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Background Next-Generation Sequencing (NGS) catalyzed breakthroughs across various scientific domains. Illumina’s sequencing by synthesis method has long been central to NGS, but new sequencing methods like Element Biosciences’ AVITI technology are emerging. AVITI is reported to offer improved signal-to-noise ratios and cost reductions. However, its reliance on rolling circle amplification, which can be affected by polymer size, raises questions about its effectiveness in sequencing small RNAs (sRNAs) such as microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), and many others. These sRNAs are crucial regulators of gene expression and involved in various biological processes. Additionally, capturing capped small RNAs (csRNA-seq) is a powerful method for mapping active or “nascent” RNA polymerase II transcription initiation in tissues and clinical samples. Results Here, we report a new protocol for seamlessly sequencing short fragments on the AVITI and demonstrate that AVITI and Illumina sequencing technologies equivalently capture human, cattle ( Bos taurus ), and bison ( Bison bison ) sRNA or csRNA sequencing libraries, increasing confidence in both sequencing approaches. Additionally, analysis of generated nascent transcription start site (TSS) data for cattle and bison revealed inaccuracies in their current genome annotations, underscoring the potential and necessity to translate small and nascent RNA sequencing methodologies to livestock. Conclusions Our accelerated and optimized protocol bridges the advantages of AVITI sequencing with critical methods that rely on sequencing short fragments. This advance bolsters the utility of AVITI technology alongside traditional Illumina platforms, offering new opportunities for NGS applications.

Background Bovine babesiosis is a tick-borne disease that poses a significant economic threat to cattle industries in tropical and subtropical areas, and Babesia bovis is the most virulent causative agent of bovine babesiosis. This apicomplexan parasite infects erythrocytes of cattle, causing severe hemolytic disease, and animals that survive an acute infection become persistently infected for life. Adult cattle (> 1 year of age) are highly susceptible and often succumb to acute infection. Protective host immunity involves peripheral blood mononuclear cells (PBMCs) including monocytes, dendritic cells (DC), natural killer (NK), T cells, and B cells, all of which act to control the pathogen. Monocytes release the cytokines interleukin (IL)-1β and tumor necrosis factor (TNF) and nitric oxide, in addition to chemokines that attract immature DCs. NK cells release IL-12, IL-18, and interferon gamma (IFNγ). Mature DC migrate to secondary lymphoid tissues to present Babesia antigens to T cells. B cells will produce antibodies against Babesia . Methods In this study, we examined the transcriptional signatures of PBMCs from adult cattle (aged > 1.5 years) experimentally infected with the B. bovis virulent strain Vir-S74-T3Bo, during the acute phase of babesiosis, at 10 days post infection (dpi), using RNA Sequencing (RNA-Seq) technology. Results Transcriptional signatures evident during the acute phase of babesiosis were cytokines and chemokines, such as IL-0, TNF, IL-1B, IL-18, CSF1, CXCL10 and CXCL16; pattern recognition receptors, such as CD14, TLR and NOD2; complement components, such as C1R, C2, C3aR1, CFB, CFI and CFP; cell adhesion molecules, such as ICAM1/2 and SELL; and apoptosis markers, such as CASP, BAX and BAK. We identified 1766 upregulated and 1508 downregulated genes, with fold changes ranging from two- to 429-fold. We discuss our findings in the context of immune responses to acute disease as a mechanism for adult host survival, with a focus on the molecular functions and biological processes involved in the response to B. bovis infection. Conclusions In this RNA-Seq analysis, we identified genes that are up- and downregulated in response to acute B. bovis infection. Gene expression of IL-10, along with that of the inflammatory cytokines IL-1β, TNFα and IL-18, suggests a non-protective response to B. bovis at 10 dpi. These results enhance our understanding of the molecular interactions between Babesia and the host immune system. Graphical Abstract

Next-Generation Sequencing (NGS) catalyzed breakthroughs across various scientific domains. Illumina's sequencing by synthesis method has long been central to NGS, but new sequencing methods like Element Biosciences' AVITI technology are emerging. AVITI is reported to offer improved signal-to-noise ratios and cost reductions. However, its reliance on rolling circle amplification, which can be affected by polymer size, raises questions about its effectiveness in sequencing small RNAs (sRNAs) such as microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), and many others. These sRNAs are crucial regulators of gene expression and involved in various biological processes. Additionally, capturing capped small RNAs (csRNA-seq) is a powerful method for mapping active or \"nascent\" RNA polymerase II transcription initiation in tissues and clinical samples. Here, we report a new protocol for seamlessly sequencing short fragments on the AVITI and demonstrate that AVITI and Illumina sequencing technologies equivalently capture human, cattle (Bos taurus), and bison (Bison bison) sRNA or csRNA sequencing libraries, increasing confidence in both sequencing approaches. Additionally, analysis of generated nascent transcription start site (TSS) data for cattle and bison revealed inaccuracies in their current genome annotations, underscoring the potential and necessity to translate small and nascent RNA sequencing methodologies to livestock. Our accelerated and optimized protocol bridges the advantages of AVITI sequencing with critical methods that rely on sequencing short fragments. This advance bolsters the utility of AVITI technology alongside traditional Illumina platforms, offering new opportunities for NGS applications.

