Therapeutic ultrasound (US) and photobiomodulation (PBM) are promising treatment modalities for restoring articular cartilage. Owing to their different mechanisms of action, individual or combined stimulation may elicit different bioeffects on chondrocytes. This study aimed to evaluate the potential of US and PBM, applied alone or combined, in augmenting chondrocytes' responses toward cartilage matrix synthesis and reverting their catabolic activities, with or without interleukin-1β (IL-1β) incubation. Human chondrocytes were cultured under basal or proinflammatory conditions (1 ng/ml IL-1β) and stimulated daily for 6 days with US, PBM, US followed by PBM, and PBM followed by US. Chondrocyte activity, along with protein and mRNA expression of cartilage synthesis- and degradation-related markers, was analyzed. IL-1β incubation did not significantly affect chondrocytes' metabolic activity or sulfated glycosaminoglycans (GAGs) production. Both US and PBM, alone or combined, increased the protein and mRNA of collagen type II (COL II) and aggrecan (ACAN) under basal and proinflammatory conditions. PBM had a stimulatory effect on GAG synthesis. While PBM did not influence the expression of cartilage destruction markers, US potentiated the chondrocytes' response to inflammation by increasing metalloproteinases and IL-1β mRNA levels. The sequential combination of US and PBM induced distinct bioeffects depending on the application order, but these were not superior to the effects of individual treatments. As this study used chondrocytes from a single donor, generalizability may be limited. While both US and PBM stimulated matrix synthesis, PBM showed a comparatively greater ability to mitigate chondrocyte degradation and may be of interest for cartilage repair strategies.

The caseinolytic protease (Clp) system has recently emerged as a promising anti-tuberculosis target. The anti-cancer drug bortezomib exhibits potent anti-mycobacterial activity and binds to Mycobacterium tuberculosis (Mtb) Clp protease complexes. We determine cryo-EM structures of Mtb ClpP1P2, ClpC1P1P2 and ClpXP1P2 complexes bound to bortezomib in different conformations. Structural and biochemical data indicate that sub-stoichiometric binding by bortezomib to the protease active sites orthosterically activates the MtbClpP1P2 complex. Bortezomib activation of MtbClpP1P2 induces structural changes promoting the recruitment of the chaperone-unfoldases, MtbClpC1 or MtbClpX, facilitating holoenzyme formation. The structures of the MtbClpC1P1P2 holoenzyme indicate that MtbClpC1 motion, induced by ATP rebinding at the MtbClpC1 spiral seam, translocates the substrate. In the MtbClpXP1P2 holoenzyme structure, we identify a specialized substrate channel gating mechanism involving the MtbClpX pore-2 loop and MtbClpP2 N-terminal domains. Our results provide insights into the intricate regulation of the Mtb Clp system and suggest that bortezomib can disrupt this regulation by sub-stoichiometric binding at the Mtb Clp protease sites.

Photobiomodulation (PBM) within red to near-infrared spectrum has been proposed as a method to restore articular cartilage injuries, due to its ability to modulate effectively chondrocyte activity, namely viability, proliferation and extracellular matrix synthesis. This in vitro study aimed to determine the most appropriate PBM dosage on human chondrocytes. Firstly, human chondrocytes were stimulated daily or every other day with light-emitting diodes (LEDs) at wavelengths of 600–940 nm and power densities of 4–17 mW/cm2, in continuous or pulsed mode for up to 4 min with temperature monitored during stimulation. After 3 days, the PBM most effective protocols enhancing metabolic activity and proliferation were further applied for seven days to assess their effect on cartilage protein production using immunocytochemistry, western blot, and alcian blue staining. Near-infrared LEDs (850 and 940 nm) at power densities of 4–17 mW/cm2, in continuous or pulsed mode, significantly increased normalized chondrocyte metabolic activity, regardless of the periodicity or operation mode applied. A dose-dependent response was demonstrated, with variations among the PBM parameters. Particularly, PBM with 17 mW/cm2 at 940 nm consistently promoted collagen type II, aggrecan, and glycosaminoglycans deposition in the chondrocyte matrix, while preserving their normal phenotype in monolayer cultures. Stimulation with LEDs did not cause a significant temperature increase. Herein, stimulation with near-infrared LEDs effectively increased the viability and secretion of cartilage proteins in human chondrocytes. This approach holds promise as a non-invasive therapeutic modality for articular cartilage repair in future preclinical and clinical research.

