Leishmaniinae: Evolutionary inferences based on protein expression profiles (PhyloQuant) congruent with phylogenetic relationships among Leishmania, Endotrypanum, Porcisia, Zelonia, Crithidia, and Leptomonas.

Evolutionary relationships among parasites of the subfamily Leishmaniinae, which comprises pathogen agents of leishmaniasis, were inferred based on differential protein expression profiles from mass spectrometry-based quantitative data using the PhyloQuant method. Evolutionary distances following identification and quantification of protein and peptide abundances using Proteome Discoverer and MaxQuant software were estimated for 11 species from six Leishmaniinae genera. Results clustered all dixenous species of the genus Leishmania, subgenera L. (Leishmania), L. (Viannia), and L. (Mundinia), sister to the dixenous species of genera Endotrypanum and Porcisia. Placed basal to the assemblage formed by all these parasites were the species of genera Zelonia, Crithidia, and Leptomonas, so far described as monoxenous of insects although eventually reported from humans. Inferences based on protein expression profiles were congruent with currently established phylogeny using DNA sequences. Our results reinforce PhyloQuant as a valuable approach to infer evolutionary relationships within Leishmaniinae, which is comprised of very tightly related trypanosomatids that are just beginning to be phylogenetically unraveled. In addition to evolutionary history, mapping of species-specific protein expression is paramount to understand differences in infection processes, tissue tropisms, potential to jump from insects to vertebrates including humans, and targets for species-specific diagnostic and drug development.

First report of Rhodnius stali Lent, Jurberg & Galvão, 1993 (Hemiptera: Reduviidae: Triatominae), vector of Trypanosoma cruzi (TcI) and Trypanosoma rangeli (TrA), in Rondônia, Southwestern Brazilian Amazonia.

Triatomines are hematophagous insects of epidemiological importance as they are vectors of Chagas disease. The first report of Rhodnius stali Lent, Jurberg & Galvão, 1993 in Rondônia, Brazil, is described. The insects were captured on palm trees of the genus Oenocarpus sp. Two adult male specimens of R. stali were identified and were found to be infected with Trypanosoma cruzi and Trypanosoma rangeli. The confirmation of this Rhodnius species in Rondônia increases the number of triatomines from nine to ten species in this state.

Genomes of Endotrypanum monterogeii from Panama and Zelonia costaricensis from Brazil: Expansion of Multigene Families in Leishmaniinae Parasites That Are Close Relatives of Leishmania spp.

The Leishmaniinae subfamily of the Trypanosomatidae contains both genus Zelonia (monoxenous) and Endotrypanum (dixenous). They are amongst the nearest known relatives of Leishmania, which comprises many human pathogens widespread in the developing world. These closely related lineages are models for the genomic biology of monoxenous and dixenous parasites. Herein, we used comparative genomics to identify the orthologous groups (OGs) shared among 26 Leishmaniinae species to investigate gene family expansion/contraction and applied two phylogenomic approaches to confirm relationships within the subfamily. The Endotrypanum monterogeii and Zelonia costaricensis genomes were assembled, with sizes of 29.9 Mb and 38.0 Mb and 9.711 and 12.201 predicted protein-coding genes, respectively. The genome of E. monterogeii displayed a higher number of multicopy cell surface protein families, including glycoprotein 63 and glycoprotein 46, compared to Leishmania spp. The genome of Z. costaricensis presents expansions of BT1 and amino acid transporters and proteins containing leucine-rich repeat domains, as well as a loss of ABC-type transporters. In total, 415 and 85 lineage-specific OGs were identified in Z. costaricensis and E. monterogeii. The evolutionary relationships within the subfamily were confirmed using the supermatrix (3384 protein-coding genes) and supertree methods. Overall, this study showed new expansions of multigene families in monoxenous and dixenous parasites of the subfamily Leishmaniinae.

Molecular detection of piroplasmids in mammals from the Superorder Xenarthra in Brazil.

