A bipartite, low-affinity roadblock domain-containing GAP complex regulates bacterial front-rear polarity.

The Ras-like GTPase MglA is a key regulator of front-rear polarity in the rod-shaped Myxococcus xanthus cells. MglA-GTP localizes to the leading cell pole and stimulates assembly of the two machineries for type IV pili-dependent motility and gliding motility. MglA-GTP localization is spatially constrained by its cognate GEF, the RomR/RomX complex, and GAP, the MglB Roadblock-domain protein. Paradoxically, RomR/RomX and MglB localize similarly with low and high concentrations at the leading and lagging poles, respectively. Yet, GEF activity dominates at the leading and GAP activity at the lagging pole by unknown mechanisms. Here, we identify RomY and show that it stimulates MglB GAP activity. The MglB/RomY interaction is low affinity, restricting formation of the bipartite MglB/RomY GAP complex almost exclusively to the lagging pole with the high MglB concentration. Our data support a model wherein RomY, by forming a low-affinity complex with MglB, ensures that the high MglB/RomY GAP activity is confined to the lagging pole where it dominates and outcompetes the GEF activity of the RomR/RomX complex. Thereby, MglA-GTP localization is constrained to the leading pole establishing front-rear polarity.

The small GTPase MglA together with the TPR domain protein SgmX stimulates type IV pili formation in M. xanthus.

Bacteria can move across surfaces using type IV pili (T4P), which undergo cycles of extension, adhesion, and retraction. The T4P localization pattern varies between species; however, the underlying mechanisms are largely unknown. In the rod-shaped Myxococcus xanthus cells, T4P localize at the leading cell pole. As cells reverse their direction of movement, T4P are disassembled at the old leading pole and then form at the new leading pole. Thus, cells can form T4P at both poles but engage only one pole at a time in T4P formation. Here, we address how this T4P unipolarity is realized. We demonstrate that the small Ras-like GTPase MglA stimulates T4P formation in its GTP-bound state by direct interaction with the tetratricopeptide repeat (TPR) domain-containing protein SgmX. SgmX, in turn, is important for polar localization of the T4P extension ATPase PilB. The cognate MglA GTPase activating protein (GAP) MglB, which localizes mainly to the lagging cell pole, indirectly blocks T4P formation at this pole by stimulating the conversion of MglA-GTP to MglA-GDP. Based on these findings, we propose a model whereby T4P unipolarity is accomplished by stimulation of T4P formation at the leading pole by MglA-GTP and SgmX and indirect inhibition of T4P formation at the lagging pole by MglB due to its MglA GAP activity. During reversals, MglA, SgmX, and MglB switch polarity, thus laying the foundation for T4P formation at the new leading pole and inhibition of T4P formation at the new lagging pole.

Protein-protein interaction network controlling establishment and maintenance of switchable cell polarity.

Cell polarity underlies key processes in all cells, including growth, differentiation and division. In the bacterium Myxococcus xanthus, front-rear polarity is crucial for motility. Notably, this polarity can be inverted, independent of the cell-cycle, by chemotactic signaling. However, a precise understanding of the protein network that establishes polarity and allows for its inversion has remained elusive. Here, we use a combination of quantitative experiments and data-driven theory to unravel the complex interplay between the three key components of the M. xanthus polarity module. By studying each of these components in isolation and their effects as we systematically reconstruct the system, we deduce the network of effective interactions between the polarity proteins. RomR lies at the root of this network, promoting polar localization of the other components, while polarity arises from interconnected negative and positive feedbacks mediated by the small GTPase MglA and its cognate GAP MglB, respectively. We rationalize this network topology as operating as a spatial toggle switch, providing stable polarity for persistent cell movement whilst remaining responsive to chemotactic signaling and thus capable of polarity inversions. Our results have implications not only for the understanding of polarity and motility in M. xanthus but also, more broadly, for dynamic cell polarity.

A polymicrobial biofilm model for testing the antimicrobial potential of a nisin-biogel for canine periodontal disease control.

