OBJECTIVE: The genus of Mycobacterium includes important clinical pathogens (M. tuberculosis). Bacteria of this genus share the unusual features of their cell cycle such as asymmetric polar cell elongation and long generation time. Markedly, control of the mycobacterial cell cycle still remains not fully understood. The main cell growth determinant in mycobacteria is the essential protein DivIVA, which is also involved in cell division. DivIVA activity is controlled by phosphorylation, but the mechanism and significance of this process are unknown. Here, we show how the previously established protein interaction partner of DivIVA in mycobacteria, the segregation protein ParA, affects the DivIVA subcellular distribution. We also demonstrate the role of a newly identified M. smegmatis DivIVA and ParA interaction partner, a protein named PapM, and we establish how their interactions are modulated by phosphorylation. Demonstrating that the tripartite interplay affects the mycobacterial cell cycle contributes to the general understanding of mycobacterial growth regulation.

This study follows a previously defined framework to investigate the impact of impairment on performance in Para dressage athletes. Twenty-one elite Para dressage athletes (grades I to V) and eleven non-disabled dressage athletes (competing at Prix St. Georges or Grand Prix) participated. Data were collected in two phases: performing a two minute custom dressage test on a riding simulator while kinematic data were synchronously collected using inertial measurement units (2000 Hz) and optical motion capture (100 Hz), and clinically assessed using a battery of impairment assessment tools administered by qualified therapists. Impairment and performance measures were compared between Para and non-disabled athletes. Significant differences between athlete groups were found for all impairment measures and two performance measures: simulator trunk harmonics (p = 0.027) and athlete trunk dynamic symmetry (p < 0.001). Impairment assessments of sitting function and muscle tone could predict 19 to 35% of the impact of impairment on performance in Para athletes but not in non-disabled athletes. These findings provide the basis for a robust, scientific evidence base, which can be used to aid in the refinement of the current classification system for Para dressage, to ensure that it is in line with the International Paralympic Committee\'s mandate for evidence-based systems of classification.

The fruit fly Drosophila melanogaster has provided important insights into how sensory information is transduced by transient receptor potential (TRP) channels in the peripheral nervous system (PNS). However, TRP channels alone have not been able to completely model mechanosensitive transduction in mechanoreceptive chordotonal neurons (CNs). Here, we show that, in addition to TRP channels, the sole voltage-gated sodium channel (NaV) in Drosophila, Para, is localized to the dendrites of CNs. Para is localized to the distal tip of the dendrites in all CNs, from embryos to adults, and is colocalized with the mechanosensitive TRP channels No mechanoreceptor potential C (NompC) and Inactive/Nanchung (Iav/Nan). Para localization also demarcates spike initiation zones (SIZs) in axons and the dendritic localization of Para is indicative of a likely dendritic SIZ in fly CNs. Para is not present in the dendrites of other peripheral sensory neurons. In both multipolar and bipolar neurons in the PNS, Para is present in a proximal region of the axon, comparable to the axonal initial segment (AIS) in vertebrates, 40-60 μm from the soma in multipolar neurons and 20-40 μm in bipolar neurons. Whole-cell reduction of para expression using RNAi in CNs of the adult Johnston\'s organ (JO) severely affects sound-evoked potentials (SEPs). However, the duality of Para localization in the CN dendrites and axons identifies a need to develop resources to study compartment-specific roles of proteins that will enable us to better understand Para\'s role in mechanosensitive transduction.

