Bronchial artery embolization using small particles is safe and effective: a single center 12-year experience.

BACKGROUND: Bronchial artery embolization (BAE) using particles is an established treatment for hemoptysis. The use of polyvinyl alcohol (PVA) with a particle size of 300 µm or larger is thought to reduce the risk of non-target embolization but may result in more proximal vessel occlusion than is ideal, resulting in a high rate of early recurrent hemorrhage. OBJECTIVE: This study evaluates the safety and efficacy of BAE using PVA particles with a size of less than 300 µm. METHODS: All patients who underwent BAE between 2010 and 2022 at a tertiary center were included. Demographic data, etiology and volume of hemoptysis, technical and clinical success, procedure-related complications, and follow-up information were collected from patients' electronic records. 150-250 µm PVA particles were used to commence embolization in all patients with the subsequent use of larger-sized particles in some individuals. The Kaplan-Meier method was used to estimate recurrence and survival rates. RESULTS: One hundred forty-four patients underwent 189 embolization procedures between 2010 and 2022 and were followed up for a median of 35 months [IQR 19-89]. 150 µm to 250 µm PVA particles were used as the sole embolic agent in 137 cases. Hemoptysis recurred within 30 days in 7%. The median time to repeat intervention was 144 days [IQR 42-441]. Seventeen out of 144 patients had a pulmonary artery branch pseudoaneurysm. The rate of major complications was 1% with no instances of stroke or spinal artery ischemia. Thirty-day mortality was 2% (4/189). CONCLUSION: BAE using 150-250 µm PVA particles is safe and effective with few complications and low rates of early hemoptysis recurrence. CLINICAL RELEVANCE STATEMENT: BAE using small particles is likely to improve outcomes, particularly the rate of early recurrence, in patients with hemoptysis, without an increase in procedural complications. KEY POINTS: BAE is a safe and effective treatment for patients with hemoptysis. Using small PVA particles in BAE has few complications and low rates of early recurrence. Pulmonary artery pseudoaneurysms should be actively sought in those with hemoptysis undergoing BAE.

Kidney Organoids Generated Using an Allelic Series of NPHS2 Point Variants Reveal Distinct Intracellular Podocin Mistrafficking.

BACKGROUND: NPHS2 variants are the most common cause of steroid-resistant nephrotic syndrome in children >1 month old. Missense NPHS2 variants were reported to cause mistrafficking of the encoded protein, PODOCIN, but this conclusion was on the basis of overexpression in some nonpodocyte cell lines. METHODS: We generated a series of human induced pluripotent stem cell (iPSC) lines bearing pathogenic missense variants of NPHS2 , encoding the protein changes p.G92C, p.P118L, p.R138Q, p.R168H, and p.R291W, and control lines. iPSC lines were also generated from a patient with steroid-resistant nephrotic syndrome (p.R168H homozygote) and a healthy heterozygous parent. All lines were differentiated into kidney organoids. Immunofluorescence assessed PODOCIN expression and subcellular localization. Podocytes were transcriptionally profiled and PODOCIN-NEPHRIN interaction interrogated. RESULTS: All variant lines revealed reduced levels of PODOCIN protein in the absence of reduced transcription. Although wild-type PODOCIN localized to the membrane, distinct variant proteins displayed unique patterns of subcellular protein trafficking, some unreported. P118L and R138Q were preferentially retained in the endoplasmic reticulum (ER); R168H and R291W accumulated in the Golgi. Podocyte profiling demonstrated minimal disease-associated transcriptional change. All variants displayed podocyte-specific apoptosis, which was not linked to ER stress. NEPHRIN-PODOCIN colocalization elucidated the variant-specific effect on NEPHRIN association and hence NEPHRIN trafficking. CONCLUSIONS: Specific variants of endogenous NPHS2 result in distinct subcellular PODOCIN localization within organoid podocytes. Understanding the effect of each variant on protein levels and localization and the effect on NEPHRIN provides additional insight into the pathobiology of NPHS2 variants. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_01_05_JASN2022060707.mp3.

Developing research skills in medical students online using an active research study.