Xenarthra mammals can be found from southern North America to southern South America, including all Brazilian biomes. Although it has been shown that Xenarthra mammals can play a role as reservoirs for several zoonotic agents, few studies investigate the diversity of piroplasmids (Apicomplexa: Piroplasmida) in this group of mammals. Taking into account that piroplasmids can cause disease in animals and humans, understanding the prevalence and diversity of piroplasmids in Xenarthra mammals would contribute to conservation efforts for this group of animals as well as to infer risk areas for transmission of emergent zoonosis. The present study aimed to investigate the occurrence and molecular identity of piroplasmids in free-living mammals of the Superorder Xenarthra from four Brazilian states (Mato Grosso do Sul, São Paulo, Rondônia, and Pará). For this, DNA was extracted from blood or spleen samples from 455 animals. A nested PCR based on the 18S rRNA gene was used as screening for piroplasmids. Of the 455 samples analyzed, 25 (5.5%) were positive. Additionally, PCR assays based on 18S rRNA near-complete, cox-1, cox-3, hsp70, cytB, ß-tubulin genes and the ITS-1 intergenic region were performed. Five out of 25 positive samples also tested positive for ITS-1-based PCR. The phylogenetic analysis positioned three 18S rRNA sequences detected in Priodontes maximus into the same clade of Babesia sp. detected in marsupials (Didelphis albiventris, Didelphis marsupialis, and Monodelphis domestica) and Amblyomma dubitatum collected from opossums and coatis in Brazil. On the other hand, the 18S rRNA sequence obtained from Dasypus novemcinctus was closely related to a Theileria sp. sequence previously detected in armadillos from Mato Grosso State, grouping in a subclade within the Theileria sensu stricto clade. In the phylogenetic analysis based on the ITS-1 region, the sequences obtained from Myrmecophaga tridactyla and Tamandua tetradactyla were placed into a single clade, apart from the other piroplasmid clades. The present study demonstrated the molecular occurrence of Piroplasmida in anteaters and Babesia sp. and Theileria sp. in armadillos from Brazil.

Molecular detection of blood-borne agents in vampire bats from Brazil, with the first molecular evidence of Neorickettsia sp. in Desmodus rotundus and Diphylla ecaudata.

Bats (Mammalia, Chiroptera) represent the second largest group of mammals. Due to their ability to fly and adapt and colonize different niches, bats act as reservoirs of several potentially zoonotic pathogens. In this context, the present work aimed to investigate, using molecular techniques, the occurrence of blood-borne agents (Anaplasmataceae, Coxiella burnetii, hemoplasmas, hemosporidians and piroplasmids) in 198 vampire bats sampled in different regions of Brazil and belonging to the species Desmodus rotundus (n = 159), Diphylla ecaudata (n = 31) and Diaemus youngii (n = 8). All vampire bats liver samples were negative in PCR assays for Ehrlichia spp., Anaplasma spp., piroplasmids, hemosporidians and Coxiella burnetii. However, Neorickettsia sp. was detected in liver samples of 1.51% (3/198) through nested PCR based on the 16S rRNA gene in D. rotundus and D. ecaudata. This is the first study to report Neorickettsia sp. in vampire bats. Hemoplasmas were detected in 6.06% (12/198) of the liver samples using a PCR based on the 16S rRNA gene. The two 16S rRNA sequences obtained from hemoplasmas were closely related to sequences previously identified in vampire and non-hematophagous bats from Belize, Peru and Brazil. The genotypic analysis identified a high diversity of bat-associated hemoplasma genotypes from different regions of the world, emphasizing the need for studies on this subject, in order to better understand the mechanisms of co-evolution between this group of bacteria and their vertebrate hosts. The role of neotropical bat-associated Neorickettsia sp. and bats from Brazil in the biological cycle of such agent warrant further investigation.

The Influence of Obesity on Melanoma and Sentinel Lymph Node Diagnosis: A Retrospective Monocentric Study in 1001 Patients.

(1) Background: While obesity is a known independent risk factor in the development of melanoma, there is no consensus on its influence on melanoma prognosis. (2) Methods: In a monocentric retrospective study, data was collected from patients who underwent sentinel lymph node (SLN) biopsy for stage IB-IIC melanoma between 2013 and 2018. Patients were divided into groups according to their body mass index (BMI). The association between BMI and melanoma features, as well as the risk factors for metastases in SLN were examined. (3) Results: Of the 1001 patients, 336 had normal weight (BMI < 25), 402 were overweight (BMI >= 25 and <30), 173 obese (BMI >= 30 and <35) and 90 extremely obese (BMI >= 35). Overweightness and obesity were associated with higher tumor thicknesses at time of diagnosis. Ulceration was not influenced by the patient's weight. Metastases in sentinel lymph node was almost twice more likely in extremely obese patients than in normal weight patients. Independent risk factors for metastases in SLN in our study were tumor thickness, ulceration, and BMI > 35. (4) Conclusions: This is the first study to show higher metastases rates in high-BMI patients with melanoma, raising important questions regarding the screening and treatment of this specific patient population.

