Protein dynamics underlying allosteric regulation.

Allostery is the mechanism by which information and control are propagated in biomolecules. It regulates ligand binding, chemical reactions, and conformational changes. An increasing level of experimental resolution and control over allosteric mechanisms promises a deeper understanding of the molecular basis for life and powerful new therapeutics. In this review, we survey the literature for an up-to-date biological and theoretical understanding of protein allostery. By delineating five ways in which the energy landscape or the kinetics of a system may change to give rise to allostery, we aim to help the reader grasp its physical origins. To illustrate this framework, we examine three systems that display these forms of allostery: allosteric inhibitors of beta-lactamases, thermosensation of TRP channels, and the role of kinetic allostery in the function of kinases. Finally, we summarize the growing power of computational tools available to investigate the different forms of allostery presented in this review.

Suspected tick-borne flavivirus meningoencephalomyelitis in dogs from the UK: six cases (2021).

OBJECTIVES: Tick-borne encephalitis virus and louping ill virus are neurotropic flaviviruses transmitted by ticks. Epidemiologically, tick-borne encephalitis is endemic in Europe whereas louping ill's predominant geographical distribution is the UK. Rarely, these flaviviruses affect dogs causing neurological signs. This case series aimed to describe the clinical, clinicopathological, and imaging findings, as well as the outcomes in six dogs with meningoencephalitis and/or meningomyelitis caused by a flavivirus in the UK in 2021. MATERIALS AND METHODS: Observational retrospective case-series study. Clinical data were retrieved from medical records of dogs with positive serological or immunohistochemical results from three different institutions from spring to winter 2021. RESULTS: Six dogs were included in the study. All dogs presented an initial phase of pyrexia and/or lethargy followed by progressive signs of spinal cord and/or intracranial disease. Magnetic resonance imaging showed bilateral and symmetrical lesions affecting the grey matter of the thalamus, pons, medulla oblongata, and thoracic or lumbar intumescences with none or mild parenchymal and meningeal contrast enhancement. Serology for tick-borne encephalitis virus was positive in five dogs with the presence of seroconversion in two dogs. The viral distinction between flaviviruses was not achieved. One dog with negative serology presented positive immunohistochemistry at post-mortem examination. Three dogs survived but presented neurological sequelae. Three dogs were euthanased due to the rapid progression of the clinical signs or static neurological signs. CLINICAL SIGNIFICANCE: These cases raise awareness of the presence of tick-borne encephalitis as an emergent disease or the increased prevalence of louping ill virus affecting dogs in the UK.

Mitochondrial electron transport chain, ceramide, and coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle.

Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Previously we showed that deficiency of coenzyme Q (CoQ) is necessary and sufficient for IR in adipocytes and skeletal muscle (Fazakerley et al., 2018). Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, CoQ deficiency, mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells result in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (mice, C57BL/6J) (under chow and high-fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.

Q1291H-CFTR molecular dynamics simulations and ex vivo theratyping in nasal epithelial models and clinical response to elexacaftor/tezacaftor/ivacaftor in a Q1291H/F508del patient.