BACKGROUND: Developing research skills and scholarship are key components of medical education. The COVID-19 pandemic necessitated that all teaching be delivered online. We introduced an approach to small group teaching in the academic year 2020-2021 online which involved students in an active (ongoing) research study to develop their research skills. METHODS: We acquired student feedback to evaluate their perspectives quantitatively on development of research and scholarship skills, teaching content and format, and tutor performance using this teaching approach. In addition, we captured free text responses from both students and tutors on the positives and negatives of our course, and their suggested improvements. We also compared summative assessment marks for the online/active research course (2020-2021) with those obtained from previous (2017-2019) and subsequent (2021-2023) teaching sessions. RESULTS: Students were largely positive about most aspects of the online course utilising an active research study (n = 13). Students agreed that they were able to acquire research skills, particularly related to data analysis, transferable skills, and giving scientific presentations. A one-way ANOVA revealed no significant difference for assessment marks across all five teaching years (two years prior and two years following the online/active research course), indicating that the course achieved the learning outcomes. Students enjoyed the convenience of online teaching and the availability of course resources, but least liked the lack of in-person interaction and laboratory training. Tutors enjoyed the collaborative aspects of online teaching, but least liked the lack of face-to-face interactions with students. CONCLUSIONS: Our study demonstrates that delivering online teaching which involves students in active research engages and motivates them to develop their research and scholarship skills. We recommend that educators consider incorporating a current research study in their undergraduate courses as this can enhance the student learning experience as well as the research project itself.

Coordinate expression loss of GKN1 and GKN2 in gastric cancer via impairment of a glucocorticoid-responsive enhancer.

Gastrokines (GKNs) are anti-inflammatory proteins secreted by gastric epithelial (surface mucous and pit) cells, with their aberrant loss of expression causally linked to premalignant inflammation and gastric cancer (GC). Transcriptional mechanisms accounting for GKN expression loss have not been elucidated. Using human clinical cohorts, mouse transgenics, bioinformatics, and transfection/reporter assays, we report a novel mechanism of GKN gene transcriptional regulation and its impairment in GC. GKN1/GKN2 loss is highly coordinated, with both genes showing parallel downregulation during human and mouse GC development, suggesting joint transcriptional control. In BAC transgenic studies, we defined a 152-kb genomic region surrounding the human GKN1/GKN2 genes sufficient to direct their tissue- and lineage-restricted expression. A screen of the 152-kb region for candidate regulatory elements identified a DNase I hypersensitive site (CR2) located 4 kb upstream of the GKN1 gene. CR2 showed overlapping enrichment of enhancer-related histone marks (H3K27Ac), a consensus binding site (GRE) for the glucocorticoid receptor (GR), strong GR occupancy in ChIP-seq data sets and, critically, exhibited dexamethasone-sensitive enhancer activity in reporter assays. Strikingly, GR showed progressive expression loss, paralleling that of GKN1/2, in human and mouse GC, suggesting desensitized glucocorticoid signaling as a mechanism underlying GKN loss. Finally, mouse adrenalectomy studies revealed a critical role for endogenous glucocorticoids in sustaining correct expression (and anti-inflammatory restraint) of GKNs in vivo. Together, these data link the coordinate expression of GKNs to a glucocorticoid-responsive and likely shared transcriptional enhancer mechanism, with its compromised activation contributing to dual GKN loss during GC progression.NEW & NOTEWORTHY Gastrokine 2 (GKN2) is an anti-inflammatory protein produced by the gastric epithelium. GKN2 expression is progressively lost during gastric cancer (GC), which is believed to play a casual role in GC development. Here, we use bacterial artificial chromosome transgenic studies to identify a glucocorticoid-responsive enhancer element that likely governs expression of GKN1/GKN2, which, via parallel expression loss of the anti-inflammatory glucocorticoid receptor, reveals a novel mechanism to explain the loss of GKN2 during GC pathogenesis.

Defining a national reference level for intraoperative radiation exposure in urological procedures: FLASH, a retrospective multicentre UK study.

