Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum.

The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.

Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy.

Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.

TBC1D2B undergoes phase separation and mediates autophagy initiation.

Small ubiquitin-like modifiers from the ATG8 family regulate autophagy initiation and progression in mammalian cells. Their interaction with LC3-interacting region (LIR) containing proteins promotes cargo sequestration, phagophore assembly, or even fusion between autophagosomes and lysosomes. Previously, we have shown that RabGAP proteins from the TBC family directly bind to LC3/GABARAP proteins. In the present study, we focus on the function of TBC1D2B. We show that TBC1D2B contains a functional canonical LIR motif and acts at an early stage of autophagy by binding to both LC3/GABARAP and ATG12 conjugation complexes. Subsequently, TBC1D2B is degraded by autophagy. TBC1D2B condensates into liquid droplets upon autophagy induction. Our study suggests that phase separation is an underlying mechanism of TBC1D2B-dependent autophagy induction.

SIK2 orchestrates actin-dependent host response upon Salmonella infection.

Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.

Transmitted drug resistance to antiretroviral drugs in Spain during the period 2019-2021.

To evaluate the prevalence of transmitted drug resistance (TDR) to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTI, NNRTI), protease inhibitors (PI), and integrase strand transfer inhibitors (INSTI) in Spain during the period 2019-2021, as well as to evaluate transmitted clinically relevant resistance (TCRR) to antiretroviral drugs. Reverse transcriptase (RT), protease (Pro), and Integrase (IN) sequences from 1824 PLWH (people living with HIV) were studied. To evaluate TDR we investigated the prevalence of surveillance drug resistance mutations (SDRM). To evaluate TCRR (any resistance level ≥ 3), and for HIV subtyping we used the Stanford v.9.4.1 HIVDB Algorithm and an in-depth phylogenetic analysis. The prevalence of NRTI SDRMs was 3.8% (95% CI, 2.8%-4.6%), 6.1% (95% CI, 5.0%-7.3%) for NNRTI, 0.9% (95% CI, 0.5%-1.4%) for PI, and 0.2% (95% CI, 0.0%-0.9%) for INSTI. The prevalence of TCRR to NRTI was 2.1% (95% CI, 1.5%-2.9%), 11.8% for NNRTI, (95% CI, 10.3%-13.5%), 0.2% (95% CI, 0.1%-0.6%) for PI, and 2.5% (95% CI, 1.5%-4.1%) for INSTI. Most of the patients were infected by subtype B (79.8%), while the majority of non-Bs were CRF02_AG (n = 109, 6%). The prevalence of INSTI and PI resistance in Spain during the period 2019-2021 is low, while NRTI resistance is moderate, and NNRTI resistance is the highest. Our results support the use of integrase inhibitors as first-line treatment in Spain. Our findings highlight the importance of ongoing surveillance of TDR to antiretroviral drugs in PLWH particularly with regard to first-line antiretroviral therapy.

Modulation of Synaptic Plasticity Genes Associated to DNA Damage in a Model of Huntington's Disease.

Huntington's disease (HD) is a disease characterized by the progressive degeneration of nerve cells in the brain. DNA damage has been implicated in many neurological disorders; however, the association between this damage and the impaired signaling related to neurodegeneration is still unclear. The transcription factor c-AMP-responsive element binding protein (CREB) has a relevant role in the neuronal plasticity process regulating the expression of several genes, including brain-derived neurotrophic factor (BDNF). Here we analyzed the direct link between DNA damage and the expression of genes involved in neuronal plasticity. The study was performed in model cell lines STHdhQ7 (wild type) and STHdhQ111 (HD model). Treatment with Etoposide (Eto) was used to induce double-strand breaks (DSBs) to evaluate the DNA damage response (DDR) and the expression of synaptic plasticity genes. Eto treatment induced phosphorylation of ATM (p-ATM) and H2AX (γH2AX), markers of DDR, in both cell lines. Interestingly, upon DNA damage, STHdhQ7 cells showed increased expression of activity-regulated cytoskeleton associated protein (Arc) and BDNF when compared to the HD cell line model. Additionally, Eto induced CREB activation with a differential localization of its co-activators in the cell types analyzed. These results suggest that DSBs impact differentially the gene expression patterns of plasticity genes in the normal cell line versus the HD model. This effect is mediated by the impaired localization of CREB-binding protein (CBP) and histone acetylation in the HD model. Our results highlight the role of epigenetics and DNA repair on HD and therefore we suggest that future studies should explore in depth the epigenetic landscape on neuronal pathologies with the goal to further understand molecular mechanisms and pinpoint therapeutic targets.

