Generation and <i>ex vivo</i> culture of murine and human pancreatic ductal adenocarcinoma tissue slice explants

Traditional 2D/3D co-culture models typically do not reflect the cellular heterogeneity of pancreatic ductal adenocarcinoma (PDAC) tumors, while in vivo models can be slow and ill-suited to mechanistic investigations. Here, we present a protocol for culturing murine PDAC explants and a corresponding human PDAC model using tissue slice explants. We describe steps for sponge production, preparation of media and materials, tissue collection, and sectioning. We then detail procedures for explant plating, daily culture, and collection of samples

Vesicular monoamine transporter 2 (SLC18A2) regulates monoamine turnover and brain development in zebrafish

Aim We aimed at identifying potential roles of vesicular monoamine transporter 2, also known as Solute Carrier protein 18 A2 (SLC18A2) (hereafter, Vmat2), in brain monoamine regulation, their turnover, behaviour and brain development using a novel zebrafish model. Methods A zebrafish strain lacking functional Vmat2 was generated with the CRISPR/Cas9 system. Larval behaviour and heart rate were monitored. Monoamines and their metabolites were analysed with high-pressure liquid chromatography. Amine synthesising and degrading enzymes, and genes essential for brain development, were analysed with quantitative PCR, in situ hybridisation and immunocytochemistry. Results The 5-bp deletion in exon 3 caused an early frameshift and was lethal within 2 weeks post-fertilisation. Homozygous mutants (hereafter, mutants) displayed normal low locomotor activity during night-time but aberrant response to illumination changes. In mutants dopamine, noradrenaline, 5-hydroxytryptamine and histamine levels were reduced, whereas levels of dopamine and 5-hydroxytryptamine metabolites were increased, implying elevated monoamine turnover. Consistently, there were fewer histamine, 5-hydroxytryptamine and dopamine immunoreactive cells. Cellular dopamine immunostaining, in wild-type larvae more prominent in tyrosine hydroxylase 1 (Th1)-expressing than in Th2-expressing neurons, was absent in mutants. Despite reduced dopamine levels, mutants presented upregulated dopamine-synthesising enzymes. Further, in mutants the number of histidine decarboxylase-expressing neurons was increased, notch1a and pax2a were downregulated in brain proliferative zones. Conclusion Lack of Vmat2 increases monoamine turnover and upregulates genes encoding amine-synthesising enzymes, including histidine decarboxylase. Notch1a and pax2a, genes implicated in stem cell development, are downregulated in mutants. The zebrafish vmat2 mutant strain may be a useful model to study how monoamine transport affects brain development and function, and for use in drug screening.Peer reviewe

Identification of a Blood-Based Protein Biomarker Panel for Lung Cancer Detection

Lung cancer is the deadliest cancer worldwide, mainly due to its advanced stage at the time of diagnosis. A non-invasive method for its early detection remains mandatory to improve patients’ survival. Plasma levels of 351 proteins were quantified by Liquid Chromatography-Parallel Reaction Monitoring (LC-PRM)-based mass spectrometry in 128 lung cancer patients and 93 healthy donors. Bootstrap sampling and least absolute shrinkage and selection operator (LASSO) penalization were used to find the best protein combination for outcome prediction. The PanelomiX platform was used to select the optimal biomarker thresholds. The panel was validated in 48 patients and 49 healthy volunteers. A 6-protein panel clearly distinguished lung cancer from healthy individuals. The panel displayed excellent performance: area under the receiver operating characteristic curve (AUC) = 0.999, positive predictive value (PPV) = 0.992, negative predictive value (NPV) = 0.989, specificity = 0.989 and sensitivity = 0.992. The panel detected lung cancer independently of the disease stage. The 6-protein panel and other sub-combinations displayed excellent results in the validation dataset. In conclusion, we identified a blood-based 6-protein panel as a diagnostic tool in lung cancer. Used as a routine test for high- and average-risk individuals, it may complement currently adopted techniques in lung cancer screening.publishedVersio

Genome sequencing of the extinct Eurasian wild aurochs, Bos primigenius, illuminates the phylogeography and evolution of cattle

