Cell constriction requires processive septal peptidoglycan synthase movement independent of FtsZ treadmilling in Staphylococcus aureus.

Bacterial cell division requires recruitment of peptidoglycan (PG) synthases to the division site by the tubulin homologue, FtsZ. Septal PG synthases promote septum growth. FtsZ treadmilling is proposed to drive the processive movement of septal PG synthases and septal constriction in some bacteria; however, the precise mechanisms spatio-temporally regulating PG synthase movement and activity and FtsZ treadmilling are poorly understood. Here using single-molecule imaging of division proteins in the Gram-positive pathogen Staphylococcus aureus, we showed that the septal PG synthase complex FtsW/PBP1 and its putative activator protein, DivIB, move with similar velocity around the division site. Impairing FtsZ treadmilling did not affect FtsW or DivIB velocities or septum constriction rates. Contrarily, PG synthesis inhibition decelerated or stopped directional movement of FtsW and DivIB, and septum constriction. Our findings suggest that a single population of processively moving FtsW/PBP1 associated with DivIB drives cell constriction independently of FtsZ treadmilling in S. aureus.

The role of GpsB in Staphylococcus aureus cell morphogenesis.

For decades, cells of the Gram-positive bacterial pathogen Staphylococcus aureus were thought to lack a dedicated elongation machinery. However, S. aureus cells were recently shown to elongate before division, in a process that requires a shape elongation division and sporulation (SEDS)/penicillin-binding protein (PBP) pair for peptidoglycan synthesis, consisting of the glycosyltransferase RodA and the transpeptidase PBP3. In ovococci and rod-shaped bacteria, the elongation machinery, or elongasome, is composed of various proteins besides a dedicated SEDS/PBP pair. To identify proteins required for S. aureus elongation, we screened the Nebraska Transposon Mutant Library, which contains transposon mutants in virtually all non-essential staphylococcal genes, for mutants with modified cell shape. We confirmed the roles of RodA/PBP3 in S. aureus elongation and identified GpsB, SsaA, and RodZ as additional proteins involved in this process. The gpsB mutant showed the strongest phenotype, mediated by the partial delocalization from the division septum of PBP2 and PBP4, two penicillin-binding proteins that synthesize and cross-link peptidoglycan. Increased levels of these PBPs at the cell periphery versus the septum result in higher levels of peptidoglycan insertion/crosslinking throughout the entire cell, possibly overriding the RodA/PBP3-mediated peptidoglycan synthesis at the outer edge of the septum and/or increasing stiffness of the peripheral wall, impairing elongation. Consequently, in the absence of GpsB, S. aureus cells become more spherical. We propose that GpsB has a role in the spatio-temporal regulation of PBP2 and PBP4 at the septum versus cell periphery, contributing to the maintenance of the correct cell morphology in S. aureus. IMPORTANCE: Staphylococcus aureus is a Gram-positive clinical pathogen, which is currently the second cause of death by antibiotic-resistant infections worldwide. For decades, S. aureus cells were thought to be spherical and lack the ability to undergo elongation. However, super-resolution microscopy techniques allowed us to observe the minor morphological changes that occur during the cell cycle of this pathogen, including cell elongation. S. aureus elongation is not required for normal growth in laboratory conditions. However, it seems to be essential in the context of some infections, such as osteomyelitis, during which S. aureus cells apparently elongate to invade small channels in the bones. In this work, we uncovered new determinants required for S. aureus cell elongation. In particular, we show that GpsB has an important role in the spatio-temporal regulation of PBP2 and PBP4, two proteins involved in peptidoglycan synthesis, contributing to the maintenance of the correct cell morphology in S. aureus.

First insights into the shortfin mako shark (Isurus oxyrinchus) fine-scale swimming behaviour.

As regional endotherms, lamnid sharks can sustain high cruising speeds and perform frequent speed bursts. However, since endothermy comes with high energetic costs, lamnids may adopt different swimming strategies to manage their energy budget. Understanding such strategies is essential to provide behavioural and physiological context to their broader movement ecology. The endangered shortfin mako (Isurus oxyrinchus) possibly has the highest energy requirements among lamnids, but our understanding of its swimming behaviour is still limited. We equipped three shortfin mako sharks with high-resolution multi-sensor tags to measure their swimming kinematics in the wild. While swimming horizontally, individuals favoured tail-beat frequencies around 0.6 Hz at speeds comparable to those of ectothermic sharks (ca 0.5 m s-1). All individuals displayed yo-yo-like diving patterns where, for a given tail-beat frequency, speeds were higher during descents, as expected for a negatively buoyant fish. Contrary to what was expected, gliding was almost absent (less than 1.31%). Speed bursts reaching up to 3.6 m s-1 were observed during the day but ceased shortly after dusk, implying a diel change in swimming behaviour. As large-scale research efforts are hindered by this species' increasing rarity, opportunistic high-resolution datasets, like the present, are fundamental to improve our understanding of shortfin mako's behaviour and ecology.