Modelos organizativos e a adesão à vacinação da gripe na população idosa / Organizational models and adherence to influenza vaccination in the elderly population

A gripe é uma das doenças mais frequentes no mundo, atingindo anualmente cinco milhões de pessoas. Tendo uma evolução na sua grande maioria benigna, encontra-se associada à mortalidade prematura em idosos e população de risco (OMS,2019). A vacinação surge como a medida de saúde pública mais importante, e a melhor de defesa contra a forma mais grave da doença. Em 2020, com o aparecimento da COVID-19, os sistemas de saúde no mundo são sujeitos a pressão sem precedentes, com um número elevado de hospitalizações e mortes (OMS,2020). Com o aparecimento da primeira vacina, surgem os centros de vacinação, com o objetivo de vacinar o maior número de pessoas, no menor tempo possível. Por orientação da DGS, desde outubro de 2021, a vacinação contra a gripe, passou a ser efetuada nestas estruturas, sendo a sua administração em conjunto com a vacina contra a COVID-19. O estudo tem como objetivos: "Conhecer o número de pessoas com 65 ou mais anos vacinadas contra a gripe no período anterior e durante a pandemia"; "Perceber como as mudanças da estrutura organizativa na administração da vacina contra a gripe, influenciaram o número de vacinas administradas à população com 65 ou mais anos". A população em estudo, é constituída por utentes com 65 ou mais anos inscritos num ACeS na Região Norte. Desde 2018 até ao final do primeiro trimestre de 2022, o número de pessoas com 65 ou mais anos aumentaram 12.8%. Em relação às taxas de vacinação, verifica-se um aumento de 47.7% para 62,9%, no período relativo às épocas vacinais de 2018-2019 a 2020-2021. Estes valores estão ainda longe, dos dados divulgados pela SPP que afirmam que na época vacinal 2021-2022, 88.3% das pessoas com 65 ou mais anos, tinha sido vacinada contra a gripe. Estes dados poderão sugerir existir diferença entre as vacinas disponibilizadas e as registadas. Verificamos que 42.4% das vacinas foram administradas fora do SNS. Esta procura tem vindo a aumentar desde 2018-2019, mas foi época 2021-2022 que teve mais impacto. Foi também nesta época, que as vacinas contra a gripe passaram a ser administradas nos CVC. Estes valores poderão ser explicados pelo facto de os utentes que recusam a vacina contra a COVID-19, procurarem outra alternativa ao CVC. Considera-se que os objetivos a que inicialmente propostos foram atingidos. Da análise dos valores obtidos no presente estudo, parecem indicar que os modelos organizativos interferem na adesão à vacinação das pessoas idosas.

Influenza is one of the most frequent diseases in the world, affecting five million people annually. Its evolution is mostly benign, but it is associated with premature mortality in the elderly and high-risk populations (WHO, 2019). Vaccination emerges as the most important public health measure, and the best defence against the most severe form of the disease. In 2020, with COVID-19´s emergence, health systems in the world are subjected to unprecedented pressure, with a high number of hospitalisations and deaths (WHO,2020). With the appearance of the first vaccine, vaccination centres emerged, with the aim of vaccinating the largest number of people, in the shortest time possible. By orientation of the DGS, since October 2021, the vaccination against influenza has been performed in these structures, and its administration was combined with the vaccine against COVID-19. This study has the following objectives: "To know the number of people aged 65 or older vaccinated against influenza in the period before and during the pandemic"; "To understand how the changes in the organizational structure of influenza vaccine administration, influenced the number of vaccines administered to people aged 65 or older". The study population, is composed by patients aged 65 or older, enrolled in an ACeS in the Northern Region. Since 2018, until the end of the first quarter of 2022, the number of people aged 65 or older increased by 12.8%. Regarding vaccination rates, there has been an increase from 47.7% to 62.9%, in the period that goes from 2018-2019 to 2020-2021 vaccination seasons. These values are still far from the data released by SPP, which states that in the 2021-2022 vaccination season, 88.3% of people aged 65 or older, had been vaccinated against influenza. These data may suggest that there is a difference between the vaccines made available and those registered. We found that 42.4% of vaccines were administered outside the SNS. This demand has been increasing since 2018-2019, but it was in the 2021-2022 season that it had the largest impact. It was also in this season, that influenza vaccines started to be administered in CVCs. These results may be explained by the fact that users who refuse the COVID-19 vaccine seek an alternative to the CVC. It is considered that the objectives which were initially proposed were achieved. From the analysis of the values obtained in this study, they seem to indicate that the organisational models interfere with the elderly people's adherence to vaccination.

