Comparing whole‐genome shotgun sequencing and DNA metabarcoding approaches for species identification and quantification of pollen species mixtures

Molecular identification of mixed‐species pollen samples has a range of applications in various fields of research. To date, such molecular identification has primarily been carried out via amplicon sequencing, but whole‐genome shotgun (WGS) sequencing of pollen DNA has potential advantages, including (1) more genetic information per sample and (2) the potential for better quantitative matching. In this study, we tested the performance of WGS sequencing methodology and publicly available reference sequences in identifying species and quantifying their relative abundance in pollen mock communities. Using mock communities previously analyzed with DNA metabarcoding, we sequenced approximately 200Mbp for each sample using Illumina HiSeq and MiSeq. Taxonomic identifications were based on the Kraken k‐mer identification method with reference libraries constructed from full‐genome and short read archive data from the NCBI database. We found WGS to be a reliable method for taxonomic identification of pollen with near 100% identification of species in mixtures but generating higher rates of false positives (reads not identified to the correct taxon at the required taxonomic level) relative to rbcL and ITS2 amplicon sequencing. For quantification of relative species abundance, WGS data provided a stronger correlation between pollen grain proportion and sequence read proportion, but diverged more from a 1:1 relationship, likely due to the higher rate of false positives. Currently, a limitation of WGS‐based pollen identification is the lack of representation of plant diversity in publicly available genome databases. As databases improve and costs drop, we expect that eventually genomics methods will become the methods of choice for species identification and quantification of mixed‐species pollen samples.     

NGSpeciesID: DNA barcode and amplicon consensus generation from long‐read sequencing data

Third‐generation sequencing technologies, such as Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), have gained popularity over the last years. These platforms can generate millions of long‐read sequences. This is not only advantageous for genome sequencing projects, but also advantageous for amplicon‐based high‐throughput sequencing experiments, such as DNA barcoding. However, the relatively high error rates associated with these technologies still pose challenges for generating high‐quality consensus sequences. Here, we present NGSpeciesID, a program which can generate highly accurate consensus sequences from long‐read amplicon sequencing technologies, including ONT and PacBio. The tool includes clustering of the reads to help filter out contaminants or reads with high error rates and employs polishing strategies specific to the appropriate sequencing platform. We show that NGSpeciesID produces consensus sequences with improved usability by minimizing preprocessing and software installation and scalability by enabling rapid processing of hundreds to thousands of samples, while maintaining similar consensus accuracy as current pipelines.     

DNA diet profiles with high‐resolution animal tracking data reveal levels of prey selection relative to habitat choice in a crepuscular insectivorous bird

Given the global decline of many invertebrate food resources, it is fundamental to understand the dietary requirements of insectivores. We give new insights into the functional relationship between the spatial habitat use, food availability, and diet of a crepuscular aerial insectivore, the European Nightjar (Caprimulgus europaeus) by relating spatial use data with high‐throughput sequencing (HTS) combined with DNA metabarcoding. Our study supports the predictions that nightjars collect a substantial part of their daily nourishment from foraging locations, sometimes at considerable distance from nesting sites. Lepidopterans comprise 65% of nightjars'' food source. Nightjars tend to select larger species of Lepidoptera (>19 mm) which suggests that nightjars optimize the efficiency of foraging trips by selecting the most energetically favorable—larger—prey items. We anticipate that our findings may shed additional light on the interactions between invertebrate communities and higher trophic levels, which is required to understand the repercussions of changing food resources on individual‐ and population‐level processes.     

