Pore-forming toxins (PFTs) are key bacterial virulence factors that disrupt host plasma membrane (PM) integrity, triggering cellular stress and initiating repair mechanisms. The cytolysin Listeriolysin O (LLO), secreted by Listeria monocytogenes, has well established roles in infection, yet the host signaling responses to LLO-induced damage remain poorly understood. Here, we identify a previously unrecognized protective pathway in which LLO triggers rapid activation of the tyrosine kinase Src, leading to phosphorylation of the non-muscle myosin II heavy chain NMHC2A at tyrosine 158. While Src activation and NMHC2A tyrosine phosphorylation have been observed during Listeria infection, we demonstrate here that both responses are directly driven by LLO. This phosphorylation event does not alter NMHC2A motor activity in vitro but is required for cytoskeletal reorganization and efficient responses to PM damage. Using Caenorhabditis elegans, we further show that phosphorylation of the NMHC2A homolog NMY-2 at the conserved tyrosine 163 is required for survival under PFT-induced stress and heat shock, revealing an evolutionarily conserved defense mechanism. Together, our findings establish Src-mediated NMHC2A phosphorylation as a critical link between PFT-induced PM damage sensing and actomyosin remodeling, advancing our understanding of host defense against bacterial toxins.

To uncover novel genetic factors required for Listeria monocytogenes cell infection, we developed an automated high-throughput microscopy screening pipeline that integrates GFP-expressing bacteria with machine learning-based image analysis. Using this approach, we screened a mariner transposon library comprising 4224 L. monocytogenes EGDe mutants and identified 58 with significantly reduced numbers of intracellular bacteria. Sequencing revealed 24 unique insertion sites corresponding to 14 genes, including previously known virulence factors and nine novel candidates not previously implicated in cell infection. These genes encode the protease chaperone ClpX, the ferric uptake regulator Fur, the sensor histidine kinase LisK, the peptide chain release factor 2 PrfB, proteins involved in proline and purine biosynthesis (ProAB, PurAB), and Lmo2217, a protein of unknown function. Among these, the targeted deletion of the adenylosuccinate synthetase gene, purA, resulted in impaired growth in minimal medium, severely reduced proliferation in epithelial and macrophage cell lines, and attenuated virulence in mice. Unexpectedly, PurA was also essential for bacterial internalization into cells. Supplementation with AMP or adenine, but not ATP, rescued the invasion capacity of the ΔpurA mutant. Mechanistically, purA deletion induced a reduction in the levels of surface-associated GAPDH, a putative plasminogen-binding protein, likely contributing to the observed invasion defect. Overall, these findings highlight the power of automated high-throughput microscopy screening to dissect host-pathogen interactions, identify novel L. monocytogenes genes required for cell infection, and uncover an unexpected role for PurA in maintaining GAPDH surface localization and promoting bacterial entry into host cells.

Wall teichoic acids (WTAs) from the major Gram-positive foodborne pathogen Listeria monocytogenes are peptidoglycan-associated glycopolymers decorated by monosaccharides that, while not essential for bacterial growth, are required for bacterial virulence and resistance to antimicrobials. Here we report the structure and function of a bacterial WTAs rhamnosyltransferase, RmlT, strictly required for L. monocytogenes WTAs rhamnosylation. In particular, we demonstrated that RmlT transfers rhamnose from dTDP-L-rhamnose to naked WTAs, and that specificity towards TDP-rhamnose is not determined by its binding affinity. Structures of RmlT with and without its substrates showed that this enzyme is a dimer, revealed the residues responsible for interaction with the substrates and that the catalytic residue pre-orients the acceptor substrate towards the nucleophilic attack to the sugar. Additionally, the structures provided indications for two potential interaction pathways for the long WTAs on the surface of RmlT. Finally, we confirmed that WTAs glycosyltransferases are promising targets for next-generation strategies against Gram-positive pathogens by showing that inactivation of the RmlT catalytic activity results in a decreased infection in vivo.

This article focuses on Teresa Noronha’s recent novel, Tornado (2021), which received the Maria Velho da Costa Award from the Portuguese Society of Authors. From Edward Said’s memoir Out of Place (1999), in which he explores the concept of exile as a way of inhabiting space with the constant awareness that one is not at home, I intend to examine how Noronha’s novel surrenders and, at the same time, expands this condition so dear to Said. At the same time, I intend to analyze the complex network of so-called racial relations in Mozambique, and how they contributed to the formation of a self marked by difference and, consequently, to its inner shattering. The feeling of estrangement leads the narrator to an inner exile and, later, an outer exile in search of herself.

