Critical care unit bed availability and postoperative outcomes: a multinational cohort study.

BACKGROUND: Critical care beds are a limited resource, yet research indicates that recommendations for postoperative critical care admission based on patient-level risk stratification are not followed. It is unclear how prioritisation decisions are made in real-world settings and the effect of this prioritisation on outcomes. METHODS: This was a prespecified analysis of an observational cohort study of adult patients undergoing inpatient surgery, conducted in 274 hospitals across the UK and Australasia during 2017. The primary outcome was postoperative morbidity at day 7. Logistic regression models were used to evaluate the relationship between critical care admission and patient and health system factors. The causal effect of critical care admission on outcome was estimated using variation in critical care occupancy as a natural experiment in an instrumental variable analysis. RESULTS: A total of 19,491 patients from 248 hospitals were eligible for analysis, of whom 2107 were directly admitted to critical care postoperatively. Postoperative morbidity occurred in 2829/19,491 (15%) patients. Increasing surgical risk was associated with critical care admission, as was increased availability of critical care beds (odds ratio (95%CI) 1.04 (1.01-1.06), p = 0.002) per available bed; however, the probability of admission varied significantly between hospitals (median odds ratio 3.05). There was no evidence of a difference in postoperative morbidity with critical care admission (odds ratio (95%CI) 0.91 (0.57-1.45), p = 0.710). DISCUSSION: Postoperative critical care admission is variable and related to bed availability. Statistical methods that adjust for unobserved confounding lowered the estimates of harm previously reported to have been associated with postoperative critical care admission. Our findings provide a rationale for a clinical trial which would evaluate any potential benefits for postoperative critical care admission for patients in whom there is no absolute indication for admission.

The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. The protein Fused in Sarcoma (FUS) undergoes LLPS and mutations in FUS have been causally linked to the motor neuron disease Amyotrophic Lateral Sclerosis (ALS-FUS). LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. However, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. Hence, we developed a method allowing for the purification of LLPS FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome, depending on its biophysical state. While non-LLPS FUS interacts mainly with factors involved in pre-mRNA processing, LLPS FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, also mitochondrial factors are strongly enriched with LLPS FUS, providing a potential explanation for the observed changes in mitochondrial gene expression in mouse models of ALS-FUS. In summary, we present a methodology to investigate the interactomes of phase separating proteins and provide evidence that LLPS shapes the FUS interactome with implications for function and disease.

Assessing the utility of long-read nanopore sequencing for rapid and efficient characterization of mobile element insertions.

Short-read next generation sequencing (NGS) has become the predominant first-line technique used to diagnose patients with rare genetic conditions. Inherent limitations of short-read technology, notably for the detection and characterization of complex insertion-containing variants, are offset by the ability to concurrently screen many disease genes. "Third-generation" long-read sequencers are increasingly being deployed as an orthogonal adjunct technology, but their full potential for molecular genetic diagnosis has yet to be exploited. Here, we describe three diagnostic cases in which pathogenic mobile element insertions were refractory to characterization by short-read sequencing. To validate the accuracy of the long-read technology, we first used Sanger sequencing to confirm the integration sites and derive curated benchmark sequences of the variant-containing alleles. Long-read nanopore sequencing was then performed on locus-specific amplicons. Pairwise comparison between these data and the previously determined benchmark alleles revealed 100% identity of the variant-containing sequences. We demonstrate a number of technical advantages over existing wet-laboratory approaches, including in silico size selection of a mixed pool of amplification products, and the relative ease with which an automated informatics workflow can be established. Our findings add to a growing body of literature describing the diagnostic utility of long-read sequencing.

Epidural Versus Transabdominal Wall Catheters: A Comparative Study of Outcomes After Pancreatic Resection.

BACKGROUND: This study compared epidural analgesia with local anesthetic administration via transabdominal wall catheters (TAWC), to determine the effect on perioperative outcomes in pancreatic surgery. MATERIALS AND METHODS: A retrospective review of patients undergoing open pancreatic surgery at Auckland City Hospital from 2015 to 2018 was undertaken. Data collected included patient demographics, type of perioperative analgesia, intravenous fluid and vasopressor use, length of high dependency unit stay, postoperative complications, and length of hospital stay. RESULTS: Seventy-two patients underwent pancreatic surgery, of which 47 had epidural analgesia and 25 TAWC. The median age was 64 y (range 29-85). Failure of analgesia method occurred in 45% of epidural patients and 28% of TAWC patients (P = 0.209). There was no significant difference in volume of intravenous fluid given or need for vasopressors in the first 3 postoperative days, length of high dependency unit stay (median 1 d, P = 0.2836), rates of postoperative pancreatic fistula (32% versus 40%, P = 0.6046), postoperative complications (38% versus 20%, P = 0.183), or mortality (0.04% versus 0.04%, P = 1.0). CONCLUSIONS: Epidural analgesia and TAWC may have comparable perioperative outcomes in patients undergoing pancreatic surgery. Further randomized studies with a larger cohort of patients are warranted to identify the best postoperative analgesic method in patients undergoing pancreatic resection.

