bEver since Carl Woese introduced the use of 16S rRNA genes for determining the phylogenetic relationships of prokaryotes, this method has been regarded as the "gold standard" in both microbial phylogeny and ecology studies. However, intragenomic heterogeneity within 16S rRNA genes has been reported in many investigations and is believed to bias the estimation of prokaryotic diversity. In the current study, 2,013 completely sequenced genomes of bacteria and archaea were analyzed and intragenomic heterogeneity was found in 952 genomes (585 species), with 87.5% of the divergence detected being below the 1% level. In particular, some extremophiles (thermophiles and halophiles) were found to harbor highly divergent 16S rRNA genes. Overestimation caused by 16S rRNA gene intragenomic heterogeneity was evaluated at different levels using the full-length and partial 16S rRNA genes usually chosen as targets for pyrosequencing. The result indicates that, at the unique level, full-length 16S rRNA genes can produce an overestimation of as much as 123.7%, while at the 3% level, an overestimation of 12.9% for the V6 region may be introduced. Further analysis showed that intragenomic heterogeneity tends to concentrate in specific positions, with the V1 and V6 regions suffering the most intragenomic heterogeneity and the V4 and V5 regions suffering the least intragenomic heterogeneity in bacteria. This is the most up-to-date overview of the diversity of 16S rRNA genes within prokaryotic genomes. It not only provides general guidance on how much overestimation can be introduced when applying 16S rRNA gene-based methods, due to its intragenomic heterogeneity, but also recommends that, for bacteria, this overestimation be minimized using primers targeting the V4 and V5 regions. F or decades, 16S rRNA genes, which encode the small subunit of rRNA in prokaryotes, have been widely used in taxonomic assignment and phylogenetic relationship determination (1, 2). The specific properties of the 16S rRNA gene, including its ubiquitous distribution, mosaic structure (3), and relative stability (4), qualify it as an optimal choice to fulfill these applications. Although some argue that 16S rRNA genes alone may not be sufficient to identify closely related species (5, 6) and the use of monocopy genes like rpoB to perform similar studies has been proposed (7), 16S rRNA genes are undoubtedly the most widely used molecular markers in microbial ecological studies due to well-maintained databases (8) and their easy accessibility.For many years, researchers have been trying to estimate the microbial diversity of complex environments, such as soil (9), marine systems (10), and animal gut systems (11, 12). Various techniques have been developed, from culture-dependent methods to 16S rRNA genes-based methods of clone library (13, 14), denaturing gradient gel electrophoresis (DGGE) (15), terminal restriction fragment length polymorphism (T-RFLP) (16), and the recently developed next-generation sequencing (17). However, the question of how diverse an e...

The outbreak of a novel coronavirus (SARS-CoV-2) since December 2019 in Wuhan, the major transportation hub in central China, became an emergency of major international concern. While several etiological studies have begun to reveal the specific biological features of this virus, the epidemic characteristics need to be elucidated. Notably, a long incubation time was reported to be associated with SARS-CoV-2 infection, leading to adjustments in screening and control policies. To