Este artigo tem como objetivo analisar a evolução do acesso à creche nos municípios brasileiros no primeiro quinquênio do Plano Nacional de Educação (PNE) em vigor. Para tanto, foram calculados indicadores de atendimento educacional de 5.565 municípios brasileiros, sendo aferidos para 2014, que representa o início do PNE, e para 2019, seu quinto ano de vigência. Para esse cálculo, foram utilizados dados de matrículas de crianças com até três anos e populacionais de mesma faixa etária. Os resultados apontaram um país com baixo acesso à creche, com municípios atendendo, em média, no quinto ano do PNE, 32,2% das crianças com até três anos. Evidenciou-se também a existência de desigualdades de acesso associadas ao nível socioeconômico, distribuição regional e localização dos municípios.

Wall teichoic acids (WTA) from the major Gram-positive foodborne pathogen Listeria monocytogenes are peptidoglycan-associated glycopolymers decorated by monosaccharides that, while not essential for bacterial growth, are required for bacterial virulence and resistance. Here we report the first structure and function of a bacterial WTA rhamnosyltransferase, RmlT, strictly required for L. monocytogenes WTA rhamnosylation. We demonstrated that RmlT transfers rhamnose from TDP-rhamnose to naked WTA, and that specificity towards TDP-rhamnose is kinetically determined. We solved the RmlT structure, identified key residues for its activity and showed that RmlT behaves as a dimer whose interface is important for its stability and function. We propose that RmlT undergoes a transition between an open and closed conformation, allowing RmlT to first bind WTA and then trapping it inside the tunnel formed by the catalytic, oligomerization and helical subdomains. Finally, we proved that mutation of a single residue in the RmlT active site leads to enzyme inactivation and decreased infection in vivo , confirming WTA glycosyltransferases as promising targets for next-generation strategies against Gram-positive pathogens.### Competing Interest StatementThe authors have declared no competing interest.

A return to homeostasis after infection-associated cellular injury can be accelerated by a rapid damage response. S. pneumoniae , a typically asymptomatic colonizer of the host upper respiratory tract, can cause serious and life-threatening infections when it gains access to the lungs and other organs. The cholesterol binding S. pneumoniae pore-forming toxin, pneumolysin (PLY), is central to the induction of host cell damage. Here, we first found that mouse lung infection by S. pneumoniae diminished pulmonary expression of CD73, a glycosylphosphatidylinositol anchored protein (GPI-AP) that modulates inflammation. Infection of the human pulmonary epithelial cell line H292 resulted in a PLY-dependent reduction of not only cell surface CD73, but also the population of surface expressed GPI-APs. The decrease in cell surface GPI-APs was rapid, required pore-forming activity, and could be recapitulated by purified PLY and other cholesterol binding cytolysins. In response to PLY-mediated insult, GPI-APs were not released from the surface of epithelial cells in extracellular vesicles but rather internalized by a mechanism dependent on the Rho GTPases RhoA and Cdc42. Internalization of GPI-APs was associated with lower levels of PLY-induced apoptosis and membrane permeabilization. These findings suggest that internalization of GPI-APs from epithelial cell membranes may constitute a rapid innate repair response to cell damage induced by PLY and other pore forming toxins that could help bacteria evade host defenses as many GPI-APs have roles in immunity.Author summary Streptococcus pneumoniae causes serious infections that can result in mortality. The pore- forming toxin, pneumolysin (PLY) produced by these bacteria is important for their ability to cause disease. Understanding how the host responds to damage by this toxin can result in better treatment against infection. In this study, we found that PLY-mediated injury results in decreased expression of glycosylphosphatidylinositol anchored proteins (GPI-AP) from the cell surface by internalization. GPI-AP co-localize in cholesterol-rich areas of the membrane where PLY inserts to form pores and cells with decreased surface GPI-APs were associated with less of PLY-induced cell death and membrane permeabilization. These results suggest that GPI-AP are internalized as part of repair mechanisms activated in response to infection-induced cell injury. As many GPI-APs have important roles in the immune response, their removal from the cell may inadvertently help the bacteria establish better infection.### Competing Interest StatementThe authors have declared no competing interest.

The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) has been proposed as a key mechanism to eliminate PFT pores and restore PM integrity. We show here that cholesterol-dependent cytolysins (CDCs), a specific family of PFTs, are at least partially eliminated through EVs release, and we hypothesize that proteins important for PM repair might be included in EVs shed by cells during repair. To identify new PM repair proteins, we collected EVs released by cells challenged with sub-lytic doses of two different bacterial CDCs, listeriolysin O and pneumolysin, and determined the EV proteomic repertoire by LC-MS/MS. Intoxicated cells release similar EVs irrespectively of the CDC used. Also, they release more and larger EVs than non-intoxicated cells. A cluster of 70 proteins including calcium-binding proteins, molecular chaperones, cytoskeletal, scaffold and membrane trafficking proteins, was detected enriched in EVs collected from intoxicated cells. While some of these proteins have well-characterized roles in repair, the involvement of others requires further study. As proof of concept, we show here that Copine-1 and Copine-3, proteins abundantly detected in EVs released by intoxicated cells, are required for efficient repair of CDC-induced PM damage. Additionally, we reveal here new proteins potentially involved in PM repair and give new insights into common mechanisms and machinery engaged by cells in response to PM damage.

