Bayesian plasma model selection for Thomson scattering.

Laser Thomson scattering (LTS) is a measurement technique that can determine electron velocity distribution functions in plasma systems. However, accurately inferring quantities of interest from an LTS signal requires the selection of a plasma physics submodel, and comprehensive uncertainty quantification (UQ) is needed to interpret the results. Automated model selection, parameter estimation, and UQ are particularly challenging for low-density, low-temperature, potentially non-Maxwellian plasmas like those created in space electric propulsion devices. This paper applies Bayesian inference and model selection to a Raman-calibrated LTS diagnostic in the context of such plasmas. Synthetic data are used to explore the performance of the method across signal-to-noise ratios and model fidelity regimes. Plasmas with Maxwellian and non-Maxwellian velocity distributions are well characterized using priors that span a range of accuracy and specificity. The model selection framework is shown to accurately detect the type of plasmas generating the electron velocity distribution submodel for signal-to-noise ratios greater than around 5. In addition, the Bayesian framework validates the widespread use of 95% confidence intervals from least-squares inversion as a conservative estimate of the uncertainty bounds. However, epistemic posterior correlations between the variables diverge between least-squares and Bayesian estimates as the number of variable parameters increases. This divergence demonstrates the need for Bayesian inference in cases where accurate correlations between electron parameters are necessary. Bayesian model selection is then applied to experimental Thomson scattering data collected in a nanosecond pulsed plasma, generated with a discharge voltage of 5 and 10 kV at a neutral argon background pressure of 7 Torr-Ar. The Bayesian maximum a posteriori estimates of the electron temperature and number density are 1.98 and 2.38 eV and 2.6 × 1018 and 2.72 × 1018 m-3, using the Maxwellian and Druyvesteyn submodels, respectively. Furthermore, for this dataset, the model selection criterion indicates strong support for the Maxwellian distribution at 10 kV discharge voltage and no strong preference between Maxwellian and Druyvesteyn distributions at 5 kV. The logarithmic Bayes' factors for these cases are -35.76 and 1.07, respectively.

An incoherent Thomson scattering system for measurements near plasma boundaries.

Laser Thomson scattering (LTS) is a minimally invasive measurement technique used for determining electron properties in plasma systems. Sheath model closure validation requires minimally invasive measurements of the electron properties that traverse the boundaries between the bulk plasma, the presheath, and the plasma sheath. Several studies have probed the radial properties along the surface of discharge electrodes with laser-based diagnostics and electrostatic probes. These measurements provide valuable insight into the electron properties in this dynamic region. However, sheath model calibration requires plasma property measurements perpendicular to plasma bounding surfaces, in this case, along the electrode normal vector between discharge electrodes. This work presents the development of a discharge plasma cell and laser Thomson scattering system with a measurement volume step of 1 mm normal to plasma bounding surfaces. The laser Thomson scattering measurements are made between a set of discharge electrodes separated by ∼25 mm that are used to generate a pulsed argon plasma. The spatial distribution of electron temperature and density is measured at several discharge voltages between 8 and 20 kV at a pressure of 8 Torr-Ar. It is determined that the system is statistically stationary and resembles a classic DC discharge plasma. The results are some of the first laser diagnostic-based "between electrode" measurements made along the plasma bounding electrode normal vector. A one-dimensional sheath model is applied to determine the near cathode electron properties, and it is determined that the edge of the presheath is probed in the high-voltage cases. As the lengths of the presheath and sheath decrease with decreasing voltage, the region recedes below the closest probed point to the cathode. To improve the performance of the diagnostic, the step size of the interrogation volume should decrease by an order of magnitude from 1 mm to less than 100 µm, and the data acquisition strategy should be revised to increase the signal-to-noise ratio.

Fat malabsorption in pancreatic cancer: Pathophysiology and management.

