Staphylococcus aureus FtsZ and PBP4 bind to the conformationally dynamic N-terminal domain of GpsB.

In the Firmicutes phylum, GpsB is a membrane associated protein that coordinates peptidoglycan synthesis with cell growth and division. Although GpsB has been studied in several bacteria, the structure, function, and interactome of Staphylococcus aureus GpsB is largely uncharacterized. To address this knowledge gap, we solved the crystal structure of the N-terminal domain of S. aureus GpsB, which adopts an atypical, asymmetric dimer, and demonstrates major conformational flexibility that can be mapped to a hinge region formed by a three-residue insertion exclusive to Staphylococci. When this three-residue insertion is excised, its thermal stability increases, and the mutant no longer produces a previously reported lethal phenotype when overexpressed in Bacillus subtilis. In S. aureus, we show that these hinge mutants are less functional and speculate that the conformational flexibility imparted by the hinge region may serve as a dynamic switch to fine-tune the function of the GpsB complex and/or to promote interaction with its various partners. Furthermore, we provide the first biochemical, biophysical, and crystallographic evidence that the N-terminal domain of GpsB binds not only PBP4, but also FtsZ, through a conserved recognition motif located on their C-termini, thus coupling peptidoglycan synthesis to cell division. Taken together, the unique structure of S. aureus GpsB and its direct interaction with FtsZ/PBP4 provide deeper insight into the central role of GpsB in S. aureus cell division.

A Novel Approach to Managing Thirst and Dry Mouth in Palliative Care: A Prospective Randomized Cross-Over Trial.

CONTEXT: Thirst and xerostomia are significant and highly distressing symptoms experienced by patients receiving palliative and end-of-life care. OBJECTIVES: Determine a reduction of thirst intensity and perceptions of dry mouth on a numerical scale following both the experimental intervention (mini mint ice cubes) and control (plain ice chips). METHODS: Cross-over Randomized Controlled Trial (RCT) to assess the effectiveness of novel intervention in the treatment of dry mouth and the sensation of thirst in palliative care patients. RESULTS: Patients rated the severity of their symptoms of dry mouth and thirst using a numeric rating scale (NRS). On commencing the study and preintervention, all patients suffered severe dry mouth and thirst (≥5/10). Mint and plain ice cubes produced improvement of symptoms immediately after interventions. Results from dry mouth ratings show, a decrease of 1.6 points for plain ice cubes (P < 0.0001), on average, ratings for mint ice cubes decreased 3.7 (P < 0.0001). For the sensation of thirst, the plain ice cube intervention group rating decreased 1.7 points (P < 0.006), ratings for mint ice cubes decreased 3.4 points (P < 0.0001). The average decrease in dry mouth and thirst intensity scores from preintervention to postintervention were significantly greater for mint ice cubes (P < 0.05) and 86.6% of patients preferred mint ice cubes. CONCLUSION: This trial found that while usual mouth care and the intervention were both able to reduce the intensity of dry mouth and the sensation of thirst, the mint intervention had a greater response.

Relieving Perception of Thirst and Xerostomia in Patients with Palliative and End-of-life Care Needs: A Rapid Review.

CONTEXT: Thirst and dry mouth are interlinked symptoms that frequently cause significant distress for patients with life-limiting conditions. OBJECTIVES: The objective of this rapid review was to identify and synthesize effective interventions that relieve perceptions of thirst and dry mouth of patients with palliative care and end-of-life care needs. METHODS: Eligible studies were undertaken in clinical settings, with patients experiencing thirst-related distress and/or dry mouth. This review of peer-reviewed literature was conducted following aspects of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines. The main outcomes of interest were: 1) efficacy of thirst and dry mouth interventions for patient, and 2) patient, caregiver, and staff acceptability and satisfaction of the interventions. Scientific journal articles were retrieved through searches in electronic databases of MEDLINE (Ovid), CINAHL (EBSCO), and AgeLine (EBSCO). RESULTS: Eleven studies were included for analysis and synthesis of the results. Most studies either focused on a dry mouth intervention or reported dry mouth outcomes within a broader thirst intervention (n = 9/11 studies). Standard oral care was the common intervention type (n = 5/11). All but one dry mouth intervention reported statistical improvement in outcomes of interest. All studies that reported on thirst were conducted in an Intensive Care Unit (ICU) setting (n = 4/4). No studies specifically addressed thirst in patients in specialist palliative care settings. CONCLUSION: Evidence from this review suggests that thirst interventions established within the ICU setting may prove effective for treatment of terminally ill patients receiving specialist palliative care.