TBK1-mediated phosphorylation of LC3C and GABARAP-L2 controls autophagosome shedding by ATG4 protease.

Autophagy is a highly conserved catabolic process through which defective or otherwise harmful cellular components are targeted for degradation via the lysosomal route. Regulatory pathways, involving post-translational modifications such as phosphorylation, play a critical role in controlling this tightly orchestrated process. Here, we demonstrate that TBK1 regulates autophagy by phosphorylating autophagy modifiers LC3C and GABARAP-L2 on surface-exposed serine residues (LC3C S93 and S96; GABARAP-L2 S87 and S88). This phosphorylation event impedes their binding to the processing enzyme ATG4 by destabilizing the complex. Phosphorylated LC3C/GABARAP-L2 cannot be removed from liposomes by ATG4 and are thus protected from ATG4-mediated premature removal from nascent autophagosomes. This ensures a steady coat of lipidated LC3C/GABARAP-L2 throughout the early steps in autophagosome formation and aids in maintaining a unidirectional flow of the autophagosome to the lysosome. Taken together, we present a new regulatory mechanism of autophagy, which influences the conjugation and de-conjugation of LC3C and GABARAP-L2 to autophagosomes by TBK1-mediated phosphorylation.

Impaired intracellular trafficking of sodium-dependent vitamin C transporter 2 contributes to the redox imbalance in Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by a glutamine expansion at the first exon of the huntingtin gene. Huntingtin protein (Htt) is ubiquitously expressed and it is localized in several organelles, including endosomes. HD is associated with a failure in energy metabolism and oxidative damage. Ascorbic acid is a powerful antioxidant highly concentrated in the brain where it acts as a messenger, modulating neuronal metabolism. It is transported into neurons via the sodium-dependent vitamin C transporter 2 (SVCT2). During synaptic activity, ascorbic acid is released from glial reservoirs to the extracellular space, inducing an increase in SVCT2 localization at the plasma membrane. Here, we studied SVCT2 trafficking and localization in HD. SVCT2 is decreased at synaptic terminals in YAC128 male mice. Using cellular models for HD (STHdhQ7 and STHdhQ111 cells), we determined that SVCT2 trafficking through secretory and endosomal pathways is altered in resting conditions. We observed Golgi fragmentation and SVCT2/Htt-associated protein-1 mis-colocalization. Additionally, we observed altered ascorbic acid-induced calcium signaling that explains the reduced SVCT2 translocation to the plasma membrane in the presence of extracellular ascorbic acid (active conditions) described in our previous results. Therefore, SVCT2 trafficking to the plasma membrane is altered in resting and active conditions in HD, explaining the redox imbalance observed during early stages of the disease.

Switch as maintenance to elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate: week 48 results in a clinical cohort.

Objectives: To assess, in a clinical cohort, the efficacy of switching current ART in virologically suppressed patients to elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate as a single-tablet regimen (STR) using the PCR signal of the plasma viral load (pVL) assay and determination of plasma drug concentration ( C 24 ). Patients and methods: This was an observational single-centre study enrolling antiretroviral-treated patients with pVL <50 copies/mL initiating elvitegravir-based STR. PCRneg was defined as an undetected PCR signal. Results: One hundred and fifty-one patients were enrolled. At STR baseline, the median time since first ART and time of virological suppression were 5 years (IQR 3-9) and 24 months (IQR 9-44), respectively. By week (W) 48, 26 (17%) of the patients had discontinued STR due to adverse events. The proportion of patients maintaining pVL <50 copies/mL on treatment was 98%, 96%, 93% and 97% at W12, W24, W36 and W48, respectively. Five patients (3.3%) experienced a virological failure and emergence of resistance was observed in two of them with the selection of M184V and N155H mutations. At baseline, W12, W24, W36 and W48, 70%, 57%, 72%, 61% and 74% of the patients with pVL <20 copies/mL had a PCRneg, respectively. The median elvitegravir plasma C 24 value was 648 ng/mL (IQR 348-989; n = 237), with 84% of elvitegravir C 24 values >45 ng/mL, the protein-adjusted IC 95 . Conclusions: In this clinical cohort of virologically suppressed patients switching to STR, most subjects had adequate elvitegravir C 24 values with a high proportion maintaining virological suppression with no residual viraemia until W48.