Exocrine pancreatic insufficiency (EPI) is common in pancreatic ductal adenocarcinoma (PDAC) and may lead to significant nutrition compromise. In the setting of cancer cachexia and gastrointestinal toxicities of cancer treatments, untreated (or undertreated) EPI exacerbates weight loss, sarcopenia, micronutrient deficiencies, and malnutrition. Together, these complications contribute to poor tolerance of oncologic therapies and negatively impact survival. Treatment of EPI in PDAC involves the addition of pancreatic enzyme replacement therapy, with titration to improve gastrointestinal symptoms. Medical nutrition therapies may also be applicable and may include fat-soluble vitamin replacement, medium-chain triglycerides, and, in some cases, enteral nutrition. Optimizing nutrition status is an important adjunct treatment approach to improve quality of life and may also improve overall survival.

Alterations in exocrine pancreatic function after acute pancreatitis.

Exocrine pancreatic dysfunction (EPD) is a malabsorptive complication of pancreatic disorders that can lead to a host of symptoms ranging from flatulence to diarrhea and contribute to weight loss and metabolic bone disease. It is increasingly recognized to occur after acute pancreatitis (AP), including episodes with mild severity. The risk of developing EPD after AP is influenced by a range of factors, including the degree of acinar cell destruction and inflammation during AP, and persistent structural derangements following AP. In this article, we discuss the epidemiology, pathophysiology, and clinical management of EPD after AP while highlighting key knowledge gaps.

Differences in Plasma Fatty Acid Composition Related to Chronic Pancreatitis: A Pilot Study.

OBJECTIVES: Chronic pancreatitis (CP) is an inflammatory disease affecting the absorption of fat-soluble nutrients. Signaling in pancreatic cells that lead to inflammation may be influenced by fatty acids (FAs) through diet and de novo lipogenesis. Here, we investigated the relationship between plasma FA composition in CP with heterogeneity of etiology and complications of CP. MATERIALS AND METHODS: Blood and clinical parameters were collected from subjects with CP (n = 47) and controls (n = 22). Plasma was analyzed for FA composition using gas chromatography and compared between controls and CP and within CP. RESULTS: Palmitic acid increased, and linoleic acid decreased in CP compared with controls. Correlations between age or body mass index and FAs are altered in CP compared with controls. Diabetes, pancreatic calcifications, and substance usage, but not exocrine pancreatic dysfunction, were associated with differences in oleic acid and linoleic acid relative abundance in CP. De novo lipogenesis index was increased in the plasma of subjects with CP compared with controls and in calcific CP compared with noncalcific CP. CONCLUSIONS: Fatty acids that are markers of de novo lipogenesis and linoleic acid are dysregulated in CP depending on the etiology or complication. These results enhance our understanding of CP and highlight potential pathways targeting FAs for treating CP.

Systemic Neutrophil Gelatinase-Associated Lipocalin Alterations in Chronic Pancreatitis: A Multicenter, Cross-Sectional Study.

BACKGROUND: Chronic pancreatitis (CP) is a progressive fibroinflammatory disorder lacking therapies and biomarkers. Neutrophil gelatinase-associated lipocalin (NGAL) is a proinflammatory cytokine elevated during inflammation that binds fatty acids (FAs) like linoleic acid. We hypothesized that systemic NGAL could serve as a biomarker for CP and, with FAs, provide insights into inflammatory and metabolic alterations. METHODS: NGAL was measured by immunoassay and FA composition was measured by gas chromatography in plasma ( n = 171) from a multicenter study, including controls ( n = 50), acute and recurrent acute pancreatitis (AP/RAP) ( n = 71), and CP ( n = 50). Peripheral blood mononuclear cells (PBMCs) from controls ( n = 16), AP/RAP ( n = 17), and CP ( n = 15) were measured by CyTOF. RESULTS: Plasma NGAL was elevated in subjects with CP compared to controls (AUC = 0.777) or AP/RAP (AUC = 0.754) in univariate and multivariate analyses with sex, age, BMI, and smoking (control AUC = 0.874; AP/RAP AUC = 0.819). NGAL was elevated in CP and diabetes compared to CP without diabetes (p < 0.001). NGAL + PBMC populations distinguished CP from controls (AUC = 0.950) or AP/RAP (AUC = 0.941). Linoleic acid was lower while dihomo-γ-linolenic and adrenic acids were elevated in CP (p < 0.05). Linoleic acid was elevated in CP with diabetes compared to CP subjects without diabetes (p = 0. 0471). CONCLUSION: Elevated plasma NGAL and differences in NGAL + PBMCs indicate an immune response shift that may serve as biomarkers of CP. The potential interaction of FAs and NGAL levels provide insights into the metabolic pathophysiology and improve diagnostic classification of CP.

