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Introduction and Objectives: The multiparametric implantable cardioverter-defibrillator HeartLogic index has proven to be a sensitive and timely predictor of impending heart failure (HF) decompensation. We evaluated the impact of a standardized follow-up protocol implemented by nursing staff and based on remote management of alerts., Methods: The algorithm was activated in HF patients at 19 Spanish centers. Transmitted data were analyzed remotely, and patients were contacted by telephone if alerts were issued. Clinical actions were implemented remotely or through outpatient visits. The primary endpoint consisted of HF hospitalizations or death. Secondary endpoints were HF outpatient visits. We compared the 12-month periods before and after the adoption of the protocol., Results: We analyzed 392 patients (aged 69±10 years, 76% male, 50% ischemic cardiomyopathy) with implantable cardioverter-defibrillators (20%) or cardiac resynchronization therapy defibrillators (80%). The primary endpoint occurred 151 times in 86 (22%) patients during the 12 months before the adoption of the protocol, and 69 times in 45 (11%) patients (P<.001) during the 12 months after its adoption. The mean number of hospitalizations per patient was 0.39±0.89 pre- and 0.18±0.57 postadoption (P<.001). There were 185 outpatient visits for HF in 96 (24%) patients before adoption and 64 in 48 (12%) patients after adoption (P<.001). The mean number of visits per patient was 0.47±1.11 pre- and 0.16±0.51 postadoption (P<.001)., Conclusions: A standardized follow-up protocol based on remote management of HeartLogic alerts enabled effective remote management of HF patients. After its adoption, we observed a significant reduction in HF hospitalizations and outpatient visits., (Copyright © 2024 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.)

Purpose: Invasive Pulmonary Fungal Infections (IPFIs) represent a diagnostic and therapeutic challenge. The exact role of surgery is not well defined. This study analyzes our experience with surgical treatment of IPFI in immunocompromised pediatric patients and, secondarily, compares IPFI caused by Aspergillus spp. with other fungal infections., Methods: This is a retrospective review (2000-2019) of patients with IPFI surgically treated at our pediatric institution. Statistical analysis was used to compare data between Aspergillus spp. and non-Aspergillus IPFI., Results: Twenty-five patients (64% female) underwent 29 lung resections. Median age at surgery was 7.19 years (1.63-19.14). The most frequent underlying condition (64%) was acute leukemia. Surgical indications included persistence or worsening of symptoms and pathological image findings (52%) or asymptomatic suspicious lesions in patients scheduled for intensive cytotoxic treatments or hematopoietic stem cell transplantation (48%). All patients underwent atypical lung resections, except one lobectomy. Aspergillus spp. was the most frequently isolated pathogen (68%). Follow-up was 4.07 years (0.07-18.07). Surgery-related mortality was 0%, but 4 patients died in the 100 days following surgery (2 due to disseminated fungal infection); the remaining 21 did not show signs of IPFI recurrence. Non-specific consolidations on CT scan were more frequent in non-Aspergillus IPFI (p < 0.05)., Conclusion: Surgical treatment of IPFI should be considered as a part of the treatment in selected pediatric immunocompromised patients, and it may have both diagnostic and therapeutic advantages over non-surgical management. When there is clinical suspicion of IPFI but CT scan shows unspecific alterations, the possibility of a non-Aspergillus IPFI should be considered., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objectives: Necrotizing enterocolitis (NEC) is a main cause of neonatal morbimortality. Gut prematurity and ischemia-reperfusion injury contribute to NEC and characterize two different scenarios: prematurity and congenital cardiopathy (CC). Our aim is to investigate whether CC worsens NEC gastrointestinal and general prognosis., Materials and Methods: NEC episodes from 2015-2023 were reviewed and classified into CC and non-CC. Patients with focal intestinal perforation were excluded. Data regarding NEC debut and management, surgical timing, intestinal segment involved and short-term outcomes were compared., Results: Out of 205 neonates, 15 were excluded for unavailable records or uncertain diagnosis. 190 cases were included, 59 with CC. Comparing CC and non-CC, no significant differences were found in weight or age at diagnosis, or NEC stage. Hemodynamic (HD) shock [38.98% vs 24.43% (p <  0.05)] and need for vasoactive support at debut were more frequent in CC patients [44.07% vs 23.66% (p < 0.05)]. No differences were found regarding need for surgery at debut, length of resected intestine or segment affected or days on parenteral nutrition. CC conditioned longer hospital stay [110.8 ± 68.4 days vs 68.4 ± 44.6 (p <  0.05)] and higher mortality [30.5 vs 11.5 (p <  0.05)]., Conclusions: NEC in CC patients presents more HD instability at debut and worse global prognosis, probably due to inherent cardiovascular compromise, but need for surgery, type of intestinal involvement and short-term outcomes are similar to non-CC NEC.

Background:  Thoracoscopic repair of esophageal atresia (EA) with tracheoesophageal fistula (TEF) is becoming an increasingly widespread technique; there is still controversy about its indication in certain patients. Our objective is to analyze if potential risk factors such as major congenital heart disease (CHD) or low birth weight (LBW) are a limitation to this approach., Methods:  Retrospective study (2017-2021) of patients with EA and distal TEF who underwent thoracoscopic repair were included. Patients with LBW less than 2,000 g or major CHD were compared with the rest., Results:  Twenty-five patients underwent thoracoscopic surgery. Nine patients (36%) had major CHD. Five of them (20%) were LBW less than 2,000 g, and only 8% (2/25) presented both risk factors. There were no differences in terms of operative time, conversion rate, tolerance evaluated with gasometric parameters (pO 2 , pCO 2 , pH) or complications (anastomotic leak and stricture, both early or during follow-up) in patients with major CHD and LBW (1,473 ± 319 vs. 2,664 ± 402 g). One conversion to thoracotomy was performed in a neonate weighing 1,050 g due to anesthetic intolerance. There was no recurrence of TEF. One patient died at the age of 9 months, due to major uncorrectable heart disease., Conclusion:  Thoracoscopic repair of EA/TEF is feasible technique in patients with CHD or LBW, with similar results to other patients. The complexity of this technique warrants individualizing the indication in each case., Level of Evidence:  IV., Competing Interests: None declared., (Thieme. All rights reserved.)

