroplasmy (or homoplasmy) for the introduced mitochondrial genomes. This caused graded variations in electron transport chain (ETC) dysfunction which were dependent upon the evolutionary divergence between donor and recipient. The protocol in generating these models involved the utilization of rhodamine-6G (R6G) to remove or eliminate endogenous mtDNA from the recipient cells. This paper will highlight the process and the implications of R6G treatment of mouse embryonic stem (ES) cells to create transmitochondrial cybrids. We summarize the history and mechanism of action of R6G as well as the future prospects for xenomitochondrial models toward increasing our understanding of mitochondrial biology and the dynamic interplay in signaling between mitochondria and the nucleus. [ABSTRACT FROM AUTHOR]

Purpose: For patients with osteosarcoma, disease-related mortality most often results from lung metastasis—a phenomenon shared with many solid tumors. While established metastatic lesions behave aggressively, very few of the tumor cells that reach the lung will survive. By identifying mechanisms that facilitate survival of disseminated tumor cells, we can develop therapeutic strategies that prevent and treat metastasis. Methods: We analyzed single cell RNA-sequencing (scRNAseq) data from murine metastasis-bearing lungs to interrogate changes in both host and tumor cells during colonization. We used these data to elucidate pathways that become activated in cells that survive dissemination and identify candidate host-derived signals that drive activation. We validated these findings through live cell reporter systems, immunocytochemistry, and fluorescent immunohistochemistry. We then validated the functional relevance of key candidates using pharmacologic inhibition in models of metastatic osteosarcoma. Results: Expression patterns suggest that the MAPK pathway is significantly elevated in early and established metastases. MAPK activity correlates with expression of anti-apoptotic genes, especially MCL1. Niche cells produce growth factors that increase ERK phosphorylation and MCL1 expression in tumor cells. Both early and established metastases are vulnerable to MCL1 inhibition, but not MEK inhibition in vivo. Combining MCL1 inhibition with chemotherapy both prevented colonization and eliminated established metastases in murine models of osteosarcoma. Conclusion: Niche-derived growth factors drive MAPK activity and MCL1 expression in osteosarcoma, promoting metastatic colonization. Although later metastases produce less MCL1, they remain dependent on it. MCL1 is a promising target for clinical trials in both human and canine patients. [ABSTRACT FROM AUTHOR]

N-glycosylation is a common posttranslational modification of secreted and membrane proteins, catalyzed by the two enzymatic isoforms of the oligosaccharyltransferase, STT3A and STT3B. Missense mutations are the most common mutations in inherited diseases; however, missense mutations that generate extra, non-native N-glycosylation sites have not been well characterized. Coagulation factor VIII (FVIII) contains five consensus N-glycosylation sites outside its functionally dispensable B domain. We developed a computer program that identified hemophilia A mutations in FVIII that can potentially create ectopic glycosylation sites. We determined that 18 of these ectopic sites indeed become N-glycosylated. These sites span the domains of FVIII and are primarily associated with a severe disease phenotype. Using STT3A and STT3B knockout cells, we determined that ectopic glycosylation exhibited different degrees of dependence on STT3A and STT3B. By separating the effects of ectopic N-glycosylation from those due to underlying amino acid changes, we showed that ectopic glycans promote the secretion of some mutants, but impair the secretion of others. However, ectopic glycans that enhanced secretion could not functionally replace a native N-glycan in the same domain. Secretion-deficient mutants, but not mutants with elevated secretion levels, show increased association with the endoplasmic reticulum chaperones BiP (immunoglobulin heavy chain-binding protein) and calreticulin. Though secreted to different extents, all studied mutants exhibited lower relative activity than wild-type FVIII. Our results reveal differential impacts of ectopic N-glycosylation on FVIII folding, trafficking and activity, which highlight complex disease-causing mechanisms of FVIII missense mutations. Our findings are relevant to other secreted and membrane proteins with mutations that generate ectopic N-glycans. [ABSTRACT FROM AUTHOR]

Background: Tumors are complex tissues containing collections of phenotypically diverse malignant and nonmalignant cells. We know little of the mechanisms that govern heterogeneity of tumor cells nor of the role heterogeneity plays in overcoming stresses, such as adaptation to different microenvironments. Osteosarcoma is an ideal model for studying these mechanisms—it exhibits widespread inter- and intra-tumoral heterogeneity, predictable patterns of metastasis, and a lack of clear targetable driver mutations. Understanding the processes that facilitate adaptation to primary and metastatic microenvironments could inform the development of therapeutic targeting strategies. Results: We investigated single-cell RNA-sequencing profiles of 47,977 cells obtained from cell line and patient-derived xenograft models as cells adapted to growth within primary bone and metastatic lung environments. Tumor cells maintained phenotypic heterogeneity as they responded to the selective pressures imposed during bone and lung colonization. Heterogenous subsets of cells defined by distinct transcriptional profiles were maintained within bone- and lung-colonizing tumors, despite high-level selection. One prominent heterogenous feature involving glucose metabolism was clearly validated using immunofluorescence staining. Finally, using concurrent lineage tracing and single-cell transcriptomics, we found that lung colonization enriches for multiple clones with distinct transcriptional profiles that are preserved across cellular generations. Conclusions: Response to environmental stressors occurs through complex and dynamic phenotypic adaptations. Heterogeneity is maintained, even in conditions that enforce clonal selection. These findings likely reflect the influences of developmental processes promoting diversification of tumor cell subpopulations, which are retained, even in the face of selective pressures. [ABSTRACT FROM AUTHOR]