The Role of Cystic Fibrosis Transmembrane Conductance Regulator Modulators After Liver Transplantation in Persons With Cystic Fibrosis.

Despite advances in treatment for cystic fibrosis (CF), liver disease remains a major contributor to morbidity and mortality for persons with CF. Therefore, liver transplantation may be considered in end-stage CF-related liver disease. We present a young patient with CF who underwent solo liver transplantation and has successfully restarted on elexacaftor/tezacaftor/ivacaftor without significant pulmonary or hepatic complications after transplant.

Microbially catalyzed conjugation of GABA and tyramine to bile acids.

Bile acids (BAs) are cholesterol-derived molecules that aid in digestion and nutrient absorption, regulate host metabolic processes, and influence physiology of the gut microbiota. Both the host and its microbiome contribute to enzymatic modifications that shape the chemical diversity of BAs in the gut. Several bacterial species have been reported to conjugate standard amino acids to BAs, but it was not known if bacteria conjugate BAs to other amine classes. Here, we show that Bacteroides fragilis strain P207, isolated from a bacterial bloom in the J-pouch of a patient with ulcerative colitis pouchitis, conjugates standard amino acids and the neuroactive amines γ-aminobutyric acid (GABA) and tyramine to deoxycholic acid. We extended this analysis to other human gut isolates and identified species that are competent to conjugate GABA and tyramine to primary and secondary BAs, and further identified diverse BA-GABA and BA-tyramine amides in human stool. A longitudinal metabolomic analysis of J-pouch contents of the patient from whom B. fragilis P207 was isolated revealed highly reduced levels of secondary bile acids and a shifting BA amide profile before, during, and after onset of pouchitis, including temporal changes in several BA-GABA amides. Treatment of pouchitis with ciprofloxacin was associated with a marked reduction of nearly all BA amides in the J-pouch. Our study expands the known repertoire of conjugated bile acids produced by bacteria to include BA conjugates to GABA and tyramine and demonstrates that these molecules are present in the human gut. IMPORTANCE BAs are modified in multiple ways by host enzymes and the microbiota to produce a chemically diverse set of molecules that assist in the digestive process and impact many physiological functions. This study reports the discovery of bacterial species that conjugate the neuroactive amines, GABA and tyramine, to primary and secondary BAs. We further present evidence that BA-GABA and BA-tyramine conjugates are present in the human gut, and document a shifting BA-GABA profile in a human pouchitis patient before, during, and after inflammation and antibiotic treatment. GABA and tyramine are common metabolic products of the gut microbiota and potent neuroactive molecules. GABA- and tyramine-conjugated BAs may influence receptor-mediated regulatory mechanisms of humans and their gut microbes, and absorption of these molecules and their entry into enterohepatic circulation may impact host physiology at distal tissue sites. This study defines new conjugated bile acids in the human gut.

A novel conjugative transposon carrying an autonomously amplified plasmid.

