ABSTRACT

Bacillus subtilis contains two known cyclic di-GMP (c-di-GMP)-dependent receptors, YdaK and DgrA, as well as three diguanylate cyclases (DGCs): soluble DgcP and membrane-integral DgcK and DgcW. DgrA regulates motility, while YdaK is responsible for the formation of a putative exopolysaccharide, dependent on the activity of DgcK. Using single-molecule tracking, we show that a majority of DgcK molecules are statically positioned in the cell membrane but significantly less so in the absence of YdaK but more so upon overproduction of YdaK. The soluble domains of DgcK and of YdaK show a direct interaction in vitro, which depends on an intact I-site within the degenerated GGDEF domain of YdaK. These experiments suggest a direct handover of a second messenger at a single subcellular site. Interestingly, all three DGC proteins contribute toward downregulation of motility via the PilZ protein DgrA. Deletion of dgrA also affects the mobility of DgcK within the membrane and also that of DgcP, which arrests less often at the membrane in the absence of DgrA. Both, DgcK and DgcP interact with DgrA in vitro, showing that divergent as well as convergent direct connections exist between cyclases and their effector proteins. Automated determination of molecule numbers in live cells revealed that DgcK and DgcP are present at very low copy numbers of 6 or 25 per cell, respectively, such that for DgcK, a part of the cell population does not contain any DgcK molecule, rendering signaling via c-di-GMP extremely efficient.

IMPORTANCE Second messengers are free to diffuse through the cells and to activate all responsive elements. Cyclic di-GMP (c-di-GMP) signaling plays an important role in the determination of the life style transition between motility and sessility/biofilm formation but involves numerous distinct synthetases (diguanylate cyclases [DGCs]) or receptor pathways that appear to act in an independent manner. Using Bacillus subtilis as a model organism, we show that for two c-di-GMP pathways, DGCs and receptor molecules operate via direct interactions, where a synthesized dinucleotide appears to be directly used for the protein-protein interaction. We show that very few DGC molecules exist within cells; in the case of exopolysaccharide (EPS) formation via membrane protein DgcK, the DGC molecules act at a single site, setting up a single signaling pool within the cell membrane. Using single-molecule tracking, we show that the soluble DGC DgcP arrests at the cell membrane, interacting with its receptor, DgrA, which slows down motility. DgrA also directly binds to DgcK, showing that divergent as well as convergent modules exist in B. subtilis. Thus, local-pool signal transduction operates extremely efficiently and specifically.

ABSTRACT

A microfluidic system coupled with fluorescence microscopy is a powerful approach for quantitative analysis of bacterial growth. Here, we measure parameters of growth and dynamic localization of the cell division initiation protein FtsZ in Bacillus subtilis. Consistent with previous reports, we found that after division, FtsZ rings remain at the cell poles, and polar FtsZ ring disassembly coincides with rapid Z-ring accumulation at the midcell. In cells mutated for minD, however, the polar FtsZ rings persist indefinitely, suggesting that the primary function of the Min system is in Z-ring disassembly. The inability to recycle FtsZ monomers in the minD mutant results in the simultaneous maintenance of multiple Z-rings that are restricted by competition for newly synthesized FtsZ. Although the parameters of FtsZ dynamics change in the minD mutant, the overall cell division time remains the same, albeit with elongated cells necessary to accumulate a critical threshold amount of FtsZ for promoting medial division. Finally, the minD mutant characteristically produces minicells composed of polar peptidoglycan shown to be inert for remodeling in the wild type. Polar peptidoglycan, however, loses its inert character in the minD mutant, suggesting that the Min system not only is important for recycling FtsZ but also may have a secondary role in the spatiotemporal regulation of peptidoglycan remodeling.

IMPORTANCE Many bacteria grow and divide by binary fission in which a mother cell divides into two identical daughter cells. To produce two equally sized daughters, the division machinery, guided by FtsZ, must dynamically localize to the midcell each cell cycle. Here, we quantitatively analyzed FtsZ dynamics during growth and found that the Min system of Bacillus subtilis is essential to disassemble FtsZ rings after division. Moreover, a failure to efficiently recycle FtsZ results in an increase in cell size. Finally, we show that the Min system has an additional role in inhibiting cell wall turnover and contributes to the "inert" property of cell walls at the poles.

Objective

Polygenic risk scores (PRSs) are used to quantify the cumulative effects of a number of genetic variants, which may individually have a very small effect on susceptibility to a disease; we used PRSs to better understand the genetic contribution to common epilepsy and its subtypes.

Objective

De novo missense mutations in the RHOBTB2 gene have been described as causative for developmental and epileptic encephalopathy.

Objective

To specifically report on ataxic-hypotonic cerebral palsy (CP) using registry data and to directly compare its features with other CP subtypes.

