Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex.

Cortical circuits are composed predominantly of pyramidal-to-pyramidal neuron connections, yet their assembly during embryonic development is not well understood. We show that mouse embryonic Rbp4-Cre cortical neurons, transcriptomically closest to layer 5 pyramidal neurons, display two phases of circuit assembly in vivo. At E14.5, they form a multi-layered circuit motif, composed of only embryonic near-projecting-type neurons. By E17.5, this transitions to a second motif involving all three embryonic types, analogous to the three adult layer 5 types. In vivo patch clamp recordings and two-photon calcium imaging of embryonic Rbp4-Cre neurons reveal active somas and neurites, tetrodotoxin-sensitive voltage-gated conductances, and functional glutamatergic synapses, from E14.5 onwards. Embryonic Rbp4-Cre neurons strongly express autism-associated genes and perturbing these genes interferes with the switch between the two motifs. Hence, pyramidal neurons form active, transient, multi-layered pyramidal-to-pyramidal circuits at the inception of neocortex, and studying these circuits could yield insights into the etiology of autism.

Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.

A tribute to Sebastián Cerdán and his key contributions to brain metabolism.

This is a tribute to Sebastián Cerdán, a brilliant and innovative NMR spectroscopist whose studies contributed greatly to the fundamental information to the understanding of brain metabolism, particularly in regard to multinuclear magnetic resonance spectroscopy (MRS) techniques. Sebastián Cerdán sadly passed away in May 2022. He was a wonderful mentor and colleague who will be greatly missed.

Segmentation of X-ray coronary angiography with an artificial intelligence deep learning model: Impact in operator visual assessment of coronary stenosis severity.

BACKGROUND: Visual assessment of the percentage diameter stenosis (%DSVE ) of lesions is essential in coronary angiography (CAG) interpretation. We have previously developed an artificial intelligence (AI) model capable of accurate CAG segmentation. We aim to compare operators' %DSVE in angiography versus AI-segmented images. METHODS: Quantitative coronary analysis (QCA) %DS (%DSQCA ) was previously performed in our published validation dataset. Operators were asked to estimate %DSVE of lesions in angiography versus AI-segmented images in separate sessions and differences were assessed using angiography %DSQCA as reference. RESULTS: A total of 123 lesions were included. %DSVE was significantly higher in both the angiography (77% ± 20% vs. 56% ± 13%, p < 0.001) and segmentation groups (59% ± 20% vs. 56% ± 13%, p < 0.001), with a much smaller absolute %DS difference in the latter. For lesions with %DSQCA of 50%-70% (60% ± 5%), an even higher discrepancy was found (angiography: 83% ± 13% vs. 60% ± 5%, p < 0.001; segmentation: 63% ± 15% vs. 60% ± 5%, p < 0.001). Similar, less pronounced, findings were observed for %DSQCA < 50% lesions, but not %DSQCA > 70% lesions. Agreement between %DSQCA /%DSVE across %DSQCA strata (<50%, 50%-70%, >70%) was approximately twice in the segmentation group (60.4% vs. 30.1%; p < 0.001). %DSVE inter-operator differences were smaller with segmentation. CONCLUSION: %DSVE was much less discrepant with segmentation versus angiography. Overestimation of %DSQCA < 70% lesions with angiography was especially common. Segmentation may reduce %DSVE overestimation and thus unwarranted revascularization.

State-of-the-Art Molecular Oncology in Brazil.

Over the past few decades, the life expectancy of humankind has increased significantly due to advancements in life sciences and medical research, particularly given our increasing success in the epidemiological and pharmacological management of bacterial, fungi, and viral infections [...].

Merging the Isonitrile-Tetrazine (4+1) Cycloaddition and the Ugi Four-Component Reaction into a Single Multicomponent Process.

Multicomponent reactions are of utmost importance at generating a unique, wide, and complex chemical space. Herein we describe a novel multicomponent approach based on the combination of the isonitrile-tetrazine (4+1) cycloaddition and the Ugi four-component reaction to generate pyrazole amide derivatives. The scope of the reaction as well as mechanistic insights governing the 4H-pyrazol-4-imine tautomerization are provided. This multicomponent process provides access to a new chemical space of pyrazole amide derivatives and offers a tool for peptide modification and stapling.

Proliferation and Invasion of Melanoma Are Suppressed by a Plant Protease Inhibitor, Leading to Downregulation of Survival/Death-Related Proteins.

