A methodology to globally assess ectodomain shedding using soluble fractions from the mouse brain.

Ectodomain shedding (ES) is a fundamental process involving the proteolytic cleavage of membrane-bound proteins, leading to the release of soluble extracellular fragments (shed ectodomains) with potential paracrine and autocrine signaling functions. In the central nervous system (CNS), ES plays pivotal roles in brain development, axonal regulation, synapse formation, and disease pathogenesis, spanning from cancer to Alzheimer's disease. Recent evidence also suggests its potential involvement in neurodevelopmental conditions like autism and schizophrenia. Past investigations of ES in the CNS have primarily relied on cell culture supernatants or cerebrospinal fluid (CSF) samples, but these methods have limitations, offering limited insights into how ES is modulated in the intact brain parenchyma. In this study, we introduce a methodology for analyzing shed ectodomains globally within rodent brain samples. Through biochemical tissue subcellular separation, mass spectrometry, and bioinformatic analysis, we show that the brain's soluble fraction sheddome shares significant molecular and functional similarities with in vitro neuronal and CSF sheddomes. This approach provides a promising means of exploring ES dynamics in the CNS, allowing for the evaluation of ES at different developmental stages and pathophysiological states. This methodology has the potential to help us deepen our understanding of ES and its role in CNS function and pathology, offering new insights and opportunities for research in this field.

Molecular Insights into the Covalent Binding of Zoxamide to the ß-Tubulin of Botrytis cinerea.

Botrytis cinerea is a fungal plant pathogen that causes significant economic losses in the agricultural industry worldwide. Fungicides that target microtubules, such as carbendazim (CBZ), diethofencarb (DEF), and zoxamide (ZOX), are widely used in crop protection against this pathogen. These groups of compounds exert their fungicidal activity by disrupting the microtubule assembly by binding to the ß-tubulin subunit, provoking cell-cycle arrest and cell death. However, with the appearance of isolates resistant to these compounds, it is necessary to search for new alternatives to control this pathogenic fungus. In this work, we gained insight into the binding and stability of these fungicides in the benzimidazole binding site of B. cinerea ß-tubulin through different computational approaches. Our molecular dynamics simulation replicas showed that R enantiomers of ZOX and its analog RH-4032 had better interaction profiles at the site compared to S enantiomers. The simulations also revealed that while the R-isomer fungicides formed H-bonds with the main chain carbonyl of V236 or the side chain residue of S314, only CBZ interacted with E198. Previous experimental data have identified key mutations in B. cinerea's ß-tubulin gene that lead to the development of resistance or, on the contrary, increased sensitivity for treatment with these fungicide compounds. In agreement with experimental findings, alchemical free energy calculations showed that E198A and E198V mutations in B. cinerea ß-tubulin have high sensitivity to (R)-ZOX, whereas the E198K mutation decreased its affinity. Similarly, the results obtained explain the resistance to CBZ of B. cinerea isolates with E198A/V/K mutations and the insensitivity of the wild-type organism to DEF. Our work provides a deeper insight into the molecular mechanism of action of these fungicides, highlighting the importance of understanding the interaction profiles to develop more effective antifungal agents.

A novel machine learning-based screening identifies statins as inhibitors of the calcium pump SERCA.

We report a novel small-molecule screening approach that combines data augmentation and machine learning to identify Food and Drug Administration (FDA)-approved drugs interacting with the calcium pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA) from skeletal (SERCA1a) and cardiac (SERCA2a) muscle. This approach uses information about small-molecule effectors to map and probe the chemical space of pharmacological targets, thus allowing to screen with high precision large databases of small molecules, including approved and investigational drugs. We chose SERCA because it plays a major role in the excitation-contraction-relaxation cycle in muscle and it represents a major target in both skeletal and cardiac muscle. The machine learning model predicted that SERCA1a and SERCA2a are pharmacological targets for seven statins, a group of FDA-approved 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors used in the clinic as lipid-lowering medications. We validated the machine learning predictions by using in vitro ATPase assays to show that several FDA-approved statins are partial inhibitors of SERCA1a and SERCA2a. Complementary atomistic simulations predict that these drugs bind to two different allosteric sites of the pump. Our findings suggest that SERCA-mediated Ca2+ transport may be targeted by some statins (e.g., atorvastatin), thus providing a molecular pathway to explain statin-associated toxicity reported in the literature. These studies show the applicability of data augmentation and machine learning-based screening as a general platform for the identification of off-target interactions and the applicability of this approach extends to drug discovery.

