G proteins and their cognate G protein-coupled receptors (GPCRs) function as critical signal transduction molecules that regulate cell survival, proliferation, motility and differentiation. The aberrant expression and/or function of these molecules have been linked to the growth, progression and metastasis of various cancers. As such, the analysis of mutations in the genes encoding GPCRs, G proteins and their downstream targets provides important clues regarding how these signaling cascades contribute to malignancy. Recent genome-wide sequencing efforts have unveiled the presence of frequent mutations in GNA13, the gene encoding the G protein Gα13, in Burkitt's lymphoma and diffuse large B-cell lymphoma (DLBCL). We found that mutations in the downstream target of Gα13, RhoA, are also present in Burkitt's lymphoma and DLBCL. By multiple complementary approaches, we now show that that these cancer-specific GNA13 and RHOA mutations are inhibitory in nature, and that the expression of wild-type Gα13 in B-cell lymphoma cells with mutant GNA13 has limited impact in vitro but results in a remarkable growth inhibition in vivo. Thus, although Gα13 and RhoA activity has previously been linked to cellular transformation and metastatic potential of epithelial cancers, our findings support a tumor suppressive role for Gα13 and RhoA in Burkitt's lymphoma and DLBCL.

The regenerative capacity of the skin, including the continuous replacement of exfoliated cells and healing of injuries relies on the epidermal stem cells and their immediate cell descendants. The relative contribution of the hair follicle stem cells and the interfollicular stem cells to dermal wound healing is an area of active investigation. Recent studies have revealed that the small GTPase Rac1, which regulates cell migration and nuclear gene expression, is required for hair follicle stem function but not for the normal homeostasis of the interfollicular skin.

The molecular mechanisms by which signaling via transforming growth factor-β (TGF-β) and interleukin 4 (IL-4) control the differentiation of CD4(+) IL-9-producing helper T cells (TH9 cells) remain incompletely understood. We found here that the DNA-binding inhibitor Id3 regulated TH9 differentiation, as deletion of Id3 increased IL-9 production from CD4(+) T cells. Mechanistically, TGF-β1 and IL-4 downregulated Id3 expression, and this process required the kinase TAK1. A reduction in Id3 expression enhanced binding of the transcription factors E2A and GATA-3 to the Il9 promoter region, which promoted Il9 transcription. Notably, Id3-mediated control of TH9 differentiation regulated anti-tumor immunity in an experimental melanoma-bearing model in vivo and also in human CD4(+) T cells in vitro. Thus, our study reveals a previously unrecognized TAK1-Id3-E2A-GATA-3 pathway that regulates TH9 differentiation.

Varicella poses an occupational risk and a nosocomial risk for susceptible healthcare personnel and patients, respectively. Patients with varicella are thought to be infectious from 1 to 2 days before rash onset until all lesions are crusted, typically 4-7 days after onset of rash. We searched Medline, Embase, Cochrane Library and CINAHL databases to assess evidence of varicella-zoster virus (VZV) transmission before varicella rash onset. Few articles (7) contributed epidemiologic evidence; no formal studies were found. Published articles reported infectiousness at variable intervals before rash onset, between <1 day to 4 days prior to rash, with 1-2 patients for each interval. Laboratory assessment of transmission before rash was also limited (10 articles). No culture-positive results were reported. VZV DNA was identified by PCR before rash onset in only one study however, PCR does not indicate infectivity of the virus. Based on available medical literature, VZV transmission before rash onset seems unlikely, although the possibility of pre-rash, respiratory transmission cannot be entirely ruled out.

