Mammalian neurotoxins, Blarina paralytic peptides, cause hyperpolarization of human T-type Ca channel hCav3.2 activation.

Among the rare venomous mammals, the short-tailed shrew Blarina brevicauda has been suggested to produce potent neurotoxins in its saliva to effectively capture prey. Several kallikrein-like lethal proteases have been identified, but the active substances of B. brevicauda remained unclear. Here, we report Blarina paralytic peptides (BPPs) 1 and 2 isolated from its submaxillary glands. Synthetic BPP2 showed mealworm paralysis and a hyperpolarization shift (-11 mV) of a human T-type Ca2+ channel (hCav3.2) activation. The amino acid sequences of BPPs were similar to those of synenkephalins, which are precursors of brain opioid peptide hormones that are highly conserved among mammals. However, BPPs rather resembled centipede neurotoxic peptides SLPTXs in terms of disulfide bond connectivity and stereostructure. Our results suggested that the neurotoxin BPPs were the result of convergent evolution as homologs of nontoxic endogenous peptides that are widely conserved in mammals. This finding is of great interest from the viewpoint of the chemical evolution of vertebrate venoms.

ALPL-1 is a target for chimeric antigen receptor therapy in osteosarcoma.

Osteosarcoma (OS) remains a dismal malignancy in children and young adults, with poor outcome for metastatic and recurrent disease. Immunotherapies in OS are not as promising as in some other cancer types due to intra-tumor heterogeneity and considerable off-target expression of the potentially targetable proteins. Here we show that chimeric antigen receptor (CAR) T cells could successfully target an isoform of alkaline phosphatase, ALPL-1, which is highly and specifically expressed in primary and metastatic OS. The target recognition element of the second-generation CAR construct is based on two antibodies, previously shown to react against OS. T cells transduced with these CAR constructs mediate efficient and effective cytotoxicity against ALPL-positive cells in in vitro settings and in state-of-the-art in vivo orthotopic models of primary and metastatic OS, without unexpected toxicities against hematopoietic stem cells or healthy tissues. In summary, CAR-T cells targeting ALPL-1 show efficiency and specificity in treating OS in preclinical models, paving the path for clinical translation.

Conformational Change of the Hairpin-like-structured Robo2 Ectodomain Allows NELL1/2 Binding.

Since neural epidermal growth factor-like-like (NELL) 2 was identified as a novel ligand for the roundabout (Robo) 3 receptor, research on NELL-Robo signaling has become increasingly important. We have previously reported that Robo2 can bind to NELL1/2 in acidic conditions but not at neutral pH. The NELL1/2-binding site that is occluded in neutral conditions is thought to be exposed by a conformational change of the Robo2 ectodomain upon exposure to acidic pH; however, the underlying structural mechanisms are not well understood. Here, we investigated the interaction between the immunoglobulin-like domains and fibronectin type III domains that form hairpin-like structure of the Robo2 ectodomain, and demonstrated that acidic pH attenuates the interaction between them. Alternative splicing isoforms of Robo2, which affect the conformation of the hairpin-like structure, were found to have distinct NELL1/2-binding affinities. We developed Förster resonance energy transfer-based indicators for monitoring conformational change of the Robo2 ectodomain by individually inserting donor and acceptor fluorescent proteins at its ends. These experiments revealed that the ends of the Robo2 ectodomain are close to each other in acidic conditions. By combining these findings with the results of size exclusion chromatography analysis, we suggest that, in acidic conditions, the Robo2 ectodomain has a compact conformation with a loose hairpin-like structure. These results may help elucidate the signaling mechanisms resulting from the interaction between Robo2 and NELL1/2 in acidic conditions.

Cryo-EM structures of thylakoid-located voltage-dependent chloride channel VCCN1.

In the light reaction of plant photosynthesis, modulation of electron transport chain reactions is important to maintain the efficiency of photosynthesis under a broad range of light intensities. VCCN1 was recently identified as a voltage-gated chloride channel residing in the thylakoid membrane, where it plays a key role in photoreaction tuning to avoid the generation of reactive oxygen species (ROS). Here, we present the cryo-EM structures of Malus domestica VCCN1 (MdVCCN1) in nanodiscs and detergent at 2.7 Å and 3.0 Å resolutions, respectively, and the structure-based electrophysiological analyses. VCCN1 structurally resembles its animal homolog, bestrophin, a Ca2+-gated anion channel. However, unlike bestrophin channels, VCCN1 lacks the Ca2+-binding motif but instead contains an N-terminal charged helix that is anchored to the lipid membrane through an additional amphipathic helix. Electrophysiological experiments demonstrate that these structural elements are essential for the channel activity, thus revealing the distinct activation mechanism of VCCN1.