The Mobile Colistin Resistance (MCR) phosphoethanolamine (PEtN) transferase is a plasmid-borne enzyme responsible for colistin antibiotic resistance in Escherichia coli, the most important antimicrobial-resistant bacterial pathogen worldwide. Bacterial PEtN transferases like MCR comprise periplasmic catalytic and integral membrane domains, with mechanistic understanding largely based on studies of the former and limited information on the full-length enzyme. Previous investigations of a Neisseria meningitidis PEtN transferase identified that the catalytic domain can effectively dissociate from the transmembrane component and instead make extensive contacts with the membrane surface. Here, we report molecular dynamics simulations of a model of full-length MCR-1 in a representative membrane comprising 80% of a PEtN donor substrate, palmitoyloleoyl phosphoethanolamine (POPE), that explore the dynamic behavior of the enzyme and the impact upon it of zinc stoichiometry and PEtN addition to the Thr285 acceptor residue. The results identify only limited movement of the two domains relative to one another, and that POPE can bind the likely "resting" state of the enzyme (monozinc with unmodified Thr285) in an orientation compatible with PEtN transfer to Thr285. Stable binding of a second zinc equivalent occurred only with application of restraints and involved Glu116 from the transmembrane domain. Mutation of this residue abolished MCR-1-mediated protection of recombinant E. coli from colistin. Our data suggest domain motions in bacterial PEtN transferases to be condition-dependent and support a proposed "ping-pong" reaction mechanism, with the monozinc enzyme competent to undertake the first stage.

A systematic review was conducted to determine the trends in devices and parameters used for brain photobiomodulation (PBM). The revised studies included clinical and cadaveric approaches, in which light stimuli were applied to the head and/or neck. PubMed, Scopus, Web of Science and Google Scholar databases were used for the systematic search. A total of 2133 records were screened, from which 97 were included in this review. The parameters that were extracted and analysed in each article were the device design, actuation area, actuation site, wavelength, mode of operation, power density, energy density, power output, energy per session and treatment time. To organize device information, 11 categories of devices were defined, according to their characteristics. The most used category of devices was laser handpieces, which relate to 21% of all devices, while 28% of the devices were not described. Studies for cognitive function and physiological characterisation are the most well defined ones and with more tangible results. There is a lack of consistency when reporting PBM studies, with several articles under defining the stimulation protocol, and a wide variety of parameters used for the same health conditions (e.g., Alzheimer's or Parkinson's disease) resulting in positive outcomes. Standardization for the report of these studies is warranted, as well as sham-controlled comparative studies to determine which parameters have the greatest effect on PBM treatments for different neurological conditions.

Introduction Acute bronchiolitis is a common reason for admission to the pediatric emergency department. Evidence has shown that most interventions do not change the natural course of the disease. Material and methods This study aimed to evaluate the economic impact of the non-compliance with the acute bronchiolitis Portuguese national guideline. A retrospective study of pediatric emergency episodes of a Portuguese hospital that had a diagnosis of acute bronchiolitis during 2019, was conducted. Results The sample included 344 emergency episodes. Non-compliance with the guideline occurred in 71.8% of the episodes, mostly due to unjustified treatment. Following guideline in the studied hospital for one year would have resulted in an estimated overall 76.6% cost reduction, with a reduction in mean direct costs per patient of 14.93 €, corresponding to a medium saving of 3.89 € for each patient and a reduction of 11.03 € for the Portuguese National Health Service. Analyzing the unjustified mean direct costs, of these 2.97 € were related to inpatient diagnostic tests and therapeutic and the remaining 11.96 € were related to outpatient therapy. Mean direct costs imputed to the patient for outpatient treatment represented only 3.31 €, therefore most of mean direct costs is paid by the National Health Service. Conclusions Compliance with guideline would allow the reduction of total estimated costs by about 76.6%, representing a waste of resources, without compromising the quality of care provided. Most of the cost associated with non-compliance with the guideline is justified by outpatient therapy, 67% of which was paid by the National Health Service.