A neurophysiological signature of dynamic emotion recognition associated with social communication skills and cortical gamma-aminobutyric acid levels in children.

Introduction: Emotion recognition is a core feature of social perception. In particular, perception of dynamic facial emotional expressions is a major feature of the third visual pathway. However, the classical N170 visual evoked signal does not provide a pure correlate of such processing. Indeed, independent component analysis has demonstrated that the N170 component is already active at the time of the P100, and is therefore distorted by early components. Here we implemented, a dynamic face emotional paradigm to isolate a more pure face expression selective N170. We searched for a neural correlate of perception of dynamic facial emotional expressions, by starting with a face baseline from which a facial expression evolved. This allowed for a specific facial expression contrast signal which we aimed to relate with social communication abilities and cortical gamma-aminobutyric acid (GABA) levels. Methods: We recorded event-related potentials (ERPs) and Magnetic Resonance (MRS) measures in 35 typically developing (TD) children, (10-16 years) sex-matched, during emotion recognition of an avatar morphing/unmorphing from neutral to happy/sad expressions. This task allowed for the elimination of the contribution low-level visual components, in particular the P100, by morphing baseline isoluminant neutral faces into specific expressions, isolating dynamic emotion recognition. Therefore, it was possible to isolate a dynamic face sensitive N170 devoid of interactions with earlier components. Results: We found delayed N170 and P300, with a hysteresis type of dependence on stimulus trajectory (morphing/unmorphing), with hemispheric lateralization. The delayed N170 is generated by an extrastriate source, which can be related to the third visual pathway specialized in biological motion processing. GABA levels in visual cortex were related with N170 amplitude and latency and predictive of worse social communication performance (SCQ scores). N170 latencies reflected delayed processing speed of emotional expressions and related to worse social communication scores. Discussion: In sum, we found a specific N170 electrophysiological signature of dynamic face processing related to social communication abilities and cortical GABA levels. These findings have potential clinical significance supporting the hypothesis of a spectrum of social communication abilities and the identification of a specific face-expression sensitive N170 which can potentially be used in the development of diagnostic and intervention tools.

Genetic diversity of Trypanosoma cruzi strains isolated from chronic chagasic patients and non-human hosts in the state of São Paulo, Brazil.

BACKGROUND: Trypanosoma cruzi shows an exuberant genetic diversity. Currently, seven phylogenetic lineages, called discrete typing units (DTUs), are recognised: TcI-TcVI and Tcbat. Despite advances in studies on T. cruzi and its populations, there is no consensus regarding its heterogeneity. OBJECTIVES: This study aimed to perform molecular characterisation of T. cruzi strains, isolated in the state of São Paulo, to identify the DTUs involved and evaluate their genetic diversity. METHODS: T. cruzi strains were isolated from biological samples of chronic chagasic patients, marsupials and triatomines through culture techniques and subjected to molecular characterisation using the fluorescent fragment length barcoding (FFLB) technique. Subsequently, the results were correlated with complementary information to enable better discrimination between the identified DTUs. FINDINGS: It was possible to identify TcI in two humans and two triatomines; TcII/VI in 19 humans, two marsupials and one triatomine; and TcIII in one human host, an individual that also presented a result for TcI, which indicated the possibility of a mixed infection. Regarding the strains characterised by the TcII/VI profile, the correlation with complementary information allowed to suggest that, in general, these parasite populations indeed correspond to the TcII genotype. MAIN CONCLUSIONS: The TcII/VI profile, associated with domestic cycles and patients with chronic Chagas disease, was the most prevalent among the identified DTUs. Furthermore, the correlation of the study results with complementary information made it possible to suggest that TcII is the predominant lineage of this work.

Vivaxin genes encode highly immunogenic, non-variant antigens on the Trypanosoma vivax cell-surface.

