Hypoxia Resistance Is an Inherent Phenotype of the Mouse Flexor Digitorum Brevis Skeletal Muscle.

The various functions of skeletal muscle (movement, respiration, thermogenesis, etc.) require the presence of oxygen (O2). Inadequate O2 bioavailability (ie, hypoxia) is detrimental to muscle function and, in chronic cases, can result in muscle wasting. Current therapeutic interventions have proven largely ineffective to rescue skeletal muscle from hypoxic damage. However, our lab has identified a mammalian skeletal muscle that maintains proper physiological function in an environment depleted of O2. Using mouse models of in vivo hindlimb ischemia and ex vivo anoxia exposure, we observed the preservation of force production in the flexor digitorum brevis (FDB), while in contrast the extensor digitorum longus (EDL) and soleus muscles suffered loss of force output. Unlike other muscles, we found that the FDB phenotype is not dependent on mitochondria, which partially explains the hypoxia resistance. Muscle proteomes were interrogated using a discovery-based approach, which identified significantly greater expression of the transmembrane glucose transporter GLUT1 in the FDB as compared to the EDL and soleus. Through loss-and-gain-of-function approaches, we determined that GLUT1 is necessary for the FDB to survive hypoxia, but overexpression of GLUT1 was insufficient to rescue other skeletal muscles from hypoxic damage. Collectively, the data demonstrate that the FDB is uniquely resistant to hypoxic insults. Defining the mechanisms that explain the phenotype may provide insight towards developing approaches for preventing hypoxia-induced tissue damage.

Isometric skeletal muscle contractile properties in common strains of male laboratory mice.

Assessing contractile function of skeletal muscle in murine models is a commonly employed laboratory technique that investigators utilize to measure the impact of genetic manipulations, drug efficacy, or other therapeutic interventions. Often overlooked is the potential for the strain of the mouse to influence the functional properties of the skeletal muscle. Thus, we sought to characterize commonly assessed isometric force measures in the hindlimb muscles across a variety of mouse strains. Using 6-8-week-old male mice, we measured isometric force, fatigue susceptibility, relaxation kinetics, muscle mass, myofiber cross-sectional area, and fiber type composition of the extensor digitorum longus (EDL) and soleus muscles in C57BL/6NJ, BALB/cJ, FVB/NJ, C57BL/6J, and C57BL/10 mice. The data demonstrate both unique differences and a number of similarities between both muscles in the various genetic backgrounds of mice. Soleus muscle specific force (i.e., force per unit size) exhibited higher variation across strains while specific force of the EDL muscle exhibited minimal variation. In contrast, absolute force differed only in a few mouse strains whereas analysis of muscle morphology revealed many distinctions when compared across all the groups. Collectively, the data suggest that the strain of the mouse can potentially influence the measured biological outcome and may possibly promote a synergistic effect with any genetic manipulation or therapeutic intervention. Thus, it is critical for the investigator to carefully consider the genetic background of the mouse used in the experimental design and precisely document the strain of mouse employed during publication.

Acinetobacter baumannii Regulates Its Stress Responses via the BfmRS Two-Component Regulatory System.

Acinetobacter baumannii is a common nosocomial pathogen that utilizes numerous mechanisms to aid its survival in both the environment and the host. Coordination of such mechanisms requires an intricate regulatory network. We report here that A. baumannii can directly regulate several stress-related pathways via the two-component regulatory system BfmRS. Similar to previous studies, results from transcriptomic analysis showed that mutation of the BfmR response regulator causes dysregulation of genes required for the oxidative stress response, the osmotic stress response, the misfolded protein/heat shock response, Csu pilus/fimbria production, and capsular polysaccharide biosynthesis. We also found that the BfmRS system is involved in controlling siderophore biosynthesis and transport, and type IV pili production. We provide evidence that BfmR binds to various stress-related promoter regions and show that BfmR alone can directly activate transcription of some stress-related genes. Additionally, we show that the BfmS sensor kinase acts as a BfmR phosphatase to negatively regulate BfmR activity. This work highlights the importance of the BfmRS system in promoting survival of A. baumannii. IMPORTANCE Acinetobacter baumannii is a nosocomial pathogen that has extremely high rates of multidrug resistance. This organism's ability to endure stressful conditions is a key part of its ability to spread in the hospital environment and cause infections. Unlike other members of the gammaproteobacteria, A. baumannii does not encode a homolog of the RpoS sigma factor to coordinate its stress response. Here, we demonstrate that the BfmRS two-component system directly controls the expression of multiple stress resistance genes. Our findings suggest that BfmRS is central to a unique scheme of general stress response regulation by A. baumannii.