Predictive Validity of Developmental Screening Questionnaires for Identifying Children With Later Cognitive or Educational Difficulties: A Systematic Review.

Context: Parent/caregiver completing developmental screening questionnaires (DSQs) for children before 5 years of age is currently recommended. The DSQs recommended by the American Academy of Pediatrics (AAP) are the Ages and Stages Questionnaires (ASQ), Parents' Evaluation of Developmental Status (PEDS), and the Survey of Well-being of Young Children (SWYC). Nevertheless, their predictive validity has not been well-established. Objective: To assess in the current literature, the value of AAP-recommended DSQs (ASQ, PEDS, SWYC) administered between 0 and 5 years of age, for predicting long-term cognitive achievement and/or school performance (CA/SP), after 1 year or more of evaluation and at/or after age 5 years, in the general population. Data Sources: Cochrane, MEDLINE PubMed, CINAHL, EMBASE, Web of Science, Scielo, and Scopus databases (until March 2021). Study Selection: Two authors selected the studies. Forward and backward citation follow-up was done; authors of DSQ were contacted to identify additional studies. Data Extraction: Cohorts were identified, and authors of selected studies were contacted to corroborate and complete extracted data. Results: Thirty-two publications, corresponding to 10 cohorts, were included. All cohorts used ASQ. Only cohort using PEDS was identified but did not meet the inclusion criteria. No cohorts conducted with SWYC were identified. Associations between ASQ and CA/SP were extracted for eight cohorts. The odds ratios were >3, and the area under the curve was 0.66-0.87. A trade-off between sensitivity and specificity was observed. Limitations: Heterogeneity in population characteristics and in DSQ adaptations. Conclusions: A positive association between ASQ and later CA/SP was found in different social, cultural, and economic settings. Additional studies are necessary to determine the impact factors in the predictive capacity of DSQs. Systematic Review Registration: PROSPERO, identifier: CRD42020183883.

[Professionalism and self-care in residents, how should we confront their training?] / Profesionalismo y auto cuidado de los residentes, ¿cómo debemos enfocar su formación?

Professionalism is a well-defined competence in the education of residents. However, it is a complex construct, sensitive to social and cultural variables. It can be defined as the necessary skill, good jud gment and appropriate behavior expected of people trained to do their jobs well. It is a competence that does not remain stable over time and declines when the professional is subjected to high levels of stress, associated with quality of care, education, ethics, moral, philosophy and humanism. It is an essential competence for the professional and therefore we must rethink the curricula to include ways to teach and evaluate professionalism. It is essential to design programs that balance the workload with the well-being of future professionals. We must generate an adequate learning environment where the trainee is an active protagonist, and self-care is made visible as an essential competence to maintain the balance between personal and professional life. This article presents a review and reflects on this topic which is becoming increasingly important in the postgraduate training of future specialists.

The structure of MgtE in the absence of magnesium provides new insights into channel gating.

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

Id2 Represses Aldosterone-Stimulated Cardiac T-Type Calcium Channels Expression.

Aldosterone excess is a cardiovascular risk factor. Aldosterone can directly stimulate an electrical remodeling of cardiomyocytes leading to cardiac arrhythmia and hypertrophy. L-type and T-type voltage-gated calcium (Ca2+) channels expression are increased by aldosterone in cardiomyocytes. To further understand the regulation of these channels expression, we studied the role of a transcriptional repressor, the inhibitor of differentiation/DNA binding protein 2 (Id2). We found that aldosterone inhibited the expression of Id2 in neonatal rat cardiomyocytes and in the heart of adult mice. When Id2 was overexpressed in cardiomyocytes, we observed a reduction in the spontaneous action potentials rate and an arrest in aldosterone-stimulated rate increase. Accordingly, Id2 siRNA knockdown increased this rate. We also observed that CaV1.2 (L-type Ca2+ channel) or CaV3.1, and CaV3.2 (T-type Ca2+ channels) mRNA expression levels and Ca2+ currents were affected by Id2 presence. These observations were further corroborated in a heart specific Id2- transgenic mice. Taken together, our results suggest that Id2 functions as a transcriptional repressor for L- and T-type Ca2+ channels, particularly CaV3.1, in cardiomyocytes and its expression is controlled by aldosterone. We propose that Id2 might contributes to a protective mechanism in cardiomyocytes preventing the presence of channels associated with a pathological state.

Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin.

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.

Interactive Guidance Intervention to Address Sustained Social Withdrawal in Preterm Infants in Chile: Protocol for a Randomized Controlled Trial.

BACKGROUND: Preterm newborns can be exposed early to significant perinatal stress, and this stress can increase the risk of altered socioemotional development. Sustained social withdrawal in infants is an early indicator of emotional distress which is expressed by low reactivity to the environment, and if persistent, is frequently associated with altered psychological development. Infants born prematurely have a higher probability of developing sustained social withdrawal (adjusted odds ratio 1.84, 95% CI 1.04-3.26) than infants born full term, and there is a correlation between weight at birth and sustained social withdrawal at 12 months of age. OBJECTIVE: The aims of this study are to compare the effect of the interactive guidance intervention to that of routine pediatric care on sustained social withdrawal in infants born moderately or late preterm and to explore the relationship between sustained social withdrawal in these infants and factors such as neonatal intensive care unit hospitalization variables, parental depression, and posttraumatic stress symptoms. METHODS: This study is designed as a multicenter randomized controlled trial. Moderate and late preterm newborns and their parents were recruited and randomized (1:1 allocation ratio) to control and experimental groups. During neonatal intensive care unit hospitalization, daily duration of skin-to-skin contact, breastfeeding, and parental visits were recorded. Also, a daily score for neonatal pain and painful invasive procedures were recorded. After discharge from neonatal intensive care, for the duration of the study, both groups will attend follow-up consultations with neonatologists at 2, 6, and 12 months of age (corrected for gestational age) and will receive routine pediatric care. Every consultation will be recorded and assessed with the Alarm Distress Baby Scale to detect sustained social withdrawal (indicated by a score of 5 or higher). The neonatologists will perform an interactive guidance intervention if an infant in the intervention group exhibits sustained social withdrawal. In each follow-up consultation, parents will fill out the Edinburgh Postnatal Depression Scale, the modified Perinatal Posttraumatic Stress Disorder Questionnaire, and the Impact of Event Scale-revised. RESULTS: Recruitment for this trial started in September 2017. As of May 2020, we have completed enrollment (N=110 infants born moderately or late preterm). We aim to publish the results by mid-2021. CONCLUSIONS: This is the first randomized controlled trial with a sample of infants born moderately or late preterm infants who will attend pediatric follow-up consultations during their first year (corrected for gestational age at birth) with neonatologists trained in the Alarm Distress Baby Scale and who will receive this interactive guidance intervention. If successful, this early intervention will show significant potential to be implemented in both public and private health care, given its low cost of training staff and that the intervention takes place during routine pediatric follow-up. TRIAL REGISTRATION: ClinicalTrials.gov NCT03212547; https://clinicaltrials.gov/ct2/show/NCT03212547. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/17943.

Association of Cow's Milk Protein Allergy Prevalence With Socioeconomic Status in a Cohort of Chilean Infants.

OBJECTIVES: The aim of the study was to compare the cow's milk protein allergy (CMPA) prevalence in 2 cohorts of children from different socioeconomic strata. METHODS: Prospective birth cohort that included patients from 2 hospitals providing care for a low- and high-income population, respectively. Healthy newborns ≥34 gestational weeks were recruited and followed up to 12 months by a monthly telephone survey. If ≥2 predefined symptoms/signs suggestive of CMPA were detected, the patient was evaluated by a pediatric gastroenterologist. Diagnosis was confirmed by exclusion diet followed by open oral food challenge. RESULTS: Overall the prevalence of CMPA was 5.2%, with a 6 times higher prevalence in the high income cohort (9.2%) compared with the low-income group (1.5%; relative risk 6.2; 95% confidence interval 1.8-20.7; P = 0.0005). All the cases were non-immunoglobulin E-mediated with predominantly gastrointestinal symptoms. High-income cohort did have higher frequency of C-section, mother's previous chronic disease, mother's history of atopy/food allergy, older age, and higher educational level of parents. Parent smoking and presence of pets at home were more frequent in the low-income cohort. Multiple logistic regression showed that the high-income cohort did have older age and higher educational level of both parents. CONCLUSION: In these cohorts the prevalence of CMPA was higher than reported previously in other developing countries and significantly higher in the high-income group. Our findings were associated with sociodemographic characteristics of the parents.