Cell division protein FtsK coordinates bacterial chromosome segregation and daughter cell separation in Staphylococcus aureus.

Unregulated cell cycle progression may have lethal consequences and therefore, bacteria have various mechanisms in place for the precise spatiotemporal control of cell cycle events. We have uncovered a new link between chromosome replication/segregation and splitting of the division septum. We show that the DNA translocase domain-containing divisome protein FtsK regulates cellular levels of a peptidoglycan hydrolase Sle1, which is involved in cell separation in the bacterial pathogen Staphylococcus aureus. FtsK interacts with a chaperone (trigger factor, TF) and establishes a FtsK-dependent TF concentration gradient that is higher in the septal region. Trigger factor binds Sle1 and promotes its preferential export at the septal region, while also preventing Sle1 degradation by the ClpXP proteolytic machinery. Upon conditions that lead to paused septum synthesis, such as DNA damage or impaired DNA replication/segregation, TF gradient is dissipated and Sle1 levels are reduced, thus halting premature septum splitting.

Fast4DReg - fast registration of 4D microscopy datasets.

Unwanted sample drift is a common issue that plagues microscopy experiments, preventing accurate temporal visualization and quantification of biological processes. Although multiple methods and tools exist to correct images post acquisition, performing drift correction of three-dimensional (3D) videos using open-source solutions remains challenging and time consuming. Here, we present a new tool developed for ImageJ or Fiji called Fast4DReg that can quickly correct axial and lateral drift in 3D video-microscopy datasets. Fast4DReg works by creating intensity projections along multiple axes and estimating the drift between frames using two-dimensional cross-correlations. Using synthetic and acquired datasets, we demonstrate that Fast4DReg can perform better than other state-of-the-art open-source drift-correction tools and significantly outperforms them in speed. We also demonstrate that Fast4DReg can be used to register misaligned channels in 3D using either calibration slides or misaligned images directly. Altogether, Fast4DReg provides a quick and easy-to-use method to correct 3D imaging data before further visualization and analysis.

Encapsulation of the septal cell wall protects Streptococcus pneumoniae from its major peptidoglycan hydrolase and host defenses.

Synthesis of the capsular polysaccharide, a major virulence factor for many pathogenic bacteria, is required for bacterial survival within the infected host. In Streptococcus pneumoniae, Wze, an autophosphorylating tyrosine kinase, and Wzd, a membrane protein required for Wze autophosphorylation, co-localize at the division septum and guarantee the presence of capsule at this subcellular location. To determine how bacteria regulate capsule synthesis, we studied pneumococcal proteins that interact with Wzd and Wze using bacterial two hybrid assays and fluorescence microscopy. We found that Wzd interacts with Wzg, the putative ligase that attaches capsule to the bacterial cell wall, and recruits it to the septal area. This interaction required residue V56 of Wzd and both the transmembrane regions and DNA-PPF domain of Wzg. When compared to the wild type, Wzd null pneumococci lack capsule at midcell, bind the peptidoglycan hydrolase LytA better and are more susceptible to LytA-induced lysis, and are less virulent in a zebrafish embryo infection model. In this manuscript, we propose that the Wzd/Wze pair guarantees full encapsulation of pneumococcal bacteria by recruiting Wzg to the division septum, ensuring that capsule attachment is coordinated with peptidoglycan synthesis. Impairing the encapsulation process, at localized subcellular sites, may facilitate elimination of bacteria by strategies that target the pneumococcal peptidoglycan.

Advancements and critical steps for statistical analyses in blood pressure response to resistance training in hypertensive older women: a methodological approach.