Structure of the Acinetobacter baumannii PmrA receiver domain and insights into clinical mutants affecting DNA binding and promoting colistin resistance.

Acinetobacter baumannii is an insidious emerging nosocomial pathogen that has developed resistance to all available antimicrobials, including the last resort antibiotic, colistin. Colistin resistance often occurs due to mutations in the PmrAB two-component regulatory system. To better understand the regulatory mechanisms contributing to colistin resistance, we have biochemically characterized the A. baumannii PmrA response regulator. Initial DNA-binding analysis shows that A. baumannii PmrA bound to the Klebsiella pneumoniae PmrA box motif. This prompted analysis of the putative A. baumannii PmrAB regulon that indicated that the A. baumannii PmrA consensus box is 5'-HTTAAD N5 HTTAAD. Additionally, we provide the first structural information for the A. baumannii PmrA N-terminal domain through X-ray crystallography and we present a full-length model using molecular modelling. From these studies, we were able to infer the effects of two critical PmrA mutations, PmrA::I13M and PmrA::P102R, both of which confer increased colistin resistance. Based on these data, we suggest structural and dynamic reasons for how these mutations can affect PmrA function and hence encourage resistive traits. Understanding these mechanisms will aid in the development of new targeted antimicrobial therapies. Graphical Abstract.

Skeletal muscle wasting: the estrogen side of sexual dimorphism.

The importance of defining sex differences across various biological and physiological mechanisms is more pervasive now than it has been over the past 15-20 years. As the muscle biology field pushes to identify small molecules and interventions to prevent, attenuate, or even reverse muscle wasting, we must consider the effect of sex as a biological variable. It should not be assumed that a therapeutic will affect males and females with equal efficacy or equivalent target affinities under conditions where muscle wasting is observed. With that said, it is not surprising to find that we have an unclear or even a poor understanding of the effects of sex or sex hormones on muscle wasting conditions. Although recent investigations are beginning to establish experimental approaches that will allow investigators to assess the impact of sex-specific hormones on muscle wasting, the field still needs rigorous scientific tools that will allow the community to address critical hypotheses centered around sex hormones. The focus of this review is on female sex hormones, specifically estrogens, and the roles that these hormones and their receptors play in skeletal muscle wasting conditions. With the overall review goal of assembling the current knowledge in the area of sexual dimorphism driven by estrogens with an effort to provide insights to interested physiologists on necessary considerations when trying to assess models for potential sex differences in cellular and molecular mechanisms of muscle wasting.

Skeletal Muscle Function Is Dependent Upon BRCA1 to Maintain Genomic Stability.

Breast Cancer gene 1 (BRCA1) is a large, multifunctional protein that regulates a variety of mechanisms in multiple different tissues. Our work established that Brca1 is expressed in skeletal muscle and localizes to the mitochondria and nucleus. Here, we propose BRCA1 expression is critical for the maintenance of force production and mitochondrial respiration in skeletal muscle.

Cross-sectional analysis of US scope of practice laws and employed physician assistants.

OBJECTIVE: This study examined if the variation in physician assistant (PA) state scope of practice (SOP) laws across states are associated with number of employed PAs, PA demographics and PA/population ratio per state. The hypothesis was that less restrictive SOP laws will increase the demand for PAs and the number of PAs in a state. DESIGN: Retrospective cross-sectional analysis at three time points: 1998, 2008, 2017. SETTING: Fifty states and the District of Columbia. PARTICIPANTS: Employed PAs in 1998, 2008, 2017. METHODS: SOP laws were categorised as permissive, average and restrictive. Three national datasets were combined to allow for descriptive analysis of employed PAs by year and SOP categories. We used linear predictive models to generate and compare PA/population ratio least square means by SOP categories for each year. Models were adjusted for percent female PA and PAs mean age. RESULTS: There was a median PA/population ratio of 23 per 100 000 population in 1998 and 33 in 2017. A heterogeneous expansion of SOP laws was seen with 17 states defined as super expanders while 15 were never adopters. In 2017, comparing restrictive to permissive states showed that in adjusted models permissive SOP laws were associated with 11.7 (p .03) increase in ratio of employed PAs per 100 000 population, demonstrating that states with permissive SOP laws have an increased PA density. CONCLUSIONS: There has been steady growth in the mean PA/population ratio since the turn of the century. At the same time, PA SOP laws in the USA have expanded, with just 10 states remaining in the restrictive category. Permissive SOP laws are associated with an increase in the ratio of employed PAs per state population. As states work to meet the projected physician need, SOP expansion may be an important policy consideration to increase the PA workforce.