A developmental delay linked missense mutation in Kalirin-7 disrupts protein function and neuronal morphology.

The Rac1 guanine exchange factor Kalirin-7 is a key regulator of dendritic spine morphology, LTP and dendritic arborization. Kalirin-7 dysfunction and genetic variation has been extensively linked to various neurodevelopmental and neurodegenerative disorders. Here we characterize a Kalirin-7 missense mutation, glu1577lys (E1577K), identified in a patient with severe developmental delay. The E1577K point mutation is located within the catalytic domain of Kalirin-7, and results in a robust reduction in Kalirin-7 Rac1 Guanosine exchange factor activity. In contrast to wild type Kalirin-7, the E1577K mutant failed to drive dendritic arborization, spine density, NMDAr targeting to, and activity within, spines. Together these results indicate that reduced Rac1-GEF activity as result of E1577K mutation impairs neuroarchitecture, connectivity and NMDAr activity, and is a likely contributor to impaired neurodevelopment in a patient with developmental delay.

Photodynamic therapy and combinatory treatments for the control of biofilm-associated infections.

The advent of antimicrobial resistance has added considerable impact to infectious diseases both in the number of infections and healthcare costs. Furthermore, the relentless emergence of multidrug-resistant bacteria, particularly in the biofilm state, has made mandatory the discovery of new alternative antimicrobial therapies that are capable to eradicate resistant bacteria and impair the development of new forms of resistance. Amongst the therapeutic strategies for treating biofilms, antimicrobial photodynamic therapy (aPDT) has shown great potential in inactivating several clinically relevant micro-organisms, including antibiotic-resistant 'priority bacteria' declared by the WHO as critical pathogens. Its antimicrobial effect is centred on the basis that harmless low-intensity light stimulates a non-toxic dye named photosensitizer, triggering the production of reactive oxygen species upon photostimulation. In addition, combination therapies of aPDT with other antimicrobial agents (e.g. antibiotics) have also drawn considerable attention, as it is a multi-target strategy. Therefore, the present review highlights the recent advances of aPDT against biofilms, also covering progress on combination therapy.

Intercellular signaling by ectodomain shedding at the synapse.

Ectodomain shedding (ES) is a post-translational protein modification process that plays key roles in health and disease. Many neuronal and synaptic membrane proteins are known to undergo ES, but the complexity of functions regulated by the shed peptides is only beginning to be unraveled. Here, we provide an overview of emerging evidence demonstrating that synaptic ES can mediate autocrine and paracrine signaling. We also discuss how advances in large-scale proteomic analyses are leading to the identification of novel synaptic proteins undergoing ES, as well as the targets and functions of their soluble ectodomains. Finally, we provide an overview of how cerebrospinal fluid (CSF) analyses of shed proteins could be used as a potential source of new biomarkers for neuropsychiatric disorders.

Changes in the potential distribution of the guava fruit fly Anastrepha striata (Diptera, Tephritidae) under current and possible future climate scenarios in Colombia.

Climate change has affected the geographical distributions of most species worldwide; in particular, insects of economic importance inhabiting tropical regions have been impacted. Current and future predictions of change in geographic distribution are frequently included in species distribution models (SDMs). The potential spatial distributions of the fruit fly Anastrepha striata Schiner, the main species of agricultural importance in guava crops, under current and possible future scenarios in Colombia were modeled, and the establishment risk was assessed for each guava-producing municipality in the country. SDMs were developed using 221 geographical records in conjunction with nine scenopoetic variables. The model for current climate conditions indicated an extensive suitable area for the establishment of A. striata in the Andean region, smaller areas in the Caribbean and Pacific, and almost no areas in the Orinoquia and Amazonian regions. A brief discussion regarding the area's suitability for the fly is offered. According to the results, altitude is one of the main factors that direct the distribution of A. striata in the tropics. The Colombian guava-producing municipalities were classified according to the degree of vulnerability to fly establishment as follows: 42 were high risk, 16 were intermediate risk, and 17 were low risk. The implementation of future integrated management plans must include optimal spatial data and must consider environmental aspects, such as those suggested by the models presented here. Control decisions should aim to mitigate the positive relationship between global warming and the increase in the dispersal area of the fruit fly.

