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The extensive genetic diversity of HIV-1 is a major challenge for the prevention and treatment of HIV-1 infections. Subtype C accounts for most of the HIV-1 infections in the world but has been mainly localized in Southern Africa, Ethiopia and India. For elusive reasons, South Brazil harbors the largest HIV-1 subtype C epidemic in the American continent that is elsewhere dominated by subtype B. To investigate this topic, we collected clinical data and viral sequences from 2611 treatment-naïve patients diagnosed with HIV-1 in Brazil. Molecular epidemiology analysis supported 35 well-delimited transmission clusters of subtype C highlighting transmission within South Brazil but also from the South to all other Brazilian regions and internationally. Individuals infected with subtype C had lower probability to be deficient in CD4+ T cells when compared to subtype B. The HIV-1 epidemics in the South was characterized by high female-to-male infection ratios and women-to-child transmission. Our results suggest that HIV-1 subtype C probably takes advantage of longer asymptomatic periods to maximize transmission and is unlikely to outcompete subtype B in settings where the infection of women is relatively less relevant. This study contributes to elucidate factors possibly underlying the geographical distribution and expansion patterns of the most spread HIV-1 subtypes.

Tuberculosis (TB), one of the deadliest threats to human health, is mainly caused by 2 highly related and human-adapted bacteria broadly known as Mycobacterium tuberculosis and Mycobacterium africanum. Whereas M. tuberculosis is widely spread, M. africanum is restricted to West Africa, where it remains a significant cause of tuberculosis. Although several differences have been identified between these 2 pathogens, M. africanum remains a lot less studied than M. tuberculosis. Here, we discuss the genetic, phenotypic, and clinical similarities and differences between strains of M. tuberculosis and M. africanum. We also discuss our current knowledge on the immune response to M. africanum and how it possibly articulates with distinct disease progression and with the geographical restriction attributed to this pathogen. Understanding the functional impact of the diversity existing in TB-causing bacteria, as well as incorporating this diversity in TB research, will contribute to the development of better, more specific approaches to tackle TB.

Almost two years have passed since COVID-19 was officially declared a pandemic by the World Health Organization. However, it still holds a tight grasp on the entire human population. Several variants of concern, one after another, have spread throughout the world. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant may become the fastest spreading virus in history. Therefore, it is more than evident that the use of personal protective equipment (PPE) will continue to play a pivotal role during the current pandemic. This work depicts an integrative approach attesting to the effectiveness of ultra-violet-C (UV-C) energy density for the sterilization of personal protective equipment, in particular FFP2 respirators used by the health care staff in intensive care units. It is increasingly clear that this approach should not be limited to health care units. Due to the record-breaking spreading rates of SARS-CoV-2, it is apparent that the use of PPE, in particular masks and respirators, will remain a critical tool to mitigate future pandemics. Therefore, similar UV-C disinfecting rooms should be considered for use within institutions and companies and even incorporated within household devices to avoid PPE shortages and, most importantly, to reduce environmental burdens., This research was funded by the Portuguese Foundation for Science and Technology (FCT), European Regional Development Fund (FEDER), Operational Program for Competitive ness Factors (COMPETE), and Portuguese Ministry of Science, Technology and Higher Education (MCTS) [UID/CTM/00264/2021]; [PTDC/CTM TEX/28295/2017]; [PTDC/CTM-TEX/1213/2020]; [UIDB/50026/2020]; [NORTE-01-0145-FEDER-072555]; [NORTE-01-0145-FEDER-000039] and con tract funding [2020.03113.CEECIND] to M.I.V. This work is a result of a project with the EXMA company in co-promotion with the University of Minho [NORTE-01-02B7-FEDER-048968] and supported by the Northern Portugal Regional Operational Program (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the FEDER. Finally, this project was funded by the FCT Research4COVID-19 special fund [011_595803006UV-Fast].

Surface disinfection currently plays a decisive role in the epidemiological situation caused by the SARS-CoV-2 coronavirus. However, most disinfection products available on the market have a high evaporation rate and only an immediate action and not continuous, creating the need for a high frequency of disinfection. To overcome this limitation, in the present work, poly(methyl methacrylate) (PMMA) microcapsules were developed with an active agent (hydrogen peroxide) encapsulated, which has the ability to inactivate/neutralize the SARS-CoV-2 virus. PMMA-H