Genome Sequences for Two Acinetobacter baumannii Strains Obtained Using the Unicycler Hybrid Assembly Pipeline.

Here, we report a complete genome sequence for Acinetobacter baumannii strain ATCC 17961, with plasmid sequences, and a high-quality (>98% complete) build for A. baumannii strain AB09-003. These genome sequences were generated by combining short-read Illumina and long-read Oxford Nanopore MinION sequencing data using the Unicycler hybrid assembly pipeline.

CsrA Supports both Environmental Persistence and Host-Associated Growth of Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic and frequently multidrug-resistant Gram-negative bacterial pathogen that primarily infects critically ill individuals. Indirect transmission from patient to patient in hospitals can drive infections, supported by this organism's abilities to persist on dry surfaces and rapidly colonize susceptible individuals. To investigate how A. baumannii survives on surfaces, we cultured A. baumannii in liquid media for several days and then analyzed isolates that lost the ability to survive drying. One of these isolates carried a mutation that affected the gene encoding the carbon storage regulator CsrA. As we began to examine the role of CsrA in A. baumannii, we observed that the growth of ΔcsrA mutant strains was inhibited in the presence of amino acids. The ΔcsrA mutant strains had a reduced ability to survive drying and to form biofilms but an improved ability to tolerate increased osmolarity compared with the wild type. We also examined the importance of CsrA for A. baumannii virulence. The ΔcsrA mutant strains had a greatly reduced ability to kill Galleria mellonella larvae, could not replicate in G. mellonella hemolymph, and also had a growth defect in human serum. Together, these results show that CsrA is essential for the growth of A. baumannii on host-derived substrates and is involved in desiccation tolerance, implying that CsrA controls key functions involved in the transmission of A. baumannii in hospitals.

Desiccation tolerance in Acinetobacter baumannii is mediated by the two-component response regulator BfmR.

For the opportunistic pathogen Acinetobacter baumannii, desiccation tolerance is thought to contribute significantly to the persistence of these bacteria in the healthcare environment. We investigated the ability of A. baumannii to survive rapid drying, and found that some strains exhibited a profoundly desiccation-resistant phenotype, characterized by the ability of a large proportion of cells to survive on a dry surface for an extended period of time. However, this phenotype was only displayed during the stationary phase of growth. Most interestingly, we found that drying resistance could be lost after extended cultivation in liquid medium. Genome sequencing of isolates that became drying-sensitive identified mutations in bfmR, which encodes a two-component response regulator that is important for A. baumannii virulence. Additionally, BfmR was necessary for the expression of stress-related proteins during stationary phase, and one of these, KatE, was important for long-term drying survival. These results suggested that BfmR may control stress responses, and we demonstrated that the ΔbfmR mutant was more sensitive to hydrogen peroxide, nutrient starvation, and increased osmolarity. We also found that cross-protection against drying could be stimulated by either starvation, which required BfmR, or increased osmolarity. These results imply that BfmR plays a role in controlling stress responses in A. baumannii which help protect cells during desiccation, and they provide a regulatory link between this organism's ability to persist in the environment and pathogenicity.

Host suppression of quorum sensing during catheter-associated urinary tract infections.