Trypanosoma vivax is a unicellular hemoparasite, and a principal cause of animal African trypanosomiasis (AAT), a vector-borne and potentially fatal livestock disease across sub-Saharan Africa. Previously, we identified diverse T. vivax-specific genes that were predicted to encode cell surface proteins. Here, we examine the immune responses of naturally and experimentally infected hosts to these unique parasite antigens, to identify immunogens that could become vaccine candidates. Immunoprofiling of host serum shows that one particular family (Fam34) elicits a consistent IgG antibody response. This gene family, which we now call Vivaxin, encodes at least 124 transmembrane glycoproteins that display quite distinct expression profiles and patterns of genetic variation. We focused on one gene (viv-ß8) that encodes one particularly immunogenic vivaxin protein and which is highly expressed during infections but displays minimal polymorphism across the parasite population. Vaccination of mice with VIVß8 adjuvanted with Quil-A elicits a strong, balanced immune response and delays parasite proliferation in some animals but, ultimately, it does not prevent disease. Although VIVß8 is localized across the cell body and flagellar membrane, live immunostaining indicates that VIVß8 is largely inaccessible to antibody in vivo. However, our phylogenetic analysis shows that vivaxin includes other antigens shown recently to induce immunity against T. vivax. Thus, the introduction of vivaxin represents an important advance in our understanding of the T. vivax cell surface. Besides being a source of proven and promising vaccine antigens, the gene family is clearly an important component of the parasite glycocalyx, with potential to influence host-parasite interactions.

Antibacterial and hemostatic capacities of cellulose nanocrystalline-reinforced poly(vinyl alcohol) electrospun mats doped with Tiger 17 and pexiganan peptides for prospective wound healing applications.

Infection is a major issue in chronic wound care. Different dressings have been developed to prevent microbial propagation, but an effective, all-in-one (cytocompatible, antimicrobial and promoter of healing) solution is still to be uncovered. In this research, polyvinyl alcohol (PVA) nanofibrous mats reinforced with cellulose nanocrystal (CNC), at 10 and 20% v/v ratios, were produced by electrospinning, crosslinked with glutaraldehyde vapor and doped with specialized peptides. Crosslinking increased the mats' fiber diameters but maintained their bead-free morphology. Miscibility between polymers was confirmed by Fourier-transform infrared spectroscopy and thermal evaluations. Despite the incorporation of CNC having reduced the mats' mechanical performance, it improved the mats' surface energy and its structural stability over time. Pexiganan with an extra cysteine group was functionalized onto the mats via hydroxyl- polyethylene glycol 2-maleimide, while Tiger 17 was physisorbed to preserve its cyclic conformation. Antimicrobial assessments demonstrated the peptide-doped mat's effectiveness against Staphylococcus aureus and Pseudomonas aeruginosa; pexiganan contributed mostly for such outcome. Tiger 17 showed excellent capacity in accelerating clotting. Cytocompatibility evaluations attested to these mats' safety. C90/10 PVA/CNC mats were deemed the most effective from the tested group and, thus, a potentially effective option for chronic wound treatments.

COVID-19 collaborative screening: An action-research project for large scale contact tracing in Northern Portugal.

In late November 2020, when Europe reached the highest 14-day incidence of COVID-19 cases, the resource-intensive and time-consuming traditional contact tracing performed by Public Health was challenged. In this context, innovative approaches were necessary to guarantee a timely interruption of disease transmission. "COVID-19 Collaborative Screening" Project was developed as a faster solution, not only because the contact tracing process is simpler for the operator, but mainly because it is possible to quickly scale up the number of operators involved. It was designed to interrupt family and social transmission chains, in a partnership with the Local Public Health Services - allowing these services to dedicate to scenarios of more complex risk assessment, using the traditional contact tracing. To perform contact tracing, this method involves Public Servants, Armed Forces and Medical Dentists. The Project also promotes participatory citizenship, by delegating to the citizen the responsibility of registering his/hers contacts with high-risk exposure in an online form, in contrast to the traditional contact tracing method which is more health professional-dependent. Until the end of January 2021, the Project has trained eight teams, enrolling a total of 213 professionals, and was implemented in eight Health Regions (with an estimated population of 1,346,150 inhabitants). The Project was successful at facing the delays in case interview and contact tracing. The strategy implemented by ColabCOVID is assembled as a sustainable, reproducible and scalable platform and is ready to be re-implemented to face the emergence of more contagious variants, as well as an eventual forthcoming health threat.

Tiger 17 and pexiganan as antimicrobial and hemostatic boosters of cellulose acetate-containing poly(vinyl alcohol) electrospun mats for potential wound care purposes.