14 páginas, 6 figuras. Supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-15832-6. Sequence data are deposited on NCBI GEO database with the accession code GSE138580, Genetic diversity of Mycobacterium tuberculosis affects immune responses and clinical outcomes of tuberculosis (TB). However, how bacterial diversity orchestrates immune responses to direct distinct TB severities is unknown. Here we study 681 patients with pulmonary TB and show that M. tuberculosis isolates from cases with mild disease consistently induce robust cytokine responses in macrophages across multiple donors. By contrast, bacteria from patients with severe TB do not do so. Secretion of IL-1β is a good surrogate of the differences observed, and thus to classify strains as probable drivers of different TB severities. Furthermore, we demonstrate that M. tuberculosis isolates that induce low levels of IL-1β production can evade macrophage cytosolic surveillance systems, including cGAS and the inflammasome. Isolates exhibiting this evasion strategy carry candidate mutations, generating sigA recognition boxes or affecting components of the ESX-1 secretion system. Therefore, we provide evidence that M. tuberculosis strains manipulate host-pathogen interactions to drive variable TB severities, This work was financed by FCT-Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Inovação grant POCI-01-0145-FEDER-028955 (to M.S.) and by the Northern Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013, to M.I.V., F.R., A.G.C., and N.S.O.). I.C.acknowledges the support of Ministerio de Ciencia, Innovación y Universidades (SAF2016-77346-R) and the European Research Council (638553-TB-ACCELERATE). H.N.B. acknowledges the support of Bolsa D. Manuel de Mello and of the Portuguese Society for Pneumology; A.B. and M.S. were also recipients of an International Exchanges Grant from the Royal Society. J.S. is funded by a research fellow NORTE-01-0145-FEDER-000012; B.C. and K.L.F. are funded by FCT Ph.D scholarships SFRH/BD/114403/2016 and SFRH/BD/114405/2016, respectively; M.I.V. is funded by FCT through DL 57/2016 (CRP) and MS through Estimulo Individual ao Emprego Científico. We thank the excellent support from the i3S scientific platforms, namely Animal facility, Advanced Light Microscopy, and BioSciences Screening, member of the national infrastructure PPBI-Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122)and Genomics (GenCore) part of the GenomePT project (POCI-01-0145-FEDER022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).

The airborne pathogen Mycobacterium tuberculosis is responsible for a present major public health problem worsened by the emergence of drug resistance. M. tuberculosis has acquired and developed streptomycin (STR) resistance mechanisms that have been maintained and transmitted in the population over the last decades. Indeed, STR resistant mutations are frequently identified across the main M. tuberculosis lineages that cause tuberculosis outbreaks worldwide. The spread of STR resistance is likely related to the low impact of the most frequent underlying mutations on the fitness of the bacteria. The withdrawal of STR from the first-line treatment of tuberculosis potentially lowered the importance of studying STR resistance. However, the prevalence of STR resistance remains very high, could be underestimated by current genotypic methods, and was found in outbreaks of multi-drug (MDR) and extensively drug (XDR) strains in different geographic regions. Therefore, the contribution of STR resistance to the problem of tuberculosis drug resistance should not be neglected. Here, we review the impact of STR resistance and detail well-known and novel candidate STR resistance mechanisms, genes, and mutations. In addition, we aim to provide insights into the possible role of STR resistance in the development of multi-drug resistant tuberculosis., This research was funded by NORTE-01-0145- FEDER-000039, NORTE-01-0145-FEDER000013, and NORTE-01-0145-FEDER-00002, supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). It was also funded through the Foundation for Science and Technology (FCT)—project FCT IF/00474/2014, UIDB/50026/2020, and UIDP/50026/2020. DR was funded by FCT Ph.D. scholarships (grant number SFRH/BD/135422/2017). DR was partially funded by the PGCD—Graduate Program Science for Development. MS is supported by FCT through the Estimulo ao Emprego Científico.

Human immunodeficiency virus (HIV) drug resistance (HIVDR) is widespread in sub-Saharan Africa. Children and pregnant women are particularly vulnerable, and laboratory testing capacity remains limited. We, therefore, used a cross-sectional design and convenience sampling to characterize HIV subtypes and resistance-associated mutations (RAMs) in these groups in Sierra Leone. In total, 96 children (age 2–9 years, 100% ART-experienced), 47 adolescents (age 10–18 years, 100% ART-experienced), and 54 pregnant women (>18 years, 72% ART-experienced) were enrolled. Median treatment durations were 36, 84, and 3 months, respectively, while the sequencing success rates were 45%, 70%, and 59%, respectively, among children, adolescents, and pregnant women. Overall, the predominant HIV-1 subtype was CRF02_AG (87.9%, 95/108), with minority variants constituting 12%. Among children and adolescents, the most common RAMs were M184V (76.6%, n = 49/64), K103N (45.3%, n = 29/64), Y181C/V/I (28.1%, n = 18/64), T215F/Y (25.0%, n = 16/64), and V108I (18.8%, n = 12/64). Among pregnant women, the most frequent RAMs were K103N (20.6%, n = 7/34), M184V (11.8%, n = 4/34), Y181C/V/I (5.9%, n = 2/34), P225H (8.8%, n = 3/34), and K219N/E/Q/R (5.9%, n = 2/34). Protease and integrase inhibitor-RAMs were relatively few or absent. Based on the genotype susceptibility score distributions, 73%, 88%, and 14% of children, adolescents, and pregnant women, respectively, were not susceptible to all three drug components of the WHO preferred first-line regimens per 2018 guidelines. These findings suggest that routine HIVDR surveillance and access to better ART choices may improve treatment outcomes in Sierra Leone., This research was funded by the Roe Green Travel Medicine and Global Health Award 2019 (Award Number J0628), University Hospitals Cleveland Medical Center (G.A.Y.), Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional-FEDER, Red Española de Investigación en SIDA (RD16/0025/0026) (E.P.), Xunta Galicia-Fondo Social Europeo (IN606A-2016/023) (E.P.) and Fundación Biomédica Galicia Sur (E.P.).