BACKGROUND: Periodontal disease (PD) in dogs is prompted by the establishment of a polymicrobial biofilm at the tooth surface and a subsequent host inflammatory response. Several strategies may be used for PD control, including dental hygiene home care procedures, like toothbrushing, special diet and chew toys that reduce dental plaque accumulation, or professional periodontal treatments. Aiming at PD control, a biogel composed by nisin and guar-gum was previously developed. This work aimed to establish an in vitro model mimicking the PD-associated biofilms and to evaluate the nisin-biogel inhibitory activity against this polymicrobial biofilm by determining its Minimum Biofilm Inhibitory (MBIC) and Eradication Concentrations (MBEC). Bacterial species tested included Neisseria zoodegmatis CCUG 52598T, Corynebacterium canis CCUG 58627T, Porphyromonas cangingivalis DSMZ VPB 4874, Peptostreptococcus canis CCUG 57081 and an Enterococcus faecalis isolate belonging to a collection of oral bacteria obtained from dogs with PD. Before establishing the biofilm, coaggregation between species was determined by optical density measurement after 2 and 24 hours. Nisin-biogel MBIC and MBEC values regarding the polymicrobial biofilm were determined using a modified version of the Calgary biofilm pin lid device, after confirming the presence of the five bacterial species by Fluorescent In Situ Hybridization. RESULTS: Only 40% of the bacterial dual suspensions were able to coaggregate at 2 hours, but all species tested exhibited a coaggregation percentage higher than 30% at 24 hours. It was possible to establish a 48 h polymicrobial biofilm model composed by the five bacterial species selected. This model was used to determine nisin-biogel MBIC (26.39 ± 5.89 µg/mL) and MBEC (62.5 ± 27.73 µg/mL) values. CONCLUSIONS: Our results showed that the nisin-biogel can inhibit and eradicate PD multispecies biofilms. As this in vitro model mimics an in vivo periodontal polymicrobial biofilm, our results reinforce the potential of the application of nisin-biogel for canine PD control.

Molecular basis and design principles of switchable front-rear polarity and directional migration in Myxococcus xanthus.

During cell migration, front-rear polarity is spatiotemporally regulated; however, the underlying design of regulatory interactions varies. In rod-shaped Myxococcus xanthus cells, a spatial toggle switch dynamically regulates front-rear polarity. The polarity module establishes front-rear polarity by guaranteeing front pole-localization of the small GTPase MglA. Conversely, the Frz chemosensory system, by acting on the polarity module, causes polarity inversions. MglA localization depends on the RomR/RomX GEF and MglB/RomY GAP complexes that localize asymmetrically to the poles by unknown mechanisms. Here, we show that RomR and the MglB and MglC roadblock domain proteins generate a positive feedback by forming a RomR/MglC/MglB complex, thereby establishing the rear pole with high GAP activity that is non-permissive to MglA. MglA at the front engages in negative feedback that breaks the RomR/MglC/MglB positive feedback allosterically, thus ensuring low GAP activity at this pole. These findings unravel the design principles of a system for switchable front-rear polarity.

In Vivo Effect of a Nisin-Biogel on the Antimicrobial and Virulence Signatures of Canine Oral Enterococci.

Periodontal disease is a relevant oral disease in dogs and nisin-biogel has been previously proposed to be used in its control. Enterococci, as inhabitants of the oral cavity with a high genetic versatility, are a reliable bacterial model for antimicrobial studies. Our goal was to evaluate the in vivo influence of the long-term dental application of the nisin-biogel on the virulence and antimicrobial signatures of canine oral enterococci. Twenty dogs were randomly allocated to one of two groups (treatment group-TG with nisin-biogel dental application, or control group-CG without treatment) and submitted to dental plaque sampling at day 0 and after 90 days (T90). Samples were processed for Enterococcus spp. isolation, quantification, identification, molecular typing and antimicrobial and virulence characterization. From a total of 140 enterococci, molecular typing allowed us to obtain 70 representative isolates, mostly identified as E. faecalis and E. faecium. No significant differences (p > 0.05) were observed in the virulence index of the isolates obtained from samples collected from the TG and CG at T90. At T90, a statistically significant difference (p = 0.0008) was observed in the antimicrobial resistance index between the isolates from the TC and CG. Oral enterococci were revealed to be reservoirs of high resistant and virulent phenotypes.