Nature has evolved diverse electron transport proteins and multiprotein assemblies essential to the generation and transduction of biological energy. However, substantially modifying or adapting these proteins for user-defined applications or to gain fundamental mechanistic insight can be hindered by their inherent complexity. De novo protein design offers an attractive route to stripping away this confounding complexity, enabling us to probe the fundamental workings of these bioenergetic proteins and systems, while providing robust, modular platforms for constructing completely artificial electron-conducting circuitry. Here, we use a set of de novo designed mono-heme and di-heme soluble and membrane proteins to delineate the contributions of electrostatic micro-environments and dielectric properties of the surrounding protein medium on the inter-heme redox cooperativity that we have previously reported. Experimentally, we find that the two heme sites in both the water-soluble and membrane constructs have broadly equivalent redox potentials in isolation, in agreement with Poisson-Boltzmann Continuum Electrostatics calculations. BioDC, a Python program for the estimation of electron transfer energetics and kinetics within multiheme cytochromes, also predicts equivalent heme sites, and reports that burial within the low dielectric environment of the membrane strengthens heme-heme electrostatic coupling. We conclude that redox cooperativity in our diheme cytochromes is largely driven by heme electrostatic coupling and confirm that this effect is greatly strengthened by burial in the membrane. These results demonstrate that while our de novo proteins present minimalist, new-to-nature constructs, they enable the dissection and microscopic examination of processes fundamental to the function of vital, yet complex, bioenergetic assemblies.

OBJECTIVE:Photobiomodulation (PBM) is not implemented in routine clinical management for knee osteoarthritis. This study aims to systematically investigate the effects of PBM in patients with knee osteoarthritis, comparing with placebo to understand its true clinical effects. METHODS:PubMed, EMBASE, Web of Science, and Cochrane databases were searched up to October 2023. Randomized placebo-controlled trials applying PBM versus placebo were included. Study characteristics, intervention parameters, and patient-reported and physical examination outcome measures were collected. The risk of bias was judged using the Cochrane risk-of-bias tool for randomized trials (version 2) and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) to interpret the certainty of results. RESULTS:Ten studies were included comprising 542 participants. All studies were judged with unclear to a high risk of bias. Meta-analysis for pain at rest (6 studies) showed that PBM significantly reduced pain at rest as compared to placebo (-0.7 [95% CI = -1.1 to -0.2]), moderate effect, very low certainty of evidence, whereas for the Timed "Up & Go" Test (three studies), no significant effect was detected. Statistically significantly within-group (PBM) mean improvement was detected for pain, Lequesne Index, and gait performance outcomes, but not always clinically relevant or significant when compared to placebo. CONCLUSION:PBM reduces pain intensity in patients with knee osteoarthritis and may improve disability. However, the very low certainty of evidence does not allow to recommend its isolated use but may be used to complement other widely recommended therapies. More rigorous clinical trials and the revision of the recommended dosage guidelines are warranted to increase the strength of evidence. IMPACT:The findings indicate that photobiomodulation can reduce pain and improve disability in patients with knee osteoarthritis. However, researchers should continue to investigate isolated photobiomodulation intervention versus placebo and extend the dosage guidelines to other types of light emitters.

Among the adjunctive procedures to accelerate orthodontic tooth movement (OTM), ultrasound (US) is a nonsurgical form of mechanical stimulus that has been explored as an alternative to the currently available treatments. This study aimed to clarify the role of US in OTM by exploring different stimulation parameters and their effects on the biological responses of cells involved in OTM. Human fetal osteoblasts and periodontal ligament fibroblasts cell lines were stimulated with US at 1.0 and 1.5 MHz central frequencies and power densities of 30 and 60 mW/cm2 in continuous mode for 5 and 10 min. Cellular proliferation, metabolic activity and protein expression were analyzed. The US parameters that significantly improved the metabolic activity were 1.0 MHz at 30 mW/cm2 for 5 min and 1.0 MHz at 60 mW/cm2 for 5 and 10 min for osteoblasts; and 1.0 MHz at 30 mW/cm2 for 5 min and 1.5 MHz at 60 mW/cm2 for 5 and 10 min for fibroblasts. By stimulating with these parameters, the expression of alkaline phosphatase was maintained, while osteoprotegerin synthesis was induced after three days of US stimulation. The US stimulation improved the biological activity of both osteoblasts and periodontal ligament fibroblasts, inducing their osteogenic differentiation.

DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including Mycobacterium tuberculosis, which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (A2B2) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of M. tuberculosis GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other functionally important regions of the GyrB ATPase domain. Specifically, our simulations identify two distinct pathways that respectively connect the GyrB ATPase site to the corynebacteria-specific C-loop, thought to interact with GyrA prior to DNA capture, and to the C-terminus of the GyrB transduction domain, which in turn contacts the C-terminal GyrB topoisomerase-primase (TOPRIM) domain responsible for interactions with GyrA and the centrally bound G-segment DNA. The connection between the ATPase site and the C-loop of dimeric GyrB is consistent with the unusual properties of M. tuberculosis DNA gyrase relative to those from other bacterial species.