Developing and validating subjective and objective risk-assessment measures for predicting mortality after major surgery: An international prospective cohort study.

BACKGROUND: Preoperative risk prediction is important for guiding clinical decision-making and resource allocation. Clinicians frequently rely solely on their own clinical judgement for risk prediction rather than objective measures. We aimed to compare the accuracy of freely available objective surgical risk tools with subjective clinical assessment in predicting 30-day mortality. METHODS AND FINDINGS: We conducted a prospective observational study in 274 hospitals in the United Kingdom (UK), Australia, and New Zealand. For 1 week in 2017, prospective risk, surgical, and outcome data were collected on all adults aged 18 years and over undergoing surgery requiring at least a 1-night stay in hospital. Recruitment bias was avoided through an ethical waiver to patient consent; a mixture of rural, urban, district, and university hospitals participated. We compared subjective assessment with 3 previously published, open-access objective risk tools for predicting 30-day mortality: the Portsmouth-Physiology and Operative Severity Score for the enUmeration of Mortality (P-POSSUM), Surgical Risk Scale (SRS), and Surgical Outcome Risk Tool (SORT). We then developed a logistic regression model combining subjective assessment and the best objective tool and compared its performance to each constituent method alone. We included 22,631 patients in the study: 52.8% were female, median age was 62 years (interquartile range [IQR] 46 to 73 years), median postoperative length of stay was 3 days (IQR 1 to 6), and inpatient 30-day mortality was 1.4%. Clinicians used subjective assessment alone in 88.7% of cases. All methods overpredicted risk, but visual inspection of plots showed the SORT to have the best calibration. The SORT demonstrated the best discrimination of the objective tools (SORT Area Under Receiver Operating Characteristic curve [AUROC] = 0.90, 95% confidence interval [CI]: 0.88-0.92; P-POSSUM = 0.89, 95% CI 0.88-0.91; SRS = 0.85, 95% CI 0.82-0.87). Subjective assessment demonstrated good discrimination (AUROC = 0.89, 95% CI: 0.86-0.91) that was not different from the SORT (p = 0.309). Combining subjective assessment and the SORT improved discrimination (bootstrap optimism-corrected AUROC = 0.92, 95% CI: 0.90-0.94) and demonstrated continuous Net Reclassification Improvement (NRI = 0.13, 95% CI: 0.06-0.20, p < 0.001) compared with subjective assessment alone. Decision-curve analysis (DCA) confirmed the superiority of the SORT over other previously published models, and the SORT-clinical judgement model again performed best overall. Our study is limited by the low mortality rate, by the lack of blinding in the 'subjective' risk assessments, and because we only compared the performance of clinical risk scores as opposed to other prediction tools such as exercise testing or frailty assessment. CONCLUSIONS: In this study, we observed that the combination of subjective assessment with a parsimonious risk model improved perioperative risk estimation. This may be of value in helping clinicians allocate finite resources such as critical care and to support patient involvement in clinical decision-making.

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription.

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes.

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.

Large-scale image-based profiling of single-cell phenotypes in arrayed CRISPR-Cas9 gene perturbation screens.

High-content imaging using automated microscopy and computer vision allows multivariate profiling of single-cell phenotypes. Here, we present methods for the application of the CISPR-Cas9 system in large-scale, image-based, gene perturbation experiments. We show that CRISPR-Cas9-mediated gene perturbation can be achieved in human tissue culture cells in a timeframe that is compatible with image-based phenotyping. We developed a pipeline to construct a large-scale arrayed library of 2,281 sequence-verified CRISPR-Cas9 targeting plasmids and profiled this library for genes affecting cellular morphology and the subcellular localization of components of the nuclear pore complex (NPC). We conceived a machine-learning method that harnesses genetic heterogeneity to score gene perturbations and identify phenotypically perturbed cells for in-depth characterization of gene perturbation effects. This approach enables genome-scale image-based multivariate gene perturbation profiling using CRISPR-Cas9.

