Search

CRISPR-Cas13 systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity in mammalian cells and mouse models. Moreover, its efficiency could be improved in vivo . Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations to enhance the CRISPR-RfxCas13d system in zebrafish. We i) used chemically modified gRNAs to allow more penetrant loss-of-function phenotypes, ii) improved nuclear RNA-targeting, and iii) compared different computational models and determined the most accurate to predict gRNA activity in vivo . Furthermore, we demonstrated that transient CRISPR-RfxCas13d can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except when targeting extremely abundant and ectopic RNAs. Finally, we implemented alternative RNA-targeting CRISPR-Cas systems with reduced or absent collateral activity. Altogether, these findings contribute to CRISPR-Cas technology optimization for RNA targeting in zebrafish through transient approaches and assist in the progression of in vivo applications., Competing Interests: Declaration of interests Kevin Holden and John A. Walker II were both employees and shareholders in Synthego Corporation at the time of this work. The rest of authors declares no competing interests.

Background: The surgical treatment of hallux valgus (HV) deformity has been the subject of countless publications but few focus on the altered windlass mechanism or analyze the biomechanical behavior immediately after surgery., Methods: Patients treated for HV between January and March 2023 were included. The surgery consisted of a L-reverse first metatarsal osteotomy. To analyze the windlass mechanism we record two different measurements; the isolated first metatarsophalangeal joint (MTPJ) dorsiflexion angle (IDA) and dynamic plantarization of the first metatarsal head when performing first MTPJ dorsiflexion imprinting a mark on a modeling foam., Results: A total of 30 patients diagnosed with symptomatic HV were included. In all patients, a change in the IDA angle was evident, being overall statistically significant. About modeling foam imprinted mark, all measurements, in all planes of space, had a clear tendency to increase, which turned out to be statistically significant (p < 0.001)., Conclusions: An altered windlass mechanism may be successfully recovered immediately after hallux valgus deformity surgery. This could be evinced by an indirect measurement analyzing the imprint of the head of the first metatarsal in a modeling foam and the IDA., Levels of Evidence Ii: None., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2024 European Foot and Ankle Society. Published by Elsevier Ltd. All rights reserved.)

Given Colombia's status as a middle-income country with healthcare challenges, leveraging telemedicine could significantly benefit hard-to-reach regions, under-resourced and underserved communities. This study provides a comprehensive overview of the country's telerehabilitation landscape, exploring the clinicians' and patients' perspectives during the COVID-19 pandemic through systematic review and qualitative analysis. Sixteen therapists and three patients were identified via snowball sampling. The literature review was scarce and scattered across various topics in the country; some studies delved into specific aspects like legislative frameworks and patient outcomes from different medical specialties. The qualitative analysis demonstrates that despite the learning curve, telerehabilitation strengthens therapeutic support, enhances patient autonomy, fosters a positive patient-provider relationship, achieves treatment goals, promotes family involvement, reduces time and costs, and ensures continuity of therapy services. This study identified research gaps, challenges, and opportunities in telerehabilitation in a Latin American country. Adopting telemedicine technologies in low- and middle-income countries could significantly enhance their healthcare systems., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2024 Sara Gabriela Pacichana-Quinayaz, Lina María Rodríguez Vélez, Daniel Sánchez Cano, Yisel Mabirlly León Sánchez, Olga Marina Hernández Orobio, Maria Ana Tovar Sánchez, Gloria Isabel Toro Córdoba, Francisco Javier Bonilla-Escobar.)

The directed differentiation of pluripotent stem cells (PSCs) from panels of genetically diverse individuals is emerging as a powerful experimental system for characterizing the impact of natural genetic variation on developing cell types and tissues. Here, we establish new PSC lines and experimental approaches for modeling embryonic development in a genetically diverse, outbred mouse stock (Diversity Outbred mice). We show that a range of inbred and outbred PSC lines can be stably maintained in the primed pluripotent state (epiblast stem cells -- EpiSCs) and establish the contribution of genetic variation to phenotypic differences in gene regulation and directed differentiation. Using pooled in vitro fertilization, we generate and characterize a genetic reference panel of Diversity Outbred PSCs (n = 230). Finally, we demonstrate the feasibility of pooled culture of Diversity Outbred EpiSCs as "cell villages", which can facilitate the differentiation of large numbers of EpiSC lines for forward genetic screens. These data can complement and inform similar efforts within the stem cell biology and human genetics communities to model the impact of natural genetic variation on phenotypic variation and disease-risk., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

An effective pulmonary hypertension (PH) treatment should combine antiproliferative and vasodilator effects. We characterized a wide-range of drugs comparing their anti-proliferative vs vasodilator effects in human and rat pulmonary artery smooth muscle cells (PASMC). Key findings: 1) Approved PH drugs (PDE5 inhibitors, sGC stimulators and PGI 2 agonists) are preferential vasodilators. 2) cGMP stimulators were more effective in cells derived from hypertensive rats. 3) Nifedipine acted equally as vasodilator and antiproliferative. 4) quercetin and imatinib were potent dual vasodilator/antiproliferative drugs. 5) Tacrolimus and levosimendan lacked antiproliferative effects. 6) Forskolin, pinacidil and hydroxyfasudil were more effective as antiproliferative in human cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Drugs that lower plasma apolipoprotein B (ApoB)-containing lipoproteins are central to treating advanced atherosclerosis and provide partial protection against clinical events. Previous research showed that lowering ApoB-containing lipoproteins stops plaque inflammation, but how these drugs affect the heterogeneous population of plaque cells derived from smooth muscle cells (SMCs) is unknown. SMC-derived cells are the main cellular component of atherosclerotic lesions and the source of structural components that determine the size of plaques and their propensity to rupture and trigger thrombosis, the proximate cause of heart attack and stroke. Using lineage tracing and single-cell techniques to investigate the full SMC-derived cellular compartment in progressing and regressing plaques in mice, here we show that lowering ApoB-containing lipoproteins reduces nuclear factor kappa-light-chain-enhancer of activated B cells signaling in SMC-derived fibromyocytes and chondromyocytes and leads to depletion of these abundant cell types from plaques. These results uncover an important mechanism through which cholesterol-lowering drugs can achieve plaque regression., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Point-of-care ultrasound (POCUS) has emerged as an indispensable diagnostic tool in cardiology, particularly within the emergency department. This narrative synthesis provides a comprehensive exploration of POCUS applications in cardiovascular diseases, elucidating its multifaceted roles and addressing challenges. The review delves into the technical attributes of POCUS, emphasizing its non-invasive nature, radiation-free qualities, and suitability for non-radiologists. It navigates through educational strategies, stressing the importance of structured programs for the seamless integration of POCUS into clinical practice. Highlighting its efficacy, the synthesis discusses POCUS applications in various scenarios such as dyspnea, chest pain, cardiac arrest, aortic dissection, pericardial effusion, and pulmonary embolism. Beyond acute care, the review explores the role of POCUS in outpatient and inpatient settings, focusing on chronic and acute heart failure, valvular heart diseases, and more. Acknowledging operator-dependent challenges and the need for continuous education, the review underscores the transformative potential of POCUS across diverse healthcare settings. This narrative synthesis accentuates POCUS as a valuable and versatile diagnostic tool in cardiology, offering efficiency, safety, and cost-effectiveness. Despite challenges, POCUS stands out as a transformative addition to clinical practices, poised to enhance patient outcomes and reshape the landscape of cardiovascular diagnostics., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Calderon Martinez et al.)