Systematic Reviews in Neonatal Respiratory Care: Are Some Conclusions Misleading?

An increasing amount of information is currently available in neonatal respiratory care. Systematic reviews are an important tool for clinical decision-making. The challenge is to combine studies that address a specific clinical question and have similar characteristics in terms of populations, interventions, comparators, and outcomes, so that their combined results provide a more precise estimate of the effect that can be validly extrapolated into clinical practice. The concept of heterogeneity is reviewed, emphasizing that it should be considered in a wider perspective and not just as a mere statistical test. A case is made of how well-designed studies of the neonatal respiratory literature, when equivocally combined, can provide very precise but potentially biased results. Systematic reviews in this field and others should be rigorously peer-reviewed before publication to avoid misleading readers to potentially biased conclusions.

Author Correction: Structural basis for the drug extrusion mechanism by a MATE multidrug transporter.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Comparison between Ages & Stages Questionnaire and Bayley Scales, to predict cognitive delay in school age.

OBJECTIVE: To compare the predictive value of the Spanish Ages & Stages Questionnaire third edition adapted for Chilean population (ASQ-Cl) and the Bayley Scale of Infant and Toddler Development 3rd edition (Bayley-III) for cognitive delay at school age, and to identify the domain predictors. METHODOLOGY: Data were collected from 306 term and preterm children of medium-high socio-economic level enrolled in a prospective cohort study. Developmental outcomes at 8, 18 and 30 months were assessed via the ASQ-Cl and Bayley-III; at 6-8 years cognitive development was assessed using the Wechsler Intelligence Scale for Children (WISC-III). The area under the curve (AUC), sensitivity, specificity and predictive values were calculated, and logistic regression analysis was used. RESULTS: Of 227 children studied, 6.6% had cognitive delay. ASQ-Cl and Bayley-III generate equivalent AUC [0.77 and 0.80]. Sensitivity 67% and 53%; specificity of 72% and 88%, positive predictive value of 14% and 24%, negative predictive values of 97% and 96% respectively. Greater predictive validity was obtained at 30 months assessment. Deficit in the communication and gross motor skills and problem-solving domains of the ASQ-Cl and all the Bayley-III domains were significantly associated with cognitive delay. CONCLUSIONS: ASQ-Cl can be used to identify children at risk for cognitive delay at 6-8 years of age, being comparable with the Bayley-III. Some domains of ASQ-Cl and all domains of Bayley-III were significant predictors for cognitive delay. These results support the use of ASQ-Cl as a screening tool for developmental delay.

RBM20 Regulates CaV1.2 Surface Expression by Promoting Exon 9* Inclusion of CACNA1C in Neonatal Rat Cardiomyocytes.

The CACNA1C gene encodes for the CaV1.2 protein, which is the pore subunit of cardiac l-type voltage-gated calcium (Ca2+) channels (l-channels). Through alternative splicing, CACNA1C encodes for various CaV1.2 isoforms with different electrophysiological properties. Splice variants of CaV1.2 are differentially expressed during heart development or pathologies. The molecular mechanisms of CACNA1C alternative splicing still remain incompletely understood. RNA sequencing analysis has suggested that CACNA1C is a potential target of the splicing factor RNA-binding protein motif 20 (RBM20). Here, we aimed at elucidating the role of RBM20 in the regulation of CACNA1C alternative splicing. We found that in neonatal rat cardiomyocytes (NRCMs), RBM20 overexpression promoted the inclusion of CACNA1C's exon 9*, whereas the skipping of exon 9* occurred upon RBM20 siRNA knockdown. The splicing of other known alternative exons was not altered by RBM20. RNA immunoprecipitation suggested that RBM20 binds to introns flanking exon 9*. Functionally, in NRCMs, RBM20 overexpression decreased l-type Ca2+ currents, whereas RBM20 siRNA knockdown increased l-type Ca2+ currents. Finally, we found that RBM20 overexpression reduced CaV1.2 membrane surface expression in NRCMs. Taken together, our results suggest that RBM20 specifically regulates the inclusion of exon 9* in CACNA1C mRNA, resulting in reduced cell-surface membrane expression of l-channels in cardiomyocytes.