BackgroundHerpes simplex virus type 1 strain 129 (H129) has represented a promising anterograde neuronal circuit tracing tool, which complements the existing retrograde tracers. However, the current H129 derived tracers are multisynaptic, neither bright enough to label the details of neurons nor capable of determining direct projection targets as monosynaptic tracer.MethodsBased on the bacterial artificial chromosome of H129, we have generated a serial of recombinant viruses for neuronal circuit tracing. Among them, H129-G4 was obtained by inserting binary tandemly connected GFP cassettes into the H129 genome, and H129-ΔTK-tdT was obtained by deleting the thymidine kinase (TK) gene and adding tdTomato coding gene to the H129 genome. Then the obtained viral tracers were tested in vitro and in vivo for the tracing capacity.ResultsH129-G4 is capable of transmitting through multiple synapses, labeling the neurons by green florescent protein, and visualizing the morphological details of the labeled neurons. H129-ΔTK-tdT neither replicates nor spreads in neurons alone, but transmits to and labels the postsynaptic neurons with tdTomato in the presence of complementary expressed TK from a helper virus. H129-ΔTK-tdT is also capable to map the direct projectome of the specific neuron type in the given brain regions in Cre transgenic mice. In the tested brain regions where circuits are well known, the H129-ΔTK-tdT tracing patterns are consistent with the previous results.ConclusionsWith the assistance of the helper virus complimentarily expressing TK, H129-ΔTK-tdT replicates in the initially infected neuron, transmits anterogradely through one synapse, and labeled the postsynaptic neurons with tdTomato. The H129-ΔTK-tdT anterograde monosynaptic tracing system offers a useful tool for mapping the direct output in neuronal circuitry. H129-G4 is an anterograde multisynaptic tracer with a labeling signal strong enough to display the details of neuron morphology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-017-0179-7) contains supplementary material, which is available to authorized users.

Poly (ADP‐ribose) polymerase (PARP) inhibitors have provided great clinical benefits to ovarian cancer patients. To date, three PARP inhibitors, namely, olaparib, rucaparib and niraparib have been approved for the treatment of ovarian cancer in the United States. Homologous recombination deficiency (HRD) and platinum sensitivity are prospective biomarkers for predicting the response to PARP inhibitors in ovarian cancers. Preclinical data have focused on identifying the gene aberrations that might generate HRD and induce sensitivity to PARP inhibitors in vitro in cancer cell lines or in vivo in patient‐derived xenografts. Clinical trials have focused on genomic scar analysis to identify biomarkers for predicting the response to PARP inhibitors. Additionally, researchers have aimed to investigate mechanisms of resistance to PARP inhibitors and strategies to overcome this resistance. Combining PARP inhibitors with HR pathway inhibitors to extend the utility of PARP inhibitors to BRCA‐proficient tumours is increasingly foreseeable. Identifying the population of patients with the greatest potential benefit from PARP inhibitor therapy and the circumstances under which patients are no longer suited for PARP inhibitor therapy are important. Further studies are required in order to propose better strategies for overcoming resistance to PARP inhibitor therapy in ovarian cancers.

Arsenic exposure is associated with hypertension, diabetes and cancer. Some mammals methylate arsenic. Saccharomyces cerevisiae hexose permeases catalyze As(OH) 3 uptake. Here we report that mammalian glucose transporter GLUT1 catalyzes As(OH) 3 and CH 3 As(OH) 2 uptake in yeast or in Xenopus laevis öocytes. Expression of GLUT1 in a yeast lacking other glucose transporters allows for growth on glucose. Yeast expressing yeast HXT1 or rat GLUT1 transport As(OH) 3 and CH 3 As (OH) 2 . The K m of GLUT1 is to 1.2 mM for CH 3 As(OH) 2 , compared to a K m of 3 mM for glucose. Inhibition between glucose and CH 3 As(OH) 2 is noncompetitive, suggesting differences between the translocation pathways of hexoses and arsenicals. Both human and rat GLUT1 catalyze uptake of both As(OH) 3 and CH 3 As(OH) 2 in öocytes. Thus GLUT1 may be a major pathway uptake of both inorganic and methylated arsenicals in erythrocytes or the epithelial cells of the blood-brain barrier, contributing to arsenic-related cardiovascular problems and neurotoxicity.Arsenic ranks first on the United States Government's Comprehensive Environmental Response, Compensation, and Liability (Superfund) Act Priority List of Hazardous Substances . In response to the Safe Water Drinking Act, the United States Environmental Protection Agency has set the allowable level of arsenic in drinking water at 10 ppb , which is less than the level in the water supplies of many U.S. municipalities . In addition, the Environmental Protection Agency's Office of Pesticide Programs is concerned with exposure to the organic arsenicals methylarsenic acid and dimethylarsenic acid, pentavalent arsenicals used as pesticides and herbicides .Health effects associated with arsenic exposure include cardiovascular and peripheral vascular disease, neurological disorders, diabetes mellitus and various cancers, including liver, bladder, kidney and skin [1][2][3]. Arsenic trioxide, which is As 2 O 3 in the solid, anhydrous form and As (OH) 3 in solution at physiological pH [4], is used clinically as a chemotherapeutic drug for treatment of acute promyelocytic leukemia [5]. Although epidemiological studies demonstrated that arsenic exposure is associated with a high frequency of circulatory and neurological problems [2,6,7], how arsenic causes non-cancer-related diseases is far from clear. Here we report that GLUT1 facilitates uptake of As(OH) 3 and CH 3 As(OH) 2 using heterologous expression in S. cerevisiae and X. laevis öocytes. S. cerevisiae is a valuable model system for structure-function studies of mammalian membrane proteins such as GLUT1 [22,23]. A yeast strain with low uptake of As(OH) 3 and CH 3 As(OH) 2 was constructed by deletion of all 18 hexose transporters, FPS1, which encodes an aquaglyceroporin that facilitates As(OH) 3 influx, and ACR3, the gene for an...