Purpose: The COVID-19 pandemic affected medical practice worldwide due to interventions to prevent spreading. Its effect on ophthalmology practices in Latin America has not yet been explored. We aimed to assess the perceptions about the pandemic from countries' ophthalmological national and subspecialty retina societies affiliated to the Pan-American Association of Ophthalmology (PAAO)., Patients and Methods: A survey-based study of leaders of national ophthalmological and retinal societies was conducted. The survey was sent by email to 30 societies, from which 20 responded (12 countries, 66.6% response rate). It included closed- and open-ended questions about (1) operational capacity and precautions, (2) telemedicine and virtual care, (3) procedures, and (4) post-pandemic considerations., Results: There was a marked decline in ophthalmology patient visits (80-95%) and elective surgeries (90%) during 2020 compared to before the pandemic. Precautions like temperature checks, mask usage, and social distancing were widely implemented while personal protective equipment (PPE) availability varied. Telemedicine use was limited due to lack of experience with it. Reopening plans focused on maintaining precautions and gradually resuming activities. Economic and security concerns were raised, and adherence to guidelines was emphasized. Respondents acknowledged the need to adapt to a "new normal". Long duration drugs, fewer imaging studies, and shorter wait times were preferred; however, availability of long duration drugs was limited., Conclusion: The pandemic impacted ophthalmology in Latin America, with reduced patient visits, procedures, and surgeries. Delayed treatment and complications were likely the result of the pandemic., Competing Interests: Dr Gerardo Garcia-Aguirre reports grants/personal fees and/or non-financial support from Alcon, AbbVie/Allergan, Bayer, Heidelberg Engineering, Nidek, Novartis, Roche Genentech, and Zeiss, outside the submitted work. The authors report no other conflicts of interest in this work., (© 2023 Bonilla-Escobar et al.)

Reduced expression and/or activity of Kv1.5 channels (encoded by KCNA5 ) is a common hallmark in human or experimental pulmonary arterial hypertension (PAH). Likewise, genetic variants in KCNA5 have been found in patients with PAH, but their functional consequences and potential impact on the disease are largely unknown. Herein, this study aimed to characterize the functional consequences of seven KCNA5 variants found in a cohort of patients with PAH. Potassium currents were recorded by patch-clamp technique in HEK293 cells transfected with wild-type or mutant Kv1.5 cDNA. Flow cytometry, Western blot, and confocal microscopy techniques were used for measuring protein expression and cell apoptosis in HEK293 and human pulmonary artery smooth muscle cells. KCNA5 variants (namely, Arg184Pro and Gly384Arg) found in patients with PAH resulted in a clear loss of potassium channel function as assessed by electrophysiological and molecular modeling analyses. The Arg184Pro variant also resulted in a pronounced reduction of Kv1.5 expression. Transfection with Arg184Pro or Gly384Arg variants decreased apoptosis of human pulmonary artery smooth muscle cells compared with the wild-type cells, demonstrating that KCNA5 dysfunction in both variants affects cell viability. Thus, in addition to affecting channel activity, both variants were associated with impaired apoptosis, a crucial process linked to the disease. The estimated prevalence of dysfunctional KCNA5 variants in the PAH population analyzed was around 1%. The data indicate that some KCNA5 variants found in patients with PAH have critical consequences for channel function, supporting the idea that KCNA5 pathogenic variants may be a causative or contributing factor for PAH.

K V 7 channels exert a pivotal role regulating vascular tone in several vascular beds. In this context, K V 7 channel agonists represent an attractive strategy for the treatment of pulmonary arterial hypertension (PAH). Therefore, in this study, we have explored the pulmonary vascular effects of the novel K V 7 channel agonist URO-K10. Consequently, the vasodilator and electrophysiological effects of URO-K10 were tested in rat and human pulmonary arteries (PA) and PA smooth muscle cells (PASMC) using myography and patch-clamp techniques. Protein expression was also determined by Western blot. Morpholino-induced knockdown of KCNE4 was assessed in isolated PA. PASMC proliferation was measured by BrdU incorporation assay. In summary, our data show that URO-K10 is a more effective relaxant of PA than the classical K V 7 activators retigabine and flupirtine. URO-K10 enhanced K V currents in PASMC and its electrophysiological and relaxant effects were inhibited by the K V 7 channel blocker XE991. The effects of URO-K10 were confirmed in human PA. URO-K10 also exhibited antiproliferative effects in human PASMC. Unlike retigabine and flupirtine, URO-K10-induced pulmonary vasodilation was not affected by morpholino-induced knockdown of the KCNE4 regulatory subunit. Noteworthy, the pulmonary vasodilator efficacy of this compound was considerably increased under conditions mimicking the ionic remodelling (as an in vitro model of PAH) and in PA from monocrotaline-induced pulmonary hypertensive rats. Taking all together, URO-K10 behaves as a KCNE4-independent K V 7 channel activator with much increased pulmonary vascular effects compared to classical K V 7 channel activators. Our study identifies a promising new drug in the context of PAH., Competing Interests: Declaration of Competing Interest None. The authors have reported that they have no relationships with industry relevant to the contents of this paper to disclose. The funders played no role in the study design; nor in the collection, analyses, interpretation of data, nor in the writing of the manuscript., (Copyright © 2023. Published by Elsevier Masson SAS.)