Pharmacokinetics, safety and efficacy of a full dose sofosbuvir-based regimen given daily in hemodialysis patients with chronic hepatitis C.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is an independent risk factor for chronic kidney disease and leads to faster liver disease progression in patients requiring hemodialysis than in those with normal renal function. Little is known about the use of a sofosbuvir-containing regimen for infected patients on hemodialysis. We aimed to describe the pharmacokinetics, safety and efficacy of sofosbuvir in 2 dosing regimens and associated antiviral agents in HCV-infected patients requiring hemodialysis. METHODS: Multicenter, prospective and observational study of patients receiving sofosbuvir, 400mg once daily (n=7) or 3 times a week (n=5), after hemodialysis with simeprevir, daclatasvir, ledipasvir or ribavirin was conducted. Drug plasma concentrations were determined by liquid chromatography-tandem mass spectrometry before and after a 4h hemodialysis and 1.5h after last drug intake at the end of hemodialysis. RESULTS: Plasma concentrations of sofosbuvir or its inactive metabolite sofosbuvir-007 did not accumulate with either regimen between hemodialysis sessions or throughout the treatment course. Sofosbuvir-007 extraction ratio (52%) was consistent with historical data. In one patient receiving the once daily regimen, sofosbuvir-007 half-life was slightly higher (38h) than for patients with normal renal function receiving a full dose. Hemodialysis did not remove any other associated anti-HCV agents. Clinical and biological tolerance was good for all patients. Two relapses occurred with the 3 times a week regimen and none with the once daily. CONCLUSIONS: A regimen including sofosbuvir, 400mg once daily, could be proposed for HCV-infected patients requiring hemodialysis and should be associated with close clinical, biological, cardiovascular, and therapeutic drug monitoring. LAY SUMMARY: Hepatitis C Virus (HCV) infection in hemodialysis patients is prevalent and aggressive. Effective anti-HCV treatment in these patients may stabilize their renal disease. However, sofosbuvir, the cornerstone of most anti-HCV-containing regimens, should not be administered to these patients until more data is available. In this pharmacokinetic study, sofosbuvir full dose (400mg once daily) administered every day with another direct antiviral agent did not accumulate in hemodialysis patients and was safe and effective.

Old Things New View: Ascorbic Acid Protects the Brain in Neurodegenerative Disorders.

Ascorbic acid is a key antioxidant of the Central Nervous System (CNS). Under brain activity, ascorbic acid is released from glial reservoirs to the synaptic cleft, where it is taken up by neurons. In neurons, ascorbic acid scavenges reactive oxygen species (ROS) generated during synaptic activity and neuronal metabolism where it is then oxidized to dehydroascorbic acid and released into the extracellular space, where it can be recycled by astrocytes. Other intrinsic properties of ascorbic acid, beyond acting as an antioxidant, are important in its role as a key molecule of the CNS. Ascorbic acid can switch neuronal metabolism from glucose consumption to uptake and use of lactate as a metabolic substrate to sustain synaptic activity. Multiple evidence links oxidative stress with neurodegeneration, positioning redox imbalance and ROS as a cause of neurodegeneration. In this review, we focus on ascorbic acid homeostasis, its functions, how it is used by neurons and recycled to ensure antioxidant supply during synaptic activity and how this antioxidant is dysregulated in neurodegenerative disorders.

Animal Performance and Carcass Characteristics of Bulls (1/2 Purunã vs 1/2 Canchim) Slaughtered at 16 and 22 Months Old, and Three Different Weights.