Poly (ADP-ribose) polymerase (PARP) inhibitors are a class of targeted agents for the treatment of solid tumors. Concurrent PARP inhibition in Breast Cancer Susceptibility Gene (BRCA)-mutated or homologous recombination-deficient tumor cells can induce “synthetic lethality”, which targets two DNA repair pathways and induces serious cytotoxicity to tumor cells without damaging normal cells. Currently, PARP inhibitors such as olaparib, rucaparib and niraparib, which improve progression-free survival, particularly in patients harboring BRCA mutations, are approved by the Food and Drug Administration (FDA) and European Medicine Agency (EMA) for the treatment of ovarian cancers. Based on the results of different clinical trials, the indications for these drugs are slightly different. PARP inhibitors have been studied both as single agents and in combination with chemotherapy, antiangiogenic agents, and ionizing radiation. This review summarizes the critical clinical trials of PARP inhibitors that have been completed, provides an overview of the ongoing trials, presents the confirmed conclusions and notes the issues that need to be addressed in future studies.

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Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na + in binding sites is beyond the resolution of the structures, two Na + binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (K d ) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na + binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two -OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium K d by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na + and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na + binding, and that Na + binding to Na2 promotes binding to Na1 and also sugar binding.I on coupled symporters, or cotransporters, such as hSGLT1 use electrochemical potential gradients to drive solutes into cells. A common finding for these transporters is that external Na + binds before the substrate. Na + binding induces a conformational change of the protein, resulting in the substrate vestibule becoming open to the external membrane surface. After substrate binding, the two ligands are transported across the membrane and are released into the cytoplasm. The atomic structures of several sodium dependent transporters have been solved [leucine transporter (LeuT), vibrio parahaemolyticus sodium glucose (vSGLT), sodium hydantoin (Mhp1), and sodium betaine (BetP)] (1-6). They share a common structural fold with a five-helix inverted repeat, the "LeuT fold". The substrate binding sites are located in the middle of the protein, isolated from the external and membrane surfaces by hydrophobic gates, and putative Na + sites have been identified. Testing these binding sites is a problem due to the fact that phenomenological kinetic constants for ligand transport (K 0.5 , half-saturation values) are interdependent on each other (7). Here we have developed a method to estimate the intrinsic Na + dissociation constants (K d ) for human sodium glucose cotransport (hSGLT1) that may be broadly applicable to other symporters. We then use this to investigate the importance of hSGLT1 residues predicted to be at or near the two Na + binding sites, Na1 and Na2.The method is based on the fact that voltage-dependent membrane proteins exhibit transient charge movements (capacitive transients) i...