Although pain is a prevalent nonmotor symptom in Parkinson’s disease (PD), it is undertreated, in part because of our limited understanding of the underlying mechanisms. Considering that the basal ganglia are implicated in pain sensation, and that their synaptic outputs are controlled by the subthalamic nucleus (STN), we hypothesized that...

Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by using the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed alternative lengthening of telomeres (ALT). Cells that use ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML bodies (APBs). APBs localize to and cluster telomeric ends together with telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can occur. However, the necessity of APBs and their function in the ALT pathway has remained unclear. Here, we used CRISPR/Cas9 to delete PML and APB components from ALT-positive cells to cleanly define the function of APBs in ALT. We found that PML is required for the ALT mechanism, and that this necessity stems from APBs’ role in localizing the BLM–TOP3A–RMI (BTR) complex to ALT telomere ends. Strikingly, recruitment of the BTR complex to telomeres in a PML-independent manner bypasses the need for PML in the ALT pathway, suggesting that BTR localization to telomeres is sufficient to sustain ALT activity.

ABSTRACT

The chemical structures of soluble fiber carbohydrates vary from source to source due to numerous possible linkage configurations among monomers. However, it has not been elucidated whether subtle structural variations might impact soluble fiber fermentation by colonic microbiota. In this study, we tested the hypothesis that subtle structural variations in a soluble polysaccharide govern the community structure and metabolic output of fermenting microbiota. We performed in vitro fecal fermentation studies using arabinoxylans (AXs) from different classes of wheat (hard red spring [AX_HRS], hard red winter [AX_HRW], and spring red winter [AX_SRW]) with identical initial microbiota. Carbohydrate analyses revealed that AX_SRW was characterized by a significantly shorter backbone and increased branching compared with those of the hard varieties. Amplicon sequencing demonstrated that fermentation of AX_SRW resulted in a distinct community structure of significantly higher richness and evenness than those of hard-AX-fermenting cultures. AX_SRW favored OTUs within Bacteroides, whereas AX_HRW and AX_HRS favored Prevotella. Accordingly, metabolic output varied between hard and soft varieties; higher propionate production was observed with AX_SRW and higher butyrate and acetate with AX_HRW and AX_HRS. This study showed that subtle changes in the structure of a dietary fiber may strongly influence the composition and function of colonic microbiota, further suggesting that physiological functions of dietary fibers are highly structure dependent. Thus, studies focusing on interactions among dietary fiber, gut microbiota, and health outcomes should better characterize the structures of the carbohydrates employed.

IMPORTANCE Diet, especially with respect to consumption of dietary fibers, is well recognized as one of the most important factors shaping the colonic microbiota composition. Accordingly, many studies have been conducted to explore dietary fiber types that could predictably manipulate the colonic microbiota for improved health. However, the majority of these studies underappreciate the vastness of fiber structures in terms of their microbial utilization and omit detailed carbohydrate structural analysis. In some cases, this causes conflicting results to arise between studies using (theoretically) the same fibers. In this investigation, by performing in vitro fecal fermentation studies using bran arabinoxylans obtained from different classes of wheat, we showed that even subtle changes in the structure of a dietary fiber result in divergent microbial communities and metabolic outputs. This underscores the need for much higher structural resolution in studies investigating interactions of dietary fibers with gut microbiota, both in vitro and in vivo.

Identifying gene variants causing monogenic diabetes (MD) increases understanding of disease etiology and allows for implementation of precision therapy to improve metabolic control and quality of life. Here, we aimed to assess the prevalence of MD in youth with diabetes in Lithuania, uncover potential diabetes-related gene variants, and prospectively introduce precision treatment. First, we assessed all pediatric and most young-adult patients with diabetes in Lithuania (n = 1,209) for diabetes-related autoimmune antibodies. We then screened all antibody-negative patients (n = 153) using targeted high-throughput sequencing of >300 potential candidate genes. In this group, 40.7% had MD, with the highest percentage (100%) in infants (diagnosis at ages 0–12 months), followed by those diagnosed at ages >1–18 years (40.3%) and >18–25 years (22.2%). The overall prevalence of MD in youth with diabetes in Lithuania was 3.5% (1.9% for GCK diabetes, 0.7% for HNF1A, 0.2% for HNF4A and ABCC8, 0.3% for KCNJ11, and 0.1% for INS). Furthermore, we identified likely pathogenic variants in 11 additional genes. Microvascular complications were present in 26% of those with MD. Prospective treatment change was successful in >50% of eligible candidates, with C-peptide >252 pmol/L emerging as the best prognostic factor.