Purpose: Several authors have described methods to predict the sural nerve pathway with non-proportional numerical distances, but none have proposed a person-proportional, reproducible method with anatomical references. The aim of this research is to describe ultrasonographically the distance and crossing zone between a surface reference line and the position of the sural nerve., Methods: Descriptive cross-sectional study, performed between January and April 2022 in patients requiring foot surgery who met inclusion criteria. The sural nerve course in the posterior leg was located and marked using ultrasound. Landmarks were drawn with a straight line from the medial femoral condyle to the tip of the fibula. Four equal zones were established in the leg by subdividing the distal half of the line. This way, areas based on simple anatomical proportions for each patient were studied. The distance between the marking and the ultrasound nerve position was measured in these 4 zones, creating intersection points and safety areas. Location and distances from the sural nerve to the proposed landmarks were assessed., Results: One-hundred and four lower limbs, 52 left and 52 right, assessed in 52 patients were included. The shortest median distance of the nerve passage was 2.9 mm from Point 2. The sural nerve intersection was 60/104 (57.7%) in Zone B, 21/104 (20.1%) in Zone C and 19/104 (18.3%) in Zone A. Safety zones were established. Average 80.5% of coincidence in sural nerve localization was found in the distal half of the leg, in relation to the surface reference line when comparing both legs of each patient., Conclusions: This study proposes a simple, reproducible, non-invasive and, for the first time, person-proportional method, that describes the distance and location of the main areas of intersection of the sural nerve with points and zones (risk and safe zones) determined by a line guided by superficial anatomical landmarks. Its application when surgeons plan and perform posterior leg approaches will help to avoid iatrogenic nerve injuries., Level of Evidence: IV., (© 2022. The Author(s).)

Quantum Key Distribution (QKD) is a cutting-edge communication method that enables secure communication between two parties. Continuous-variable QKD (CV-QKD) is a promising approach to QKD that has several advantages over traditional discrete-variable systems. Despite its potential, CV-QKD systems are highly sensitive to optical and electronic component impairments, which can significantly reduce the secret key rate. In this research, we address this challenge by modeling a CV-QKD system to simulate the impact of individual impairments on the secret key rate. The results show that laser frequency drifts and small imperfections in electro-optical devices such as the beam splitter and the balanced detector have a negative impact on the secret key rate. This provides valuable insights into strategies for optimizing the performance of CV-QKD systems and overcome limitations caused by component impairments. By offering a method to analyze them, the study enables the establishment of quality standards for the components of CV-QKD systems, driving the development of advanced technologies for secure communication in the future., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Background: Description of a large cohort of Akin osteotomies without fixation (286 feet), to depict the mean angular correction, to analyze complication rates and to pull over with rates described in the literature with other techniques., Methods: Retrospective single-center cohort study. Five radiologic measurements analyzed preoperatively and postoperatively until fusion was completed. Evaluation of all peri- and postoperative complication rates. Evaluation of correlation between complications and the presence of diabetes mellitus (DM), smoking and rheumatic disease., Results: Between 2011 and 2018, 222 patients, 286 feet (147 left, 139 right) met the inclusion criteria. We found an average difference between pre-op and post-op at 3 months of distal articular set angle (DASA) of 7.0 degrees and average interphalangeal joint obliquity angle (IPOA) of 12.0 degrees (p < 0.001). All cases achieved fusion but in 5.9 % (17/289 cases) of cases, we observed delayed consolidation. The average union time in these cases was 22.1 weeks. Hyperextension of the distal fragment (mal union in plantar flexion) was observed in 7 cases (2.4 %). None of the 7 cases required correction. Out of 286 surgeries, 8 (2.7 %) required reoperation, but only one case for a hypocorrection required Akin's re osteotomy. Regarding the inter- and intra-observer correlations, good and excellent reliability are observed for all parameters under consideration., Conclusion: The absence of internal fixation would appear not to impair osteotomy healing and complication rates compared with techniques with fixation would not appear to be worse. The results are encouraging; nevertheless, we believe that further studies need to be performed in order to confirm the data., Level of Evidence: IV retrospective cases series., Competing Interests: Competing interests The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Despite increasing evidence suggesting that pulmonary arterial hypertension (PAH) is a complex disease involving vasoconstriction, thrombosis, inflammation, metabolic dysregulation and vascular proliferation, all the drugs approved for PAH mainly act as vasodilating agents. Since excessive TGF-β signaling is believed to be a critical factor in pulmonary vascular remodeling, we hypothesized that blocking TGFβ-activated kinase 1 (TAK-1), alone or in combination with a vasodilator therapy (i.e., riociguat) could achieve a greater therapeutic benefit. Methods: PAH was induced in male Wistar rats by a single injection of the VEGF receptor antagonist SU5416 (20 mg/kg) followed by exposure to hypoxia (10%O 2 ) for 21 days. Two weeks after SU5416 administration, vehicle, riociguat (3 mg/kg/day), the TAK-1 inhibitor 5Z-7-oxozeaenol (OXO, 3 mg/kg/day), or both drugs combined were administered for 7 days. Metabolic profiling of right ventricle (RV), lung tissues and PA smooth muscle cells (PASMCs) extracts were performed by magnetic resonance spectroscopy, and the differences between groups analyzed by multivariate statistical methods. Results: In vitro , riociguat induced potent vasodilator effects in isolated pulmonary arteries (PA) with negligible antiproliferative effects and metabolic changes in PASMCs. In contrast, 5Z-7-oxozeaenol effectively inhibited the proliferation of PASMCs characterized by a broad metabolic reprogramming but had no acute vasodilator effects. In vivo, treatment with riociguat partially reduced the increase in pulmonary arterial pressure (PAP), RV hypertrophy (RVH), and pulmonary vascular remodeling, attenuated the dysregulation of inosine, glucose, creatine and phosphocholine (PC) in RV and fully abolished the increase in lung IL-1β expression. By contrast, 5Z-7-oxozeaenol significantly reduced pulmonary vascular remodeling and attenuated the metabolic shifts of glucose and PC in RV but had no effects on PAP or RVH. Importantly, combined therapy had an additive effect on pulmonary vascular remodeling and induced a significant metabolic effect over taurine, amino acids, glycolysis, and TCA cycle metabolism via glycine-serine-threonine metabolism. However, it did not improve the effects induced by riociguat alone on pulmonary pressure or RV remodeling. None of the treatments attenuated pulmonary endothelial dysfunction and hyperresponsiveness to serotonin in isolated PA. Conclusion: Our results suggest that inhibition of TAK-1 induces antiproliferative effects and its addition to short-term vasodilator therapy enhances the beneficial effects on pulmonary vascular remodeling and RV metabolic reprogramming in experimental PAH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Morales-Cano, Izquierdo-García, Barreira, Esquivel-Ruiz, Callejo, Pandolfi, Villa-Valverde, Rodríguez, Cogolludo, Ruiz-Cabello, Perez-Vizcaino and Moreno.)

The Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its sublineages pose a new challenge to healthcare systems worldwide due to its ability to efficiently spread in immunized populations and its resistance to currently available therapies. COVID-19, although targeting primarily the respiratory system, is also now well established that later affects every organ in the body. Most importantly, despite the available therapy and vaccine-elicited protection, the long-term consequences of viral infection in breakthrough and asymptomatic individuals are areas of concern. In the past two years, investigators accumulated evidence on how the virus triggers our immune system and the molecular signals involved in the cross-talk between immune cells and structural cells in the pulmonary vasculature to drive pathological lung complications such as endothelial dysfunction and thrombosis. In the review, we emphasize recent updates on the pathophysiological inflammatory and immune responses associated with SARS-CoV-2 infection and their potential long-term consequences that may consequently lead to the development of pulmonary vascular diseases., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kumar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Directed differentiation of pluripotent stem cells (PSCs) is a powerful model system for deconstructing embryonic development. Although mice are the most advanced mammalian model system for genetic studies of embryonic development, state-of-the-art protocols for directed differentiation of mouse PSCs into defined lineages require additional steps and generates target cell types with lower purity than analogous protocols for human PSCs, limiting their application as models for mechanistic studies of development. Here, we examine the potential of mouse epiblast stem cells cultured in media containing Wnt pathway inhibitors as a starting point for directed differentiation. As a proof of concept, we focused our efforts on two specific cell/tissue types that have proven difficult to generate efficiently and reproducibly from mouse embryonic stem cells: definitive endoderm and neural organoids. We present new protocols for rapid generation of nearly pure definitive endoderm and forebrain-patterned neural organoids that model the development of prethalamic and hippocampal neurons. These differentiation models present new possibilities for combining mouse genetic tools with in vitro differentiation to characterize molecular and cellular mechanisms of embryonic development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Background: Aim of this study was to investigate immune cells and subsets in different stages of human coronary artery disease with a novel multiplex immunohistochemistry (mIHC) technique., Methods: Human left anterior descending coronary artery specimens were analyzed: eccentric intimal thickening (N = 11), pathological intimal thickening (N = 10), fibroatheroma (N = 9), and fibrous plaque (N = 9). Eccentric intimal thickening was considered normal, and pathological intimal thickening, fibroatheroma, and fibrous plaque were considered diseased coronary arteries. Two mIHC panels, consisting of six and five primary antibodies, autofluoresence, and DAPI, were used to detect adaptive and innate immune cells. Via semi-automated analysis, (sub)types of immune cells in whole plaques and specific plaque regions were quantified., Results: Increased numbers of CD3 + T cells (P < 0.001), CD20 + B cells (P = 0.013), CD68 + macrophages (P = 0.003), CD15 + neutrophils (P = 0.017), and CD31 + endothelial cells (P = 0.024) were identified in intimas of diseased coronary arteries compared to normal. Subset analyses of T cells and macrophages showed that diseased coronary arteries contained an abundance of CD3 + CD8 - non-cytotoxic T cells and CD68 + CD206 - non-M2-like macrophages. Proportions of CD3 + CD45RO + memory T cells were similar to normal coronary arteries. Among pathological intimal thickening, fibroatheroma, and fibrous plaque, all immune cell numbers and subsets were similar., Conclusions: The type of immune response does not differ substantially between different stages of plaque development and may provide context for mechanistic research into immune cell function in atherosclerosis. We provide the first comprehensive map of immune cell subtypes across plaque types in coronary arteries demonstrating the potential of mIHC for vascular research., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

HIV and Schistosoma infections have been individually associated with pulmonary vascular disease. Co-infection with these pathogens is very common in tropical areas, with an estimate of six million people co-infected worldwide. However, the effects of HIV and Schistosoma co-exposure on the pulmonary vasculature and its impact on the development of pulmonary vascular disease are largely unknown. Here, we have approached these questions by using a non-infectious animal model based on lung embolization of Schistosoma mansoni eggs in HIV-1 transgenic (HIV) mice. Schistosome-exposed HIV mice but not wild-type (Wt) counterparts showed augmented pulmonary arterial pressure associated with markedly suppressed endothelial-dependent vasodilation, increased endothelial remodeling and vessel obliterations, formation of plexiform-like lesions and a higher degree of perivascular fibrosis. In contrast, medial wall muscularization was similarly increased in both types of mice. Moreover, HIV mice displayed an impaired immune response to parasite eggs in the lung, as suggested by decreased pulmonary leukocyte infiltration, small-sized granulomas, and augmented residual egg burden. Notably, vascular changes in co-exposed mice were associated with increased expression of proinflammatory and profibrotic cytokines, including IFN-γ and IL-17A in CD4 + and γδ T cells and IL-13 in myeloid cells. Collectively, our study shows for the first time that combined pulmonary persistence of HIV proteins and Schistosoma eggs, as it may occur in co-infected people, alters the cytokine landscape and targets the vascular endothelium for aggravated pulmonary vascular pathology. Furthermore, it provides an experimental model for the understanding of pulmonary vascular disease associated with HIV and Schistosoma co-morbidity.

Five National Metrology Institutes (NMIs) worked together to validate the assessment of laser tracker's (LT) uncertainty for large scale dimensional metrology in subsequent measurements using the network method. The LT uncertainty is assessed by measuring a set of a fixed network of targets from different LT positions. Afterward, we must perform a "bundled adjustment" of all the measurements to determine the transformations of the LT positions that minimize the residuals (differences) between a computed "virtual" group of targets, called composite points, and the redundant targets coming from the LT positions transformed to a unique LT position. Each residual is weighted with the LT's Maximum Permissible Error (MPE) specified by the LT manufacturer. The standard deviation of the weighted residuals becomes LT uncertainty. Afterward, with Monte-Carlo simulation, we propagate the LT uncertainty to the position of every target in the network and 3D distances between them. LT uncertainty is valid if 3D distance's uncertainties computed with composite points is greater than the error computed with these distances minus the same distances calibrated with the line of sight method (LOS) or if the absolute normalized error is less or equal to one. LOS method uses only the LT's interferometer for calibration, which dispense the use of a calibrated scale bar. Based on the network method, the NMIs developed two methods to improve LT geometry error parameters. The first improves the LT geometry error parameters by fitting the composite points to the LT geometry error model. The second evaluates the parameters placing the LT geometry error model in the minimization of the bundled adjustment's residual. LT geometry error parameters are valid if the parameter's uncertainties are negligent.

