Historic records indicate that in 1909 the remains of 17 Maronite patriarchs were exhumed from their primary burial location and transferred to a collective secondary burial inside the Saint Marina chapel in an underground cave at the Qanubin monastery in the Northern Lebanese mountains. We used Church records, iconography, archaeology, anthropology, and ancient DNA (aDNA) analyses to investigate whether the remains found in the chapel might belong to the patriarchs. Further, we hoped to identify the remains of patriarch Estephan El Douaihy, one of the 17 patriarchs who was among those said to be buried in the chapel and who is in the process of being canonized by the Vatican. The entire secondary burial was excavated by horizontal ‘décapage’. Pairing of bones, and reconstruction were undertaken, and the Minimum Number of Individuals (MNI) was identified. Age at death was determined through senescence indicators and sex determination was determined from pelvic bone observations. There were only 16 complete crania represented in the collection, and these were targeted for aDNA analyses. The complete mitochondrial genomes were sequenced for all 16 samples and Y-chromosome haplogroups were able to be determined for four individuals. The evidence from the funerary stele, historical church records, osteological analyses and aDNA analyses when combined provide strong evidence to suggest that the 16 complete skulls present in the burial likely belong to 16 of the patriarchs exhumed in 1909.

Herpes simplex virus 1 (HSV-1) encodes a family B DNA polymerase (Pol) capable of exonucleolytic proofreading whose functions have been extensively studied in the past. Early studies on the in vitro activity of purified Pol protein found that the enzymatic functions of the holoenzyme are largely separate. Consequently, exonuclease activity can be reduced or abolished by certain point mutations within catalytically important regions, with no or only minor effects on polymerase activity. Despite unimpaired polymerase activity, the recovery of HSV-1 mutants with a catalytically inactive exonuclease has been so far unsuccessful. Hence, mutations such as D368A, which abolish exonuclease activity, are believed to be lethal. Here, we show that HSV-1 can be recovered in the absence of Pol intrinsic exonuclease activity and demonstrate that a lack of proofreading causes the rapid accumulation of likely detrimental mutations. Although mutations that abolish exonuclease activity do not appear to be lethal, the lack of proofreading yields viruses with a suicidal phenotype that cease to replicate within few passages following reconstitution. Hence, we conclude that high replication fidelity conferred by proofreading is essential to maintain HSV-1 genome integrity and that a lack of exonuclease activity produces an initially viable but rapidly suicidal phenotype. However, stably replicating viruses with reduced exonuclease activity and therefore elevated mutation rates can be generated by mutating a catalytically less important site located within a conserved exonuclease domain. IMPORTANCE Recovery of fully exonuclease-deficient herpes simplex virus 1 (HSV-1) DNA polymerase mutants has been so far unsuccessful. However, exonuclease activity is not known to be directly essential for virus replication, and the lethal phenotype of certain HSV-1 polymerase mutants is thus attributed to factors other than exonuclease activity. Here, we showed that the recovery of a variety of exonuclease-deficient HSV-1 polymerase mutants is possible and that these mutants are initially replication competent. We, however, observed a progressive loss of mutant viability upon cell culture passaging, which coincided with the rapid accumulation of mutations in exonuclease-deficient viruses. We thus concluded that a lack of DNA proofreading in exonuclease-deficient viruses causes an initially viable but rapidly suicidal hypermutator phenotype and, consequently, the extinction of mutant viruses within few generations following recovery. This would make the absence of exonuclease activity the primary reason for the long-reported difficulties in culturing exonuclease-deficient HSV-1 mutants.

The Istituti Zooprofilattici Sperimentali (IZSs) are public health institutes dealing with the aetiology and path-ogenesis of infectious diseases of domestic and wild animals. During Coronavirus Disease 2019 epidemic, the Italian Ministry of Health appointed the IZSs to carry out diagnostic tests for the detection of SARS-CoV-2 in human samples. In particular, the IZS of Abruzzo and Molise (IZS-Teramo) was involved in the diagnosis of SARS-CoV-2 through testing nasopharyngeal swabs by Real Time RT-PCR. Activities and infrastructures were reor-ganised to the new priorities, in a “One Health” framework, based on interdisciplinary, laboratory promptness, accreditation of the test for the detection of the RNA of SARS-CoV-2 in human samples, and management of confidentiality of sensitive data. The laboratory information system-SILAB-was implemented with a One Health module for managing data of human origin, with tools for the automatic registration of information improving the quality of the data. Moreover, the “National Reference Centre for Whole Genome Sequencing of microbial pathogens-database and bioinformatics analysis”-GENPAT-formally established at the IZS-Teramo, developed bioinformatics workflows and IT dashboard with ad hoc surveillance tools to support the metagenomics-based SARS-CoV-2 surveillance, providing molecular sequencing analysis to quickly intercept the variants circulating in the area. This manuscript describes the One Health system developed by adapting and integrating both SILAB and GENPAT tools for supporting surveillance during COVID-19 epidemic in the Abruzzo region, southern Italy. The developed dashboard permits the health authorities to observe the SARS-CoV-2 spread in the region, and by combining spatio-temporal information with metagenomics provides early evidence for the identification of emerging space-time clusters of variants at the municipality level. The implementation of the One Health module was designed to be easily modelled and adapted for the management of other diseases and future hypothetical events of pandemic nature.

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.