Genetic Inactivation of the ß1 adrenergic receptor prevents Cerebral Cavernous Malformations in zebrafish.

Propranolol reduces experimental murine cerebral cavernous malformations (CCMs) and prevents embryonic caudal venous plexus (CVP) lesions in zebrafish that follow mosaic inactivation of ccm2. Because morpholino silencing of the ß1 adrenergic receptor (adrb1) prevents the embryonic CVP lesion, we proposed that adrb1 plays a role in CCM pathogenesis. Here we report that adrb1 -/- zebrafish exhibited 86% fewer CVP lesions and 87% reduction of CCM lesion volume relative to wild type brood mates at 2dpf and 8-10 weeks stage, respectively. Treatment with metoprolol, a ß1 selective antagonist, yielded a similar reduction in CCM lesion volume. Adrb1 -/- zebrafish embryos exhibited reduced heart rate and contractility and reduced CVP blood flow. Similarly, slowing the heart and eliminating the blood flow in CVP by administration of 2,3-BDM suppressed the CVP lesion. In sum, our findings provide genetic and pharmacological evidence that the therapeutic effect of propranolol on CCM is achieved through ß1 receptor antagonism.

Transcriptomic signatures of individual cell types in cerebral cavernous malformation.

Cerebral cavernous malformation (CCM) is a hemorrhagic neurovascular disease with no currently available therapeutics. Prior evidence suggests that different cell types may play a role in CCM pathogenesis. The contribution of each cell type to the dysfunctional cellular crosstalk remains unclear. Herein, RNA-seq was performed on fluorescence-activated cell sorted endothelial cells (ECs), pericytes, and neuroglia from CCM lesions and non-lesional brain tissue controls. Differentially Expressed Gene (DEG), pathway and Ligand-Receptor (LR) analyses were performed to characterize the dysfunctional genes of respective cell types within CCMs. Common DEGs among all three cell types were related to inflammation and endothelial-to-mesenchymal transition (EndMT). DEG and pathway analyses supported a role of lesional ECs in dysregulated angiogenesis and increased permeability. VEGFA was particularly upregulated in pericytes. Further pathway and LR analyses identified vascular endothelial growth factor A/ vascular endothelial growth factor receptor 2 signaling in lesional ECs and pericytes that would result in increased angiogenesis. Moreover, lesional pericytes and neuroglia predominantly showed DEGs and pathways mediating the immune response. Further analyses of cell specific gene alterations in CCM endorsed potential contribution to EndMT, coagulation, and a hypoxic microenvironment. Taken together, these findings motivate mechanistic hypotheses regarding non-endothelial contributions to lesion pathobiology and may lead to novel therapeutic targets. Video Abstract.

Biocatalytic reductive amination as a route to isotopically labelled amino acids suitable for analysis of large proteins by NMR.

We demonstrate an atom-efficient and easy to use H2-driven biocatalytic platform for the enantioselective incorporation of 2H-atoms into amino acids. By combining the biocatalytic deuteration catalyst with amino acid dehydrogenase enzymes capable of reductive amination, we synthesised a library of multiply isotopically labelled amino acids from low-cost isotopic precursors, such as 2H2O and 15NH4+. The chosen approach avoids the use of pre-labeled 2H-reducing agents, and therefore vastly simplifies product cleanup. Notably, this strategy enables 2H, 15N, and an asymmetric centre to be introduced at a molecular site in a single step, with full selectivity, under benign conditions, and with near 100% atom economy. The method facilitates the preparation of amino acid isotopologues on a half-gram scale. These amino acids have wide applicability in the analytical life sciences, and in particular for NMR spectroscopic analysis of proteins. To demonstrate the benefits of the approach for enabling the workflow of protein NMR chemists, we prepared l-[α-2H,15N, ß-13C]-alanine and integrated it into a large (>400 kDa) heat-shock protein oligomer, which was subsequently analysable by methyl-TROSY techniques, revealing new structural information.

Targeted Reversible Covalent Modification of a Noncatalytic Lysine of the Krev Interaction Trapped 1 Protein Enables Site-Directed Screening for Protein-Protein Interaction Inhibitors.

