Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images.
Objectives Omicron lineages BA.1/2 are considered to cause mild clinical courses. Nevertheless, fatal cases after those infections are recognized but little is known about risk factors. Methods Twenty-three full and three partial autopsies in deceased with known Omicron BA.1/2 infections have been consecutively performed. The investigations included histology, blood analyses and molecular virus detection. Results COVID-19-associated diffuse alveolar damage (DAD) was found in only eight cases (31%). This rate is significantly lower compared to previous studies, including non-Omicron variants, where rates between 69% and 92% were observed. Neither vaccination nor known risk factors were significantly associated with a direct cause of death by COVID-19. Only those patients who were admitted to the clinic due to COVID-19 but not for other reasons had a significant association with a direct COVID-19 caused death (P > 0.001).). Conclusions DAD still occurred in the Omicron BA.1/BA.2 era but at considerably lower frequency than seen with previous variants of concern. None of the known risk factors discriminated the cases with COVID-19-caused death from those that died due to a different disease. Therefore, the host’s genomics might play a key role in this regard. Further studies should elucidate the existence of such a genomic risk factor.
Although Borneo has received more attention from biologists than most other islands in the Malay Archipelago, many questions regarding the systematic relationships of Bornean mammals remain. Using next-generation sequencing technology, we obtained mitochondrial DNA sequences from the holotype of Suncus ater, the only known specimen of this shrew. Several shrews collected recently in Sarawak are closely aligned, both morphologically and mitochondrially, with the holotype of S. ater. Phylogenetic analyses of mitochondrial sequences indicate that the S. ater holotype and new Sarawak specimens do not belong to the genus Suncus, but instead are most closely related to Palawanosorex muscorum. Until now Palawanosorex has been known only from the neighboring Philippine island of Palawan. Additional sequences from nuclear ultra-conserved elements from the new Sarawak specimens strongly support a sister relationship to P. muscorum. We therefore transfer ater to Palawanosorex. The new specimens demonstrate that P. ater is more widespread in northern Borneo than previously recorded. Continued sampling of Bornean mammal diversity and reexamination of type material are critical in understanding the evolutionary history of the biologically rich Malay Archipelago.
Abstract Background Karyotype dynamics driven by chromosomal rearrangements has long been considered as a fundamental question in the evolutionary genetics. Saccharum spontaneum , the most primitive and complex species in the genus Saccharum , has reportedly undergone at least two major chromosomal rearrangements, however, its karyotypic evolution remains unclear. Results In this study, four representative accessions, i.e., hypothetical diploid sugarcane ancestor (sorghum, x = 10), Sa. spontaneum Np-X (x = 10, tetraploid), 2012–46 (x = 9, hexaploid) and AP85–441 (x = 8, tetraploid), were selected for karyotype evolution studies. A set of oligonucleotide (oligo)-based barcode probes was developed based on the sorghum genome, which allowed universal identification of all chromosomes from sorghum and Sa. spontaneum . By comparative FISH assays, we reconstructed the karyotype evolutionary history and discovered that although chromosomal rearrangements resulted in greater variation in relative lengths of some chromosomes, all chromosomes maintained a conserved metacentric structure. Additionally, we found that the barcode oligo probe was not applicable for chromosome identification in both Sa. robustum and Sa. officinarum species, suggesting that sorghum is more distantly related to Sa. robustum and Sa. officinarum compared with Sa. spontaneum species. Conclusions Our study demonstrated that the barcode oligo-FISH is an efficient tool for chromosome identification and karyotyping research, and expanded our understanding of the karyotypic and chromosomal evolution in the genus Saccharum .
Falsified medicines are a major threat to global health. Antimalarial drugs have been particularly targeted by criminals. As DNA analysis has revolutionized forensic criminology, we hypothesized that these techniques could also be used to investigate the origins of falsified medicines. Medicines may contain diverse adventitious biological contamination, and the sealed nature of blister-packages may capture and preserve genetic signals from the manufacturing processes allowing identification of production source(s). We conducted a blinded pilot study to determine if such environmental DNA (eDNA) could be detected in eleven samples of falsified and genuine artesunate antimalarial tablets, collected in SE Asia, which could be indicative of origin. Massively Parallel Sequencing (MPS) was used to characterize microbial and eukaryote diversity. Two mitochondrial DNA analysis approaches were explored to detect the presence of human DNA. Trace eDNA from these low biomass samples demonstrated sample specific signals using two target markers. Significant differences in bacterial and eukaryote DNA community structures were observed between genuine and falsified tablets and between different packaging types of falsified artesunate. Human DNA, which was indicative of likely east Asian ancestry, was found in falsified tablets. This pilot study of the ‘pharmabiome’ shows the potential of environmental DNA as a powerful forensic tool to assist with the identification of the environments, and hence location and timing, of the source and manufacture of falsified medicines, establish links between seizures and complement existing tools to build a more complete picture of criminal trade routes. The finding of human DNA in tablets raises important ethical issues that need to be addressed.
