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We also characterize the exonuclease stereospecificity using phosphorothioate-modified DNA, provide a homology model for the DNA primer strand in the exonuclease active site, and propose a dynamic structural model for the transfer of DNA from the polymerase to the exonuclease active site based on MD simulations.The bacterial second messenger bis-(3'-5')-cyclic diguanylate monophosphate (c-di-GMP) controls various cellular processes, including motility, toxin production, and biofilm formation. c-di-GMP is enzymatically synthesized by GGDEF domain-containing diguanylate cyclases and degraded by HD-GYP domain-containing phosphodiesterases (PDEs) to 2 GMP or by EAL domain-containing PDE-As to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). Since excess pGpG feedback inhibits PDE-A activity and thereby can lead to the uncontrolled accumulation of c-di-GMP, a PDE that degrades pGpG to 2 GMP (PDE-B) has been presumed to exist. To date, the only enzyme known to hydrolyze pGpG is oligoribonuclease Orn, which degrades all kinds of oligoribonucleotides. Here, we identified a pGpG-specific PDE, which we named PggH, using biochemical approaches in the gram-negative bacteria Vibrio cholerae. Biochemical experiments revealed that PggH exhibited specific PDE activity only toward pGpG, thus differing from the previously reported Orn. Furthermore, the high-resolution structure of PggH revealed the basis for its PDE activity and narrow substrate specificity. Finally, we propose that PggH could modulate the activities of PDE-As and the intracellular concentration of c-di-GMP, resulting in phenotypic changes including in biofilm formation.

Tumor-only sequencing, implemented for the identification of somatic variants, is oftentimes used for the detection of actionable germline variants. We sought to determine whether tumor-only sequencing assays are suitable for detection of actionable germline variants, given their importance for the delivery of targeted therapies and risk-reducing measures.

The detection of germline variants affecting moderate- and high-penetrance cancer susceptibility genes (CSGs) by tumor-only sequencing was compared to clinical germline testing in 21 333 cancer patients who underwent tumor and germline testing using the Food and Drug Administration (FDA)-authorized Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Targets (MSK-IMPACT) assay. Seven homologous recombination deficiency (HRD), two DNA damage response (DDR) and four mismatch repair (MMR) genes, as well as NF1, RB1 and TP53 were included in the analysis. FDA-authorized and New York State Department of Health-approved sequencing methods for h-risk patients with negative tumor-only sequencing results, clinical genetic testing could be considered given the impact of these variants on therapy and genetic counseling.

Tumor-only sequencing is adequate for the detection of clinically actionable germline variants, particularly for SNVs and small indels; however, a small subset of alterations affecting HRD, DDR and MMR genes may not be detected optimally. Therefore, for high-risk patients with negative tumor-only sequencing results, clinical genetic testing could be considered given the impact of these variants on therapy and genetic counseling.

Airway epithelial cells can actively participate in the defense against environmental pathogens to elicit local or systemic inflammation. Diesel exhaust particles (DEP), a main component of urban air pollution with particulate matter, are associated with the occurrence of acute and chronic upper airway inflammatory diseases.

We sought to investigate the effect of DEP alone or in combination with lipopolysaccharide on the secretome in the primary human nasal epithelium (PHNE) and to find potential biomarkers to relate DEP exposure to upper airway inflammatory diseases.

PHNE was cultured at an air-liquid interface to create a differentiated invivo-like model. Secreted proteins (secretome) on the bottom media of the PHNE were analyzed by mass spectrometry-based label-free quantitative proteomics and ELISA.

Considerably more differentially expressed secreted proteins were identified in response to DEP plus lipopolysaccharide than to DEP alone. Some canonical pathways related to inflammation and cancer such as the p53, β-catenin, and extracellular signal-regulated kinase 1/2 pathways were involved. Among differentially expressed secreted proteins, leukemia inhibitory factor was also detected at a high level in the middle ear effusions of otitis media patients, and the leukemia inhibitory factor level was significantly correlated with daily mean mass concentrations of atmospheric particulate matter averaged over 8 days before sample collection.

Apical stimulation with DEP and lipopolysaccharide can significantly alter the basal secretome in PHNE, and this alteration can be reflected by surrounding inflammation with effusion of fluids invivo such as middle ear effusions in otitis media patients.

Apical stimulation with DEP and lipopolysaccharide can significantly alter the basal secretome in PHNE, and this alteration can be reflected by surrounding inflammation with effusion of fluids in vivo such as middle ear effusions in otitis media patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic and contagious coronavirus that caused a global pandemic with 5.2 million fatalities to date. Questions concerning serologic features of long-term immunity, especially dominant epitopes mediating durable antibody responses after SARS-CoV-2 infection, remain to be elucidated.