Next-Generation Sequencing (NGS) catalyzed breakthroughs across various scientific domains. Illumina's sequencing by synthesis method has long been essential for NGS but emerging technologies like Element Biosciences' sequencing by avidity (AVITI) represent a novel approach. It has been reported that AVITI offers improved signal-to-noise ratios and cost reductions. However, the method relies on rolling circle amplification which can be impacted by polymer size, raising questions about its efficacy sequencing small RNAs (sRNA) molecules including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and others that are crucial regulators of gene expression and involved in various biological processes. In addition, capturing capped small RNAs (csRNA-seq) has emerged as a powerful method to map active or \"nascent\" RNA polymerase II transcription initiation in tissues and clinical samples. Here, we report a new protocol for seamlessly sequencing short DNA fragments on the AVITI and demonstrate that AVITI and Illumina sequencing technologies equivalently capture human, cattle ( ) and the bison ( ) sRNA or csRNA sequencing libraries, augmenting the confidence in both approaches. Additionally, analysis of generated nascent transcription start sites (TSSs) data for cattle and bison revealed inaccuracies in their current genome annotations and highlighted the possibility and need to translate small RNA sequencing methodologies to livestock. Our accelerated and optimized protocol therefore bridges the advantages of AVITI sequencing and critical methods that rely on sequencing short DNA fragments.

Mutant KRAS (KM), the most common oncogene in lung cancer (LC), regulates fatty acid (FA) metabolism. However, the role of FA in LC tumorigenesis is still not sufficiently characterized. Here, we show that KMLC has a specific lipid profile, with high triacylglycerides and phosphatidylcholines (PC). We demonstrate that FASN, the rate-limiting enzyme in FA synthesis, while being dispensable in EGFR-mutant or wild-type KRAS LC, is required for the viability of KMLC cells. Integrating lipidomic, transcriptomic and functional analyses, we demonstrate that FASN provides saturated and monounsaturated FA to the Lands cycle, the process remodeling oxidized phospholipids, such as PC. Accordingly, blocking either FASN or the Lands cycle in KMLC, promotes ferroptosis, a reactive oxygen species (ROS)- and iron-dependent cell death, characterized by the intracellular accumulation of oxidation-prone PC. Our work indicates that KM dictates a dependency on newly synthesized FA to escape ferroptosis, establishing a targetable vulnerability in KMLC. Mutant KRAS (KM) is associated with poor prognosis in lung cancer and reported to promote lipid metabolism. Here, the authors show that fatty acid synthesis, which provides lipids to repair oxidized phospholipids through the FASN-Lands cycle axis, is a specific vulnerability for KM lung cancer.

The pregnancy vaginal microbiome contributes to risk of preterm birth, the primary cause of death in children under 5 years of age. Here we describe direct on-swab metabolic profiling by Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) for sample preparation-free characterisation of the cervicovaginal metabolome in two independent pregnancy cohorts (VMET, n  = 160; 455 swabs; VMET II, n  = 205; 573 swabs). By integrating metataxonomics and immune profiling data from matched samples, we show that specific metabolome signatures can be used to robustly predict simultaneously both the composition of the vaginal microbiome and host inflammatory status. In these patients, vaginal microbiota instability and innate immune activation, as predicted using DESI-MS, associated with preterm birth, including in women receiving cervical cerclage for preterm birth prevention. These findings highlight direct on-swab metabolic profiling by DESI-MS as an innovative approach for preterm birth risk stratification through rapid assessment of vaginal microbiota-host dynamics. Here, the authors apply DESI-MS, a sample preparation-free, direct on-swab mass spectrometry analytical tool, to profile the cervicovaginal metabolome of two independent cohorts of pregnant women and, combined with matched metataxonomic and immuno-profiling data, show that DESI-MS predicts vaginal microbiota composition and local inflammatory status associated with preterm birth and clinical interventions used during pregnancy.

