ABSTRACT

Candida auris has emerged globally as a multidrug-resistant yeast that can spread via nosocomial transmission. An initial phylogenetic study of isolates from Japan, India, Pakistan, South Africa, and Venezuela revealed four populations (clades I, II, III, and IV) corresponding to these geographic regions. Since this description, C. auris has been reported in more than 30 additional countries. To trace this global emergence, we compared the genomes of 304 C. auris isolates from 19 countries on six continents. We found that four predominant clades persist across wide geographic locations. We observed phylogeographic mixing in most clades; clade IV, with isolates mainly from South America, demonstrated the strongest phylogeographic substructure. C. auris isolates from two clades with opposite mating types were detected contemporaneously in a single health care facility in Kenya. We estimated a Bayesian molecular clock phylogeny and dated the origin of each clade within the last 360 years; outbreak-causing clusters from clades I, III, and IV originated 36 to 38 years ago. We observed high rates of antifungal resistance in clade I, including four isolates resistant to all three major classes of antifungals. Mutations that contribute to resistance varied between the clades, with Y132F in ERG11 as the most widespread mutation associated with azole resistance and S639P in FKS1 for echinocandin resistance. Copy number variants in ERG11 predominantly appeared in clade III and were associated with fluconazole resistance. These results provide a global context for the phylogeography, population structure, and mechanisms associated with antifungal resistance in C. auris.

IMPORTANCE In less than a decade, C. auris has emerged in health care settings worldwide; this species is capable of colonizing skin and causing outbreaks of invasive candidiasis. In contrast to other Candida species, C. auris is unique in its ability to spread via nosocomial transmission and its high rates of drug resistance. As part of the public health response, whole-genome sequencing has played a major role in characterizing transmission dynamics and detecting new C. auris introductions. Through a global collaboration, we assessed genome evolution of isolates of C. auris from 19 countries. Here, we described estimated timing of the expansion of each C. auris clade and of fluconazole resistance, characterized discrete phylogeographic population structure of each clade, and compared genome data to sensitivity measurements to describe how antifungal resistance mechanisms vary across the population. These efforts are critical for a sustained, robust public health response that effectively utilizes molecular epidemiology.

Multienvironment trials (METs) are widely used to assess the performance of promising crop germplasm. Though seldom designed to elucidate genetic mechanisms, MET data sets are often much larger than could be duplicated for genetic research and, given proper interpretation, may offer valuable insights into the genetics of adaptation across time and space. The Cooperative Dry Bean Nursery (CDBN) is a MET for common bean (Phaseolus vulgaris) grown for > 70 years in the United States and Canada, consisting of 20–50 entries each year at 10–20 locations. The CDBN provides a rich source of phenotypic data across entries, years, and locations that is amenable to genetic analysis. To study stable genetic effects segregating in this MET, we conducted genome-wide association studies (GWAS) using best linear unbiased predictions derived across years and locations for 21 CDBN phenotypes and genotypic data (1.2 million SNPs) for 327 CDBN genotypes. The value of this approach was confirmed by the discovery of three candidate genes and genomic regions previously identified in balanced GWAS. Multivariate adaptive shrinkage (mash) analysis, which increased our power to detect significant correlated effects, found significant effects for all phenotypes. Mash found two large genomic regions with effects on multiple phenotypes, supporting a hypothesis of pleiotropic or linked effects that were likely selected on in pursuit of a crop ideotype. Overall, our results demonstrate that statistical genomics approaches can be used on MET phenotypic data to discover significant genetic effects and to define genomic regions associated with crop improvement.

Single-cross hybrids have been critical to the improvement of maize (Zea mays L.), but the characterization of their genetic architectures remains challenging. Previous studies of hybrid maize have shown the contribution of within-locus complementation effects (dominance) and their differential importance across functional classes of loci. However, they have generally considered panels of limited genetic diversity, and have shown little benefit from genomic prediction based on dominance or functional enrichments. This study investigates the relevance of dominance and functional classes of variants in genomic models for agronomic traits in diverse populations of hybrid maize. We based our analyses on a diverse panel of inbred lines crossed with two testers representative of the major heterotic groups in the U.S. (1106 hybrids), as well as a collection of 24 biparental populations crossed with a single tester (1640 hybrids). We investigated three agronomic traits: days to silking (DTS), plant height (PH), and grain yield (GY). Our results point to the presence of dominance for all traits, but also among-locus complementation (epistasis) for DTS and genotype-by-environment interactions for GY. Consistently, dominance improved genomic prediction for PH only. In addition, we assessed enrichment of genetic effects in classes defined by genic regions (gene annotation), structural features (recombination rate and chromatin openness), and evolutionary features (minor allele frequency and evolutionary constraint). We found support for enrichment in genic regions and subsequent improvement of genomic prediction for all traits. Our results suggest that dominance and gene annotations improve genomic prediction across diverse populations in hybrid maize.

Replication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early- and late-replicating regions of the genome, a process known as replication timing (RT). RT changes during development to ensure accurate genome duplication and maintain genome stability. To understand the relative contributions that cell lineage, cell cycle, and replication initiation regulators have on RT, we utilized the powerful developmental systems available in Drosophila melanogaster. We generated and compared RT profiles from mitotic cells of different tissues and from mitotic and endocycling cells of the same tissue. Our results demonstrate that cell lineage has the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little to no effect on RT. Additionally, we demonstrate that the RT differences we observed in all cases are largely independent of transcriptional differences. We also employed a genetic approach in these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, has on RT control. Our results demonstrate that Rif1 can function in a tissue-specific manner to control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for Rif1 to regulate RT. Together, our data support a model in which the RT program is primarily driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT programs.

During meiotic prophase, concurrent transcription, recombination, and chromosome synapsis place substantial topological strain on chromosomal DNA, but the role of topoisomerases in this context remains poorly defined. Here, we analyzed the roles of topoisomerases I and II (Top1 and Top2) during meiotic prophase in Saccharomyces cerevisiae. We show that both topoisomerases accumulate primarily in promoter-containing intergenic regions of actively transcribing genes, including many meiotic double-strand break (DSB) hotspots. Despite the comparable binding patterns, top1 and top2 mutations have different effects on meiotic recombination. TOP1 disruption delays DSB induction and shortens the window of DSB accumulation by an unknown mechanism. By contrast, temperature-sensitive top2-1 mutants exhibit a marked delay in meiotic chromosome remodeling and elevated DSB signals on synapsed chromosomes. The problems in chromosome remodeling were linked to altered Top2 binding patterns rather than a loss of Top2 catalytic activity, and stemmed from a defect in recruiting the chromosome remodeler Pch2/TRIP13 to synapsed chromosomes. No chromosomal defects were observed in the absence of TOP1. Our results imply independent roles for Top1 and Top2 in modulating meiotic chromosome structure and recombination.