Current study aimed to evaluate the performance of bulls (1/2 Purunã vs 1/2 Canchim) slaughtered at two ages and three different weights. One hundred and thirteen bulls were divided into two slaughter ages (16 and 22 months) and three different slaughter weights (light, 422 kg; medium, 470 kg; and heavy, 550 kg). The body weight was higher for bulls slaughtered at 16 months. Daily gain, carcass weight and dressing were higher for bulls slaughtered at 16 months. Feed intake was higher for bulls slaughtered at 22 months although feed efficiency was better for bulls slaughtered at 16 months. Carcass characteristics were better for bulls slaughtered at 16 months. The percentages of muscle, fat and bone and meat characteristics were similar between two slaughter ages. Feed intake and animal performance was lower for lighter animals. Feed conversion and carcass dressing were similar in the three slaughter weights. Muscle percentage was higher for heavier animals but fat and bone percentages were lower. Slaughter weight had no effect on meat characteristics.

Knowledge on dental trauma management and caries prevention.

PURPOSE: This study evaluated the knowledge of dental assistants (DA) and hygienists (DH) on dental trauma management and dental caries prevention. Secondarily, other aims were 2-fold: (1) to update the assistants/hygienists through courses supported on scientific evidence-based information and (2) to evaluate their job satisfaction. METHODS: A cross-sectional study was conducted with 46 participants, DHs and Das, working in the Secretary of Education of the city of Brasilia (Federal District, Brazil). The information was gathered through 2 self-administered questionnaires during 2 training meetings. The first questionnaire was applied before the first meeting and the second questionnaire after the last meeting. All participants were women older than 38 years and with more than 15 years of experience. RESULTS: The participants demonstrated knowledge on dental trauma types but lack of knowledge on dental trauma first-aid approaches. Concerning to dental caries, the professional demonstrated knowledge on both etiology and prevention. CONCLUSION: Lectures, periodical training, and inclusion in multidisciplinary training groups aiming to caries prevention and dental trauma are necessary.

Functional Capacity of Oldest Old Living in a Long-stay Institution in Rio De Janeiro, Brazil.

[Purpose] A significant increase in the number of oldest old has occurred worldwide. The aim of this study was to characterize the functional capacity of the oldest old residents in a long-stay institution in Rio de Janeiro, Brazil. [Subjects and Methods] All participants were evaluated according to the following metrics: anthropometry, body composition (bioelectrical impedance), handgrip strength, balance (Berg scale and stabilometry), ankle mobility (electrogoniometry), physical capacity (six-minute walk test), quality of life (WHOQOL-OLD), and dietary habits (questionnaire). [Results] Twenty elderly subjects with a mean age of 85.75 ± 5.22 years and a mean fat percentage of 39.02 ± 5.49% participated in the study. The group at risk of falls (n = 8) had a lower handgrip strength and walked a shorter distance over the course of six minutes compared with the group not at risk of falls. The obese group (n = 15) had higher values for stabilometric variables than the nonobese group. There was a positive and significant correlation between ankle joint mobility and physical capacity (r = 0.47). [Conclusion] High values for obesity and low values for handgrip strength and physical capacity were associated with worse body balance. Low values for ankle mobility were also associated with worse physical capacity in this population.

Perception of graduates about the education of Nursing Technicians.

OBJECTIVES: to interpret the perception of graduates from the nursing technician program on the learning process developed during their education. METHODS: a qualitative study was conducted from March to September 2021, based on interviews with 20 graduates from a nursing technician program at a school in the Midwest of São Paulo, using thematic analysis and the NVivo tool. RESULTS: the following themes were identified: traditional teaching methods, active learning strategies, valuing proactivity and experiences in professional practice. FINAL CONSIDERATIONS: according to the graduates, the teaching process is essentially based on traditional methods, although there are initiatives to implement active strategies and recognition of the importance of advancing student proactivity and practical experience.

Impact of mesenchymal stromal cell-derived vesicular cargo on B-cell acute lymphoblastic leukemia progression.