In this research, we propose to engineer a nanostructured mat that can simultaneously kill bacteria and promote an environment conducive to healing for prospective wound care. Polyvinyl alcohol (PVA) and cellulose acetate (CA) were combined at different polymer ratios (100/0, 90/10, 80/20% v/v), electrospun and crosslinked with glutaraldehyde vapor. Crosslinked fibers increased in diameter (from 194 to 278 nm), retaining their uniform structure. Fourier-transform infrared spectroscopy and thermal analyses proved the excellent miscibility between polymers. CA incorporation incremented the fibers swelling capacity and reduced the water vapor and air permeabilities of the mats, preventing the excessive drying of wounds. The antimicrobial peptide cys-pexiganan and the immunoregulatory peptide Tiger 17 were incorporated onto the mats via polyethylene glycol spacer (hydroxyl-PEG2-maleimide) and physisorbed, respectively. Time-kill kinetics evaluations revealed the mats effectiveness against Staphylococcus aureus and Pseudomonas aeruginosa. Tiger 17 played a major role in accelerating clotting of re-calcified plasma. Data reports for the first time the collaborative effect of pexiganan and Tiger 17 against bacterial infections and in boosting hemostasis. Cytocompatibility data verified the peptide-modified mats safety. Croslinked 90/10 PVA/CA mats were deemed the most promising combination due to their moderate hydrophilicity and permeabilities, swelling capacity, and high yields of peptide loading.

Acute Chagas Disease Caused by Trypanosoma cruzi TcIV and Transmitted by Panstrongylus geniculatus: Molecular Epidemiological Insights Provided by the First Documented Autochthonous Case in Rondônia, Southwestern Amazonia, Brazil.

Recurrent outbreaks of oral infection and isolated cases characterize the new epidemiological scenario of Chagas disease (CD) in the Brazilian Amazon. Acute Chagas disease (ACD) is common in Pará and Amazonas, Northeastern and Northwestern Brazilian Amazonia. In the present study, we describe the first molecularly characterized autochthonous case of ACD in Rondônia, Southwestern Amazonia. The patient, a 39-year-old male resident in the small city of Cujubim, presented typical ACD symptoms: fever, asthenia, myalgia, progressive dyspnea, swelling of the legs, and tiredness at minimal efforts, all compatible with ACD and indicative of cardiac involvement. A thick blood drop test revealed trypomastigote forms of Trypanosoma cruzi genotyped as TcIV. An epidemiological investigation ruled out oral infection, and support for vectorial transmission included the finding of Panstrongylus geniculatus positive for T. cruzi (TcIII and TcIV) inside the tent used by the patient when harvesting forest timber, and a circular cutaneous lesion resembling a chagoma of inoculation. Treatment with benznidazole led to blood parasite clearance as confirmed by molecular tests. Altogether, our findings fitted well into the ecological scenario where deforestation and colonization of forested areas represent an important risk factor to the adaptation of P. geniculatus to human habitats, favoring vectorial transmission of CD in the Amazonian region.

Molecular investigation of haemotropic mycoplasmas and Coxiella burnetii in free-living Xenarthra mammals from Brazil, with evidence of new haemoplasma species.

Although mammals of the superorder Xenarthra are considered hosts of a wide range of zoonotic agents, works aiming at investigating the role of these animals as hosts for bacteria with zoonotic potential are rare. The present study aimed to investigate the occurrence and molecularly characterize Coxiella burnetii and haemoplasma (haemotropic mycoplasmas) DNA in blood and spleen samples from 397 free-living Xenarthra mammals (233 sloths, 107 anteaters and 57 armadillos) in five Brazilian states (Mato Grosso do Sul, São Paulo, Pará, Rondônia and Rio Grande do Sul). All biological samples from Xenarthra were negative in the qPCR for Coxiella burnetii based on the IS1111 gene. The absence of C. burnetii DNA in blood and spleen samples from Xenarthra suggests that these mammals may not act as possible hosts for this agent in the locations studied. When performed conventional PCR assays for the endogenous (gapdh) mammalian gene, 386 samples were positive. When screened by molecular assays based on the 16S rRNA gene of haemoplasmas, 81 samples were positive, of which 15.54% (60/386) were positive by conventional PCR and 5.44% (21/386) were positive by real-time PCR; three samples were positive in both assays. Of these, 39.74% (31/78) were also positive for the 23S rRNA gene and 7.69% (6/78) for the haemoplasma RNAse P gene. Among the samples positive for haemoplasmas, 25.64% (20/78) were obtained from anteaters (Tamandua tetradactyla and Myrmecophaga tridactyla), 39.74% (31/78) from sloths (Bradypus tridactylus, Bradypus sp. and Choloepus sp.) 34.61% (27/78) from armadillos (Priodontes maximus, Euphractus sexcinctus and Dasypus novemcinctus). A haemoplasma 16S rRNA sequence closely related and showing high identity (99.7%) to Mycoplasma wenyonii was detected, for the first time, in B. tridactylus. Based on the low identity and phylogenetic positioning of 16S rRNA and 23S rRNA sequences of haemoplasmas detected in anteaters and armadillos, the present study showed, for the first time, the occurrence of putative new Candidatus haemotropic Mycoplasma spp. ("Candidatus Mycoplasma haematotetradactyla" and "Candidatus Mycoplasma haematomaximus") in Xenarthra mammals from Brazil.