Setting: University-affiliated hospital located in Porto, North Portugal, an area with a low to intermediate incidence of tuberculosis (TB). Objective: To identify predictors and outcomes of disseminated TB (dTB). Design: A cohort of patients diagnosed with TB between 2007 and 2013 was retrospectively analysed. Patients with dTB criteria were characterized and compared to single organ TB cases. Factors independently associated with dTB were determined by multivariate logistic regression analysis. Results: A total of 744 patients were analysed, including 145 with dTB. Independent risk factors for dTB were pharmacological immunosuppression (OR 5.6, 95% CI 2.8–11.3), HIV infection (OR 5.1, 95% CI 3.1–8.3), chronic liver failure or cirrhosis (OR 2.3, 95% CI 1.4–4.1) and duration of symptoms (OR 2.3, 95% CI 1.4–3.8). Compared to single organ TB, the clinical presentation of dTB patients differed by the absence of haemoptysis (OR 3.2, 95% CI 1.3–8.4) and of dyspnoea (OR 1.9, 95% CI 1.2–3.1), presence of weight loss (OR 1.8, 95% CI 1.1–2.9), night sweats (OR 1.7, 95% CI 1.1–2.7) and bilateral lung involvement (OR 4.4, 95% CI 2.8–7.1). Mortality and time until culture conversion were higher for dTB patients, although not reaching statistical significance. Conclusion: Immunosuppressive conditions and chronic liver failure or cirrhosis were associated with increased risk of dTB. The haematogenous spread may be dependent on longer symptomatic disease and usually progresses with bilateral lung involvement., HNB acknowledges the receipt of research scholarships from Bolsa D. Manuel de Mello and the Portuguese Society for Pneumology. NSO acknowledges FCT IF/00474/2014. The MS lab is financed by FEDER --- Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 --- Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT in the framework of the project ‘‘Institute for Research and Innovation in Health Sciences’’ (POCI-01-0145-FEDER-007274).

The already enormous burden caused by Mycobacterium tuberculosis and Human Immunodeficiency Virus type 1 (HIV-1) alone is aggravated by co-infection. Despite obvious differences in the rate of evolution comparing these two human pathogens, genetic diversity plays an important role in the success of both. The extreme evolutionary dynamics of HIV-1 is in the basis of a robust capacity to evade immune responses, to generate drug-resistance and to diversify the populationlevel reservoir of M group viral subtypes. Compared to HIV-1 and other retroviruses, M. tuberculosis generates minute levels of genetic diversity within the host. However, emerging whole-genome sequencing data show that the M. tuberculosis complex contains at least nine human-adapted phylogenetic lineages. This level of genetic diversity results in differences in M. tuberculosis interactions with the host immune system, virulence and drug resistance propensity. In co-infected individuals, HIV-1 and M. tuberculosis are likely to co-colonize host cells. However, the evolutionary impact of the interaction between the host, the slowly evolving M. tuberculosis bacteria and the HIV-1 viral “mutant cloud” is poorly understood. These evolutionary dynamics, at the cellular niche of monocytes/macrophages, are also discussed and proposed as a relevant future research topic in the context of single-cell sequencing., This work has been funded by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020; by the projects NORTE-01- 0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and by Gilead Génese PGG/009/2017. ASP, CM, and PMMA were funded by FCT PhD scholarships PD/BD/127827/2016, SFRH/BD/132797/2017 and PDE/BDE/113599/2015, respectively.

[Excerto] A família Coronoviridae inclui uma panóplia de vírus compostos por envelope e RNA de cadeia simples que infetam aves e mamíferos (Payne, 2017). Existem pelo menos sete coronavírus humanos (HCoV), nomeadamente o HCoV-229E, o HCoV-OC43, o HCoV-NL63, o HCoV-HKU1, o coronavírus da síndrome respiratória aguda severa (SARS-CoV), o coronavírus da síndrome respiratória do Médio Oriente (MERS-CoV) e o mais recente coronavírus da síndrome respiratória aguda severa (SARS-CoV-2). Ao contrário dos SARS-CoV, MERS-CoV e SARS-CoV-2, os HCoV “comuns” geralmente causam doença pouco severa das vias respiratórias superiores e contribuem de forma sazonal para 15% a 30% dos casos de “gripe comum” em adultos (Liu, Liang, & Fung, 2020; Woo, Lau, Yip, Huang, & Yuen, 2009). A primeira epidemia severa causada por coronavírus remonta a novembro de 2002, com a transmissão entre pessoas do vírus SARS-CoV. Os sintomas de SARS incluem febre, mal-estar, dor de cabeça, diarreia, entre outros, não havendo um quadro de sintomas específico que permita per se o diagnóstico da doença. A origem do surto de SARS terá, presumivelmente, sido a infeção de pessoas a partir de morcegos num mercado com animais vivos em Guangdong, na China (Zhong et al., 2003). Este coronavírus altamente contagioso, e causador de elevada taxa de mortalidade (9.6%), infetou entre 2002 e 2003 mais de 8000 pessoas, matou mais de 700 e propagou-se a pelo menos 26 países (J. T. Wang & Chang, 2004). Constrangeu períodos de quarentena e de restrições a viagens que conduziram a perdas económicas estimadas num total de 40 mil milhões de dólares, correspondentes a prejuízos na ordem dos 2,6% do PIB na China, 1,05% em Hong Kong e 0,15% no Canadá (Website, 2020). O período de maior verosimilhança de uma pessoa infetada transmitir SARS-CoV foi descrito na segunda semana de doença, aquando do pico de cargas virais no hospedeiro e no qual os sintomas eram regularmente evidentes (Petersen et al., 2020). Esta característica do vírus pode ter ajudado na implementação de práticas adequadas de proteção, tento o notável esforço realizado permitido o controlo total desta epidemia no final de maio de 2003. Desde então, a transmissão entre pessoas do SARS-CoV parece ter parado, com exceção de casos esporádicos resultantes de acidentes laboratoriais (Lim et al., 2004). [...]

© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/., In response to infection, macrophages adapt their metabolism rapidly to enhance glycolysis and fuel specialized antimicrobial effector functions. Here we show that fungal melanin is an essential molecule required for the metabolic rewiring of macrophages during infection with the fungal pathogen Aspergillus fumigatus. Using pharmacological and genetic tools, we reveal a molecular link between calcium sequestration by melanin inside the phagosome and induction of glycolysis required for efficient innate immune responses. By remodeling the intracellular calcium machinery and impairing signaling via calmodulin, melanin drives an immunometabolic signaling axis towards glycolysis with activation of hypoxia-inducible factor 1 subunit alpha (HIF-1α) and phagosomal recruitment of mammalian target of rapamycin (mTOR). These data demonstrate a pivotal mechanism in the immunometabolic regulation of macrophages during fungal infection and highlight the metabolic repurposing of immune cells as a potential therapeutic strategy., This work was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013), the Fundação para a Ciência e Tecnologia (FCT) (SFRH/BD/136814/2018 to S.M.G., SFRH/BD/141127/2018 to C.D.O., PD/BD/137680/2018 to D.A., IF/00474/2014 to N.S.O., IF/01390/2014 to E.T., IF/00959/2014 to S.C., IF/00021/2014 to R.S., PTDC/SAU-SER/29635/2017 and CEECIND/04601/2017 to C.C., and CEECIND/03628/2017 to A.C.), the Institut Mérieux (Mérieux Research Grant 2017 to C.C.), and the European Society of Clinical Microbiology and Infectious Diseases (ESCMID Research Grant 2017 to A.C.). M.G.N. was supported by a Spinoza grant of the Netherlands Organization for Scientific Research. A.A.B. was supported by the Deutsche Forschungsgemeinschaft Collaborative Research Center/Transregio TR124 FungiNet (project A1). G.D.B. was funded by the Wellcome Trust (102705), the MRC Centre for Medical Mycology and the University of Aberdeen (MR/N006364/1)

Human immunodeficiency virus 1 (HIV-1) genome sequencing is routinely done for drug resistance monitoring in hospitals worldwide. Subtyping these extensive datasets of HIV-1 sequences is a critical first step in molecular epidemiology and evolution studies. The clinical relevance of HIV-1 subtypes is increasingly recognized. Several studies suggest subtype-related differences in disease progression, transmission route efficiency, immune evasion, and even therapeutic outcomes. HIV-1 subtyping is mainly done using web-servers. These tools have limitations in scalability and potential noncompliance with data protection legislation. Thus, the aim of this work was to develop an efficient method for large-scale local HIV-1 subtyping. We designed SNAPPy: a snakemake pipeline for scalable HIV-1 subtyping by phylogenetic pairing. It contains several tasks of phylogenetic inference and BLAST queries, which can be executed sequentially or in parallel, taking advantage of multiple-core processing units. Although it was built for subtyping, SNAPPy is also useful to perform extensive HIV-1 alignments. This tool facilitates large-scale sequence-based HIV-1 research by providing a local, resource efficient and scalable alternative for HIV-1 subtyping. It is capable of analyzing full-length genomes or partial HIV-1 genomic regions (GAG, POL, and ENV) and recognizes more than ninety circulating recombinant forms. SNAPPy is freely available at: https://github.com/PMMAraujo/snappy/releases., FEDER, COMPETE, and FCT by the projects NORTE-01-0145-FEDER-000013, POCI-01-0145- FEDER-007038, and IF/00474/2014; FCT PhD scholarship PDE/ BDE/113599/2015; FCT contract IF/00474/2014

bioRxiv - the Preprint Server for Biology., Neoepitopes generated by amino acid variants specifically found in pathogens or cancer cells are gaining momentum in immunotherapy development. HABIT (HLA Binding InTelligence) is a web platform designed to generate and analyse machine learning-based T cell epitope predictions for improved neoepitope discovery. Availability and Implementation: HABIT is available at http://habit.evobiomed.com . Peptide-HLA binding predictions by NetMHCpan 4.0 and NetMHCIIpan 3.1 were implemented in a web application for interactive data exploration using shiny package powered by RStudio., info:eu-repo/semantics/publishedVersion