Tetracyclines serve as broad-spectrum antibiotics to treat bacterial infections. The discovery of new tetracycline resistance genes has led to new questions about the underlying mechanisms of resistance, gene transfer, and their relevance to human health. We tracked changes in the abundance of a 55-kbp conjugative transposon (CTn214) carrying tetQ, a tetracycline resistance gene, within a Bacteroides fragilis metagenome-assembled genome derived from shotgun sequencing of microbial DNA extracted from the ileal pouch of a patient with ulcerative colitis. The mapping of metagenomic reads to CTn214 revealed the multi-copy nature of a 17,044-nt region containing tetQ in samples collected during inflammation and uninflamed visits. B. fragilis cultivars isolated from the same patient during periods of inflammation harbored CTn214 integrated into the chromosome or both a circular, multi-copy, extrachromosomal region of the CTn214 containing tetQ and the corresponding integrated form. The tetracycline-dependent mechanism for the transmission of CTn214 is nearly identical to a common conjugative transposon found in the genome of B. fragilis (CTnDOT), but the autonomously amplified nature of a circular 17,044-nt region of CTn214 that codes for tetQ and the integration of the same sequence in the linear chromosome within the same cell is a novel observation. Genome and transcriptome sequencing of B. fragilis cultivars grown under different concentrations of tetracycline and ciprofloxacin indicates that tetQ in strains containing the circular form remains actively expressed regardless of treatment, while the expression of tetQ in strains containing the linear form increases only in the presence of tetracycline.IMPORTANCEThe exchange of antibiotic production and resistance genes between microorganisms can lead to the emergence of new pathogens. In this study, short-read mapping of metagenomic samples taken over time from the illeal pouch of a patient with ulcerative colitis to a Bacteroides fragilis metagenome-assembled genome revealed two distinct genomic arrangements of a novel conjugative transposon, CTn214, that encodes tetracycline resistance. The autonomous amplification of a plasmid-like circular form from CTn214 that includes tetQ potentially provides consistent ribosome protection against tetracycline. This mode of antibiotic resistance offers a novel mechanism for understanding the emergence of pathobionts like B. fragilis and their persistence for extended periods of time in patients with inflammatory bowel disease.

Open-label, clinical trial extension: Two-year safety and efficacy results of seladelpar in patients with primary biliary cholangitis.

BACKGROUND: Seladelpar is a potent and selective peroxisome proliferator-activated receptor-δ agonist that targets multiple cell types involved in primary biliary cholangitis (PBC), leading to anti-cholestatic, anti-inflammatory and anti-pruritic effects. AIMS: To evaluate the long-term safety and efficacy of seladelpar in patients with PBC. METHODS: In an open-label, international, long-term extension study, patients with PBC completing seladelpar lead-in studies continued treatment. Seladelpar was taken orally once daily at doses of 5 or 10 mg with dose adjustment permitted for safety or tolerability. The primary analysis was for safety and the secondary efficacy analysis examined biochemical markers of cholestasis and liver injury. The study was terminated early due to the unexpected histological findings in a concurrent study for non-alcoholic steatohepatitis, which were subsequently found to predate treatment. Safety and efficacy data were analysed through 2 years. RESULTS: There were no serious treatment-related adverse events observed among 106 patients treated with seladelpar for up to 2 years. There were four discontinuations for safety, one possibly related to seladelpar. Among 53 patients who completed 2 years of seladelpar, response rates increased from years 1 to 2 for the composite endpoint (alkaline phosphatase [ALP] <1.67 × ULN, ≥15% decrease in ALP, and total bilirubin ≤ULN) and ALP normalisation from 66% to 79% and from 26% to 42%, respectively. In those with elevated bilirubin at baseline, 43% achieved normalisation at year 2. CONCLUSIONS: Seladelpar was safe, and markedly improved biochemical markers of cholestasis and liver injury in patients with PBC. These effects were maintained or improved throughout the second year. CLINICALTRIALS: gov: NCT03301506; Clinicaltrialsregister.eu: 2017-003910-16.

Head-related transfer functions of rabbits within the front horizontal plane.

The head-related transfer function (HRTF) describes the direction-dependent acoustic filtering by the head that occurs between a source signal in free-field space and the signal at the tympanic membrane. HRTFs contain information on sound source location via interaural differences of their magnitude or phase spectra and via the shapes of their magnitude spectra. The present study characterized HRTFs for source locations in the front horizontal plane for nine rabbits, which are a species commonly used in studies of the central auditory system. HRTF magnitude spectra shared several features across individuals, including a broad spectral peak at 2.6kHz that increased gain by 12 to 23dB depending on source azimuth; and a notch at 7.6kHz and peak at 9.8kHz visible for most azimuths. Overall, frequencies above 4kHz were amplified for sources ipsilateral to the ear and progressively attenuated for frontal and contralateral azimuths. The slope of the magnitude spectrum between 3 and 5kHz was found to be an unambiguous monaural cue for source azimuths ipsilateral to the ear. Average interaural level difference (ILD) between 5 and 16kHz varied monotonically with azimuth over ±31dB despite a relatively small head size. Interaural time differences (ITDs) at 0.5kHz and 1.5kHz also varied monotonically with azimuth over ±358 µs and ±260 µs, respectively. Remeasurement of HRTFs after pinna removal revealed that the large pinnae of rabbits were responsible for all spectral peaks and notches in magnitude spectra and were the main contribution to high-frequency ILDs (5-16kHz), whereas the rest of the head was the main contribution to ITDs and low-frequency ILDs (0.2-1.5kHz). Lastly, inter-individual differences in magnitude spectra were found to be small enough that deviations of individual HRTFs from an average HRTF were comparable in size to measurement error. Therefore, the average HRTF may be acceptable for use in neural or behavioral studies of rabbits implementing virtual acoustic space when measurement of individualized HRTFs is not possible.