Aims: Heart failure (HF) guidelines recommend treating all patients with HF and reduced ejection fraction (HFrEF) with quadruple therapy, although they do not establish how to start it. This study aimed to evaluate the implementation of these recommendations, analyzing the efficacy and safety of the different therapeutic schedules., Methods and Results: Prospective, observational, and multicenter registry that evaluated the treatment initiated in patients with newly diagnosed HFrEF and its evolution at 3 months. Clinical and analytical data were collected, as well as adverse reactions and events during follow-up. Five hundred and thirty-three patients were included, selecting four hundred and ninety-seven, aged 65.5 ± 12.9 years (72% male). The most frequent etiologies were ischemic (25.5%) and idiopathic (21.1%), with a left ventricular ejection fraction of 28.7 ± 7.4%. Quadruple therapy was started in 314 (63.2%) patients, triple in 120 (24.1%), and double in 63 (12.7%). Follow-up was 112 days [IQI 91; 154], with 10 (2%) patients dying. At 3 months, 78.5% had quadruple therapy (p < 0.001). There were no differences in achieving maximum doses or reducing or withdrawing drugs (< 6%) depending on the starting scheme. Twenty-seven (5.7%) patients had any emergency room visits or admission for HF, less frequent in those with quadruple therapy (p = 0.02)., Conclusion: It is possible to achieve quadruple therapy in patients with newly diagnosed HFrEF early. This strategy makes it possible to reduce admissions and visits to the emergency room for HF without associating a more significant reduction or withdrawal of drugs or significant difficulty in achieving the target doses., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Myocarditis is defined as myocardial inflammation and its etiology is highly diverse, including infectious agents, drugs, and autoimmune diseases. The clinical presentation also varies widely, extending beyond the classic clinical picture of acute chest pain, and includes cases of cardiomyopathy of unknown cause whose etiology may be inflammatory. Because certain patients may benefit from targeted treatments, the search for the etiology should begin when myocarditis is first suspected. There remain several areas of uncertainty in the diagnosis and treatment of this disease. Consequently, this consensus document aims to provide clear recommendations for its diagnosis and treatment. Hence, a diagnostic algorithm is proposed, specifying when non-invasive diagnosis with cardiac MR is appropriate vs a noninvasive approach with endomyocardial biopsy. In addition, more novel aspects are discussed, such as when to suspect an underlying genetic etiology. The recommendations cover the management of myocarditis and inflammatory cardiomyopathy, both for general complications and specific clinical entities., (Copyright © 2024 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.)

Purpose: Split abdominal wall muscle flap (SAWMF) is a technique to repair large defects in congenital diaphragmatic hernia (CDH). A possible objection to this intervention could be any associated abdominal muscle weakness. Our aim is to analyze the evolution of this abdominal muscle wall weakness., Methods: Retrospective review of CDH repair by SAWMF (internal oblique muscle and transverse) from 2004 to 2023 focusing on the evolution of muscle wall weakness., Results: Eighteen neonates of 148 CDH patients (12,1%) were repaired using SAWMF. Mean gestational age and birth weight were 35.7 ± 3.5 weeks and 2587 ± 816 g. Mean lung-to-head ratio was 1.49 ± 0.28 and 78% liver-up. Seven patients (38%) were prenatally treated by tracheal occlusion. Ninety-four percent of the flaps were used for primary repair and one to repair a recurrence. One patient (5.6%) experienced recurrence. Abdominal muscle wall weakness was present in the form of a bulge. Resolution of weakness at 1, 2 and 3 years was 67%, 89% and 94%, respectively. No patient required treatment for weakness or died., Conclusions: Abdominal muscular weakness after a split abdominal wall muscle flap repair is not a limitation for its realization since it is asymptomatic and presents a prompt spontaneous resolution., Level of Evidence: IV., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The superficial circumflex iliac artery (SCIA) perforator (SCIP) flap has been used for scrotal reconstruction after Fournier's gangrene, skin cancer, or infections. However, there are few publications with regard to penoscrotal reconstruction after a traumatic injury with this flap. In this article, we propose a new SCIP flap variation, the "extended" or "direct" SCIP flap, to effectively reconstruct a wide scrotal defect after a traumatic injury. The "extended" SCIP flap is designed medial and cranial to the anterosuperior iliac spine (ASIS) using the superficial branch of the SCIA as the main pedicle., Competing Interests: Conflict of Interest None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. ( https://creativecommons.org/licenses/by/4.0/ ).)

The Klebsiella pneumoniae species complex (KpSC) is a major source of nosocomial infections globally with high rates of resistance to antimicrobials. Consequently, there is growing interest in understanding virulence factors and their association with cellular metabolic processes for developing novel anti-KpSC therapeutics. Phenotypic assays have revealed metabolic diversity within the KpSC, but metabolism research has been neglected due to experiments being difficult and cost-intensive. Genome-scale metabolic models (GSMMs) represent a rapid and scalable in silico approach for exploring metabolic diversity, which compile genomic and biochemical data to reconstruct the metabolic network of an organism. Here we use a diverse collection of 507 KpSC isolates, including representatives of globally distributed clinically relevant lineages, to construct the most comprehensive KpSC pan-metabolic model to date, KpSC pan v2. Candidate metabolic reactions were identified using gene orthology to known metabolic genes, prior to manual curation via extensive literature and database searches. The final model comprised a total of 3550 reactions, 2403 genes and can simulate growth on 360 unique substrates. We used KpSC pan v2 as a reference to derive strain-specific GSMMs for all 507 KpSC isolates, and compared these to GSMMs generated using a prior KpSC pan-reference (KpSC pan v1) and two single-strain references. We show that KpSC pan v2 includes a greater proportion of accessory reactions (8.8 %) than KpSC pan v1 (2.5 %). GSMMs derived from KpSC pan v2 also generate more accurate growth predictions, with high median accuracies of 95.4 % (aerobic, n =37 isolates) and 78.8 % (anaerobic, n =36 isolates) for 124 matched carbon substrates. KpSC pan v2 is freely available at https://github.com/kelwyres/KpSC-pan-metabolic-model, representing a valuable resource for the scientific community, both as a source of curated metabolic information and as a reference to derive accurate strain-specific GSMMs. The latter can be used to investigate the relationship between KpSC metabolism and traits of interest, such as reservoirs, epidemiology, drug resistance or virulence, and ultimately to inform novel KpSC control strategies.