OBJECTIVES: To define reference levels for intraoperative radiation during stent insertion, ureteroscopy (URS), and percutaneous nephrolithotomy (PCNL); to identify variation in radiation exposure between individual hospitals across the UK, between low- and high-volume PCNL centres, and between grade of lead surgeon. PATIENTS/SUBJECTS AND METHODS: In all, 3651 patients were identified retrospectively across 12 UK hospitals over a 1-year period. Radiation exposure was defined in terms of total fluoroscopy time (FT) and dose area product (DAP). The 75th percentiles of median values for each hospital were used to define reference levels for each procedure. RESULTS: Reference levels: ureteric stent insertion/replacement (DAP, 2.3 Gy/cm2 ; FT, 49 s); URS (DAP, 2.8 Gy/cm2 ; FT, 57 s); PCNL (DAP, 24.1 Gy/cm2 ; FT, 431 s). Significant variations in the median DAP and FT were identified between individual centres for all procedures (P < 0.001). For PCNL, there was a statistically significant difference between DAP for low- (<50 cases/annum) and high-volume centres (>50 cases/annum), at a median DAP of 15.0 Gy/cm2 vs 4.2 Gy/cm2 (P < 0.001). For stent procedures, the median DAP and FT differed significantly between grade of lead surgeon: Consultant (DAP, 2.17 Gy/cm2 ; FT, 41 s) vs Registrar (DAP, 1.38 Gy/cm2 ; FT, 26 s; P < 0.001). CONCLUSION: This multicentre study is the largest of its kind. It provides the first national reference level to guide fluoroscopy use in urological procedures, thereby adding a quantitative and objective value to complement the principles of keeping radiation exposure 'as low as reasonably achievable'. This snapshot of real-time data shows significant variation around the country, as well as significant differences between low- and high-volume centres for PCNL, and grade of lead surgeon for stent procedures.

Identification of an Immortalized Human Airway Epithelial Cell Line with Dyskinetic Cilia.

Primary ciliary dyskinesia is an inherited, currently incurable condition. In the respiratory system, primary ciliary dyskinesia causes impaired functioning of the mucociliary escalator, leading to nasal congestion, cough, and recurrent otitis media, and commonly progresses to cause more serious and permanent damage, including hearing deficits, chronic sinusitis, and bronchiectasis. New treatment options for the condition are thus necessary. In characterizing an immortalized human bronchial epithelial cell line (BCi-NS1.1) grown at an air-liquid interface to permit differentiation, we have identified that these cells have dyskinetic motile cilia. The cells had a normal male karyotype, and phenotypic markers of epithelial cell differentiation emerged, as previously shown. Ciliary beat frequency (CBF) as assessed by high-speed videomicroscopy was lower than normal (4.4 Hz). Although changes in CBF induced by known modulators were as expected, the cilia displayed a dyskinetic, circular beat pattern characteristic of central microtubular agenesis with outer doublet transposition. This ultrastructural defect was confirmed by electron microscopy. We propose that the BCi-NS1.1 cell line is a useful model system for examination of modulators of CBF and more specifically could be used to screen for novel drugs with the ability to enhance CBF and perhaps repair a dyskinetic ciliary beat pattern.

Contribution of the two genes encoding histone variant h3.3 to viability and fertility in mice.

Histones package DNA and regulate epigenetic states. For the latter, probably the most important histone is H3. Mammals have three near-identical H3 isoforms: canonical H3.1 and H3.2, and the replication-independent variant H3.3. This variant can accumulate in slowly dividing somatic cells, replacing canonical H3. Some replication-independent histones, through their ability to incorporate outside S-phase, are functionally important in the very slowly dividing mammalian germ line. Much remains to be learned of H3.3 functions in germ cell development. Histone H3.3 presents a unique genetic paradigm in that two conventional intron-containing genes encode the identical protein. Here, we present a comprehensive analysis of the developmental effects of null mutations in each of these genes. H3f3a mutants were viable to adulthood. Females were fertile, while males were subfertile with dysmorphic spermatozoa. H3f3b mutants were growth-deficient, dying at birth. H3f3b heterozygotes were also growth-deficient, with males being sterile because of arrest of round spermatids. This sterility was not accompanied by abnormalities in sex chromosome inactivation in meiosis I. Conditional ablation of H3f3b at the beginning of folliculogenesis resulted in zygote cleavage failure, establishing H3f3b as a maternal-effect gene, and revealing a requirement for H3.3 in the first mitosis. Simultaneous ablation of H3f3a and H3f3b in folliculogenesis resulted in early primary oocyte death, demonstrating a crucial role for H3.3 in oogenesis. These findings reveal a heavy reliance on H3.3 for growth, gametogenesis, and fertilization, identifying developmental processes that are particularly susceptible to H3.3 deficiency. They also reveal partial redundancy in function of H3f3a and H3f3b, with the latter gene being generally the most important.