Background Domestication of the now-extinct wild aurochs, Bos primigenius, gave rise to the two major domestic extant cattle taxa, B. taurus and B. indicus. While previous genetic studies have shed some light on the evolutionary relationships between European aurochs and modern cattle, important questions remain unanswered, including the phylogenetic status of aurochs, whether gene flow from aurochs into early domestic populations occurred, and which genomic regions were subject to selection processes during and after domestication. Here, we address these questions using whole-genome sequencing data generated from an approximately 6,750-year-old British aurochs bone and genome sequence data from 81 additional cattle plus genome-wide single nucleotide polymorphism data from a diverse panel of 1,225 modern animals. Results Phylogenomic analyses place the aurochs as a distinct outgroup to the domestic B. taurus lineage, supporting the predominant Near Eastern origin of European cattle. Conversely, traditional British and Irish breeds share more genetic variants with this aurochs specimen than other European populations, supporting localized gene flow from aurochs into the ancestors of modern British and Irish cattle, perhaps through purposeful restocking by early herders in Britain. Finally, the functions of genes showing evidence for positive selection in B. taurus are enriched for neurobiology, growth, metabolism and immunobiology, suggesting that these biological processes have been important in the domestication of cattle. Conclusions This work provides important new information regarding the origins and functional evolution of modern cattle, revealing that the interface between early European domestic populations and wild aurochs was significantly more complex than previously thought

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Rapid identification of bovine MHCI haplotypes in genetically divergent cattle populations Using Next-Generation Sequencing

The major histocompatibility complex (MHC) region contains many genes that are key regulators of both innate and adaptive immunity including the polymorphic MHCI and MHCII genes. Consequently, the characterisation of the repertoire of MHC genes is critical to understanding the variation that determines the nature of immune responses. Our current knowledge of the bovine MHCI repertoire is limited with only the Holstein-Friesian breed having been studied in any depth. Traditional methods of MHCI genotyping are of low resolution and laborious and this has been a major impediment to a more comprehensive analysis of the MHCI repertoire of other cattle breeds. Next-generation sequencing (NGS) technologies have been used to enable high throughput and much higher resolution MHCI typing in a number of species. In this study we have developed a MiSeq platform approach and requisite bioinformatics pipeline to facilitate typing of bovine MHCI repertoires. The method was validated initially on a cohort of Holstein-Friesian animals and then demonstrated to enable characterisation of MHCI repertoires in African cattle breeds, for which there was limited or no available data. During the course of these studies we identified >140 novel classical MHCI genes and defined 62 novel MHCI haplotypes, dramatically expanding the known bovine MHCI repertoire

Researching COVID to enhance recovery (RECOVER) tissue pathology study protocol: Rationale, objectives, and design.

ImportanceSARS-CoV-2 infection can result in ongoing, relapsing, or new symptoms or organ dysfunction after the acute phase of infection, termed Post-Acute Sequelae of SARS-CoV-2 (PASC), or long COVID. The characteristics, prevalence, trajectory and mechanisms of PASC are poorly understood. The objectives of the Researching COVID to Enhance Recovery (RECOVER) tissue pathology study (RECOVER-Pathology) are to: (1) characterize prevalence and types of organ injury/disease and pathology occurring with PASC; (2) characterize the association of pathologic findings with clinical and other characteristics; (3) define the pathophysiology and mechanisms of PASC, and possible mediation via viral persistence; and (4) establish a post-mortem tissue biobank and post-mortem brain imaging biorepository.MethodsRECOVER-Pathology is a cross-sectional study of decedents dying at least 15 days following initial SARS-CoV-2 infection. Eligible decedents must meet WHO criteria for suspected, probable, or confirmed infection and must be aged 18 years or more at the time of death. Enrollment occurs at 7 sites in four U.S. states and Washington, DC. Comprehensive autopsies are conducted according to a standardized protocol within 24 hours of death; tissue samples are sent to the PASC Biorepository for later analyses. Data on clinical history are collected from the medical records and/or next of kin. The primary study outcomes include an array of pathologic features organized by organ system. Causal inference methods will be employed to investigate associations between risk factors and pathologic outcomes.DiscussionRECOVER-Pathology is the largest autopsy study addressing PASC among US adults. Results of this study are intended to elucidate mechanisms of organ injury and disease and enhance our understanding of the pathophysiology of PASC

Using toxin-producing bacteria to treat explants and autochthonous mouse models of pancreatic cancer

