An ancient bacterial zinc acquisition system identified from a cyanobacterial exoproteome.

Bacteria have developed fine-tuned responses to cope with potential zinc limitation. The Zur protein is a key player in coordinating this response in most species. Comparative proteomics conducted on the cyanobacterium Anabaena highlighted the more abundant proteins in a zur mutant compared to the wild type. Experimental evidence showed that the exoprotein ZepA mediates zinc uptake. Genomic context of the zepA gene and protein structure prediction provided additional insights on the regulation and putative function of ZepA homologs. Phylogenetic analysis suggests that ZepA represents a primordial system for zinc acquisition that has been conserved for billions of years in a handful of species from distant bacterial lineages. Furthermore, these results show that Zur may have been one of the first regulators of the FUR family to evolve, consistent with the scarcity of zinc in the ecosystems of the Archean eon.

Clinicopathological correlates in the frontotemporal lobar degeneration-motor neuron disease spectrum.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease (MND) that shares a common clinical, genetic and pathologic spectrum with frontotemporal dementia (FTD). It is highly heterogeneous in its presentation and features. Up to 50% of patients with MND develop cognitive-behavioural symptoms during the course of the disease, meeting criteria for FTD in 10%-15% of cases. In the absence of a precise biomarker, neuropathology is still a valuable tool to understand disease nosology, reach a definite diagnostic confirmation and help define specific subgroups of patients with common phenotypic, genetic and biomarker profiles. However, few neuropathological series have been published, and the frequency of frontotemporal lobar degeneration (FTLD) in MND is difficult to estimate. In this work we describe a large clinicopathological series of MND patients, analysing the frequency of concurrent FTLD changes and trying to define specific subgroups of patients based on their clinical, genetic and pathological characteristics. We performed an observational, retrospective, multicentre case study. We included all cases meeting neuropathological criteria for MND from the Neurological Tissue Bank of the FRCB-IDIBAPS-Hospital Clínic Barcelona Biobank between 1994 and 2022, regardless of their last clinical diagnosis. While brain donation is encouraged in all patients, it is performed in very few, and representativeness of the cohort might not be precise for all patients with MND. We retrospectively reviewed clinical and neuropathological data and describe the main clinical, genetic and pathogenic features, comparing neuropathologic groups between MND with and without FTLD changes and aiming to define specific subgroups. We included brain samples from 124 patients, 44 of whom (35.5%) had FTLD neuropathologic features (i.e. FTLD-MND). Pathologic TDP-43 aggregates were present in 93.6% of the cohort and were more extensive (higher Brettschneider stage) in those with concurrent FTLD (P < 0.001). Motor symptom onset was more frequent in the bulbar region in FTLD-MND cases than in those with isolated MND (P = 0.023), with no differences in survival. We observed a better clinicopathological correlation in the MND group than in the FTLD-MND group (93.8% versus 61.4%; P < 0.001). Pathogenic genetic variants were more common in the FTLD-MND group, especially C9orf72. We describe a frequency of FTLD of 35.5% in our series of neuropathologically confirmed cases of MND. The FTLD-MND spectrum is highly heterogeneous in all aspects, especially in patients with FTLD, in whom it is particularly difficult to define specific subgroups. In the absence of definite biomarkers, neuropathology remains a valuable tool for a definite diagnosis, increasing our knowledge in disease nosology.

Sound-based cough peak flow estimation in patients with neuromuscular disorders.

INTRODUCTION/AIMS: Cough impairment is common in individuals with neuromuscular disorders and is associated with respiratory infections and shorter survival. Cough strength is assessed by measuring cough peak flow (CPF) using a flow meter, but this method requires a complex device setup and trained staff. The aim of the study is to evaluate the reliability of a smartphone app to estimate CPF based on cough sounds in a cohort of individuals with neuromuscular disorders. METHODS: Individuals with neuromuscular disorders underwent CPF measurement with a flow meter and a smartphone app. A CPF <270 L/min was considered abnormal. RESULTS: Of the 50 patients studied, 26 had amyotrophic lateral sclerosis (52%), 15 had hereditary myopathies (30%), and 9 had myasthenia gravis (18%). The intraclass correlation coefficient (ICC) between the CPF measured with a flow meter and CPF estimated with cough sounds was 0.774 (p < .001) even if the patients had orofacial weakness (ICC = 0.806, p < .001). The smartphone app had 94.4% sensitivity and 100% specificity to detect patients with CPF of less than 270 L/min. DISCUSSION: Our findings suggest that sounds measured with a smartphone app provide a reliable estimate of CPF in patients with neuromuscular disorders, even in the presence of with orofacial weakness. This may be a convenient way to monitor respiratory involvement in patients with neuromuscular disorders, but larger studies of more diverse patient cohorts are needed.