A phase I clinical trial of navitoclax, a targeted high-affinity Bcl-2 family inhibitor, in combination with gemcitabine in patients with solid tumors.

PURPOSE: To investigate the safety, optimal dosing, pharmacokinetics and clinical activity of a regimen of navitoclax (ABT-263) combined with gemcitabine in patients with solid tumors. EXPERIMENTAL DESIGN: Patients with solid tumors for which gemcitabine was deemed an appropriate therapy were enrolled into one of two different dosing schedules (21-day dosing schedule: navitoclax administered orally on days 1-3 and 8-10,; and gemcitabine 1,000 mg/m(2) on days 1 and 8; 28-day dosing schedule: navitoclax administrated orally on days 1-3, 8-10, and 15-17; and gemcitabine 1,000 mg/m(2) on days 1, 8 and 15). Navitoclax doses were escalated from 150 to 425 mg. An expanded safety cohort was conducted for the 21-day dosing schedule at the maximum tolerated dose (MTD) of navitoclax. RESULTS: Forty-six patients were enrolled at three U.S. centers. The most common adverse events included: hematologic abnormalities (thrombocytopenia, neutropenia, and anemia), liver enzyme elevations (ALT and AST), and gastrointestinal disturbances (diarrhea, nausea, and vomiting). Dose-limiting toxicities (DLTs) observed in cycle 1 were grade 4 thrombocytopenia (2 patients), grade 4 neutropenia (1 patient), and grade 3 AST elevation (2 patients). The MTD of navitoclax was 325 mg co-administered with gemcitabine 1,000 mg/m(2) for the 21-day schedule. No clinically significant pharmacokinetic drug-drug interactions were observed. There were no objective responses. Stable disease, reported at the end of cycle 2, was the best response in 54 % of evaluable patients (n = 39). CONCLUSIONS: The combination of navitoclax 325 mg with gemcitabine 1,000 mg/m(2) was generally well tolerated and exhibited a favorable safety profile in patients with advanced solid tumors.

Prospective Study of Non-Contrast, Abbreviated MRI for Hepatocellular Carcinoma Surveillance in Patients with Suboptimal Hepatic Visualisation on Ultrasound.

BACKGROUND: Biannual ultrasound (US) is recommended for hepatocellular carcinoma (HCC) surveillance in patients with cirrhosis. However, US has limited sensitivity for early-stage HCC, particularly in overweight cohorts, where hepatic visualisation is often inadequate. Currently there are no robust imaging surveillance strategies in patients with inadequate US visualisation. We investigated the ability of non-contrast, abbreviated magnetic resonance imaging (aMRI) to adequately visualise the liver for HCC surveillance in patients with previously inadequate US. METHODS: Patients undergoing US surveillance, where liver visualisation was inadequate (LI-RADS VIS-B and VIS-C), were prospectively recruited. Patients underwent non-contrast T2-weighted and diffusion-weighted aMRI. The images were reviewed and reported by an expert liver radiologist. Three independent, blinded radiologists assessed the aMRI visualisation quality using a binary score assessing five parameters (parenchymal definition, vascular definition, coverage of the liver, uniformity of liver appearance and signal-to-noise ratio). RESULTS: Thirty patients completed the aMRI protocol. The majority (90%) had underlying cirrhosis and were overweight (93.3%), with 50% obese and 20% severely obese. A total of 93.3% of the aMRI scans were of satisfactory quality. Six patients (20%) had hepatic abnormalities detected with aMRI that were not seen on their US: one HCC, one haemangioma and three clinically insignificant lesions. For the aMRI visualisation quality assessment, the coverage of the liver, vascular definition and parenchymal definition were consistently rated to be of sufficient quality by all three radiologists. CONCLUSIONS: Non-contrast aMRI provided good visualisation of the liver and detection of abnormalities in patients with inadequate US. aMRI should be further explored in a larger, prospective study as an alternative surveillance strategy in patients with inadequate US.