Species delimitation is a rich scientific field that often uses different sources of data to identify independently evolving lineages that might be recognized as species. Here, we use an integrative approach based on morphometrics, COI-barcoding, and phylogenomics using ultraconserved elements (UCEs) to investigate whether the orchid bee species Eulaema cingulata (Fabricius, 1804) and E. pseudocingulata Oliveira, 2006 represent a single variable taxon or two different species. We analyzed 126 specimens across the geographical range of these nominal species to test species hypotheses using the general lineage concept. We found substantial overlap in wing and head morphometrics, and both taxa form one phylogenetic lineage based on COI mitochondrial and UCE data. Our results support the recognition of both forms as members of the same evolutionary unit and E. pseudocingulata is herein recognized as a junior synonym of E. cingulata.
Eastern black walnut (Juglans nigra L.), one of the most valuable timber and veneer trees in North America, provides nut shells with unique industrial uses and nut kernels with distinctive culinary attributes. A mature F1 full-sib progeny orchard of 248 individuals from the cross of two eastern black walnut cultivars provides a long-term resource for discovering genetic mechanisms controlling life history, quality traits, and stress resistance. The genetic linkage map, constructed with 356 single nucleotide polymorphism (SNP) markers and 62 expressed sequence tag simple sequence repeats (EST-SSRs), is 1645.7 cM in length, distributed across the expected 16 linkage groups. In this first application of QTL mapping in J. nigra, we report QTL for budbreak, peak pistillate bloom, peak staminate bloom, and heterodichogamy. A dominant major QTL for heterodichogamy is reported, the sequence for which is syntenic with the heterodichogamy QTL on chromosome 11 of Persian walnut (J. regia L.). The mapping population parents are both protogynous, and segregation suggests a Mendelian component, with a 3:1-like inheritance pattern from heterozygous parents. Mapping the sequenced EST-SSR markers to the J. regia “Chandler” V2.0 genome sequence revealed evidence for collinearity and structural changes on two of the sixteen chromosomes. The inclusion of sequenced EST-SSR markers enables the direct comparison of this and subsequent J. nigra maps and other Juglandaceae genetic maps. This investigation initiates long-term QTL detection studies for quality and stress resistance traits in black walnut.
Coronavirus disease 2019 (COVID-19) is known to cause multi-organ dysfunction1–3 during acute infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with some patients experiencing prolonged symptoms, termed post-acute sequelae of SARS-CoV-2 (refs. 4,5). However, the burden of infection outside the respiratory tract and time to viral clearance are not well characterized, particularly in the brain3,6–14. Here we carried out complete autopsies on 44 patients who died with COVID-19, with extensive sampling of the central nervous system in 11 of these patients, to map and quantify the distribution, replication and cell-type specificity of SARS-CoV-2 across the human body, including the brain, from acute infection to more than seven months following symptom onset. We show that SARS-CoV-2 is widely distributed, predominantly among patients who died with severe COVID-19, and that virus replication is present in multiple respiratory and non-respiratory tissues, including the brain, early in infection. Further, we detected persistent SARS-CoV-2 RNA in multiple anatomic sites, including throughout the brain, as late as 230 days following symptom onset in one case. Despite extensive distribution of SARS-CoV-2 RNA throughout the body, we observed little evidence of inflammation or direct viral cytopathology outside the respiratory tract. Our data indicate that in some patients SARS-CoV-2 can cause systemic infection and persist in the body for months.
As genome resources for wheat (Triticum L.) expand at a rapid pace, it is important to update targeted sequencing tools to incorporate improved sequence assemblies and regions of previously unknown significance. Here, we developed an updated regulatory region enrichment capture for wheat and other Triticeae species. The core target space includes sequences from 2-Kbp upstream of each gene predicted in the Chinese Spring wheat genome (IWGSC RefSeq Annotation v1.0) and regions of open chromatin identified with an assay for transposase-accessible chromatin using sequencing from wheat leaf and root samples. To improve specificity, we aggressively filtered candidate repetitive sequences using a combination of nucleotide basic local alignment search tool (BLASTN) searches to the Triticeae Repetitive Sequence Database (TREP), identification of regions with read over-coverage from previous target enrichment experiments, and k-mer frequency analyses. The final design comprises 216.5 Mbp of predicted hybridization space in hexaploid wheat and showed increased specificity and coverage of targeted sequences relative to previous protocols. Test captures on hexaploid and tetraploid wheat and other diploid cereals show that the assay has broad potential utility for cost-effective promoter and open chromatin resequencing and general-purpose genotyping of various Triticeae species.
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