Polyamines and linear DNA mediate bacterial threat assessment of bacteriophage infection.

Monitoring the extracellular environment for danger signals is a critical aspect of cellular survival. However, the danger signals released by dying bacteria and the mechanisms bacteria use for threat assessment remain largely unexplored. Here, we show that lysis of Pseudomonas aeruginosa cells releases polyamines that are subsequently taken up by surviving cells via a mechanism that relies on Gac/Rsm signaling. While intracellular polyamines spike in surviving cells, the duration of this spike varies according to the infection status of the cell. In bacteriophage-infected cells, intracellular polyamines are maintained at high levels, which inhibits replication of the bacteriophage genome. Many bacteriophages package linear DNA genomes and linear DNA is sufficient to trigger intracellular polyamine accumulation, suggesting that linear DNA is sensed as a second danger signal. Collectively, these results demonstrate how polyamines released by dying cells together with linear DNA allow P. aeruginosa to make threat assessments of cellular injury.

Exploring oral care practices, barriers, and facilitators in an inpatient stroke unit: a thematic analysis.

PURPOSE: Oral care is essential to support the health and rehabilitation of stroke survivors but is often performed poorly in the inpatient setting. This article examines the current practices, barriers and facilitators to oral care on an Australian stroke unit to inform future knowledge translation interventions within this setting. METHODS: Qualitative semi-structured interviews were conducted with nursing staff members and speech-language pathologists with experience delivering oral care to stroke survivors. Reflexive thematic analysis was used to analyse the data. RESULTS: Three themes were developed from the data. In the first theme, participants described current oral care practices within their stroke unit and acknowledged that there is room for improvement. In the second theme, participants identified the multiple overlapping barriers to oral care delivery on the ward. Finally, the third theme explored participants' suggestions for improving oral care, including both local and organisational change. CONCLUSIONS: This study illustrates the complexities of oral care in the inpatient stroke setting and gives voice to the perspectives of nursing and speech-language pathology staff. It is clear that future oral health interventions in this setting require a comprehensive approach to addressing barriers and should prioritise the concerns of staff delivering the care.IMPLICATIONS FOR REHABILITATIONThe delivery of good oral care post-stroke is essential, but in practice is difficult due to limited time and supplies, inadequate education, fear of harm to patient and self, and perceived low priority of the care.Speech-language pathologists are often involved in oral care, but their role is not clearly delineated, and they are not provided with formal training in oral care provision.Oral care interventions should be comprehensive, including organisational policies, clear practice guidelines, structured multi-disciplinary education, and appropriate supplies.

A unique class of Zn2+-binding serine-based PBPs underlies cephalosporin resistance and sporogenesis in Clostridioides difficile.

Treatment with ß-lactam antibiotics, particularly cephalosporins, is a major risk factor for Clostridioides difficile infection. These broad-spectrum antibiotics irreversibly inhibit penicillin-binding proteins (PBPs), which are serine-based enzymes that assemble the bacterial cell wall. However, C. difficile has four different PBPs (PBP1-3 and SpoVD) with various roles in growth and spore formation, and their specific links to ß-lactam resistance in this pathogen are underexplored. Here, we show that PBP2 (known to be essential for vegetative growth) is the primary bactericidal target for ß-lactams in C. difficile. PBP2 is insensitive to cephalosporin inhibition, and this appears to be the main basis for cephalosporin resistance in this organism. We determine crystal structures of C. difficile PBP2, alone and in complex with ß-lactams, revealing unique features including ligand-induced conformational changes and an active site Zn2+-binding motif that influences ß-lactam binding and protein stability. The Zn2+-binding motif is also present in C. difficile PBP3 and SpoVD (which are known to be essential for sporulation), as well as in other bacterial taxa including species living in extreme environments and the human gut. We speculate that this thiol-containing motif and its cognate Zn2+ might function as a redox sensor to regulate cell wall synthesis for survival in adverse or anaerobic environments.