Objective: Atheromatous fibrous caps are produced by smooth muscle cells (SMCs) that are recruited to the subendothelial space. We tested whether the recruitment mechanisms are the same as in embryonic artery development, which relies prominently on Notch signaling to form the subendothelial medial SMC layers., Approach and Results: Notch elements were expressed in regions of fibrous cap in human and mouse plaques. To assess the causal role of Notch signaling in cap formation, we studied atherosclerosis in mice where the Notch pathway was inactivated in SMCs by conditional knockout of the essential effector transcription factor RBPJ (recombination signal-binding protein for immunoglobulin kappa J region). The recruitment of cap SMCs was significantly reduced without major effects on plaque size. Lineage tracing revealed the accumulation of SMC-derived plaque cells in the cap region was unaltered but that Notch-defective cells failed to re-acquire the SMC phenotype in the cap. Conversely, to analyze whether the loss of Notch signaling is required for SMC-derived cells to accumulate in atherogenesis, we studied atherosclerosis in mice with constitutive activation of Notch signaling in SMCs achieved by conditional expression of the Notch intracellular domain. Forced Notch signaling inhibited the ability of medial SMCs to contribute to plaque cells, including both cap SMCs and osteochondrogenic cells, and significantly reduced atherosclerosis development., Conclusions: Sequential loss and gain of Notch signaling is needed to build the cap SMC population. The shared mechanisms with embryonic arterial media assembly suggest that the cap forms as a neo-media that restores the connection between endothelium and subendothelial SMCs, transiently disrupted in early atherogenesis.

Background: Vitamin D (vitD) deficiency is highly prevalent in patients with pulmonary arterial hypertension (PAH). Moreover, PAH-patients with lower levels of vitD have worse prognosis. We hypothesize that recovering optimal levels of vitD in an animal model of PAH previously depleted of vitD improves the hemodynamics, the endothelial dysfunction and the ionic remodeling. Methods: Male Wistar rats were fed a vitD-free diet for five weeks and then received a single dose of Su5416 (20 mg/Kg) and were exposed to vitD-free diet and chronic hypoxia (10% O 2 ) for three weeks to induce PAH. Following this, vitD deficient rats with PAH were housed in room air and randomly divided into two groups: (a) continued on vitD-free diet or (b) received an oral dose of 100,000 IU/Kg of vitD plus standard diet for three weeks. Hemodynamics, pulmonary vascular remodeling, pulmonary arterial contractility, and K + currents were analyzed. Results: Recovering optimal levels of vitD improved endothelial function, measured by an increase in the endothelium-dependent vasodilator response to acetylcholine. It also increased the activity of TASK-1 potassium channels. However, vitD supplementation did not reduce pulmonary pressure and did not ameliorate pulmonary vascular remodeling and right ventricle hypertrophy. Conclusions: Altogether, these data suggest that in animals with PAH and severe deficit of vitD, restoring vitD levels to an optimal range partially improves some pathophysiological features of PAH.

Objective: To describe the case of a patient with subcutaneous inguinal recurrence of penile cancer without lymph node involvement., Methods: Description of a clinical case and review of the literature on the subject., Results: We present the case of a 72-year-old man with penile cancer and extranodal inguinal extension that affected subcutaneous cell tissue, with a history of negative sentinel lymph node and subsequently without invasion of the regional lymph nodes in lymphadenectomy after chemotherapy. The patient presented disease progression despite multimodal treatment., Conclusion: Extranodal inguinal involvement in penile cancer may occur despite a history of negative sentinel lymph node. The evolution of the patient we presented was disastrous despite the multimodal treatment carried out.

Current approved therapies for pulmonary hypertension (PH) aim to restore the balance between endothelial mediators in the pulmonary circulation. These drugs may exert vasodilator effects on poorly oxygenated vessels. This may lead to the derivation of blood perfusion towards low ventilated alveoli, i.e., producing ventilation-perfusion mismatch, with detrimental effects on gas exchange. The aim of this study is to analyze the oxygen-sensitivity in vitro of 25 drugs currently used or potentially useful for PH. Additionally, the study analyses the effectiveness of these vasodilators in the pulmonary vs the systemic vessels. Vasodilator responses were recorded in pulmonary arteries (PA) and mesenteric arteries (MA) from rats and in human PA in a wire myograph under different oxygen concentrations. None of the studied drugs showed oxygen selectivity, being equally or more effective as vasodilators under conditions of low oxygen as compared to high oxygen levels. The drugs studied showed low pulmonary selectivity, being equally or more effective as vasodilators in systemic than in PA. A similar behavior was observed for the members within each drug family. In conclusion, none of the drugs showed optimal vasodilator profile, which may limit their therapeutic efficacy in PH.

Inositol polyphosphates are vital metabolic and secondary messengers, involved in diverse cellular functions. Therefore, tight regulation of inositol polyphosphate metabolism is essential for proper cell physiology. Here, we describe an early-onset neurodegenerative syndrome caused by loss-of-function mutations in the multiple inositol-polyphosphate phosphatase 1 gene (MINPP1). Patients are found to have a distinct type of Pontocerebellar Hypoplasia with typical basal ganglia involvement on neuroimaging. We find that patient-derived and genome edited MINPP1 -/- induced stem cells exhibit an inefficient neuronal differentiation combined with an increased cell death. MINPP1 deficiency results in an intracellular imbalance of the inositol polyphosphate metabolism. This metabolic defect is characterized by an accumulation of highly phosphorylated inositols, mostly inositol hexakisphosphate (IP 6 ), detected in HEK293 cells, fibroblasts, iPSCs and differentiating neurons lacking MINPP1. In mutant cells, higher IP 6 level is expected to be associated with an increased chelation of intracellular cations, such as iron or calcium, resulting in decreased levels of available ions. These data suggest the involvement of IP 6 -mediated chelation on Pontocerebellar Hypoplasia disease pathology and thereby highlight the critical role of MINPP1 in the regulation of human brain development and homeostasis.

Vitamin D (VitD) receptor regulates the expression of several genes involved in signaling pathways affected in pulmonary hypertension (PH). VitD deficiency is highly prevalent in PH, and low levels are associated with poor prognosis. We investigated if VitD deficiency may predispose to or exacerbate PH. Male Wistar rats were fed with a standard or a VitD-free diet for 5 wk. Next, rats were further divided into controls or PH, which was induced by a single dose of Su-5416 (20 mg/kg) and exposure to hypoxia (10% O 2 ) for 2 wk. VitD deficiency had no effect on pulmonary pressure in normoxic rats, indicating that, by itself, it does not trigger PH. However, it induced several moderate but significant changes characteristic of PH in the pulmonary arteries, such as increased muscularization, endothelial dysfunction, increased survivin, and reduced bone morphogenetic protein ( Bmp ) 4 , Bmp6 , DNA damage-inducible transcript 4 , and K + two - pore domain channel subfamily K member 3 ( Kcnk3 ) expression. Myocytes isolated from pulmonary arteries from VitD-deficient rats had a reduced whole voltage-dependent potassium current density and acid-sensitive (TASK-like) potassium currents. In rats with PH induced by Su-5416 plus hypoxia, VitD-free diet induced a modest increase in pulmonary pressure, worsened endothelial function, increased the hyperreactivity to serotonin, arterial muscularization, decreased total and TASK-1 potassium currents, and further depolarized the pulmonary artery smooth muscle cell membrane. In human pulmonary artery smooth muscle cells from controls and patients with PH, the active form of VitD calcitriol significantly increased KCNK3 mRNA expression. Altogether, these data strongly suggest that the deficit in VitD induces pulmonary vascular dysfunction.

