Rho-type GTPases are key regulators that control eukaryotic cell polarity, but their role in fungal morphogenesis is only beginning to emerge. In this study, we investigate the role of the CDC-42 - RAC - CDC-24 module in Neurospora crassa. rac and cdc-42 deletion mutants are viable, but generate highly compact colonies with severe morphological defects. Double mutants carrying conditional and loss of function alleles of rac and cdc-42 are lethal, indicating that both GTPases share at least one common essential function. The defects of the GTPase mutants are phenocopied by deletion and conditional alleles of the guanine exchange factor (GEF) cdc-24, and in vitro GDP-GTP exchange assays identify CDC-24 as specific GEF for both CDC-42 and RAC. In vivo confocal microscopy shows that this module is organized as membrane-associated cap that covers the hyphal apex. However, the specific localization patterns of the three proteins are distinct, indicating different functions of RAC and CDC-42 within the hyphal tip. CDC-42 localized as confined apical membrane-associated crescent, while RAC labeled a membrane-associated ring excluding the region labeled by CDC42. The GEF CDC-24 occupied a strategic position, localizing as broad apical membrane-associated crescent and in the apical cytosol excluding the Spitzenkörper. RAC and CDC-42 also display distinct localization patterns during branch initiation and germ tube formation, with CDC-42 accumulating at the plasma membrane before RAC. Together with the distinct cellular defects of rac and cdc-42 mutants, these localizations suggest that CDC-42 is more important for polarity establishment, while the primary function of RAC may be maintaining polarity. In summary, this study identifies CDC-24 as essential regulator for RAC and CDC-42 that have common and distinct functions during polarity establishment and maintenance of cell polarity in N. crassa.

The spleen contains a myriad of conventional dendritic cell (cDC) subsets that protect against systemic pathogen dissemination by bridging antigen detection to the induction of adaptive immunity. How cDC subsets differentiate in the splenic environment is poorly understood. Here, we report that LTα1β2-expressing Rorgt+ ILC3s, together with B cells, control the splenic cDC niche size and the terminal differentiation of Sirpα+CD4+Esam+ cDC2s, independently of the microbiota and of bone marrow pre-cDC output. Whereas the size of the splenic cDC niche depended on lymphotoxin signaling only during a restricted time frame, the homeostasis of Sirpα+CD4+Esam+ cDC2s required continuous lymphotoxin input. This latter property made Sirpα+CD4+Esam+ cDC2s uniquely susceptible to pharmacological interventions with LTβR agonists and antagonists and to ILC reconstitution strategies. Together, our findings demonstrate that LTα1β2-expressing Rorgt+ ILC3s drive splenic cDC differentiation and highlight the critical role of ILC3s as perpetual regulators of lymphoid tissue homeostasis.

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.

We report the draft genome sequence of Mortierella alpina isolate CDC-B6842. M. alpina is a nonpathogenic member of the Mucoromycotina subphylum of fungi that is an important model for understanding the molecular mechanisms of lipid production and metabolism.

Segniliparus rotundus Butler 2005 is the type species of the genus Segniliparus, which is currently the only genus in the corynebacterial family Segniliparaceae. This family is of large interest because of a novel late-emerging genus-specific mycolate pattern. The type strain has been isolated from human sputum and is probably an opportunistic pathogen. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of the family Segniliparaceae. The 3,157,527 bp long genome with its 3,081 protein-coding and 52 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Dendritic cell (DC) specification and differentiation are controlled by a circuit of transcription factors, which regulate the expression of DC effector genes as well as the transcription factors themselves. E proteins are a widely expressed basic helix-loop-helix family of transcription factors whose activity is suppressed by their inhibitors, ID proteins. Loss-of-function studies have demonstrated the essential role of both E and ID proteins in different aspects of DC development. In this study, we employed a gain-of-function approach to illustrate the importance of the temporal control of E protein function in maintaining balanced differentiation of conventional DC (cDC) subsets, cDC1 and cDC2. We expressed an E protein mutant, ET2, which dimerizes with endogenous E proteins to overcome inhibition by ID proteins and activate the transcription of E protein targets. Induction of ET2 expression at the hematopoietic progenitor stage led to a dramatic reduction in cDC2 precursors (pre-cDC2s) with little impact on pre-cDC1s. Consequently, we observed decreased numbers of cDC2s in the spleen and lung, as well as in FLT3L-driven bone marrow-derived DC cultures. Furthermore, in mice bearing ET2, we detected increased expression of the IRF8 transcription factor in cDC2s, in which IRF8 is normally down-regulated and IRF4 up-regulated. This aberrant expression of IRF8 induced by ET2 may contribute to the impairment of cDC2 differentiation. In addition, analyses of the transcriptomes of splenic cDC1s and cDC2s revealed that ET2 expression led to a shift, at least in part, of the transcriptional profile characteristic of cDC2s to that of cDC1. Together, these results suggest that a precise control of E protein activity is crucial for balanced DC differentiation.