The covalent reversible modification of proteins is a validated strategy for the development of probes and candidate therapeutics. However, the covalent reversible targeting of noncatalytic lysines is particularly challenging. Herein, we characterize the 2-hydroxy-1-naphthaldehyde (HNA) fragment as a targeted covalent reversible ligand of a noncatalytic lysine (Lys720) of the Krev interaction trapped 1 (KRIT1) protein. We show that the interaction of HNA with KRIT1 is highly specific, results in prolonged residence time of >8 h, and inhibits the Heart of glass 1 (HEG1)-KRIT1 protein-protein interaction (PPI). Screening of HNA derivatives identified analogs exhibiting similar binding modes as the parent fragment but faster target engagement and stronger inhibition activity. These results demonstrate that HNA is an efficient site-directing fragment with promise in developing HEG1-KRIT1 PPI inhibitors. Further, the aldimine chemistry, when coupled with templating effects that promote proximity, can produce a long-lasting reversible covalent modification of noncatalytic lysines.

IL-2 can signal via chemokine receptors to promote regulatory T cells' suppressive function.

Canonical interleukin-2 (IL-2) signaling via the high-affinity CD25-containing IL-2 receptor-Janus kinase (JAK)1,3-signal transducer and activator of transcription 5 (STAT5) pathway is essential for development and maintenance of CD4+CD25HiFoxp3+ regulatory T cells (Tregs) that support immune homeostasis. Here, we report that IL-2 signaling via an alternative CD25-chemokine receptor pathway promotes the suppressive function of Tregs. Using an antibody against CD25 that biases IL-2 signaling toward this alternative pathway, we establish that this pathway increases the suppressive activity of Tregs and ameliorates murine experimental autoimmune encephalomyelitis (EAE). Furthermore, heparan sulfate, an IL-2-binding element of cell surfaces and extracellular matrix, or an engineered IL-2 immunocytokine can also direct IL-2 signaling toward this alternative pathway. Overall, these data reveal a non-canonical mechanism for IL-2 signaling that promotes suppressive functions of Tregs, further elucidates how IL-2 supports immune homeostasis, and suggests approaches to promote or suppress Treg functions.

Circulating Plasma miRNA Homologs in Mice and Humans Reflect Familial Cerebral Cavernous Malformation Disease.

Patients with familial cerebral cavernous malformation (CCM) inherit germline loss of function mutations and are susceptible to progressive development of brain lesions and neurological sequelae during their lifetime. To date, no homologous circulating molecules have been identified that can reflect the presence of germ line pathogenetic CCM mutations, either in animal models or patients. We hypothesize that homologous differentially expressed (DE) plasma miRNAs can reflect the CCM germline mutation in preclinical murine models and patients. Herein, homologous DE plasma miRNAs with mechanistic putative gene targets within the transcriptome of preclinical and human CCM lesions were identified. Several of these gene targets were additionally found to be associated with CCM-enriched pathways identified using the Kyoto Encyclopedia of Genes and Genomes. DE miRNAs were also identified in familial-CCM patients who developed new brain lesions within the year following blood sample collection. The miRNome results were then validated in an independent cohort of human subjects with real-time-qPCR quantification, a technique facilitating plasma assays. Finally, a Bayesian-informed machine learning approach showed that a combination of plasma levels of miRNAs and circulating proteins improves the association with familial-CCM disease in human subjects to 95% accuracy. These findings act as an important proof of concept for the future development of translatable circulating biomarkers to be tested in preclinical studies and human trials aimed at monitoring and restoring gene function in CCM and other diseases.

Study of Metakaolinite Geopolymeric Mortar with Plastic Waste Replacing the Sand: Effects on the Mechanical Properties, Microstructure, and Efflorescence.

In this study, the production of a mortar was proposed in which plastic waste replaced sand by 0%, 50%, and 100% in order to create a sustainable alternative for construction. The performance of the mortars was tested with two types of activators, one with NaOH, as a simple activator, and the other with NaOH and Na2SiO3, as a compound activator. The effects of the LDPE plastic bag waste and the activators on compressive strength, porosity, microstructure analysis, and efflorescence formation were correlated and discussed. The results showed that the replacement of sand with plastic waste at 50% and 100% proportionally reduced the compressive strength due to the increase in porosity caused by the waste, especially in the group of mortars with the simple activator, and included the formation of efflorescence. On the other hand, the compound activator increased the packing of the particles in the mortar, as observed in the images of the microstructure. This reduced porosity inhibited efflorescence and resulted in higher resistances that reached a maximum value of 22.68 MPa at 28 days in the group of 50% mortars with the compound activator. Therefore, the study showed that there is potential for the replacement of sand with plastic waste for the production of mortars, which can be considered a more sustainable building material.

