Omicron Waves in Argentina: Dynamics of SARS-CoV-2 Lineages BA.1, BA.2 and the Emerging BA.2.12.1 and BA.4/BA.5.

The COVID-19 pandemic has lately been driven by Omicron. This work aimed to study the dynamics of SARS-CoV-2 Omicron lineages during the third and fourth waves of COVID-19 in Argentina. Molecular surveillance was performed on 3431 samples from Argentina, between EW44/2021 and EW31/2022. Sequencing, phylogenetic and phylodynamic analyses were performed. A differential dynamic between the Omicron waves was found. The third wave was associated with lineage BA.1, characterized by a high number of cases, very fast displacement of Delta, doubling times of 3.3 days and a low level of lineage diversity and clustering. In contrast, the fourth wave was longer but associated with a lower number of cases, initially caused by BA.2, and later by BA.4/BA.5, with doubling times of about 10 days. Several BA.2 and BA.4/BA.5 sublineages and introductions were detected, although very few clusters with a constrained geographical distribution were observed, suggesting limited transmission chains. The differential dynamic could be due to waning immunity and an increase in population gatherings in the BA.1 wave, and a boosted population (for vaccination or recent prior immunity for BA.1 infection) in the wave caused by BA2/BA.4/BA.5, which may have limited the establishment of the new lineages.

Robust and scalable barcoding for massively parallel long-read sequencing.

Nucleic-acid barcoding is an enabling technique for many applications, but its use remains limited in emerging long-read sequencing technologies with intrinsically low raw accuracy. Here, we apply so-called NS-watermark barcodes, whose error correction capability was previously validated in silico, in a proof of concept where we synthesize 3840 NS-watermark barcodes and use them to asymmetrically tag and simultaneously sequence amplicons from two evolutionarily distant species (namely Bordetella pertussis and Drosophila mojavensis) on the ONT MinION platform. To our knowledge, this is the largest number of distinct, non-random tags ever sequenced in parallel and the first report of microarray-based synthesis as a source for large oligonucleotide pools for barcoding. We recovered the identity of more than 86% of the barcodes, with a crosstalk rate of 0.17% (i.e., one misassignment every 584 reads). This falls in the range of the index hopping rate of established, high-accuracy Illumina sequencing, despite the increased number of tags and the relatively low accuracy of both microarray-based synthesis and long-read sequencing. The robustness of NS-watermark barcodes, together with their scalable design and compatibility with low-cost massive synthesis, makes them promising for present and future sequencing applications requiring massive labeling, such as long-read single-cell RNA-Seq.

Assessing Genetic Diversity for a Pre-Breeding Program in Piaractus mesopotamicus by SNPs and SSRs.

The pacu (Piaractus mesopotamicus) is a Neotropical fish with remarkable productive performance for aquaculture. Knowledge of genetic resources in Neotropical fish is essential for their applications in breeding programs. The aim of this study was to characterize the genetic diversity of seven farmed populations of pacu which will constitute the basis for a broodstock foundation for coming breeding programs in Brazil. Analysis of one wild population (Paraná River) was used as a reference to compare genetic parameters in the farmed populations. The analyses were performed using 32 single-nucleotide polymorphisms (SNP) and 8 simple sequence repeat (SSR) markers. No significant differences in genetic diversity between populations estimated through the number of alleles and allelic richness, observed heterozygosity, expected heterozygosity, and minimum allele frequency were detected (p > 0.05). Low genetic diversity was observed in all farmed stocks and the wild population. Moreover, we detected low genetic structure when comparing farmed and wild populations for SNPs (FST = 0.07; K = 3) and SSRs (FST = 0.08; K = 2). Analysis of molecular variance (AMOVA) demonstrated that genetic variation was mostly within populations. Kinship analysis showed that most fish farms included related individuals at a proportion of at least 25%. Our results suggest that the basal broodstock for pacu breeding programs should be founded with individuals from different fish farms for higher genetic diversity and to avoid inbreeding risks.

First DNA Barcode Reference Library for the Identification of South American Freshwater Fish from the Lower Paraná River.

Valid fish species identification is essential for biodiversity conservation and fisheries management. Here, we provide a sequence reference library based on mitochondrial cytochrome c oxidase subunit I for a valid identification of 79 freshwater fish species from the Lower Paraná River. Neighbour-joining analysis based on K2P genetic distances formed non-overlapping clusters for almost all species with a ≥99% bootstrap support each. Identification was successful for 97.8% of species as the minimum genetic distance to the nearest neighbour exceeded the maximum intraspecific distance in all these cases. A barcoding gap of 2.5% was apparent for the whole data set with the exception of four cases. Within-species distances ranged from 0.00% to 7.59%, while interspecific distances varied between 4.06% and 19.98%, without considering Odontesthes species with a minimum genetic distance of 0%. Sequence library validation was performed by applying BOLDs BIN analysis tool, Poisson Tree Processes model and Automatic Barcode Gap Discovery, along with a reliable taxonomic assignment by experts. Exhaustive revision of vouchers was performed when a conflicting assignment was detected after sequence analysis and BIN discordance evaluation. Thus, the sequence library presented here can be confidently used as a benchmark for identification of half of the fish species recorded for the Lower Paraná River.