Complex Polymorphisms in the Plasmodium falciparum Multidrug Resistance Protein 2 Gene and Its Contribution to Antimalarial Response, Plasmodium falciparum has the capacity to escape the actions of essentially all antimalarial drugs. ATP-binding cassette (ABC) transporter proteins are known to cause multidrug resistance in a large range of organisms, including the Apicomplexa parasites. P. falciparum genome analysis has revealed two genes coding for the multidrug resistance protein (MRP) type of ABC transporters: Pfmrp1, previously associated with decreased parasite drug susceptibility, and the poorly studied Pfmrp2. The role of Pfmrp2 polymorphisms in modulating sensitivity to antimalarial drugs has not been established. We herein report a comprehensive account of the Pfmrp2 genetic variability in 46 isolates from Thailand. A notably high frequency of 2.8 single nucleotide polymorphisms (SNPs)/kb was identified for this gene, including some novel SNPs. Additionally, we found that Pfmrp2 harbors a significant number of microindels, some previously not reported. We also investigated the potential association of the identified Pfmrp2 polymorphisms with altered in vitro susceptibility to several antimalarials used in artemisinin-based combination therapy and with parasite clearance time. Association analysis suggested Pfmrp2 polymorphisms modulate the parasite's in vitro response to quinoline antimalarials, including chloroquine, piperaquine, and mefloquine, and association with in vivo parasite clearance. In conclusion, our study reveals that the Pfmrp2 gene is the most diverse ABC transporter known in P. falciparum with a potential role in antimalarial drug resistance., This work was supported by project grants from the Swedish Development Cooperation Agency, Department for Research Cooperation (SWE 2007-174 and SWE-2009-165). M.I.V. and N.S.O. are recipients of post-doctoral fellowship from Fundacao para a Ciencia e Tecnologia (FCT)/Ministerio da Ciencia e Ensino Superior, Portugal, MCES (SFRH/BPD/76614/2011 and UMINHO/BPD/15/2014, respectively). The Shoklo Malaria Research Unit is part of the Mahidol Oxford University Tropical Medicine Research Unit and is supported by the Wellcome Trust of Great Britain.

The already enormous burden caused by tuberculosis (TB) will be further aggravated by the association of this disease with modern epidemics, as human immunodeficiency virus and diabetes. Furthermore, the increasingly aging population and the wider use of suppressive immune therapies hold the potential to enhance the incidence of TB. New preventive and therapeutic strategies based on recent advances on our understanding of TB are thus needed. In particular, understanding the intricate network of events modulating inflammation in TB will help to build more effective vaccines and host-directed therapies to stop TB. This review integrates the impact of host, pathogen, and extrinsic factors on inflammation and the almost scientifically unexplored complexity emerging from the interactions between these three factors. We highlight the exciting data showing a contribution of this troika for the clinical outcome of TB and the need of incorporating it when developing novel strategies to rewire the immune response in TB.

A striking feature of tuberculosis (TB) is the variability of infection outcomes. The heterogeneity observed has been classically attributed to environmental and host determinants. However, recent studies uncovering Mycobacterium tuberculosis complex (MTBC) genomic diversity have shown the potential importance of pathogen-related factors. Nonetheless, the role of this variability in the clinical setting is still underappreciated. In the present work, 193 patients without known predictor or precipitator TB factors were selected from a cohort of 681 pulmonary TB cases, and stratified into TB severity groups. The MTBC infecting strains from these patients were isolated and genotyped using single nucleotide polymorphisms (SNPs) as stable genetic markers to define the different phylogenetic lineages and sublineages. SNP-typing revealed that the large majority (n= 94, 96.9%) of the infecting isolates belong to Lineage 4 (L4 or Euro-American) and that the sublineage LAM (Latin American-Mediterranean) represented 72.2% of the L4 isolates. Disease severity was not exclusively related to any specific phylogenetic taxa. In conclusion, we show a highly homogeneous phylogenetic composition of MTBC in a population of north of Portugal, where L4 and LAM sublineage dominate. Furthermore, mutations implicated in disease severity are not associated with lineage defining variability, but could be related to recent microevolution events within host. The methods used in this study appear well suited to monitor the molecular epidemiology regarding MTBC population structure.

This study was conducted to develop an assessment tool to stratify mortality risk in pulmonary tuberculosis (PTB) patients. A derivation cohort of 681 PTB cases was reviewed to generate a model based on multiple logistic regression analysis of prognostic variables with 6-month mortality as outcome measure. A clinical scoring system was developed and tested against a validation cohort of 103 patients. Five risk features were included in the model: hypoxemic respiratory failure (OR 4.7, 95%CI 2.8-7.9), ≥50 years (OR 2.9, 95%CI 1.7-4.8), bilateral lung involvement (OR 2.5, 95%CI 1.4-4.4), ≥1 significant comorbidity (OR 2.3, 95%CI 1.3-3.8) and hemoglobin