K + channels play a fundamental role regulating membrane potential of pulmonary artery (PA) smooth muscle cells and their impairment is a common feature in pulmonary arterial hypertension (PAH). K + voltage-gated channel subfamily Q ( KCNQ1-5 ) or Kv7 channels and their regulatory subunits subfamily E (KCNE) regulatory subunits are known to regulate vascular tone, but whether Kv7 channel function is impaired in PAH and how this can affect the rationale for targeting Kv7 channels in PAH remains unknown. Here, we have studied the role of Kv7/KCNE subunits in rat PA and their possible alteration in PAH. Using the patch-clamp technique, we found that the total K + current is reduced in PA smooth muscle cells from pulmonary hypertension animals (SU5416 plus hypoxia) and Kv7 currents made a higher contribution to the net K + current. Likewise, enhanced vascular responses to Kv7 channel modulators were found in pulmonary hypertension rats. Accordingly, KCNE4 subunit was highly upregulated in lungs from pulmonary hypertension animals and patients. Additionally, Kv7 channel activity was enhanced in the presence of Kv1.5 and TASK-1 channel inhibitors and this was associated with an increased KCNE4 membrane abundance. Compared with systemic arteries, PA showed a poor response to Kv7 channel modulators which was associated with reduced expression and membrane abundance of Kv7.4 and KCNE4. Our data indicate that Kv7 channel function is preserved and KCNE4 is upregulated in PAH. Therefore, compared with other downregulated channels, the contribution of Kv7 channels is increased in PAH resulting in an enhanced sensitivity to Kv7 channel modulators. This study provides insight into the potential usefulness of targeting Kv7 channels in PAH.

Combining the quantum optical properties of single-photon emitters with the strong near-field interactions available in nanophotonic and plasmonic systems is a powerful way of creating quantum manipulation and metrological functionalities. The ability to actively and dynamically modulate emitter-environment interactions is of particular interest in this regard. While thermal, mechanical and optical modulation have been demonstrated, electrical modulation has remained an outstanding challenge. Here we realize fast, all-electrical modulation of the near-field interactions between a nanolayer of erbium emitters and graphene, by in-situ tuning the Fermi energy of graphene. We demonstrate strong interactions with a  >1000-fold increased decay rate for  ~25% of the emitters, and electrically modulate these interactions with frequencies up to 300 kHz - orders of magnitude faster than the emitter's radiative decay (~100 Hz). This constitutes an enabling platform for integrated quantum technologies, opening routes to quantum entanglement generation by collective plasmon emission or photon emission with controlled waveform.

Objective: Pulmonary arterial hypertension (PAH), one of the major complications of systemic sclerosis (SSc), is a rare disease with unknown etiopathogenesis and noncurative treatments. As mice deficient in P-selectin glycoprotein ligand 1 (PSGL-1) develop a spontaneous SSc-like syndrome, we undertook this study to analyze whether they develop PAH and to examine the molecular mechanisms involved., Methods: Doppler echocardiography was used to estimate pulmonary pressure, immunohistochemistry was used to assess vascular remodeling, and myography of dissected pulmonary artery rings was used to analyze vascular reactivity. Angiotensin II (Ang II) levels were quantified by enzyme-linked immunosorbent assay, and Western blotting was used to measure Ang II type 1 receptor (AT 1 R), AT 2 R, endothelial cell nitric oxide synthase (eNOS), and phosphorylated eNOS expression in lung lysates. Flow cytometry allowed us to determine cytokine production by immune cells and NO production by endothelial cells. In all cases, there were 4-8 mice per experimental group., Results: PSGL-1 -/- mice showed lung vessel wall remodeling and a reduced mean ± SD expression of pulmonary AT 2 R (expression ratio [relative to β-actin] in female mice age >18 months: wild-type mice 0.799 ± 0.508 versus knockout mice 0.346 ± 0.229). With aging, female PSGL-1 -/- mice had impaired up-regulation of estrogen receptor α (ERα) and developed lung vascular endothelial dysfunction coinciding with an increase in mean ± SEM pulmonary Ang II levels (wild-type 48.70 ± 5.13 pg/gm lung tissue versus knockout 78.02 ± 28.09 pg/gm lung tissue) and a decrease in eNOS phosphorylation, leading to reduced endothelial NO production. These events led to a reduction in the pulmonary artery acceleration time:ejection time ratio in 33% of aged female PSGL-1 -/- mice, indicating pulmonary hypertension. Importantly, we found expanded populations of interferon-γ-producing PSGL-1 -/- T cells and B cells and a reduced presence of regulatory T cells., Conclusion: The absence of PSGL-1 induces a reduction in Treg cells, NO production, and ERα expression and causes an increase in Ang II in the lungs of female mice, favoring the development of PAH., (© 2019 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology.)

Aim: Intensity-modulated radiotherapy (IMRT) has demonstrated improved local control in extremity soft tissue sarcoma (STS) after limb-sparing surgery compared with three-dimensional conformal radiation therapy. Our purpose was to evaluate sliding-window IMRT (SW-IMRT) and volumetric arc therapy (VMAT) in planning target volume (PTV) coverage and dose to organs-at-risk (OAR)., Methods: Sixteen patients undergoing postoperative RT for lower extremity STS were included. For each patient, one VMAT plan and one SW-IMRT plan were proposed. Both were evaluated using cumulative dose-volume histogram data for OAR and PTVs. Prescribed dose was 66 Gy (2 Gy/fraction) to PTV1 and 56 Gy (1.69 Gy/fraction) to PTV2. OARs contoured were femur, neurovascular bundle, minimum tissue corridor, normal tissue outside PTV2, joint and genitalia. T-Student test was performed., Results: Eleven male (69%) and five female patients (31%) were analyzed. Mean age was 60 years. Both techniques showed optimal target coverage, conformity index (CI) and homogeneity index (HI). VMAT PTV2 CI was 1.13 (mean) ± 0.08 (standard deviation) versus 1.19 ± 0.10 SW-IMRT PTV2 CI (P < 0.05). VMAT PTV1 HI was 0.09 ± 0.01 versus 0.08 ± 0.01 SW-IMRT PTV1 HI (P < 0.05). Regarding OARs, VMAT delivered lower dose to femur, genitalia, normal tissue outside PTV2 and joints. SW-IMRT spared tissue corridor mean dose (10.4 Gy ± 6.8 Gy) versus (14.7 ± 6.5 Gy) VMAT (P < 0.05)., Conclusions: Both techniques achieved great conformity, homogeneity and coverage of PTV. VMAT produced lower dose to OARS and SW-IMRT was superior in sparing dose to normal-tissue-corridor, which could reduce risk of lymphedema., (© 2019 John Wiley & Sons Australia, Ltd.)