Zoonotic parasitic diseases are considered a global threat to public health. In this sense, canines and felines may be infected by different cosmopolitan parasites, with playgrounds serving as an important focus of infection for humans, as well as domestic or wild animals. Knowledge of the epidemiological situation of parasites in animal reservoirs integrated into the environment, identifying the spread pathways, is a key element for an effective response to this threat. Thus, the aim of this study was to assess the frequency of intestinal parasites with zoonotic potential in 120 playgrounds in the Malaga province (Spain). Samples were processed and analysed following standard parasitological procedures. Some 36.7% of playgrounds were parasite-positive with one or more zoonotic parasites. The most common parasites recovered were nematodes (60.0%), followed by protozoan species (33.3%) and cestodes (6.7%). In the parasite-positive playgrounds, Toxocara spp. (17.0 ± 3.5%) and Giardia duodenalis (17.0 ± 3.4%) were the most predominant parasites. In addition, 34.1% of playgrounds were infected with multiple parasites. Our results show a high presence of parasitic forms with zoonotic potential in playgrounds in Malaga, Spain. Due to the close contact between pets and humans in playgrounds, the potential zoonotic risk may increase if prevention and control measures are not designed.

Background: Anastomosis near the ileocecal valve (ICV) are controversial due to the increased pressure on the suture; in this situation, the valve could be removed at a first stage or at the moment of stoma closure. However, preservation of the ICV has proved important benefits in the long term. The aim of this study is to evaluate its feasibility in neonates with focal intestinal perforation (FIP)., Methods: Retrospective study (2010-2019) of neonates with FIP who underwent intestinal resection and primary anastomosis. Patients were divided into group A (anastomosis less than 5 cm from ICV) and group B (more than 5 cm)., Results: Forty patients were treated. Patients ostomized or with resection of ICV were excluded. Finally, 24 patients (birth weight 1043 ± 594 g (520-3000), age 8.8 ± 7.8 days (2-39)) were included for analysis. Patent ductus arteriosus was present in 75%. There were 6 patients in group A (25%) and 18 in group B (75%). Groups were comparable in terms of gestational age, birth weight, and age at the time of surgery ( p  > .05). There were no cases of dehiscence nor stenosis of the anastomosis. There were no differences in reoperation rate, infectious complications, time to enteral feeding, days of parenteral nutrition, hospital stay nor survival ( p  > .05)., Conclusion: Ileo-ileal anastomosis closer to the ileocecal junction, in neonates with focal intestinal perforation, is an effective and safe option which also allows the preservation of the ICV avoiding the complications derived from its absence in a group of patients with high morbidity.

The Klebsiella group, found in humans, livestock, plants, soil, water and wild animals, is genetically and ecologically diverse. Many species are opportunistic pathogens and can harbour diverse classes of antimicrobial resistance genes. Healthcare-associated Klebsiella pneumoniae clones that are non-susceptible to carbapenems can spread rapidly, representing a high public health burden. Here we report an analysis of 3,482 genome sequences representing 15 Klebsiella species sampled over a 17-month period from a wide range of clinical, community, animal and environmental settings in and around the Italian city of Pavia. Northern Italy is a hotspot for hospital-acquired carbapenem non-susceptible Klebsiella and thus a pertinent setting to examine the overlap between isolates in clinical and non-clinical settings. We found no genotypic or phenotypic evidence for non-susceptibility to carbapenems outside the clinical environment. Although we noted occasional transmission between clinical and non-clinical settings, our data point to a limited role of animal and environmental reservoirs in the human acquisition of Klebsiella spp. We also provide a detailed genus-wide view of genomic diversity and population structure, including the identification of new groups., (© 2022. The Author(s).)

Aims: Hyperkalaemia (HK) is common in heart failure (HF) patients, related to renal dysfunction and medical treatment. It limits medical therapy optimization, which impacts prognosis. New potassium (K) binders help control HK, allowing better medical management of HF., Methods and Results: A retrospective multicentre register included all outpatients with HF and HK (K ≥ 5.1 mEq/L) treated with patiromer according to current recommendations. We evaluated analytic and clinical parameters before starting the treatment and at 7, 30 and 90 days, as well as adverse events related to patiromer and treatment optimization. We included 74 patients (71.6% male) with a mean age of 70.8 years (SD 9.2). Sixty-seven patients (90.5%) presented HK in the previous year. Forty patients (54.1%) underwent down-titration of a renin-angiotensin-aldosterone inhibitor (RAASi) or a mineralocorticoid receptor antagonist (MRA), and 27 (36.5%) stopped any of them due to HK. Initial K was 5.5 mEq/L (SD 0.6), with a significantly reduction at 7 days (4.9 mEq/L (SD 0.8); P < 0.001), maintained at 90 days (4.9 mEq/L (SD 0.8); P < 0.001). There were no other electrolyte disturbances, with a slight improvement in renal function [glomerular filtration rate 39.6 mL/min (SD 20.4) to 42.7 mL/min (SD 23.2); P = 0.005]. Adverse events were reported in 33.9% of patients, the most common being hypomagnesaemia (16.3%), gastrointestinal disturbances (14.9%) and HK (2.8%). Withdrawal of patiromer was uncommon (12.2%) due to gastrointestinal disturbances in 66.7% of cases. Nine patients (12.2%) started on a RAASi, and 15 patients (20.3%) on an MRA during the follow-up. Forty-five patients (60.8%) increased the dose of RAASi or MRA, increasing to target doses in 5.4 and 10.8% of patients, respectively. At 90 days, NTproBNP values were reduced from 2509.5 pg/mL [IQR 1311-4,249] to 1396.0 pg/mL [IQR 804-4263]; P = 0.003, but the reduction was only observed in those who optimized HF medical treatment [NTproBNP from 1950.5 pg/mL (IQR 1208-3403) to 1349.0 pg/mL (IQR 804-2609); P < 0.01]. NYHA functional class only improved in 7.5% of patients, corresponding with those who optimized HF medical treatment. Compared with the previous 3 months before patiromer treatment, the rate of hospitalization was reduced from 28.4 to 10.9% (P < 0.01), and the emergency room visits from 18.9 to 5.4% (P < 0.01)., Conclusions: In a real-life cohort of patients with HF, patiromer reduced and maintained K levels during 3 months of follow-up. The most common adverse events were hypomagnesaemia and gastrointestinal disturbances. Patiromer helps optimize medical treatment, increasing the percentage of patients treated with RAASi and MRA at target doses. At the end of follow-up, natriuretic peptides values and hospital visits were reduced, suggesting the benefit of optimizing HF medical treatment., (© 2022 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Introduction and Objectives: Optimal medical therapy decreases mortality and heart failure (HF) hospitalizations in HF patients with reduced left ventricular ejection fraction. Women have been underrepresented in clinical trials and not specifically evaluated. This study aimed to compare the safety and effectiveness of drug titration in women vs men., Methods: This post hoc gender study of the ETIFIC multicenter randomized trial included hospitalized patients with new-onset HF with reduced ejection fraction and New York Heart Association II-III and no contraindications to beta-blockers. A structured 4-month titration process was implemented in HF clinics. The primary endpoint was the mean relative dose (% of target dose) of beta-blockers achieved by women vs men. Secondary endpoints included the mean relative doses of angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, and mineralocorticoid receptor antagonists, adverse events, and other clinical outcomes at 6 months., Results: A total of 320 patients were included, 83 (25.93%) women and 237 (74.06%) men (76 vs 213 analyzed). The mean±standard deviation of the relative doses achieved by women vs men were as follows: beta-blockers 62.08%±30.72% vs 64.4%±32.77%, with a difference of-2.32% (95%CI,-10.58-5.94), P = .580; and mineralocorticoid receptor antagonists 79.85%±27.72% vs 67.29%±31.43%, P =.003. No other differences in drug dosage were found. Multivariate analysis showed nonsignificant differences. CV mortality was 1 (1.20%) vs 3 (1.26%), P=1, and HF hospitalizations 0 (0.00%) vs 10 (4.22%), P=.125., Conclusions: In a post hoc analysis from the HF-titration ETIFIC trial, we found nonsignificant gender differences in drug dosage, cardiovascular mortality, and HF hospitalizations. Trial registry number: NCT02546856., (Copyright © 2021 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.)