BACKGROUND: Repeated measures analysis of covariance and three-way analysis of variance with repeated measures are common statistical methods. For a valid interpretation of blood pressure (BP) response to exercise, a variety of additional statistical methods must be implemented. Four additional statistical methods are presented: technical error of measurement (SEM), smallest real difference (SRD), magnitude-based inference and mixed effect modeling technique (MEM). The aim of this perspective article is to demonstrate how to apply already known statistical analyses regarding BP responsiveness in order to improve interpretation and achieve higher reliability for future studies in exercise science. METHODS: A total of 27 hypertensive older women (aged 68.37 ± 5.55 years) participated in the present study. A whole-body resistance training (RT) program was performed on two nonconsecutive days per week for 10 weeks. BP was monitored during the 10-week RT intervention and after 15 weeks of detraining. First, individuals were classified as high and low responders, then statistical methods to analyze data included the use of SEM, SRD, magnitude-based inference and MEM. RESULTS: When magnitude-based inference was used to classify responsiveness, most participants displayed a trivial response. Decrements in SBP between 1 and 10 mmHg were not clinically meaningful but fell within the measurement error of the SBP measurements. Baseline SBP and time of training predicted post-SBP response. CONCLUSION: Changes over time and declines in SBP might not be a SRD and fell in the SEM. Moreover, SBP responsiveness was the result of inappropriate control of covariates such as period of training.

eHooke: A tool for automated image analysis of spherical bacteria based on cell cycle progression.

Fluorescence microscopy is a critical tool for cell biology studies on bacterial cell division and morphogenesis. Because the analysis of fluorescence microscopy images evolved beyond initial qualitative studies, numerous images analysis tools were developed to extract quantitative parameters on cell morphology and organization. To understand cellular processes required for bacterial growth and division, it is particularly important to perform such analysis in the context of cell cycle progression. However, manual assignment of cell cycle stages is laborious and prone to user bias. Although cell elongation can be used as a proxy for cell cycle progression in rod-shaped or ovoid bacteria, that is not the case for cocci, such as Staphylococcus aureus. Here, we describe eHooke, an image analysis framework developed specifically for automated analysis of microscopy images of spherical bacterial cells. eHooke contains a trained artificial neural network to automatically classify the cell cycle phase of individual S. aureus cells. Users can then apply various functions to obtain biologically relevant information on morphological features of individual cells and cellular localization of proteins, in the context of the cell cycle.

Reassessment of the distinctive geometry of Staphylococcus aureus cell division.

Staphylococcus aureus is generally thought to divide in three alternating orthogonal planes over three consecutive division cycles. Although this mode of division was proposed over four decades ago, the molecular mechanism that ensures this geometry of division has remained elusive. Here we show, for three different strains, that S. aureus cells do not regularly divide in three alternating perpendicular planes as previously thought. Imaging of the divisome shows that a plane of division is always perpendicular to the previous one, avoiding bisection of the nucleoid, which segregates along an axis parallel to the closing septum. However, one out of the multiple planes perpendicular to the septum which divide the cell in two identical halves can be used in daughter cells, irrespective of its orientation in relation to the penultimate division plane. Therefore, division in three orthogonal planes is not the rule in S. aureus.

SEDS-bPBP pairs direct lateral and septal peptidoglycan synthesis in Staphylococcus aureus.

Peptidoglycan (PGN) is the major component of the bacterial cell wall, a structure that is essential for the physical integrity and shape of the cell. Bacteria maintain cell shape by directing PGN incorporation to distinct regions of the cell, namely, through the localization of late-stage PGN synthesis proteins. These include two key protein families, SEDS transglycosylases and bPBP transpeptidases, proposed to function in cognate pairs. Rod-shaped bacteria have two SEDS-bPBP pairs, involved in elongation and division. Here, we elucidate why coccoid bacteria, such as Staphylococcus aureus, also possess two SEDS-bPBP pairs. We determined that S. aureus RodA-PBP3 and FtsW-PBP1 probably constitute cognate pairs of interacting proteins. A lack of RodA-PBP3 resulted in more spherical cells due to deficient sidewall PGN synthesis, whereas depletion of FtsW-PBP1 arrested normal septal PGN incorporation. Although PBP1 is an essential protein, a mutant lacking PBP1 transpeptidase activity is viable, showing that this protein has a second function. We propose that the FtsW-PBP1 pair has a role in stabilizing the divisome at midcell. In the absence of these proteins, the divisome appears as multiple rings or arcs that drive lateral PGN incorporation, leading to cell elongation. We conclude that RodA-PBP3 and FtsW-PBP1 mediate sidewall and septal PGN incorporation, respectively, and that their activity must be balanced to maintain coccoid morphology.