Endothelial dysfunction plays a central role in the pathophysiology of pulmonary arterial hypertension (PAH). MicroRNAs (miRNAs) are small single-strand and non-coding RNAs that negatively regulate gene function by binding to the 3'-untranslated region (3'-UTR) of specific mRNAs. microRNA-1 (miR-1) is upregulated in plasma from idiopathic PAH patients and in lungs from an experimental model of PAH. However, the role of miRNA-1 on endothelial dysfunction is unknown. The aim of this study was to analyze the effects of miR-1 on endothelial function in rat pulmonary arteries (PA). Endothelial function was studied in PA from PAH or healthy animals and mounted in a wire myograph. Some PA from control animals were transfected with miR-1 or scramble miR. Superoxide anion production by miR-1 was quantified by dihydroethidium (DHE) fluorescence in rat PA smooth muscle cells (PASMC). Bioinformatic analysis identified superoxide dismutase-1 (SOD1), connexin-43 (Cx43), caveolin 2 (CAV2) and Krüppel-like factor 4 (KLF4) as potential targets of miR-1. The expression of SOD1, Cx43, CAV2, and KLF4 was determined by qRT-PCR and western blot in PASMC. PA incubated with miR-1 presented decreased endothelium-dependent relaxation to acetylcholine. We also found an increase in the production of O 2 - and decreased expression of SOD1, Cx43, CAV2, and KLF4 in PASMC induced by miR-1, which may contribute to endothelial dysfunction. In conclusion, these data indicate that miR-1 induces endothelial dysfunction, suggesting a pathophysiological role in PAH.

Scope: The aim of the present study is to investigate the potential protective effect of a cocoa-rich diet on functional and structural vascular alterations in diabetes and the mechanism involved., Methods and Results: Male Zucker diabetic fatty (ZDF) rats are fed on a standard (ZDF-C) or cocoa-rich diet (ZDF-Co) from week 10 to 20 of life. Diabetic ZDF-C rats showed increased blood pressure and enhanced aortic stiffness, as demonstrated by the increased pulse pressure and the augmented aortic medial thickness with loss and disruption of elastic fibres. Interestingly, cocoa intake strongly avoided all these adverse effects and reduced aortic oxidative stress. Mechanistically, cocoa diet prevented sirtuin-1 (SIRT-1) depletion and increased NADPH oxidases (NOXs) and reactive oxygen species production as well as reduced active nuclear factor E2 related factor 2 (Nrf2) and their antioxidant products., Conclusion: The results demonstrate for the first time that a cocoa-rich diet strongly prevents aortic stiffening and remodeling in diabetic animals and avoids aortic oxidative stress. It is suggested that this effect could be mediated via its effects on SIRT-1, NOXs, and Nrf2., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background Inflammation underlies many forms of pulmonary hypertension (PH), including that resulting from Schistosoma infection, a major cause of PH worldwide. Schistosomiasis-associated PH is proximately triggered by embolization of parasite eggs into the lungs, resulting in localized type 2 inflammation. However, the role of CD4 + T cells in this disease is not well defined. Methods and Results We used a mouse model of schistosomiasis-associated PH, induced by intraperitoneal egg sensitization followed by intravenous egg challenge, with outcomes including right ventricle systolic pressure measured by cardiac catheterization, and cell density and phenotype assessed by flow cytometry. We identified that embolization of Schistosoma eggs into lungs of egg-sensitized mice increased the perivascular density of T-helper 2 (Th2) CD4 + T cells by recruitment of cells from the circulation and triggered type 2 inflammation. Parabiosis confirmed that egg embolization is required for localized type 2 immunity. We found Th2 CD4 + T cells were necessary for Schistosoma-induced PH, given that deletion of CD4 + T cells or inhibiting their Th2 function protected against type 2 inflammation and PH following Schistosoma exposure. We also observed that adoptive transfer of Schistosoma-sensitized CD4 + Th2 cells was sufficient to drive type 2 inflammation and PH. Conclusions Th2 CD4 + T cells are a necessary and sufficient component for the type 2 inflammation-induced PH following Schistosoma exposure.

Background and Purpose: The NO/cGMP pathway represents a major physiological signalling controlling tone in pulmonary arteries (PA), and drugs activating this pathway are used to treat pulmonary arterial hypertension. K v channels expressed in PA smooth muscle cells (PASMCs) are key determinants of vascular tone. We aimed to analyse the contribution of K v 1.5 and K v 7 channels in the electrophysiological and vasodilating effects evoked by NO donors and the GC stimulator riociguat in PA., Experimental Approach: K v currents were recorded in isolated rat PASMCs using the patch-clamp technique. Vascular reactivity was assessed in a wire myograph., Key Results: The NO donors diethylamine NONOate diethylammonium (DEA-NO) and sodium nitroprusside hyperpolarized the membrane potential and induced a bimodal effect on K v currents (augmenting the current between -40 and -10 mV and decreasing it at more depolarized potentials). The hyperpolarization and the enhancement of the current were suppressed by K v 7 channel inhibitors and by the GC inhibitor ODQ but preserved when K v 1.5 channels were inhibited. Additionally, DEA-NO enhanced K v 7.5 currents in COS7 cells expressing the KCNQ5 gene. Riociguat increased K v currents at all potentials ≥-40 mV and induced membrane hyperpolarization. Both effects were prevented by K v 7 inhibition. Likewise, PA relaxation induced by NO donors and riociguat was attenuated by K v 7 inhibitors., Conclusions and Implications: NO donors and riociguat enhance K v 7 currents, leading to PASMC hyperpolarization. This mechanism contributes to NO/cGMP-induced PA vasodilation. Our study identifies K v 7 channels as a novel mechanism of action of vasodilator drugs used in the treatment of pulmonary arterial hypertension., (© 2019 The British Pharmacological Society.)

Competing Interests: Competing interests: None declared.