Resumo Este artigo apresenta uma sistematização e análise do debate acadêmico acerca de avaliações em larga escala no Brasil, sob a forma de um continuum argumentativo. O objetivo é sintetizar o embate discursivo no campo da avaliação da escola básica em zonas argumentativas – apoio incondicional, apoio crítico, recusa propositiva e recusa categórica – que são gradações de um continuum com diferentes graus de apoio e recusa às avaliações em larga escala. Da perspectiva deste trabalho, o embate ideológico entre dois discursos-fonte (ou discursos-geradores) – o discurso economicista e o discurso crítico – gerou ao longo do tempo esses quatro posicionamentos político-educacionais. Metodologicamente, a apreciação dos discursos gravitantes da temática orientou-se pela noção de polifonia (BAKHTIN, 2005; MAINGUENEAU, 2002), ou seja, pelo esforço de abordar, de forma simultânea e equipolente, diferentes vozes que se pronunciaram em relação à controvérsia em tela – um recorte de um conjunto mais amplo de discursos acadêmicos que circulam no país. Ao evidenciar que o debate a respeito da avaliação externa da escola básica no país não se resume à simples polarização entre proponentes e opositores, acredita-se que tal continuum argumentativo – construído com base em produções de autores nacionais – constitui uma síntese (possível) da controvérsia acerca de avaliações em larga escala no Brasil, mimetizando em abstrata miniatura uma realidade heterogênea e complexa de lutas discursivas concretas.

Taking into account the prominent role of the theoretical field of memory studies that has provided us with relevant lenses to examine not only autobiographies, but fictional narratives that emphasize the autobiographical memories of the narrator-characters, this article aims to explore the power of resistance contained in narratives of a personal nature. To this end, the books 'Os Cus de Judas' (1979) by Antonio Lobo Antunes and 'Caderno de Memorias Coloniais' (2009) by Isabela Figueiredo will be analyzed in comparison, also taking into account the element "time" in the construction of a memory about the Portuguese colonial empire. In other words, does temporal distancing imply a less emotionally compromised and more pragmatic memory of past events?

Resumo O objetivo deste artigo é apresentar possibilidades de monitoramento do acesso à creche no nível municipal. Como estratégia metodológica foram calculadas taxas de atendimento escolar utilizando-se dados de matrículas obtidos no censo escolar e dados populacionais calculados a partir da metodologia de correlação de razões. Os resultados demonstraram que, em média, os municípios atendem 32,2% das crianças com até três anos, sendo evidenciadas desigualdades no acesso relacionadas à localização e ao contexto socioeconômico. A metodologia utilizada revelou potencial para auxiliar no monitoramento anual do acesso à creche em municípios do país.

Non-muscle myosin 2A (NM2A) is a key cytoskeletal enzyme that, along with actin, assembles into actomyosin filaments inside cells. NM2A is fundamental for cell adhesion and motility, playing important functions in different stages of development and during the progression of viral and bacterial infections. Phosphorylation events regulate the activity and the cellular localization of NM2A. We previously identified the tyrosine phosphorylation of residue 158 (pTyr(158)) in the motor domain of the NM2A heavy chain. This phosphorylation can be promoted by Listeria monocytogenes infection of epithelial cells and is dependent on Src kinase; however, its molecular role is unknown. Here, we show that the status of pTyr(158) defines cytoskeletal organization, affects the assembly/disassembly of focal adhesions, and interferes with cell migration. Cells overexpressing a non-phosphorylatable NM2A variant or expressing reduced levels of Src kinase display increased stress fibers and larger focal adhesions, suggesting an altered contraction status consistent with the increased NM2A activity that we also observed. We propose NM2A pTyr(158) as a novel layer of regulation of actomyosin cytoskeleton organization.

