[Prevalence of Outpatient Use of Medicines by the Elderly Population in Portugal: A Cross-Sectional Study]. / Prevalência da Dispensa de Medicamentos em Ambulatório na População Idosa em Portugal: Um Estudo Transversal.

INTRODUCTION: Like in other countries, the age pyramid in Portugal has been changing considerably, with a substantial increase in the size of the older population and a significant reduction in the number of young people. With aging, co-occurrence of several conditions becomes frequent, often leading to the use of multiple medications (polypharmacy). Polypharmacy in the older population is particularly relevant considering the physiological changes of the ageing process, which increase the risk of drug interactions, poor adherence to treatment, and adverse drug reactions, especially in the oldest-old population (85 years or older). As the size of the older population is likely to increase significantly, it is important to characterize the pattern of medicines' use by the elderly while also identifying cases of polypharmacy in order to obtain evidence that can be used to develop specific measures to tackle the high prevalence of use and its associated risks. To this end, the aim of this study was to characterize medication use by older individuals in Portugal. METHODS: Cross-sectional study with data from the National Health System's Control and Monitoring Center on reimbursed medicines that were prescribed and dispensed to individuals aged 65 years or older in 2019 in all community pharmacies of the Portuguese mainland. We performed a demographic and geographic analysis of the data by international nonproprietary name and therapeutic group. The number of reimbursed packages and the number of reimbursed packages per capita were the metrics used (data from Instituto Nacional de Estatística). RESULTS: A higher consumption of medicines was observed in women, increasing with age, except in the oldest olds, in which the sex difference tended to shrink. Use per capita showed an opposite trend, with the oldest-old men surpassing the oldest-old women (mean reimbursed packages: 55.5 in men versus 55.1 in women). In women, consumption was led by cardiovascular medicines (31%), followed by central nervous system medications (30%) and antidiabetics (13%); in men, 37% of TOP 10 consumption was due to cardiovascular medications, antidiabetics (16%) and drugs for benign prostatic hypertrophy (14%). CONCLUSION: In the elderly, there were sex differences in the pattern of medicines' use, and there were also significant age-related differences in 2019. To the best of our knowledge, our study is the first nationwide analysis of reimbursed medicines' consumption data in the elderly, which is essential to characterize the use of medicines in this age group in Portugal.

Introdução: À semelhança de outros países, a pirâmide etária em Portugal tem sofrido alterações profundas, com um expressivo aumento na dimensão da população idosa. A multimorbilidade que surge com o envelhecimento leva, frequentemente, à utilização concomitante de vários medicamentos. A polimedicação é particularmente importante no idoso devido às alterações fisiológicas associadas ao processo de envelhecimento, que aumentam o risco de interações medicamentosas, de fraca adesão à terapêutica e de reações adversas à medicação, em particular nos indivíduos muito idosos (85 ou mais anos). Dado que a dimensão da população idosa poderá aumentar significativamente, importa caracterizar o padrão de consumo de medicamentos pelos idosos, identificando também os casos de polifarmácia, de forma a gerar-se evidência que permita o desenvolvimento de medidas específicas de combate à elevada prevalência de utilização e riscos associados. Assim, o objetivo desta análise preliminar foi determinar a prevalência e caracterizar do padrão de utilização de medicamentos pelos idosos em Portugal, desagregando por faixa etária, sexo e localização geográfica. Métodos: Estudo transversal com dados relativos aos medicamentos comparticipados e dispensados nas farmácias comunitárias de Portugal Continental em 2019, aos utentes com mais de 65 anos. Efetuou-se análise descritiva demográfica e geográfica, por denominação comum internacional e grupo terapêutico. A utilização foi estudada através do número de embalagens comparticipadas dispensadas e número de embalagens comparticipadas dispensadas per capita (dados do Instituto Nacional de Estatística). Resultados: Observou-se uma dispensa superior nas mulheres, a qual foi aumentando com a idade, à exceção dos idosos 85+, nos quais a diferença tendeu a diminuir. No que diz respeito ao número de embalagens comparticipadas dispensadas per capita, a tendência foi inversa, com os homens muito idosos a ultrapassarem as mulheres 85+ (média de embalagens: 55,5 nos homens versus 55,1 nas mulheres). Nas mulheres, os medicamentos mais consumidos foram os do foro cardiovascular (31%), seguidos dos prescritos para o sistema nervoso central (30%) e antidiabéticos (13%). Nos homens, o ranking foi liderado também pelos medicamentos para o aparelho cardiovascular (37%); contudo, em segundo lugar surgem os antidiabéticos (16%), seguidos dos medicamentos para a hiperplasia benigna da próstata (14%). Conclusão: Existiram diferenças de sexo e idade relevantes no padrão de dispensa de medicamentos comparticipados nos idosos portugueses em 2019. Esta é a primeira análise publicada de âmbito nacional à dispensa de medicamentos em idosos, sendo essencial para caracterizar o perfil de utilização de medicamentos pelos seniores em Portugal.