Long coronavirus disease (COVID) is emerging as a common clinical entity in the current era. Autonomic dysfunction is one of the frequently reported post-COVID complications. We hypothesize a bi-directional relationship between the autonomic function and the COVID course. This postulation has been inadequately addressed in the literature. A retrospective cohort (pre and post-comparison) study was conducted on 30 young adults whose pre-COVID autonomic function test results were available. They were divided into case and control groups based on whether they tested reverse transcription polymerase chain reaction positive for COVID-19. Autonomic function tests were performed in both the case and control groups. COVID infection in healthy young adults shifts the sympatho-vagal balance from the pre-disease state. Postural orthostatic tachycardia syndrome was present in 35% of the COVID-affected group. COVID course parameters were found to be associated with parasympathetic reactivity and the baroreflex function. Baseline autonomic function (parasympathetic reactivity represented by Δ heart rate changes during deep breathing and 30:15 ratio during lying-to-standing test) was also associated with the COVID course, the post-COVID symptoms and the post-COVID autonomic function profile. Additionally, multiple regression analysis found that the baseline parasympathetic reactivity was a very important determinant of the clinical course of COVID, the post-COVID symptoms and the post-COVID autonomic profile. Sympatho-vagal balance shifts to parasympathetic withdrawal with sympathetic predominance due to COVID infection in healthy young adults. There is a bi-directional relationship between the autonomic function and the COVID course.

UNASSIGNED: We previously reported that >50% of postoperative opioids prescribed at our institution went unused for common otolaryngologic procedures. Based on these findings, we instituted multimodal, evidence-based guidelines for postoperative pain management. In the second part of our multiphasic study, we evaluated the effects of these guidelines on (1) quantity of unused opioids, (2) patient satisfaction, and (3) institutional perceptions toward the opioid epidemic and prescribing guidelines.
UNASSIGNED: Standardized, procedure-specific opioid prescription guidelines were created using prospective data from the first phase of our study and evidence from current literature. Again, we examined sialendoscopy, parotidectomy, parathyroidectomy/thyroidectomy, and transoral robotic surgery (TORS). Patients were surveyed at their first postoperative appointment. Groups from Phases I and II were compared. Attending physicians were surveyed before the start of the multiphasic project and after prescribing guidelines were implemented.
UNASSIGNED: Prescribing guidelines led to an average reduction in prescribed morphine milligram equivalents (MME) per patient by: 48% (sialendoscopy), 63% (parotidectomy), 60% (para/thyroidectomy), and 42% (TORS). Average used MME per patient for parotidectomy was significantly reduced (64%). The proportion of unused MME per patient and patient satisfaction scores did not significantly change after guidelines were implemented.
UNASSIGNED: Implementation of opioid-prescribing guidelines and the use of multimodal analgesia substantially reduced the amount of opioids prescribed across all procedures without impacting patient satisfaction.
UNASSIGNED: 2.

Maintaining proper chromosome inheritance after the completion of each cell cycle is paramount for bacterial survival. Mechanistic details remain incomplete for how bacteria manage to retain complete chromosomes after each cell cycle. In this study, we examined the potential roles of the partitioning protein ParA on chromosomal maintenance that go beyond triggering the onset of chromosome segregation in Caulobacter crescentus. Our data revealed that increasing the levels of ParA result in cells with multiple origins of replication in a DnaA-ATP-dependent manner. This ori supernumerary is retained even when expressing variants of ParA that are deficient in promoting chromosome segregation. Our data suggest that in Caulobacter ParA\'s impact on replication initiation is likely indirect, possibly through the effect of other cell cycle events. Overall, our data provide new insights into the highly interconnected network that drives the forward progression of the bacterial cell cycle. IMPORTANCE The successful generation of a daughter cell containing a complete copy of the chromosome requires the exquisite coordination of major cell cycle events. Any mistake in this coordination can be lethal, making these processes ideal targets for novel antibiotics. In this study, we focused on the coordination between the onset of chromosome replication, and the partitioning protein ParA. We demonstrate that altering the cellular levels of ParA causes cells to accumulate multiple origins of replication in Caulobacter crescentus. Our work provides important insights into the complex regulation involved in the coordination of the bacterial cell cycle.