Low-flow vascular malformations are congenital overgrowths composed of abnormal blood vessels potentially causing pain, bleeding and obstruction of different organs. These diseases are caused by oncogenic mutations in the endothelium, which result in overactivation of the PI3K/AKT pathway. Lack of robust in vivo preclinical data has prevented the development and translation into clinical trials of specific molecular therapies for these diseases. Here, we demonstrate that the Pik3ca H1047R activating mutation in endothelial cells triggers a transcriptome rewiring that leads to enhanced cell proliferation. We describe a new reproducible preclinical in vivo model of PI3K-driven vascular malformations using the postnatal mouse retina. We show that active angiogenesis is required for the pathogenesis of vascular malformations caused by activating Pik3ca mutations. Using this model, we demonstrate that the AKT inhibitor miransertib both prevents and induces the regression of PI3K-driven vascular malformations. We confirmed the efficacy of miransertib in isolated human endothelial cells with genotypes spanning most of human low-flow vascular malformations., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

The Klebsiella pneumoniae species complex (KpSC) is a set of seven Klebsiella taxa that are found in a variety of niches and are an important cause of opportunistic health care-associated infections in humans. Because of increasing rates of multi-drug resistance within the KpSC, there is a growing interest in better understanding the biology and metabolism of these organisms to inform novel control strategies. We collated 37 sequenced KpSC isolates isolated from a variety of niches, representing all seven taxa. We generated strain-specific genome-scale metabolic models (GEMs) for all 37 isolates and simulated growth phenotypes on 511 distinct carbon, nitrogen, sulfur, and phosphorus substrates. Models were curated and their accuracy was assessed using matched phenotypic growth data for 94 substrates (median accuracy of 96%). We explored species-specific growth capabilities and examined the impact of all possible single gene deletions using growth simulations in 145 core carbon substrates. These analyses revealed multiple strain-specific differences, within and between species, and highlight the importance of selecting a diverse range of strains when exploring KpSC metabolism. This diverse set of highly accurate GEMs could be used to inform novel drug design, enhance genomic analyses, and identify novel virulence and resistance determinants. We envisage that these 37 curated strain-specific GEMs, covering all seven taxa of the KpSC, provide a valuable resource to the Klebsiella research community., (© 2022 Hawkey et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: The lack of dystrophin expression in Duchenne muscular dystrophy (DMD) induces muscle fibre and replacement by fibro-adipose tissue. Although the role of some growth factors in the process of fibrogenesis has been studied, pathways activated by PDGF-AA have not been described so far. Our aim was to study the molecular role of PDGF-AA in the fibrotic process of DMD., Methods: Skeletal muscle fibro-adipogenic progenitor cells (FAPs) from three DMD treated with PDGF-AA at 50 ng/mL were analysed by quantitative mass spectrometry-based proteomics. Western-blot, immunofluorescence, and G-LISA were used to confirm the mass spectrometry results. We evaluated the effects of PDGF-AA on the activation of RhoA pathway using two inhibitors, C3-exoenzyme and fasudil. Cell proliferation and migration were determined by BrdU and migration assay. Actin reorganization and collagen synthesis were measured by phalloidin staining and Sircol assay, respectively. In an in vivo proof of concept study, we treated dba/2J-mdx mice with fasudil for 6 weeks. Muscle strength was assessed with the grip strength. Immunofluorescence and flow cytometry analyses were used to study fibrotic and inflammatory markers in muscle tissue., Results: Mass spectrometry revealed that RhoA pathway proteins were up-regulated in treated compared with non-treated DMD FAPs (n = 3, mean age = 8 ± 1.15 years old). Validation of proteomic data showed that Arhgef2 expression was significantly increased in DMD muscles compared with healthy controls by a 7.7-fold increase (n = 2, mean age = 8 ± 1.14 years old). In vitro studies showed that RhoA/ROCK2 pathway was significantly activated by PDGF-AA (n = 3, 1.88-fold increase, P < 0.01) and both C3-exoenzyme and fasudil blocked that activation (n = 3, P < 0.05 and P < 0.001, respectively). The activation of RhoA pathway by PDGF-AA promoted a significant increase in proliferation and migration of FAPs (n = 3, P < 0.001), while C3-exoenzyme and fasudil inhibited FAPs proliferation at 72 h and migration at 48 and 72 h (n = 3, P < 0.001). In vivo studies showed that fasudil improved muscle function (n = 5 non-treated dba/2J-mdx and n = 6 treated dba/2J-mdx, 1.76-fold increase, P < 0.013), and histological studies demonstrated a 23% reduction of collagen-I expression area (n = 5 non-treated dba/2J-mdx and n = 6 treated dba/2J-mdx, P < 0.01)., Conclusions: Our results suggest that PDGF-AA promotes the activation of RhoA pathway in FAPs from DMD patients. This pathway could be involved in FAPs activation promoting its proliferation, migration, and actin reorganization, which represents the beginning of the fibrotic process. The inhibition of RhoA pathway could be considered as a potential therapeutic target for muscle fibrosis in patients with muscular dystrophies., (© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Hyperkalaemia is a growing concern in the treatment of patients with heart failure and reduced ejection fraction (HFrEF) as it limits the use of some prognostic-modifying drugs and has a negative impact on prognosis. The objective of the present study was to estimate the prevalence of hyperkalaemia in outpatients with HFrEF and its impact on achieving optimal medical treatment. For this purpose, a multicentre, prospective, and observational study was carried out on consecutive HFrEF patients who were monitored as outpatients in heart failure (HF) units and who, in the opinion of their doctor, received optimal medical treatment. A total of 565 HFrEF patients were included from 16 specialised HF units. The mean age was 66 ± 12 years, 78% were male, 45% had an ischemic cause, 39% had atrial fibrillation, 43% were diabetic, 42% had a glomerular filtration rate < 60 mL/min/1.7 m2, and the mean left ventricular ejection fraction was 31 ± 7%. Treatment at the study entry included: 76% on diuretics, 13% on ivabradine, 7% on digoxin, 18.9% on angiotensin-conversing enzyme inhibitors (ACEi), 11.3% on angiotensin receptors blockers (ARBs), 63.8% on angiotensin-neprilysin inhibitors (ARNi), 78.5% on mineralocorticoid receptor antagonists (MRAs), and 92.9% on beta-blockers. Potassium levels in the baseline analysis were: ≤5 mEq/L = 80.5%, 5.1−5.4 mEq/L = 13.8%, 5.5−5.9 mEq/L = 4.6%, and ≥6 mEq/L = 1.06%. Hyperkalaemia was the reason for not prescribing or reaching the target dose of an MRAs in 34.8% and 12.5% of patients, respectively. The impact of hyperkalaemia on not prescribing or dropping below the target dose in relation to ACEi, ARBs, and ARNi was significantly less. In conclusion, hyperkalaemia is a frequent problem in the management of patients with HFrEF and a limiting factor in the optimisation of medical treatment.