Leukocyte Adhesion Deficiency I (LAD-I) is a primary immunodeficiency caused by single gene mutations in the CD18 subunit of β2 integrins which result in defective transmigration of neutrophils into the tissues. Affected patients suffer from recurrent life threatening infections and severe oral disease (periodontitis). Microbial communities in the local environment (subgingival plaque) are thought to be the triggers for inflammatory periodontitis, yet little is known regarding the microbial communities associated with LAD-I periodontitis. Here we present the first comprehensive characterization of the subgingival communities in LAD-I, using a 16S rRNA gene-based microarray, and investigate the relationship of this tooth adherent microbiome to the local immunopathology of periodontitis. We show that the LAD subgingival microbiome is distinct from that of health and Localized Aggressive Periodontitits. Select periodontitis-associated species in the LAD microbiome included Parvimonas micra, Porphyromonas endodontalis, Eubacterium brachy and Treponema species. Pseudomonas aeruginosa, a bacterium not typically found in subgingival plaque is detected in LAD-I. We suggest that microbial products from LAD-associated communities may have a role in stimulating the local inflammatory response. We demonstrate that bacterial LPS translocates into the lesions of LAD-periodontitis potentially triggering immunopathology. We also show in in vitro assays with human macrophages and in vivo in animal models that microbial products from LAD-associated subgingival plaque trigger IL-23-related immune responses, which have been shown to dominate in patient lesions. In conclusion, our current study characterizes the subgingival microbial communities in LAD-periodontitis and supports their role as triggers of disease pathogenesis.

The eukaryotic translation factor, eIF5A, is a translation factor essential for protein synthesis, cell growth and animal development. By use of a adenoviral eIF5A shRNA, we have achieved an effective depletion of eIF5A in HeLa cells and undertook in vivo comprehensive proteomic analyses to examine the effects of eIF5A depletion on the total proteome and to identify cellular pathways influenced by eIF5A. The proteome of HeLa cells transduced with eIF5A shRNA was compared with that of scramble shRNA-transduced counterpart by the iTRAQ method. We identified 972 proteins consistently detected in three iTRAQ experiments and 104 proteins with significantly altered levels (protein ratio ≥1.5 or ≤0.66, p-value ≤0.05) at 72 h and/or 96 h of Ad-eIF5A-shRNA transduction. The altered expression levels of key pathway proteins were validated by western blotting. Integration of functional ontology with expression data of the 104 proteins revealed specific biological processes that are prominently up- or down-regulated. Heatmap analysis and Cytoscape visualization of biological networks identified protein folding as the major cellular process affected by depletion of eIF5A. Our unbiased, quantitative, proteomic data demonstrate that the depletion of eIF5A leads to endoplasmic reticulum stress, an unfolded protein response and up-regulation of chaperone expression in HeLa cells.

Colitis-associated colorectal cancers are an etiologically distinct subgroup of colon cancers that occur in individuals suffering from inflammatory bowel disease and arise as a consequence of persistent exposure of hyperproliferative epithelial stem cells to an inflammatory microenvironment. An intrinsic defect in the intestinal epithelial barrier has been proposed to be one of several factors that contribute to the inappropriate immune response to the commensal microbiota that underlies inflammatory bowel disease. Matriptase is a membrane-anchored serine protease encoded by Suppression of Tumorigenicity-14 (ST14) that strengthens the intestinal epithelial barrier by promoting tight junction formation. Here, we show that intestinal epithelial-specific ablation of St14 in mice causes formation of colon adenocarcinoma with very early onset and high penetrance. Neoplastic progression is preceded by a chronic inflammation of the colon that resembles human inflammatory bowel disease and is promoted by the commensal microbiota. This study demonstrates that inflammation-associated colon carcinogenesis can be initiated and promoted solely by an intrinsic intestinal permeability barrier perturbation, establishes St14 as a critical tumor-suppressor gene in the mouse gastrointestinal tract and adds matriptase to the expanding list of pericellular proteases with tumor-suppressive functions.

Syphilis is a complex, multistage, sexually transmitted infection (STI) caused by the bacterium Treponema pallidum subspecies pallidum (TP). New diagnostic tools are needed to minimize transmission. In this study, we aimed to assess the additional value of an investigational transcription-mediated amplification test for TP (TP-TMA) for routine diagnostics.

Orolabial lymphogranuloma venereum was diagnosed for a man in Michigan, USA, who had sex with men, some infected with HIV. High index of suspicion for lymphogranuloma venereum led to accurate diagnosis, successful therapy, and description of an L2b variant with a unique genetic mutation.