Para football is currently played in impairment-specific formats by thousands of people worldwide. To date, there have been no prospective longitudinal injury surveillance studies. This study aimed to implement a prospective injury surveillance study within elite English Para football and analyse the injury risk within the England Blind and Cerebral Palsy (CP) squads. Match and training injury data based on a \'time loss\' definition were collected and analysed for each squad including incidence per 1,000 player hours, severity, injury location and associated event of injury. Injury incidence were lower in training than matches (CP 67.6/1000 player match hours (CI 33.8-135.2) and 5.7/1000 training hours (CI 3.8-8.7) and Blind 44.0/1000 player match hours (CI 26.1-74.3) and 5.5/1000 training hours (CI 3.5-8.6). Training injuries were more severe than match injuries across both squads (CP median 12 days lost in matches and 16 training and Blind median days 5 matches and 12 training). 73% Blind and 74% CP footballer injuries were to the lower limb and 17% head and neck equally across both Para football squads. \'Muscle and tendon injuries\' (51%) represented the greatest proportion of injuries for CP footballers, and \'joint (non-bone)/ligament injuries\' (43%) for Blind footballers. Collaboration and implementation of higher quality surveillance methodology and data collection in Para sport with greater athlete numbers are needed to inform injury prevention strategies.

Replication and segregation of the genetic information is necessary for a cell to proliferate. In Bacillus subtilis, the Par system (ParA/Soj, ParB/Spo0J and parS) is required for segregation of the chromosome origin (oriC) region and for proper control of DNA replication initiation. ParB binds parS sites clustered near the origin of replication and assembles into sliding clamps that interact with ParA to drive origin segregation through a diffusion-ratchet mechanism. As part of this dynamic process, ParB stimulates ParA ATPase activity to trigger its switch from an ATP-bound dimer to an ADP-bound monomer. In addition to its conserved role in DNA segregation, ParA is also a regulator of the master DNA replication initiation protein DnaA. We hypothesized that in B. subtilis the location of the Par system proximal to oriC would be necessary for ParA to properly regulate DnaA. To test this model, we constructed a range of genetically modified strains with altered numbers and locations of parS sites, many of which perturbed chromosome origin segregation as expected. Contrary to our hypothesis, the results show that regulation of DNA replication initiation by ParA is maintained when a parS site is separated from oriC. Because a single parS site is sufficient for proper control of ParA, the results are consistent with a model where ParA is efficiently regulated by ParB sliding clamps following loading at parS.

Multiplexed CRISPR technologies have great potential for pathway engineering and genome editing. However, their applications are constrained by complex, laborious and time-consuming cloning steps. In this research, we developed a novel method, PARA, which allows for the one-step assembly of multiple guide RNAs (gRNAs) into a CRISPR vector with up to 18 gRNAs. Here, we demonstrate that PARA is capable of the efficient assembly of transfer RNA/Csy4/ribozyme-based gRNA arrays. To aid in this process and to streamline vector construction, we developed a user-friendly PARAweb tool for designing PCR primers and component DNA parts and simulating assembled gRNA arrays and vector sequences.

Most bacteria use the ParABS system to segregate their newly replicated chromosomes. The two protein components of this system from various bacterial species share their biochemical properties: ParB is a CTPase that binds specific centromere-like parS sequences to assemble a nucleoprotein complex, while the ParA ATPase forms a dimer that binds DNA non-specifically and interacts with ParB complexes. The ParA-ParB interaction incites the movement of ParB complexes toward the opposite cell poles. However, apart from their function in chromosome segregation, both ParAB may engage in genus-specific interactions with other protein partners. One such example is the polar-growth controlling protein DivIVA in Actinomycetota, which binds ParA in Mycobacteria while interacts with ParB in Corynebacteria. Here, we used heterologous hosts to investigate whether the interactions between DivIVA and ParA or ParB are maintained across phylogenic classes. Specifically, we examined interactions of proteins from four bacterial species, two belonging to the Gram positive Actinomycetota phylum and two belonging to the Gram-negative Pseudomonadota. We show that while the interactions between ParA and ParB are preserved for closely related orthologs, the interactions with polarly localised protein partners are not conferred by orthologous ParABs. Moreover, we demonstrate that heterologous ParA cannot substitute for endogenous ParA, despite their high sequence similarity. Therefore, we conclude that ParA orthologs are fine-tuned to interact with their partners, especially their interactions with polarly localised proteins are adjusted to particular bacterial species demands.