Bile acid fitness determinants of a Bacteroides fragilis isolate from a human pouchitis patient.

IMPORTANCE: The Gram-negative bacterium Bacteroides fragilis is a common member of the human gut microbiota that colonizes multiple host niches and can influence human physiology through a variety of mechanisms. Identification of genes that enable B. fragilis to grow across a range of host environments has been impeded in part by the relatively limited genetic tractability of this species. We have developed a high-throughput genetic resource for a B. fragilis strain isolated from a UC pouchitis patient. Bile acids limit microbial growth and are altered in abundance in UC pouches, where B. fragilis often blooms. Using this resource, we uncovered pathways and processes that impact B. fragilis fitness in bile and that may contribute to population expansions during bouts of gut inflammation.

Efficacy and Safety of Elafibranor in Primary Biliary Cholangitis.

BACKGROUND: Primary biliary cholangitis is a rare, chronic cholestatic liver disease characterized by the destruction of interlobular bile ducts, leading to cholestasis and liver fibrosis. Whether elafibranor, an oral, dual peroxisome proliferator-activated receptor (PPAR) α and δ agonist, may have benefit as a treatment for primary biliary cholangitis is unknown. METHODS: In this multinational, phase 3, double-blind, placebo-controlled trial, we randomly assigned (in a 2:1 ratio) patients with primary biliary cholangitis who had had an inadequate response to or unacceptable side effects with ursodeoxycholic acid to receive once-daily elafibranor, at a dose of 80 mg, or placebo. The primary end point was a biochemical response (defined as an alkaline phosphatase level of <1.67 times the upper limit of the normal range, with a reduction of ≥15% from baseline, and normal total bilirubin levels) at week 52. Key secondary end points were normalization of the alkaline phosphatase level at week 52 and a change in pruritus intensity from baseline through week 52 and through week 24, as measured on the Worst Itch Numeric Rating Scale (WI-NRS; scores range from 0 [no itch] to 10 [worst itch imaginable]). RESULTS: A total of 161 patients underwent randomization. A biochemical response (the primary end point) was observed in 51% of the patients (55 of 108) who received elafibranor and in 4% (2 of 53) who received placebo, for a difference of 47 percentage points (95% confidence interval [CI], 32 to 57; P<0.001). The alkaline phosphatase level normalized in 15% of the patients in the elafibranor group and in none of the patients in the placebo group at week 52 (difference, 15 percentage points; 95% CI, 6 to 23; P = 0.002). Among patients who had moderate-to-severe pruritus (44 patients in the elafibranor group and 22 in the placebo group), the least-squares mean change from baseline through week 52 on the WI-NRS did not differ significantly between the groups (-1.93 vs. -1.15; difference, -0.78; 95% CI, -1.99 to 0.42; P = 0.20). Adverse events that occurred more frequently with elafibranor than with placebo included abdominal pain, diarrhea, nausea, and vomiting. CONCLUSIONS: Treatment with elafibranor resulted in significantly greater improvements in relevant biochemical indicators of cholestasis than placebo. (Funded by GENFIT and Ipsen; ELATIVE ClinicalTrials.gov number, NCT04526665.).

Are the non-weight bearing guidelines for the after treatment of calcaneal fractures still decisive? A Dutch survey among orthopaedic and trauma surgeons.