Specification of the Drosophila Orcokinin A neurons by combinatorial coding.

The central nervous system contains a daunting number of different cell types. Understanding how each cell acquires its fate remains a major challenge for neurobiology. The developing embryonic ventral nerve cord (VNC) of Drosophila melanogaster has been a powerful model system for unraveling the basic principles of cell fate specification. This pertains specifically to neuropeptide neurons, which typically are stereotypically generated in discrete subsets, allowing for unambiguous single-cell resolution in different genetic contexts. Here, we study the specification of the OrcoA-LA neurons, characterized by the expression of the neuropeptide Orcokinin A and located laterally in the A1-A5 abdominal segments of the VNC. We identified the progenitor neuroblast (NB; NB5-3) and the temporal window (castor/grainyhead) that generate the OrcoA-LA neurons. We also describe the role of the Ubx, abd-A, and Abd-B Hox genes in the segment-specific generation of these neurons. Additionally, our results indicate that the OrcoA-LA neurons are "Notch Off" cells, and neither programmed cell death nor the BMP pathway appears to be involved in their specification. Finally, we performed a targeted genetic screen of 485 genes known to be expressed in the CNS and identified nab, vg, and tsh as crucial determinists for OrcoA-LA neurons. This work provides a new neuropeptidergic model that will allow for addressing new questions related to neuronal specification mechanisms in the future.

Ultrasonographic and manometric study of the tongue as biomarkers of dysphagia in patients with amyotrophic lateral sclerosis.

BACKGROUND: The possibility of having methods to assess dysphagia in amyotrophic lateral sclerosis (ALS) patients in a minimally invasive manner could facilitate follow-up and allow performing of therapeutic interventions at earlier stages of the disease. The aim of the study was to analyze the role of tongue strength and thickness in ALS patients and their correlation with dysphagia and bulbar function. METHODS: A sample of outpatients with ALS was evaluated for demographic and clinical features. Tongue thickness and strength have been measured for each patient, and quantitative and qualitative data of the videofluoroscopy swallow study have been analyzed. RESULTS: Of the 38 ALS patients studied, 47.4% were women, and 26.3% had bulbar onset. The median time between symptom onset and the study was 24 months (IQR 11.5-48), and 55.3% of the patients were carriers of non-invasive mechanical ventilation. Tongue strength identified patients with impaired oral and pharyngeal transit and those with bolus residue scale (BRS) > 1 or penetration-aspiration scale (PAS) ≥ 3. In contrast, tongue thickness is only associated with impaired oral transit. Finally, anterior tongue strength ≤ 34 kPa and posterior tongue strength ≤ 34.5 kPa detected ALS penetrators/aspirators (PAS ≥ 3) and patients with ALS with post-swallow residue (BRS > 1). CONCLUSIONS: Our results suggest that measures that assess the functionality (strength) of the tongue are more valuable than morphological measurements (thickness) for the follow-up of patients with ALS. Alterations of the anterior and posterior lingual strength correlate with the presence of bronchoaspiration and post-swallowing residue (BRS > 1).

Role of a cryptic tRNA gene operon in survival under translational stress.

As compared to eukaryotes, bacteria have a reduced tRNA gene set encoding between 30 and 220 tRNAs. Although in most bacterial phyla tRNA genes are dispersed in the genome, many species from distinct phyla also show genes forming arrays. Here, we show that two types of arrays with distinct evolutionary origins exist. This work focuses on long tRNA gene arrays (L-arrays) that encompass up to 43 genes, which disseminate by horizontal gene transfer and contribute supernumerary tRNA genes to the host. Although in the few cases previously studied these arrays were reported to be poorly transcribed, here we show that the L-array of the model cyanobacterium Anabaena sp. PCC 7120, encoding 23 functional tRNAs, is largely induced upon impairment of the translation machinery. The cellular response to this challenge involves a global reprogramming of the transcriptome in two phases. tRNAs encoded in the array are induced in the second phase of the response, directly contributing to cell survival. Results presented here show that in some bacteria the tRNA gene set may be partitioned between a housekeeping subset, which constantly sustains translation, and an inducible subset that is generally silent but can provide functionality under particular conditions.