Age-at-onset is one of the critical traits in cohort studies of age-related diseases. Large-scale genome-wide association studies (GWAS) of age-at-onset traits can provide more insights into genetic effects on disease progression and transitions between stages. Moreover, proportional hazards (or Cox) regression models can achieve higher statistical power in a cohort study than a case-control trait using logistic regression. Although mixed-effects models are widely used in GWAS to correct for sample dependence, application of Cox mixed-effects models (CMEMs) to large-scale GWAS is so far hindered by intractable computational cost. In this work, we propose COXMEG, an efficient R package for conducting GWAS of age-at-onset traits using CMEMs. COXMEG introduces fast estimation algorithms for general sparse relatedness matrices including, but not limited to, block-diagonal pedigree-based matrices. COXMEG also introduces a fast and powerful score test for dense relatedness matrices, accounting for both population stratification and family structure. In addition, COXMEG generalizes existing algorithms to support positive semidefinite relatedness matrices, which are common in twin and family studies. Our simulation studies suggest that COXMEG, depending on the structure of the relatedness matrix, is orders of magnitude computationally more efficient than coxme and coxph with frailty for GWAS. We found that using sparse approximation of relatedness matrices yielded highly comparable results in controlling false-positive rate and retaining statistical power for an ethnically homogeneous family-based sample. By applying COXMEG to a study of Alzheimer’s disease (AD) with a Late-Onset Alzheimer’s Disease Family Study from the National Institute on Aging sample comprising 3456 non-Hispanic whites and 287 African Americans, we identified the APOE 4 variant with strong statistical power (P = 1e–101), far more significant than that reported in a previous study using a transformed variable and a marginal Cox model. Furthermore, we identified novel SNP rs36051450 (P = 2e–9) near GRAMD1B, the minor allele of which significantly reduced the hazards of AD in both genders. These results demonstrated that COXMEG greatly facilitates the application of CMEMs in GWAS of age-at-onset traits.

Key Points

The past decade has increased our understanding of how genome topology controls RAG endonuclease-mediated assembly of lymphocyte AgR genes. New technologies have illuminated how the large IgH, Ig, TCRα/, and TCRβ loci fold into compact structures that place their numerous V gene segments in similar three-dimensional proximity to their distal recombination center composed of RAG-bound (D)J gene segments. Many studies have shown that CTCF and cohesin protein–mediated chromosome looping have fundamental roles in lymphocyte lineage- and developmental stage–specific locus compaction as well as broad usage of V segments. CTCF/cohesin–dependent loops have also been shown to direct and restrict RAG activity within chromosome domains. We summarize recent work in elucidating molecular mechanisms that govern three-dimensional chromosome organization and in investigating how these dynamic mechanisms control V(D)J recombination. We also introduce remaining questions for how CTCF/cohesin–dependent and –independent genome architectural mechanisms might regulate compaction and recombination of AgR loci.

Programmed death-ligand 1 (PD-L1/B7-H1) serves as a cosignaling molecule in cell-mediated immune responses and contributes to chronicity of inflammation and the escape of tumor cells from immunosurveillance. Here, we investigated the molecular mechanisms leading to PD-L1 upregulation in human oral carcinoma cells and in primary human gingival keratinocytes in response to infection with Porphyromonas gingivalis (P. gingivalis), a keystone pathogen for the development of periodontitis. The bacterial cell wall component peptidoglycan uses bacterial outer membrane vesicles to be taken up by cells. Internalized peptidoglycan triggers cytosolic receptors to induce PD-L1 expression in a myeloid differentiation primary response 88 (Myd88)-independent and receptor-interacting serine/threonine-protein kinase 2 (RIP2)-dependent fashion. Interference with the kinase activity of RIP2 or mitogen-activated protein (MAP) kinases interferes with inducible PD-L1 expression.

In mammalian cells, alphavirus replication complexes are anchored to the plasma membrane. This interaction with lipid bilayers is mediated through the viral methyl/guanylyltransferase nsP1 and reinforced by palmitoylation of cysteine residue(s) in the C-terminal region of this protein. Lipid content of membranes supporting nsP1 anchoring remains poorly studied. Here, we explore the membrane binding capacity of nsP1 with regard to cholesterol. Using the medically important chikungunya virus (CHIKV) as a model, we report that nsP1 cosegregates with cholesterol-rich detergent-resistant membrane microdomains (DRMs), also called lipid rafts. In search for the critical factor for cholesterol partitioning, we identify nsP1 palmitoylated cysteines as major players in this process. In cells infected with CHIKV or transfected with CHIKV trans-replicase plasmids, nsP1, together with the other nonstructural proteins, are detected in DRMs. While the functional importance of CHIKV nsP1 preference for cholesterol-rich membrane domains remains to be determined, we observed that U18666A- and imipramine-induced sequestration of cholesterol in late endosomes redirected nsP1 to these compartments and simultaneously dramatically decreased CHIKV genome replication. A parallel study of Sindbis virus (SINV) revealed that nsP1 from this divergent alphavirus displays a low affinity for cholesterol and only moderately segregates with DRMs. Behaviors of CHIKV and SINV with regard to cholesterol, therefore, match with the previously reported differences in the requirement for nsP1 palmitoylation, which is dispensable for SINV but strictly required for CHIKV replication. Altogether, this study highlights the functional importance of nsP1 segregation with DRMs and provides new insight into the functional role of nsP1 palmitoylated cysteines during alphavirus replication.

IMPORTANCE Functional alphavirus replication complexes are anchored to the host cell membranes through the interaction of nsP1 with the lipid bilayers. In this work, we investigate the importance of cholesterol for such an association. We show that nsP1 has affinity for cholesterol-rich membrane microdomains formed at the plasma membrane and identify conserved palmitoylated cysteine(s) in nsP1 as the key determinant for cholesterol affinity. We demonstrate that drug-induced cholesterol sequestration in late endosomes not only redirects nsP1 to this compartment but also dramatically decreases genome replication, suggesting the functional importance of nsP1 targeting to cholesterol-rich plasma membrane microdomains. Finally, we show evidence that nsP1 from chikungunya and Sindbis viruses displays different sensitivity to cholesterol sequestering agents that parallel with their difference in the requirement for nsP1 palmitoylation for replication. This research, therefore, gives new insight into the functional role of palmitoylated cysteines in nsP1 for the assembly of functional alphavirus replication complexes in their mammalian host.

The TEAD family of transcription factors requires associating cofactors to induce gene expression. TEAD1 is known to activate the early promoter of human papillomavirus (HPV), but the precise mechanisms of TEAD1-mediated transactivation of the HPV promoter, including its relevant cofactors, remain unexplored. Here, we reveal that VGLL1, a TEAD-interacting cofactor, contributes to HPV early gene expression. Knockdown of VGLL1 and/or TEAD1 led to a decrease in viral early gene expression in human cervical keratinocytes and cervical cancer cell lines. We identified 11 TEAD1 target sites in the HPV16 long control region (LCR) by in vitro DNA pulldown assays; 8 of these sites contributed to the transcriptional activation of the early promoter in luciferase reporter assays. VGLL1 bound to the HPV16 LCR via its interaction with TEAD1 both in vitro and in vivo. Furthermore, introducing HPV16 and HPV18 whole genomes into primary human keratinocytes led to increased levels of VGLL1, due in part to the upregulation of TEADs. These results suggest that multiple VGLL1/TEAD1 complexes are recruited to the LCR to support the efficient transcription of HPV early genes.

IMPORTANCE Although a number of transcription factors have been reported to be involved in HPV gene expression, little is known about the cofactors that support HPV transcription. In this study, we demonstrate that the transcriptional cofactor VGLL1 plays a prominent role in HPV early gene expression, dependent on its association with the transcription factor TEAD1. Whereas TEAD1 is ubiquitously expressed in a variety of tissues, VGLL1 displays tissue-specific expression and is implicated in the development and differentiation of epithelial lineage tissues, where HPV gene expression occurs. Our results suggest that VGLL1 may contribute to the epithelial specificity of HPV gene expression, providing new insights into the mechanisms that regulate HPV infection. Further, VGLL1 is also critical for the growth of cervical cancer cells and may represent a novel therapeutic target for HPV-associated cancers.

