Brazil is the third country most affected by Covid-19 pandemic. In spite of this, viral evolution in municipality resolution is poorly understood in Brazil and it is crucial to understand the epidemiology of viral spread. We identified four main circulating lineages in Esteio (Southern Brazil) and their relationship with global, national and regional lineages using phylogenetics and phylodynamics inferences from 21 SARS-CoV-2 genome sequences. We provided a comprehensive view of viral mutations from a time- and age-representative sampling from May to October 2020, in Esteio (RS, Brazil), highlighting two frequent mutations in Spike glycoprotein (D614G and V1176F), an emergent mutation (E484K) in Spike Receptor Binding Domain (RBD) characteristic of the South African lineage B.1.351, and the adjacent replacement of 2 amino acids in Nucleocapsid phosphoprotein (R203K and G204R). A significant viral diversity was evidenced with the identification of 80 different SNPs. The E484K replacement was found in two genomes (9.5%) from samples obtained in mid-October, which is to our best knowledge the earliest description of E484K harboring SARS-CoV-2 in South Brazil. This mutation identified in a small municipality from the RS state demonstrates that it was probably widely distributed in the Brazilian territory, but went unnoticed so far by the lack of genomic surveillance in Brazil. The introduction of E484K mutants shows temporal correlation with later increases in new cases in our state. Importantly, since it has been associated with immune evasion and enhanced interaction with hACE-2, lineages containing this substitution must be the subject of intense surveillance. Our date demonstrates multiple introductions of the most prevalent lineages (B.1.1.33 and B.1.1.248) and the major role of community transmission in viral spreading and the establishment of Brazilian lineages. This represents an important contribution to the epidemiology of SARS-CoV-2.
22Regular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, 23 hard to reproduce, and produce low yields. In an attempt to improve the protocols used for 24 EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii 25 and C. neoformans on solid media. Nanoparticle tracking analysis in combination with 26 transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in 27 solid media. These results were reproduced with an acapsular mutant of C. neoformans, as 28 well as with isolates of Candida albicans, Histoplasma capsulatum, and Saccharomyces 29 cerevisiae. Cryptococcal EVs produced in solid media were biologically active and contained 30 regular vesicular components, including the major polysaccharide glucuronoxylomannan 31 (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular 32 methods used for the isolation of fungal EVs, we asked if it would be applicable to address 33 fundamental questions related to cryptococcal secretion. On the basis that polysaccharide 34 export in Cryptococcus requires highly organized membrane traffic culminating with EV 35 release, we analyzed the participation of a putative scramblase (Aim25, CNBG_3981) in EV-36 mediated GXM export and capsule formation in C. gattii. EVs from a C. gattii aim25 strain 37 differed from those obtained from wild-type (WT) cells in physical-chemical properties and 38 cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, 39EVs obtained from the aim25 mutant were more efficiently used as a source of capsular 40 polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and 41 increased capsular dimensions. These results associate the description of a novel protocol 42 for the isolation of fungal EVs with the identification of a previously unknown regulator of 43 polysaccharide release. 44 45 IMPORTANCE. Extracellular vesicles (EVs) are fundamental components of the physiology of 46 cells from all kingdoms. In pathogenic fungi, they participate in important mechanisms of 47 results describe a fast and reliable method for the study of fungal EVs and reveal the 53 participation of scramblase, a phospholipid translocating enzyme, in secretory processes of 54 C. gattii. 55 56 57
To the Editor -From the onset of the COVID-19 pandemic, we expected that people, especially women, caring for children, elderly, people with disabilities or other family members, would be the most impacted in academia; data proved this to be the case [1][2][3] . This issue is central to the long-standing problem of low female representation in science, since women across all employment sectors persist as the main caregivers.With attempts to return to normal lives following more than a year of remote working and home schooling in many countries, a question arises: how many institutions have implemented effective action to mitigate the pandemic's negative impacts on the academic careers of caregivers? These include reducing teaching or administrative workloads; extending deadlines for caregivers when necessary (for grants, scholarships or career advancement assessments); including specific criteria to fairly assess caregivers' productivity when compared to their peers; creating specific grants to support caregivers in their academic progress; and enforcing policies to increase diversity in academia, especially towards women of colour.Worldwide, the most common measure to offset the pandemic's negative effects on academics was the extension of deadlines, especially for career advancement and tenure track assessments (for example, stopping the tenure clock). While positive, these actions were not specifically tailored to caregivers -those who were hit the hardest. A study published before the pandemic demonstrated that the adoption of neutral tenure clock-stopping policies substantially reduced female tenure rates and substantially increased male tenure rates 4 . Hence, an in-depth assessment with a gender perspective is needed of the consequences of such policies specific to the pandemic. In the specific case of caregivers, actions taken so far are scarce.
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