Tuberculosis imposes high human and economic tolls, including in Europe. This study was conducted to develop a severity assessment tool for stratifying mortality risk in pulmonary tuberculosis (PTB) patients. A derivation cohort of 681 PTB cases was retrospectively reviewed to generate a model based on multiple logistic regression analysis of prognostic variables with 6-month mortality as the outcome measure. A clinical scoring system was developed and tested against a validation cohort of 103 patients. Five risk features were selected for the prediction model: hypoxemic respiratory failure (OR 4.7, 95% CI 2.8-7.9), age >= 50 years (OR 2.9, 95% CI 1.7-4.8), bilateral lung involvement (OR 2.5, 95% CI 1.44.4), >= 1 significant comorbidity-HIV infection, diabetes mellitus, liver failure or cirrhosis, congestive heart failure and chronic respiratory disease-(OR 2.3, 95% CI 1.3-3.8), and hemoglobin < 12 g/dL (OR 1.8, 95% CI 1.1-3.1). A tuberculosis risk assessment tool (TReAT) was developed, stratifying patients with low (score = 6) mortality risk. The mortality associated with each group was 2.9%, 22.9% and 53.9%, respectively. The model performed equally well in the validation cohort. We provide a new, easy-to-use clinical scoring system to identify PTB patients with high-mortality risk in settings with good healthcare access, helping clinicians to decide which patients are in need of closer medical care during treatment., This work was supported by Fundacao Amelia de Mello/Jose de Mello Saude and Sociedade Portuguesa de Pneumologia (SPP). This work was developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). NSO is a FCT (Fundacao para a Ciencia e Tecnologia) investigator. MS is an Associate FCT Investigator. The fundershad no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1 .pdf file of 46 Pags. with text and 7 Figs. and 5 .pdfs files of Supplementary Tables. The definitve version is available at: http://gbe.oxfordjournals.org/, The advent of whole-genome sequencing has provided an unprecedented detail about the evolution and genetic significance of species-specific variations across the whole Mycobacterium tuberculosis Complex. However, little attention has been focused on understanding the functional roles of these variations in the protein coding sequences. In this work, we compare the coding sequences from 74 sequenced mycobacterial species including M. africanum, M. bovis, M. canettii, M. caprae, M. orygis, and M. tuberculosis. Results show that albeit protein variations affect all functional classes, those proteins involved in lipid and intermediary metabolism and respiration have accumulated mutations during evolution. To understand the impact of these mutations on protein functionality, we explored their implications on protein ductility/disorder, a yet unexplored feature of mycobacterial proteomes. In agreement with previous studies, we found that a Gly71Ile substitution in the PhoPR virulence system severely affects the ductility of its nearby region in M. africanum and animal-adapted species. In the same line of evidence, the SmtB transcriptional regulator shows amino acid variations specific to the Beijing lineage, which affects the flexibility of the N-terminal trans-activation domain. Furthermore, despite the fact that MTBC epitopes are evolutionary hyperconserved, we identify strain- and lineage-specific amino acid mutations affecting previously known T-cell epitopes such as EsxH and FbpA (Ag85A). Interestingly, in silico studies reveal that these variations result in differential interaction of epitopes with the main HLA haplogroups., This work was supported by Gobierno de Aragón (DGA-GC B18 and B25), the Spanish Ministry of Science and Competitiveness (BIO2014-52580P, CSIC13-4E-2490), Instituto de Salud Carlos III (PI12/01970) and the European Commission Horizon 2020 (H2020-PHC-643381). Some of these grants were partially financed by the EU FEDER Program. This work was also supported by Fundação para a Ciência e Tecnologia, Portugal (IF/00474/2014) and cofunded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER).

We thank Rosana Puccia for providing isolate Pb02., Paracoccidioides brasiliensis budding pattern and polymorphic growth were previously shown to be closely linked to the expression of PbCDC42 and to infl uence the pathogenesis of the fungus. In this work we conducted a detailed morphogenetic evaluation of the yeast-forms of 11 different clinical and environmental P. brasiliensis isolates comprising four phylogenetic lineages (S1, PS2, PS3 and Pb01-like), as well as a PbCDC42 knockdown strain. High variations in the shape and size of mother and bud cells of each isolate were observed but we did not fi nd a characteristic morphologic profi le for any of the phylogenetic groups. In all isolates studied, the bud size and shape were demonstrated to be highly dependent on the mother cell. Importantly, we found strong correlations between PbCDC42 expression and both the shape of mother and bud cells and the size of the buds in all isolates and the knock-down strain. Our results suggested that PbCDC42 expression can explain approximately 80% of mother and bud cell shape and 19% of bud cell size. This data support PbCDC42 expression level as being a relevant predictor of P. brasiliensis morphology. Altogether, these fi ndings quantitatively describe the polymorphic nature of the P. brasiliensis yeast form and provide additional support for the key role of PbCDC42 expression on yeast cell morphology., J.F.M. was supported by Fundação para a Ciência e Tecnologia (FCT), Portugal (SFRH/BD/33446/2008).This work was supported by a grant from FCT (PTDC/BIA-MIC/108309/2008).

Juvenile neuronal ceroid lipofuscinosis (JNCL), also known as Batten disease, is a fatal inherited neurodegenerative disorder. The major clinical features of this disease are vision loss, seizures and progressive cognitive and motor decline starting in childhood. Mutations in CLN3 are known to cause the disease, allowing the generation of mouse models that are powerful tools for JNCL research. In this study, we applied behavioural phenotyping protocols to test for early behavioural alterations in Cln3(Deltaex7/8) knock-in mice, a genetic model that harbours the most common disease-causing CLN3 mutation. We found delayed acquisition of developmental milestones, including negative geotaxis, grasping, wire suspension time and postural reflex in both homozygous and heterozygous Cln3(Deltaex7/8) preweaning pups. To further investigate the consequences of this neurodevelopmental delay, we studied the behaviour of juvenile mice and found that homozygous and heterozygous Cln3(Deltaex7/8) knock-in mice also exhibit deficits in exploratory activity. Moreover, when analysing motor behaviour, we observed severe motor deficits in Cln3(Deltaex7/8) homozygous mice, but only a mild impairment in motor co-ordination and ambulatory gait in Cln3(Deltaex7/8) heterozygous animals. This study reveals previously overlooked behaviour deficits in neonate and young adult Cln3(Deltaex7/8) mice indicating neurodevelopmental delay as a putative novel component of JNCL., Fundação para a Ciência e Tecnologia (FCT) project PTDC/SAU-NEU/70161/2006, NIH R01NS044310 and the BeatBatten Foundation. N.S.O. was supported by the PhD grant15318 from FC

Introduction: Of the estimated 9 million new cases of tuberculosis (TB) each year, 1.1 million are co-infected with HIV. This infection plays an important role in TB, with major clinical impact. Aims: Compare the differences between HIV and non-HIV co-infected TB patients. Methods: A retrospective analysis was made of all the patients diagnosed with TB during a period of 7 years at Centro Hospitalar de Sao Joao. Demographic, clinical, analytical and radiological characteristics were assessed. Results: Out of a total of 714 patients with TB diagnosis, 470 patients met the inclusion criteria (age ≥18 years, HIV serology tested). A total of 105 (20.3%) patients were HIV+. The age at diagnosis was significantly lower at the HIV+ group (43.0±13.2 vs 51.9±18.7; p Conclusions: The patients with HIV co-infection have a set of significant clinical, analytical, radiological and diagnostic particularities. HIV infection poses as risk for the development of extrapulmonary and disseminated TB.