O artigo caracteriza e analisa iniciativas de avaliação educacional desenvolvidas no estado do Ceará que se revestiram de finalidades e atributos distintos do Sistema Permanente de Avaliação da Educação Básica, o qual tem sido objeto de diversos estudos. São tratadas três iniciativas, implementadas no decurso dos anos 1980, 1990 e 2000: i) avaliação de cursos de formação de profissionais; ii) avaliação institucional; e iii) avaliação de uma vertente da política educacional. A retrospectiva histórica dessas iniciativas se realizou por meio da consulta a documentos que registram seus objetivos e escopo. Intenta-se, com a difusão dessas proposições, iluminar perspectivas de ação que venham a se integrar ao sistema estadual de avaliação desse e de outros entes federados.

this article takes into account the particularity of "geography also [as the protagonist of] the novelistic construction" in the novel Margens e travessias by Boaventura Cardoso, intending to analyze its eco-critical perspective. The "critical" of the word is highlighted, which established itself as an enunciator of a set of theories about the relationship between literature and nature to emphasize the sharpness of the narrative gaze in a book in which little or nothing escapes in the path through the History of Angola, in which the reader is immersed. Through the course of rivers and descriptions of nature, linked to a world prior to colonialism and, consequently, to capitalist exploitation, the book's true intention is to re-write History and better understand the past, inviting the reader to have attention to the mutual dialogue between man and nature, dominator and dominated, and oppressor and victim, in view of a possible policy of conciliation in the future of Angola.

Vitamin B12 deficiency has a wide spectrum of clinical presentation with a variety of neurological symptoms and signs. Early diagnosis and treatment are crucial to prevent the irreversible damage to the nervous system. We present a case report of a 72-year-old woman with 2 weeks history of progressive bilateral hand and feet numbness followed by paresthesia with subsequent development of weakness, and ataxia accompanied with weight loss, vomitus, and diarrhea. The neurological assessment revealed glove and-stocking paresthesia with impaired joint position and vibration sensation in toes and fingers and hyperreflexia. Full blood count revealed macrocytic anemia. Further study showed severe vitamin B12 deficiency and positive for autoantibodies against intrinsic factor and parietal cells. Gastroscopy showed gastric mucosa atrophy with compatible biopsy results. Magnetic resonance imaging (MRI) of the spine revealed an increased T2-weighted signal in the posterior columns of cervical spinal cord, extending from C1 to C7 level, without any gadolinium enhancement on T1-weighted contrast images. These findings were consistent with subacute combined degeneration and autoimmune gastritis with pernicious anemia. Clinical improvement occurred over the ensuing months with parenteral B12 therapy. Neurological symptoms in vitamin B12 deficiency are frequent. Early spinal MR imaging assists in the early diagnosis and treatment.

INTRODUCTIONCoronavirus Disease 2019 (COVID-19) generally appears to have milder clinical symptoms and fewer laboratory abnormalities in children. It remains unknown whether children and young people with inflammatory chronic diseases who acquire SARS-CoV-2 infection have a more severe course, due to either underlying disease or immunosuppressive treatments.OBJECTIVESTo assess the epidemiological features and clinical outcomes of children and young people with inflammatory chronic diseases followed at Pediatric Rheumatology Clinics who were infected with SARS-CoV-2.METHODSA multicentric prospective observational study was performed. Data on demographic variables, clinical features and treatment were collected between March 2020 and September 2021, using the Rheumatic Diseases Portuguese Register (Reuma.pt) and complemented with data from the hospital clinical records.RESULTSThirty-four patients were included, 62% were female, with a median age of 13 [8-16] years and a median time of inflammatory chronic disease of 6 [3-10] years. The most common diagnoses were juvenile idiopathic arthritis (n=22, 64.7%), juvenile dermatomyositis (n=3, 8.8%) and idiopathic uveitis (n=3, 8.8%). Twenty patients were on conventional synthetic disease modifying drugs (csDMARDs) and 10 on biologic DMARDs (bDMARDs). Five patients had an active inflammatory disease at the time of infection (low activity). Seven patients had an asymptomatic infection while 27 patients (79%) had symptoms: cough (n=12), fever (n=11), rhinorrhea (n=10), headache (n=8), malaise (n=8), fatigue (n=7), anosmia (n=5), myalgia (n=5),dysgeusia (n=4), odynophagia (n=4), chest pain (n=2), diarrhea (n=2), arthralgia (n=1), vomiting (n=1) and conjunctivitis (n=1). No patient required hospitalization or directed treatment, and all recovered without sequelae. In 8 patients there was a change in the baseline medication during the infection: suspension of bDMARDs (n=4), reduction of bDMARDs (n=1), suspension of csDMARDs (n=4) and reduction of csDMARDs (n=2). Only in one patient with juvenile dermatomyositis (who discontinued bDMARDs and csDMARDs), the underlying disease worsened.CONCLUSIONSThis is the first study involving children with inflammatory chronic diseases followed at Rheumatology Clinics and SARS-CoV-2 infection in Portugal. In our cohort, mild illness was predominant, which is consistent with the literature. There was no need for hospitalization or specific treatment, and, in most cases, no worsening of the underlying disease was identified.