GpsB Coordinates Cell Division and Cell Surface Decoration by Wall Teichoic Acids in Staphylococcus aureus.

Bacterial cell division is a complex and highly regulated process requiring the coordination of many different proteins. Despite substantial work in model organisms, our understanding of the systems regulating cell division in noncanonical organisms, including critical human pathogens, is far from complete. One such organism is Staphylococcus aureus, a spherical bacterium that lacks known cell division regulatory proteins. Recent studies on GpsB, a protein conserved within the Firmicutes phylum, have provided insight into cell division regulation in S. aureus and other related organisms. It has been revealed that GpsB coordinates cell division and cell wall synthesis in multiple species. In S. aureus, we have previously shown that GpsB directly regulates FtsZ polymerization. In this study, using Bacillus subtilis as a tool, we isolated spontaneous suppressors that abrogate the lethality of S. aureus GpsB overproduction in B. subtilis. Through characterization, we identified several residues important for the function of GpsB. Furthermore, we discovered an additional role for GpsB in wall teichoic acid (WTA) biosynthesis in S. aureus. Specifically, we show that GpsB directly interacts with the WTA export protein TarG. We also identified a region in GpsB that is crucial for this interaction. Analysis of TarG localization in S. aureus suggests that WTA machinery is part of the divisome complex. Taken together, this research illustrates how GpsB performs an essential function in S. aureus by directly linking the tightly regulated cell cycle processes of cell division and WTA-mediated cell surface decoration. IMPORTANCE Cytokinesis in bacteria involves an intricate orchestration of several key cell division proteins and other factors involved in building a robust cell envelope. Presence of teichoic acids is a signature characteristic of the Gram-positive cell wall. By characterizing the role of Staphylococcus aureus GpsB, an essential cell division protein in this organism, we have uncovered an additional role for GpsB in wall teichoic acid (WTA) biosynthesis. We show that GpsB directly interacts with TarG of the WTA export complex. We also show that this function of GpsB may be conserved in other GpsB homologs as GpsB and the WTA exporter complex follow similar localization patterns. It has been suggested that WTA acts as a molecular signal to control the activity of autolytic enzymes, especially during the separation of conjoined daughter cells. Thus, our results reveal that GpsB, in addition to playing a role in cell division, may also help coordinate WTA biogenesis.

Phenogenomic Characterization of a Newly Domesticated and Novel Species from the Genus Verrucosispora.

The concept of bacterial dark matter stems from our inability to culture most microbes and represents a fundamental gap in our knowledge of microbial diversity. Here, we present the domestication of such an organism: a previously uncultured, novel species from the rare Actinomycetes genus Verrucosispora. Although initial recovery took >4 months, isolation of phenotypically distinct, domesticated generations occurred within weeks. Two isolates were subjected to phenogenomic analyses, revealing domestication correlated with enhanced growth rates in nutrient-rich media but diminished capacity to metabolize diverse amino acids. This is seemingly mediated by genomic atrophy through a mixed approach of pseudogenization and reversion of pseudogenization of amino acid metabolism genes. Conversely, later generational strains had enhanced spore germination rates, potentially through the reversion of a sporulation-associated kinase from pseudogene to true gene status. We observed that our most wild-type isolate had the greatest potential for antibacterial activity, which correlated with extensive mutational attrition of biosynthetic gene clusters in domesticated strains. Comparative analyses revealed wholesale genomic reordering in strains, with widespread single nucleotide polymorphism, indel, and pseudogene-impactful mutations observed. We hypothesize that domestication of this previously unculturable organism resulted from the shedding of genomic flexibility required for life in a dynamic marine environment, parsing out genetic redundancy to allow for a newfound cultivable amenability. IMPORTANCE The majority of environmental bacteria cannot be cultured within the laboratory. Understanding why only certain environmental isolates can be recovered is key to unlocking the abundant microbial dark matter that is widespread on our planet. In this study, we present not only the culturing but domestication of just such an organism. Although initial recovery took >4 months, we were able to isolate distinct, subpassaged offspring from the originating colony within mere weeks. A phenotypic and genotypic analysis of our generational strains revealed that adaptation to life in the lab occurred as a result of wholesale mutational changes. These permitted an enhanced ability for growth in nutrient rich media but came at the expense of reduced genomic flexibility. We suggest that without dynamic natural environmental stressors our domesticated strains effectively underwent genomic atrophy as they adapted to static conditions experienced in the laboratory.