The Epstein-Barr virus (EBV) causes human cancers, and epidemiological studies have shown that lytic replication is a risk factor for some of these tumors. This fits with the observation that EBV M81, which was isolated from a Chinese patient with nasopharyngeal carcinoma, induces potent virus production and increases the risk of genetic instability in infected B cells. To find out whether this property extends to viruses found in other parts of the world, we investigated 22 viruses isolated from Western patients. While one-third of the viruses hardly replicated, the remaining viruses showed variable levels of replication, with three isolates replicating at levels close to that of M81 in B cells. We cloned one strongly replicating virus into a bacterial artificial chromosome (BAC); the resulting recombinant virus (MSHJ) retained the properties of its nonrecombinant counterpart and showed similarities to M81, undergoing lytic replication in vitro and in vivo after 3 weeks of latency. In contrast, B cells infected with the nonreplicating Western B95-8 virus showed early but abortive replication accompanied by cytoplasmic BZLF1 expression. Sequencing confirmed that rMSHJ is a Western virus, being genetically much closer to B95-8 than to M81. Spontaneous replication in rM81- and rMSHJ-infected B cells was dependent on phosphorylated Btk and was inhibited by exposure to ibrutinib, opening the way to clinical intervention in patients with abnormal EBV replication. As rMSHJ contains the complete EBV genome and induces lytic replication in infected B cells, it is ideal to perform genetic analyses of all viral functions in Western strains and their associated diseases.

IMPORTANCE The Epstein-Barr virus (EBV) infects the majority of the world population but causes different diseases in different countries. Evidence that lytic replication, the process that leads to new virus progeny, is linked to cancer development is accumulating. Indeed, viruses such as M81 that were isolated from Far Eastern nasopharyngeal carcinomas replicate strongly in B cells. We show here that some viruses isolated from Western patients, including the MSHJ strain, share this property. Moreover, replication of both M81 and of MSHJ was sensitive to ibrutinib, a commonly used drug, thereby opening an opportunity for therapeutic intervention. Sequencing of MSHJ showed that this virus is quite distant from M81 and is much closer to nonreplicating Western viruses. We conclude that Western EBV strains are heterogeneous, with some viruses being able to replicate more strongly and therefore being potentially more pathogenic than others, and that the virus sequence information alone cannot predict this property.

Influenza D virus (IDV) was initially isolated in the United States in 2011. IDV is distributed worldwide and is one of the causative agents of the bovine respiratory disease complex (BRDC), which causes high morbidity and mortality in feedlot cattle. The molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics systems are vital tools not only for studying the biology of viruses, but also for use in applications such as recombinant vaccine viruses. Here, we report the establishment of a plasmid-based reverse genetics system for IDV. We first verified that the 3'-terminal nucleotide of each 7-segmented genomic RNA contained uracil (U), contrary to previous reports, and we were then able to successfully generate recombinant IDV by cotransfecting 7 plasmids containing these genomic RNAs along with 4 plasmids expressing polymerase proteins and nucleoprotein into human rectal tumor 18G (HRT-18G) cells. The recombinant virus had a growth deficit compared to the wild-type virus, and we determined the reason for this growth difference by examining the genomic RNA content of the viral particles. We found that the recombinant virus incorporated an unbalanced ratio of viral RNA segments into particles compared to that of the wild-type virus, and thus we adjusted the amount of each plasmid used in transfection to obtain a recombinant virus with the same replicative capacity as the wild-type virus. Our work here in establishing a reverse genetics system for IDV will have a broad range of applications, including uses in studies focused on better understanding IDV replication and pathogenicity, as well as in those contributing to the development of BRDC countermeasures.

IMPORTANCE The bovine respiratory disease complex (BRDC) causes high mortality and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is considered to be a causative agent of the BRDC. Here, we developed a reverse genetics system that allows for the generation of IDV from cloned cDNAs and the introduction of mutations into the IDV genome. This reverse genetics system will become a powerful tool for use in studies related to understanding the molecular mechanisms of viral replication and pathogenicity and will also lead to the development of new countermeasures against the BRDC.

Nuclear import of viral genomes is an important step during the life cycle of adenoviruses (AdV), requiring soluble cellular factors as well as proteins of the nuclear pore complex (NPC). We addressed the role of the cytoplasmic nucleoporin Nup358 during adenoviral genome delivery by performing depletion/reconstitution experiments and time-resolved quantification of adenoviral genome import. Nup358-depleted cells displayed reduced efficiencies of nuclear import of adenoviral genomes, and the nuclear import receptor transportin 1 became rate limiting under these conditions. Furthermore, we identified a minimal N-terminal region of Nup358 that was sufficient to compensate for the import defect. Our data support a model where Nup358 functions as an assembly platform that promotes the formation of transport complexes, allowing AdV to exploit a physiological protein import pathway for accelerated transport of its DNA.

IMPORTANCE Nuclear import of viral genomes is an essential step to initiate productive infection for several nuclear replicating DNA viruses. On the other hand, DNA is not a physiological nuclear import substrate; consequently, viruses have to exploit existing physiological transport routes. Here, we show that adenoviruses use the nucleoporin Nup358 to increase the efficiency of adenoviral genome import. In its absence, genome import efficiency is reduced and the transport receptor transportin 1 becomes rate limiting. We show that the N-terminal half of Nup358 is sufficient to drive genome import and identify a transportin 1 binding region. In our model, adenovirus genome import exploits an existing protein import pathway and Nup358 serves as an assembly platform for transport complexes.

A novel lytic bacteriophage, ValSw3-3, which efficiently infects pathogenic strains of Vibrio alginolyticus, was isolated from sewage water and characterized by microbiological and in silico genomic analyses. Transmission electron microscopy indicated that ValSw3-3 has the morphology of siphoviruses. This phage can infect four species in the Vibrio genus and has a latent period of 15 min and a burst size of 95 ± 2 PFU/infected bacterium. Genome sequencing results show that ValSw3-3 has a 39,846-bp double-stranded DNA genome with a GC content of 43.1%. The similarity between the genome sequences of ValSw3-3 and those of other phages recorded in the GenBank database was below 50% (42%), suggesting that ValSw3-3 significantly differs from previously reported phages at the DNA level. Multiple genome comparisons and phylogenetic analysis based on the major capsid protein revealed that phage ValSw3-3 is grouped in a clade with five other phages, including Listonella phage phiHSIC (GenBank accession no. NC_006953.1), Vibrio phage P23 (MK097141.1), Vibrio phage pYD8-B (NC_021561.1), Vibrio phage 2E1 (KX507045.1), and Vibrio phage 12G5 (HQ632860.1), and is distinct from all known genera within the Siphoviridae family that have been ratified by the International Committee on Taxonomy of Viruses (ICTV). An in silico proteomic comparison of diverse phages from the Siphoviridae family supported this clustering result and suggested that ValSw3-3, phiHSIC, P23, pYD8-B, 2E1, and 12G5 should be classified as a novel genus cluster of Siphoviridae. A subsequent analysis of core genes also revealed the common genes shared within this new cluster. Overall, these results provide a characterization of Vibrio phage ValSw3-3 and support our proposal of a new viral genus within the family Siphoviridae.