Random transposon mutagenesis is a powerful and unbiased genetic approach to answer fundamental biological questions. Here, we introduce an improved mariner-based transposon system with enhanced stability during propagation and versatile applications in mutagenesis. We used a low-copy-number plasmid as a transposon delivery vehicle, which affords a lower frequency of unintended recombination during vector construction and propagation in Escherichia coli. We generated a variety of transposons allowing for gene disruption or artificial overexpression, each in combination with one of four different antibiotic resistance markers. In addition, we provide transposons that will report gene/protein expression due to transcriptional or translational coupling. We believe that the TnFLX system will help enhance the flexibility of future transposon modification and application in Bacillus and other organisms.

IMPORTANCE The stability of transposase-encoding vectors during cloning and propagation is crucial for the reliable application of transposons. Here, we increased the stability of the mariner delivery vehicle in E. coli. Moreover, the TnFLX transposon system will improve the application of forward genetic methods with an increased number of antibiotic resistance markers and the ability to generate unbiased green fluorescent protein (GFP) fusions to report on protein translation and subcellular localization.

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.

Etravirine (ETR) is a nonnucleoside reverse transcriptase inhibitor (NNRTI) used in treatment-experienced individuals. Genotypic resistance test-interpretation systems can predict ETR resistance; however, genotype-based algorithms are derived primarily from HIV-1 subtype B and may not accurately predict resistance in non-B subtypes. The frequency of ETR resistance among recombinant subtype C HIV-1 and the accuracy of genotypic interpretation systems were investigated. HIV-1_LAI containing full-length RT from HIV-1 subtype C-positive individuals experiencing virologic failure (>10,000 copies/ml and >1 NNRTI resistance-associated mutation) were phenotyped for ETR susceptibility. Fold change (FC) was calculated against a composite 50% effective concentration (EC₅₀) from treatment-naive individuals and three classifications were assigned: (i) <2.9-FC, susceptible; (ii) ≥2.9- to 10-FC, partially resistant; and (iii) >10-FC, fully resistant. The Stanford HIVdb-v8.4 was used for genotype predictions merging the susceptible/potential low-level and low-level/intermediate groups for 3 x 3 comparison. Fifty-four of a hundred samples had reduced ETR susceptibility (≥2.9-FC). The FC correlated with HIVdb-v8.4 (Spearman’s rho = 0.62; P < 0.0001); however, 44% of samples were partially (1 resistance classification difference) and 4% completely discordant (2 resistance classification differences). Of the 34 samples with an FC of >10, 26 were HIVdb-v8.4 classified as low-intermediate resistant. Mutations L100I, Y181C, or M230L were present in 27/34 (79%) of samples with an FC of >10 but only in 2/46 (4%) of samples with an FC of <2.9. No other mutations were associated with ETR resistance. Viruses containing the mutation K65R were associated with reduced ETR susceptibility, but 65R reversions did not increase ETR susceptibility. Therefore, genotypic interpretation systems were found to misclassify ETR susceptibility in HIV-1 subtype C samples. Modifications to genotypic algorithms are needed to improve the prediction of ETR resistance for the HIV-1 subtype C.

We report the diagnostic challenges and the clinical course of a patient with an extraordinary presentation of B-lymphoblastic leukemia (B-ALL) with eosinophilia. We identified a novel ZBTB20-JAK2 gene fusion as a chimeric RNA transcript using the Archer platform. This gene fusion from the same patient was recently identified by Peterson et al. (2019) at the genomic level using a different sequencing technology platform. The configuration of this gene fusion predicts the production of a kinase-activating JAK2 fusion protein, which would normally lead to a diagnosis of Philadelphia chromosome–like B-ALL (Ph-like B-ALL). However, the unusual presentation of eosinophilia led us to demonstrate the presence of this gene fusion in nonlymphoid hematopoietic cells by fluorescence in situ hybridization (FISH) studies with morphologic correlation. Therefore, we believe this disease, in fact, represents blast crisis arising from an underlying myeloid neoplasm with JAK2 rearrangements. This case illustrates the difficulty in differentiating Ph-like B-ALL and myeloid/lymphoid neoplasm with eosinophilia and gene rearrangements (MLN-EGR) in blast crisis. As currently defined, the diagnosis of MLN-EGR relies on the hematologic presentations and the identification of marker gene fusions (including PCM1-JAK2, ETV6-JAK2, and BCR-JAK2). However, these same gene fusions, when limited to B-lymphoblasts, also define Ph-like B-ALL. Yet, our case does not conform to either condition. Therefore, the assessment for lineage restriction of gene rearrangements to reflect the pathophysiologic difference between B-ALL and MLN-EGR in blast crisis is likely a more robust diagnostic approach and allows the inclusion of MLN-EGR with novel gene fusions.

Background:

Lipid traits have been inconsistently linked to risk of non-Hodgkin lymphoma (NHL). We examined the association of genetically predicted lipid traits with risk of diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and marginal zone lymphoma (MZL) using Mendelian randomization (MR) analysis.