PURPOSE: The current rehabilitation for patients with surgically treated displaced intra-articular calcaneal fractures (DIACFs) consists of non-weightbearing for 8-12 weeks. The purpose of the present survey was to investigate the current pre-, peri- and post-operative practices among Dutch foot and ankle surgeons. Moreover, it aims to analyze whether surgeons comply to the Arbeitsgemeinschaft für Osteosynthesefragen (AO) guidelines and which decision criteria were used in the determination of the start of weightbearing. METHODS: A survey was distributed among Dutch trauma and orthopaedic surgeons to determine the most common practices in postoperative weightbearing in patients with DIACFs. RESULTS: 75 surgeons responded to the survey. 33% of the respondents adhered to the AO guidelines. 4% of the respondents strictly followed non-weightbearing guidelines, while 96% interpret the AO guidelines or their local protocol freely, in any frequency. When respondents tended to deviate from the AO guidelines or local protocol, a good patients' compliance to therapy was expected. 83% of the respondents started weightbearing on the fracture, based on reported patient complaints. 87% of the respondents did not see any relation between early weightbearing and the occurrence of complications, including loosening of osteosynthesis materials. CONCLUSION: This study demonstrates that there is limited consensus on the rehabilitation for DIACFs. Moreover, it shows that most surgeons are inclined to interpret the current (AO) guideline or their own local protocol freely. New guidelines, supported with well-founded literature, could help surgeons in a more appropriate daily practice in weightbearing for the rehabilitation of calcaneal fractures.

Are periprosthetic hip fractures more severe than native hip fractures? A systematic review of outcomes and resource utilization.

BACKGROUND: There is a paucity of data comparing periprosthetic hip fracture (PPHFx) outcomes and resource utilization to native fractures. Many surgeons consider periprosthetic hip fractures to be more severe injuries than native fractures. The aim of this systematic review is to characterize the outcomes of PPHFx and assess their severity relative to native hip fractures (NHFx). METHODS: A Preferred Reporting Items for Systematic Reviews and Meta-Analysis systematic review was conducted using Medline, Biosis, and Cinahl. Primary outcomes were time to surgery, length of stay (LOS), cost of management, disposition, complication rates, readmission rates, and mortality. RESULTS: 14 articles (13,489 patients) from 2010 to 2018 were included in the study. Study quality was generally low. Patient follow-up ranged from 1 month to 3.2 years. LOS ranged from 5.2 to 38 days. US cost of management was best estimated at $53,669 ± 19,817. Discharge to skilled nursing facilities ranged from 64.5 to 74.5%. Time to surgery ranged from 1.9 to 5.7 days. Readmission rates ranged from 12 to 32%. Per Clavien-Dindo classification, 33.9% suffered minor complications; 14.3% suffered major complications. 1 month and 1 year mortality ranged from 2.9% to 10% and 9.7% to 45%, respectively. CONCLUSION: Time to surgery and LOS were longer for PPHFx relative to NHFx. Complications' rates were higher for PPHFx compared to NHFx. There is no evidence for differences in LOS, cost, discharge, readmission rates, or mortality between PPHFx and NHFx. These results may serve as a baseline in future evaluation of PPHFx management.

'Paleofloodscapes': Application of sediment source fingerprinting to track flood regime change over space and time at the Peace-Athabasca Delta, Canada.