Cochlear ribbon synapse maturation requires Nlgn1 and Nlgn3.

Hearing depends on precise synaptic transmission between cochlear inner hair cells and spiral ganglion neurons through afferent ribbon synapses. Neuroligins (Nlgns) facilitate synapse maturation in the brain, but they have gone unstudied in the cochlea. We report Nlgn3 and Nlgn1 knockout (KO) cochleae have fewer ribbon synapses and have impaired hearing. Nlgn3 KO is more vulnerable to noise trauma with limited activity at high frequencies one day after noise. Furthermore, Nlgn3 KO cochleae have a 5-fold reduction in synapse number compared to wild type after two weeks of recovery. Double KO cochlear phenotypes are more prominent than the KOs, for example, 5-fold smaller synapses, 25% reduction in synapse density, and 30% less synaptic output. These observations indicate Nlgn3 and Nlgn1 are essential to cochlear ribbon synapse maturation and function.

Direct Binding of Rap1 to Talin1 and to MRL Proteins Promotes Integrin Activation in CD4+ T Cells.

Agonist-induced Rap1 GTP loading results in integrin activation involved in T cell trafficking and functions. MRL proteins Rap1-interacting adapter molecule (RIAM) and lamellipodin (LPD) are Rap1 effectors that can recruit talin1 to integrins, resulting in integrin activation. Recent work also implicates direct Rap1-talin1 interaction in integrin activation. Here, we analyze in mice the connections between Rap1 and talin1 that support integrin activation in conventional CD4+ T (Tconv) and CD25HiFoxp3+CD4+ regulatory T (Treg) cells. Talin1(R35E, R118E) mutation that disrupts both Rap1 binding sites results in a partial defect in αLß2, α4ß1, and α4ß7 integrin activation in both Tconv and Treg cells with resulting defects in T cell homing. Talin1(R35E,R118E) Tconv manifested reduced capacity to induce colitis in an adoptive transfer mouse model. Loss of RIAM exacerbates the defects in Treg cell function caused by the talin1(R35E,R118E) mutation, and deleting both MRL proteins in combination with talin1(R35E,R118E) phenocopy the complete lack of integrin activation observed in Rap1a/b-null Treg cells. In sum, these data reveal the functionally significant connections between Rap1 and talin1 that enable αLß2, α4ß1, and α4ß7 integrin activation in CD4+ T cells.

Non-dipper effect in hypertense patients after renal transplantation by 24-hour ambulatory blood pressure monitoring.

OBJECTIVE: Determinate instant and after 1-month non-dipper effect in hypertense patients after renal transplant by 24-hour ambulatory blood pressure monitoring in Hospital General de Zona No. 50, San Luis Potosí, Mexico. METHOD: Descriptive, longitudinal and prospective cohort study of a non-probability convenience sampling in post-transplant patients with hypertension. We collected data from MAPA and includes age, sex, cardiovascular risk factors in variables. Use of central tendency and dispersion measures for descriptive analysis and t Student for inferential analysis. RESULTS: 19 patients were included, 11 male (57.9%) and 8 females (42.1%), with age range 20 to 49 years (median of 30.2 years ± 7.7). Where the non-dipper effect in the first take was 89.5% and in the second take 84.2%. CONCLUSIONS: There is a high frequency of the non-dipper pattern in patients at one month of kidney transplant, the persistence of this hypertension may be, among others, by the use of immunosuppressants. A new category for non-dipper classification is described.