Current and Emerging Techniques for Diagnosis and MRD Detection in AML: A Comprehensive Narrative Review.

Acute myeloid leukemia (AML) comprises a group of hematologic neoplasms characterized by abnormal differentiation and proliferation of myeloid progenitor cells. AML is associated with poor outcome due to the lack of efficient therapies and early diagnostic tools. The current gold standard diagnostic tools are based on bone marrow biopsy. These biopsies, apart from being very invasive, painful, and costly, have low sensitivity. Despite the progress uncovering the molecular pathogenesis of AML, the development of novel detection strategies is still poorly explored. This is particularly important for patients that check the criteria for complete remission after treatment, since they can relapse through the persistence of some leukemic stem cells. This condition, recently named as measurable residual disease (MRD), has severe consequences for disease progression. Hence, an early and accurate diagnosis of MRD would allow an appropriate therapy to be tailored, improving a patient's prognosis. Many novel techniques with high potential in disease prevention and early detection are being explored. Among them, microfluidics has flourished in recent years due to its ability at processing complex samples as well as its demonstrated capacity to isolate rare cells from biological fluids. In parallel, surface-enhanced Raman scattering (SERS) spectroscopy has shown outstanding sensitivity and capability for multiplex quantitative detection of disease biomarkers. Together, these technologies can allow early and cost-effective disease detection as well as contribute to monitoring the efficiency of treatments. In this review, we aim to provide a comprehensive overview of AML disease, the conventional techniques currently used for its diagnosis, classification (recently updated in September 2022), and treatment selection, and we also aim to present how novel technologies can be applied to improve the detection and monitoring of MRD.

Spatiotemporal regulation of switching front-rear cell polarity.

Bacterial cells are spatiotemporally highly organised with proteins localising dynamically to distinct subcellular regions. Motility in the rod-shaped Myxococcus xanthus cells represents an example of signal-induced spatiotemporal regulation of cell polarity. M. xanthus cells move across surfaces with defined front-rear polarity; occasionally, they invert polarity and, in parallel, reverse direction of movement. The polarity module establishes front-rear polarity between reversals and consists of the Ras-like GTPase MglA and its cognate GEF and GAP, that all localise asymmetrically to the cell poles. The Frz chemosensory system constitutes the polarity inversion module and interfaces with the proteins of the polarity module, thereby triggering their polar repositioning. As a result, the polarity proteins, over time, toggle between the cell poles causing cells to oscillate irregularly. Here, we review recent progress in how front-rear polarity is established by the polarity module and inverted by the Frz system and highlight open questions for future studies.

In Vivo Evaluation of the Efficacy of a Nisin-Biogel as a New Approach for Canine Periodontal Disease Control.

Periodontal disease (PD) is a common oral disease in dogs. Recent in vitro research revealed that nisin−biogel is a promising compound for canine PD control. In this work, a clinical trial was developed to assess the in vivo efficacy of nisin−biogel in dogs by determining the dental plaque index (DPI), gingivitis index (GI), and periodontal pocket depth (PPD) after dental administration. The biogel's influence on aerobic bacteria counts was also evaluated, as well as its acceptance/adverse effects in dogs. Twenty animals were allocated to one of two groups: a treatment group (TG) subjected to a dental topical application of nisin−biogel for 90 days and a control group (CG) with no treatment. Besides daily monitoring, on day 1 (T0) and at the end of the assay (T90), animals were subjected to blood analysis, periodontal evaluation, dental plaque sampling, scaling, and polishing. Statistical analysis with mixed models showed a significant reduction in mean PPD (estimate = −0.371, p-value < 0.001) and DPI (estimate = −0.146, p-value < 0.05) in the TG animals at T90. A reduction in the GI (estimate = −0.056, p-value > 0.05) was also observed but with no statistical significance. No influence on total bacterial counts was observed, and no adverse effects were detected. The nisin−biogel was revealed to be a promising compound for canine PD control.

Evaluation of Four Clinical Metrology Instruments for the Assessment of Osteoarthritis in Dogs.