Background: Cystic fibrosis (CF) is caused by a wide spectrum of mutations in the CF transmembrane conductance regulator (CFTR) gene, with some leading to non-classical clinical presentations. We present an integrated in vivo, in silico and in vitro investigation of an individual with CF carrying the rare Q1291H-CFTR allele and the common F508del allele. At age 56 years, the participant had obstructive lung disease and bronchiectasis, qualifying for Elexacaftor/Tezacaftor/Ivacaftor (ETI) CFTR modulator treatment due to their F508del allele. Q1291H CFTR incurs a splicing defect, producing both a normally spliced but mutant mRNA isoform and a misspliced isoform with a premature termination codon, causing nonsense mediated decay. The effectiveness of ETI in restoring Q1291H-CFTR is largely unknown. Methods: We collected clinical endpoint measurements, including forced expiratory volume in 1 s percent predicted (FEV1pp) and body mass index (BMI), and examined medical history. In silico simulations of the Q1291H-CFTR were compared to Q1291R, G551D, and wild-type (WT)-CFTR. We quantified relative Q1291H CFTR mRNA isoform abundance in patient-derived nasal epithelial cells. Differentiated pseudostratified airway epithelial cell models at air liquid interface were created and ETI treatment impact on CFTR was assessed by electrophysiology assays and Western blot. Results: The participant ceased ETI treatment after 3 months due to adverse events and no improvement in FEV1pp or BMI. In silico simulations of Q1291H-CFTR identified impairment of ATP binding similar to known gating mutants Q1291R and G551D-CFTR. Q1291H and F508del mRNA transcripts composed 32.91% and 67.09% of total mRNA respectively, indicating 50.94% of Q1291H mRNA was misspliced and degraded. Mature Q1291H-CFTR protein expression was reduced (3.18% ± 0.60% of WT/WT) and remained unchanged with ETI. Baseline CFTR activity was minimal (3.45 ± 0.25 µA/cm2) and not enhanced with ETI (5.73 ± 0.48 µA/cm2), aligning with the individual's clinical evaluation as a non-responder to ETI. Conclusion: The combination of in silico simulations and in vitro theratyping in patient-derived cell models can effectively assess CFTR modulator efficacy for individuals with non-classical CF manifestations or rare CFTR mutations, guiding personalized treatment strategies and optimizing clinical outcomes.

Mitochondrial electron transport chain, ceramide and Coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle.

Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, deficiency of coenzyme Q (CoQ), mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells results in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (under chow and high fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial Ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.

S945L-CFTR molecular dynamics, functional characterization and tezacaftor/ivacaftor efficacy in vivo and in vitro in matched pediatric patient-derived cell models.

Cystic Fibrosis (CF) results from over 400 different disease-causing mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. These CFTR mutations lead to numerous defects in CFTR protein function. A novel class of targeted therapies (CFTR modulators) have been developed that can restore defects in CFTR folding and gating. This study aimed to characterize the functional and structural defects of S945L-CFTR and interrogate the efficacy of modulators with two modes of action: gating potentiator [ivacaftor (IVA)] and folding corrector [tezacaftor (TEZ)]. The response to these modulators in vitro in airway differentiated cell models created from a participant with S945L/G542X-CFTR was correlated with in vivo clinical outcomes of that participant at least 12 months pre and post modulator therapy. In this participants' airway cell models, CFTR-mediated chloride transport was assessed via ion transport electrophysiology. Monotherapy with IVA or TEZ increased CFTR activity, albeit not reaching statistical significance. Combination therapy with TEZ/IVA significantly (p = 0.02) increased CFTR activity 1.62-fold above baseline. Assessment of CFTR expression and maturation via western blot validated the presence of mature, fully glycosylated CFTR, which increased 4.1-fold in TEZ/IVA-treated cells. The in vitro S945L-CFTR response to modulator correlated with an improvement in in vivo lung function (ppFEV1) from 77.19 in the 12 months pre TEZ/IVA to 80.79 in the 12 months post TEZ/IVA. The slope of decline in ppFEV1 significantly (p = 0.02) changed in the 24 months post TEZ/IVA, becoming positive. Furthermore, there was a significant improvement in clinical parameters and a fall in sweat chloride from 68 to 28 mmol/L. The mechanism of dysfunction of S945L-CFTR was elucidated by in silico molecular dynamics (MD) simulations. S945L-CFTR caused misfolding of transmembrane helix 8 and disruption of the R domain, a CFTR domain critical to channel gating. This study showed in vitro and in silico that S945L causes both folding and gating defects in CFTR and demonstrated in vitro and in vivo that TEZ/IVA is an efficacious modulator combination to address these defects. As such, we support the utility of patient-derived cell models and MD simulations in predicting and understanding the effect of modulators on CFTR function on an individualized basis.

Molecular Dynamics and Theratyping in Airway and Gut Organoids Reveal R352Q-CFTR Conductance Defect.