Leukemia cells reciprocally interact with their surrounding bone marrow microenvironment (BMM), rendering it hospitable to leukemia cell survival, for instance through the release of small extracellular vesicles (sEVs). In contrast, we show here that BMM deficiency of pleckstrin homology domain family M member 1 (PLEKHM1), which serves as a hub between fusion and secretion of intracellular vesicles and is important for vesicular secretion in osteoclasts, accelerates murine BCR-ABL1+ B-cell acute lymphoblastic leukemia (B-ALL) via regulation of the cargo of sEVs released by BMM-derived mesenchymal stromal cells (MSCs). PLEKHM1-deficient MSCs and their sEVs carry increased amounts of syntenin and syndecan-1, resulting in a more immature B-cell phenotype and an increased number/function of leukemia-initiating cells (LICs) via focal adhesion kinase and AKT signaling in B-ALL cells. Ex vivo pretreatment of LICs with sEVs derived from PLEKHM1-deficient MSCs led to a strong trend toward acceleration of murine and human BCR-ABL1+ B-ALL. In turn, inflammatory mediators such as recombinant or B-ALL cell-derived tumor necrosis factor α or interleukin-1ß condition murine and human MSCs in vitro, decreasing PLEKHM1, while increasing syntenin and syndecan-1 in MSCs, thereby perpetuating the sEV-associated circuit. Consistently, human trephine biopsies of patients with B-ALL showed a reduced percentage of PLEKHM1+ MSCs. In summary, our data reveal an important role of BMM-derived sEVs for driving specifically BCR-ABL1+ B-ALL, possibly contributing to its worse prognosis compared with BCR-ABL1- B-ALL, and suggest that secretion of inflammatory cytokines by cancer cells in general may similarly modulate the tumor microenvironment.

An atypical GABARAP binding module drives the pro-autophagic potential of the AML-associated NPM1c variant.

The nucleolar scaffold protein NPM1 is a multifunctional regulator of cellular homeostasis, genome integrity, and stress response. NPM1 mutations, known as NPM1c variants promoting its aberrant cytoplasmic localization, are the most frequent genetic alterations in acute myeloid leukemia (AML). A hallmark of AML cells is their dependency on elevated autophagic flux. Here, we show that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB, thereby coordinating the expression of lysosomal proteins and autophagy regulators. Importantly, both NPM1 and NPM1c bind to autophagy modifiers of the GABARAP subfamily through an atypical binding module preserved within its N terminus. The propensity of NPM1c to induce autophagy depends on this module, likely indicating that NPM1c exerts its pro-autophagic activity by direct engagement with GABARAPL1. Our data report a non-canonical binding mode of GABARAP family members that drives the pro-autophagic potential of NPM1c, potentially enabling therapeutic options.

18F-FDG PET/CT in Patients with Vulvar and Vaginal Cancer: A Preliminary Study of 20 Cases.

INTRODUCTION: Despite the growing evidence supporting the use of 2-[F-18]-fluor-2-desoxi-D-glucose positron emission tomography/computed tomography in cervical and ovarian malignant tumours, data on vulvar and vaginal cancer is sparse. Our aim was to assess the role of 2-[F-18]-fluor-2-desoxi-D-glucose positron emission tomography/computed tomography in patients with vulvar and vaginal cancer. MATERIAL AND METHODS: A retrospective study was conducted on a cohort of 20 patients with biopsy-proven vulvar (n = 17) and vaginal (n = 3) cancer who performed 2-[F-18]-fluor-2-desoxi-D-glucose positron emission tomography/computed tomography, between January 2013 and April 2018. We collected the clinical data of all patients, as well as the indication for 2-[F-18]-fluor-2-desoxi-D-glucose positron emission tomography/computed tomography, its results, and the main lesion maximum standard uptake value (SUVmax). In addition, we correlated the results of 2-[F-18]-fluor-2-desoxi-D-glucose positron emission tomography/computed tomography with other diagnostic modalities, namely histological findings, computed tomography and magnetic resonance imaging. Patients were divided into two groups, one with newly diagnosed disease and another with recurrent disease. RESULTS: Six patients had newly diagnosed disease and 14 had recurrent disease. The main lesion was detected by 2-[F-18]-fluor-2-desoxi-D-glucose positron emission tomography/computed tomography in five out of six patients with newly diagnosed disease and in all 14 patients with recurrent disease. Additional sites of 2-[F-18]-fluor-2-desoxi-D-glucose uptake were identified in inguinal and iliac lymph nodes and in distant lesions. Magnetic resonance imaging and computed tomography were performed in 12 cases. In four patients with recurrent disease, abnormalities (main lesion/ metastatic lymph nodes) identified by 2-[F-18]-fluor-2-desoxi-D-glucose positron emission tomography/computed tomography were not detected as suspicious by computed tomography. DISCUSSION: In our study, 2-[F-18]-fluor-2-desoxi-D-glucose positron emission tomography/computed tomography identified abnormalities more often than conventional computed tomography scans in recurrent disease. In comparison with histology, 2-[F-18]-fluor-2-desoxi-D-glucose positron emission tomography/computed tomography had a sensitivity of 95% and a positive predictive value of 100% in identifying the primary tumour and the recurrent main lesion. Little data is available regarding the usefulness of 2-[F-18]-fluor-2-desoxi-D-glucose positron emission tomography/computed tomography in the management of vulvar and vaginal cancers. The existing evidence supports a high accuracy in detecting lymph node metastases and a change of 36.0% - 61.5% in patient management. Our findings reinforce the usefulness of this technique in vulvar and vaginal cancer. Limitations of our study include its retrospective nature and the rareness of both vulvar and vaginal cancer, which leads to a small sample size and few comparative imaging tests. CONCLUSION: In this preliminary study, 2-[F-18]-fluor-2-desoxi-D-glucose positron emission tomography/computed tomography demonstrated it can be a useful method in patients with vulvar and vaginal cancers, namely in defining the extent of disease and contributing to accurate staging and restaging.