Osteoarthritis (OA) is the most commonly diagnosed joint disease in companion animals, and proper tools are necessary to assess patients and response to treatment. We aimed to perform the psychometric evaluation of several clinical metrology instruments (CMI), developed to evaluate pain and assess outcome. Fifty police working dogs with bilateral hip OA were assessed in a prospective, randomised, double-blinded study. Patients were evaluated using a stance analyser in six different moments divided over a 180-day period. Pedometer step count, weight-bearing symmetry index and deviation from normal weight-bearing were calculated and used for criterion validity. In each evaluation moment, a copy of the Hudson Visual Analogue Scale (HVAS), Canine Brief Pain Inventory (CBPI), Liverpool Osteoarthritis in Dogs (LOAD) and Canine Orthopaedic Index (COI) were completed by the dogs' handlers. Correlations between CMIs were evaluated as construct validity. Further evaluation was performed with the Kaiser-Meyer-Olin measure of sampling adequacy, Eigenvalue and scree-plot analysis. Internal consistency was tested with Cronbach's α. Significant weak correlation was found between all CMIs and stance analysis symmetry index measure and deviation, indicating criterion validity. Significant weak correlation was also found between pedometer count and LOAD plus COI. Cronbach's α was 0.80 for HVAS, 0.98 for CBPI, 0.97 for LOAD and 0.98 for COI. Significant strong correlation was observed between CMIs, indicating construct validity. We present criterion and construct validity of these CMIs, which are able to capture various dimensions of OA. They can be used for the evaluation of osteoarthritis and response to treatment in dogs.

Management of Osteoarthritis Using 1 Intra-articular Platelet Concentrate Administration in a Canine Osteoarthritis Model.

BACKGROUND: Osteoarthritis (OA) represents a significant burden to societies, as it affects quality of life and performance and implies a large cost in terms of health care. Autologous platelets are a regenerative treatment modality for OA that are thought to be a potential stimulation of the natural healing cascade. PURPOSE: To describe the effect of the platelet concentrate V-PET in the management of OA in a naturally occurring canine model, using several outcome assessment modalities. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 40 joints of active working police dogs with hip OA were randomly assigned to a control group (CG) and a platelet concentrate group (PCG; treatment) and evaluated. At treatment day (T0) and 8, 15, 30, 90, and 180 days after treatment, weight distribution, joint range of motion at flexion and extension, thigh girth, digital thermography, radiographic signs, 4 clinical metrology instruments, and synovial fluid interleukin 1 and C-reactive protein levels were recorded. Results were compared using repeated-measures analysis of variance with a Huynh-Feldt correction, paired-samples t test, or Wilcoxon signed rank test, with P < .05. RESULTS: Dogs were 6.5 ± 2.4 years old (mean ± SD) and had a body weight of 26.7 ± 5.2 kg. At T0, 32 (80%) joints were graded as having mild OA, 6 (15%) as moderate, and 2 (5%) as severe. No differences were found between groups at T0. Between the PCG and CG, the symmetry index showed significant improvements in the PCG from 8 days (P = .01) to 180 days (P = .01). Joint flexion also improved in the PCG up to 90 days (P < .05) and extension improved up to 180 days (P < .01). Several clinical metrology instrument scores also improved up to 90 to 180 days after treatment. In the CG, radiographic signs progressed, while the PCG showed some improved signs. In both groups, increasing body weight and age corresponded with worse clinical and laboratory findings. CONCLUSION: A single injection of platelet concentrate had a positive effect, lasting up to 6 months, on several clinical, imaging, and laboratory signs in a naturally occurring canine OA model. CLINICAL RELEVANCE: We characterized the effects of this platelet concentrate in dogs, considered the gold standard of the study of OA, with a group of working animals with similar high demands as athletes.

Spatial control of the GTPase MglA by localized RomR-RomX GEF and MglB GAP activities enables Myxococcus xanthus motility.