We aimed to dissect the kinetics and longevity of immune responses in coronavirus disease 2019 (COVID-19) patients, as well as the epitopes responsible for sustained long-term humoral immunity against SARS-CoV-2.

We assessed SARS-CoV-2 immune dynamics up to 180 to 220 days after disease onset in 31 individuals who predominantly experienced moderate symptoms of COVID-19, then performed a proteome-wide profiling of dominant epitopes responsible for persistent humoral immune responses.

Longitudinal analysis revealed sustained SARS-CoV-2 spike protein-specific antibodies and neutralizing antibodies in COVID-19 patients, along with activation of cytokina path to aid rational vaccine design and diagnostic development.Medical and recreational cannabis legalization lead to increased cannabis use among adults. There is concern that legalization has negative implications for minors via effects on parents. We conducted a systematic review of studies examining legalization in the United States. Web of Science, PsycInfo, and PubMed were searched through May 2021, studies examining effects of legalization on maternal cannabis and other substance use during pregnancy and postpartum, perinatal outcomes, parental cannabis and other substance use and attitudes, parenting, and child outcomes were identified, and two independent reviewers extracted information on study designs, samples, and outcomes, and assessed classification of evidence and risk of bias. Forty-one studies met inclusion criteria; only 6 (15%) used the most causally informative study design (difference in differences). It is likely legalization increases maternal cannabis use during pregnancy and postpartum, parental cannabis use, and approval of adult cannabis use. Legalization may increase some adverse perinatal outcomes, though findings were inconsistent. It is likely legalization increases unintentional pediatric cannabis exposure. There is insufficient evidence for effects of legalization on child abuse and neglect, and there have been no studies examining effects of legalization on other aspects of parenting or on child adjustment. There is a critical lack of causally informative epidemiological studies examining effects of legalization on parenting and young children. Additional causally informative research is needed. Studies of parental cannabis use in a legal context are particularly needed. Commonsense guidelines must recognize the shifting national landscape around legalization while seeking to minimize potential harm to minors.The short stature homeobox-containing (SHOX) is the most frequently analysed gene in patients classified as short stature patients (ISS) or diagnosed with Leri-Weill dyschondrosteosis (LWD), Langer mesomelic dysplasia (LMD), or Madelung deformity (MD). However, clinical testing of this gene focuses primarily on single nucleotide variants (SNV) in its coding sequences and copy number variants (CNV) overlapping SHOX gene. This review summarizes the clinical impact of variants in noncoding regions of SHOX. RECENT FINDINGS CNV extending exclusively into the regulatory elements (i.e., not interrupting the coding sequence) are found more frequently in downstream regulatory elements of SHOX. Further, duplications are more frequent than deletions. Interestingly, downstream duplications are more common than deletions in patients with ISS or LWD but no such differences exist for upstream CNV. Moreover, the presence of specific CNVs in the patient population suggests the involvement of additional unknown factors. Some of its intronic variants, notably NM_000451.3(SHOX)c.-9delG and c.-65C>A in the 5'UTR, have unclear clinical roles. Dihydroethidium nmr However, these intronic SNV may increase the probability that other CNV will arise de novo in the SHOX gene based on homologous recombination or incorrect splicing of mRNA. SUMMARY This review highlights the clinical impact of noncoding changes in the SHOX gene and the need to apply new technologies and genotype-phenotype correlation in their analysis.Metastasis, the fatal hallmark of breast cancer (BC), is a serious hurdle for therapy. Current prognostic approaches are not sufficient to predict the metastasis risk for BC patients. Therefore, in the present study, we analyzed gene expression data from GSE139038 and TCGA database to develop predictive markers for BC metastasis. Initially, the data from GSE139038 which contained 65 samples consisting of 41 breast tumor tissues, 18 paired morphologically normal tissues and 6 from non-malignant breast tissues were analyzed for differentially expressed genes (DEGs). DEGs were obtained from three different comparisons paired morphologically normal (MN) versus tumor samples (C), apparently normal (AN) versus tumor samples (C), and paired morphologically normal (MN) versus apparently normal samples (AN). Multiple bioinformatic methods were employed to evaluate metastasis, EMT and triple negative breast cancer (TNBC) specific genes. Further, regulation of gene expression, clinicopathological factors and DNA methylation patterns of DEGs in BC were validated with TCGA datasets. Our bioinformatic analysis showed that 40 genes were upregulated and 294 were found to be downregulated between AN vs C; 124 were upregulated and 760 genes were downregulated between MN vs C; 4 were upregulated and 13 were downregulated between MN vs AN. Analysis using TCGA dataset revealed 18 genes were significantly altered in nodal positive BC patients compared to nodal negative BC patients. Our study showed novel candidate genes as predictive markers for BC metastasis which can also be used for therapeutic targets for BC treatment.