Imbalance in Bone Morphogenic Proteins 2 and 7 Is Associated with Renal and Cardiovascular Damage in Chronic Kidney Disease.

Arterial stiffness is a major vascular complication of chronic kidney disease (CKD). The development of renal damage, hypertension, and increased pulse wave velocity (PWV) in CKD might be associated with an imbalance in bone morphogenetic proteins (BMP)-2 and BMP-7. Plasma BMP-2 and BMP-7 were determined by ELISA in CKD patients (stages I-III; n = 95) and Munich Wistar Frömter (MWF) rats. Age-matched Wistar rats were used as a control. The expression of BMP-2, BMP-7, and profibrotic and calcification factors was determined in kidney and perivascular adipose tissues (PVAT). BMP-2 was higher in stage III CKD patients compared to control subjects. BMP-7 was lower at any CKD stage compared to controls, with a significant further reduction in stage III patients. A similar imbalance was observed in MWF rats together with the increase in systolic (SBP) and diastolic blood pressure (DBP), or pulse wave velocity (PWV). MWF exhibited elevated urinary albumin excretion (UAE) and renal expression of BMP-2 or kidney damage markers, Kim-1 and Ngal, whereas renal BMP-7 was significantly lower than in Wistar rats. SBP, DBP, PWV, UAE, and plasma creatinine positively correlated with the plasma BMP-2/BMP-7 ratio. Periaortic and mesenteric PVAT from MWF rats showed an increased expression of BMP-2 and profibrotic and calcification markers compared to Wistar rats, together with a reduced BMP-7 expression. BMP-2 and BMP-7 imbalance in plasma, kidney, and PVATs is associated with vascular damage, suggesting a profibrotic/pro-calcifying propensity associated with progressive CKD. Thus, their combined analysis stratified by CKD stages might be of clinical interest to provide information about the degree of renal and vascular damage in CKD.

Prevalence of Hyponatremia and Its Associated Morbimortality in Hospitalized Patients Receiving Parenteral Nutrition.

BACKGROUND: The objective of the study was to determine the prevalence of hyponatremia (HN) and its associated morbimortality in hospitalized patients receiving parenteral nutrition (PN). METHODS: A retrospective study including 222 patients receiving total PN (parenteral nutrition group [PNG]) over a 7-month period in a tertiary hospital and 176 matched to 179 control subjects without PN (control subjects group [CSG]). Demographic data, Charlson Comorbidity Index (CCI), date of HN detection-(serum sodium or SNa <135 mmol/L)-intrahospital mortality, and hospital length-of-stay (LOS) were registered. In the PNG, body mass index (BMI) and SNa before, during, and after PN were recorded. RESULTS: HN was more prevalent in the PNG: 52.8 vs. 35.8% (p = 0.001), and independent of age, gender, or CCI (OR 1.8 [95% CI 1.1-2.8], p = 0.006). In patients on PN, sustained HN (75% of all intraindividual SNa <135 mmol/L) was associated with a higher mortality rate independent of age, gender, CCI, or BMI (OR 7.38 [95% CI 1.07-50.8], p = 0.042). The absence of HN in PN patients was associated with a shorter hospital LOS (<30 days) and was independent of other comorbidities (OR 3.89 [95% CI 2.11-7.18], p = 0.001). CONCLUSIONS: HN is more prevalent in patients on PN. Sustained HN is associated with a higher intrahospital mortality rate. Absence of HN is associated with a shorter hospital LOS.

Trans-oligomerization of duplicated aminoacyl-tRNA synthetases maintains genetic code fidelity under stress.

Aminoacyl-tRNA synthetases (aaRSs) play a key role in deciphering the genetic message by producing charged tRNAs and are equipped with proofreading mechanisms to ensure correct pairing of tRNAs with their cognate amino acid. Duplicated aaRSs are very frequent in Nature, with 25,913 cases observed in 26,837 genomes. The oligomeric nature of many aaRSs raises the question of how the functioning and oligomerization of duplicated enzymes is organized. We characterized this issue in a model prokaryotic organism that expresses two different threonyl-tRNA synthetases, responsible for Thr-tRNA(Thr) synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNA(Thr). Low zinc promotes dissociation of dimeric T1 into monomers deprived of aminoacylation activity and simultaneous induction of T2, which is active for aminoacylation under low zinc. T2 either forms homodimers or heterodimerizes with T1 subunits that provide essential proofreading activity in trans. These findings evidence that in organisms with duplicated genes, cells can orchestrate the assemblage of aaRSs oligomers that meet the necessities of the cell in each situation. We propose that controlled oligomerization of duplicated aaRSs is an adaptive mechanism that can potentially be expanded to the plethora of organisms with duplicated oligomeric aaRSs.