Background: Arrhythmogenic cardiomyopathy (AC) is a life-threatening disease which predispose to malignant arrhythmias and sudden cardiac death (SCD) in the early stages of the disease. Risk stratification relies on the electrical, genetic, and imaging data. Our study aimed to investigate how myocardial deformation parameters may identify the subjects at risk of known predictors of major ventricular arrhythmias. Methods: A cohort of 45 subjects with definite or borderline diagnosis of AC was characterized using the advanced transthoracic echocardiography (TTE) and cardiac magnetic resonance (CMR) and divided into the groups according to the potential arrhythmic risk markers, such as non-sustained ventricular tachycardia (NSVT), late gadolinium enhancement (LGE), and genetic status. Layer-specific global longitudinal strain (GLS) by TTE 2D speckle tracking was compared in patients with and without these arrhythmic risk markers. Results: In this study, 23 (51.1%) patients were men with mean age of 43 ± 16 years. Next-generation sequencing identified a potential pathogenic mutation in 39 (86.7%) patients. Thirty-nine patients presented LGE (73.3%), mostly located at the subepicardial-to-mesocardial layers. A layer-specific-GLS analysis showed worse GLS values at the epicardial and mesocardial layers in the subjects with NSVT and LGE. The epicardial GLS values of -15.4 and -16.1% were the best cut-off values for identifying the individuals with NSVT and LGE, respectively, regardless of left ventricular ejection fraction (LVEF). Conclusions: The layer-specific GLS assessment identified the subjects with high-risk arrhythmic features in AC, such as NSVT and LGE. An epicardial GLS may emerge as a potential instrument for detecting the subjects at risk of SCD in AC., 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 © 2021 Segura-Rodríguez, Bermúdez-Jiménez, González-Camacho, Moreno Escobar, García-Orta, Alcalá-López, Bautista Pavés, Oyonarte-Ramírez, López-Fernández, Álvarez, Tercedor and Jiménez-Jáimez.)

Skeletal muscle contains multiple cell types that work together to maintain tissue homeostasis. Among these, satellite cells (SC) and fibroadipogenic progenitors cells (FAPs) are the two main stem cell pools. Studies of these cells using animal models have shown the importance of interactions between these cells in repair of healthy muscle, and degeneration of dystrophic muscle. Due to the unavailability of fresh patient muscle biopsies, similar analysis of interactions between human FAPs and SCs is limited especially among the muscular dystrophy patients. To address this issue here we describe a method that allows the use of frozen human skeletal muscle biopsies to simultaneously isolate and grow SCs and FAPs from healthy or dystrophic patients. We show that while the purified SCs differentiate into mature myotubes, purified FAPs can differentiate into adipocytes or fibroblasts demonstrating their multipotency. We find that these FAPs can be immortalized and the immortalized FAPs (iFAPs) retain their multipotency. These approaches open the door for carrying out personalized analysis of patient FAPs and interactions with the SCs that lead to muscle loss., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Laparoscopy is widely accepted for the surgical treatment of benign ovarian lesions in adult women, with results similar to an open approach when spillage is avoided by different means such as endobag retrieval. This approach is controversial in children. Materials and Methods: Retrospective study (2009-2018) of all pediatric ovarian tumors with histological confirmation treated in a tertiary center. Data regarding preoperative selection criteria, surgical technique, and follow-up were registered. Laparoscopy was used only in tumors without malignant features and whose size allowed retrieving them through a small incision without morcellating. Emphasis was set in ovarian sparing surgery (OSS), comparing the results of open (OP) and laparoscopic (LPC) approach. Results: During the study period, 48 suspicious lesions in 45 patients were operated. Thirty cases (32 tumors) showed benign features (10 cystadenomas, 14 mature teratomas, 6 nontumor lesions); treatment consisted in oophorectomy in 13 (OP 6, LPC 7) and OSS in 17 (OP 5, LPC 12). There was one conversion in the LPC group. Mean follow-up was 3.44 years (range 0.03-8.84), based on annual ultrasound (US) and tumor markers during at least 5 years, recommending a lifetime follow-up by a gynecologist. There was only one relapse in a laparoscopically enucleated cystadenoma, and 2 patients developed contralateral metacronous teratomas. No procedure-related complications happened. Conclusions: Our series, although small and with a limited follow-up, suggest that laparoscopy could be an option of treatment in a subset of highly selected patients, although laparotomy remains the gold standard approach if malignancy is suspected.