The management of slow or non-healing ulcerations constitutes an increasing clinical challenge in the developed world because of the ageing of the population and the pandemic rise in type II diabetes. Recent studies suggest that molecular circuitries deployed by tumor cells to promote cancerous growth may also contribute to tissue regeneration. Here, we exploited this emerging information to search for novel molecular targets to accelerate wound healing.

Background: The diagnosis of SARS-CoV-2 infection relies on RT-PCR from nasopharyngeal swabs. The pre-analytical value of different methods of material harvesting for SARS-CoV-2 are unknown. Methods: We conducted a comprehensive investigation of the pre-analytical performance for different pharyngeal sampling procedures in hospitalized patients with confirmed SARS-CoV-2 infection. In addition to swabs taken simultaneously from different locations, saliva and pharyngeal lavages were also analyzed using RT-PCR. Results: In 10 COVID-19 patients, standard nasopharyngeal swabs detected 8 out of 10 positive patients, whereas swabs taken from the palatoglossal arch resulted in 9 correct-positive results. Brushing the posterior pharynx wall with swabs resulted in detection of 9 out of 10 positive patients with no difference using either dry swabs or liquid Amies medium. A strong correlation between Ct values of both swab materials was observed. Pharyngeal lavages yielded 6 out of 10 positive results in concordance with 85% of nasopharyngeal swabs in late-stage COVID-19 patients. Investigating 23 patients with early SARS-CoV-2 infection, pharyngeal lavages showed a concordance rate of 100% compared to nasopharyngeal swabs. Conclusions: The diagnostic performance of swabs taken from the palatoglossal arch in detecting SARS-CoV-2 infection is similar to that of specimens taken from the nasopharyngeal region. However, the former sampling method is associated with less discomfort and much easier to perform. Pharyngeal lavages may replace swabs for mass screening in early stages of SARS-CoV-2 infection. The predictive values are comparable, and the procedure is performed without exposing healthcare workers to transmission risks.

Zenker carcinoma is still being treated empirically because of the lack of evidence- based guidelines. We report for the first time about the genetic examination of this rare entity. The revealed mutations show genetic similarities with HPV(-)HNSCC which suggests that well-known therapeutic strategies may be applicable for this disease.

Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (CT) can facilitate transmission of HIV. Men who have sex with men (MSM) may harbour infections at genital and extragenital sites. Data regarding extragenital GC and CT infections in military populations are lacking. We examined the prevalence and factors associated with asymptomatic GC and CT infection among this category of HIV-infected military personnel.

Anthrax lethal toxin (LT) is an A-B type toxin secreted by Bacillus anthracis, consisting of the cellular binding moiety, protective antigen (PA), and the catalytic moiety, lethal factor (LF). To target cells, PA binds to cell-surface receptors and is then proteolytically processed forming a LF-binding competent PA oligomer where each LF binding site is comprised of three subsites on two adjacent PA monomers. We previously generated PA-U2-R200A, a urokinase-activated PA variant with LF-binding subsite II residue Arg200 mutated to Ala, and PA-L1-I210A, a matrix metalloproteinase-activated PA variant with subsite III residue Ile210 mutated to Ala. PA-U2-R200A and PA-L1-I210A displayed reduced cytotoxicity when used singly. However, when combined, they formed LF-binding competent heterogeneous oligomers by intermolecular complementation, and achieved high specificity in tumor targeting. Nevertheless, each of these proteins, in particular PA-L1-I210A, retained residual LF-binding ability. In this work, we screened a library containing all possible amino acid substitutions for LF-binding site to find variants with activity strictly dependent upon intermolecular complementation. PA-I207R was identified as an excellent replacement for the original clockwise-side variant, PA-I210A. Consequently, the new combination of PA-L1-I207R and PA-U2-R200A showed potent anti-tumor activity and low toxicity, exceeding the performance of the original combination, and warranting further investigation.