IMPORTANCE Phage therapy has been considered a potential alternative to antibiotic therapy in treating bacterial infections. For controlling the vibriosis-causing pathogen Vibrio alginolyticus, well-documented phage candidates are still lacking. Here, we characterize a novel lytic Vibrio phage, ValSw3-3, based on its morphology, host range and infectivity, growth characteristics, stability under various conditions, and genomic features. Our results show that ValSw3-3 could be a potent candidate for phage therapy to treat V. alginolyticus infections due to its stronger infectivity and better pH and thermal stability than those of previously reported Vibrio phages. Moreover, genome sequence alignments, phylogenetic analysis, in silico proteomic comparison, and core gene analysis all support that this novel phage, ValSw3-3, and five unclassified phages form a clade distant from those of other known genera ratified by the ICTV. Thus, we propose a new viral genus within the Siphoviridae family to accommodate this clade, with ValSw3-3 as a representative member.

Genetic variants within complement factor H (CFH), a major alternative complement pathway regulator, are associated with the development of age-related macular degeneration (AMD) and other complementopathies. This is explained with the reduced binding of CFH or its splice variant factor H-like protein 1 (FHL-1) to self-ligands or altered self-ligands (e.g.,...

The apparent detection of an exoplanet orbiting Fomalhaut was announced in 2008. However, subsequent observations of Fomalhaut b raised questions about its status: Unlike other exoplanets, it is bright in the optical and nondetected in the infrared, and its orbit appears to cross the debris ring around the star without...

Recently, abundant evidence has clarified that long noncoding RNAs (lncRNAs) play an oncogenic or anticancer role in the tumorigenesis and development of diverse human cancers. Described as a crucial regulator in some cancers, MIR22HG has not yet been studied in laryngocarcinoma and therefore the underlying regulatory role of MIR22HG in laryngocarcinoma is worth detecting. In this study, MIR22HG expression in laryngocarcinoma cells was confirmed to be downregulated, and upregulated MIR22HG expression led to suppressive effects on laryngocarcinoma cell proliferation and migration. Molecular mechanism assays revealed that MIR22HG sponges miR-5000-3p in laryngocarcinoma cells. Besides, decreased expression of miR-5000-3p suppressed laryngocarcinoma cell proliferation and migration. Moreover, the FBXW7 gene was reported to be a downstream target gene of miR-5000-3p in laryngocarcinoma cells. More importantly, rescue assays verified that FBXW7 depletion or miR-5000-3p upregulation countervailed the repressive effects of MIR22HG overexpression on laryngocarcinoma progression. In addition, E2F6 was proved to be capable of inhibiting MIR22HG transcription in laryngocarcinoma cells. To sum up, E2F6-induced downregulation of MIR22HG promotes laryngocarcinoma progression through the miR-5000-3p/FBXW7 axis.

Leukocyte recruitment is a universal feature of tissue inflammation and regulated by the interactions of chemokines with their G protein–coupled receptors. Activation of CC chemokine receptor 2 (CCR2) by its cognate chemokine ligands, including CC chemokine ligand 2 (CCL2), plays a central role in recruitment of monocytes in several inflammatory diseases. In this study, we used phosphoproteomics to conduct an unbiased characterization of the signaling network resulting from CCL2 activation of CCR2. Using data-independent acquisition MS analysis, we quantified both the proteome and phosphoproteome in FlpIn-HEK293T cells stably expressing CCR2 at six time points after activation with CCL2. Differential expression analysis identified 699 significantly regulated phosphorylation sites on 441 proteins. As expected, many of these proteins are known to participate in canonical signal transduction pathways and in the regulation of actin cytoskeleton dynamics, including numerous guanine nucleotide exchange factors and GTPase-activating proteins. Moreover, we identified regulated phosphorylation sites in numerous proteins that function in the nucleus, including several constituents of the nuclear pore complex. The results of this study provide an unprecedented level of detail of CCR2 signaling and identify potential targets for regulation of CCR2 function.

Groundwater is best described as an open access good: as a common pool resource others cannot be excluded from using and as a finite resource, its consumption leaves less for others to enjoy. In addition to its obvious benefits to humans and animals, it contributes in sustaining the hydro-environment and life on the planet. Scarcity causes groundwater to become an economic good and have an economic value. By and large, economic valuations respond to changes in the quantity and quality of groundwater. They are based on willingness to pay for maintaining its benefits or willingness to accept compensation for giving them up. There have been some 50 published groundwater valuation studies worldwide in the last 30 years, mostly focusing on groundwater quality and contamination. There is sparsity of valuations in Africa and Asia. The results suggest median economic values of 60–160 and mean values of 70–480 US dollars per household per year. Generally, values are higher in the USA followed by Europe, and lowest in Asia. People's income is a major factor affecting values, but this does not mean that they value groundwater less. Economic valuations and cost–benefit analysis are useful in highlighting people's priorities and choice of options. However, economics alone should not dictate actions for protecting groundwater resources from degradation and depletion.

Leprosy is caused by Mycobacterium leprae, and it remains underdiagnosed in Burkina Faso. We investigated the use of fluorescent in situ hybridization (FISH) for detecting M. leprae in 27 skin samples (skin biopsy samples, slit skin samples, and skin lesion swabs) collected from 21 patients from Burkina Faso and three from Côte d’Ivoire who were suspected of having cutaneous leprosy. In all seven Ziehl-Neelsen-positive skin samples (four skin biopsy samples and three skin swabs collected from the same patient), FISH specifically identified M. leprae, including one FISH-positive skin biopsy sample that remained negative after testing with PCR targeting the rpoB gene and with the GenoType LepraeDR assay. Twenty other skin samples and three negative controls all remained negative for Ziehl-Neelsen staining, FISH, and rpoB PCR. These data indicate the usefulness of a microscopic examination of skin samples after FISH for first-line diagnosis of cutaneous leprosy. Accordingly, FISH represents a potentially useful point-of-care test for the diagnosis of cutaneous leprosy.

Klebsiella species are problematic pathogens in neonatal units and may cause outbreaks, for which the sources of transmission may be challenging to elucidate. We describe the use of whole-genome sequencing (WGS) to investigate environmental sources of transmission during an outbreak of extended-spectrum-β-lactamase (ESBL)-producing Klebsiella michiganensis colonizing neonates. Ceftriaxone-resistant Klebsiella spp. isolated from neonates (or their mothers) and the hospital environment were included. Short-read sequencing (Illumina) and long-read sequencing (MinION; Oxford Nanopore Technologies) were used to confirm species taxonomy, to identify antimicrobial resistance genes, and to determine phylogenetic relationships using single-nucleotide polymorphism profiling. A total of 21 organisms (10 patient-derived isolates and 11 environmental isolates) were sequenced. Standard laboratory methods identified the outbreak strain as an ESBL-producing Klebsiella oxytoca, but taxonomic assignment from WGS data suggested closer identity to Klebsiella michiganensis. Strains isolated from multiple detergent-dispensing bottles were either identical or closely related by single-nucleotide polymorphism comparison. Detergent bottles contaminated by K. michiganensis had been used for washing milk expression equipment. No new cases were identified once the detergent bottles were removed. Environmental reservoirs may be an important source in outbreaks of multidrug-resistant organisms. WGS, in conjunction with traditional epidemiological investigation, can be instrumental in revealing routes of transmission and guiding infection control responses.

Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens due to its particular action on the persistent forms of Mycobacterium tuberculosis. However, no drug susceptibility test (DST) is considered sufficiently reliable for routine application. Although molecular tests are endorsed, their application is limited to known PZA resistance associated mutations. Microbiological DSTs for PZA have been restricted by technical limitations, especially the necessity for an acidic pH. Here, for the first time, MODS culture at neutral pH was evaluated using high PZA concentrations (400 and 800 μg/ml) to determine PZA susceptibility directly from sputum samples. Sputum samples were cultured with PZA for up to 21 days at 37°C. Plate reading was performed at two time points: R1 (mean, 10 days) and R2 (mean, 13 days) for each PZA concentration. A consensus reference test, composed of MGIT-PZA, pncA sequencing, and the classic Wayne test, was used. A total of 182 samples were evaluated. The sensitivity and specificity for 400 μg/ml ranged from 76.9 to 89.7 and from 93.0 to 97.9%, respectively, and for 800 μg/ml ranged from 71.8 to 82.1 and from 95.8 to 98.6%, respectively. Compared to MGIT-PZA, our test showed a similar turnaround time (medians of 10 and 12 days for PZA-sensitive and -resistant isolates, respectively). In conclusion, MODS-PZA is presented as a fast, simple, and low-cost DST that could complement the MODS assay to evaluate resistance to the principal first-line antituberculosis drugs. Further optimization of test conditions would be useful in order to increase its performance.

Serological testing for nasopharyngeal carcinoma (NPC) has recently been reinvigorated by the implementation of novel Epstein-Barr virus (EBV)-specific IgA and IgG antibodies from a proteome array. Although proteome arrays are well suited for comprehensive antigen selection, they are not applicable for large-scale studies. We adapted a 13-marker EBV antigen signature for NPC risk identified by proteome arrays to multiplex serology to establish an assay for large-scale studies. Taiwanese NPC cases (n = 175) and matched controls (n = 175) were used for assay validation. Spearman’s correlation was calculated, and the diagnostic value of all multiplex markers was assessed independently using the area under the receiver operating characteristic curve (AUC). Two refined signatures were identified using stepwise logistic regression and internally validated with 10-fold cross validation. Array and multiplex serology showed strong correlation for each individual EBV marker, as well as for a 13-marker combined model on continuous data. Two refined signatures with either four (LF2 and BGLF2 IgG, LF2 and BMRF1 IgA) or two (LF2 and BGLF2 IgG) antibodies on dichotomous data were identified as the most parsimonious set of serological markers able to distinguish NPC cases from controls with AUCs of 0.992 (95% confidence interval [CI], 0.983 to 1.000) and 0.984 (95% CI, 0.971 to 0.997), respectively. Neither differed significantly from the 13-marker model (AUC, 0.992; 95% CI, 0.982 to 1.000). All models were internally validated. Multiplex serology successfully validated the original EBV proteome microarray data. Two refined signatures of four and two antibodies were capable of detecting NPC with 99.2% and 98.4% accuracy.

Large-scale metagenomic and metatranscriptomic data analyses are often restricted by their gene-centric approach, limiting the ability to understand organismal and community biology. De novo assembly of large and mosaic eukaryotic genomes from complex meta-omics data remains a challenging task, especially in comparison with more straightforward bacterial and archaeal systems. Here, we use a transcriptome reconstruction method based on clustering co-abundant genes across a series of metagenomic samples. We investigated the co-abundance patterns of ~37 million eukaryotic unigenes across 365 metagenomic samples collected during the Tara Oceans expeditions to assess the diversity and functional profiles of marine plankton. We identified ~12,000 co-abundant gene groups (CAGs), encompassing ~7 million unigenes, including 924 metagenomics-based transcriptomes (MGTs, CAGs larger than 500 unigenes). We demonstrated the biological validity of the MGT collection by comparing individual MGTs with available references. We identified several key eukaryotic organisms involved in dimethylsulfoniopropionate (DMSP) biosynthesis and catabolism in different oceanic provinces, thus demonstrating the potential of the MGT collection to provide functional insights on eukaryotic plankton. We established the ability of the MGT approach to capture interspecies associations through the analysis of a nitrogen-fixing haptophyte-cyanobacterial symbiotic association. This MGT collection provides a valuable resource for analyses of eukaryotic plankton in the open ocean by giving access to the genomic content and functional potential of many ecologically relevant eukaryotic species.

Hepatocellular carcinomas (HCC) are adapted to survive extreme genomic stress conditions imposed by hyperactive DNA replication and genotoxic drug treatment. The underlying mechanisms remain unclear, but may involve intensified DNA damage response/repair programs. Here, we investigate a new role of nucleostemin (NS) in allowing HCC to survive its own malignancy, as NS was previously shown to promote liver regeneration via a damage repair mechanism. We first established that a higher NS transcript level correlates with high-HCC grades and poor prognostic signatures, and is an independent predictor of shorter overall and progression-free survival specifically for HCC and kidney cancer but not for others. Immunostaining confirmed that NS is most abundantly expressed in high-grade and metastatic HCCs. Genome-wide analyses revealed that NS is coenriched with MYC target and homologous recombination (HR) repair genes in human HCC samples and functionally intersects with those involved in replication stress response and HR repair in yeasts. In support, NS-high HCCs are more reliant on the replicative/oxidative stress response pathways, whereas NS-low HCCs depend more on the mTOR pathway. Perturbation studies showed NS function in protecting human HCC cells from replication- and drug-induced DNA damage. Notably, NS depletion in HCC cells increases the amounts of physical DNA damage and cytosolic double-stranded DNA, leading to a reactive increase of cytokines and PD-L1. This study shows that NS provides an essential mechanism for HCC to adapt to high genomic stress for oncogenic maintenance and propagation. NS deficiency sensitizes HCC cells to chemotherapy but also triggers tumor immune responses.

Cholangiocarcinoma (CCA) is a malignant tumor that arises from the epithelial cells of the bile duct and is notorious for its poor prognosis. The clinical outcome remains disappointing, and thus more effective therapeutic options are urgently required. Cordycepin, a traditional Chinese medicine, provides multiple pharmacological strategies in antitumors, but its mechanisms have not been fully elucidated. In this study, we reported that cordycepin inhibited the viability and proliferation capacity of CCA cells in a time- and dose-dependent manner determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and colony formation assay. Flow cytometry and Hoechst dye showed that cordycepin induced cancer cell apoptosis via extracellular signal-regulated kinase (ERK) 1/2 deactivation. Moreover, cordycepin significantly reduced the angiogenetic capabilities of CCA in vitro as examined by tube formation assay. We also discovered that cordycepin inhibited DEK expression by using Western blot assay. DEK serves as an oncogenic protein that is overexpressed in various gastrointestinal tumors. DEK silencing inhibited CCA cell viability and angiogenesis but not apoptosis induction determined by Western blot and flow cytometry. Furthermore, cordycepin significantly inhibited tumor growth and angiogenic capacities in a xenograft model by downregulating the expression of DEK, phosphorylated ERK1/2 CD31 and von Willebrand factor (vWF). Taken together, we demonstrated that cordycepin inhibited CCA cell proliferation and angiogenesis with a DEK interaction via downregulation in ERK signaling. These data indicate that cordycepin may serve as a novel agent for CCA clinical treatment and prognosis improvement.