Introduction:  Repair of large congenital diaphragmatic hernias (CDHs) is challenging. As primary repair is not always feasible, patches are commonly used. An alternative treatment is split abdominal wall muscle flap repair, which uses vascularized autologous tissue. The aim of this study was to analyze the long-term outcome of large CDH defects undergoing split abdominal wall muscle repair., Materials and Methods:  This is a retrospective review (2003-2016) of large CDH treated by split abdominal wall muscle flap repair., Results:  In a total of 107 CDH patients, the abdominal muscle flap technique was used in 10 (9.3%); 7 had been prenatally treated with tracheal occlusion. Two patients experienced recurrence at 2 months and 6 years, respectively. Only one patient required abdominoplasty due to abdominal wall muscle weakness. Two patients developed progressive scoliosis; one of them required orthopaedic treatment. Minor chest wall deformities were detected in seven, but only one required orthopaedic treatment. The lung-to-head ratio was 0.79 in patients developing musculoskeletal deformities, and 1.5 in those without this complication ( p  < 0.05). Median follow-up was 11.2 years (3.5-14.2), and all patients were alive at the time of writing this article., Conclusion:  The split abdominal wall muscle flap technique is a valid option for repair of large CDH. Associated musculoskeletal deformities seem to be influenced not only by the repair technique used but also by the degree of pulmonary hypoplasia and inherent pathophysiological changes., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Aims: Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a life-threatening entity with a highly heterogeneous genetic background. Cardiac magnetic resonance (CMR) imaging can identify fibrofatty scar by late gadolinium enhancement (LGE). Our aim is to investigate genotype-phenotype correlation in ARVC/D mutation carriers, focusing on CMR-LGE and myocardial fibrosis patterns., Methods and Results: A cohort of 44 genotyped patients, 33 with definite and 11 with borderline ARVC/D diagnosis, was characterized using CMR and divided into groups according to their genetic condition (desmosomal, non-desmosomal mutation, or negative). We collected information on cardiac volumes and function, as well as LGE pattern and extension. In addition, available ventricular myocardium samples from patients with pathogenic gene mutations were histopathologically analysed. Half of the patients were women, with a mean age of 41.6 ± 17.5 years. Next-generation sequencing identified a potential pathogenic mutation in 71.4% of the probands. The phenotype varied according to genetic status, with non-desmosomal male patients showing lower left ventricular (LV) systolic function. LV fibrosis was similar between groups, but distribution in non-desmosomal patients was frequently located at the posterolateral LV wall; a characteristic LV subepicardial circumferential LGE pattern was significantly associated with ARVC/D caused by desmin mutation. Histological analysis showed increased fibrillar connective tissue and intercellular space in all the samples., Conclusion: Desmosomal and non-desmosomal mutation carriers showed different morphofunctional features but similar LV LGE presence. DES mutation carriers can be identified by a specific and extensive LV subepicardial circumferential LGE pattern. Further studies should investigate the specificity of LGE in ARVC/D., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Introduction and Objectives: Hyperkalemia is a growing concern in the treatment of patients with heart failure and reduced ejection fraction because it limits the use of effective drugs. We report estimates of the magnitude of this problem in routine clinical practice in Spain, as well as changes in potassium levels during follow-up and associated factors., Methods: This study included patients with acute (n=881) or chronic (n=3587) heart failure recruited in 28 Spanish hospitals of the European heart failure registry of the European Society of Cardiology and followed up for 1 year. Various outcomes were analyzed, including changes in serum potassium levels and their impact on treatment., Results: Hyperkalemia (K + > 5.4 mEq/L) was identified in 4.3% (95%CI, 3.7%-5.0%) and 8.2% (6.5%-10.2%) of patients with chronic and acute heart failure, respectively, and was responsible for 28.9% of all cases of contraindication to mineralocorticoid receptor antagonist use and for 10.8% of all cases of failure to reach the target dose. Serum potassium levels were not recorded in 291 (10.8%) of the 2693 chronic heart failure patients with reduced ejection fraction. During follow-up, potassium levels increased in 179 of 1431 patients (12.5%, 95%CI, 10.8%-14.3%). This increase was directly related to age, diabetes, and history of stroke and was inversely related to history of hyperkalemia., Conclusions: This study highlights the magnitude of the problem of hyperkalemia in patients with heart failure in everyday clinical practice and the need to improve monitoring of this factor in these patients due to its interference with the possibility of receiving optimal treatment., (Copyright © 2019 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.)