Hematopoietic Stem Cell Transplantation to Treat Leukodystrophies: Clinical Practice Guidelines from the Hunter's Hope Leukodystrophy Care Network.

The leukodystrophies are a heterogeneous group of inherited diseases characterized by progressive demyelination of the central nervous system leading to devastating neurologic symptoms and premature death. Hematopoietic stem cell transplantation (HSCT) has been successfully used to treat certain leukodystrophies, including adrenoleukodystrophy, globoid leukodystrophy (Krabbe disease), and metachromatic leukodystrophy, over the past 30 years. To date, these complex patients have primarily been transplanted at a limited number of pediatric centers. As the number of cases identified through pregnancy and newborn screening is increasing, additional centers will be required to treat these children. Hunter's Hope created the Leukodystrophy Care Network in part to create and standardize high-quality clinical practice guidelines to guide the care of affected patients. In this report the clinical guidelines for the care of pediatric patients with leukodystrophies undergoing treatment with HSCT are presented. The initial transplant evaluation, determination of patient eligibility, donor selection, conditioning, supportive care, and post-transplant follow-up are discussed. Throughout these guidelines the need for early detection and treatment and the role of the partnership between families and multidisciplinary providers are emphasized.

¡vIVA la DivIVA!

Reproduction in the bacterial kingdom predominantly occurs through binary fission-a process in which one parental cell is divided into two similarly sized daughter cells. How cell division, in conjunction with cell elongation and chromosome segregation, is orchestrated by a multitude of proteins has been an active area of research spanning the past few decades. Together, the monumental endeavors of multiple laboratories have identified several cell division and cell shape regulators as well as their underlying regulatory mechanisms in rod-shaped Escherichia coli and Bacillus subtilis, which serve as model organisms for Gram-negative and Gram-positive bacteria, respectively. Yet our understanding of bacterial cell division and morphology regulation is far from complete, especially in noncanonical and non-rod-shaped organisms. In this review, we focus on two proteins that are highly conserved in Gram-positive organisms, DivIVA and its homolog GpsB, and attempt to summarize the recent advances in this area of research and discuss their various roles in cell division, cell growth, and chromosome segregation in addition to their interactome and posttranslational regulation.

Extended-Duration Use of Direct Oral Anticoagulants to Prevent VTE in Acutely III Medical Patients.

OBJECTIVE: To evaluate current clinical evidence for the use of direct oral anticoagulants (DOACs) for extended-duration thromboprophylaxis in the acutely ill medical population for venous thromboembolism (VTE) and bleeding events.
DATA SOURCES: Were obtained through a MEDLINE/PubMed search for clinical trials conducted from March 2008 to 2018 using relevant key words. Limitations of English and human subjects were applied to search results.
STUDY SELECTION AND DATA EXTRACTION: Forty-one articles were identified, and abstracts reviewed by the authors for inclusion of the study population (acutely ill medical patients) and VTE outcomes. Clinical studies evaluating the use of DOACs for extended duration of VTE prevention in acutely ill medical patients were included in the review, resulting in three clinical trials and two subgroup analyses. The participants enrolled had an overall mean age of 71.4 years.
DATA SYNTHESIS: The DOAC trials collectively demonstrated a positive outcome in composite endpoints of VTE prevention with extended-duration thromboprophylaxis in acutely ill medical patients compared with enoxaparin. As for safety, rivaroxaban and apixaban trials reported more major bleeding events compared with enoxaparin. The betrixaban trial demonstrated no difference in bleeding compared with enoxaparin.
CONCLUSION: DOACs reduced the number of VTE events in acutely ill medical patients on extended-duration thromboprophylaxis, but with an overall increased bleeding risk. An individualized patient approach based on risk factors should be utilized for treatment with extended-duration DOAC in the older adult population with recent hospitalization.