Authentication of beef cuts by multielement and machine learning approaches.

BACKGROUND: Brazil has consolidated a relevant position in the world market, being the largest exporter and second producer of beef. Genetics, feeding system, geographic origin and climate influence the multielement profile of beef. The feasibility of combining classification algorithms with major and trace elements was evaluated as a tool for authentication of beef cuts. METHODS: Animals of Angus, Nelore and Wagyu crossbreeds, raised in a vertically integrated system, were sampled at the slaughterhouse for chuck steak, rump cap and sirloin steak. Supervised learning algorithms i.e. Classification and Regression Tree (CART), Multilayer Perceptron (MLP), Naïve Bayes (NB), Random Forest (RF) and Sequential Minimal Optimization (SMO) were used to build classification models based on the multielement profile of beef determined by neutron activation analysis. RESULTS: Br, Co, Cs, Fe, K, Na, Rb, Se and Zn were determined in the beef samples. The classification accuracy values obtained for the beef cuts were 96% (MLP), 95% (SMO), 91% (RF), 86% (NB) and 70% (CART). CONCLUSION: The Multilayer Perceptron algorithm provided the best classification performance towards authentication of beef cuts on basis of major and trace element mass fractions.

Contralateral Afferent Input to Lumbar Lamina I Neurons as a Neural Substrate for Mirror-Image Pain.

Mirror-image pain arises from pathologic alterations in the nociceptive processing network that controls functional lateralization of the primary afferent input. Although a number of clinical syndromes related to dysfunction of the lumbar afferent system are associated with the mirror-image pain, its morphophysiological substrate and mechanism of induction remain poorly understood. Therefore, we used ex vivo spinal cord preparation of young rats of both sexes to study organization and processing of the contralateral afferent input to the neurons in the major spinal nociceptive projection area Lamina I. We show that decussating primary afferent branches reach contralateral Lamina I, where 27% of neurons, including projection neurons, receive monosynaptic and/or polysynaptic excitatory drive from the contralateral Aδ-fibers and C-fibers. All these neurons also received ipsilateral input, implying their involvement in the bilateral information processing. Our data further show that the contralateral Aδ-fiber and C-fiber input is under diverse forms of inhibitory control. Attenuation of the afferent-driven presynaptic inhibition and/or disinhibition of the dorsal horn network increased the contralateral excitatory drive to Lamina I neurons and its ability to evoke action potentials. Furthermore, the contralateral Aßδ-fibers presynaptically control ipsilateral C-fiber input to Lamina I neurons. Thus, these results show that some lumbar Lamina I neurons are wired to the contralateral afferent system whose input, under normal conditions, is subject to inhibitory control. A pathologic disinhibition of the decussating pathways can open a gate controlling contralateral information flow to the nociceptive projection neurons and, thus, contribute to induction of hypersensitivity and mirror-image pain.SIGNIFICANCE STATEMENT We show that contralateral Aδ-afferents and C-afferents supply lumbar Lamina I neurons. The contralateral input is under diverse forms of inhibitory control and itself controls the ipsilateral input. Disinhibition of decussating pathways increases nociceptive drive to Lamina I neurons and may cause induction of contralateral hypersensitivity and mirror-image pain.

The Naked Neck Gene in the Domestic Chicken: A Genetic Strategy to Mitigate the Impact of Heat Stress in Poultry Production-A Review.

The poultry sector is one of the most important food industries in the world. Poultry production generates high-value protein products (meat and eggs) that are produced efficiently without the need for large areas. In poultry production, especially in the tropics, environmental factors, such as temperature and humidity, play a major role. Heat stress (HS) causes behavioral, physical, and physiological changes in poultry, with severe financial impacts. Therefore, it is important to find strategies to minimize it. The naked neck (Na) is an autosomal, incompletely dominant gene. Compared with normal feathered birds, these animals are known for their ability to adapt, perform, and reproduce under hot and humid climate conditions. Due to the absence of feathers on the neck, these animals increase heat dissipation, alleviating adverse heat effects, especially on productive performance. Genetic improvement of heat tolerance may provide a low-cost solution, of particular interest for developing countries in the tropics. The focus of this review is to evaluate the impact of HS in poultry with a special emphasis on the advantages of using the Na gene.