Robo2 contains a cryptic binding site for neural EGFL-like (NELL) protein 1/2.

The signaling pathways that are mediated by Slit ligands and their Roundabout (Robo) family of receptors play multifunctional roles in the development of the nervous system and other organs. A recent study identified neural epidermal growth factor-like (NEL)-like 2 (NELL2) as a novel ligand for Robo3. In this study, we carried out a comprehensive analysis of the interaction between NELL1 and the Robo family of receptors and demonstrated that Robo2 contains a cryptic binding site for both NELL1 and NELL2. NELL1/2 binds to the first fibronectin type III (FNIII) domain of Robo2 but not to intact Robo2. Mutation analysis revealed that several amino acids within the first FNIII domain are critical for NELL1 binding to Robo2 but not to Robo1. The Robo2 deletion mutants without the fourth immunoglobulin domain and single amino acid substitution mutants that can influence the architecture of the ectodomain facilitated binding to NELL1/2. Acidic conditions increased the binding affinity of Robo2 for NELL1. These results suggest that Robo2 functions as a receptor for NELL1/2, particularly under circumstances where Robo2 undergoes proteolytic digestion. If this is not the case, conformational changes of the ectodomain of Robo2 may unmask the binding site for NELL1/2.

Structural Basis of H+-Dependent Conformational Change in a Bacterial MATE Transporter.

Multidrug and toxic compound extrusion (MATE) transporters efflux toxic compounds using a Na+ or H+ gradient across the membrane. Although the structures of MATE transporters have been reported, the cation-coupled substrate transport mechanism remains controversial. Here we report crystal structures of VcmN, a Vibrio cholerae MATE transporter driven by the H+ gradient. High-resolution structures in two distinct conformations associated with different pHs revealed that the rearrangement of the hydrogen-bonding network around the conserved Asp35 induces the bending of transmembrane helix 1, as in the case of the H+-coupled Pyrococcus furiosus MATE transporter. We also determined the crystal structure of the D35N mutant, which captured a unique conformation of TM1 facilitated by an altered hydrogen-bonding network. Based on the present results, we propose a common step in the transport cycle shared among prokaryotic H+-coupled MATE transporters.

MicroRNA-204 Is Necessary for Aldosterone-Stimulated T-Type Calcium Channel Expression in Cardiomyocytes.

Activation of the mineralocorticoid receptor (MR) in the heart is considered to be a cardiovascular risk factor. MR activation leads to heart hypertrophy and arrhythmia. In ventricular cardiomyocytes, aldosterone induces a profound remodeling of ion channel expression, in particular, an increase in the expression and activity of T-type voltage-gated calcium channels (T-channels). The molecular mechanisms immediately downstream from MR activation, which lead to the increased expression of T-channels and, consecutively, to an acceleration of spontaneous cell contractions in vitro, remain poorly investigated. Here, we investigated the putative role of a specific microRNA in linking MR activation to the regulation of T-channel expression and cardiomyocyte beating frequency. A screening assay identified microRNA 204 (miR-204) as one of the major upregulated microRNAs after aldosterone stimulation of isolated neonatal rat cardiomyocytes. Aldosterone significantly increased the level of miR-204, an effect blocked by the MR antagonist spironolactone. When miR-204 was overexpressed in isolated cardiomyocytes, their spontaneous beating frequency was significantly increased after 24 h, like upon aldosterone stimulation, and messenger RNAs coding T-channels (CaV3.1 and CaV3.2) were increased. Concomitantly, T-type calcium currents were significantly increased upon miR-204 overexpression. Specifically repressing the expression of miR-204 abolished the aldosterone-induced increase of CaV3.1 and CaV3.2 mRNAs, as well as T-type calcium currents. Finally, aldosterone and miR-204 overexpression were found to reduce REST-NRSF, a known transcriptional repressor of CaV3.2 T-type calcium channels. Our study thus strongly suggests that miR-204 expression stimulated by aldosterone promotes the expression of T-channels in isolated rat ventricular cardiomyocytes, and therefore, increases the frequency of the cell spontaneous contractions, presumably through the inhibition of REST-NRSF protein.