A significant challenge to making targeted cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies accessible to all individuals with cystic fibrosis (CF) are many mutations in the CFTR gene that can cause CF, most of which remain uncharacterized. Here, we characterized the structural and functional defects of the rare CFTR mutation R352Q, with a potential role contributing to intrapore chloride ion permeation, in patient-derived cell models of the airway and gut. CFTR function in differentiated nasal epithelial cultures and matched intestinal organoids was assessed using an ion transport assay and forskolin-induced swelling assay, respectively. CFTR potentiators (VX-770, GLPG1837, and VX-445) and correctors (VX-809, VX-445, with or without VX-661) were tested. Data from R352Q-CFTR were compared with data of 20 participants with mutations with known impact on CFTR function. R352Q-CFTR has residual CFTR function that was restored to functional CFTR activity by CFTR potentiators but not the corrector. Molecular dynamics simulations of R352Q-CFTR were carried out, which indicated the presence of a chloride conductance defect, with little evidence supporting a gating defect. The combination approach of in vitro patient-derived cell models and in silico molecular dynamics simulations to characterize rare CFTR mutations can improve the specificity and sensitivity of modulator response predictions and aid in their translational use for CF precision medicine.

Molecular dynamics and functional characterization of I37R-CFTR lasso mutation provide insights into channel gating activity.

Characterization of I37R, a mutation located in the lasso motif of the CFTR chloride channel, was conducted by theratyping several CFTR modulators from both potentiator and corrector classes. Intestinal current measurements in rectal biopsies, forskolin-induced swelling (FIS) in intestinal organoids, and short circuit current measurements in organoid-derived monolayers from an individual with I37R/F508del CFTR genotype demonstrated that the I37R-CFTR results in a residual function defect amenable to treatment with potentiators and type III, but not type I, correctors. Molecular dynamics of I37R using an extended model of the phosphorylated, ATP-bound human CFTR identified an altered lasso motif conformation which results in an unfavorable strengthening of the interactions between the lasso motif, the regulatory (R) domain, and the transmembrane domain 2 (TMD2). Structural and functional characterization of the I37R-CFTR mutation increases understanding of CFTR channel regulation and provides a potential pathway to expand drug access to CF patients with ultra-rare genotypes.

Global redox proteome and phosphoproteome analysis reveals redox switch in Akt.

Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than their absolute activation state, highlighting an unappreciated interplay between these modifications. Mechanistic analysis of the redox regulation of Akt identified two cysteine residues in the pleckstrin homology domain (C60 and C77) to be reversibly oxidized. Oxidation at these sites affected Akt recruitment to the plasma membrane by stabilizing the PIP3 binding pocket. Our data provide insights into the interplay between oxidative stress-derived redox signalling and protein phosphorylation networks and serve as a resource for understanding the contribution of cellular oxidation to a range of diseases.

Microparticle-embedded fibroin/alginate beads for prolonged local release of simvastatin hydroxyacid to mesenchymal stem cells.

In the present work, we propose silk fibroin/alginate (SF/Alg) beads embedding simvastatin-loaded biodegradable microparticles as a versatile platform capable of tuning SVA release and in so doing osteogenic effects. In a first part of the study, microparticles of poly(lactic-co-glycolic) acid incorporating simvastatin either as lactone (SVL) or as hydroxyacid form (SVA) were prepared by spray-drying. While SVA-loaded microparticles released the drug in three days, long-term release of SVA could be obtained from SVL-loaded microparticles. In this latter case, SVL was promptly transformed to the osteogenic active SVA during release. When tested on mesenchymal stem cells, a time- and dose-dependent effect of SVL-loaded microparticles on cell proliferation and alkaline phosphatase (ALP) activity was found. Thereafter, SVL-loaded microparticles were embedded in SF/Alg beads to limit the initial simvastatin burst and to achieve easier implantation as well. Microparticle-embedded beads showed no cytotoxicity while ALP activity increased. If correctly exploited, the developed system may be suitable as osteogenic polymer scaffolds releasing correct amount of the drug locally for long time-frames.