Computed tomography and radioactive 32P detected phosphorus impairment in metabolism, reduced bones density and animal performance caused by mixed infection of Haemonchus contortus and Trichostrongylus colubriformis in sheep.

Phosphorus (P) is one of the main minerals present in the animal body and exerts crucial functions in the organism. P is present at all cell membranes and integrates the structure of bones, being necessary its supplementation in ruminants due to the deficiency of this mineral in the pastures. One of the principal factors that compromise its metabolization are gastrointestinal nematodes (GIN). Thus, the objective of this study was evaluate the performance and metabolism of P through its distribution in the animal body, density of bones and muscles, dynamic fluxes, biological availability and half live of P, concentration of P in tissues and bones of lambs simultaneously infected with the most prevalent GIN to sheep, in tropical or subtropical areas, (Haemonchus contortus and Trichostrongylus colubriformis) using the isotopic dilution technique with 32P radioisotope. Twenty Santa Ines sheep with seven months of age and averaging initial weight of 30.8 ± 6.41 kg were used and allocated to one of two treatments. Ten animals were orally infected (a single dose of 30,000 L3 larvae of T. colubriformis + 10,000 L3 larvae of H. contortus), and ten animals were not infected (control group). During the experimental, samples of blood, feces, urine, and diet refusals were collected and weighting were performed. A computed tomography was performed twice, before infection and at the end of the experiment, to evaluate changes in body composition. On 64-d after experimental infection, animals received an intravenous injection of 32P solution, and 7-d after they received radioisotope injection. The experimental animals were slaughtered, and tissue and bones were collected for P concentrations. The results showed that the parasitic infection compromised the absorption of P, impairing the metabolism, decreasing the mineral bioavailability increasing P bones reabsorption, and reducing bones density, also negatively compromising the infected animal performance.

Trichostrongylus colubriformis infection damages intestine brush board cells and could negatively impact postabsorptive parameters of Santa Ines lambs.

This study aimed to evaluate histological, digestive and postabsorptive physiological parameters in Santa Ines lambs infected with Trichostrongylus colubriformis and fed different levels of phosphorus. Therefore, eighteen Santa Ines, castrated male, six-month old, healthy lambs (initial body weight 22.4 ± 2.7 kg) were distributed in one of four treatments arranged in a 2 × 2 split-plot arrangement: Sufficient dietary P level and uninfected (SPui; n = 4), Sufficient dietary P level and infected (SPi; n = 5), Deficient dietary P level and uninfected (DPui; n = 4), Deficient dietary P level and infected (DPi; n = 5). Infected lambs received, orally, a single pulse dose of 40.000 T. colubriformis infective larval stage (L3). Animals were fed Tifton 85 hay (Cynodon ssp.; 60%), and cassava meal and maize gluten meal (40%). Measurement of nutrient apparent digestibility and nitrogen metabolism were performed in individual metabolic stalls. To achieve the trial results, it was measured methane emissions in respiratory chambers, urine purine derivatives, ruminal short-chain fatty acids (SCFA), histological cuts of duodenal mucosal tissues and passage rates fluxes, analyzed by external (Yb, Cr, and Co) and internal (iNDF) markers. Statistical procedures were performed in R studio. The fixed main effects of treatment and the interactions were tested by ANOVA, and means compared by Duncan's test at 5% significance. Apparent digestibility was not affected by treatments, however, nitrogen retained decreased (P < 0.01) and urinary nitrogen losses increased (P < 0.01) in infected animals. Small intestine digesta content, empty segment weight, and length were higher in infected animals (P < 0.05). Passage rate was not majorly affected by infection or dietary P levels. Methane emissions, SCFA concentrations, and purine derivative excretion were also not affected by treatments. Regarding the histology, the vilosity weight (P < 0.05), and crypt depth (P < 0.01) decreased in infected animals. In conclusion, T. colubriformis infection can damage intestinal mucosa and affect nitrogen metabolism, but did not affect the digesta transit, and nutrient digestibility. The P dietary levels did not promote any modification in GIT physiological parameters tested in this study.

Elemental profile of dietary supplements and agricultural byproducts evaluated by neutron activation analysis.