Cell adhesion and migration are crucial for cancer progression and malignancy. Drugs available for the treatment of metastatic melanoma are expensive and unfit for certain patients. Therefore, there is still a need to identify new drugs that block tumor cell development. We investigated the effects of Enterolobium contortisiliquum trypsin inhibitor (EcTI), a protease inhibitor, on cell viability, cell migration, invasion, cell adhesion, and cell death (hallmarks of cancer) in vitro using human melanoma cells (SK-MEL-28 and CHL-1). Although EcTI did not affect non-tumor cells, it significantly inhibited the proliferation, migration, invasion, and adhesion of melanoma cells. Investigation of the underlying mechanisms revealed that EcTI triggered apoptosis and nuclear shrinkage, increased PI uptake, activated effector caspases-3/7, and produced reactive oxygen species (ROS). Furthermore, EcTI disrupted the mitochondrial membrane potential, altered calcium homeostasis, and modified proteins associated with survival and apoptosis/autophagy regulation. Acridine orange staining indicated acidic vesicular organelle formation upon EcTI treatment, demonstrating a cell death display. Electronic microscopy corroborated the apoptotic pattern by allowing the visualization of apoptotic bodies, mitochondrial cristae disorganization, and autophagic vesicles. Taken together, these results provide new insights into the anti-cancer properties of the natural EcTI protein, establishing it as a promising new therapeutic drug for use in melanoma treatment.

Chemoproteomics-Enabled Identification of 4-Oxo-ß-Lactams as Inhibitors of Dipeptidyl Peptidases 8 and 9.

Dipeptidyl peptidases 8 and 9 (DPP8/9) have gathered interest as drug targets due to their important roles in biological processes like immunity and tumorigenesis. Elucidation of their distinct individual functions remains an ongoing task and could benefit from the availability of novel, chemically diverse and selective chemical tools. Here, we report the activity-based protein profiling (ABPP)-mediated discovery of 4-oxo-ß-lactams as potent, non-substrate-like nanomolar DPP8/9 inhibitors. X-ray crystallographic structures revealed different ligand binding modes for DPP8 and DPP9, including an unprecedented targeting of an extended S2' (eS2') subsite in DPP8. Biological assays confirmed inhibition at both target and cellular levels. Altogether, our integrated chemical proteomics and structure-guided small molecule design approach led to novel DPP8/9 inhibitors with alternative molecular inhibition mechanisms, delivering the highest selectivity index reported to date.

Visual Analogue Score for Urinary Symptoms-VASUS, validation of a visual scale for lower urinary tract symptoms (LUTS) in an African country.

PURPOSE: Validate a visual scale to assess LUTS, especially in developing countries, as an alternative to IPSS. VASUS consist of five questions, where Q1 and Q2 assess urinary stream quality, Q3 nocturia, Q4 incomplete emptying and Q5 QoL. METHODS: Between 2014 and 2017, we carried out a study in the male population over 30 years from São Tomé and Príncipe, a Portuguese speaking African Country. A stratified sample (age and district) of subjects completed IPSS, VASUS and a free flowmetry. RESULTS: We obtained 812 valid responses (average age: 50.72, range: 30-95 years old). In the comparison between IPSS and VASUS, we found positive correlations, with p value < 0.0001, for all variables analyzed and negative correlation for all urodynamic variables. Upon verifying the association of VASUS with IPSS, namely when comparing questions with similar objectives such as nocturia (VASUS-Q3 and IPSS-Q7), the stream quality (VASUS-Q1 and Q2 and IPSS-Q5) or the quality of life (VASUS-Q5 and IPSS-Q8), strong positive correlations were found. CONCLUSION: VASUS is a visual alternative to IPSS allowing evaluation of LUTS and having correlation with IPSS and flowmetry. Its use in developing countries with low levels of literacy will be an asset. The authors believe that widespread use of a scale such as VASUS in urology consultations is warranted, to increase daily practice objectification of LUTS.

Augmenting Adaptive Machine Learning with Kinetic Modeling for Reaction Optimization.

We combine random sampling and active machine learning (ML) to optimize the synthesis of isomacroin, executing only 3% of all possible Friedländer reactions. Employing kinetic modeling, we augment machine intuition by extracting mechanistic knowledge and verify that a global optimum was obtained with ML. Our study contributes evidence on the potential of multiscale approaches to expedite the access to chemical matter, further democratizing organic chemistry in a data-motivated fashion.