Key Points: The expression of miR-1 is increased in lungs from the Hyp/Su5416 PAH rat model. Pulmonary artery smooth muscle cells from this animal model are more depolarized and show decreased expression and activity of voltage-dependent potassium channel (Kv)1.5. miR-1 directly targets Kv1.5 channels, reduces Kv1.5 activity and induces membrane depolarization. Antagomir-1 prevents Kv1.5 channel downregulation and the depolarization induced by hypoxia/Su5416 exposition., Abstract: Impairment of the voltage-dependent potassium channel (Kv) plays a central role in the development of cardiovascular diseases, including pulmonary arterial hypertension (PAH). MicroRNAs are non-coding RNAs that regulate gene expression by binding to the 3'-untranslated region region of specific mRNAs. The present study aimed to analyse the effects of miR-1 on Kv channel function in pulmonary arteries (PA). Kv channel activity was studied in PA from healthy animals transfected with miR-1 or scrambled-miR. Kv currents were studied using the whole-cell configuration of the patch clamp technique. The characterization of the Kv1.5 currents was performed with the selective inhibitor DPO-1. miR-1 expression was increased and Kv1.5 channels were decreased in lungs from a rat model of PAH induced by hypoxia and Su5416. miR-1 transfection increased cell capacitance, reduced Kv1.5 currents and induced membrane depolarization in isolated pulmonary artery smooth muscle cells. A luciferase reporter assay indicated that KCNA5, which encodes Kv1.5 channels, is a direct target gene of miR-1. Incubation of PA with Su5416 and hypoxia (3% O 2 ) increased miR-1 and induced a decline in Kv1.5 currents, which was prevented by antagomiR-1. In conclusion, these data indicate that miR-1 induces pulmonary artery smooth muscle cell hypertrophy and reduces the activity and expression of Kv channels, suggesting a pathophysiological role in PAH., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Diabetes is a very strong predictor of chronic systemic vascular diseases and acute cardiovascular events. Recently, associations between metabolic disorders and pulmonary hypertension have also been reported in both humans and animal models. In order to get some further insight into the relationship of pulmonary hypertension with obesity, insulin resistance and hyperglycemia, herein we have used the Zucker diabetic fatty rats (ZDF/clr-lepr fa) at 20 weeks fed a standard diet and compared to their lean Zucker littermates (ZL). ZDF rats were obese, had elevated plasma glucose levels and insulin resistance, i.e. a clinically relevant model of type 2 diabetes. They presented elevated systolic, diastolic and mean pulmonary arterial pressures and a parallel increase in the Fulton index. Systemic arterial pressures were also increased but the left ventricle plus septum weight was similar in both groups and the heart rate was reduced. Wall media thickening was observed in the small pulmonary arteries from the ZDF rats. Isolated pulmonary arteries mounted in a wire myograph showed similar vasoconstrictor responses to phenylephrine and 5-HT and similar responses to the endothelium-dependent vasodilator acetylcholine. However, the iNOS inhibitor 1400W enhanced the vasoconstrictor responses in ZDF but not in ZL rats. The protein expression of eNOS and iNOS was not significantly different in the lungs of the two groups. The lung expression of Bmpr2 mRNA was downregulated. However, the mRNA expression of Kcna5, Kcnk3, Kcnq1, Kcnq4 or Kcnq5, which encode for the potassium channels Kv1.5, TASK-1, Kv7.1, Kv7.4 and Kv7.5, respectively, was similar in ZL and ZDF rats. In conclusion, ZDF rats show increased pulmonary arterial pressure, right ventricular hypertrophy, pulmonary arterial medial thickening and downregulated lung Bmpr2 despite leptin resistance. These changes were mild but are consistent with the view that diabetes is a risk factor for pulmonary hypertension., Competing Interests: The authors have declared that no competing interests exist.

Human immunodeficiency virus (HIV) infection is an established risk factor for pulmonary arterial hypertension (PAH); however, the pathogenesis of HIV-related PAH remains unclear. Since K + channel dysfunction is a common marker in most forms of PAH, our aim was to analyze whether the expression of HIV proteins is associated with impairment of K + channel function in the pulmonary vascular bed. HIV transgenic mice (Tg26) expressing seven of the nine HIV viral proteins and wild-type (WT) mice were used. Hemodynamic assessment was performed by echocardiography and catheterization. Vascular reactivity was studied in endothelium-intact pulmonary arteries. K + currents were recorded in freshly isolated pulmonary artery smooth muscle cells (PASMC) using the patch-clamp technique. Gene expression was assessed using quantitative RT-PCR. PASMC from Tg26 mice had reduced K + currents and were more depolarized than those from WT. Whereas voltage-gated K + channel 1.5 (Kv1.5) currents were preserved, pH-sensitive noninactivating background currents ( I KN ) were nearly abolished in PASMC from Tg26 mice. Tg26 mice had reduced lung expression of Kv7.1 and Kv7.4 channels and decreased responses to the Kv7.1 channel activator L-364,373 assessed by vascular reactivity and patch-clamp experimental approaches. Although we found pulmonary vascular remodeling and endothelial dysfunction in Tg26 mice, this was not accompanied by changes in hemodynamic parameters. In conclusion, the expression of HIV proteins in vivo impairs pH-sensitive I KN and Kv7 currents. This negative impact of HIV proteins in K + channels was not sufficient to induce PAH, at least in mice, but may play a permissive or accessory role in the pathophysiology of HIV-associated PAH.

Proper brain development relies highly on protein N-glycosylation to sustain neuronal migration, axon guidance and synaptic physiology. Impairing the N-glycosylation pathway at early steps produces broad neurological symptoms identified in congenital disorders of glycosylation. However, little is known about the molecular mechanisms underlying these defects. We generated a cerebellum specific knockout mouse for Srd5a3 , a gene involved in the initiation of N-glycosylation. In addition to motor coordination defects and abnormal granule cell development, Srd5a3 deletion causes mild N-glycosylation impairment without significantly altering ER homeostasis. Using proteomic approaches, we identified that Srd5a3 loss affects a subset of glycoproteins with high N-glycans multiplicity per protein and decreased protein abundance or N-glycosylation level. As IgSF-CAM adhesion proteins are critical for neuron adhesion and highly N-glycosylated, we observed impaired IgSF-CAM-mediated neurite outgrowth and axon guidance in Srd5a3 mutant cerebellum. Our results link high N-glycan multiplicity to fine-tuned neural cell adhesion during mammalian brain development., Competing Interests: DM, EU, LN, MN, JL, JB, CT, FF, NL, CF, LC, IG, VC No competing interests declared, (© 2018, Medina-Cano et al.)