OBJETIVO: Determinar el efecto non-dipper inmediato y posterior a 1 mes en pacientes adultos hipertensos postrasplante renal con monitoreo continuo de la presión arterial de 24 horas, en el Hospital General de Zona No. 50 de San Luis Potosí, México. MÉTODO: Estudio de tipo cohorte, longitudinal, prospectivo, con muestreo no probabilístico por conveniencia de casos consecutivos en pacientes receptores de trasplante renal con hipertensión arterial. Se recogieron los siguientes datos: edad, sexo, factores de riesgo cardiovascular, uso de antihipertensivos o inmunosupresores, y monitoreo ambulatorio de la presión arterial de 24 horas. Se aplicaron medidas de tendencia central y de dispersión para análisis descriptivo, y prueba t de Student para análisis inferencial. RESULTADOS: Se incluyeron 11 hombres (57.9%) y 8 mujeres (42.1%), con una edad de 20 a 49 años (media 30.2 ± 7.7), en los que el efecto non-dipper inmediato fue del 89.5% y posterior a 1 mes fue del 84.2%. CONCLUSIONES: Existe una alta frecuencia del patrón non-dipper en pacientes a 1 mes del trasplante renal. La persistencia de la hipertensión puede ser, entre otras causas, por el uso de inmunosupresores. Se describe una nueva categoría para la clasificación non-dipper.

Trivalent Subunit Vaccine Candidates for COVID-19 and Their Delivery Devices.

The COVID-19 pandemic highlights the need for platform technologies enabling rapid development of vaccines for emerging viral diseases. The current vaccines target the SARS-CoV-2 spike (S) protein and thus far have shown tremendous efficacy. However, the need for cold-chain distribution, a prime-boost administration schedule, and the emergence of variants of concern (VOCs) call for diligence in novel SARS-CoV-2 vaccine approaches. We studied 13 peptide epitopes from SARS-CoV-2 and identified three neutralizing epitopes that are highly conserved among the VOCs. Monovalent and trivalent COVID-19 vaccine candidates were formulated by chemical conjugation of the peptide epitopes to cowpea mosaic virus (CPMV) nanoparticles and virus-like particles (VLPs) derived from bacteriophage Qß. Efficacy of this approach was validated first using soluble vaccine candidates as solo or trivalent mixtures and subcutaneous prime-boost injection. The high thermal stability of our vaccine candidates allowed for formulation into single-dose injectable slow-release polymer implants, manufactured by melt extrusion, as well as microneedle (MN) patches, obtained through casting into micromolds, for prime-boost self-administration. Immunization of mice yielded high titers of antibodies against the target epitope and S protein, and data confirms that antibodies block receptor binding and neutralize SARS-CoV and SARS-CoV-2 against infection of human cells. We present a nanotechnology vaccine platform that is stable outside the cold-chain and can be formulated into delivery devices enabling single administration or self-administration. CPMV or Qß VLPs could be stockpiled, and epitopes exchanged to target new mutants or emergent diseases as the need arises.

Cerebral Cavernous Malformation: From Mechanism to Therapy.

Cerebral cavernous malformations are acquired vascular anomalies that constitute a common cause of central nervous system hemorrhage and stroke. The past 2 decades have seen a remarkable increase in our understanding of the pathogenesis of this vascular disease. This new knowledge spans genetic causes of sporadic and familial forms of the disease, molecular signaling changes in vascular endothelial cells that underlie the disease, unexpectedly strong environmental effects on disease pathogenesis, and drivers of disease end points such as hemorrhage. These novel insights are the integrated product of human clinical studies, human genetic studies, studies in mouse and zebrafish genetic models, and basic molecular and cellular studies. This review addresses the genetic and molecular underpinnings of cerebral cavernous malformation disease, the mechanisms that lead to lesion hemorrhage, and emerging biomarkers and therapies for clinical treatment of cerebral cavernous malformation disease. It may also serve as an example for how focused basic and clinical investigation and emerging technologies can rapidly unravel a complex disease mechanism.

E. coli Nickel-Iron Hydrogenase 1 Catalyses Non-native Reduction of Flavins: Demonstration for Alkene Hydrogenation by Old Yellow Enzyme Ene-reductases*.

A new activity for the [NiFe] uptake hydrogenase 1 of Escherichia coli (Hyd1) is presented. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H2 as a simple, completely atom-economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25-70 °C) and extended reaction times. The utility of this system as a simple, easy to implement FMNH2 or FADH2 regenerating system is then demonstrated by supplying reduced flavin to Old Yellow Enzyme "ene-reductases" to support asymmetric alkene reductions with up to 100 % conversion. Hyd1 turnover frequencies up to 20.4 min-1 and total turnover numbers up to 20 200 were recorded during flavin recycling.

