Specific nucleic acid sequences can be detected in individual cells by in situ hybridization. However, when very few copies of a target sequence are present per cell, its signal is undetectable by flow cytometry. Although various approaches have been developed to increase fluorescence signals for in situ hybridization, flow cytometric detection of specific genomic DNA sequences has not been established. Here, we present a flow cytometry assay for detection of single-copy genomic sequences in human lymphocytes using in situ PCR with universal energy transfer-labelled primers.Study question What is the role of pigment epithelium-derived factor (PEDF) from decidual natural killer (dNK) cells during early pregnancy? Summary answer PEDF from dNK cells limits the lipopolysaccharide (LPS)-induced apoptosis and inflammation of decidual stromal cells (DSCs) to maintain DSCs homoeostasis and immune balance at the maternal-foetal interface during early pregnancy. What is known already dNK cells, which secrete PEDF, play critical roles during pregnancy via a series of key regulators. PEDF, a multifunctional endogenous glycoprotein, exhibits a wide range of biological actions upon angiogenesis, inflammation, metabolic homoeostasis, immunomodulation etc., providing potential clinical applications. Study design, size, duration Natural killer (NK) cells from decidua and peripheral blood as well as DSCs isolated from normal pregnancy (NP) during the first trimester (6-10 weeks) and the matched patients suffering recurrent miscarriage (RM) were studied. RNA-sequencing analysis of dNK cells was pece and Technology Grant from NHC Key Laboratory of Reproduction Regulation (CX2017-2), the Programme of Shanghai Academic/Technology Research Leader (17XD1400900) and the Key Project of Shanghai Basic Research from Shanghai Municipal Science and Technology Commission (STCSM; 12JC1401600). None of the authors has any conflict of interest to declare.Environmental variation experienced by a species across space and time can promote the maintenance of genetic diversity that may be adaptive in future global change conditions. Selection experiments have shown that purple sea urchin, Strongylocentrotus purpuratus, populations have adaptive genetic variation for surviving pH conditions at the „edge” (pH 7.5) of conditions experienced in nature. However, little is known about whether populations have genetic variation for surviving low-pH events beyond those currently experienced in nature or how variation in pH conditions affects organismal and genetic responses. Here, we quantified survival, growth, and allele frequency shifts in experimentally selected developing purple sea urchin larvae in static and variable conditions at three pH levels pH 8.1 (control), pH 7.5 (edge-of-range), and pH 7.0 (extreme). Variable treatments recovered body size relative to static treatments, but resulted in higher mortality, suggesting a potential tradeoff between survival and t increased acidification does not result in a linear response but elicits unique physiological stresses and survival mechanisms.CD4+ T cells may induce potent antitumor immune responses through interaction with antigen-presenting cells within the tumor microenvironment. Using a murine model of multiple myeloma, we demonstrated that adoptive transfer of idiotype-specific CD4+ T cells may elicit curative responses against established multifocal myeloma in bone marrow. This finding indicates that the myeloma bone marrow niche contains antigen-presenting cells that may be rendered tumoricidal. Given the complexity of the bone marrow microenvironment, the mechanistic basis of such immunotherapeutic responses is not known. Through a functional characterization of antitumor CD4+ T-cell responses within the bone marrow microenvironment, we found that killing of myeloma cells is orchestrated by a population of bone marrow-resident CD11b+F4/80+MHC-IIHigh macrophages that have taken up and present secreted myeloma protein. The present results demonstrate the potential of resident macrophages as powerful mediators of tumor killing within the bone marrow and provide a basis for novel therapeutic strategies against multiple myeloma and other malignancies that affect the bone marrow.Background In South Africa, Neisseria gonorrhoeae (NG) is the predominant cause of male urethritis syndrome (MUS). The national MUS treatment guidelines recommend gentamicin as salvage therapy for ceftriaxone treatment failures. We ascertained and compared gentamicin MICs obtained by agar dilution and Etest for clinical isolates of NG. Methods Gentamicin MICs for NG culture isolates obtained from 272 MUS cases in 2017 were determined using agar dilution, as per CLSI agar dilution methods, and Etest® (bioMérieux, Marcy-l’Étoile, France). Previously published interpretive criteria were used MIC ≤4 mg/L, susceptible (S); MIC 8-16 mg/L, intermediately resistant (IR); and MIC ≥32 mg/L, resistant (R). WHO 2008 NG reference strains were used as comparison standards. Results Gentamicin agar dilution versus Etest MIC results (mg/L) were as follows MIC50 = 16 versus 4; MIC90 = 16 versus 8; minimum MIC = 4 versus 1; and maximum MIC = 32 versus 16. Interpretive categories for agar dilution versus Etest were as follows S, 4.4% versus 86.8%; IR, 86.0% versus 13.4%; and R, 9.6% versus 0%. The gentamicin MIC50 by agar dilution was significantly higher than by Etest (sign test P value less then 0.001); overall MIC agreement was 7.4% [kappa statistic (κ) = -0.014 (95% CI -0.039 to 0.010)]. Correlation with expected MICs for WHO reference strains was consistently better with Etest than with agar dilution. Conclusions There was a significant discordance between NG gentamicin MICs by agar dilution versus Etest. NG gentamicin AST methodology must be standardized and interpretive criteria established to optimize the monitoring of susceptibility trends.The developmental environment can exert powerful effects on animal phenotype. Recently epigenetic modifications have emerged as one mechanism that can modulate developmentally