Methionyl-tRNA synthetase synthetic and proofreading activities are determinants of antibiotic persistence.

Bacterial antibiotic persistence is a phenomenon where bacteria are exposed to an antibiotic and the majority of the population dies while a small subset enters a low metabolic, persistent, state and are able to survive. Once the antibiotic is removed the persistent population can resuscitate and continue growing. Several different molecular mechanisms and pathways have been implicated in this phenomenon. A common mechanism that may underly bacterial antibiotic persistence is perturbations in protein synthesis. To investigate this mechanism, we characterized four distinct metG mutants for their ability to increase antibiotic persistence. Two metG mutants encode changes near the catalytic site of MetRS and the other two mutants changes near the anticodon binding domain. Mutations in metG are of particular interest because MetRS is responsible for aminoacylation both initiator tRNAMet and elongator tRNAMet indicating that these mutants could impact translation initiation and/or translation elongation. We observed that all the metG mutants increased the level of antibiotic persistence as did reduced transcription levels of wild type metG. Although, the MetRS variants did not have an impact on MetRS activity itself, they did reduce translation rates. It was also observed that the MetRS variants affected the proofreading mechanism for homocysteine and that these mutants' growth is hypersensitive to homocysteine. Taken together with previous findings, our data indicate that both reductions in cellular Met-tRNAMet synthetic capacity and reduced proofreading of homocysteine by MetRS variants are positive determinants for bacterial antibiotic persistence.

Revisional surgery for malnutrition after SADI-S: prevalence, indications, techniques and outcomes.

KEY POINTS: SADIS with short common limb (< 250 cm) is a malabsorptive operation. Reoperation is advised in patients requiring admission for severe malnutrition. Elongation of the common channel is the preferred revisional technique Introduction: Single-Anastomosis Duodeno-Ileal bypass with Sleeve gastrectomy (SADI-S) is a modification of the duodenal switch. Initial common channel's length was 200, and after malnutrition was detected in some patients, it was elongated to 250 or 300 cm. The present study analyzes presentation and treatment of malnutrition after SADI-S. MATERIALS: Three hundred and thirty-three consecutive patients undergoing SADI-S between May 2007 and February 2019 were included. The common limb length was 200 cm in 50 cases, 250 cm in 211, 300 in 71 and 350 in 1. Thirty-one patients were admitted for severe hypoalbuminemia and 17 patients were submitted to revisional surgery, and constitute the series of our study. Mean weight before reoperation was 57 kg and mean body mass index (BMI) was 21 kg/m2. Mean number of daily bowel movements was 5,6. RESULTS: Mean time to reoperation was 56 months. The limb was found shorter than expected in 6 cases. Revisional surgery was conversion into a Roux en Y duodenal switch in 3 cases, elongation of the common limb in 11 patients, duodeno-duodenostomy in 1 and duodeno-jejunostomy to the first jejunal loop in 2. Mean weight regain was 14 kg, and mean final BMI 26 kg/m2. Daily bowel movements were reduced to 1,3. Factors related to hypoalbuminemia were hypertension, poor-controlled diabetes, shorter common limb and liver-test alterations. CONCLUSION: SADI-S is expected to be less malabsorptive than previous biliopancreatic diversions. However, caution must be taken with certain patients to avoid postoperative malnutrition. Adequate follow up with long-term supplementation is required.

Regulation of internal promoters in a zinc-responsive operon is influenced by transcription from upstream promoters.