Shed CNTNAP2 ectodomain is detectable in CSF and regulates Ca2+ homeostasis and network synchrony via PMCA2/ATP2B2.

Although many neuronal membrane proteins undergo proteolytic cleavage, little is known about the biological significance of neuronal ectodomain shedding (ES). Here, we show that the neuronal sheddome is detectable in human cerebrospinal fluid (hCSF) and is enriched in neurodevelopmental disorder (NDD) risk factors. Among shed synaptic proteins is the ectodomain of CNTNAP2 (CNTNAP2-ecto), a prominent NDD risk factor. CNTNAP2 undergoes activity-dependent ES via MMP9 (matrix metalloprotease 9), and CNTNAP2-ecto levels are reduced in the hCSF of individuals with autism spectrum disorder. Using mass spectrometry, we identified the plasma membrane Ca2+ ATPase (PMCA) extrusion pumps as novel CNTNAP2-ecto binding partners. CNTNAP2-ecto enhances the activity of PMCA2 and regulates neuronal network dynamics in a PMCA2-dependent manner. Our data underscore the promise of sheddome analysis in discovering neurobiological mechanisms, provide insight into the biology of ES and its relationship with the CSF, and reveal a mechanism of regulation of Ca2+ homeostasis and neuronal network synchrony by a shed ectodomain.

Utility of lateral flow tests in SARS-CoV-2 infection monitorization.

OBJECTIVE: The diagnosis of SARS-CoV-2 infection is crucial for medical and public health reasons, to allow the best treatment of cases and the best control of the pandemic. Serology testing allows for the detection of asymptomatic infections and 19-COVID cases once the virus has been cleared. We analyzed the usefulness of the SARS-CoV-2 rapid test of Autobio and tried to correlate its pattern with the severity of COVID19 infection. METHODS: We analyzed the accuracy and clinical usefulness of a point-of-care IgM and/or IgG test for SARS-CoV-2 in 35 COVID-19 patients [12 (34.3%) mild-moderate and 23 (65.7%) severe-critical] admitted to a field hospital in Madrid, as well as in 5 controls. RESULTS: The mean time from the first day of symptoms to the antibody test was 28 days (SD: 8.7), similar according to the severity of the disease. All patients with SARS-CoV-2 PCR+ showed the corresponding IgG positivity, while these results were negative in all control individuals. A total of 26 (74%) cases also presented with positive IgM, 19 (83%) were severe-critical cases and 7 (58%) were mild-moderate cases. The IgM response lasted longer in the severe critical cases (mean: 29.7 days; SD: 8.4) compared to the moderate cases (mean: 21.2 days; SD: 2.0).. CONCLUSIONS: Rapid serology tests are useful for the diagnosis of patients with COVID-19 (mainly IgG detection) and may also be correlated with the severity of the infection (based on IgM detection).

Utility of neutrophil-to-lymphocyte ratio plus C-reactive protein for infection in systemic lupus erythematosus.

OBJECTIVE: To analyze the utility of neutrophil-to-lymphocyte ratio (NLR) plus C-reactive protein (CRP) to differentiate between infection and active disease in patients with SLE. METHODS: A cross-sectional study of a cohort of patients with SLE was carried out. Blood samples from four groups (patients without infection or active disease, patients with infection, patients with active disease, and patients with both infection and active disease) before therapeutic interventions were analyzed. We excluded patients with current malignancy, pregnancy, ischemic heart disease or use of antimicrobials during previous 7 days. Hematological cell count, CRP and cultures were obtained. We constructed receiver operating characteristic curves; sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated. RESULTS: Forty patients were included. NLR cut-off ≥6.3 had sensitivity 70%, specificity 85%, PPV 83% and NPV 74% to detect patients with non-viral infections. A CRP cut-off ≥7.5 mg/L had sensitivity 90%, specificity 75%, PPV 78% and NPV 88% to detect infections regardless of SLE activity. Combination of CRP plus NLR improves the specificity to 90% and PPV to 88%. Excluding the group with both infection and active disease, CRP plus NLR expands specificity to 95% and NPV to 90%. CONCLUSION: In our experience, levels of CRP, particularly CRP plus NLR, were useful in differentiating patients with SLE from those with suspected non-viral infection regardless of the activity of the disease.