This systematic review aims to assess the impact of high (>30 Hz) and low (≤30 Hz) frequency vibrations on orthodontic tooth movement (OTM). Several articles were collected through a systematic search in the databases MEDLINE and SCOPUS, following PRISMA methodology and using a PICO question. Relevant information on selected articles was extracted, and the quality of each study was assessed by the quality assessment tools EPHPP, ROBINS-1 and STAIR. Out of 350 articles, 30 were chosen. Low-frequency vibrations did not seem to accelerate OTM with aligners or fixed appliances, despite some positive outcomes in certain studies. Conversely, high-frequency vibrations were linked to increased aligner change, tooth movement, and space closure with fixed appliances. In vivo studies reported favourable results with high-frequency vibrations (60 Hz to 120 Hz), which stimulate bone biomarkers, facilitating alveolar bone remodelling. The results suggest that high-frequency vibration effectively speeds up orthodontic tooth movement, showing promise in both in vivo and clinical studies. Larger-scale research is needed to strengthen its potential in orthodontics.

Numerous pieces of evidence have supported the therapeutic potential of photobiomodulation (PBM) to modulate bone remodeling on mechanically stimulated teeth, proving PBM's ability to be used as a coadjuvant treatment to accelerate orthodontic tooth movement (OTM). However, there are still uncertainty and discourse around the optimal PBM protocols, which hampers its optimal and consolidated clinical applicability. Given the differential expression and metabolic patterns exhibited in the tension and compression sides of orthodontically stressed teeth, it is plausible that different types of irradiation may be applied to each side of the teeth. In this sense, this study aimed to design and implement an optimization protocol to find the most appropriate PBM parameters to stimulate specific bone turnover processes. To this end, three levels of wavelength (655, 810 and 940 nm), two power densities (5 and 10 mW/cm2) and two regimens of single and multiple sessions within three consecutive days were tested. The biological response of osteoblasts and periodontal ligament (PDL) fibroblasts was addressed by monitoring the PBM's impact on the cellular metabolic activity, as well as on key bone remodeling mediators, including alkaline phosphatase (ALP), osteoprotegerin (OPG) and receptor activator of nuclear factor κ-B ligand (RANK-L), each day. The results suggest that daily irradiation of 655 nm delivered at 10 mW/cm2, as well as 810 and 940 nm light at 5 mW/cm2, lead to an increase in ALP and OPG, potentiating bone formation. In addition, irradiation of 810 nm at 5 mW/cm2 delivered for two consecutive days and suspended by the third day promotes a downregulation of OPG expression and a slight non-significant increase in RANK-L expression, being suitable to stimulate bone resorption. Future studies in animal models may clarify the impact of PBM on bone formation and resorption mediators for longer periods and address the possibility of testing different stimulation periodicities. The present in vitro study offers valuable insights into the effectiveness of specific PBM protocols to promote osteogenic and osteoclastogenesis responses and therefore its potential to stimulate bone formation on the tension side and bone resorption on the compression side of orthodontically stressed teeth.

The dynamical approach to nonequilibrium molecular dynamics (D-NEMD), conceptualised by Ciccotti et al. in the 1970s, has seen resurgence in recent years. In the biomolecular simulation field, the technique provides novel utility in the study of signal propagation and allosteric effects in biological macromolecules. Through comparison of equilibrium MD simulations and perturbed nonequilibrium simulations, the D-NEMD approach provides clear maps of the time-dependent structural response of proteins to a perturbation, and straightforward assessment of the statistical significance of the responses. D-NEMD has recently been shown to complement various equilibrium-based allosteric analysis techniques, such as shortest path maps and distance fluctuation analyses. Here, we review recent applications of D-NEMD to biomolecular systems. D-NEMD simulations identify allosteric 'hotspots' in the oncotarget K-Ras4B; an allosteric binding site, and sites associated with drug resistance in the SARS-CoV-2 main protease. In the SARS-CoV-2 spike, D-NEMD simulations showed the fatty acid binding site connects to distant, functionally relevant sites, and have probed the effects of pH changes. D-NEMD identified a general mechanism of signal propagation in nicotinic acetylcholine receptors. In class A beta-lactamases, reveal the communication networks between allosteric and active sites, and pinpoint sites which, when mutated, alter antibiotic resistance spectrum of activity.