Subtractive immunization is useful for directing the immune response away from dominant epitopes to allow antibodies against less immunogenic antigens to be developed. It is also useful when there is a need to downplay the response to carriers added to peptides and recombinant proteins, such as Gst and Ig, to aid in production and purification. Subtractive immunization protocols also can be helpful if the target protein is part of a mixture (such as a cell lysate) or present on the surface of transfected cells.

BACKGROUND AND PURPOSE:

Not all tandem occlusions diagnosed on traditional vascular imaging modalities, such as MRA, represent actual complete ICA occlusion. This study aimed to explore the utility of high-resolution vessel wall imaging in identifying true ICA tandem occlusions and screening patients for their suitability for endovascular recanalization.

[Having appointment with real estate experts #5] While buying a home is quite easy for high-income households, it is another story for the middle-class. With a wise and specific financial plan, the chances of owning a home can still lie within reach even if an average income.

Get ready for the first scored event in DARPA's latest Grand Challenge

Roboticists share what they've learned so far from DARPA's Subterranean Challenge, and how they readied their bots for the next event—the Tunnel Circuit

How autonomous robots in the underground scavenger hunt pick up points for their teams

Supporting long-range communications inside of a mine is extremely difficult, so DARPA SubT teams are trying out some creative approaches

It's about more than just first place as teams look toward more competitions in 2020

Mark Lawson and Dan Milmo discuss the sustainability of the streaming service. Plus: Lara Spirit on why you should register to vote before Tuesday’s deadline

Netflix has risen from obscurity to be one of the most powerful media companies in the world with more than 150 million global subscribers. It has launched critically acclaimed hits such as House of Cards, The Crown and Unbelievable, as well as showcasing the back catalogues of popular television series. But as part of its rapid growth, the company has racked up huge debts.

Joining Anushka Asthana to discuss the long-term sustainability of Netflix are the TV critic Mark Lawson and the Guardian’s deputy business editor Dan Milmo.

What is likely to be Kimi Raikkonen's final Formula 1 victory at the 2018 US Grand Prix is being live-streamed by F1 today. The high point of a disappointing second spell at Ferrari, it nonetheless gave the 2007 world champion a chance to prove a point.

Shuttered stores worsened Montreal-based company’s already struggling business

Follow our live coronavirus updates here Coronavirus: the symptoms

The boss of Airbus has warned that it is "bleeding cash" at such a rapid rate during the coronavirus lockdowns that its survival is in doubt.

The Hawaii-based Gemini telescope produces a super-sharp picture of the gas giant in the infrared.

Covid-19 could be 'antidote to complacency' among those on the fence about vaccines, scientists say

The falling rupee will severely affect the small companies, whereas the big ones will be impacted moderately. Get rid of Debt | Adopt correct investment strategies

Prime Minister Justin Trudeau described Canada’s current economic situation as a recession on Friday, and that should come as no surprise, says Finance Minister Bill Morneau, as the latest economic figures show two consecutive months of major job losses.

'If I'm not acting, I'm not sure who I am,' the True Blood star said

'The Mindy Project' star and writer talks to Priya Arora about breaking boundaries with her new Netflix show 'Never Have I Ever', a coming-of-age comedy about a modern-day first-generation Indian American teenage girl, much like younger herself

'Without subtitles and the British accent, its a no from me'

Deep recessions have long shadows and already there is a gaping hole opening up in our pandemic response

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The great recession was followed by Brexit, the election of Donald Trump and the rise of authoritarianism particularly in Europe.

Big economic events have big political consequences.

Hakim Ziyech insists his Chelsea future was never in doubt and claims he was always intent on joining the club in July.

Welcome to the Evening Standard's LIVE coverage as the coronavirus crisis continues to heavily impact sport across the globe.

BT Sport will broadcast a special programme on Saturday to mark David Beckham's 45th birthday, showing some of the Manchester United legend's finest Champions League moments.

Kevin De Bruyne has revealed the outcome of Manchester City's appeal against their Champions League ban may determine his future at the club.

Up to 50 Premier League players are planning to revolt against plans to restart the season, according to reports.

Half of parents with young children will struggle to make ends meet in the next three months, study suggests.

Perth Airport is using heavy machinery and company vehicles to block a number of Virgin Australia aircraft from taking off over what it says are $16 million in unpaid debts.

Australia's second-largest bank says its upcoming results will suffer a massive hit due to COVID-19, on another volatile day for the ASX.

It is one of the most popular and important websites for cattle producers, rangers and carbon farmers in northern Australia, but its funding is about to run out.

One year after releasing its plan to drag the NT budget out of structural deficit, the Labor Government says a third of its promises have been implemented in full. But some key measures are still in the works and the Opposition is demanding a better look at the books.