Dietary supplements and agricultural byproducts were characterized by neutron activation analysis. The nutritional potential of supplements was evaluated according to alternative and commercial categories, using analysis of variance and cluster analysis, and recommended dietary intake for children. The results indicated statistically significant differences between both categories for the elements Cs, K, Na, and Rb. For the nutritional elements Ca, Co, Fe, K, Na, and Zn, the categories were similar in cluster analysis. The similarity between elemental profiles of alternative supplements and agricultural byproducts was calculated using a dissimilarity matrix, showing that rice and wheat are the predominant ingredients.

Labeling on a Chip of Cellular Fibronectin and Matrix Metallopeptidase-9 in Human Serum.

We present a microfluidic chip for protein labeling in the human serum-based matrix. Serum is a complex sample matrix that contains a variety of proteins, and a matrix is used in many clinical tests. In this study, the device performance was tested using commercial serum samples from healthy donors spiked with the following target proteins: cellular fibronectin (c-Fn) and matrix metallopeptidase 9 (MMP9). The microfluidic molds were fabricated using micro milling on acrylic and using stereolithography (SLA) three-dimensional (3D) printing for an alternative method and comparison. A simple quality control was performed for both fabrication mold methods to inspect the channel height of the chip that plays a critical role in the labeling process. The fabricated microfluidic chip shows a good reproducibility and repeatability of the performance for the optimized channel height of 150 µm. The spiked proteins of c-Fn and MMP9 in the human serum-based matrix, were successfully labeled by the functionalized magnetic nanoparticles (MNPs). The biomarker labeling occurring in the serum was compared using a simple matrix sample: phosphate buffer. The measured signals obtained by using a magnetoresistive (MR) biochip platform showed that the labeling using the proposed microfluidic chip is in good agreement for both matrixes, i.e., the analytical performance (sensitivity) obtained with the serum, near the relevant cutoff values, is within the uncertainty of the measurements obtained with a simple and more controlled matrix: phosphate buffer. This finding is promising for stroke patient stratification where these biomarkers are found at high concentrations in the serum.

Quantitative spatial analysis reveals that the local axons of lamina I projection neurons and interneurons exhibit distributions that predict distinct roles in spinal sensory processing.

Our knowledge about the detailed wiring of neuronal circuits in the spinal dorsal horn (DH), where initial sensory processing takes place, is still very sparse. While a substantial amount of data is available on the somatodendritic morphology of DH neurons, the laminar and segmental distribution patterns and consequential function of individual axons are much less characterized. In the present study, we fully reconstructed the axonal and dendritic processes of 10 projection neurons (PNs) and 15 interneurons (INs) in lamina I of the rat, to reveal quantitative differences in their distribution. We also performed whole-cell patch-clamp recordings to test the predicted function of certain axon collaterals. In line with our earlier qualitative description, we found that lamina I INs in the lateral aspect of the superficial DH send axon collaterals toward the medial part and occupy mostly laminae I-III, providing anatomical basis for a lateromedial flow of information within the DH. Local axon collaterals of PNs were more extensively distributed including dorsal commissural axon collaterals that might refer to those reported earlier linking the lateral aspect of the left and right DHs. PN collaterals dominated the dorsolateral funiculus and laminae IV-VI, suggesting propriospinal and ventral connections. Indeed, patch-clamp recordings confirmed the existence of a dorsoventral excitatory drive upon activation of neurokinin-1 receptors that, although being expressed in various lamina I neurons, are specifically enriched in PNs. In summary, lamina I PNs and INs have almost identical dendritic input fields, while their segmental axon collateral distribution patterns are distinct. INs, whose somata reside in lamina I, establish local connections, may show asymmetry, and contribute to bridging the medial and lateral halves of the DH. PNs, on the other hand, preferably relay their integrated dendritic input to deeper laminae of the spinal gray matter where it might be linked to other ascending pathways or the premotor network, resulting in a putative direct contribution to the nociceptive withdrawal reflex.

Sensory neurons have an axon initial segment that initiates spontaneous activity in neuropathic pain.