Cleft palate is a common birth defect, occurring in approximately 1 in 1000 live births worldwide. Known etiological mechanisms of cleft palate include defects within developing palate shelf tissues, defects in mandibular growth and defects in spontaneous fetal mouth movement. Until now, experimental studies directly documenting fetal mouth immobility as an underlying cause of cleft palate have been limited to models lacking neurotransmission. This study extends the range of anomalies directly demonstrated to have fetal mouth movement defects correlated with cleft palate. Here, we show that mouse embryos deficient in retinoic acid (RA) have mispatterned pharyngeal nerves and skeletal elements that block spontaneous fetal mouth movement in utero Using X-ray microtomography, in utero ultrasound video, ex vivo culture and tissue staining, we demonstrate that proper retinoid signaling and pharyngeal patterning are crucial for the fetal mouth movement needed for palate formation. Embryos with deficient retinoid signaling were generated by stage-specific inactivation of retinol dehydrogenase 10 (Rdh10), a gene crucial for the production of RA during embryogenesis. The finding that cleft palate in retinoid deficiency results from a lack of fetal mouth movement might help elucidate cleft palate etiology and improve early diagnosis in human disorders involving defects of pharyngeal development.

Upon SARS-CoV-2 infection, most individuals develop neutralizing antibodies and T-cell immunity. However, some individuals reportedly remain SARS-CoV-2 PCR positive by pharyngeal swabs weeks after recovery. Whether viral RNA in these persistent carriers is contagious and stimulates SARS-CoV-2-specific immune responses is unknown.

The miniature pig is an optimal animal model for studying nervous system disease because of its physiologic and pathologic features. However, the rete mirabile composed of arteries and veins at the skull base limits their application as a model of ischemic stroke by middle cerebral artery occlusion. The present study investigated the possibility of establishing an ischemic stroke model in the miniature pig by blocking the skull base retia with sodium alginate microspheres. Three Bama miniature pigs were used. Using the monitor of C-arm X-ray machine, sodium alginate microspheres (100-300 μm), a novel embolic material, were injected through the femoral artery, aortic arch, common carotid artery, ascending pharyngeal artery and the retia. Results were evaluated using carotid arteriography, MRI, behavior observation and histology. The unilateral rete mirabile was completely blocked, resulting in disturbance in blood supply to the basal ganglia, astasia of the right hind limb and salivation. MRI and hematoxylin-eosin staining showed an evident infarction focus in the basal ganglia. These findings indicate that sodium alginate microspheres are a suitable embolic material for blocking the skull base retia in miniature pigs to establish an ischemic stroke models.

Chronic granulomatous disease (CGD) is caused by defects in nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) complex subunits (gp91(phox) (a.k.a. Nox2), p47(phox), p67(phox), p22(phox), p40(phox)) leading to reduced phagocyte-derived reactive oxygen species production. Almost half of patients with CGD develop inflammatory bowel disease, and the involvement of the intestinal microbiome in relation to this predisposing immunodeficiency has not been explored.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic. Diagnostic testing for SARS-CoV-2 has continuously been challenged due to several variants with diverse spike (S) and nucleocapsid (N) protein mutations []. SARS-CoV-2 variant proliferation potentially affects N protein-targeted rapid antigen testing. In this study, rapid antigen and reverse transcription PCR (RT-PCR) tests were performed simultaneously in patients with suspected coronavirus disease 2019 (COVID-19). Direct whole genome sequencing was performed to determine the N protein variations, and the viral assemblies were uploaded to GISAID. The genomes were then compared with those of global virus strains from GISAID. These isolates belonged to the B.1.1.7 variant, exhibiting several amino acid substitutions, including D3L, R203K, G204R, and S235F N protein mutations. The T135I mutation was also identified in one variant case in which the rapid antigen test and RT-PCR test were discordantly negative and positive, respectively. These findings suggest that the variants undetected by the Panbio COVID-19 rapid antigen test may be due to the T135I mutation in the N protein, posing a potential diagnostic risk for commercially available antigen tests. Hence, we recommend concomitant paired rapid antigen tests and molecular diagnostic methods to detect SARS-CoV-2. False-negative results could be rapidly corrected using confirmatory RT-PCR results to prevent future COVID-19 outbreaks.

Streptococcus pyogenes causes a profound global burden of morbidity and mortality across its diverse clinical spectrum. To support a new controlled human infection ('challenge') model seeking to accelerate S. pyogenes vaccine development, we aimed to develop an accurate and reliable molecular method for quantifying bacterial load from pharyngeal swabs collected during experimental human pharyngitis.