Cysteine proteases as potential targets for anti-trypanosomatid drug discovery.

Leishmaniasis and trypanosomiasis are endemic neglected disease in South America and Africa and considered a significant public health problem, mainly in poor communities. The limitations of the current available therapeutic options, including the lack of specificity, relatively high toxicity, and the drug resistance acquiring, drive the constant search for new targets and therapeutic options. Advances in knowledge of parasite biology have revealed essential enzymes involved in the replication, survival, and pathogenicity of Leishmania and Trypanosoma species. In this scenario, cysteine proteases have drawn the attention of researchers and they are being proposed as promising targets for drug discovery of antiprotozoal drugs. In this systematic review, we will provide an update on drug discovery strategies targeting the cysteine proteases as potential targets for chemotherapy against protozoal neglected diseases.

Clinical impact of MultiPoint pacing in responders to cardiac resynchronization therapy.

BACKGROUND: Cardiac resynchronization therapy demonstrated benefits in heart failure. However, only 60-70% are responders and only 22% are super-responders. MultiPoint pacing (MPP) improves structural remodeling, but data in responder patients is scarce. METHODS: A prospective, randomized study of the efficacy of MPP was conducted in patients who were CRT responders after 6 months of bi-ventricular (BiV) therapy. At 6 months, responder patients (LV end-systolic volume [LVESV] reduction ≥15%) were randomized to either continued BiV therapy or to MPP programmed with wide anatomical separation ≥30 mm, and followed until 12 months. Efficacy was determined by 6-12 month changes in LVESV and LV ejection fraction (LVEF). Evaluations of super-responder rate (LVESV reduction ≥30%) and quality of life (NYHA, EQ-5D, MLHFQ) were also performed. RESULTS: From February 2017 to February 2019, 73 CRTs with Quartet LV leads were implanted (42.9% female, 65.7 ± 10.8 years old, 79.5% dilated cardiomyopathy). At 6 months, 74.2% responded to BiV and were randomized to BiV (n = 25) or MPP (n = 24). MPP versus BiV delivered greater LVESV improvement (8.3% decrease in MPP vs. 10.3% increase in BiV patients, p = .047), greater increase in LVEF (7.7% vs. 1.8%, p = .008), and higher 0-12 month super-responder rate (86.4% vs. 56.0%, p = .027). More MPP vs. BiV patients experienced an improvement in NYHA (84.6% vs. 50.0%, p = .047) and EQ-5D (94.4% vs. 54.0%, p = .006). CONCLUSIONS: MPP with wide anatomical spacing in CRT responder patients resulted in improved LV reverse remodeling with higher rates of super-responders, and better quality of life metrics.

Pulmonary embolism and COVID-19: A comparative analysis of different diagnostic models performance.

OBJECTIVE: Pulmonary embolism (PE) is a common complication of SARS-CoV-2 infection. Several diagnostic prediction rules based on pretest probability and D-dimer have been validated in non-COVID patients, but it remains unclear if they can be safely applied in COVID-19 patients. We aimed to compare the diagnostic accuracy of the standard approach based on Wells and Geneva scores combined with a standard D-dimer cut-off of 500 ng/mL with three alternative strategies (age-adjusted, YEARS and PEGeD algorithms) in COVID-19 patients. METHODS: This retrospective study included all COVID-19 patients admitted to the Emergency Department (ED) who underwent computed tomography pulmonary angiography (CTPA) due to PE suspicion. The diagnostic prediction rules for PE were compared between patients with and without PE. RESULTS: We included 300 patients and PE was confirmed in 15%. No differences were found regarding comorbidities, traditional risk factors for PE and signs and symptoms between patients with and without PE. Wells and Geneva scores showed no predictive value for PE occurrence, whether a standard or an age-adjusted cut-off was considered. YEARS and PEGeD algorithms were associated with increased specificity (19% CTPA reduction) but raising non-diagnosed PE. Despite elevated in all patients, those with PE had higher D-dimer levels. However, incrementing thresholds to select patients for CTPA was also associated with a substantial decrease in sensitivity. CONCLUSION: None of the diagnostic prediction rules are reliable predictors of PE in COVID-19. Our data favour the use of a D-dimer threshold of 500 ng/mL, considering that higher thresholds increase specificity but limits this strategy as a screening test.