The axon initial segment is a specialized compartment of the proximal axon of CNS neurons where action potentials are initiated. However, it remains unknown whether this domain is assembled in sensory dorsal root ganglion neurons, in which spikes are initiated in the peripheral terminals. Here we investigate whether sensory neurons have an axon initial segment and if it contributes to spontaneous activity in neuropathic pain. Our results demonstrate that myelinated dorsal root ganglion neurons assemble an axon initial segment in the proximal region of their stem axon, enriched in the voltage-gated sodium channels Nav1.1 and Nav1.7. Using correlative immunofluorescence and calcium imaging, we demonstrate that the Nav1.7 channels at the axon initial segment are associated with spontaneous activity. Computer simulations further indicate that the axon initial segment plays a key role in the initiation of spontaneous discharges by lowering their voltage threshold. Finally, using a Cre-based mouse model for time-controlled axon initial segment disassembly, we demonstrate that this compartment is a major source of spontaneous discharges causing mechanical allodynia in neuropathic pain. Thus, an axon initial segment domain is present in sensory neurons and facilitates their spontaneous activity. This study provides a new insight in the cellular mechanisms that cause pathological pain and identifies a new potential target for chronic pain management.

Glycoproteogenomics characterizes the CD44 splicing code associated with bladder cancer invasion.

Rationale: Bladder cancer (BC) management demands the introduction of novel molecular targets for precision medicine. Cell surface glycoprotein CD44 has been widely studied as a potential biomarker of BC aggressiveness and cancer stem cells. However, significant alternative splicing and multiple glycosylation generate a myriad of glycoproteoforms with potentially distinct functional roles. The lack of tools for precise molecular characterization has led to conflicting results, delaying clinical applications. Addressing these limitations, we have interrogated the transcriptome and glycoproteome of a large BC patient cohort for splicing signatures. Methods:CD44 gene and its splicing variants were assessed by Real Time-Polymerase Chain Reaction (RT-PCR) and RNAseq in tumor tissues. The co-localization of CD44 and short O-glycans was evaluated by proximity ligation assay (PLA), immunohistochemistry and double-immunofluorescence. An innovative glycoproteogenomics approach, integrating transcriptomics-customized datasets and glycomics for protein annotation from nanoLC-ESI-MS/MS experiments, was developed and implemented to identify CD44 variants and associated glycosignatures. The impact of CD44 silencing on proliferation and invasion of BC cell lines and glycoengineered cells was determined by BrdU ELISA and Matrigel invasion assays, respectively. Antibody phosphoarrays were used to investigate the role of CD44 and its glycoforms in the activation of relevant oncogenic signaling pathways. Results: Transcriptomics analysis revealed remarkable CD44 isoforms heterogeneity in bladder cancer tissues, as well as associations between short CD44 standard splicing isoform (CD44s), invasion and poor prognosis. We further demonstrated that targeting short O-glycoforms such as the Tn and sialyl-Tn antigens was key to overcome the lack of cancer specificity presented by CD44. Glycoproteogenomics allowed, for the first time, the comprehensive characterization of CD44 splicing code at the protein level. The concept was applied to invasive human BC cell lines, glycoengineered cells, and tumor tissues, enabling unequivocal CD44s identification as well as associated glycoforms. Finally, we confirmed the link between CD44 and invasion in CD44s-enriched cells in vitro by small interfering RNA (siRNA) knockdown, supporting findings from BC tissues. The key role played by short-chain O-glycans in CD44-mediated invasion was also demonstrated through glycoengineered cell models. Conclusions: Overall, CD44s emerged as biomarker of poor prognosis and CD44-Tn/ Sialyl-Tn (STn) as promising molecular signatures for targeted interventions. This study materializes the concept of glycoproteogenomics and provides a key vision to address the cancer splicing code at the protein level, which may now be expanded to better understand CD44 functional role in health and disease.

Combined detection of molecular and serological signatures of viral infections: The dual assay concept.

The recent worldwide spread of viral infections has highlighted the need for accurate, fast, and inexpensive disease diagnosis and monitorization methods. Current diagnostics tend to focus either on molecular or serological testing. In this work we propose a dual detection assay approach for viral diseases, where both serological and molecular assays are combined in a single analysis performed on a magnetoresistive system. This type of assay guarantees an accurate assessment of the infection phase, saving time and costs associated with multiple independent tests. Zika and dengue viruses were used as model diseases for the validation of the system. Human IgG anti-zika and anti-dengue antibodies were successfully detected in infected patients' serum, using a novel approach combining competitive and sandwich strategies in a magnetoresistive portable platform. Specificity and sensitivity values of 100% were obtained. Calibration curves with dynamic ranges between 10 ng/mL and 1 µg/mL were established achieving LODs of 1.26 and 1.38 nM for IgG anti-ZIKV and anti-DENV antibodies, respectively. Viral RNA detection down to a few hundreds of pM was also successfully carried out after the design of specific oligo probes and primers for RT-PCR amplification. Dual assays were performed for both viruses, where viral RNA and anti-virus antibodies in serum samples were simultaneously detected. The results obtained for the detection of the molecular and serological targets in the dual assay format show no significant difference between the ones obtained individually, proving the feasibility and accuracy of the dual detection assay. This assay format represents a new paradigm in viral infections diagnostics.