Relative Handgrip Strength as a Simple Tool to Evaluate Impaired Heart Rate Recovery and a Low Chronotropic Index in Obese Older Women.

The aim of the present study was to compare differences in heart rate response following a treadmill exercise test in elderly obese women categorized into groups based on relative handgrip strength. Eighty-eight obese elderly women who were between the ages of 60 and 87 participated in the study and were categorized and enrolled to one of two groups based on lower (< 1.51 m2) or higher (≥ 1.51 m2) relative handgrip strength, respectively. The heart rate recovery in the first and second minutes following the treadmill exercise test and the chronotropic index were compared between groups. The higher relative handgrip strength group presented a significantly higher peak heart rate during exercise and a quicker heart rate recovery following exercise versus the lower relative handgrip strength group (p<0.05). Furthermore, there was a tendency (p=0.059) toward a significantly greater chronotropic index in the higher versus the lower relative handgrip strength group. In conclusion, elderly women with greater relative handgrip strength also demonstrated a better heart rate response during and following exercise, possibly indicating better autonomic balance. The relative handgrip strength might be an important and inexpensive tool for the elderly obese women to indirect assess cardiovascular health.

Blood pressure response to resistance training in hypertensive and normotensive older women.

PURPOSE: The purpose of the present study was to identify the variability of blood pressure response to a 10-week resistance training (RT) program in hypertensive and normotensive elderly women. PARTICIPANTS AND METHODS: Twenty-seven untrained hypertensive and 12 normotensive elderly women participated in the present study. A whole-body RT program was performed on two nonconsecutive days per week for 10 weeks. The responsiveness of resting systolic blood pressure (SBP) was determined based on the percent decline between the pre- and post-training time points T1 and T4. The term responders were used to describe subjects who exhibited a percent SBP decline ≥-2.58% and the term nonresponders for subjects who exhibited a percent SBP decline <-2.58%, respectively. RESULTS: Both the responders and nonresponders in the hypertensive group presented significant changes in SBP (-7.83 ± 5.70 mmHg vs 3.78 ± 7.42 mmHg), respectively. Moreover, the responders and nonresponders in the normotensive group presented significant changes in SBP as well (-8.58 ± 5.52 mmHg vs 5.71 ± 3.84 mmHg). CONCLUSION: SBP presents a heterogeneous response to a controlled RT program in hypertensive and normotensive elderly women. A different modality of training and additional therapies should be used for nonresponders in order to decrease resting SBP.

Peptidoglycan synthesis drives an FtsZ-treadmilling-independent step of cytokinesis.

Peptidoglycan is the main component of the bacterial wall and protects cells from the mechanical stress that results from high intracellular turgor. Peptidoglycan biosynthesis is very similar in all bacteria; bacterial shapes are therefore mainly determined by the spatial and temporal regulation of peptidoglycan synthesis rather than by the chemical composition of peptidoglycan. The form of rod-shaped bacteria, such as Bacillus subtilis or Escherichia coli, is generated by the action of two peptidoglycan synthesis machineries that act at the septum and at the lateral wall in processes coordinated by the cytoskeletal proteins FtsZ and MreB, respectively. The tubulin homologue FtsZ is the first protein recruited to the division site, where it assembles in filaments-forming the Z ring-that undergo treadmilling and recruit later divisome proteins. The rate of treadmilling in B. subtilis controls the rates of both peptidoglycan synthesis and cell division. The actin homologue MreB forms discrete patches that move circumferentially around the cell in tracks perpendicular to the long axis of the cell, and organize the insertion of new cell wall during elongation. Cocci such as Staphylococcus aureus possess only one type of peptidoglycan synthesis machinery, which is diverted from the cell periphery to the septum in preparation for division. The molecular cue that coordinates this transition has remained elusive. Here we investigate the localization of S. aureus peptidoglycan biosynthesis proteins and show that the recruitment of the putative lipid II flippase MurJ to the septum, by the DivIB-DivIC-FtsL complex, drives peptidoglycan incorporation to the midcell. MurJ recruitment corresponds to a turning point in cytokinesis, which is slow and dependent on FtsZ treadmilling before MurJ arrival but becomes faster and independent of FtsZ treadmilling after peptidoglycan synthesis activity is directed to the septum, where it provides additional force for cell envelope constriction.