Indirect Estimation of Breathing Rate from Heart Rate Monitoring System during Running.

Recent advances in wearable technologies integrating multi-modal sensors have enabled the in-field monitoring of several physiological metrics. In sport applications, wearable devices have been widely used to improve performance while minimizing the risk of injuries and illness. The objective of this project is to estimate breathing rate (BR) from respiratory sinus arrhythmia (RSA) using heart rate (HR) recorded with a chest belt during physical activities, yielding additional physiological insight without the need of an additional sensor. Thirty-one healthy adults performed a run at increasing speed until exhaustion on an instrumented treadmill. RR intervals were measured using the Polar H10 HR monitoring system attached to a chest belt. A metabolic measurement system was used as a reference to evaluate the accuracy of the BR estimation. The evaluation of the algorithms consisted of exploring two pre-processing methods (band-pass filters and relative RR intervals transformation) with different instantaneous frequency tracking algorithms (short-term Fourier transform, single frequency tracking, harmonic frequency tracking and peak detection). The two most accurate BR estimations were achieved by combining band-pass filters with short-term Fourier transform, and relative RR intervals transformation with harmonic frequency tracking, showing 5.5% and 7.6% errors, respectively. These two methods were found to provide reasonably accurate BR estimation over a wide range of breathing frequency. Future challenges consist in applying/validating our approaches during in-field endurance running in the context of fatigue assessment.

The Quorum Sensing Auto-Inducer 2 (AI-2) Stimulates Nitrogen Fixation and Favors Ethanol Production over Biomass Accumulation in Zymomonas mobilis.

Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates expression of genes involved in a series of alternative mechanisms, when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, and production of public goods, or pathogenicity factors, among others). Contrary to most autoinducers, AI-2 can induce QS responses in both Gram-negative and Gram-positive bacteria, and has been suggested to constitute a trans-specific system of bacterial communication, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Zymomonas mobilis (a non-AI-2 producer) responds to exogenous AI-2 by modulating expression of genes involved in mechanisms typically associated with QS in other bacteria, such as motility, DNA repair, and nitrogen fixation. Interestingly, the metabolism of AI-2-induced Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high-energy demand of N2 fixation. This opens the possibility of employing AI-2 during the industrial production of second-generation ethanol, as a way to boost N2 fixation by these bacteria, which could reduce costs associated with the use of nitrogen-based fertilizers, without compromising ethanol production in industrial plants.

Impairment of adenosinergic system in Rett syndrome: Novel therapeutic target to boost BDNF signalling.

Rett syndrome (RTT; OMIM#312750) is mainly caused by mutations in the X-linked MECP2 gene (methyl-CpG-binding protein 2 gene; OMIM*300005), which leads to impairments in the brain-derived neurotrophic factor (BDNF) signalling. The boost of BDNF mediated effects would be a significant breakthrough but it has been hampered by the difficulty to administer BDNF to the central nervous system. Adenosine, an endogenous neuromodulator, may accomplish that role since through A2AR it potentiates BDNF synaptic actions in healthy animals. We thus characterized several hallmarks of the adenosinergic and BDNF signalling in RTT and explored whether A2AR activation could boost BDNF actions. For this study, the RTT animal model, the Mecp2 knockout (Mecp2-/y) (B6.129P2 (C)-Mecp2tm1.1Bird/J) mouse was used. Whenever possible, parallel data was also obtained from post-mortem brain samples from one RTT patient. Ex vivo extracellular recordings of field excitatory post-synaptic potentials in CA1 hippocampal area were performed to evaluate synaptic transmission and long-term potentiation (LTP). RT-PCR was used to assess mRNA levels and Western Blot or radioligand binding assays were performed to evaluate protein levels. Changes in cortical and hippocampal adenosine content were assessed by liquid chromatography with diode array detection (LC/DAD). Hippocampal ex vivo experiments revealed that the facilitatory actions of BDNF upon LTP is absent in Mecp2-/y mice and that TrkB full-length (TrkB-FL) receptor levels are significantly decreased. Extracts of the hippocampus and cortex of Mecp2-/y mice revealed less adenosine amount as well as less A2AR protein levels when compared to WT littermates, which may partially explain the deficits in adenosinergic tonus in these animals. Remarkably, the lack of BDNF effect on hippocampal LTP in Mecp2-/y mice was overcome by selective activation of A2AR with CGS21680. Overall, in Mecp2-/y mice there is an impairment on adenosinergic system and BDNF signalling. These findings set the stage for adenosine-based pharmacological therapeutic strategies for RTT, highlighting A2AR as a therapeutic target in this devastating pathology.