Introdução: Apesar da crescente evidência que suporta o uso da tomografia por emissão de positrões/ tomografia computadorizada com 2-[F-18]-fluor-2-desoxi-D-glucose em tumores malignos do colo do útero e do ovário, os dados sobre o carcinoma da vulva e da vagina são escassos. O nosso objetivo foi avaliar o papel da tomografia por emissão de positrões/ tomografia computadorizada com 2-[F-18]-fluor-2-desoxi-D-glucose em doentes com carcinoma da vulva e da vagina. Material e Métodos: Entre janeiro de 2013 e abril de 2018 foi realizado um estudo retrospetivo numa coorte de 20 doentes com carcinoma da vulva (n = 17) e da vagina (n = 3), comprovados por biópsia, que efetuaram tomografia por emissão de positrões/ tomografia computadorizada com 2-[F-18]-fluor-2-desoxi-D-glucose. Recolheram-se os dados clínicos de todos os doentes, bem como a indicação clínica para a realização da tomografia por emissão de positrões/ tomografia computadorizada com 2-[F-18]-fluor-2-desoxi-D-glucose, os seus resultados e o valor de captação padronizado máximo da lesão principal (SUVmax). Para além disso, correlacionaram-se os resultados da tomografia por emissão de positrões/ tomografia computadorizada com 2-[F-18]-fluor-2-desoxi-D-glucose com os de outras modalidades diagnósticas, nomeadamente com os achados histológicos, a tomografia computadorizada e a ressonância magnética. Os doentes foram divididos em dois grupos, um com doença recém diagnosticada e outro com doença recorrente. Resultados: Seis doentes tinham doença recém diagnosticada e 14 tinham doença recorrente. A lesão principal foi detetada em cinco dos seis doentes com doença recém diagnosticada e nos 14 com doença recorrente. Foram identificados outros locais de captação de 2-[F-18]-fluor-2-desoxi-D-glucose, nomeadamente gânglios linfáticos ilíacos e inguinais, e lesões à distância. Em 12 casos foram realizadas ressonância magnética e tomografia computadorizada. Em quatro casos com doença recorrente, as anomalias (lesão principal /gânglios linfáticos metastáticos) identificadas na tomografia por emissão de positrões/ tomografia computadorizada com 2-[F-18]-fluor-2-desoxi-D-glucose não haviam sido descritas como suspeitas pela tomografia computadorizada. Discussão: No nosso estudo, a tomografia por emissão de positrões/ tomografia computadorizada com 2-[F-18]-fluor-2-desoxi-D-glucose identificou mais anomalias que a tomografia computadorizada na doença recorrente. Comparando com os resultados histológicos, a tomografia por emissão de positrões/ tomografia computadorizada com 2-[F-18]-fluor-2-desoxi-D-glucose apresentou sensibilidade de 95% e um valor preditivo positivo de 100% na identificação do tumor primário/ lesão principal recorrente. Poucos dados estão disponíveis sobre a utilidade da tomografia por emissão de positrões/ tomografia computadorizada com 2-[F-18]-fluor-2-desoxi-D-glucose no seguimento de carcinomas da vulva e da vagina. As evidências atuais sugerem uma elevada exatidão na deteção de metástases ganglionares e uma mudança de 36,0% - 61,5% no tratamento destes doentes. Os nossos achados reforçam a utilidade desta técnica no carcinoma da vulva e da vagina. As limitações do nosso estudo decorrem da sua natureza retrospetiva e da raridade das patologias estudadas, o que condiciona o tamanho da amostra e a quantidade de exames de imagem comparativos. Conclusão: Neste estudo preliminar, a tomografia por emissão de positrões/ tomografia computadorizada com 2-[F-18]-fluor-2-desoxi-D-glucose demonstrou poder ser um método útil em doentes com carcinoma da vulva e da vagina, nomeadamente na definição da extensão da doença e na contribuição para o estadiamento e restadiamento precisos.