A retrospective analysis of EBV‐DNA status with the prognosis of lymphoma

Epstein–Barr virus (EBV) infection is proved to be associated with clinicopathology of lymphoma. However, little is known about the relationship between EBV‐DNA status after treatment and prognosis. In this study, real‐time polymerase chain reaction (PCR) was used for quantitative detection of EBV‐DNA load in peripheral blood of all 26,527 patients with lymphoma, and the clinical characteristics and prognosis of 202 patients were retrospectively analysed, including 100 patients with positive EBV‐DNA and 102 randomly selected patients with negative EBV‐DNA. We found that the average rate of EBV‐DNA positivity in lymphomas was 0.376%, and EBV‐DNA‐positive patients presented higher risk with elevated lactate dehydrogenase (LDH) and β2‐MG level, B symptoms, secondary hemophagocytic syndrome and lower objective response rate compared to EBV‐DNA‐negative patients. Multivariate analysis revealed EBV‐DNA‐positive patients had inferior progression‐free survival (PFS) and overall survival (OS) and EBV‐DNA level before treatment was related to PFS but not OS of T/NK cell lymphoma. In T/NK cell lymphoma, EBV‐DNA converting negative after treatment was correlated with better PFS but not OS, and second‐line therapy could induce more EBV‐DNA‐negative conversion compared to CHOP‐based therapy. In all, EBV‐DNA positivity before treatment can be a biomarker representing the tumour burden and an independent prognostic factor. EBV‐DNA‐negative conversion after treatment is a good prognostic factor for T/NK cell lymphomas.     

Bioinspiration as a method of problem‐based STEM education: A case study with a class structured around the COVID‐19 crisis

Bioinspiration is a promising lens for biology instruction as it allows the instructor to focus on current issues, such as the COVID‐19 pandemic. From social distancing to oxygen stress, organisms have been tackling pandemic‐related problems for millions of years. What can we learn from such diverse adaptations in our own applications? This review uses a seminar course on the COVID‐19 crisis to illustrate bioinspiration as an approach to teaching biology content. At the start of the class, students mind‐mapped the entire problem; this range of subproblems was used to structure the biology content throughout the entire class. Students came to individual classes with a brainstormed list of biological systems that could serve as inspiration for a particular problem (e.g., absorptive leaves in response to the problem of toilet paper shortages). After exploration of relevant biology content, discussion returned to the focal problem. Students dug deeper into the literature in a group project on mask design and biological systems relevant to filtration and transparency. This class structure was an engaging way for students to learn principles from ecology, evolution, behavior, and physiology. Challenges with this course design revolved around the interdisciplinary and creative nature of the structure; for instance, the knowledge of the participants was often stretched by engineering details. While the present class was focused on the COVID‐19 crisis, a course structured through a bioinspired approach can be applied to other focal problems, or subject areas, giving instructors a powerful method to deliver interdisciplinary content in an integrated and inquiry‐driven way.     

Structural basis for the interaction of SARS‐CoV‐2 virulence factor nsp1 with DNA polymerase α–primase

The molecular mechanisms that drive the infection by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)—the causative agent of coronavirus disease 2019 (COVID‐19)—are under intense current scrutiny to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated. Recent proteomic screens of the interactions between viral and host proteins have identified the human proteins targeted by SARS‐CoV‐2. The DNA polymerase α (Pol α)–primase complex or primosome—responsible for initiating DNA synthesis during genomic duplication—was identified as a target of nonstructural protein 1 (nsp1), a major virulence factor in the SARS‐CoV‐2 infection. Here, we validate the published reports of the interaction of nsp1 with the primosome by demonstrating direct binding with purified recombinant components and providing a biochemical characterization of their interaction. Furthermore, we provide a structural basis for the interaction by elucidating the cryo‐electron microscopy structure of nsp1 bound to the primosome. Our findings provide biochemical evidence for the reported targeting of Pol α by the virulence factor nsp1 and suggest that SARS‐CoV‐2 interferes with Pol α''s putative role in the immune response during the viral infection.     

Diet of breeding Eleonora's falcon Falco eleonorae in Algeria: Insights for the autumn trans‐Mediterranean avian migration

How environmental changes are affecting bird population dynamics is one of the most challenging conservation issues. Dietary studies of top avian predators could offer scope to monitor anthropogenic drivers of ecosystem changes. We investigated the diet of breeding Eleonora''s falcon in an area of Northeastern Algeria in the years 2010–2012. Feathers and insect remains originating from prey plucking behavior were analyzed, providing insights into the seasonally changing diet of this raptor, as well as the trans‐Mediterranean avian migration. A total of 77 species of birds (16 Sylviidae, 11 Turdidae, and 4 Emberizidae), 3 species of insects, and 1 lizard were identified among prey remains, reflecting a diverse diet. Diet composition and prey abundance varied seasonally, faithfully correlating with the passage of migrant birds as recorded from bird ring recoveries. Our findings suggest that dietary studies of predators might be deployed to investigate changes in bird migration. We discuss our results in the context of trans‐Mediterranean migration, with early‐season prey mainly comprising trans‐Saharan migrants (Apus apus and Merops apiaster) and late‐season prey being dominated by Mediterranean winter migrants (Erithacus rubecula, Turdus philomelos, Sylvia atricapilla, and Sturnus vulgaris). Notably, we observed a significant reduction in species richness of passerine remains in 2012, potentially highlighting a decline in the diversity of avian migrants.     