Successful nuclear migration through constricted spaces between cells or in the extracellular matrix relies on the ability of the nucleus to deform. Little is known about how this takes place in vivo. We have studied confined nuclear migration in Caenorhabditis elegans larval P cells, which is mediated by the LINC complex to pull nuclei towards the minus ends of microtubules. Null mutations of the LINC component unc-84 lead to a temperature-dependent phenotype, suggesting a parallel pathway for P-cell nuclear migration. A forward genetic screen for enhancers of unc-84 identified cgef-1 (CDC-42 guanine nucleotide exchange factor). Knockdown of CDC-42 in the absence of the LINC complex led to a P-cell nuclear migration defect. Expression of constitutively active CDC-42 partially rescued nuclear migration in cgef-1; unc-84 double mutants, suggesting that CDC-42 functions downstream of CGEF-1. The Arp2/3 complex and non-muscle myosin II (NMY-2) were also found to function parallel to the LINC pathway. In our model, CGEF-1 activates CDC-42, which induces actin polymerization through the Arp2/3 complex to deform the nucleus during nuclear migration, and NMY-2 helps to push the nucleus through confined spaces.

Caenorhabditis elegans possesses two p97/VCP/Cdc48p homologues, named CDC-48.1 (C06A1.1) and CDC-48.2 (C41C4.8), and their expression patterns and levels are differently regulated. To clarify the regulatory mechanisms of differential expression of two p97 proteins of C. elegans, we performed detailed deletion analysis of their promoter regions. We found that the promoter of cdc-48.1 contains two regions necessary for embryonic and for post-embryonic expression, while the promoter of cdc-48.2 contains the single region necessary for embryonic expression. In particular, two elements (Element A and Element B) and three conserved boxes (Box a, Box b and Box c) were essential for cdc-48.1 expression in embryos and at post-embryonic stages, respectively. By using South-Western blotting and MALDI-TOF MS analysis, we identified HMG-12 and CAR-1 as proteins that bind to Element A and Element B, respectively, from the embryonic nuclear extract. Importantly, we found the decreased expression of p97 in embryos prepared from hmg-12(RNAi) or car-1(RNAi) worms. These results indicate that both HMG-12 and CAR-1 play important roles in embryonic expression of cdc-48.1.

Agonistic αCD40 therapy has been shown to inhibit cancer progression in only a fraction of patients. Understanding the cancer cell-intrinsic and microenvironmental determinants of αCD40 therapy response is therefore crucial to identify responsive patient populations and to design efficient combinatorial treatments. Here, we show that the therapeutic efficacy of αCD40 in subcutaneous melanoma relies on preexisting, type 1 classical dendritic cell (cDC1)-primed CD8+ T cells. However, after administration of αCD40, cDC1s were dispensable for antitumor efficacy. Instead, the abundance of activated cDCs, potentially derived from cDC2 cells, increased and further activated antitumor CD8+ T cells. Hence, distinct cDC subsets contributed to the induction of αCD40 responses. In contrast, lung carcinomas, characterized by a high abundance of macrophages, were resistant to αCD40 therapy. Combining αCD40 therapy with macrophage depletion led to tumor growth inhibition only in the presence of strong neoantigens. Accordingly, treatment with immunogenic cell death-inducing chemotherapy sensitized lung tumors to αCD40 therapy in subcutaneous and orthotopic settings. These insights into the microenvironmental regulators of response to αCD40 suggest that different tumor types would benefit from different combinations of therapies to optimize the clinical application of CD40 agonists.