Relationship between adiposity and heart rate recovery following an exercise stress test in obese older women / Relação entre a adiposidade e a recuperação da frequência cardíaca após teste de esforço em mulheres idosas obesas

Abstract The aim of the present study was to compare differences in heart rate (HR) response during and following exercise in obese older women with different percent body fat levels. Ninety older, obese women aged 60-87 years participated in the study, were categorized, and enrolled to one of two groups based on a lower percent body fat (LPBF ≤ 41.10 %) or higher percent body fat (HPBF > 41.10 %) as measured by dual-energy x-ray absorptiometry. The peak HR during exercise and in the first and second minutes of recovery period were compared between groups. The HPBF group presented a lower peak HR during exercise (p =.001) and an impaired HR recovery (p =.001) when compared to LPBF group. The present study demonstrated that older women who were in exceedingly obese level have an impaired heart rate response during exercise and in the recovery period, indicating possible autonomic dysfunction.

Resumo O objetivo do presente estudo foi comparar a reposta da frequência cardíaca (FC) durante e após um teste de esforço entre mulheres idosas obesas com diferentes níveis de percentual de gordura corporal. Noventa idosas obesas com idade entre 60-87 anos participaram desse estudo e foram separadas em dois grupos com base no baixo percentual (LPBF ≤ 41.10 %) e alto percentual de gordura corporal (HPBF > 41.10 %) medido por absorciometria de raio-x de dupla energia. A FC pico durante o teste e no primeiro e segundo minuto de recuperação foram comparadas entre os grupos. O grupo HPBF apresentou FC pico inferior durante o teste (p = 0.001) e também após o período de recuperação (p = 0.001) quando comparado com o grupo LPBF. Os dados desse estudo demonstraram que mulheres idosas com alto percentual de gordura corporal apresentaram FC pico inferior e menor recuperação da FC durante o período de recuperação, indicando possivelmente uma disfunção autonômica.

Effects of exercise on cardiac autonomic modulation in children: literature update / Efeitos do exercício sobre a modulação autonômica em crianças: atualização de literatura

AbstractIntroduction It is known that physical exercise is beneficial and precipitates adjustments to the autonomic nervous system. However, the effect of exercise on cardiac autonomic modulation in children, despite its importance, is poorly investigated.Objective To bring together current information about the effects of exercise on heart rate variability in healthy and obese children.Methods The literature update was performed through a search for articles in the following databases; PubMed, PEDro, SciELO and Lilacs, using the descriptors “exercise” and “child” in conjunction with the descriptors “autonomic nervous system”, “sympathetic nervous system”, “parasympathetic nervous system” and also with no descriptor, but the key word of this study, “heart rate variability”, from January 2005 to December 2012.Results After removal of items that did not fit the subject of the study, a total of 9 articles were selected, 5 with healthy and 4 with obese children.Conclusion The findings suggest that exercise can act in the normalization of existing alterations in the autonomic nervous system of obese children, as well as serve as a preventative factor in healthy children, enabling healthy development of the autonomic nervous system until the child reaches adulthood.

ResumoIntrodução Sabe-se que a prática de exercícios físicos é benéfica e proporciona adaptações ao sistema nervoso autônomo. No entanto, o efeito do exercício sobre a modulação autonômica cardíaca em crianças, apesar de ser uma condição importante, é pouco investigada.Objetivo Reunir informações atuais a respeito dos efeitos do exercício físico sobre a variabilidade da frequência cardíaca de crianças saudáveis e obesas.Método A atualização da literatura foi realizada por meio de uma busca dos artigos nas bases de dados PubMed, PEDro, Scielo e Lilacs — por meio dos descritores “exercício” e “criança” em cruzamento com os descritores “sistema nervoso autônomo”, “sistema nervoso simpático”, “sistema nervoso parassimpático” e também com o não descritor, mas palavra chave deste estudo, “variabilidade da frequência cardíaca” — publicados no período de janeiro de 2005 a dezembro de 2012.Resultados Após a eliminação dos artigos que não se enquadravam no tema do artigo foi selecionado um total de nove artigos, sendo cinco com crianças saudáveis e quatro com obesas.Conclusão Os achados sugerem que o exercício físico pode atuar na normalização das alterações existentes no sistema nervoso autonômico de crianças obesas, assim como servir como um fator de prevenção em crianças saudáveis, para que ocorra um desenvolvimento saudável do sistema nervoso autonômico até que a crianças atinja a idade adulta.