In the cyanobacterium Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120), a zinc-responsive operon (all4725-all4721) has been described, which contains 4 distinct promoters. The two most upstream ones bind Zur with high affinity, whereas the other two do not or do so with a very low affinity. In this paper, a detailed characterization of the four promoters is presented, showing that all four were induced by metal depletion, and they were constitutively derepressed in a zur mutant, despite the two downstream promoters not being direct targets for this regulator. Crucially, induction by metal depletion of the two downstream promoters was abrogated when transcription initiated at the upstream promoters was interrupted by a polar insertion midway in the operon. In contrast, insertion of a nitrogen-responsive promoter at a roughly similar position provoked the two downstream promoters to adopt a regulatory pattern mimicking that of the inserted promoter. Thus, regulation of the two downstream promoters is apparently influenced by transcription from promoters upstream. Evidence is presented indicating that the activity of the two downstream promoters is kept basal in Anabaena by repression. A regulatory model compatible with these results is proposed, where promoters controlled by repression in bacterial operons may be subjected to a hierarchical regulation depending on their position in the operon. According to this model, internal promoters may respond to stimuli governing the activity of promoters upstream by an indirect regulation and to specific stimuli by a direct regulation.

Characterization of the response to zinc deficiency in the cyanobacterium Anabaena sp. strain PCC 7120.

Zur regulators control zinc homeostasis by repressing target genes under zinc-sufficient conditions in a wide variety of bacteria. This paper describes how part of a survey of duplicated genes led to the identification of the open reading frame all2473 as the gene encoding the Zur regulator of the cyanobacterium Anabaena sp. strain PCC 7120. All2473 binds to DNA in a zinc-dependent manner, and its DNA-binding sequence was characterized, which allowed us to determine the relative contribution of particular nucleotides to Zur binding. A zur mutant was found to be impaired in the regulation of zinc homeostasis, showing sensitivity to elevated concentrations of zinc but not other metals. In an effort to characterize the Zur regulon in Anabaena, 23 genes containing upstream putative Zur-binding sequences were identified and found to be regulated by Zur. These genes are organized in six single transcriptional units and six operons, some of them containing multiple Zur-regulated promoters. The identities of genes of the Zur regulon indicate that Anabaena adapts to conditions of zinc deficiency by replacing zinc metalloproteins with paralogues that fulfill the same function but presumably with a lower zinc demand, and with inducing putative metallochaperones and membrane transport systems likely being involved in the scavenging of extracellular zinc, including plasma membrane ABC transport systems and outer membrane TonB-dependent receptors. Among the Zur-regulated genes, the ones showing the highest induction level encode proteins of the outer membrane, suggesting a primary role for components of this cell compartment in the capture of zinc cations from the extracellular medium.

ChREBP expression in the liver, adipose tissue and differentiated preadipocytes in human obesity.

ChREBP is an essential transcription factor for lipogenesis. Its physiological role in adipose tissue has been studied only to a small extent and the control of its expression remains unknown in human adipocytes. We have studied ChREBP mRNA and protein expression levels in the liver and the omental (OM) and subcutaneous (SC) adipose tissues from obese and lean subjects, as well as in human differentiated preadipocytes. Liver and OM and SC adipose tissue biopsies were obtained from lean and obese patients. Human preadipocytes were isolated from the adipose tissues from obese patients and differentiated under adipogenic conditions. ChREBP expression levels were quantified by RT-PCR and Western blot analysis. We found opposing results in terms of ChREBP regulation in the liver and adipose samples. ChREBP increased in the liver from obese compared to lean subjects, whereas the expression decreased in both adipose tissues. The mRNAs of other adipogenic markers were checked in these tissues. The pattern of FASN was similar to the one for ChREBP, ADCY3 decreased in both adipose tissues from obese patients, AP2 decreased only in OM adipose tissue of obese patients and ATGL did not change. The levels of ChREBP mRNA and protein showed dramatic increases during the differentiation of human OM and SC preadipocytes. In conclusion, ChREBP expression has an opposite regulation in the liver and adipose tissue from obese subjects which is compatible with the increased hepatic lipogenesis and decreased adipocytic lipogenesis found in these patients. The dramatic increase of ChREBP mRNA and protein levels during preadipocyte differentiation suggests a role in adipogenesis.

Ophthalmological Manifestations of Hereditary Myopathies.

BACKGROUND: Myopathies are neuromuscular disorders of the skeletal muscles, in which the main symptom is muscle weakness due to muscle fiber dysfunction. Myopathies may be classified into two main categories: inherited and acquired. Hereditary myopathies are a heterogeneous group of diseases that include congenital myopathies, mitochondrial myopathies, myotonic syndromes, muscular dystrophies, and other myopathies. PURPOSE: The objective of this paper is to review the ophthalmological findings and genetic patterns of hereditary myopathies. METHODS: This review is based on articles obtained by a relevant search of the PubMed database. CONCLUSION: Ophthalmoplegia with or without ptosis and pupil sparing appeared to be the most frequent ophthalmological manifestation of myopathies. The identification of the main ophthalmological features can help in the diagnosis and treatment of these muscular diseases.