Dendritic cells (DCs) play critical roles in regulating the innate and adaptive immune responses, and have long been a major focus of cancer immunotherapy. Accumulating evidence suggests that conventional type 1 DCs (cDC1s) excel in cross-presentation of exogenous antigens on MHC-I molecules and induction of antitumor CD8+ T cell immunity; however, obtaining large numbers of cDC1s is difficult. The use of reprogramming and differentiation technology is advantageous for obtaining unlimited numbers of autologous cDC1s especially for therapeutic interventions where repeated vaccinations are required. However, generation of cDC1s from human induced pluripotent stem cells (iPSCs) remains elusive.

Thirty Clostridioides difficile isolates collected in 2016 through the Centers for Disease Control and Prevention Emerging Infections Program were selected for reference antimicrobial susceptibility testing and whole-genome sequencing. Here, we present the genetic characteristics of these isolates and announce their availability in the CDC & FDA Antibiotic Resistance Isolate Bank.

Kinase-catalyzed phosphorylation plays a crucial role in pathological cardiac hypertrophy. Here, we show that CDC-like kinase 4 (CLK4) is a critical regulator of cardiomyocyte hypertrophy and heart failure. Knockdown of Clk4 leads to pathological cardiomyocyte hypertrophy, while overexpression of Clk4 confers resistance to phenylephrine-induced cardiomyocyte hypertrophy. Cardiac-specific Clk4-knockout mice manifest pathological myocardial hypertrophy with progressive left ventricular systolic dysfunction and heart dilation. Further investigation identifies nexilin (NEXN) as the direct substrate of CLK4, and overexpression of a phosphorylation-mimic mutant of NEXN is sufficient to reverse the hypertrophic growth of cardiomyocytes induced by Clk4 knockdown. Importantly, restoring phosphorylation of NEXN ameliorates myocardial hypertrophy in mice with cardiac-specific Clk4 deletion. We conclude that CLK4 regulates cardiac function through phosphorylation of NEXN, and its deficiency may lead to pathological cardiac hypertrophy. CLK4 is a potential intervention target for the prevention and treatment of heart failure.

In Caenorhabditis elegans hermaphrodites, physiological germline apoptosis is higher in cdc-25.3 mutants than in wild-type. The elevated germline apoptosis in cdc-25.3 mutants seems to be induced by accumulation of double-stranded DNA breaks (DSBs). Both DNA damage and synapsis checkpoint genes are required to increase the germline apoptosis. Notably, the number of germ cells that lose P-granule components, PGL-1 and PGL-3, increase in cdc-25.3 mutants, and the increase in germline apoptosis requires the activity of SIR-2.1, a Sirtuin orthologue. These results suggest that elevation of germline apoptosis in cdc-25.3 mutants is induced by accumulation of DSBs, leading to a loss of PGL-1 and PGL-3 in germ cells, which promotes cytoplasmic translocation of SIR-2.1, and finally activates the core apoptotic machinery.

LIN-12/Notch signaling is important for cell-cell interactions during development, and mutations resulting in constitutive LIN-12/Notch signaling can cause cancer. Loss of negative regulators of lin-12/Notch activity has the potential for influencing cell fate decisions during development and the genesis or aggressiveness of cancer.

Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL) has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC) due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH). We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested ex vivo in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.