Presynaptic Interactions between Trigeminal and Cervical Nociceptive Afferents Supplying Upper Cervical Lamina I Neurons.

Cervical and trigeminal afferents innervate neighboring cranial territories, and their convergence on upper cervical dorsal horn neurons provides a potential substrate for pain referral in primary headache syndromes. Lamina I neurons are central to this mechanism, as they relay convergent nociceptive input to supraspinal pain centers. Unfortunately, little is known about the interactions between trigeminal and cervical afferents supplying Lamina I neurons. Here, we used rats of both sexes to show that cervical and trigeminal afferents interact via presynaptic inhibition, where monosynaptic inputs to Lamina I neurons undergo unidirectional as well as reciprocal presynaptic control. This means that afferent-driven presynaptic inhibition shapes the way trigeminal and cervical Aδ-fiber and C-fiber input reaches Lamina I projection neurons (PNs) and local-circuit neurons (LCNs). We propose that this inhibition provides a feedforward control of excitatory drive to Lamina I neurons that regulates their convergent and cervical-specific or trigeminal-specific processing modes. As a consequence, disruption of the trigeminal and cervical afferent-driven presynaptic inhibition may contribute to development of primary headache syndromes.SIGNIFICANCE STATEMENT Cervical and trigeminal afferents innervate neighboring cranial territories, and their convergence on upper cervical dorsal horn neurons provides a potential substrate for pain referral in primary headache syndromes. Lamina I neurons are central to this mechanism as they relay convergent nociceptive input to supraspinal pain centers. Here, we show that cervical and trigeminal afferents interact via presynaptic inhibition, where inputs to Lamina I neurons undergo unidirectional as well as reciprocal control. The afferent-driven presynaptic inhibition shapes the trigeminocervical Aδ-fiber and C-fiber input to Lamina I neurons. This inhibition provides control of excitatory drive to Lamina I neurons that regulates their convergent and cervical-specific or trigeminal-specific processing modes. Disruption of this control may contribute to development of primary headache syndromes.

A pump-free microfluidic device for fast magnetic labeling of ischemic stroke biomarkers.

This research proposes a low-cost and simple operation microfluidic chip to enhance the magnetic labeling efficiency of two ischemic stroke biomarkers: cellular fibronectin (c-Fn) and matrix metallopeptidase 9 (MMP9). This fully portable and pump-free microfluidic chip is operated based on capillary attractions without any external power source and battery. It uses an integrated cellulose sponge to absorb the samples. At the same time, a magnetic field is aligned to hold the target labeled by the magnetic nanoparticles (MNPs) in the pre-concentrated chamber. By using this approach, the specific targets are labeled from the beginning of the sampling process without preliminary sample purification. The proposed study enhanced the labeling efficiency from 1 h to 15 min. The dynamic interactions occur in the serpentine channel, while the crescent formation of MNPs in the pre-concentrated chamber, acting as a magnetic filter, improves the biomarker-MNP interaction. The labeling optimization by the proposed device influences the dynamic range by optimizing the MNP ratio to fit the linear range across the clinical cutoff value. The limits of detection (LODs) of 2.8 ng/mL and 54.6 ng/mL of c-Fn measurement were achieved for undiluted and four times dilutions of MNP, respectively. While for MMP9, the LODs were 11.5 ng/mL for undiluted functionalized MNP and 132 ng/mL for four times dilutions of functionalized MNP. The results highlight the potential use of this device for clinical sample preparation and specific magnetic target labeling. When combined with a detection system, it could also be used as an integrated component of a point-of-care platform.

Processing of trigeminocervical nociceptive afferent input by neuronal circuity in the upper cervical lamina I.