Generation of a human ACTA1-tdTomato reporter iPSC line using CRISPR/Cas9 editing.

We used gene editing to introduce DNA sequences encoding the tdTomato fluorescent protein into the α -skeletal actin 1 (ACTA1) locus to develop an ACTA1-tdTomato induced pluripotent stem cell reporter line for monitoring differentiation of skeletal muscle. This cell line will be used to better understand skeletal muscle maturation and development in vitro as well as provide a useful tool for drug screening and the evaluation of novel therapeutics for the treatment of skeletal muscle disease.

How temperature shapes the biosynthesis of polyhydroxyalkanoates in mixed microbial cultures.

Three sequential batch reactors were operated for the enrichment in microbial communities able to store polyhydroxyalkanoates (PHAs) using activated sludge as inoculum. They ran simultaneously under the same operational conditions (organic loading rate, hydraulic and solids retention time, cycle length, C/N ratio) just with the solely difference of the working temperature: psychrophilic (15°C), mesophilic (30°C), and thermophilic (48°C). The microbial communities enriched showed different behaviors in terms of consumption and production rates. In terms of PHA accumulation, the psychrophilic community was able to accumulate an average amount of 17.7 ± 5.7 wt% poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the mesophilic 40.3 ± 7.0 wt% PHBV, and the thermophilic 14.8 ± 0.3 wt% PHBV in dry weight over total solids. The average PHBV production yields for each selected community were 0.41 ± 0.12 CmmolPHBV /CmmolVFA at 15°C, 0.64 ± 0.05 CmmolPHBV /CmmolVFA at 30°C, and 0.39 ± 0.14 CmmolPHBV /CmmolVFA at 48°C. The overall performance of the mesophilic reactor was better than the other two, and the copolymers obtained at this temperature contained a higher PHV fraction. The physico-chemical properties of the obtained biopolymers at each temperature were also measured, and major differences were found in the molecular weight, following an increasing trend with temperature. PRACTITIONER POINTS: PHBV molecular weight is influenced by the operational temperature increasing with it. Increasing temperatures promote the production of HB over HV. The best accumulation performance was found at 30°C for the tested operational conditions.

Reflections on the Teaching Symposium at the Microbiology Society Annual Conference 2023.

The Microbiology Society Education and Outreach Network (EON) recently hosted the Teaching Symposium at the Microbiology Society Annual Conference, sponsored by Access Microbiology. The presence of the Symposium as an established parallel session within the wider Annual Conference reflects the importance of high-quality, contemporary microbiology education and outreach delivered in an enthusiastic and inclusive manner. At the 2023 Symposium, a variety of pedagogical research projects in higher education learning, teaching and assessment, as well as public engagement projects, were showcased through invited talks, offered talks, flash talks and posters. The event was attended by up to 70 delegates. Several themes were noted throughout the day: engaging with Gen Z (Generation Z, those born between 1996 and 2010), active learning, art in science and engaging with non-higher education (HE) audiences. Inclusivity was a key driver in the organization of the Symposium; the room was set up to encourage discussion and participants could ask questions using an online platform as well as speaking in the room. We now encourage all speakers to consider publishing their work as a peer-reviewed article for further dissemination and impact.

Generation of iPSC lines from peripheral blood mononuclear cells from five healthy donors.

We describe the generation and characterisation of five human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) of healthy adult individuals. The PBMCs were reprogrammed using non-integrating Sendai viruses containing the reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC. The iPSC lines exhibited a normal karyotype, and pluripotency was validated by flow cytometry and immunofluorescence of pluripotency markers, and their differentiation into cells representative of the three embryonic germ layers. These iPSC lines can be used as controls in studying disease mechanisms.