plastic responses to environmental variability. For example, the DNA methylation profile at promoters of hormone receptor genes can affect their expression and patterns of hormone release. Across taxonomic groups, epigenetic alterations have been linked to changes in glucocorticoid (GC) physiology. GCs are metabolic hormones that influence growth, development, transitions between life-history stages, and thus fitness. To date, relatively few studies have examined epigenetic effects on phenotypic traits in wild animals, especially in amphibians. Here, we examined the effects of exposure to predation threat and experimentally manipulated DNA methylation on corticosterone (CORT) levels in tadpoles and metamorphs of the invasive cane toad (Rhinella marina). We included offspring of toads sampled from populations across the species’ Australi (NR3C1) promoter region in response to alarm cue or zebularine exposure. In both alarm cue and zebularine-exposed individuals, we found differentially methylated DNA in the suppressor of cytokine signaling 3 gene (SOCS3), which may be involved in predator avoidance behavior. In total, our data reveal that alarm cues have significant impacts on tadpole physiology, but show only weak links between DNA methylation and CORT levels. We also identify genes containing differentially methylated regions in tadpoles exposed to alarm cues and zebularine, particularly in range-edge populations, that warrant further investigation.The COVID-19 pandemic and antimicrobial resistance are parallel and interacting health emergencies with opportunity for mutual learning. As their measures and consequences are comparable, the COVID-19 pandemic helps to illustrate the potential long-term impact of AMR, which is less acute but not less crucial. They may also impact each other as there is a push to resort to existing antimicrobials in critically ill COVID-19 patients in the absence of specific treatments, while attempts to manage the spread of COVID-19 may also lead to a slow down AMR. Understanding how COVID-19 affects AMR trends and what we can expect if these remain the same or worsen, will help us plan next steps to tackle AMR. Researchers should now start collecting data to measure the impact of current COVID-19 policies and programs on AMR.Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide adaptive immunity to prokaryotes against invading phages and plasmids. As a countermeasure, phages have evolved anti-CRISPR (Acr) proteins that neutralize the CRISPR immunity. AcrIIA5, isolated from a virulent phage of Streptococcus thermophilus, strongly inhibits diverse Cas9 homologs, but the molecular mechanism underlying the Cas9 inhibition remains unknown. Here, we report the solution structure of AcrIIA5, which features a novel α/β fold connected to an N-terminal intrinsically disordered region (IDR). Remarkably, truncation of the N-terminal IDR abrogates the inhibitory activity against Cas9, revealing that the IDR is essential for Cas9 inhibition by AcrIIA5. Progressive truncations and mutations of the IDR illustrate that the disordered region not only modulates the association between AcrIIA5 and Cas9-sgRNA, but also alters the catalytic efficiency of the inhibitory complex. The length of IDR is critical for the Cas9-sgRNA recognition by AcrIIA5, whereas the charge content of IDR dictates the inhibitory activity. Conformational plasticity of IDR may be linked to the broad-spectrum inhibition of Cas9 homologs by AcrIIA5. Identification of the IDR as the main determinant for Cas9 inhibition expands the inventory of phage anti-CRISPR mechanisms.DNA2 is an essential nuclease-helicase implicated in DNA repair, lagging-strand DNA synthesis, and the recovery of stalled DNA replication forks (RFs). In Saccharomyces cerevisiae, dna2Δ inviability is reversed by deletion of the conserved helicase PIF1 and/or DNA damage checkpoint-mediator RAD9. It has been suggested that Pif1 drives the formation of long 5′-flaps during Okazaki fragment maturation, and that the essential function of Dna2 is to remove these intermediates. In the absence of Dna2, 5′-flaps are thought to accumulate on the lagging strand, resulting in DNA damage-checkpoint arrest and cell death. In line with Dna2’s role in RF recovery, we find that the loss of Dna2 results in severe chromosome under-replication downstream of endogenous and exogenous RF-stalling. Importantly, unfaithful chromosome replication in Dna2-mutant cells is exacerbated by Pif1, which triggers the DNA damage checkpoint along a pathway involving Pif1’s ability to promote homologous recombination-coupled replication. We propose that Dna2 fulfils its essential function by promoting RF recovery, facilitating replication completion while suppressing excessive RF restart by recombination-dependent replication (RDR) and checkpoint activation. The critical nature of Dna2’s role in controlling the fate of stalled RFs provides a framework to rationalize the involvement of DNA2 in Seckel syndrome and cancer.Cobalamin riboswitches encompass a structurally diverse group of cis-acting, gene regulatory elements found mostly in bacterial messenger RNA and are classified into subtypes based on secondary and tertiary characteristics. An unusual variant of the cobalamin riboswitch with predicted structural features was identified in Bacillus subtilis over a decade ago, but its structure and mechanisms of cobalamin selectivity and translational control have remained unsolved. We present the crystal structure of the aptamer domain of this atypical cobalamin riboswitch and a model for the complete riboswitch, including its expression platform domain. We demonstrate that this riboswitch binds to multiple cobalamin derivatives and correlate its promiscuous behavior to its structure and unique arrangement of peripheral elements. Comparative structural analyses between conventional cobalamin riboswitches and the B. subtilis cobalamin riboswitch reveal that the likely basis for this promiscuous ligand binding is intrinsic structural adaptability encoded in the RNA structure.