In vitro approaches for predicting drug-drug interactions (DDIs) caused by alterations in transporter protein regulation are not well established. However, reports of transporter regulation via nuclear receptor (NR) modulation by drugs are increasing. This study examined alterations in transporter protein levels in sandwich-cultured human hepatocytes (SCHH; n = 3 donors) measured by liquid chromatography–tandem mass spectrometry–based proteomic analysis after treatment with N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide (T0901317), the first described synthetic liver X receptor agonist. T0901317 treatment (10 μM, 48 hours) decreased the levels of organic cation transporter (OCT) 1 (0.22-, 0.43-, and 0.71-fold of control) and organic anion transporter (OAT) 2 (0.38-, 0.38-, and 0.53-fold of control) and increased multidrug resistance protein (MDR) 1 (1.37-, 1.48-, and 1.59-fold of control). The induction of NR downstream gene expression supports the hypothesis that T0901317 off-target effects on farnesoid X receptor and pregnane X receptor activation are responsible for the unexpected changes in OCT1, OAT2, and MDR1. Uptake of the OCT1 substrate metformin in SCHH was decreased by T0901317 treatment. Effects of decreased OCT1 levels on metformin were simulated using a physiologically-based pharmacokinetic (PBPK) model. Simulations showed a clear decrease in metformin hepatic exposure resulting in a decreased pharmacodynamic effect. This DDI would not be predicted by the modest changes in simulated metformin plasma concentrations. Altogether, the current study demonstrated that an approach combining SCHH, proteomic analysis, and PBPK modeling is useful for revealing tissue concentration–based DDIs caused by unexpected regulation of hepatic transporters by NR modulators.

The emergence of drug resistance is a major obstacle for the success of targeted therapy in melanoma. Additionally, conventional chemotherapy has not been effective as drug-resistant cells escape lethal DNA damage effects by inducing growth arrest commonly referred to as cellular dormancy. We present a therapeutic strategy termed "targeted chemotherapy" by depleting protein phosphatase 2A (PP2A) or its inhibition using a small molecule inhibitor (1,10-phenanthroline-5,6-dione [phendione]) in drug-resistant melanoma. Targeted chemotherapy induces the DNA damage response without causing DNA breaks or allowing cellular dormancy. Phendione treatment reduces tumor growth of BRAF^V600E-driven melanoma patient-derived xenografts (PDX) and diminishes growth of NRAS^Q61R-driven melanoma, a cancer with no effective therapy. Remarkably, phendione treatment inhibits the acquisition of resistance to BRAF inhibition in BRAF^V600E PDX highlighting its effectiveness in combating the advent of drug resistance.

Introduction

Immunotherapy has become the standard of care in advanced non-small cell lung cancer (NSCLC). We aimed to quantify the economic impact, in France, of anti-PD-1 therapy for NSCLC.

ABSTRACT

In numerous instances, tracking the biological significance of a nucleic acid sequence can be augmented through the identification of environmental niches in which the sequence of interest is present. Many metagenomic data sets are now available, with deep sequencing of samples from diverse biological niches. While any individual metagenomic data set can be readily queried using web-based tools, meta-searches through all such data sets are less accessible. In this brief communication, we demonstrate such a meta-metagenomic approach, examining close matches to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in all high-throughput sequencing data sets in the NCBI Sequence Read Archive accessible with the "virome" keyword. In addition to the homology to bat coronaviruses observed in descriptions of the SARS-CoV-2 sequence (F. Wu, S. Zhao, B. Yu, Y. M. Chen, et al., Nature 579:265–269, 2020, https://doi.org/10.1038/s41586-020-2008-3; P. Zhou, X. L. Yang, X. G. Wang, B. Hu, et al., Nature 579:270–273, 2020, https://doi.org/10.1038/s41586-020-2012-7), we note a strong homology to numerous sequence reads in metavirome data sets generated from the lungs of deceased pangolins reported by Liu et al. (P. Liu, W. Chen, and J. P. Chen, Viruses 11:979, 2019, https://doi.org/10.3390/v11110979). While analysis of these reads indicates the presence of a similar viral sequence in pangolin lung, the similarity is not sufficient to either confirm or rule out a role for pangolins as an intermediate host in the recent emergence of SARS-CoV-2. In addition to the implications for SARS-CoV-2 emergence, this study illustrates the utility and limitations of meta-metagenomic search tools in effective and rapid characterization of potentially significant nucleic acid sequences.

IMPORTANCE Meta-metagenomic searches allow for high-speed, low-cost identification of potentially significant biological niches for sequences of interest.

There is a pressing need for new exploration tools to target and vector towards mineralization in covered terrains. Groundwater provides a valuable and under-utilized geochemical sampling medium, and represents an important and cost-effective tool to expose covered terrains to systematic exploration. For Au exploration, researchers agree the best hydrogeochemistry pathfinder is dissolved Au itself, with additional potential from other pathfinders (albeit non-unique) such as As, Ag, W and Mo. Despite Au's relatively low solubility, with rigorous field protocols and appropriate analytical methods, explorers can respond to dissolved Au directly with robust parts per trillion (ppt)-level analyses.

Even with ppt-level analyses, a practical implication of Au's low solubility is that a deposit's dissolved Au signature is generally weaker than seen in other more mobile pathfinders, producing a smaller detectable footprint, which must be considered when designing exploration programmes. Using purpose-drilled groundwater sampling bores, explorers can collect groundwater samples at the density required to respond to dissolved Au where existing borehole coverage is otherwise insufficient. In addition to its use at the regional scale, with even tighter sample density, hydrogeochemistry also shows promise at the project scale, allowing the 3D modelling of pathfinder dispersion.

For hydrogeochemistry to be widely adopted for Au exploration, explorers need confidence in ppt-level dissolved Au analyses, and the context to understand their significance. This paper aims to address these topics and provide a straightforward starting point for Au explorers interested in applying hydrogeochemistry by: (i) summarizing examples of regional sampling programmes and more focused case studies to illustrate how covered Au deposits create measurable dissolved Au footprints distinguishable from background; and (ii) sharing examples of dissolved Au analyses that are being integrated into exploration at the regional and project scales.

As seen in the results, the distributions of dissolved Au in the regional- and project-scale programmes show remarkably similar and easy to interpret high-contrast, low-frequency anomalies against relatively low backgrounds. These are desirable attributes of any geochemical pathfinder. When combined with the benefits of hydrogeochemistry v. other geochemical exploration tools (e.g. groundwater can create larger footprints requiring fewer samples to detect, and groundwater can recharge from depth to reflect deeper mineralization), dissolved Au is a powerful pathfinder ideally suited for Au exploration in covered terrains.

While this paper focuses on the use of dissolved Au, additional pathfinders can provide valuable information, including indications of lithological changes, hydrothermal alteration and different styles of mineralization, as well as opportunities to use secondary pathfinders when sample density or local conditions may not result in detectable dissolved Au signatures.

Thematic collection: This article is part of the Exploration 17 collection available at: https://www.lyellcollection.org/cc/exploration-17

Viruses and mobile genetic elements are molecular parasites or symbionts that coevolve with nearly all forms of cellular life. The route of virus replication and protein expression is determined by the viral genome type. Comparison of these routes led to the classification of viruses into seven "Baltimore classes" (BCs) that define the major features of virus reproduction. However, recent phylogenomic studies identified multiple evolutionary connections among viruses within each of the BCs as well as between different classes. Due to the modular organization of virus genomes, these relationships defy simple representation as lines of descent but rather form complex networks. Phylogenetic analyses of virus hallmark genes combined with analyses of gene-sharing networks show that replication modules of five BCs (three classes of RNA viruses and two classes of reverse-transcribing viruses) evolved from a common ancestor that encoded an RNA-directed RNA polymerase or a reverse transcriptase. Bona fide viruses evolved from this ancestor on multiple, independent occasions via the recruitment of distinct cellular proteins as capsid subunits and other structural components of virions. The single-stranded DNA (ssDNA) viruses are a polyphyletic class, with different groups evolving by recombination between rolling-circle-replicating plasmids, which contributed the replication protein, and positive-sense RNA viruses, which contributed the capsid protein. The double-stranded DNA (dsDNA) viruses are distributed among several large monophyletic groups and arose via the combination of distinct structural modules with equally diverse replication modules. Phylogenomic analyses reveal the finer structure of evolutionary connections among RNA viruses and reverse-transcribing viruses, ssDNA viruses, and large subsets of dsDNA viruses. Taken together, these analyses allow us to outline the global organization of the virus world. Here, we describe the key aspects of this organization and propose a comprehensive hierarchical taxonomy of viruses.