Las implicancias del juego para el periodo de latencia / The implications of playing for the latency period

La pregunta que convoca este escrito surge a partir de la gran incidencia de casos que se presentan, en nuestra práctica clínica, de pacientes que no se encuentran dentro de lo esperable con respecto a la entrada en el período de latencia, según lo conceptualizado por Freud (1905), en su escrito Tres ensayos de teoría sexual. Se trata de niños de entre 6 y 12 años aproximadamente, que sostienen modos de satisfacción desfasados en cuanto al momento del desarrollo que se encuentran atravesando. Observamos que los mismos presentan características del orden del autoerotismo. En consideración de lo dicho, nos preguntamos acerca de las implicancias que puede tener el juego, en nuestro tiempo, como posibilitador de la construcción de recursos subjetivos para la entrada en el período de latencia sexual infantil.

The question convened in this paper arises from the high incidence of cases that occur, in our clinical practice, of patients who are not within the expected regarding the entry into the latency period, as conceptualized by Freud (1905). These are children between 6 and 12 years old,who maintain outdated modes of satisfaction in terms of the moment of development they are going through. We observe that they present characteristics of the order of autoerotism.In consideration of what has been said, we ask ourselves about the implications that playing may have, in our time, as an enabler of the construction of subjective resources for the entry into the period of infantile sexual latency.

Neuropsychological alterations and neuroradiological findings in patients with post-traumatic concussion: Results of a pilot study. / Alteraciones neuropsicológicas y hallazgos neurorradiológicos en pacientes con conmoción cerebral postraumática. Resultados de un estudio piloto.

INTRODUCTION: Mild traumatic brain injury (mTBI) has traditionally been considered to cause no significant brain damage since symptoms spontaneously remit after a few days. However, this idea is facing increasing scrutiny. The purpose of this study is to demonstrate the presence of early cognitive alterations in a series of patients with mTBI and to link these findings to different markers of brain damage. METHODS: We conducted a prospective study of a consecutive series of patients with mTBI who were evaluated over a 12-month period. Forty-one (3.7%) of the 1144 included patients had experienced a concussion. Patients underwent a routine clinical evaluation and a brain computed tomography (CT) scan, and were also administered a standardised test for post-concussion symptoms within the first 24hours of mTBI and also 1 to 2 weeks later. The second assessment also included a neuropsychological test battery. The results of these studies were compared to those of a control group of 28 healthy volunteers with similar characteristics. Twenty patients underwent an MRI scan. RESULTS: Verbal memory and learning were the cognitive functions most affected by mTBI. Seven out of the 20 patients with normal CT findings displayed structural alterations on MR images, which were compatible with diffuse axonal injury in 2 cases. CONCLUSIONS: Results from this pilot study suggest that early cognitive alterations and structural brain lesions affect a considerable percentage of patients with post-concussion syndrome following mTBI.

La incidencia de la época actual en la precarización de la subjetividad. Un aporte desde el psicoanálisis a la clínica con niños / The incidence of the present time in the precarization of subjectivity. A contribution from psychoanalysis to the clinic with children

La pregunta que convoca este escrito surge a partir de la gran incidencia de casos que se presentan, en la práctica institucional, de pacientes que no se encuentran dentro de lo esperable con respecto a la adquisición del lenguaje y a todo lo que implica el armado del aparataje simbólico. Esto es: la posibilidad de la constitución de un yo-cuerpo libidinal como una unidad diferenciada del otro (Freud, 1923, p.27), y el ordenamiento pulsional consecuente en el sentido de un armado del mundo habitable para el sujeto, todo lo cual implica la construcción de la subjetividad. Se trata de casos en donde no se construyó la posibilidad de una cierta autonomía en el niño: el certificado de discapacidad denota esto. En este estado de cosas se requiere de una dependencia permanente del otro, muchas veces el Estado, a través de las instituciones estatales o privadas. Todo esto nos lleva a pensar, a modo de hipótesis, en la incidencia de las variables de la época actual en lo que ubicamos como una precarización en la construcción de la subjetividad en los casos descriptos.

The question that summons this paper arises from the great incidence of cases that are presented, in the institutional practice, of patients that are not within the expectable regarding the acquisition of the language and all that implies the building of the symbolic apparatus. This is: the possibility of the constitution of a libidinal self-body as a differentiated unit from the other (Freud, 1923, p.27), and the consequent instinental ordering in the sense the disposition of an habitable world for the subject, all of which involves the construction of subjectivity. These are cases where the possibility of a certain autonomy in the child was not built: the certificate of disability denotes this. In this state of things, the patient requires a permanent dependence on the other, often the State, through state or private institutions. All this leads us to think, as a hypothesis, in the incidence of the variables of the present time on what we place as a precarization in the construction of subjectivity in the cases described.