Development of inhalable hyaluronan/mannitol composite dry powders for flucytosine repositioning in local therapy of lung infections.

Flucytosine (5-fluorocytosine, 5-FC) is a fluorinated analogue of cytosine currently approved for the systemic treatment of fungal infections, which has recently demonstrated a very promising antivirulence activity against the bacterial pathogen Pseudomonas aeruginosa. In this work, we propose novel inhalable hyaluronic acid (HA)/mannitol composite dry powders for repositioning 5-FC in the local treatment of lung infections, including those affecting cystic fibrosis (CF) patients. Different dry powders were produced in one-step by spray-drying. Powder composition and process conditions were selected after in depth formulation studies aimed at selecting the 5-FC/HA/mannitol formulation with convenient aerosolization properties and drug release profile in simulated lung fluids. The optimized 5-FC/HA/mannitol powder for inhalation (HyaMan_FC#3) was effectively delivered from different breath-activated dry powder inhalers (DPI) already available to CF patients. Nevertheless, the aerodynamic assessment of fine particles suggested that the developed formulation well fit with a low-resistance DPI. HyaMan_FC#3 inhibited the growth of the fungus Candida albicans and the production of the virulence factor pyoverdine by P. aeruginosa at 5-FC concentrations that did not affect the viability of both wild type (16HBE14o-) and CF (CFBE41o-) human bronchial epithelial cells. Finally, pharmacokinetics of HyaMan_FC#3 inhalation powder and 5-FC solution after intratracheal administration in rats were compared. In vivo results clearly demonstrated that, when formulated as dry powder, 5-FC levels in both bronchoalveolar lavage fluid and lung tissue were significantly higher and sustained over time as compared to those obtained with the 5-FC solution. Of note, when the same 5-FC amount was administered intravenously, no significant drug amount was found in the lung at each time point from the injection. To realize a 5-FC lung concentration similar to that obtained by using HyaMan_FC#3, a 6-fold higher dose of 5-FC should be administered intravenously. Taken together, our data demonstrate the feasibility to deliver 5-FC by the pulmonary route likely avoiding/reducing the well-known side effects associated to the high systemic 5-FC doses currently used in humans. Furthermore, our results highlight that an appropriate formulation design can improve the persistence of the drug at lungs, where microorganisms causing severe infections are located.

NerveCheck: An inexpensive quantitative sensory testing device for patients with diabetic neuropathy.

AIMS: Sensory neuropathy is central to the development of painful neuropathy, and foot ulceration in patients with diabetes. Currently, available QST devices take considerable time to perform and are expensive. NerveCheck is the first inexpensive ($500), portable QST device to perform both vibration and thermal testing and hence evaluate diabetic peripheral neuropathy (DPN). This study was undertaken to establish the reproducibility and diagnostic validity of NerveCheck for detecting neuropathy. METHODS: 130 subjects (28 with DPN, 46 without DPN and 56 control subjects) underwent QST assessment with NerveCheck; vibration perception and thermal testing. DPN was defined according to the Toronto criteria. RESULTS: NerveCheck's intra correlation coefficient for vibration, cold and warm sensation testing was 0.79 (95% LOA: -4.20 to 6.60), 0.86 (95% LOA: -1.38 to 2.72) and 0.71 (95% LOA: -2.36 to 3.83), respectively. The diagnostic accuracy (AUC) for vibration, cold and warm sensation testing was 86% (SE: 0.038, 95% CI 0.79-0.94), 79% (SE: 0.058, 95% CI 0.68-0.91) and 72% (SE: 0.058, 95% CI 0.60-0.83), respectively. CONCLUSIONS: This study shows that NerveCheck has good reproducibility and comparable diagnostic accuracy to established QST equipment for the diagnosis of DPN.

Sonochemical assisted synthesis of SrFe12O19 nanoparticles.