Chronic bacterial infections on medical devices, including catheter-associated urinary tract infections (CAUTI), are associated with bacterial biofilm communities that are refractory to antibiotic therapy and resistant to host immunity. Previously, we have shown that Pseudomonas aeruginosa can cause CAUTI by forming a device-associated biofilm that is independent of known biofilm exopolysaccharides. Here, we show by RNA-seq that host urine alters the transcriptome of P. aeruginosa by suppressing quorum sensing regulated genes. P. aeruginosa produces acyl homoserine lactones (AHLs) in the presence of urea, but cannot perceive AHLs. Repression of quorum sensing by urine implies that quorum sensing should be dispensable during infection of the urinary tract. Indeed, mutants defective in quorum sensing are able to colonize similarly to wild-type in a murine model of CAUTI. Quorum sensing-regulated processes in clinical isolates are also inhibited by urea. These data show that urea in urine is a natural anti-quorum sensing mechanism in mammals.

PsrA controls the synthesis of the Pseudomonas aeruginosa quinolone signal via repression of the FadE homolog, PA0506.

Pseudomonas aeruginosa is a ubiquitous, Gram-negative opportunistic pathogen that can cause disease in various sites within the human body. This bacterium is a major source of nosocomial infections that are often difficult to treat due to high intrinsic antibiotic resistance and coordinated virulence factor production. P. aeruginosa utilizes three cell-to-cell signaling systems to regulate numerous genes in response to cell density. One of these systems utilizes the small molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal [PQS]) as a signal that acts as a co-inducer for the transcriptional regulator PqsR. Quinolone signaling is required for virulence in multiple infection models, and PQS is produced during human infections, making this system an attractive target for potential drug development. In this study we have examined the role of a TetR-type transcriptional regulator, PsrA, in the regulation of PQS production by P. aeruginosa. Previous studies showed that PsrA regulates genes of the fatty acid ß-oxidation pathway, including PA0506, which encodes a FadE homolog. In this report, we show that deletion of psrA resulted in a large decrease in PQS production and that co-deletion of PA0506 allowed PQS production to be restored to a wild type level. We also found that PQS production could be restored to the psrA mutant by the addition of oleic or octanoic acid. Taken together, our data suggest that psrA positively affects PQS production by repressing the transcription of PA0506, which leads to a decrease in the conversion of acyl-CoA compounds to enoyl-CoA compounds, thereby allowing some octanoyl-CoA to escape the ß-oxidation pathway and serve as a PQS precursor.

Redox Sensing within the Genus Shewanella.

A novel bacterial behavior called congregation was recently described in Shewanella oneidensis MR-1 as the accumulation of cells around insoluble electron acceptors (IEA). It is the result of a series of "run-and-reversal" events enabled by modulation of swimming speed and direction. The model proposed that the swimming cells constantly sense their surroundings with specialized outer membrane cytochromes capable of extracellular electron transport (EET). Up to this point, neither the congregation nor attachment behavior have been studied in any other strains. In this study, the wild type of S. oneidensis MR-1 and several deletion mutants as well as eight other Shewanella strains (Shewanella putrefaciens CN32, S. sp. ANA-3, S. sp. W3-18-1, Shewanella amazonensis SB2B, Shewanella loihica PV-4, Shewanella denitrificans OS217, Shewanella baltica OS155, and Shewanella frigidimarina NCIMB400) were screened for the ability to congregate. To monitor congregation and attachment, specialized cell-tracking techniques, as well as a novel cell accumulation after photo-bleaching (CAAP) confocal microscopy technique were utilized in this study. We found a strong correlation between the ability of strain MR-1 to accumulate on mineral surface and the presence of key EET genes such as mtrBC/omcA (SO_1778, SO_1776, and SO_1779) and gene coding for methyl-accepting protein (MCPs) with Ca+ channel chemotaxis receptor (Cache) domain (SO_2240). These EET and taxis genes were previously identified as essential for characteristic run and reversal swimming around IEA surfaces. CN32, ANA-3, and PV-4 congregated around both Fe(OH)3 and MnO2. Two other Shewanella spp. showed preferences for one oxide over the other: preferences that correlated with the metal content of the environments from which the strains were isolated: e.g., W3-18-1, which was isolated from an iron-rich habitat congregated and attached preferentially to Fe(OH)3, while SB2B, which was isolated from a MnO2-rich environment, preferred MnO2.

Distal and proximal promoters co-regulate pqsR expression in Pseudomonas aeruginosa.