Adiponectin and sporadic Alzheimer's disease: Clinical and molecular links.

Obesity has been consistently associated with Alzheimer's disease (AD) though the exact mechanisms by which it influences cognition are still elusive and subject of current research. Adiponectin, the most abundant adipokine in circulation, is inversely correlated with adipose tissue dysfunction and seems to be a central player in this association. In fact, different signalling pathways are shared by adiponectin and proteins involved in AD pathophysiology and considerable amount of evidence supports its direct and indirect influence on ß-amyloid and tau aggregates formation. In this paper we present a critical review of cellular, animal and clinical studies which have contributed to a more thorough understanding of the extent to which adiponectin influences the risk of developing AD as well as its progression. Finally, the effect of acetylcholinesterase inhibitors on circulating adiponectin levels, possible therapeutic applications and future research strategies are also discussed.

Severity of prepregnancy diabetes on the fetal malformations and viability associated with early embryos in rats†.

Preexisting/pregestational diabetes enhances the risk of birth defects. Several factors have been involved during the implantation process, such as cytokines (granulocyte-macrophage-colony-stimulating factor [GM-CSF]). The objective was to evaluate the effects of two levels of diabetes on the redox status of preimplantation embryos during the implantation process to comprehend how both are involved in embryo and fetal viability against maternal diabetes. Female Sprague-Dawley rats received streptozotocin at birth (mild diabetes [MD]) or at adulthood (severe diabetes [SD]) to obtain two experimental diabetes intensities. After confirming the diabetic status, the nondiabetic and diabetic groups were mated around day 110 of life. At gestational day (GD) 21, fetuses were assessed for viability and malformations and ovaries for embryo loss before implantation. Other pregnant nondiabetic and diabetic rats were sacrificed at GD2-4 for maternal and preimplantation embryo oxidative stress markers, maternal serum insulin, uterine fluid GM-CSF, and preimplantation embryo morphological analysis. MD and SD caused abnormal redox levels, lower GM-CSF and insulin levels during the preimplantation period, and embryonic loss before implantation. SD caused lower fetal viability and higher fetal malformation percentages at GD21. The SD dam-derived preimplantation embryos presented lower glutathione levels and higher thiobarbituric acid reactive substances concentration at GD3 and an increased frequency of abnormal preimplantation embryos at GD4. In conclusion, preexisting diabetes leads to complications in the implantation process. Furthermore, maternal oxidative stress and other metabolic changes alter the redox state and morphological structure of preimplantation embryos, contributing to damaged growth and development in late pregnancy.

GLP-1 improves adipose tissue glyoxalase activity and capillarization improving insulin sensitivity in type 2 diabetes.

Methylglyoxal was shown to impair adipose tissue capillarization and insulin sensitivity in obese models. We hypothesized that glyoxalase-1 (GLO-1) activity could be diminished in the adipose tissue of type 2 diabetic obese patients. Moreover, we assessed whether such activity could be increased by GLP-1-based therapies in order to improve adipose tissue capillarization and insulin sensitivity. GLO-1 activity was assessed in visceral adipose tissue of a cohort of obese patients. The role of GLP-1 in modulating GLO-1 was assessed in type 2 diabetic GK rats submitted to sleeve gastrectomy or Liraglutide treatment, in the adipose tissue angiogenesis assay and in the HUVEC cell line. Glyoxalase-1 activity was decreased in visceral adipose tissue of pre-diabetic and diabetic obese patients, together with other markers of adipose tissue dysfunction and correlated with increased HbA1c levels. Decreased adipose tissue GLO-1 levels in GK rats were increased by sleeve gastrectomy and Liraglutide, being associated with overexpression of angiogenic and vasoactive factors, as well as insulin receptor phosphorylation (Tyr1161). Moreover, GLP-1 increased adipose tissue capillarization and HUVEC proliferation in a glyoxalase-dependent manner. Lower adipose tissue GLO-1 activity was observed in dysmetabolic patients, being a target for GLP-1 in improving adipose tissue capillarization and insulin sensitivity.