Aim: The optimum surgical approach of neonates with right-sided aortic arch (RAA) and esophageal atresia (EA)/tracheoesophageal fistula (TEF) is still an unsolved question. In order to propose an operative algorithm in the era of endoscopic surgery, we performed an international survey to know the current practice between pediatric endoscopic surgeons. Two of the most important societies in endoscopic pediatric surgery were queried: the International Pediatric Endosurgery Group (IPEG) and the European Society of Paediatric Endoscopic Surgeons (ESPES)., Materials and Methods: During December 2016, an anonymous online-based survey was sent to all IPEG and ESPES members, collecting data regarding perioperative management and surgical repair of EA/TEF with RAA., Results: 144 surgeons from 23 countries completed the questionnaire. 69.2% of respondents were IPEG members, 30.8% were ESPES members. 71.5% of members who answered the survey had more than 10years of surgical experience. A preoperative echocardiography was almost uniformly performed (93.1%). 31.9% of the surveyed surgeons had never treated an EA/TEF with RAA. The remaining 98 surveyed surgeons had managed 279 cases of EA/TEF with RAA. Thoracotomy was considered the preferred approach for 54.2% of the surgeons, and 51.9% chose a right-sided approach. When RAA was an intraoperative finding, 76% would perform a contralateral thoracotomy if difficulties arose. Thoracoscopy was preferred by 45.8% of surgeons. If RAA was suspected preoperatively, 63.1% preferred to attempt a left-sided thoracoscopy and only 24.2% would change their approach to a thoracotomy. If RAA was an intraoperative finding and a safe surgical repair could not be achieved through right-sided thoracoscopy, 51.5% of them chose to perform a left sided thoracoscopy, rather than convert to thoracotomy., Conclusions: Preoperative echocardiography performed by experienced examiners helps in surgical planning. Preoperative diagnosis of RAA should not discourage thoracoscopic repair, which is increasingly becoming more popular for the correction of EA/TEF. In case of an unexpected intraoperative diagnosis of RAA or operative difficulties when approaching through the right side, thoracoscopy offers a less aggressive approach., Level of Evidence: V., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Introduction and Objectives: To analyze the prognostic value of nocturnal hypoxemia measured with portable nocturnal pulse-oximetry in patients hospitalized due to heart failure and its relation to mortality and hospital readmission., Methods: We included 38 patients who were admitted consecutively to our unit with the diagnosis of decompensated heart failure. Pulse-oximetry was considered positive for hypoxemia when more than 10 desaturations per hour were recorded during sleep. Follow-up was performed for 30.3 (standard deviation [SD] 14.2) months, the main objective being a combined endpoint of all-cause mortality and hospital readmission due to heart failure., Results: The average age was 70.7 (SD 10.7) years, 63.3% were males. Pulse-oximetry was considered positive for hypoxemia in 27 (71%) patients. Patients with positive pulse-oximetry had the most frequent endpoint (9.1% [1] vs. 61.5% [16], P = 0.003). After multivariate analysis, continuous nocturnal hypoxemia was related to the combined endpoint (HR = 8.37, 1.19-68.4, P = 0.03)., Discussion: Patients hospitalized for heart failure and nocturnal hypoxemia measured with portable pulse-oximeter have an increased risk of hospital readmission and death., (Copyright © 2017 Elsevier España, S.L.U. All rights reserved.)

Background: Desmin ( DES ) mutations cause severe skeletal and cardiac muscle disease with heterogeneous phenotypes. Recently, DES mutations were described in patients with inherited arrhythmogenic right ventricular cardiomyopathy/dysplasia, although their cellular and molecular pathomechanisms are not precisely known. Our aim is to describe clinically and functionally the novel DES -p.Glu401Asp mutation as a cause of inherited left ventricular arrhythmogenic cardiomyopathy/dysplasia., Methods: We identified the novel DES mutation p.Glu401Asp in a large Spanish family with inherited left ventricular arrhythmogenic cardiomyopathy/dysplasia and a high incidence of adverse cardiac events. A full clinical evaluation was performed on all mutation carriers and noncarriers to establish clinical and genetic cosegregation. In addition, desmin, and intercalar disc-related proteins expression were histologically analyzed in explanted cardiac tissue affected by the DES mutation. Furthermore, mesenchymal stem cells were isolated and cultured from 2 family members with the DES mutation (1 with mild and 1 with severe symptomatology) and a member without the mutation (control) and differentiated ex vivo to cardiomyocytes. Then, important genes related to cardiac differentiation and function were analyzed by real-time quantitative polymerase chain reaction. Finally, the p.Glu401Asp mutated DES gene was transfected into cell lines and analyzed by confocal microscopy., Results: Of the 66 family members screened for the DES -p.Glu401Asp mutation, 23 of them were positive, 6 were obligate carriers, and 2 were likely carriers. One hundred percent of genotype-positive patients presented data consistent with inherited arrhythmogenic cardiomyopathy/dysplasia phenotype with variable disease severity expression, high-incidence of sudden cardiac death, and absence of skeletal myopathy or conduction system disorders. Immunohistochemistry was compatible with inherited arrhythmogenic cardiomyopathy/dysplasia, and the functional study showed an abnormal growth pattern and cellular adhesion, reduced desmin RNA expression, and some other membrane proteins, as well, and desmin aggregates in transfected cells expressing the mutant desmin., Conclusions: The DES -p.Glu401Asp mutation causes predominant inherited left ventricular arrhythmogenic cardiomyopathy/dysplasia with a high incidence of adverse clinical events in the absence of skeletal myopathy or conduction system disorders. The pathogenic mechanism probably corresponds to an alteration in desmin dimer and oligomer assembly and its connection with membrane proteins within the intercalated disc., (© 2017 American Heart Association, Inc.)

Introduction and Objectives: Nonischemic sudden cardiac death (SCD) is predominantly caused by cardiomyopathies and channelopathies. There are many diagnostic tests, including some complex techniques. Our aim was to analyze the diagnostic yield of a systematic diagnostic protocol in a specialized unit., Methods: The study included 56 families with at least 1 index case of SCD (resuscitated or not). Survivors were studied with electrocardiogram, advanced cardiac imaging, exercise testing, familial study, genetic testing and, in some cases, pharmacological testing. Families with deceased probands were studied using the postmortem findings, familial evaluation, and molecular autopsy with next-generation sequencing (NGS)., Results: A positive diagnosis was obtained in 80.4% of the cases, with no differences between survivors and nonsurvivors (P=.53). Cardiac channelopathies were more prevalent among survivors than nonsurvivors (66.6% vs 40%, P=.03). Among the 30 deceased probands, the definitive diagnosis was given by autopsy in 7. A diagnosis of cardiomyopathy tended to be associated with a higher event rate in the family. Genetic testing with NGS was performed in 42 index cases, with a positive result in 28 (66.6%), with no differences between survivors and nonsurvivors (P=.21)., Conclusions: There is a strong likelihood of reaching a diagnosis in SCD after a rigorous protocol, with a more prevalent diagnosis of channelopathy among survivors and a worse familial prognosis in cardiomyopathies. Genetic testing with NGS is useful and its value is increasing with respect to the Sanger method., (Copyright © 2017 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.)

ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step of bacterial glycogen and plant starch biosynthesis, the most common carbon storage polysaccharides in nature. A major challenge is to understand how AGPase activity is regulated by metabolites in the energetic flux within the cell. Here we report crystal structures of the homotetrameric AGPase from Escherichia coli in complex with its physiological positive and negative allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP, and sucrose in the active site. FBP and AMP bind to partially overlapping sites located in a deep cleft between glycosyltransferase A-like and left-handed β helix domains of neighboring protomers, accounting for the fact that sensitivity to inhibition by AMP is modulated by the concentration of the activator FBP. We propose a model in which the energy reporters regulate EcAGPase catalytic activity by intra-protomer interactions and inter-protomer crosstalk, with a sensory motif and two regulatory loops playing a prominent role., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Introduction and Objectives: To estimate the percentage of heart failure patients in Spain that received the European Society of Cardiology recommended treatments, and in those that did not, to determine the reasons why., Methods: The study included 2834 consecutive ambulatory patients with heart failure from 27 Spanish hospitals. We recorded general information, the treatment indicated, and the reasons why it was not prescribed in some cases. In patients who met the criteria to receive a certain drug, true undertreatment was defined as the percentage of patients who, without justification, did not receive the drug., Results: In total, 92.6% of ambulatory patients with low ejection fraction received angiotensin converting enzyme inhibitors or angiotensin receptor blockers, 93.3% beta-blockers, and 74.5% mineralocorticoid receptor antagonists. The true undertreatment rates were 3.4%, 1.8%, and 19.0%, respectively. Target doses were reached in 16.2% of patients receiving angiotensin converting enzyme inhibitors, 23.3% of those with angiotensin receptor blockers, 13.2% of those prescribed beta-blockers, and 23.5% of those with mineralocorticoid receptor antagonists. Among patients who could benefit from ivabradine, 29.1% received this drug. In total, 36% of patients met the criteria for defibrillator implantation and 90% of them had received the device or were scheduled for implantation, whereas 19.6% fulfilled the criteria for resynchronization therapy and 88.0% already had or would soon have the device. In patients who met the criteria, but did not undergo device implantation, the reasons were not cost-related., Conclusions: When justified reasons for not administering heart failure drugs were taken into account, adherence to the guideline recommendations was excellent. Exclusive use of the percentage of treated patients is a poor indicator of the quality of healthcare in heart failure. Measures should be taken to improve the attainment of optimal dosing in each patient., (Copyright © 2015 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.)

Background: Advanced heart failure (HF) is associated with high morbidity and mortality; it represents a major burden for the health system. Episodes of acute decompensation requiring frequent and prolonged hospitalizations account for most HF-related expenditure. Inotropic drugs are frequently used during hospitalization, but rarely in out-patients. The LAICA clinical trial aims to evaluate the effectiveness and safety of monthly levosimendan infusion in patients with advanced HF to reduce the incidence of hospital admissions for acute HF decompensation., Methods: The LAICA study is a multicenter, prospective, randomized, double-blind, placebo-controlled, parallel group trial. It aims to recruit 213 out-patients, randomized to receive either a 24-h infusion of levosimendan at 0.1 μg/kg/min dose, without a loading dose, every 30 days, or placebo., Results: The main objective is to assess the incidence of admission for acute HF worsening during 12 months. Secondarily, the trial will assess the effect of intermittent levosimendan on other variables, including the time in days from randomization to first admission for acute HF worsening, mortality and serious adverse events., Conclusions: The LAICA trial results could allow confirmation of the usefulness of intermittent levosimendan infusion in reducing the rate of hospitalization for HF worsening in advanced HF outpatients.

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of peripheral membrane-associated GTs for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here we used single molecule force spectroscopy techniques to study the mechanical and conformational properties of PimA. In our studies, we engineered a polyprotein containing PimA flanked by four copies of the well characterized I27 protein, which provides an unambiguous mechanical fingerprint. We found that PimA exhibits weak mechanical stability albeit displaying β-sheet topology expected to unfold at much higher forces. Notably, PimA unfolds following heterogeneous multiple step mechanical unfolding pathways at low force akin to molten globule states. Interestingly, the ab initio low resolution envelopes obtained from small angle x-ray scattering of the unliganded PimA and the PimA·GDP complexed forms clearly demonstrate that not only the "open" and "closed" conformations of the GT-B enzyme are largely present in solution, but in addition, PimA experiences remarkable flexibility that undoubtedly corresponds to the N-terminal "Rossmann fold" domain, which has been proved to participate in protein-membrane interactions. Based on these results and on our previous experimental data, we propose a model wherein the conformational transitions are important for the mannosyltransferase to interact with the donor and acceptor substrates/membrane.

Considerable progress has been made in recent years in our understanding of the structural basis of glycosyl transfer. Yet the nature and relevance of the conformational changes associated with substrate recognition and catalysis remain poorly understood. We have focused on the glucosyl-3-phosphoglycerate synthase (GpgS), a "retaining" enzyme, that initiates the biosynthetic pathway of methylglucose lipopolysaccharides in mycobacteria. Evidence is provided that GpgS displays an unusually broad metal ion specificity for a GT-A enzyme, with Mg(2+), Mn(2+), Ca(2+), Co(2+), and Fe(2+) assisting catalysis. In the crystal structure of the apo-form of GpgS, we have observed that a flexible loop adopts a double conformation L(A) and L(I) in the active site of both monomers of the protein dimer. Notably, the L(A) loop geometry corresponds to an active conformation and is conserved in two other relevant states of the enzyme, namely the GpgS·metal·nucleotide sugar donor and the GpgS·metal·nucleotide·acceptor-bound complexes, indicating that GpgS is intrinsically in a catalytically active conformation. The crystal structure of GpgS in the presence of Mn(2+)·UDP·phosphoglyceric acid revealed an alternate conformation for the nucleotide sugar β-phosphate, which likely occurs upon sugar transfer. Structural, biochemical, and biophysical data point to a crucial role of the β-phosphate in donor and acceptor substrate binding and catalysis. Altogether, our experimental data suggest a model wherein the catalytic site is essentially preformed, with a few conformational changes of lateral chain residues as the protein proceeds along the catalytic cycle. This model of action may be applicable to a broad range of GT-A glycosyltransferases.