Glycosomal bromodomain factor 1 from Trypanosoma cruzi enhances trypomastigote cell infection and intracellular amastigote growth.

Acetylation is a ubiquitous protein modification present in prokaryotic and eukaryotic cells that participates in the regulation of many cellular processes. The bromodomain is the only domain known to bind acetylated lysine residues. In the last few years, many bromodomain inhibitors have been developed in order to treat diseases caused by aberrant acetylation of lysine residues and have been tested as anti-parasitic drugs. In the present paper, we report the first characterization of Trypanosoma cruzi bromodomain factor 1 (TcBDF1). TcBDF1 is expressed in all life cycle stages, but it is developmentally regulated. It localizes in the glycosomes directed by a PTS2 (peroxisome-targeting signal 2) sequence. The overexpression of wild-type TcBDF1 is detrimental for epimastigotes, but it enhances the infectivity rate of trypomastigotes and the replication of amastigotes. On the other hand, the overexpression of a mutated version of TcBDF1 has no effect on epimastigotes, but it does negatively affect trypomastigotes' infection and amastigotes' replication.

Trypanosoma cruzi bromodomain factor 3 binds acetylated α-tubulin and concentrates in the flagellum during metacyclogenesis.

Bromodomains are highly conserved acetyl-lysine binding domains found mainly in proteins associated with chromatin and nuclear acetyltransferases. The Trypanosoma cruzi genome encodes at least four bromodomain factors (TcBDFs). We describe here bromodomain factor 3 (TcBDF3), a bromodomain-containing protein localized in the cytoplasm. TcBDF3 cytolocalization was determined, using purified antibodies, by Western blot and immunofluorescence analyses in all life cycle stages of T. cruzi. In epimastigotes and amastigotes, it was detected in the cytoplasm, the flagellum, and the flagellar pocket, and in trypomastigotes only in the flagellum. Subcellular localization of TcBDF3 was also determined by digitonin extraction, ultrastructural immunocytochemistry, and expression of TcBDF3 fused to cyan fluorescent protein (CFP). Tubulin can acquire different posttranslational modifications, which modulate microtubule functions. Acetylated α-tubulin has been found in the axonemes of flagella and cilia, as well as in the subpellicular microtubules of trypanosomatids. TcBDF3 and acetylated α-tubulin partially colocalized in isolated cytoskeletons and flagella from T. cruzi epimastigotes and trypomastigotes. Interaction between the two proteins was confirmed by coimmunoprecipitation and far-Western blot assays with synthetic acetylated α-tubulin peptides and recombinant TcBDF3.

Characterization of TcHMGB, a high mobility group B family member protein from Trypanosoma cruzi.

High mobility group B (HMGB) proteins are highly abundant non-histone chromatin proteins that play important roles in the execution and control of many nuclear functions. Based on homology searches, we identified the coding sequence for the TcHMGB protein, an HMGB family member from Trypanosoma cruzi. TcHMGB has two HMG box domains, similar to mammalian HMGBs, but lacks the typical C-terminal acidic tail. Instead, it contains a 110 amino acid long N-terminal domain. The TcHMGB N-terminal domain is conserved between the TriTryp sequences (70-80% similarity) and seems to be characteristic of kinetoplastid HMGBs. Despite these differences, TcHMGB maintains HMG box architectural functions: we demonstrated that the trypanosomatid HMGB binds distorted DNA structures such as cruciform DNA in gel shift assays. TcHMGB is also able to bend linear DNA as determined by T4 ligase circularization assays, similar to other HMGB family members. Immunofluorescence and western blot assays showed that TcHMGB is a nuclear protein expressed in all life cycle stages. Protein levels, however, seem to vary throughout the life cycle, which may be related to previously described changes in heterochromatin distribution and transcription rates.

Trypanosoma cruzi bromodomain factor 2 (BDF2) binds to acetylated histones and is accumulated after UV irradiation.

Histone tail post-translational modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) regulate many cellular processes. Among these modifications, phosphorylation, methylation and acetylation have already been described in trypanosomatid histones. Bromodomains, together with chromodomains and histone-binding SANT domains, were proposed to be responsible for "histone code" reading. The Trypanosoma cruzi genome encodes four coding sequences (CDSs) that contain a bromodomain, named TcBDF1-4. Here we show that one of those, TcBDF2, is expressed in discrete regions inside the nucleus of all the parasite life cycle stages and binds H4 and H2A purified histones from T. cruzi. Immunolocalization experiments using both anti-histone H4 acetylated peptides and anti-TcBDF2 antibodies determined that TcBDF2 co-localizes with histone H4 acetylated at lysines K10 and K14. TcDBF2 and K10 acetylated H4 interaction was confirmed by co-immunoprecipitation. It is also shown that TcBDF2 was accumulated after UV irradiation of T. cruzi epimastigotes. These results suggest that TcBDF2 could be taking part in a chromatin remodelling complex in T. cruzi.