The near-failure on September 16, 2008, of American International Group (AIG) was an iconic moment in the financial crisis. Two large bets on real estate made with funding vulnerable to bank-run-like dynamics pushed AIG to the brink of bankruptcy. AIG used securities lending to transform insurance company assets into residential mortgage-backed securities and collateralized debt obligations, ultimately losing at least$21 billion and threatening the solvency of the life insurance companies. AIG also sold insurance on multisector collateralized debt obligations, backed by real estate assets, ultimately losing more than $ 30 billion. These activities were apparently motivated by a belief that AIG's real estate bets would not suffer defaults and were “money-good.” We find that these securities have in fact suffered write-downs and that the stark “money-good” claim can be rejected. Ultimately, both liquidity and solvency were issues for AIG.

The 2013–2016 Ebola virus (EBOV) outbreak in West Africa was the largest and most complex outbreak ever, with a total number of cases and deaths higher than in all previous EBOV outbreaks combined. The outbreak was characterized by rapid spread of the infection in nations that were weakly prepared to handle it. EBOV ribonucleic acid (RNA) is known to persist in body fluids following disease recovery, and studying this persistence is crucial for controlling such epidemics. Observational cohort studies investigating EBOV persistence in semen require following up recently recovered survivors of Ebola virus disease (EVD), from recruitment to the time when their semen tests negative for EBOV, the endpoint being time-to-event. Because recruitment of EVD survivors takes place weeks or months following disease recovery, the event of interest may have already occurred. Survival analysis methods are the best suited for the estimation of the virus persistence in body fluids but must account for left- and interval-censoring present in the data, which is a more complex problem than that of presence of right censoring alone. Using the Sierra Leone Ebola Virus Persistence Study, we discuss study design issues, endpoint of interest and statistical methodologies for interval- and right-censored non-parametric and parametric survival modelling. Using the data from 203 EVD recruited survivors, we illustrate the performance of five different survival models for estimation of persistence of EBOV in semen. The interval censored survival analytic methods produced more precise estimates of EBOV persistence in semen and were more representative of the source population than the right censored ones. The potential to apply these methods is enhanced by increased availability of statistical software to handle interval censored survival data. These methods may be applicable to diseases of a similar nature where persistence estimation of pathogens is of interest.

Oxidative phosphorylation (OXPHOS) defects caused by somatic mitochondrial DNA (mtDNA) mutations increase with age in human colorectal epithelium and are prevalent in colorectal tumours, but whether they actively contribute to tumorigenesis remains unknown. Here we demonstrate that mtDNA mutations causing OXPHOS defects are enriched during the human adenoma/carcinoma sequence, suggesting they may confer a metabolic advantage. To test this we deleted the tumour suppressor Apc in OXPHOS deficient intestinal stem cells in mice. The resulting tumours were larger than in control mice due to accelerated cell proliferation and reduced apoptosis. We show that both normal crypts and tumours undergo metabolic remodelling in response to OXPHOS deficiency by upregulating the serine synthesis pathway (SSP). Moreover, normal human colonic crypts upregulate the SSP in response to OXPHOS deficiency prior to tumorigenesis. Our data show that age-associated OXPHOS deficiency causes metabolic remodelling that can functionally contribute to accelerated intestinal cancer development.

Little is known about the prevalence and best management of needle fear in adults with chronic disease, who may experience frequent and long-term exposure to needles for lifesaving therapies such as renal dialysis and cancer treatment. Identifying interventions that assist in management of needle fear and associated distress is essential to support these patients with repeated needle and cannula exposure. We followed the PRISMA methodology for scoping reviews and systematically searched PsychINFO, PubMed (MEDLINE), ProQuest, Embase and grey literature and reference lists between 1989 and October 2020 for articles related to needle discomfort, distress, anxiety, fear or phobia. The following chronic diseases were included: arthritis, asthma, chronic back pain, cancer, cardiovascular disease, chronic obstructive pulmonary disease, diabetes, and mental illness, or kidney failure. Literature concerning dentistry, vaccination, intravenous drug users and paediatric populations were excluded. We identified 32 papers reporting prevalence (n = 24), management (n = 5) or both (n = 3). Needle fear prevalence varied in disease cohorts: 17-52% (cancer), 25-47% (chronic kidney disease) and 0.2-80% (diabetes). Assessment methods varied across studies. Management strategies had poor evidence-base, but included needle-specific education, decorated devices, cognitive-behavioural stress management techniques, distraction, and changing the therapy environment or modality. Although needle fear is common there is a paucity of evidence regarding interventions to address it among adults living with chronic disease. This scoping review has highlighted the need for improved identification of needle fear in adults and development of interventions are required for these cohorts.