Recommendations for the diagnosis and treatment of hypoglycaemia after bariatric surgery.

Postprandial hyperinsulinaemic hypoglycaemia is a common complication of bariatric surgery. Although in general its evolution is mild and self-limited, it can lead to neuroglycopaenia and compromise the patient's safety and quality of life. The aim of this document is to offer some recommendations to facilitate the clinical care of these complex patients, reviewing the aetiopathogenesis, its diagnosis and treatment that, sequentially, will include dietary and pharmacological measures and surgery in refractory cases. In the absence of high-quality studies, the diagnostic and therapeutic approach proposed is based on the consensus of experts of the Grupo de Obesidad de la Sociedad Española de Endocrinología y Nutrición [Obesity Group of the Spanish Society of Endocrinology and Nutrition], GOSEEN. Those undergoing bariatric surgery should be informed of the possibility of developing this complication.

The Relationship between Serum Adipokines, miR-222-3p, miR-103a-3p and Glucose Regulation in Pregnancy and Two to Three Years Post-Delivery in Women with Gestational Diabetes Mellitus Adhering to Mediterranean Diet Recommendations.

The San Carlos Gestational Diabetes Mellitus (GDM) prevention study, a nutritional intervention RCT based on a Mediterranean Diet (MedDiet), has been shown to reduce the incidence of GDM. The objective of this study is to investigate the relationship of leptin, adiponectin, interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α), insulin and HOMA-IRand circulating miRNAs (miR-29a-3p, miR-103a-3p, miR-132-3p, miR-222-3p) with the appearance of GDM and with MedDiet-based nutritional intervention, at 24−28 gestational weeks (GW), and in glucose regulation 2−3 years post-delivery (PD). A total of 313 pregnant women, 77 with GDM vs. 236 with normal glucose tolerance (NGT), 141 from the control group (CG, MedDiet restricting the consumption of dietary fat including EVOO and nuts during pregnancy) vs. 172 from the intervention group (IG, MedDiet supplemented with extra virgin olive oil (EVOO) and pistachios during pregnancy) were compared at Visit 1 (8−12 GW), Visit 2 (24−28 GW) and Visit 3 (2−3 years PD). Expression of miRNAs was determined by the Exiqon miRCURY LNA RT-PCR system. Leptin, adiponectin, IL-6 and TNF-α, were measured by Milliplex® immunoassays on Luminex 200 and insulin by RIA. Women with GDM vs. NTG had significantly higher leptin median (Q1−Q3) levels (14.6 (9.2−19.4) vs. 9.6 (6.0−15.1) ng/mL; p < 0.05) and insulin levels (11.4 (8.6−16.5) vs. 9.4 (7.0−12.8) µUI/mL; p < 0.001) and lower adiponectin (12.9 (9.8−17.2) vs. 17.0 (13.3−22.4) µg/mL; p < 0.001) at Visit 2. These findings persisted in Visit 3, with overexpression of miR-222-3p (1.45 (0.76−2.21) vs. 0.99 (0.21−1.70); p < 0.05)) and higher levels of Il-6 and TNF-α. When the IG is compared with the CG lower levels of insulin, HOMA-IR-IR, IL-6 levels at Visit 2 and 3 and leptin levels only at Visit 2 were observed. An overexpression of miR-222-3p and miR-103a-3p were also observed in IG at Visit 2 and 3. The miR-222-3p and miR103a-3p expression correlated with insulin levels, HOMA-IR, IL-6 and TNF-α at Visit 2 (all p < 0.05). These data support the association of leptin, adiponectin and insulin/HOMA-IR with GDM, as well as the association of insulin/HOMA-IR and IL-6 and miR-222-3p and miR-103a-3p expression with a MedDiet-based nutritional intervention.

An Integrative Analysis of DNA Methylation Pattern in Myotonic Dystrophy Type 1 Samples Reveals a Distinct DNA Methylation Profile between Tissues and a Novel Muscle-Associated Epigenetic Dysregulation.