The existing data support Portugal as the western European country with the highest HIV-1 subtype diversity. However, detailed phylogenetic studies of Portuguese HIV-1 epidemics are still scarce. Thus, our main goal was to analyze the phylodynamics of a local HIV-1 infection in the Portuguese region of Minho. Molecular epidemiological analysis was applied to data from 289 HIV-1-infected individuals followed at the reference hospital of the province of Minho, Portugal, at which isolated viruses had been sequenced between 2000 and 2012. Viruses of the G (29.1%) and B (27.0%) subtypes were the most frequent, followed by recombinant forms (17.6%) and the C (14.5%), F1 (7.3%), and A1 (4.2%) subtypes. Multinomial logistic regression revealed that the odds of being infected with the A1 and F1 subtypes increased over the years compared with those with B, G, or C subtypes or recombinant viruses. As expected, polyphyletic patterns suggesting multiple and old introductions of the B and G subtypes were found. However, transmission clusters of non-B and non-G viruses among native individuals were also found, with the dates of the most recent common ancestor estimated to be in the early 2000s. Our study supports that the HIV-1 subtype diversity in the Portuguese region of Minho is high and has been increasing in a manner that is apparently driven by factors other than immigration and international travel. Infections with A1 and F1 viruses in the region of Minho are becoming established and are mainly found in sexually transmitted clusters, reinforcing the need for more efficacious control measures targeting this infection route.

undisputable. However, a combination of these two aspects in teaching and learning human anatomy seems to be unexplored. The Medical Council of India does not mandate the use of AV in the dissection hall (DH) and no study was available in current literature on the effectiveness of AV aids in the DH. Therefore, the current study was undertaken. What was tried? Audiovisual aids were introduced in our department to assist students in cadaveric dissection via live streaming and projection. It comprised of two high-definition Sony! video cameras, wireless microphones with audio-amplifiers and speakers. The video switcher was connected to ten 34-inch LCD panels mounted adjacent to dissection tables. Each dissection session of 2 hours was divided into a pre-dissection workshop of 15 minutes (for stepwise demonstration of dissection in a pre-dissected cadaver), a dissection session of 1.5 hours and a debriefing session of 15 minutes (for discussing the region dissected in nutshell). The effectiveness of AV aids was assessed by two methods in 127 medical undergraduates; first by questionnaire and second by comparing their performance in term-end examinations with that of a previous group, who were taught without using AV aids. Written consent was obtained from students. What lessons were learned? Responding to the questionnaire, 125 (98%) students said that the AV system in the DH facilitated the overall understanding of human anatomy, 119 (93.7%) felt that both the pre-dissection workshops and the post-dissection debriefing are useful and 99 (78%) wanted it to be used in every DH session. Common drawbacks were difficulty in orientation to cadavers (63/49.60%) and difficulty in comprehension (20/15.74%). Although 126 (99.2%) students said image and sound qualities were good, 66 (55.9%) felt that camera and microphone handling by instructors requires more expertise. Feedback from students was used to improve the use of the system. Students performed better in both theory (mean scores: 46.82 ! 9.41% and 51.03 ! 8.79%) and practical examinations (mean scores: 49.14 ! 8.82% and 51.91 ! 8.35%) when AV aids were used. Student’s t-test revealed that the difference in performance was statistically significant at p < 0.05. Hence, DH teaching of human anatomy can be rendered more effective by use of AV aids; especially in the current scenario of teacher to student ratios in India and for the time that is available to medical undergraduates for mastering human anatomy. REFERENCE