Predictors of mortality in hospitalized COVID-19 patients: A Mexican population-based cohort study.

OBJECTIVES: COVID-19 outbreak brings a challenge to healthcare systems. The sex, age, and cardiometabolic comorbidities have been considered risk factors for disease severity. To evaluate the association between risk factors with death as well the risk of death in hospitalized COVID-19 patients. METHODS: The present cross-sectional cohort study, includes hospitalized SARS-CoV-2 confirmed cases. Data analysis was performed using the National COVID-19 Cases Report Database. Pearson's χ2 test and odds ratios (95% CI) were calculated to determine the association between variables. Thereafter, risk of death was evaluated by Cox proportional hazards model. RESULTS: A total of 67 328 inpatients were included; mean age 55.29 years (±15.97). Of total, 42 164 (62.62%) were men, 6 349 (9.43%) were intubated, and 23 873 (35.46%) died. Male sex, age older than 60 years, and cardiometabolic comorbidities were associated with death. Hazard ratio for death in older intubated patients was lower than in non-intubated (HR 1.242, 95%CI, 1.167-1.322; P < 0.001) and (HR 2.128, 95%CI, 2.066-2.193; P < 0.001) respectively. CONCLUSIONS: Tracheal intubation or not is the most important predictor for death in COVID-19 infected patients in this Mexican cohort. Already known risk factors for COVID-19 severity may become less relevant once patients require tracheal intubation.

E. coli Nickel-Iron Hydrogenase 1 Catalyses Non-native Reduction of Flavins: Demonstration for Alkene Hydrogenation by Old Yellow Enzyme Ene-reductases.

A new activity for the [NiFe] uptake hydrogenase 1 of Escherichia coli (Hyd1) is presented. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H2 as a simple, completely atom-economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25-70 °C) and extended reaction times. The utility of this system as a simple, easy to implement FMNH2 or FADH2 regenerating system is then demonstrated by supplying reduced flavin to Old Yellow Enzyme "ene-reductases" to support asymmetric alkene reductions with up to 100 % conversion. Hyd1 turnover frequencies up to 20.4 min-1 and total turnover numbers up to 20 200 were recorded during flavin recycling.

Noise Exposures Causing Hearing Loss Generate Proteotoxic Stress and Activate the Proteostasis Network.

Exposure to excessive sound causes noise-induced hearing loss through complex mechanisms and represents a common and unmet neurological condition. We investigate how noise insults affect the cochlea with proteomics and functional assays. Quantitative proteomics reveals that exposure to loud noise causes proteotoxicity. We identify and confirm hundreds of proteins that accumulate, including cytoskeletal proteins, and several nodes of the proteostasis network. Transcriptomic analysis reveals that a subset of the genes encoding these proteins also increases acutely after noise exposure, including numerous proteasome subunits. Global cochlear protein ubiquitylation levels build up after exposure to excess noise, and we map numerous posttranslationally modified lysines residues. Several collagen proteins decrease in abundance, and Col9a1 specifically localizes to pillar cell heads. After two weeks of recovery, the cochlea selectively elevates the abundance of the protein synthesis machinery. We report that overstimulation of the auditory system drives a robust cochlear proteotoxic stress response.

Catastrophic Failure of Reverse Shoulder Arthroplasty from a Broken Screw: A Case Report.

CASE: A 63-year-old woman presented with pain and limited range of motion 2 years after undergoing reverse shoulder arthroplasty (RSA). She underwent revision RSA for catastrophic implant failure caused by central screw breakage, subsequent polyethylene wear, and glenosphere loosening, and achieved excellent outcomes. CONCLUSION: This is a unique case of a salvage procedure with satisfactory outcomes for a rare complication of RSA. It demonstrates the importance of clinical vigilance notwithstanding the absence of radiographic abnormalities in long-term monitoring of patient implant failure.

Management of irreparable massive rotator cuff tears: a systematic review and meta-analysis of patient-reported outcomes, reoperation rates, and treatment response.