Vermamoeba vermiformis is a predominant free-living amoeba in human environments and amongst the most common amoebae that can cause severe infections in humans. It is a niche for numerous amoeba-resisting microorganisms such as bacteria and giant viruses. Differences in the susceptibility to these giant viruses have been observed. V. vermiformis and amoeba-resisting microorganisms share a sympatric lifestyle that can promote exchanges of genetic material. This work analyzed the first draft genome sequence of a V. vermiformis strain (CDC-19) through comparative genomic, transcriptomic and phylogenetic analyses. The genome of V. vermiformis is 59.5 megabase pairs in size, and 22,483 genes were predicted. A high proportion (10% (n = 2,295)) of putative genes encoded proteins showed the highest sequence homology with a bacterial sequence. The expression of these genes was demonstrated for some bacterial homologous genes. In addition, for 30 genes, we detected best BLAST hits with members of the Candidate Phyla Radiation. Moreover, 185 genes (0.8%) best matched with giant viruses, mostly those related to the subfamily Klosneuvirinae (101 genes), in particular Bodo saltans virus (69 genes). Lateral sequence transfers between V. vermiformis and amoeba-resisting microorganisms were strengthened by Sanger sequencing, transcriptomic and phylogenetic analyses. This work provides important insights and genetic data for further studies about this amoeba and its interactions with microorganisms.

Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV). There are four genetically distinct DENVs (DENV-1-4) that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1-4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA) for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1-4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1-4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases.

Early-life immune development is critical to long-term host health. However, the mechanisms that determine the pace of postnatal immune maturation are not fully resolved. Here, we analyzed mononuclear phagocytes (MNPs) in small intestinal Peyer's patches (PPs), the primary inductive site of intestinal immunity. Conventional type 1 and 2 dendritic cells (cDC1 and cDC2) and RORgt+ antigen-presenting cells (RORgt+ APC) exhibited significant age-dependent changes in subset composition, tissue distribution, and reduced cell maturation, subsequently resulting in a lack in CD4+ T cell priming during the postnatal period. Microbial cues contributed but could not fully explain the discrepancies in MNP maturation. Type I interferon (IFN) accelerated MNP maturation but IFN signaling did not represent the physiological stimulus. Instead, follicle-associated epithelium (FAE) M cell differentiation was required and sufficient to drive postweaning PP MNP maturation. Together, our results highlight the role of FAE M cell differentiation and MNP maturation in postnatal immune development.

Pythium insidiosum ATCC 200269 strain CDC-B5653, an isolate from necrotizing lesions on the mouth and eye of a 2-year-old boy in Memphis, Tennessee, USA, was sequenced using a combination of Illumina MiSeq (300 bp paired-end, 14 millions reads) and PacBio (10  Kb fragment library, 356,001 reads). The sequencing data were assembled using SPAdes version 3.1.0, yielding a total genome size of 45.6 Mb contained in 8992 contigs, N50 of 13 Kb, 57% G + C content, and 17,867 putative protein-coding genes. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JRHR00000000.

Transcriptional regulation of ribosome and tRNA synthesis plays a central role in determining protein synthetic capacity and is tightly controlled in response to nutrient availability and cellular stress. In Saccharomyces cerevisiae, the regulation of ribosome and tRNA synthesis was recently shown to involve the Cdc-like kinase Kns1 and the GSK-3 kinase Mck1. In this study, we explored additional roles for these conserved kinases in processes connected to the target of rapamycin complex 1 (TORC1). We conducted a synthetic chemical-genetic screen in a kns1Δ mck1Δ strain and identified many novel rapamycin-hypersensitive genes. Gene ontology analysis showed enrichment for TORC1-regulated processes (vesicle-mediated transport, autophagy, and regulation of cell size) and identified new connections to protein complexes including the protein kinase CK2. CK2 is considered to be a constitutively active kinase and in budding yeast, the holoenzyme comprises two regulatory subunits, Ckb1 and Ckb2, and two catalytic subunits, Cka1 and Cka2. We show that Ckb1 is differentially phosphorylated in vivo and that Kns1 mediates this phosphorylation when nutrients are limiting and under all tested stress conditions. We determined that the phosphorylation of Ckb1 does not detectably affect the stability of the CK2 holoenzyme but correlates with the reduced occupancy of Ckb1 on tRNA genes after rapamycin treatment. Thus, the differential occupancy of tRNA genes by CK2 is likely to modulate its activation of RNA polymerase III transcription. Our data suggest that TORC1, via its effector kinase Kns1, may regulate the association of CK2 with some of its substrates by phosphorylating Ckb1.