The rod-shaped Myxococcus xanthus cells move with defined front-rear polarity using polarized motility systems. A polarity module consisting of the small GTPase MglA, its cognate GTPase activating protein (GAP) MglB and RomR establishes this polarity. Agl-Glt gliding motility complexes assemble and disassemble at the leading and lagging pole, respectively. These processes are stimulated by MglA-GTP at the leading and MglB at the lagging pole. Here, we identify RomX as an integral component of the polarity module. RomX and RomR form a complex that has MglA guanine nucleotide exchange factor (GEF) activity and also binds MglA-GTP. In vivo RomR recruits RomX to the leading pole forming the RomR-RomX complex that stimulates MglA-GTP formation and binding, resulting in a high local concentration of MglA-GTP. The spatially separated and opposing activities of the RomR-RomX GEF at the leading and the MglB GAP at the lagging cell pole establish front-rear polarity by allowing the spatially separated assembly and disassembly of Agl-Glt motility complexes. Our findings uncover a regulatory system for bacterial cell polarity that incorporates a nucleotide exchange factor as well as an NTPase activating protein for regulation of a nucleotide-dependent molecular switch and demonstrate a spatial organization that is conserved in eukaryotes.

Five years of teleconsultation: experience of the Cardiology Department of Coimbra Pediatric Hospital.

BACKGROUND: Telemedicine is an excellent tool to expand specialized medical care, providing better access for patients and lowering costs for their families. The objective of this work was to evaluate the reliability of teleconsultation in pediatric cardiology, with similar results to traditional consultation. It can be a useful tool to upgrade technical skills among the staff involved and to improve the quality of life of patients and families. METHODS: The authors present their 5 years' experience of pediatric cardiology teleconsultation involving Coimbra Central Pediatric Hospital and district hospitals in the central region of Portugal. During this period 1761 consultations, involving 1056 patients, were given with a pre-established weekly schedule for each hospital and joint online reports were produced, with data confidentiality assured. RESULTS: Cardiac murmur was the most common reason for consultation (73%). No heart disease was diagnosed in 49% of the patients. Among septal defects, accounting for 43%, ventricular septal defect was the most frequent (20%). Only 10% of all patients had to be seen in the Pediatric Hospital Cardiology Department. Six emergency consultations were requested. CONCLUSIONS: Pediatric cardiology teleconsultation is a reliable procedure using the Medigraf platform, with a similar error rate to consultations by traditional methods. The costs are lower, especially for the families, who do not have to travel far from home or take time off work. The waiting time for a consultation is shorter and therapeutic decisions are faster. A pediatric cardiologist can cover a larger geographic area and the cooperative work during sessions is useful for exchanging knowledge and experiences.

Microbiological and physicochemical properties of fresh retail cuts of beef packaged under an advanced vacuum skin system and stored at 4 degrees C.

The effect of an advanced vacuum skin packaging system on the microbiological and physicochemical properties of fresh retail cuts of beef (including meat portions from six different anatomical regions) stored at 4 degrees C was compared with the effect of traditional vacuum packaging. The vacuum skin packaging system whose effect on meat quality was evaluated in this work displayed two remarkable features: (i) the instantaneous heating of the lower surface of the upper film of the package before the film descended over the meat surface and (ii) the tight disposition of the plastic film on the meat surface, which avoided wrinkles and purges. Throughout storage at 4 degrees C, rates of bacterial growth were statistically significantly slower in beef portions processed with the vacuum skin packaging system than in those processed with traditional vacuum packaging, with average differences of 2.07, 1.60, and 1.25 log CFU/g in counts of aerobic mesophiles, anaerobes, and lactic acid bacteria, respectively. pH values were statistically significantly lower for beef portions packaged with the vacuum skin system than for those that were vacuum packaged in the traditional manner, probably because of the relative predominance of lactic acid bacteria observed in such samples, which coincided with both higher meat firmness values and a slower meat tenderization process. The vacuum skin system prevented the appearance of undesirable coloration on the meat surface and also significantly improved the commercial color of the meat as determined on the basis of luminosity (L*) and the redness (a*). Overall, the quality (as determined by microbiological and physicochemical analyses and by visual examination) of fresh retail cuts of beef packaged with the vacuum skin system and stored at 4 degrees C was higher than that of meat samples processed with the traditional vacuum-packaging system.