We present the synthesis of M-type strontium hexaferrite by sonochemistry and annealing. The effects of the sonication time and thermal energy on the crystal structure and magnetic properties of the obtained powders are presented. Strontium hexagonal ferrite (SrFe12O19) was successfully prepared by the ultrasonic cavitation (sonochemistry) of a complexed polyol solution of metallic acetates and diethylene glycol. The obtained materials were subsequently annealed at temperatures from 300 to 900 °C. X-ray diffraction analysis shows that the sonochemical process yields an amorphous phase containing Fe(3+), Fe(2+) and Sr(2+) ions. This amorphous phase transforms into an intermediate phase of maghemite (γ-Fe2O3) at 300 °C. At 500 °C, the intermediate species is converted to hematite (α-Fe2O3) by a topotactic transition. The final product of strontium hexaferrite (SrFe12O19) is generated at 800 °C. The obtained strontium hexaferrite shows a magnetization of 62.3 emu/g, which is consistent with pure hexaferrite obtained by other methods, and a coercivity of 6.25 kOe, which is higher than expected for this hexaferrite. The powder morphology is composed of aggregates of rounded particles with an average particle size of 60 nm.

Alginate-hyaluronan composite hydrogels accelerate wound healing process.

In this paper we propose polysaccharide hydrogels combining alginate (ALG) and hyaluronan (HA) as biofunctional platform for dermal wound repair. Hydrogels produced by internal gelation were homogeneous and easy to handle. Rheological evaluation of gelation kinetics of ALG/HA mixtures at different ratios allowed understanding the HA effect on ALG cross-linking process. Disk-shaped hydrogels, at different ALG/HA ratio, were characterized for morphology, homogeneity and mechanical properties. Results suggest that, although the presence of HA does significantly slow down gelation kinetics, the concentration of cross-links reached at the end of gelation is scarcely affected. The in vitro activity of ALG/HA dressings was tested on adipose derived multipotent adult stem cells (Ad-MSC) and an immortalized keratinocyte cell line (HaCaT). Hydrogels did not interfere with cell viability in both cells lines, but significantly promoted gap closure in a scratch assay at early (1 day) and late (5 days) stages as compared to hydrogels made of ALG alone (p<0.01 and 0.001 for Ad-MSC and HaCaT, respectively). In vivo wound healing studies, conducted on a rat model of excised wound indicated that after 5 days ALG/HA hydrogels significantly promoted wound closure as compared to ALG ones (p<0.001). Overall results demonstrate that the integration of HA in a physically cross-linked ALG hydrogel can be a versatile strategy to promote wound healing that can be easily translated in a clinical setting.

Spray-by-spray in situ cross-linking alginate hydrogels delivering a tea tree oil microemulsion.

In this paper we propose an in situ forming ionically cross-linked alginate (Alg) hydrogel delivering a Tea Tree Oil microemulsion (MeTTO) and potentially useful as an advanced dressing for infected wounds. Alg hydrogels were prepared by a spray-by-spray deposition method with the aim to minimize the discomforts during application. From pseudoternary phase diagrams, it was found that proper combination of TTO, water, polysorbate 80 and ethanol gave stable spherical MeTTO with good antimicrobial activity. On this basis, MeTTO at 20% TTO was selected for further inclusion in an Alg hydrogel prepared by alternating sprays of Alg/MeTTO and calcium chloride solutions. Homogeneous dispersion of MeTTO inside cross-linked Alg was assessed by different macroscopic and microscopic methods demonstrating the superior propensity of MeTTO to be integrated in the water-based hydrogel as compared to TTO. Antimicrobial effect of Alg/MeTTO hydrogels on Escherichia Coli strains was remarkable, highlighting the potential of the system as bioactive wound dressing.

[Possible denosumab-induced jaw osteonecrosis in the treatment of osteoporosis. A case report]. / Posible osteonecrosis mandibular inducida por denosumab en el tratamiento de la osteoporosis. A propósito de un caso.

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Posible osteonecrosis mandibular inducida por denosumab en el tratamiento de la osteoporosis. A propósito de un caso / Posible denosumab-induced jaw osteonecrosis in the treatment of osteoporosis. A case report

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