Myotonic dystrophy type 1 (DM1) is a progressive, non-treatable, multi-systemic disorder. To investigate the contribution of epigenetics to the complexity of DM1, we compared DNA methylation profiles of four annotated CpG islands (CpGis) in the DMPK locus and neighbouring genes, in distinct DM1 tissues and derived cells, representing six DM1 subtypes, by bisulphite sequencing. In blood, we found no differences in CpGi 74, 43 and 36 in DNA methylation profile. In contrast, a CTCF1 DNA methylation gradient was found with 100% methylation in congenital cases, 50% in childhood cases and 13% in juvenile cases. CTCF1 methylation correlated to disease severity and CTG expansion size. Notably, 50% of CTCF1 methylated cases showed methylation in the CTCF2 regions. Additionally, methylation was associated with maternal transmission. Interestingly, the evaluation of seven families showed that unmethylated mothers passed on an expansion of the CTG repeat, whereas the methylated mothers transmitted a contraction. The analysis of patient-derived cells showed that DNA methylation profiles were highly preserved, validating their use as faithful DM1 cellular models. Importantly, the comparison of DNA methylation levels of distinct DM1 tissues revealed a novel muscle-specific epigenetic signature with methylation of the CTCF1 region accompanied by demethylation of CpGi 43, a region containing an alternative DMPK promoter, which may decrease the canonical promoter activity. Altogether, our results showed a distinct DNA methylation profile across DM1 tissues and uncovered a novel and dual epigenetic signature in DM1 muscle samples, providing novel insights into the epigenetic changes associated with DM1.

Obesity in Patients with Type 1 Diabetes: Links, Risks and Management Challenges.

Obesity affects large numbers of patients with type 1 diabetes (T1D) across their lifetime, with rates ranging between 2.8% and 37.1%. Patients with T1D and obesity are characterized by the presence of insulin resistance, of high insulin requirements, have a greater cardiometabolic risk and an enhanced risk of developing chronic complications when compared to normal-weight persons with T1D. Dual treatment of obesity and T1D is challenging and no specific guidelines for improving outcomes of both glycemic control and weight management have been established for this population. Nevertheless, although evidence is scarce, a comprehensive approach based on a balanced hypocaloric diet, physical activity and cognitive behavioral therapy by a multidisciplinary team, expert in both obesity and diabetes, remains as the best clinical practice. However, weight loss responses with lifestyle changes alone are limited, so in the "roadmap" of the treatment of obesity in T1D, it will be helpful to include anti-obesity pharmacotherapy despite at present there is a lack of evidence since T1D patients have been excluded from anti-obesity drug clinical trials. In case of severe obesity, bariatric surgery has proven to be of benefit in obtaining a substantial and long-term weight loss and reduction in cardiovascular risk. The near future looks promising with the development of new and more effective anti-obesity treatments and strategies to improve insulin resistance and oxidative stress. Advances in precision medicine may help individualize and optimize the medical management and care of these patients. This review, by gathering current evidence, highlights the need of solid knowledge in all facets of the treatment of patients with obesity and T1D that can only be obtained through high quality well-designed studies.

Quantification of Cover Test Prior and Post Pyridostigmine in Diagnosis of Myasthenia Gravis.

Objective: To assess the value of measuring diplopia before and after pyridostigmine intake to differentiate myasthenia gravis from sagging eye syndrome.To establish a threshold for a positive response to pyridostigmine in the diagnosis of myasthenia gravis.Methods: 15 patients with myasthenia gravis and 15 with sagging eye syndrome diplopia were evaluated. Diplopia was measured in five positions (upgaze, downgaze, right gaze, left gaze, and primary position). After baseline measurements, the patient received a single dose (60 mg) of pyridostigmine. After 60 minutes the prism measure was performed again in five positions. Horizontal deviation, vertical deviation at distance was compared before and after a single dose of pyridostigmine in each gaze. Ocular deviations were compared between the two groups to identify the threshold with the highest sensitivity and specificity.Results: Differences between pretest deviations and posttest deviations in any gaze were found to be statistically significant only in the MG group. The optimum threshold for a positive response to pyridostigmine was a reduction of 2 prism diopters in any component in any gaze. Sensitivity for the detection of myasthenia diplopia was 80.00% and specificity was 86.67%.Conclusions: Our results suggest that measuring diplopia with prisms before and after pyridostigmine administration can help to detect patients with suspected myasthenia.