Inhibition of Escherichia Virus MS2, Surrogate of SARS-CoV-2, via Essential Oils-Loaded Electrospun Fibrous Mats: Increasing the Multifunctionality of Antivirus Protection Masks.

One of the most important measures implemented to reduce SARS-CoV-2 transmission has been the use of face masks. Yet, most mask options available in the market display a passive action against the virus, not actively compromising its viability. Here, we propose to overcome this limitation by incorporating antiviral essential oils (EOs) within polycaprolactone (PCL) electrospun fibrous mats to be used as intermediate layers in individual protection masks. Twenty EOs selected based on their antimicrobial nature were examined for the first time against the Escherichia coli MS2 virus (potential surrogate of SARS-CoV-2). The most effective were the lemongrass (LGO), Niaouli (NO) and eucalyptus (ELO) with a virucidal concentration (VC) of 356.0, 365.2 and 586.0 mg/mL, respectively. PCL was processed via electrospinning, generating uniform, beadless fibrous mats. EOs loading was accomplished via two ways: (1) physisorption on pre-existing mats (PCLaEOs), and (2) EOs blending with the polymer solution prior to fiber electrospinning (PCLbEOs). In both cases, 10% v/v VC was used as loading concentration, so the mats' stickiness and overwhelming smell could be prevented. The EOs presence and release from the mats were confirmed by UV-visible spectroscopy (≈5257-631 µg) and gas chromatography-mass spectrometry evaluations (average of ≈14.3% EOs release over 4 h), respectively. PCLbEOs mats were considered the more mechanically and thermally resilient, with LGO promoting the strongest bonds with PCL (PCLbLGO). On the other hand, PCLaNO and PCLaELO were deemed the least cohesive combinations. Mats modified with the EOs were all identified as superhydrophobic, capable of preventing droplet penetration. Air and water-vapor permeabilities were affected by the mats' porosity (PCL < PCLaEOs < PCLbEOs), exhibiting a similar tendency of increasing with the increase of porosity. Antimicrobial testing revealed the mats' ability to retain the virus (preventing infiltration) and to inhibit its action (log reduction averaging 1). The most effective combination against the MS2 viral particles was the PCLbLGO. These mats' scent was also regarded as the most pleasant during sensory evaluation. Overall, data demonstrated the potential of these EOs-loaded PCL fibrous mats to work as COVID-19 active barriers for individual protection masks.

Saccharomyces cerevisiae Cells Lacking the Zinc Vacuolar Transporter Zrt3 Display Improved Ethanol Productivity in Lignocellulosic Hydrolysates.

Yeast-based bioethanol production from lignocellulosic hydrolysates (LH) is an attractive and sustainable alternative for biofuel production. However, the presence of acetic acid (AA) in LH is still a major problem. Indeed, above certain concentrations, AA inhibits yeast fermentation and triggers a regulated cell death (RCD) process mediated by the mitochondria and vacuole. Understanding the mechanisms involved in AA-induced RCD (AA-RCD) may thus help select robust fermentative yeast strains, providing novel insights to improve lignocellulosic ethanol (LE) production. Herein, we hypothesized that zinc vacuolar transporters are involved in vacuole-mediated AA-RCD, since zinc enhances ethanol production and zinc-dependent catalase and superoxide dismutase protect from AA-RCD. In this work, zinc limitation sensitized wild-type cells to AA-RCD, while zinc supplementation resulted in a small protective effect. Cells lacking the vacuolar zinc transporter Zrt3 were highly resistant to AA-RCD, exhibiting reduced vacuolar dysfunction. Moreover, zrt3Δ cells displayed higher ethanol productivity than their wild-type counterparts, both when cultivated in rich medium with AA (0.29 g L-1 h-1 versus 0.11 g L-1 h-1) and in an LH (0.73 g L-1 h-1 versus 0.55 g L-1 h-1). Overall, the deletion of ZRT3 emerges as a promising strategy to increase strain robustness in LE industrial production.

Leishmania diversity in bats from an endemic area for visceral and cutaneous leishmaniasis in Southeastern Brazil.