Mortality in people with schizophrenia: a systematic review and meta‐analysis of relative risk and aggravating or attenuating factors

People with schizophrenia die 15‐20 years prematurely. Understanding mortality risk and aggravating/attenuating factors is essential to reduce this gap. We conducted a systematic review and random‐effects meta‐analysis of prospective and retrospective, nationwide and targeted cohort studies assessing mortality risk in people with schizophrenia versus the general population or groups matched for physical comorbidities or groups with different psychiatric disorders, also assessing moderators. Primary outcome was all‐cause mortality risk ratio (RR); key secondary outcomes were mortality due to suicide and natural causes. Other secondary outcomes included any other specific‐cause mortality. Publication bias, subgroup and meta‐regression analyses, and quality assessment (Newcastle‐Ottawa Scale) were conducted. Across 135 studies spanning from 1957 to 2021 (schizophrenia: N=4,536,447; general population controls: N=1,115,600,059; other psychiatric illness controls: N=3,827,955), all‐cause mortality was increased in people with schizophrenia versus any non‐schizophrenia control group (RR=2.52, 95% CI: 2.38‐2.68, n=79), with the largest risk in first‐episode (RR=7.43, 95% CI: 4.02‐13.75, n=2) and incident (i.e., earlier‐phase) schizophrenia (RR=3.52, 95% CI: 3.09‐4.00, n=7) versus the general population. Specific‐cause mortality was highest for suicide or injury‐poisoning or undetermined non‐natural cause (RR=9.76‐8.42), followed by pneumonia among natural causes (RR=7.00, 95% CI: 6.79‐7.23), decreasing through infectious or endocrine or respiratory or urogenital or diabetes causes (RR=3 to 4), to alcohol or gastrointestinal or renal or nervous system or cardio‐cerebrovascular or all natural causes (RR=2 to 3), and liver or cerebrovascular, or breast or colon or pancreas or any cancer causes (RR=1.33 to 1.96). All‐cause mortality increased slightly but significantly with median study year (beta=0.0009, 95% CI: 0.001‐0.02, p=0.02). Individuals with schizophrenia <40 years of age had increased all‐cause and suicide‐related mortality compared to those ≥40 years old, and a higher percentage of females increased suicide‐related mortality risk in incident schizophrenia samples. All‐cause mortality was higher in incident than prevalent schizophrenia (RR=3.52 vs. 2.86, p=0.009). Comorbid substance use disorder increased all‐cause mortality (RR=1.62, 95% CI: 1.47‐1.80, n=3). Antipsychotics were protective against all‐cause mortality versus no antipsychotic use (RR=0.71, 95% CI: 0.59‐0.84, n=11), with largest effects for second‐generation long‐acting injectable anti­psychotics (SGA‐LAIs) (RR=0.39, 95% CI: 0.27‐0.56, n=3), clozapine (RR=0.43, 95% CI: 0.34‐0.55, n=3), any LAI (RR=0.47, 95% CI: 0.39‐0.58, n=2), and any SGA (RR=0.53, 95% CI: 0.44‐0.63, n=4). Antipsychotics were also protective against natural cause‐related mortality, yet first‐generation antipsychotics (FGAs) were associated with increased mortality due to suicide and natural cause in incident schizophrenia. Higher study quality and number of variables used to adjust the analyses moderated larger natural‐cause mortality risk, and more recent study year moderated larger protective effects of antipsychotics. These results indicate that the excess mortality in schizophrenia is associated with several modifiable factors. Targeting comorbid substance abuse, long‐term maintenance antipsychotic treatment and appropriate/earlier use of SGA‐LAIs and clozapine could reduce this mortality gap.     