Report of the 2018-2019 Student Affairs Standing Committee.

The 2018-2019 Student Affairs Standing Committee addressed charges related to examining the institutional leadership models and professional development needs of faculty and staff to optimize achievement of Strategic Priority #1 on the applicant pipeline. The report provides five recommendations to AACP and twelve suggestions for colleges and schools of pharmacy. The committee focused on the need to develop tailored leadership training and mentoring programs for admissions personnel on relevant topics, including change management, holistic thinking, leadership, problem solving, technical knowledge, professional development, paths for promotion, conflict resolution, networking, persuasive communication, and strategic planning. Rather than develop new resources, the committee identified existing professional competencies and assessment resources developed by other organizations for student affairs and admissions personnel that could spur enhanced strategic marketing and professional development opportunities in pharmacy education. It also reaffirmed the need for student diversity and the use of data to drive strategic decisions in recruitment. To identify gaps in knowledge among AACP member institutions, the committee analyzed the results of its fall 2018 survey on the current depth and breadth of student recruitment activities and their perceived effectiveness. The committee also recommended ways institutions can encourage faculty and others outside of the admissions office to participate in student recruitment activities. Finally, the committee concluded that it will be necessary for colleges and schools to collaborate across the academy to promote the benefits of pharmacy profession to prospective students, rather than individual colleges and schools of pharmacy, and be more responsive to the expectations of Gen Z students before the tide in applications will shift in a positive direction.

Respiratory sinus arrhythmia and adaptive emotion regulation as predictors of nonsuicidal self-injury in young adults.

Research suggests that both respiratory sinus arrhythmia (RSA) and emotion regulation are related to maladaptive outcomes. However, little is known about how these factors jointly contribute to nonsuicidal self-injury (NSSI). We examined how resting RSA and RSA withdrawal to stress predicted NSSI among young adults, and whether adaptive emotion regulation strategies moderated this effect. We examined this relation in a sample of 70 young adults (Mage = 19.25, SDage = 0.92, 94% female), 35 with a recent history of NSSI and 35 who had never engaged in NSSI. Participants completed a baseline questionnaire and lab visit where physiological responses to a social stressor were collected. Results indicated that resting RSA did not predict recent NSSI engagement. However, greater RSA withdrawal to social stress significantly predicted recent NSSI (ß = 1.35 [0.57], Wald χ2 = 5.52 p = 0.02). Further, problem-solving moderated the relation between RSA withdrawal and recent NSSI such that individuals with greater levels of RSA withdrawal were significantly more likely to engage in NSSI if they also reported low problem-solving (ß = -1.44 [0.64], Wald χ2 = 5.16, p = 0.02). In contrast, the effect of greater RSA withdrawal on NSSI was buffered by high problem-solving. Cognitive reappraisal did not moderate the relation between RSA withdrawal and NSSI. These findings suggest that problem-solving may be an important moderator in the relation between physiological reactivity and NSSI. Interventions should help clients with poor self-regulation develop adaptive problem-solving strategies.

Radiation dosimetry in cell biology: comparison of calculated and measured absorbed dose for a range of culture vessels and clinical beam qualities.

PURPOSE: Cell culture studies are frequently used to evaluate the effects of cancer treatments such as radiotherapy, hormone therapy, chemotherapy, nanoparticle enhancement, and to determine any synergies between the treatments. To achieve valid results, the absorbed dose of each therapy needs to be well known and controlled. In this study, we aim to determine the uncertainty associated with radiation exposure in different experimental conditions. MATERIALS AND METHODS: We have performed an in-depth evaluation of the absorbed dose and dose distribution that would be delivered to a cell sample when cultivated in a number of the more popular designs of culture vessels. We focus on exposure to two beam types: a kilovoltage x-ray beam and a megavoltage photon beam, both of which are routinely used to treat cancer patients in the clinical environment. CONCLUSIONS: Our results identify large variations of up to 16% in the absorbed dose across multi-well culture plates, which if ignored in radiobiological experiments, have the potential to lead to erroneous conclusions.