ABSTRACT: Afferents from the C2 spinal nerve (SN) and trigeminal nerve (TN) innervate neighboring cranial territories, and their convergence on the upper cervical dorsal horn neurons represents neural substrate of pain referral in primary headache disorders. Unfortunately, little is known about trigeminocervical input to the major spinal nociceptive projection area lamina I. Here, we used ex vivo brainstem-cervical cord preparation for the visually guided whole-cell recording from the upper cervical lamina I neurons. We show that 50% of them receive convergent monosynaptic input from both nerves, whereas 35% and 11% of neurons receive specific supply from the C2 SN and TN, respectively. Altogether, 10 distinct patterns of synaptic input from the C2 SN and TN to lamina I neurons could be identified. Although stimulation of both nerves evoked excitatory/inhibitory responses, more numerous pure inhibitory inputs arose from the TN. We show that cervical and trigeminal nociceptors converge on to lamina I projection and inhibitory neurons. Thus, trigeminocervical input in lamina I is processed in both nerve-specific and convergent circuitries. Afferent convergence on to inhibitory interneurons serves as a feedforward mechanism balancing excitatory drive to projection neurons. Disruption of this balance may cause pain in primary headache syndromes.

Taxonomy and Functional Diversity in the Fecal Microbiome of Beef Cattle Reared in Brazilian Traditional and Semi-Intensive Production Systems.

The importance of beef production for economy of Brazil and the growing demand for animal protein across the globe warrant an improvement in the beef production system. Although most attention has been on modulation of the rumen microbiome to improve ruminant production, the role of the lower gut microbiome in host health and nutrition remains relatively unexplored. This work aimed to investigate the taxonomy and functional variations in the fecal microbiome of Brazilian beef cattle reared in two different production systems using a metagenomic approach. Sixty male beef cattle from six farms representing semi-intensive (I, n = 2) and traditional (T, n = 4) Brazilian beef production systems were enrolled in the study. Shotgun sequencing was used to characterize taxonomic and functional composition and diversity of the microbiome in fecal samples collected from each animal. Fecal samples were analyzed for copper (Cu), lead (Pb), nitrogen (N), phosphorous (P), selenium (Se), and zinc (Zn) and stable isotopes of carbon (13C) and nitrogen (15N). The fecal microbiome was influenced by the beef production systems with greater functional and lower taxonomic diversity in beef cattle feces from I systems compared with that from T systems. The concentration of N, P, and Zn was higher in beef cattle feces from I systems compared with that from T systems and was associated with taxonomic and functional profile of fecal microbiome in I system, suggesting the role of fecal nutrients in shaping system-specific microbiome. Semi-intensive management practices led to a more complex but less connected fecal microbiome in beef cattle. The microbial community in beef cattle feces from I systems was characterized by greater abundance of beneficial bacteria (phylum Firmicutes and butyrate-producing bacteria family Lachnospiraceae and genera Anaerostipes, Blautia, Butyrivibrio, Eubacterium, Roseburia, and Ruminococcus). In addition, the fecal abundance of microbial genes related to immune system, nutrient metabolism, and energy production was greater in beef cattle raised under I systems compared with that under T systems. Findings of the current study suggest that semi-intensive management practices could facilitate the development of a healthier and more efficient fecal microbiome in beef cattle by driving an increase in the abundance of beneficial bacteria and functional genes.

Suicidal behaviour after first-episode psychosis: results from a 1-year longitudinal study in Portugal.

BACKGROUND: Suicide is one of the main causes of excess of premature death in psychotic patients. Published studies found that suicide risk begins in ultra-high risk of psychosis and continues in early years of the disease. Previous studies identifying predictive and risk factors associated with suicidality in first-episode psychosis (FEP) are highly inconsistent. Also, there are relatively few longitudinal studies on suicidal behaviour in FEP. The aim of this study was to examine prevalence, evolution and predictors of suicidal behaviour at baseline and the 12-month follow-up in patients presenting with FEP. METHODS: One hundred and eighteen patients presenting with FEP were recruited from two early psychosis units in Portugal. A comprehensive assessment examining socio-demographic and clinical characteristics was administered at baseline and the 12-month follow-up. Odds ratio were calculated using logistic regression analyses. McNemar test was used to evaluate the evolution of suicidal behaviour and depression prevalence from baseline to 12 months of follow-up. RESULTS: Follow-up data were available for 60 participants from the 118 recruited. Approximately 25.4% of the patients had suicidal behaviour at the baseline evaluation, with a significant reduction during the follow-up period to 13.3% (p = 0.035). A multivariate binary logistic regression showed that a history of suicidal behaviour and depression at baseline independently predicted suicidal behaviour at baseline, and a history of suicidal behaviour and low levels of total cholesterol predicted suicidal behaviour at the 12-month follow-up. A significant proportion of patients also had depression at the baseline evaluation (43.3%), with the last month of suicidal behaviour at baseline independently predicting depression at this time. CONCLUSIONS: The findings of our study indicate that suicidal behaviour was prevalent on the year after FEP. Patients with a history of suicidal behaviour, depression at baseline and low levels of cholesterol should undergo close evaluation, monitoring and possible intervention in order to reduce suicide risk in the early phases of psychosis.