Sex‐related differential susceptibility to ponatinib cardiotoxicity and differential modulation of the Notch1 signalling pathway in a murine model

Ponatinib (PON), a tyrosine kinase inhibitor approved in chronic myeloid leukaemia, has proven cardiovascular toxicity. We assessed mechanisms of sex‐related PON‐induced cardiotoxicity and identified rescue strategies in a murine model. PON+scrambled siRNA‐treated male mice had a higher number of TUNEL‐positive cells (%TdT+6.12 ± 0.17), higher percentage of SA‐β‐gal‐positive senescent cardiac area (%SA‐β‐gal 1.41 ± 0.59) and a lower reactivity degree (RD) for the survival marker Bmi1 [Abs (OD) 5000 ± 703] compared to female (%TdT+3.75 ± 0.35; %SA‐β‐gal 0.77 ± 0.02; Bmi1 [Abs (OD) 8567 ± 2173]. Proteomics analysis of cardiac tissue showed downstream activation of cell death in PON+siRNA scrambled compared to vehicle or PON+siRNA‐Notch1‐treated male mice. Upstream analysis showed beta‐oestradiol activation, and downstream analysis showed activation of cell survival and inhibition of cell death in PON+scrambled siRNA compared to vehicle or PON+siRNA‐Notch1‐treated female mice. PON+scrambled siRNA‐treated mice also had a downregulation of cardiac actin—more marked in males—and vessel density—more marked in females. Female hearts showed greater cardiac fibrosis than their male counterparts at baseline, with no significant change after PON treatment. PON+siRNA‐scrambled mice had less fibrosis than vehicle or PON+siRNA‐Notch1‐treated mice. The left ventricular systolic dysfunction showed by PON+scrambled siRNA‐treated mice (male %EF 28 ± 9; female %EF 36 ± 7) was reversed in both PON+siRNA‐Notch1‐treated male (%EF 53 ± 9) and female mice (%EF 52 ± 8). We report sex‐related differential susceptibility and Notch1 modulation in PON‐induced cardiotoxicity. This can help to identify biomarkers and potential mechanisms underlying sex‐related differences in PON‐induced cardiotoxicity.     

Documenting the short‐tailed albatross (Phoebastria albatrus) clades historically present in British Columbia,Canada, through ancient DNA analysis of archaeological specimens

The short‐tailed albatross (Phoebastria albatrus) is a threatened seabird whose present‐day range encompasses much of the North Pacific. Within this species, there are two genetic clades (Clades 1 and 2) that have distinctive morphologies and foraging ecologies. Due to a global population collapse in the late 19th and early 20th centuries, the frequency of these clades among the short‐tailed albatross population that historically foraged off British Columbia, Canada, is unclear. To document the species'' historical genetic structure in British Columbia, we applied ancient DNA (aDNA) analysis to 51 archaeological short‐tailed albatross specimens from the Yuquot site (Borden site number: DjSp‐1) that span the past four millennia. We obtained a 141 bp cytochrome b sequence from 43 of the 51 (84.3%) analyzed specimens. Analyses of these sequences indicate 40 of the specimens belong to Clade 1, while 2 belong to Clade 2. We also identified a single specimen with a novel cytochrome b haplotype. Our results indicate that during the past four millennia most of the short‐tailed albatrosses foraging near Yuquot belonged to Clade 1, while individuals from other lineages made more limited use of the area. Comparisons with the results of previous aDNA analyses of archaeological albatrosses from Japanese sites suggest the distribution of Clades 1 and 2 differed. While both albatross clades foraged extensively in the Northwest Pacific, Clade 1 albatrosses appear to have foraged along the west coast of Vancouver Island to a greater extent. Due to their differing distributions, these clades may be exposed to different threats.     

Premature aging is associated with higher levels of 8‐oxoguanine and increased DNA damage in the Polg mutator mouse

Mitochondrial dysfunction plays an important role in the aging process. However, the mechanism by which this dysfunction causes aging is not fully understood. The accumulation of mutations in the mitochondrial genome (or “mtDNA”) has been proposed as a contributor. One compelling piece of evidence in support of this hypothesis comes from the Polg ^D257A/D257A mutator mouse (Polg ^mut/mut ). These mice express an error‐prone mitochondrial DNA polymerase that results in the accumulation of mtDNA mutations, accelerated aging, and premature death. In this paper, we have used the Polg ^mut/mut model to investigate whether the age‐related biological effects observed in these mice are triggered by oxidative damage to the DNA that compromises the integrity of the genome. Our results show that mutator mouse has significantly higher levels of 8‐oxoguanine (8‐oxoGua) that are correlated with increased nuclear DNA (nDNA) strand breakage and oxidative nDNA damage, shorter average telomere length, and reduced mtDNA integrity. Based on these results, we propose a model whereby the increased level of reactive oxygen species (ROS) associated with the accumulation of mtDNA mutations in Polg ^mut/mut mice results in higher levels of 8‐oxoGua, which in turn lead to compromised DNA integrity and accelerated aging via increased DNA fragmentation and telomere shortening. These results suggest that mitochondrial play a central role in aging and may guide future research to develop potential therapeutics for mitigating aging process.     