Despite the importance of the Bering Sea for subarctic oceanography and climate, relatively little is known of the foraminifera from the extensive Aleutian Basin. We report the occurrence of modern deep-water agglutinated foraminifera collected at seven sites cored during Integrated Ocean Drilling Program (IODP) Expedition 323 in the Bering Sea. Assemblages collected from core-top samples contained 32 genera and 50 species and are described and illustrated here for the first time. Commonly occurring species include typical deep-water Rhizammina, Reophax, Rhabdammina, Recurvoides and Nodulina. Assemblages from the northern sites also consist of accessory Cyclammina, Eggerelloides and Glaphyrammina, whilst those of the Bowers Ridge sites consist of other tubular genera and Martinottiella. Of the studied stations with the lowest dissolved oxygen concentrations, the potentially Bering Sea endemic Eggerelloides sp. 1 inhabits the northern slope, which has the highest primary productivity, and the potentially endemic Martinottiella sp. 3 inhabits Bowers Ridge, which has the lowest oxygen concentrations but relatively low annual productivity. Martinottiella sp. 3, with open pores on its test surface, has previously been reported in Pliocene to Recent material from Bowers Ridge. Despite relatively small sample sizes, ecological constraints may imply that the Bering Sea experienced high productivity and reduced oxygen at times since at least the Pliocene. We note the partially endemic nature of the agglutinated foraminiferal assemblages, which may at least in part be due to basin restriction, the geologically long time period of reduced oxygen, and high organic carbon flux. Our results indicate the importance of gathering further surface sample data from the Aleutian Basin.

To identify genomic alterations contributing to the pathogenesis of high-risk chronic lymphocytic leukemia (CLL) beyond the well-established role of TP53 aberrations, we comprehensively analyzed 75 relapsed/refractory and 71 treatment-naïve high-risk cases from prospective clinical trials by single nucleotide polymorphism arrays and targeted next-generation sequencing. Increased genomic complexity was a hallmark of relapsed/refractory and treatment-naïve high-risk CLL. In relapsed/refractory cases previously exposed to the selective pressure of chemo(immuno)therapy, gain(8)(q24.21) and del(9)(p21.3) were particularly enriched. Both alterations affect key regulators of cell-cycle progression, namely MYC and CDKN2A/B. While homozygous CDKN2A/B loss has been directly associated with Richter transformation, we did not find this association for heterozygous loss of CDKN2A/B. Gains in 8q24.21 were either focal gains in a MYC enhancer region or large gains affecting the MYC locus, but only the latter type was highly enriched in relapsed/refractory CLL (17%). In addition to a high frequency of NOTCH1 mutations (23%), we found recurrent genetic alterations in SPEN (4% mutated), RBPJ (8% deleted) and SNW1 (8% deleted), all affecting a protein complex that represses transcription of NOTCH1 target genes. We investigated the functional impact of these alterations on HES1, DTX1 and MYC gene transcription and found derepression of these NOTCH1 target genes particularly with SPEN mutations. In summary, we provide new insights into the genomic architecture of high-risk CLL, define novel recurrent DNA copy number alterations and refine knowledge on del(9p), gain(8q) and alterations affecting NOTCH1 signaling. This study was registered at ClinicalTrials.gov with number NCT01392079.

The Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study demonstrated that intensive glucose control reduced the risk of developing diabetic peripheral neuropathy (DPN) and cardiovascular autonomic neuropathy (CAN). We evaluated multiple risk factors and phenotypes associated with DPN and CAN in this large, well-characterized cohort of participants with type 1 diabetes, followed for >23 years. DPN was defined by symptoms, signs, and nerve conduction study abnormalities in ≥2 nerves; CAN was assessed using standardized cardiovascular reflex tests. Generalized estimating equation models assessed the association of DPN and CAN with individual risk factors measured repeatedly. During DCCT/EDIC, 33% of participants developed DPN and 44% CAN. Higher mean HbA_1c was the most significant risk factor for DPN, followed by older age, longer duration, greater height, macroalbuminuria, higher mean pulse rate, β-blocker use, and sustained albuminuria. The most significant risk factor for CAN was older age, followed by higher mean HbA_1c, sustained albuminuria, longer duration of type 1 diabetes, higher mean pulse rate, higher mean systolic blood pressure, β-blocker use, estimated glomerular filtration rate <60 mL/min/1.73 m², higher most recent pulse rate, and cigarette smoking. These findings identify risk factors and phenotypes of participants with diabetic neuropathy that can be used in the design of new interventional trials and for personalized approaches to neuropathy prevention.

Congenital anomalies of the kidneys and urinary tracts (CAKUT) are disorders caused by defects in the development of the kidneys and their outflow tracts. The formation of the kidneys begins at week 3 and nephrogenesis continues until week 36, therefore, the kidneys and outflow tracts are susceptible to environmental risk factors that perturb development throughout gestation. Many genes have been implicated in kidney and outflow tract development, and mutations have been identified in patients with CAKUT. In severe cases of CAKUT, when the kidneys do not form, the fetus will not survive. However, in less severe cases, the baby can survive with combined kidney and outflow tract defects or they may only be identified in adulthood. In this review, we will cover the clinical presentation of CAKUT, its epidemiology, and its long-term outcomes. We will then discuss risk factors for CAKUT, including genetic and environmental contributions. Although severe CAKUT is rare, low nephron number is a much more common disorder with its effect on kidney function increasingly apparent as a person ages. Low nephron number appears to arise by the same mechanisms as CAKUT, but it differs in terms of the magnitude of the insult and the timing of when it occurs during gestation. By understanding the causes of CAKUT and low nephron number, we can begin to identify preventive treatments and establish clinical guidelines for how these patients should be followed.

Merkel cell carcinoma (MCC) is often caused by persistent expression of Merkel cell polyomavirus (MCPyV) T-antigen (T-Ag). These non-self proteins comprise about 400 amino acids (AA). Clinical responses to immune checkpoint inhibitors, seen in about half of patients, may relate to T-Ag–specific T cells. Strategies to increase CD8⁺ T-cell number, breadth, or function could augment checkpoint inhibition, but vaccines to augment immunity must avoid delivery of oncogenic T-antigen domains. We probed MCC tumor-infiltrating lymphocytes (TIL) with an artificial antigen-presenting cell (aAPC) system and confirmed T-Ag recognition with synthetic peptides, HLA-peptide tetramers, and dendritic cells (DC). TILs from 9 of 12 (75%) subjects contained CD8⁺ T cells recognizing 1–8 MCPyV epitopes per person. Analysis of 16 MCPyV CD8⁺ TIL epitopes and prior TIL data indicated that 97% of patients with MCPyV⁺ MCC had HLA alleles with the genetic potential that restrict CD8⁺ T-cell responses to MCPyV T-Ag. The LT AA 70–110 region was epitope rich, whereas the oncogenic domains of T-Ag were not commonly recognized. Specific recognition of T-Ag–expressing DCs was documented. Recovery of MCPyV oncoprotein–specific CD8⁺ TILs from most tumors indicated that antigen indifference was unlikely to be a major cause of checkpoint inhibition failure. The myriad of epitopes restricted by diverse HLA alleles indicates that vaccination can be a rational component of immunotherapy if tumor immune suppression can be overcome, and the oncogenic regions of T-Ag can be modified without impacting immunogenicity.