The ubiquitous bacterium Pseudomonas aeruginosa is an opportunistic pathogen that can cause serious infections in immunocompromised individuals. P. aeruginosa virulence is controlled partly by intercellular communication, and the transcription factor PqsR is a necessary component in the P. aeruginosa cell-to-cell signaling network. PqsR acts as the receptor for the Pseudomonas quinolone signal, and it controls the production of 2-alkyl-4-quinolone molecules which are important for pathogenicity. Previous studies showed that the expression of pqsR is positively controlled by the quorum-sensing regulator LasR, but it was unclear how LasR is able to induce pqsR transcription. In this report, we further investigated the control of pqsR, and discovered two separate promoter sites that contribute to pqsR expression. LasR-mediated activation occurs at the distal promoter site, but this activation can be antagonized by the regulator CysB. The proximal promoter site also contributes to pqsR transcription, but initiation at this site is inhibited by a negative regulatory sequence element, and potentially by the H-NS family members MvaT and MvaU. We propose a model where positive and negative regulatory influences at each promoter site are integrated to modify pqsR expression. This arrangement could allow for information from both environmental signals and cell-to-cell communication to influence PqsR levels.

Xpert MTB/RIF Assay Shows Faster Clearance of Mycobacterium tuberculosis DNA with Higher Levels of Rifapentine Exposure.

The Xpert MTB/RIF assay is both sensitive and specific as a diagnostic test. Xpert also reports quantitative output in cycle threshold (CT) values, which may provide a dynamic measure of sputum bacillary burden when used longitudinally. We evaluated the relationship between Xpert CT trajectory and drug exposure during tuberculosis (TB) treatment to assess the potential utility of Xpert CT for treatment monitoring. We obtained serial sputum samples from patients with smear-positive pulmonary TB who were consecutively enrolled at 10 international clinical trial sites participating in study 29X, a CDC-sponsored Tuberculosis Trials Consortium study evaluating the tolerability, safety, and antimicrobial activity of rifapentine at daily doses of up to 20 mg/kg of body weight. Xpert was performed at weeks 0, 2, 4, 6, 8, and 12. Longitudinal CT data were modeled using a nonlinear mixed effects model in relation to rifapentine exposure (area under the concentration-time curve [AUC]). The rate of change of CT was higher in subjects receiving rifapentine than in subjects receiving standard-dose rifampin. Moreover, rifapentine exposure, but not assigned dose, was significantly associated with rate of change in CT (P = 0.02). The estimated increase in CT slope for every additional 100 µg · h/ml of rifapentine drug exposure (as measured by AUC) was 0.11 CT/week (95% confidence interval [CI], 0.05 to 0.17). Increasing rifapentine exposure is associated with a higher rate of change of Xpert CT, indicating faster clearance of Mycobacterium tuberculosis DNA. These data suggest that the quantitative outputs of the Xpert MTB/RIF assay may be useful as a dynamic measure of TB treatment response.

Designed Small-Molecule Inhibitors of the Anthranilyl-CoA Synthetase PqsA Block Quinolone Biosynthesis in Pseudomonas aeruginosa.

The Gram-negative bacterial pathogen Pseudomonas aeruginosa uses three interconnected intercellular signaling systems regulated by the transcription factors LasR, RhlR, and MvfR (PqsR), which mediate bacterial cell-cell communication via small-molecule natural products and control the production of a variety of virulence factors. The MvfR system is activated by and controls the biosynthesis of the quinolone quorum sensing factors HHQ and PQS. A key step in the biosynthesis of these quinolones is catalyzed by the anthranilyl-CoA synthetase PqsA. To develop inhibitors of PqsA as novel potential antivirulence antibiotics, we report herein the design and synthesis of sulfonyladeonsine-based mimics of the anthranilyl-AMP reaction intermediate that is bound tightly by PqsA. Biochemical, microbiological, and pharmacological studies identified two potent PqsA inhibitors, anthranilyl-AMS (1) and anthranilyl-AMSN (2), that decreased HHQ and PQS production in P. aeruginosa strain PA14. However, these compounds did not inhibit production of the virulence factor pyocyanin. Moreover, they exhibited limited bacterial penetration in compound accumulation studies. This work provides the most potent PqsA inhibitors reported to date and sets the stage for future efforts to develop analogues with improved cellular activity to investigate further the complex relationships between quinolone biosynthesis and virulence factor production in P. aeruginosa and the therapeutic potential of targeting PqsA.