Generation of induced pluripotent stem cell lines from three individuals with autism spectrum disorder.

Uncovering the molecular mechanisms of autism spectrum disorder (autism) necessitates development of relevant experimental models that are capable of recapitulating features of the clinical phenotype. Using non-integrative episomal vectors, peripheral blood mononuclear cells derived from three unrelated individuals diagnosed with autism were reprogrammed to induced pluripotent stem cells (iPSCs). The resultant lines exhibited the expected cellular morphology, karyotype, and evidence of pluripotency. These iPSCs constitute a valuable resource to support investigations of the underlying aetiology of autism.

Transcript profiling and inference of Escherichia coli K-12 ArcA activity across the range of physiologically relevant oxygen concentrations.

Oxygen availability is the major determinant of the metabolic modes adopted by Escherichia coli. Although much is known about E. coli gene expression and metabolism under fully aerobic and anaerobic conditions, the intermediate oxygen tensions that are encountered in natural niches are understudied. Here, for the first time, the transcript profiles of E. coli K-12 across the physiologically significant range of oxygen availabilities are described. These suggested a progressive switch to aerobic respiratory metabolism and a remodeling of the cell envelope as oxygen availability increased. The transcriptional responses were consistent with changes in the abundance of cytochrome bd and bo' and the outer membrane protein OmpW. The observed transcript and protein profiles result from changes in the activities of regulators that respond to oxygen itself or to metabolic and environmental signals that are sensitive to oxygen availability (aerobiosis). A probabilistic model (TFInfer) was used to predict the activity of the indirect oxygen-sensing two-component system ArcBA across the aerobiosis range. The model implied that the activity of the regulator ArcA correlated with aerobiosis but not with the redox state of the ubiquinone pool, challenging the idea that ArcA activity is inhibited by oxidized ubiquinone. The amount of phosphorylated ArcA correlated with the predicted ArcA activities and with aerobiosis, suggesting that fermentation product-mediated inhibition of ArcB phosphatase activity is the dominant mechanism for regulating ArcA activity under the conditions used here.

Dynamics of a starvation-to-surfeit shift: a transcriptomic and modelling analysis of the bacterial response to zinc reveals transient behaviour of the Fur and SoxS regulators.

We describe a hybrid transcriptomic and modelling analysis of the dynamics of a bacterial response to stress, namely the addition of 200 µM Zn to Escherichia coli growing in severely Zn-depleted medium and of cells growing at different Zn concentrations at steady state. Genes that changed significantly in response to the transition were those reported previously to be associated with zinc deficiency (zinT, znuA, ykgM) or excess (basR, cpxP, cusF). Cellular Zn levels were confirmed by ICP-AES to be 14- to 28-fold greater after Zn addition but there was also 6- to 8-fold more cellular Fe 30 min after Zn addition. Statistical modelling of the transcriptomic data generated from the Zn shift focused on the role of ten key regulators; ArsR, BaeR, CpxR, CusR, Fur, OxyR, SoxS, ZntR, ZraR and Zur. The data and modelling reveal a transient change in the activity of the iron regulator Fur and of the oxidative stress regulator SoxS, neither of which is evident from the steady-state transcriptomic analyses. We hypothesize a competitive binding mechanism that combines these observations and existing data on the physiology of Zn and Fe uptake. Formalizing the mechanism in a differential equation model shows that it can reproduce qualitatively the behaviour seen in the data. This gives new insights into the interplay of these two fundamental metal ions in gene regulation and bacterial physiology, as well as highlighting the importance of dynamic studies to reverse-engineer systems behaviour.

Rapid evolution of mouse Y centromere repeat DNA belies recent sequence stability.