HES3 is a basic helix-loop-helix transcription factor that regulates neural stem cell renewal during development. HES3 overexpression is predictive of reduced overall survival in patients with fusion-positive rhabdomyosarcoma, a pediatric cancer that resembles immature and undifferentiated skeletal muscle. However, the mechanisms of HES3 cooperation in fusion-positive rhabdomyosarcoma are unclear and are likely related to her3 / HES3's role in neurogenesis. To investigate HES3's function during development , we generated a zebrafish CRISPR/Cas9 null mutation of her3 , the zebrafish ortholog of HES3. Loss of her3 is not embryonic lethal and adults exhibit expected Mendelian ratios. Embryonic her3 zebrafish mutants exhibit dysregulated neurog1 expression, a her3 target gene, and the mutant her3 fails to bind the neurog1 promoter sequence. Further, her3 mutants are significantly smaller than wildtype and a subset present with lens defects as adults. Transcriptomic analysis of her3 mutant embryos indicates that genes involved in organ development, such as pctp and grinab, are significantly downregulated. Further, differentially expressed genes in her3 null mutant embryos are enriched for Hox and Sox10 motifs. Several cancer-related gene pathways are impacted, including the inhibition of matrix metalloproteinases. Altogether, this new model is a powerful system to study her3/HES3 -mediated neural development and its misappropriation in cancer contexts. [Display omitted] • Generated zebrafish her3 CRISPR/Cas9 knockouts with loss-of-function mutations. • her3 mutant is unable to bind known target, neurog1 , causing dysregulated expression. • her3 loss causes abnormal expression of core subset of developmental regulatory genes. • Sox10 DNA binding motifs are enriched in her3 knockout differentially expressed genes. • Tumor microenvironment and matrix metalloprotease pathways are enriched in her3 knockout. [ABSTRACT FROM AUTHOR]

Understanding mosquito host choice is important for assessing vector competence or identifying disease reservoirs. Unfortunately, the availability of an unbiased method for comprehensively evaluating the composition of insect blood meals is very limited, as most current molecular assays only test for the presence of a few pre-selected species. These approaches also have limited ability to identify the presence of multiple mammalian hosts in a single blood meal. Here, we describe a novel high-throughput sequencing method that enables analysis of 96 mosquitoes simultaneously and provides a comprehensive and quantitative perspective on the composition of each blood meal. We validated in silico that universal primers targeting the mammalian mitochondrial 16S ribosomal RNA genes (16S rRNA) should amplify more than 95% of the mammalian 16S rRNA sequences present in the NCBI nucleotide database. We applied this method to 442 female Anopheles punctulatus s. l. mosquitoes collected in Papua New Guinea (PNG). While human (52.9%), dog (15.8%) and pig (29.2%) were the most common hosts identified in our study, we also detected DNA from mice, one marsupial species and two bat species. Our analyses also revealed that 16.3% of the mosquitoes fed on more than one host. Analysis of the human mitochondrial hypervariable region I in 102 human blood meals showed that 5 (4.9%) of the mosquitoes unambiguously fed on more than one person. Overall, analysis of PNG mosquitoes illustrates the potential of this approach to identify unsuspected hosts and characterize mixed blood meals, and shows how this approach can be adapted to evaluate inter-individual variations among human blood meals. Furthermore, this approach can be applied to any disease-transmitting arthropod and can be easily customized to investigate non-mammalian host sources. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax resistance to chloroquine (CQ) has been reported worldwide, although the World Health Organization clinical drug efficacy studies protocol does not permit classification of patient outcomes.Methods: We enrolled 40 patients with P. vivax malaria in northeastern Cambodia, where >17% treatment failures were previously reported. Patients were treated with CQ (30 mg/kg) and followed for 2 months, with frequent clinical examination and capillary blood sample collection for microscopy, molecular parasite detection and genotyping, and drug concentration measurements. Reinfections were prevented by relocating patients to a transmission-free area.Results: P. vivax parasites were eliminated in all patients by day 3. Genomic analyses revealed that all clones in polyclonal infections were cleared at the same rate, indicating their equal susceptibility to CQ. CQ blood concentrations were below the therapeutic level in all recurrent infections (24 of 40 patients), which were efficiently cleared by a second course of CQ treatment. Genotyping (128 SNPs barcode) and sequences of entire parasite genome (Whole-Genome Sequencing, Illumina) indicated that two thirds (6 of 8) of the recurrent parasites resulted from heterologous relapses whose 50% are from by sibling/recombinant clones.Conclusions: No evidence of CQ resistance was observed. Our data suggest that P. vivax antimalarial drug resistance is likely overestimated and that the current guidelines for clinical drug studies of P. vivax malaria need to be revised. [ABSTRACT FROM AUTHOR]