This study aimed to determine the occurrence of Leishmania infection in bats in urban and wild areas in an endemic municipality for visceral and cutaneous leishmaniasis in Minas Gerais, Brazil. Between April 2014 to April 2015, 247 bats were captured and classified into 26 species belonging to Phyllostomidae (90.7%), Vespertilionidae (8.1%) and Molossidae (1.2%) families. Blood samples from 247 bats were collected and submitted to nested-PCR, targeting the variable V7-V8 region of the SSU rRNA gene, followed by sequencing of the PCR product. The overall infection rate of Leishmania spp. in bats was 4.4%. Of the eleven bats infected, ten were frugivorous bats: Artibeus planirostris (8/11), Artibeus lituratus (1/11) and Artibeus cinereus (1/11) and one a nectarivorous bat (Glossophaga soricina). None of the individuals exhibited macroscopic alterations in the skin, spleen or liver. Phylogenetic analysis separated Leishmania species in clades corresponding to the subgenera Viannia, Leishmania, and Mundinia, and supported that the isolates characterized in the present study clustered closely with Leishmania (Viannia) sp., Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis. Here we report for the first time the bat Artibeus cinereus as a host of Leishmania (Leishmania) amazonensis. In the study we found that the mean abundance of bats did not differ in wild habitats and urban areas and that bat-parasite interactions were similarly distributed in the two environments. On the other hand, further studies should be conducted in more recent times to verify whether there have been changes in these parameters.

Molecular epidemiological insights into Trypanosoma vivax in Argentina: From the endemic Gran Chaco to outbreaks in the Pampas.

Argentina is a home to millions of beef and dairy cattle and is one of the world's major exporters of meat. In the present study, Trypanosoma vivax was prevalent (2016-2018) in two major livestock farming regions, the Gran Chaco and the Pampas. In the Gran Chaco, 29% and 51% of animals (n = 72, taurine x zebuine crossbreed) were, respectively, positive by TviCATL-PCR and the more sensitive fluorescent fragment length barcoding (FFLB) method. While 18.4/38.8% of breeding cows (n = 49) tested positive by PCR/FFLB, infection increased to 52.2/78.3% in an outbreak of acute infection in steers (n = 23, taurine breed) brought from a non-endemic area. In the Pampas, overall infection rates in dairy cows (n = 54, taurine breed) were comparable (p > .01) between PCR (66.7%) and FFLB (62.9%) and showed a remarkable increase (PCR / FFLB) from 48.3/44.8% in 2017 to 88/84% in 2018. Infected dairy cattle exhibited anaemia, fever, anorexia, enlarged lymph nodes, emaciation and neurological signs. In contrast, beef cows (taurine x zebuine crossbreed) from the Pampas (n = 30) were asymptomatic despite exhibiting 16.7% (PCR) and 53.3% (FFLB) infection rates. Microsatellite genotyping revealed a remarkable microheterogeneity, seven genotypes in the Gran Chaco, nine in the Pampas and five shared between both regions, consistent with regular movement of T. vivax infected livestock. Data gathered in our study support the Gran Chaco being an endemic area for T. vivax, whereas the Pampas emerged as an outbreak area of acute infection in dairy cattle with critical negative impact in milk production. To the best of our knowledge, this is the first molecular study of T. vivax in Argentina, and results indicated the need for preventive measures to control T. vivax spread from the Gran Chaco to vast livestock farming areas across Argentina.

Remarkable kinetoplast, cytostome-cytopharynx complex, and storage-related structures as dissected by three-dimensional reconstruction of Trypanosoma sp. 858 isolated from a toad (Amphibia: Anura).

In Brazil, the Trypanosoma sp. 858 was isolated from a toad (Anura: Bufonidae: Rhinella ictericus) and successfully maintained in cultures. We previously demonstrated that this trypanosome is different but tightly clustered phylogenetically with other trypanosomes from anurans. In this study, we addressed the ultrastructural features of cultured epimastigotes of this new trypanosome. Our results showed very long and thin free motile forms exhibiting a long flagellum and remarkable large and loose K-DNA network. In addition, the anterior portion contained many acidocalcisomes and a well-developed spongiome tubules-contractile vacuole system. One of the main morphological features of this anuran trypanosome was the presence of a complex cytostome-cytopharynx with a specialized membrane coating at the entrance, which is often hidden by the flagellum. Other conspicuous features are the presence of lipid-like droplets, lamellar membrane limited inclusions, and one very large reservosome, all at the posterior portion of the cell body. This new trypanosome may constitute an excellent model for organelles studies related to endocytosis and lipid storage, as demonstrated herein using scanning and transmission electron microscopy and three-dimensional models obtained by either electron microscopy tomography or dual-beam slice and view series.