The Y centromere sequence of house mouse, Mus musculus, remains unknown despite our otherwise significant knowledge of the genome sequence of this important mammalian model organism. Here, we report the complete molecular characterization of the C57BL/6J chromosome Y centromere, which comprises a highly diverged minor satellite-like sequence (designated Ymin) with higher-order repeat (HOR) sequence organization previously undescribed at mouse centromeres. The Ymin array is approximately 90 kb in length and resides within a single BAC clone that provides sequence information spanning an endogenous animal centromere for the first time. By exploiting direct patrilineal inheritance of the Y chromosome, we demonstrate stability of the Y centromere DNA structure spanning at least 175 inbred generations to beyond the time of domestication of the East Asian M.m. molossinus "fancy" mouse through which the Y chromosome was first introduced into the classical inbred laboratory mouse strains. Despite this stability, at least three unequal genetic exchange events have altered Ymin HOR unit length and sequence structure since divergence of the ancestral Mus musculus subspecies around 900,000 yr ago, with major turnover of the HOR arrays driving rapid divergence of sequence and higher-order structure at the mouse Y centromere. A comparative sequence analysis between the human and chimpanzee centromeres indicates a similar rapid divergence of the primate Y centromere. Our data point to a unique DNA sequence and organizational architecture for the mouse Y centromere that has evolved independently of all other mouse centromeres.

Beyond Basic Diversity Estimates-Analytical Tools for Mechanistic Interpretations of Amplicon Sequencing Data.

Understanding microbial ecology through amplifying short read regions, typically 16S rRNA for prokaryotic species or 18S rRNA for eukaryotic species, remains a popular, economical choice. These methods provide relative abundances of key microbial taxa, which, depending on the experimental design, can be used to infer mechanistic ecological underpinnings. In this review, we discuss recent advancements in in situ analytical tools that have the power to elucidate ecological phenomena, unveil the metabolic potential of microbial communities, identify complex multidimensional interactions between species, and compare stability and complexity under different conditions. Additionally, we highlight methods that incorporate various modalities and additional information, which in combination with abundance data, can help us understand how microbial communities respond to change in a typical ecosystem. Whilst the field of microbial informatics continues to progress substantially, our emphasis is on popular methods that are applicable to a broad range of study designs. The application of these methods can increase our mechanistic understanding of the ongoing dynamics of complex microbial communities.

A cost-benefit analysis of the ENIGMA trial.

BACKGROUND: The ENIGMA trial was a prospective, randomized, multicenter study that evaluated the clinical consequences of including N2O in general anesthesia. Patients who were given a N2O-free anesthetic when undergoing major surgery for which the expected hospital stay was at least 3 days had lower rates of some postoperative complications. This suggests that, despite a higher consumption of potent inhalational agent, there could be a financial benefit when N2O is avoided in such settings. METHODS: A retrospective cost analysis of the 2,050 patients recruited to the ENIGMA trial was performed. We measured costs from the perspective of an implementing hospital. Direct health care costs include the costs for maintaining anesthesia, daily medications, hospitalization, and complications. The primary outcome was the net financial savings from avoiding N2O in major noncardiac surgery. Comparisons between groups were analyzed using Student t test and bootstrap methods. Sensitivity analyses were also performed. RESULTS: Rates of some serious complications were higher in the N2O group. Total costs in the N2O group were $16,203 and in the N2O-free group $13,837, mean difference of $2,366 (95% CI: 841-3,891); P = 0.002. All sensitivity analyses retained a significant difference in favor of the N2O-free group (all P ≤ 0.005). CONCLUSIONS: Despite N2O reducing the consumption of more expensive potent inhalational agent, there were marked additional costs associated with its use in adult patients undergoing major surgery because of an increased rate of complications. There is no cogent argument to continue using N2O on the basis that it is an inexpensive drug.

Generating an iPSC line (with isogenic control) from the PBMCs of an ACTA1 (p.Gly148Asp) nemaline myopathy patient.

To produce an in vitro model of nemaline myopathy, we reprogrammed the peripheral blood mononuclear cells (PBMCs) of a patient with a heterozygous p.Gly148Asp mutation in exon 3 of the ACTA1 gene to iPSCs. Using CRISPR/Cas9 gene editing we corrected the mutation to generate an isogenic control line. Both the mutant and control show a normal karyotype, express pluripotency markers and could differentiae into the three cell states that represent embryonic germ layers (endoderm, mesoderm and neuroectoderm) and the dermomyotome (precursor of skeletal muscle). When differentiated these cell lines will be used to explore disease mechanisms and evaluate novel therapeutics.