Deltas are hydrologically dynamic landscapes where river floodwaters create a mosaic of productive ecosystems that provide important services. The flood regime, however, is vulnerable to upstream anthropogenic activities, climate change and geomorphic processes. Deciphering the roles of multiple potential stressors on flood regime change is critical for developing appropriate adaptive and mitigative strategies but requires knowledge of hydrological variability at broader scales of space and time than is typically available from instrumental and observational records. At the globally recognized Peace-Athabasca Delta (Canada), the timing, magnitude and causes of reduced flooding and drawdown of perched basin water levels remain an intense focus of investigation. Here we employ novel 'paleofloodscapes', generated from geospatial interpolation of Bayesian mixing model fingerprinting of sediment elemental concentrations, to quantify variation in the delta's flood regime during the past ~140 years. Results reveal that flooding of the delta began to decline several decades before hydroelectric regulation of Peace River flow, not coincident with it, and the influence of floodwaters from the unregulated Athabasca River has declined more than the regulated Peace River. A key discovery is that widespread flooding of perched basins occurs when ice-jam events on the river(s) coincide with a relatively high water-plane in the delta's open-drainage network. Without knowledge of open-drainage water levels, inferred change to the flood regime of perched basins may be inaccurate when derived solely from analyses of Peace River hydrometric data and climatic records. The paleofloodscapes illustrate that rising sediment delivery caused by a natural river avulsion in 1982 may undermine the intended purpose of a proposed weir installation. The most recent paleofloodscape, developed from lake surface sediment sampling shortly after widespread flooding, demonstrates the value of the approach as a landscape hydrological monitoring tool, and is readily transferrable to other floodplains to track flood regime change.

Neotelomeres and Telomere-Spanning Chromosomal Arm Fusions in Cancer Genomes Revealed by Long-Read Sequencing.

Alterations in the structure and location of telomeres are key events in cancer genome evolution. However, previous genomic approaches, unable to span long telomeric repeat arrays, could not characterize the nature of these alterations. Here, we applied both long-read and short-read genome sequencing to assess telomere repeat-containing structures in cancers and cancer cell lines. Using long-read genome sequences that span telomeric repeat arrays, we defined four types of telomere repeat variations in cancer cells: neotelomeres where telomere addition heals chromosome breaks, chromosomal arm fusions spanning telomere repeats, fusions of neotelomeres, and peri-centromeric fusions with adjoined telomere and centromere repeats. Analysis of lung adenocarcinoma genome sequences identified somatic neotelomere and telomere-spanning fusion alterations. These results provide a framework for systematic study of telomeric repeat arrays in cancer genomes, that could serve as a model for understanding the somatic evolution of other repetitive genomic elements.

Germline ablation achieved via CRISPR/Cas9 targeting of NANOS3 in bovine zygotes.

NANOS3 is expressed in migrating primordial germ cells (PGCs) to protect them from apoptosis, and it is known to be a critical factor for germline development of both sexes in several organisms. However, to date, live NANOS3 knockout (KO) cattle have not been reported, and the specific role of NANOS3 in male cattle, or bulls, remains unexplored. This study generated NANOS3 KO cattle via cytoplasmic microinjection of the CRISPR/Cas9 system in vitro produced bovine zygotes and evaluated the effect of NANOS3 elimination on bovine germline development, from fetal development through reproductive age. The co-injection of two selected guide RNA (gRNA)/Cas9 ribonucleoprotein complexes (i.e., dual gRNA approach) at 6 h post fertilization achieved a high NANOS3 KO rate in developing embryos. Subsequent embryo transfers resulted in a 31% (n = 8/26) pregnancy rate. A 75% (n = 6/8) total KO rate (i.e., 100% of alleles present contained complete loss-of-function mutations) was achieved with the dual gRNA editing approach. In NANOS3 KO fetal testes, PGCs were found to be completely eliminated by 41-day of fetal age. Importantly, despite the absence of germ cells, seminiferous tubule development was not impaired in NANOS3 KO bovine testes during fetal, perinatal, and adult stages. Moreover, a live, NANOS3 KO, germline-ablated bull was produced and at sexual maturity he exhibited normal libido, an anatomically normal reproductive tract, and intact somatic gonadal development and structure. Additionally, a live, NANOS3 KO, germline-ablated heifer was produced. However, it was evident that the absence of germ cells in NANOS3 KO cattle compromised the normalcy of ovarian development to a greater extent than it did testes development. The meat composition of NANOS3 KO cattle was unremarkable. Overall, this study demonstrated that the absence of NANOS3 in cattle leads to the specific deficiency of both male and female germ cells, suggesting the potential of NANOS3 KO cattle to act as hosts for donor-derived exogenous germ cell production in both sexes. These findings contribute to the understanding of NANOS3 function in cattle and have valuable implications for the development of novel breeding technologies using germline complementation in NANOS3 KO germline-ablated hosts.