Vaccinia virus (VACV) is a double-stranded DNA virus that devotes a large portion of its 200 kbp genome to suppressing and manipulating the immune response of its host. Here, we investigated how targeted removal of immunomodulatory genes from the VACV genome impacted immune cells in the tumor microenvironment with the intention of improving the therapeutic efficacy of VACV in breast cancer. We performed a head-to-head comparison of six mutant oncolytic VACVs, each harboring deletions in genes that modulate different cellular pathways, such as nucleotide metabolism, apoptosis, inflammation, and chemokine and interferon signaling. We found that even minor changes to the VACV genome can impact the immune cell compartment in the tumor microenvironment. Viral genome modifications had the capacity to alter lymphocytic and myeloid cell compositions in tumors and spleens, PD-1 expression, and the percentages of virus-targeted and tumor-targeted CD8⁺ T cells. We observed that while some gene deletions improved responses in the nonimmunogenic 4T1 tumor model, very little therapeutic improvement was seen in the immunogenic HER2/neu TuBo model with the various genome modifications. We observed that the most promising candidate genes for deletion were those that interfere with interferon signaling. Collectively, this research helped focus attention on the pathways that modulate the immune response in the context of VACV oncolytic virotherapy. They also suggest that the greatest benefits to be obtained with these treatments may not always be seen in "hot tumors."

The Stenotrophomonas maltophilia complex (Smc) comprises opportunistic environmental Gram-negative bacilli responsible for a variety of infections in both humans and animals. Beyond its large genetic diversity, its genetic organization in genogroups was recently confirmed through the whole-genome sequencing of human and environmental strains. As they are poorly represented in these analyses, we sequenced the whole genomes of 93 animal strains to determine their genetic background and characteristics. Combining these data with 81 newly sequenced human strains and the genomes available from RefSeq, we performed a genomic analysis that included 375 nonduplicated genomes with various origins (animal, 104; human, 226; environment, 30; unknown, 15). Phylogenetic analysis and clustering based on genome-wide average nucleotide identity confirmed and specified the genetic organization of Smc in at least 20 genogroups. Two new genogroups were identified, and two previously described groups were further divided into two subgroups each. Comparing the strains isolated from different host types and their genogroup affiliation, we observed a clear disequilibrium in certain groups. Surprisingly, some antimicrobial resistance genes, integrons, and/or clusters of attC sites lacking integron-integrase (CALIN) sequences targeting antimicrobial compounds extensively used in animals were mainly identified in animal strains. We also identified genes commonly found in animal strains coding for efflux systems. The result of a large whole-genome analysis performed by us supports the hypothesis of the putative contribution of animals as a reservoir of Stenotrophomonas maltophilia complex strains and/or resistance genes for strains in humans.

IMPORTANCE Given its naturally large antimicrobial resistance profile, the Stenotrophomonas maltophilia complex (Smc) is a set of emerging pathogens of immunosuppressed and cystic fibrosis patients. As it is group of environmental microorganisms, this adaptation to humans is an opportunity to understand the genetic and metabolic selective mechanisms involved in this process. The previously reported genomic organization was incomplete, as data from animal strains were underrepresented. We added the missing piece of the puzzle with whole-genome sequencing of 93 strains of animal origin. Beyond describing the phylogenetic organization, we confirmed the genetic diversity of the Smc, which could not be estimated through routine phenotype- or matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF)-based laboratory tests. Animals strains seem to play a key role in the diversity of Smc and could act as a reservoir for mobile resistance genes. Some genogroups seem to be associated with particular hosts; the genetic support of this association and the role of the determinants/corresponding genes need to be explored.

Biofilm formation by Pseudomonas aeruginosa contributes to its survival on surfaces and represents a major clinical threat because of the increased tolerance of biofilms to disinfecting agents. This study aimed to investigate the efficacy of 405-nm light-emitting diode (LED) illumination in eliminating P. aeruginosa biofilms formed on stainless steel coupons under different temperatures. Time-dependent killing assays using planktonic and biofilm cells were used to determine the antimicrobial and antibiofilm activities of LED illumination. We also evaluated the effects of LED illumination on the disinfectant susceptibility, biofilm structure, extracellular polymeric substance (EPS) structure and composition, and biofilm-related gene expression of P. aeruginosa biofilm cells. Results showed that the abundance of planktonic P. aeruginosa cells was reduced by 0.88, 0.53, and 0.85 log CFU/ml following LED treatment for 2 h compared with untreated controls at 4, 10, and 25°C, respectively. For cells in biofilms, significant reductions (1.73, 1.59, and 1.68 log CFU/cm²) were observed following LED illumination for 2 h at 4, 10, and 25°C, respectively. Moreover, illuminated P. aeruginosa biofilm cells were more sensitive to benzalkonium chloride or chlorhexidine than untreated cells. Scanning electron microscopy and confocal laser scanning microscopic observation indicated that both the biofilm structure and EPS structure were disrupted by LED illumination. Further, reverse transcription-quantitative PCR revealed that LED illumination downregulated the transcription of several genes associated with biofilm formation. These findings suggest that LED illumination has the potential to be developed as an alternative method for prevention and control of P. aeruginosa biofilm contamination.

IMPORTANCE Pseudomonas aeruginosa can form biofilms on medical implants, industrial equipment, and domestic surfaces, contributing to high morbidity and mortality rates. This study examined the antibiofilm activity of 405-nm light-emitting diode (LED) illumination against mature biofilms formed on stainless steel coupons. We found that the disinfectant susceptibility, biofilm structure, and extracellular polymeric substance structure and composition were disrupted by LED illumination. We then investigated the transcription of several critical P. aeruginosa biofilm-related genes and analyzed the effect of illumination temperature on the above characteristics. Our results confirmed that LED illumination could be developed into an effective and safe method to counter P. aeruginosa biofilm contamination. Further research will be focused on the efficacy and application of LED illumination for elimination of complicated biofilms in the environment.

Purpose:

Unleashing the immune system by PD-1 and/or CTLA-4 blockade can cause severe immune-related toxicity necessitating immunosuppressive treatment. Whether immunosuppression for toxicity impacts survival is largely unknown.

Purpose:

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer, which can be effectively controlled by immunotherapy with PD-1/PD-L1 checkpoint inhibitors. However, a significant proportion of patients are characterized by primary therapy resistance. Predictive biomarkers for response to immunotherapy are lacking.

Purpose:

To provide a better understanding of the interplay between the immune system and brain metastases to advance therapeutic options for this life-threatening disease.

Purpose:

The development of leptomeningeal melanoma metastases (LMM) is a rare and devastating complication of the late-stage disease, for which no effective treatments exist. Here, we performed a multi-omics analysis of the cerebrospinal fluid (CSF) from patients with LMM to determine how the leptomeningeal microenvironment shapes the biology and therapeutic responses of melanoma cells.

In this retrospective study, whole-genome sequencing (WGS) data generated on an Ion Torrent platform was used to predict phenotypic drug resistance profiles for first- and second-line drugs among Swedish clinical Mycobacterium tuberculosis isolates from 2016 to 2018. The accuracy was ~99% for all first-line drugs and 100% for four second-line drugs. Our analysis supports the introduction of WGS into routine diagnostics, which might, at least in Sweden, replace phenotypic drug susceptibility testing in the future.