Pancreatic cancer is the 10th most common cancer diagnosis and 4th most common cause of cancer mortality in the United States, highlighting a disparity between disease prevalence and outcome. Ineffective drug delivery to these tumors contributes to the poor prognosis for this disease, as intravenous drug delivery is hampered by poor vascularity within these tumors. Bacterial therapy, or the use of bacterial components to treat disease, is thought to be able to overcome such drug delivery challenges; through a combination of tumor homing and long-term colonization, bacteria can be utilized to produce anti-cancer molecules directly within the cores of tumors. As such, here, we interrogate the feasibility of bacterial cancer therapy for pancreatic ductal adenocarcinoma (PDAC). Before delving too deeply into bacterial therapy design, it was important to first address one major limitation in therapeutic screening models. As a therapeutic should be effective against the entirety of the tumor, without a specific emphasis on the malignant epithelia, we developed and characterized a novel protocol for culturing ex vivo (explant) murine PDAC tissue with a corresponding protocol for human PDAC tissue. We demonstrated that these tumor slice explants retain the complex cellular architecture and population complexity throughout culture, making them a useful resource for not only therapeutic screens, but also paracrine interactions, which are infeasible to explore with in vitro and in vivo models. Use of these murine and human PDAC explant models assisted in the selection of a potent, bacterial-derived cytotoxin, theta toxin, as a potential therapeutic candidate for PDAC, in both bacteria lysate and live bacteria contexts. Ultimately, we employed a strain of a probiotic bacteria, E. coli Nissle 1917, as a ‘living drug’ to selectively produce theta toxin within the confines of a PDAC tumor in a mouse model of pancreatic cancer. In in vivo studies, we demonstrated that live bacteria preferentially colonize tumor tissue following a single, direct, intratumoral injection into the primary PDAC tumor. We found that not only did the bacteria colonize the injected tumor, but also translocated to distant regions of metastasis and secondary tumors such as anogenital papillomas. However, the long-term efficacy of this strategy is in question, as bacterial colonization and therapeutic capability waned after several weeks. Despite the limited time scale of the bacterial colonization, treatment with a single dose of live, theta toxin-producing bacteria provided a nearly 3-fold improvement in overall survival compared to vehicle and standard of care chemotherapy (gemcitabine) treatment arms. Preliminary evidence suggests that this improvement is due to a combination of the direct cytotoxic effect of the theta toxin and an inherently immunostimulatory capacity of these bacteria, resulting in an influx of anti-tumor immune cells and an overall reduction in immunosuppression phenotype markers. These findings suggest that bacterial therapy could be a useful tool for the treatment of pancreatic cancer, not solely due to the direct cytotoxic effect on the tumor, but with the potential for a combination treatment with immunotherapies

Respondent-driven sampling for an adolescent health study in vulnerable urban settings: A multi-country study

The global adolescent population is larger than ever before and is rapidly urbanizing. Global surveillance systems to monitor youth health typically use household- and school-based recruitment methods. These systems risk not reaching the most marginalized youth made vulnerable by conditions of migration, civil conflict, and other forms of individual and structural vulnerability. We describe the methodology of the Well-Being of Adolescents in Vulnerable Environments survey, which used respondent-driven sampling (RDS) to recruit male and female youth aged 15–19 years and living in economically distressed urban settings in Baltimore, MD; Johannesburg, South Africa; Ibadan, Nigeria; New Delhi, India; and Shanghai, China (migrant youth only) for a cross-sectional study. We describe a shared recruitment and survey administration protocol across the five sites, present recruitment parameters, and illustrate challenges and necessary adaptations for use of RDS with youth in disadvantaged urban settings. We describe the reach of RDS into populations of youth who may be missed by traditional household- and school-based sampling. Across all sites, an estimated 9.6% were unstably housed; among those enrolled in school, absenteeism was pervasive with 29% having missed over 6 days of school in the past month. Overall findings confirm the feasibility, efficiency, and utility of RDS in quickly reaching diverse samples of youth, including those both in and out of school and those unstably housed, and provide direction for optimizing RDS methods with this population. In our rapidly urbanizing global landscape with an unprecedented youth population, RDS may serve as a valuable tool in complementing existing household- and school-based methods for health-related surveillance that can guide policy

Respondent-Driven Sampling for an Adolescent Health Study in Vulnerable Urban Settings: A Multi-Country Study

The global adolescent population is larger than ever before and is rapidly urbanizing. Global surveillance systems to monitor youth health typically use household- and school-based recruitment methods. These systems risk not reaching the most marginalized youth made vulnerable by conditions of migration, civil conflict and other forms of individual and structural vulnerability. We describe the methodology of the Well Being of Adolescents in Vulnerable Environments (WAVE) survey, which used respondent-driven sampling (RDS) to recruit male and female youth aged 15 to 19 years and living in economically distressed urban settings in Baltimore, USA, Johannesburg, South Africa, Ibadan, Nigeria, Delhi, India and Shanghai, China (migrant youth only) for a cross-sectional study. We describe a shared recruitment and survey administration protocol across the five sites, present recruitment parameters, and illustrate challenges and necessary adaptations for use of RDS with youth in disadvantaged urban settings. We describe the reach of RDS into populations of youth who may be missed by traditional householdbased and school-based sampling. Across all sites, an estimated 9.6% were unstably housed; among those enrolled in school, absenteeism was pervasive with 29% having missed over 6 days of school in the past month. Overall findings confirm the feasibility, efficiency and utility of RDS in quickly reaching diverse samples of youth, including those both in and out of school and those unstably housed, and provide direction for optimizing RDS methods with this population. In our rapidly urbanizing global landscape with an unprecedented youth population, RDS may serve as a valuable tool in complementing existing household- and school-based methods for health-related surveillance that can guide policy