Glycoproteomics identifies HOMER3 as a potentially targetable biomarker triggered by hypoxia and glucose deprivation in bladder cancer.

BACKGROUND: Muscle invasive bladder cancer (MIBC) remains amongst the deadliest genitourinary malignancies due to treatment failure and extensive molecular heterogeneity, delaying effective targeted therapeutics. Hypoxia and nutrient deprivation, oversialylation and O-glycans shortening are salient features of aggressive tumours, creating cell surface glycoproteome fingerprints with theranostics potential. METHODS: A glycomics guided glycoproteomics workflow was employed to identify potentially targetable biomarkers using invasive bladder cancer cell models. The 5637 and T24 cells O-glycome was characterized by mass spectrometry (MS), and the obtained information was used to guide glycoproteomics experiments, combining sialidase, lectin affinity and bottom-up protein identification by nanoLC-ESI-MS/MS. Data was curated by a bioinformatics approach developed in-house, sorting clinically relevant molecular signatures based on Human Protein Atlas insights. Top-ranked targets and glycoforms were validated in cell models, bladder tumours and metastases by MS and immunoassays. Cells grown under hypoxia and glucose deprivation disclosed the contribution of tumour microenvironment to the expression of relevant biomarkers. Cancer-specificity was validated in healthy tissues by immunohistochemistry and MS in 20 types of tissues/cells of different individuals. RESULTS: Sialylated T (ST) antigens were found to be the most abundant glycans in cell lines and over 900 glycoproteins were identified potentially carrying these glycans. HOMER3, typically a cytosolic protein, emerged as a top-ranked targetable glycoprotein at the cell surface carrying short-chain O-glycans. Plasma membrane HOMER3 was observed in more aggressive primary tumours and distant metastases, being an independent predictor of worst prognosis. This phenotype was triggered by nutrient deprivation and concomitant to increased cellular invasion. T24 HOMER3 knockdown significantly decreased proliferation and, to some extent, invasion in normoxia and hypoxia; whereas HOMER3 knock-in increased its membrane expression, which was more pronounced under glucose deprivation. HOMER3 overexpression was associated with increased cell proliferation in normoxia and potentiated invasion under hypoxia. Finally, the mapping of HOMER3-glycosites by EThcD-MS/MS in bladder tumours revealed potentially targetable domains not detected in healthy tissues. CONCLUSION: HOMER3-glycoforms allow the identification of patients' subsets facing worst prognosis, holding potential to address more aggressive hypoxic cells with limited off-target effects. The molecular rationale for identifying novel bladder cancer molecular targets has been established.

ST6Gal1 targets the ectodomain of ErbB2 in a site-specific manner and regulates gastric cancer cell sensitivity to trastuzumab.

The clinical performance of the therapeutic monoclonal antibody trastuzumab in the treatment of ErbB2-positive unresectable gastric cancer (GC) is severely hampered by the emergence of molecular resistance. Trastuzumab's target epitope is localized within the extracellular domain of the oncogenic cell surface receptor tyrosine kinase (RTK) ErbB2, which is known to undergo extensive N-linked glycosylation. However, the site-specific glycan repertoire of ErbB2, as well as the detailed molecular mechanisms through which specific aberrant glycan signatures functionally impact the malignant features of ErbB2-addicted GC cells, including the acquisition of trastuzumab resistance, remain elusive. Here, we demonstrate that ErbB2 is modified with both α2,6- and α2,3-sialylated glycan structures in GC clinical specimens. In-depth mass spectrometry-based glycomic and glycoproteomic analysis of ErbB2's ectodomain disclosed a site-specific glycosylation profile in GC cells, in which the ST6Gal1 sialyltransferase specifically targets ErbB2 N-glycosylation sites occurring within the receptor's trastuzumab-binding domain. Abrogation of ST6Gal1 expression reshaped the cellular and ErbB2-specific glycomes, expanded the cellular half-life of the ErbB2 receptor, and sensitized ErbB2-dependent GC cells to trastuzumab-induced cytotoxicity through the stabilization of ErbB dimers at the cell membrane, and the decreased activation of both ErbB2 and EGFR RTKs. Overall, our data demonstrates that ST6Gal1-mediated aberrant α2,6-sialylation actively tunes the resistance of ErbB2-driven GC cells to trastuzumab.