Inference for epidemic models with time‐varying infection rates: Tracking the dynamics of oak processionary moth in the UK

1. Invasive pests pose a great threat to forest, woodland, and urban tree ecosystems. The oak processionary moth (OPM) is a destructive pest of oak trees, first reported in the UK in 2006. Despite great efforts to contain the outbreak within the original infested area of South‐East England, OPM continues to spread.
2. Here, we analyze data consisting of the numbers of OPM nests removed each year from two parks in London between 2013 and 2020. Using a state‐of‐the‐art Bayesian inference scheme, we estimate the parameters for a stochastic compartmental SIR (susceptible, infested, and removed) model with a time‐varying infestation rate to describe the spread of OPM.
3. We find that the infestation rate and subsequent basic reproduction number have remained constant since 2013 (with R0 between one and two). This shows further controls must be taken to reduce R0 below one and stop the advance of OPM into other areas of England.
4. Synthesis. Our findings demonstrate the applicability of the SIR model to describing OPM spread and show that further controls are needed to reduce the infestation rate. The proposed statistical methodology is a powerful tool to explore the nature of a time‐varying infestation rate, applicable to other partially observed time series epidemic data.

     

LncRNA‐AK137033 inhibits the osteogenic potential of adipose‐derived stem cells in diabetic osteoporosis by regulating Wnt signaling pathway via DNA methylation

ObjectivesBone tissue engineering based on adipose‐derived stem cells (ASCs) is expected to become a new treatment for diabetic osteoporosis (DOP) patients with bone defects. However, compared with control ASCs (CON‐ASCs), osteogenic potential of DOP‐ASCs is decreased, which increased the difficulty of bone reconstruction in DOP patients. Moreover, the cause of the poor osteogenesis of ASCs in a hyperglycemic microenvironment has not been elucidated. Therefore, this study explored the molecular mechanism of the decline in the osteogenic potential of DOP‐ASCs from the perspective of epigenetics to provide a possible therapeutic target for bone repair in DOP patients with bone defects.Materials and methodsAn animal model of DOP was established in mice. CON‐ASCs and DOP‐ASCs were isolated from CON and DOP mice, respectively. {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 small interfering RNA (SiRNA) and an {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 overexpression plasmid were used to regulate the expression of {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 in CON‐ASCs and DOP‐ASCs in vitro. Lentiviruses that carried shRNA‐{"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 or {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 cDNA were used to knockdown or overexpress {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033, respectively, in CON‐ASCs and DOP‐ASCs in vivo. Hematoxylin and eosin (H&E), Masson''s, alizarin red, and alkaline phosphatase (ALP) staining, micro‐computed tomography (Micro‐CT), flow cytometry, qPCR, western blotting, immunofluorescence, and bisulfite‐specific PCR (BSP) were used to analyze the functional changes of ASCs.ResultsThe DOP mouse model was established successfully. Compared with CON‐ASCs, {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 expression, the DNA methylation level of the sFrp2 promoter region, Wnt signaling pathway markers, and the osteogenic differentiation potential were decreased in DOP‐ASCs. In vitro experiments showed that {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 silencing inhibited the Wnt signaling pathway and osteogenic ability of CON‐ASCs by reducing the DNA methylation level in the sFrp2 promoter region. Additionally, overexpression of {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 in DOP‐ASCs rescued these changes caused by DOP. Moreover, the same results were obtained in vivo.ConclusionsLncRNA‐{"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 inhibits the osteogenic potential of DOP‐ASCs by regulating the Wnt signaling pathway via modulating the DNA methylation level in the sFrp2 promoter region. This study provides an important reference to find new targets for the treatment of bone defects in DOP patients.