Tuberculosis associates with a wide spectrum of disease outcomes. The Beijing (Bj) lineage of Mycobacterium tuberculosis (Mtb) is suggested to be more virulent than other Mtb lineages and prone to elicit non-protective immune responses. However, highly heterogeneous immune responses were reported upon infection of innate immune cells with Bj strains or stimulation with their glycolipids. Using both in vitro and in vivo mouse models of infection, we here report that the molecular mechanism for this heterogeneity may be related to distinct TLR activations. Among this Mtb lineage, we found strains that preferentially activate TLR2, and others that also activate TLR4. Recognition of Mtb strains by TLR4 resulted in a distinct cytokine profile in vitro and in vivo, with specific production of type I IFN. We also uncover a novel protective role for TLR4 activation in vivo. Thus, our findings contribute to the knowledge of the molecular basis underlying how host innate immune cells handle different Mtb strains, in particular the intricate host-pathogen interaction with strains of the Mtb Bj lineage., This work has been funded by Fundacao para a Ciencia e Tecnologia, Portugal. Project grants: PTDC/SAU-MII/101977/2008 and PTDC/BIA-BCM/102776/2008. Personal grants: SFRH/BD/35981/2007 to JC; SFRH/BPD/3306/2007 to AC; SFRH/BPD/77399/2011 to LMT; SFRH/BI/33456/2008 to CS; and SFRH/BPD/33959/2009 to NSO. MS is a Ciencia 2007 fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Tuberculosis (TB) is a global health problem estimated to kill 1.4 million people per year. Recent advances in the genomics of the causative agents of TB, bacteria known as the Mycobacterium tuberculosis complex (MTBC), have allowed a better comprehension of its population structure and provided the foundation for molecular evolution analyses. These studies are crucial for a better understanding of TB, including the variation of vaccine efficacy and disease outcome, together with the emergence of drug resistance. Starting from the analysis of 73 publicly available genomes from all the main MTBC lineages, we have screened for evidences of positive selection, a set of 576 genes previously associated with drug resistance or encoding membrane proteins. As expected, because antibiotics constitute strong selective pressure, some of the codons identified correspond to the position of confirmed drug-resistance-associated substitutions in the genes embB, rpoB, and katG. Furthermore, we identified diversifying selection in specific codons of the genes Rv0176 and Rv1872c coding for MCE1-associated transmembrane protein and a putative L-lactate dehydrogenase, respectively. Amino acid sequence analyses showed that in Rv0176, sites undergoing diversifying selection were in a predicted antigen region that varies between “modern” lineages and “ancient” MTBC/BCG strains. In Rv1872c, some of the sites under selection are predicted to impact protein function and thus might result from metabolic adaptation. These results illustrate that diversifying selection in MTBC is happening as a consequence of both antibiotic treatment and other evolutionary pressures., N.S.O. is supported by the Portuguese Foundation for Science and Technology (FCT) SFRH/BPD/33959/2009, and M.S. is a Ciência 2007 Fellow. I.C. is supported by Marie Curie FP7 action 272086. This work was partially supported by Ministerio de Economia y Competitividad (MINECO), Spain, project BFU2011-24112.

Nitric oxide (NO) is a small molecule with distinct roles in diverse physiological functions in biological systems, among them the control of the apoptotic signalling cascade. By combining proteomic, genetic and biochemical approaches we demonstrate that NO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are crucial mediators of yeast apoptosis. Using indirect methodologies and a NO-selective electrode, we present results showing that H2O2-induced apoptotic cells synthesize NO that is associated to a nitric oxide synthase (NOS)-like activity as demonstrated by the use of a classical NOS kit assay. Additionally, our results show that yeast GAPDH is a target of extensive proteolysis upon H2O2-induced apoptosis and undergoes S-nitrosation. Blockage of NO synthesis with Nomega-nitro-L-arginine methyl ester leads to a decrease of GAPDH S-nitrosation and of intracellular reactive oxygen species (ROS) accumulation, increasing survival. These results indicate that NO signalling and GAPDH S-nitrosation are linked with H2O2-induced apoptotic cell death. Evidence is presented showing that NO and GAPDH S-nitrosation also mediate cell death during chronological life span pointing to a physiological role of NO in yeast apoptosis., This work was supported by a grant from FCT-Fundação para a Ciência e a Tecnologia (POCI/BIA-BCM/57364/2004). B.A. has a fellowship from FCT (SFRH/BD/15317/2005). We are also grateful to FWF for a Lipotox grant to F.M. and B.M. and for grant no. S-9304-B05 to F.M. and S.B.

The juvenile form of neuronal ceroid lipofuscinoses (JNCLs), or Batten disease, results from mutations in the CLN3 gene, and it is characterized by the accumulation of lipopigments in the lysosomes of several cell types and by extensive neuronal death. We report that the yeast model for JNCL (btn1-Delta) that lacks BTN1, the homologue to human CLN3, has increased resistance to menadione-generated oxidative stress. Expression of human CLN3 complemented the btn1-Delta phenotype, and equivalent Btn1p/Cln3 mutations correlated with JNCL severity. We show that the previously reported decreased levels of L-arginine in btn1-Delta limit the synthesis of nitric oxide (.NO) in both physiological and oxidative stress conditions. This defect in .NO synthesis seems to suppress the signaling required for yeast menadione-induced apoptosis, thus explaining btn1-Delta phenotype of increased resistance. We propose that in JNCL, a limited capacity to synthesize .NO directly caused by the absence of Cln3 function may contribute to the pathology of the disease., Fundação para a Ciência e a Tecnologia (FCT) grant POCI/BIA-BCM/57364/2004, PTDC/ SAU-NEU/70161/2006, and National Institutes of Health grant R01 NS36610. N.S.O., A.C., and A.J.A. are supported by scholarships from the FCT

In humans, allelic variants in Toll-like receptors (TLRs) associate with several pathologies. However, the underlying cellular and molecular mechanisms of this association remain largely unknown. Analysis of the human TLR9 promoter revealed that the C allele of the rs5743836 polymorphism generates several regulatory sites, including an IL-6-responding element. Here, we show that, in mononuclear cells carrying the TC genotype of rs5743836, IL-6 up-regulates TLR9 expression, leading to exacerbated cellular responses to CpG, including IL-6 production and B-cell proliferation. Our study uncovers a role for the rs5743836 polymorphism in B-cell biology with implications on TLR9-mediated diseases and on the therapeutic usage of TLR9 agonists/antagonists., Fundação para a Ciência e Tecnologia (FCT) This study was supported by FCT - Grant Number PIC/IC/83313/2007.