Dietary probiotic supplementation improves growth and the intestinal morphology of Nile tilapia.

Probiotic administration can be a nutritional strategy to improve the immune response and growth performance of fish. The current study aimed to evaluate the effects of a probiotic blend (Bacillus sp., Pediococcus sp., Enterococcus sp., Lactobacillus sp.) as a dietary supplement on growth performance, feed utilization, innate immune and oxidative stress responses and intestinal morphology in juvenile Nile tilapia (Oreochromis niloticus). The probiotic was incorporated into a basal diet at three concentrations: 0 g/kg (A0: control), 3 g/kg (A1: 1.0×106 colony forming unit (CFU)/g) and 6 g/kg (A2: 2.3×106 CFU/g diet). After 8 weeks of probiotic feeding, weight and specific growth rate where significantly higher in fish-fed A1 diet than in fish-fed A0. Alternative complement in plasma was significantly enhanced in fish-fed A2 when compared with A0. The hepatic antioxidant indicators were not affected by probiotic supplementation. Villi height and goblet cell counts increased significantly in the intestine of fish-fed A1 and A2 diets compared with A0. The dietary probiotic supplementation was maintained until 20 weeks of feeding. Then the selected immune parameters, digestive enzymes and apparent digestibility of diets were studied. No effect of probiotic feeding was observed after that longer period supplementation. The dietary supplementation of mixed species probiotic may constitute a valuable nutritional approach towards a sustainable tilapia aquaculture. The improvement of the immune responses and intestinal morphology play an important role in increasing growth performance, nutrient absorption and disease resistance in fish, important outcomes in such a competitive and developing aquaculture sector.

Prediction of fetal growth restriction using estimated fetal weight vs a combined screening model in the third trimester.

OBJECTIVES: To compare the performance of third-trimester screening, based on estimated fetal weight centile (EFWc) vs a combined model including maternal baseline characteristics, fetoplacental ultrasound and maternal biochemical markers, for the prediction of small-for-gestational-age (SGA) neonates and late-onset fetal growth restriction (FGR). METHODS: This was a nested case-control study within a prospective cohort of 1590 singleton gestations undergoing third-trimester (32 + 0 to 36 + 6 weeks' gestation) evaluation. Maternal baseline characteristics, mean arterial pressure, fetoplacental ultrasound and circulating biochemical markers (placental growth factor (PlGF), lipocalin-2, unconjugated estriol and inhibin A) were assessed in all women who subsequently delivered a SGA neonate (n = 175), defined as birth weight < 10th centile according to customized standards, and in a control group (n = 875). Among SGA cases, those with birth weight < 3rd centile and/or abnormal uterine artery pulsatility index (UtA-PI) and/or abnormal cerebroplacental ratio (CPR) were classified as FGR. Logistic regression predictive models were developed for SGA and FGR, and their performance was compared with that obtained using EFWc alone. RESULTS: In SGA cases, EFWc, CPR Z-score and maternal serum concentrations of unconjugated estriol and PlGF were significantly lower, while mean UtA-PI Z-score and lipocalin-2 and inhibin A concentrations were significantly higher, compared with controls. Using EFWc alone, 52% (area under receiver-operating characteristics curve (AUC), 0.82 (95% CI, 0.77-0.85)) of SGA and 64% (AUC, 0.86 (95% CI, 0.81-0.91)) of FGR cases were predicted at a 10% false-positive rate. A combined screening model including a-priori risk (maternal characteristics), EFWc, UtA-PI, PlGF and estriol (with lipocalin-2 for SGA) achieved a detection rate of 61% (AUC, 0.86 (95% CI, 0.83-0.89)) for SGA cases and 77% (AUC, 0.92 (95% CI, 0.88-0.95)) for FGR. The combined model for the prediction of SGA and FGR performed significantly better than did using EFWc alone (P < 0.001 and P = 0.002, respectively). CONCLUSIONS: A multivariable integrative model of maternal characteristics, fetoplacental ultrasound and maternal biochemical markers modestly improved the detection of SGA and FGR cases at 32-36 weeks' gestation when compared with screening based on EFWc alone. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.