New insight into the role of PTCH1 protein in serous ovarian carcinomas.

The Hedgehog (Hh) signaling pathway is essential for normal embryonic development, while its hyperactivation in the adult organism is associated with the development of various cancers. The role of the Hh signaling pathway in ovarian cancer has not been sufficiently investigated. Therefore, the present study investigated the role of protein patched homolog 1 (PTCH1), a component of the Hh signaling pathway, and changes in the promoter methylation status of the corresponding gene in a cohort of low­(LGSC) and high­grade serous ovarian carcinomas (HGSC) and HGSC cell lines (OVCAR8 and OVSAHO). PTCH1 protein expression level was analyzed using immunohistochemistry in tissue samples and immunofluorescence and western blotting in cell lines. DNA methylation patterns of the PTCH1 gene were analyzed using methylation­specific PCR. PTCH1 protein expression was significantly higher in HGSCs and LGSCs compared with controls (healthy ovaries and fallopian tubes). Similarly, ovarian cancer cell lines exhibited significantly higher PTCH1 protein expression compared with a normal fallopian tube non­ciliated epithelial cell line (FNE1). PTCH1 protein fragments of different molecular weights were detected in all cell lines, indicating possible proteolytic cleavage of this protein, resulting in the generation of soluble N­terminal fragments that are translocated to the nucleus. DNA methylation of the PTCH1 gene promoter was exclusively detected in a proportion of HGSC (13.5%) but did not correlate with protein expression. PTCH1 protein was highly expressed in serous ovarian carcinoma tissues and cell lines, while PTCH1 promoter methylation was only detected in HGSC. Further investigation is required to elucidate the possible mechanisms of PTCH1 activation in serous ovarian carcinomas.

Incidence and Severity of SARS-CoV-2 Infection in HIV-Infected Individuals During the First Year of the Pandemic.

BACKGROUND: We aim to investigate the infection rate, the clinical characteristics and outcomes of COVID-19-disease in a cohort of people living with HIV in Madrid (Spain), during the first year of pandemics. SETTING: Observational single-center study, in which we included all HIV-infected patients (aged ≥ 18 years) with laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection as of February 28, 2021, at the Hospital Universitario 12 de Octubre. METHODS: Confirmed disease was defined as any patient with a positive antigen test, reverse transcriptase polymerase chain reaction, or serology for SARS-CoV-2. We compared the characteristics of patients with mild disease (asymptomatic included) with those with moderate or severe disease (requiring admission). RESULTS: Of 2344 HIV-infected patients, 158 (82.9% male; median age, 46.5 years) were diagnosed with SARS-CoV-2 (infection rate, 6.74%; 95% confidence interval, 5.79 to 7.83). Thirty-nine individuals (24.7%) had moderate or severe disease, 43.7% had mild disease, and 31.6% were asymptomatic. Hypertension (23.4%) and obesity (15.8%) were the most prevalent comorbidities; 12.7% had at least 2 comorbidities. One hundred forty-five patients (97.3%) had RNA-HIV viral load of <50 copies per milliliter, and only 3 had CD4 cell count of <200 cells per cubic millimeter before infection. Of those admitted to hospital, 59% required oxygen support and 15.4%, invasive mechanical ventilation. Five patients died. None of the patient taking tenofovir-disoproxil-fumarate required admission. In the multivariate analysis, age remained as the only independent factor for moderate-severe disease (odds ratio, 1.09; 95% confidence interval 1.04 to 1.14; P < 0.001). CONCLUSIONS: People living with HIV are at risk of severe SARS-CoV-2 infection. Age was the only variable with an independent association with moderate-severe disease, after adjusting by comorbidities and other factors.