In situ Detection of Microbial Life in the Deep Biosphere in Igneous Ocean Crust.

The deep biosphere is a major frontier to science. Recent studies have shown the presence and activity of cells in deep marine sediments and in the continental deep biosphere. Volcanic lavas in the deep ocean subsurface, through which substantial fluid flow occurs, present another potentially massive deep biosphere. We present results from the deployment of a novel in situ logging tool designed to detect microbial life harbored in a deep, native, borehole environment within igneous oceanic crust, using deep ultraviolet native fluorescence spectroscopy. Results demonstrate the predominance of microbial-like signatures within the borehole environment, with densities in the range of 10(5) cells/mL. Based on transport and flux models, we estimate that such a concentration of microbial cells could not be supported by transport through the crust, suggesting in situ growth of these communities.

CysB Negatively Affects the Transcription of pqsR and Pseudomonas Quinolone Signal Production in Pseudomonas aeruginosa.

UNLABELLED: Pseudomonas aeruginosa is a Gram-negative bacterium that is ubiquitous in the environment, and it is an opportunistic pathogen that can infect a variety of hosts, including humans. During the process of infection, P. aeruginosa coordinates the expression of numerous virulence factors through the production of multiple cell-to-cell signaling molecules. The production of these signaling molecules is linked through a regulatory network, with the signal N-(3-oxododecanoyl) homoserine lactone and its receptor LasR controlling the induction of a second acyl-homoserine lactone signal and the Pseudomonas quinolone signal (PQS). LasR-mediated control of PQS occurs partly by activating the transcription of pqsR, a gene that encodes the PQS receptor and is necessary for PQS production. We show that LasR interacts with a single binding site in the pqsR promoter region and that it does not influence the transcription of the divergently transcribed gene, nadA. Using DNA affinity chromatography, we identified additional proteins that interact with the pqsR-nadA intergenic region. These include the H-NS family members MvaT and MvaU, and CysB, a transcriptional regulator that controls sulfur uptake and cysteine biosynthesis. We show that CysB interacts with the pqsR promoter and that CysB represses pqsR transcription and PQS production. Additionally, we provide evidence that CysB can interfere with the activation of pqsR transcription by LasR. However, as seen with other CysB-regulated genes, pqsR expression was not differentially regulated in response to cysteine levels. These findings demonstrate a novel role for CysB in influencing cell-to-cell signal production by P. aeruginosa. IMPORTANCE: The production of PQS and other 4-hydroxy-2-alkylquinolone (HAQs) compounds is a key component of the P. aeruginosa cell-to-cell signaling network, impacts multiple physiological functions, and is required for virulence. PqsR directly regulates the genes necessary for HAQ production, but little is known about the regulation of pqsR. We identified CysB as a novel regulator of pqsR and PQS production, but, unlike other CysB-controlled genes, it does not appear to regulate pqsR in response to cysteine. This implies that CysB functions as both a cysteine-responsive and cysteine-unresponsive regulator in P. aeruginosa.

Post-transcriptional regulation of gene PA5507 controls Pseudomonas quinolone signal concentration in P. aeruginosa.

Pseudomonas aeruginosa can sense and respond to a myriad of environmental signals and utilizes a system of small molecules to communicate through intercellular signaling. The small molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal [PQS]) is one of these signals and its synthesis is important for virulence. Previously, we identified an RpiR-type transcriptional regulator, QapR, that positively affects PQS production by repressing the qapR operon. An in-frame deletion of this regulator caused P. aeruginosa to produce a greatly reduced concentration of PQS. Here, we report that QapR translation is linked to the downstream gene PA5507. We found that introduction of a premature stop codon within qapR eliminates transcriptional autorepression of the qapR operon as expected but has no effect on PQS concentration. This was investigated with a series of lacZ reporter fusions which showed that translation of QapR must terminate at, or close to, the native qapR stop codon in order for translation of PA5507 to occur. Also, it was shown that truncation of the 5' end of the qapR transcript permitted PA5507 translation without translation of QapR. Our findings led us to conclude that PA5507 transcription and translation are both tightly controlled by QapR and this control is important for PQS homeostasis.