BACKGROUND: There is no consensus on the treatment of irreparable massive rotator cuff tears. The goal of this systematic review and meta-analysis was to (1) compare patient-reported outcome scores, (2) define failure and reoperation rates, and (3) quantify the magnitude of patient response across treatment strategies. METHODS: The MEDLINE, Embase, CENTRAL (Cochrane Central Register of Controlled Trials), and Scopus databases were searched for studies including physical therapy and operative treatment of massive rotator cuff tears. The criteria of the Methodological Index for Non-randomized Studies were used to assess study quality. Primary outcome measures were patient-reported outcome scores as well as failure, complication, and reoperation rates. To quantify patient response to treatment, we compared changes in the Constant-Murley score and American Shoulder and Elbow Surgeons (ASES) score with previously reported minimal clinically important difference (MCID) thresholds. RESULTS: No level I or II studies that met the inclusion and exclusion criteria were found. Physical therapy was associated with a 30% failure rate among the included patients, and another 30% went on to undergo surgery. Partial repair was associated with a 45% retear rate and 10% reoperation rate. Only graft interposition was associated with a weighted average change that exceeded the MCID for both the Constant-Murley score and ASES score. Latissimus tendon transfer techniques using humeral bone tunnel fixation were associated with a 77% failure rate. Superior capsular reconstruction with fascia lata autograft was associated with a weighted average change that exceeded the MCID for the ASES score. Reverse arthroplasty was associated with a 10% prosthesis failure rate and 8% reoperation rate. CONCLUSION: There is a lack of high-quality comparative studies to guide treatment recommendations. Compared with surgery, physical therapy is associated with less improvement in perceived functional outcomes and a higher clinical failure rate.

Novel Approaches of Nanoceria with Magnetic, Photoluminescent, and Gas-Sensing Properties.

The modification of CeO2 with rare-earth elements opens up a wide range of applications as biomedical devices using infrared emission as well as magnetic and gas-sensing devices, once the structural, morphological, photoluminescent, magnetic, electric, and gas-sensing properties of these systems are strongly correlated to quantum electronic transitions between rare-earth f-states among defective species. Quantitative phase analysis revealed that the nanopowders are free from secondary phases and crystallize in the fluorite-type cubic structure. Magnetic coercive field measurements on the powders indicate that the substitution of cerium with lanthanum (8 wt %), in a fluorite-type cubic structure, created oxygen vacancies and led to a decrease in the fraction of Ce species in the 3+ state, resulting in a stronger room-temperature ferromagnetic response along with high coercivity (160 Oe). In addition to the magnetic and photoluminescent behavior, a fast response time (5.5 s) was observed after CO exposure, indicating that the defective structure of ceria-based materials corresponds to the key of success in terms of applications using photoluminescent, magnetic, or electrical behaviors.

Frontline Science: A flexible kink in the transmembrane domain impairs ß2 integrin extension and cell arrest from rolling.

ß2 integrins are the main adhesion molecules in neutrophils and other leukocytes and are rapidly activated by inside-out signaling, which results in conformational changes that are transmitted through the transmembrane domain (TMD). Here, we investigated the biologic effect of introducing a proline mutation in the ß2 integrin TMD to create a flexible kink that uncouples the topology of the inner half of the TMD from the outer half and impairs integrin activation. The ß2 integrin alpha chains, αL, αM, αX, and αD, all contain an inserted (I) domain with homology to von Willebrand factor A domain. ß2 activation was monitored in a homogenous binding assay of 2 reporter monoclonal antibodies: KIM127 reporting extension (E+ ) and mAb24 reporting the high-affinity (H+ ) conformation of the ß2 I-like domain. The proline mutation partially diminished chemokine-induced extension, but not the high-affinity conformation. The proline mutation in the TMD of ß2 completely inhibited arrest of rolling HL-60 cells in response to the chemokine IL-8. TMD mutant HL-60 cells rolling on P-selectin and ICAM-1 were unable to reduce their rolling velocity in response to IL-8. Quantitative dynamic footprinting live-cell imaging showed that blocking TMD topology transmission impaired the chemokine-induced activation of ß2, limiting the appearance of extended high-affinity (E+ H+ ) ß2. This also resulted in a defect in early spreading (3 min after arrest), which could be overcome by forced integrin activation using Mn2+ . We conclude that the TMD proline mutation severely impairs ß2 integrin extension, cell arrest, and early spreading.