Postoperative critical care and high-acuity care provision in the United Kingdom, Australia, and New Zealand.

BACKGROUND: Decisions to admit high-risk postoperative patients to critical care may be affected by resource availability. We aimed to quantify adult ICU/high-dependency unit (ICU/HDU) capacity in hospitals from the UK, Australia, and New Zealand (NZ), and to identify and describe additional 'high-acuity' beds capable of managing high-risk patients outside the ICU/HDU environment. METHODS: We used a modified Delphi consensus method to design a survey that was disseminated via investigator networks in the UK, Australia, and NZ. Hospital- and ward-level data were collected, including bed numbers, tertiary services offered, presence of an emergency department, ward staffing levels, and the availability of critical care facilities. RESULTS: We received responses from 257 UK (response rate: 97.7%), 35 Australian (response rate: 32.7%), and 17 NZ (response rate: 94.4%) hospitals (total 309). Of these hospitals, 91.6% reported on-site ICU or HDU facilities. UK hospitals reported fewer critical care beds per 100 hospital beds (median=2.7) compared with Australia (median=3.7) and NZ (median=3.5). Additionally, 31.1% of hospitals reported having high-acuity beds to which high-risk patients were admitted for postoperative management, in addition to standard ICU/HDU facilities. The estimated numbers of critical care beds per 100 000 population were 9.3, 14.1, and 9.1 in the UK, Australia, and NZ, respectively. The estimated per capita high-acuity bed capacities per 100 000 population were 1.2, 3.8, and 6.4 in the UK, Australia, and NZ, respectively. CONCLUSIONS: Postoperative critical care resources differ in the UK, Australia, and NZ. High-acuity beds may have developed to augment the capacity to deliver postoperative critical care.

A conceptual framework for capacity strengthening of health research in conflict: the case of the Middle East and North Africa region.

BACKGROUND: In conflict settings, research capacities have often been de-prioritized as resources are diverted to emergency needs, such as addressing elevated morbidity, mortality and health system challenges directly and/or indirectly associated to war. This has had an adverse long-term impact in such protracted conflicts such as those found in the Middle East and North Africa region (MENA), where research knowledge and skills have often been compromised. In this paper, we propose a conceptual framework for health research capacity strengthening that adapts existing models and frameworks in low- and middle-income countries and uses our knowledge of the MENA context to contextualise them for conflict settings. METHODS: The framework was synthesized using "best fit" framework synthesis methodology. Relevant literature, available in English and Arabic, was collected through PubMed, Google Scholar and Google using the keywords: capacity building; capacity strengthening; health research; framework and conflict. Grey literature was also assessed. RESULTS: The framework is composed of eight principal themes: "structural levels", "the influence of the external environment", "funding, community needs and policy environment", "assessing existing capacity and needs", "infrastructure and communication", "training, leadership and partnership", "adaptability and sustainability", and "monitoring and evaluation"; with each theme being supported by examples from the MENA region. Our proposed framework takes into consideration safety, infrastructure, communication and adaptability as key factors that affect research capacity strengthening in conflict. As it is the case more generally, funding, permissible political environments and sustainability are major determinants of success for capacity strengthening for health research programmes, though these are significantly more challenging in conflict settings. Nonetheless, health research capacity strengthening should remain a priority. CONCLUSION: The model presented is the first framework that focuses on strengthening health research capacity in conflict with a focus on the MENA region. It should be viewed as a non-prescriptive reference tool for health researchers and practitioners, from various disciplines, involved in research capacity strengthening to evaluate, use, adapt and improve. It can be further extended to include representative indicators and can be later evaluated by assessing its efficacy for interventions in conflict settings.

Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep.

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in ~ 50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.

Robustly detecting differential expression in RNA sequencing data using observation weights.

A popular approach for comparing gene expression levels between (replicated) conditions of RNA sequencing data relies on counting reads that map to features of interest. Within such count-based methods, many flexible and advanced statistical approaches now exist and offer the ability to adjust for covariates (e.g. batch effects). Often, these methods include some sort of 'sharing of information' across features to improve inferences in small samples. It is important to achieve an appropriate tradeoff between statistical power and protection against outliers. Here, we study the robustness of existing approaches for count-based differential expression analysis and propose a new strategy based on observation weights that can be used within existing frameworks. The results suggest that outliers can have a global effect on differential analyses. We demonstrate the effectiveness of our new approach with real data and simulated data that reflects properties of real datasets (e.g. dispersion-mean trend) and develop an extensible framework for comprehensive testing of current and future methods. In addition, we explore the origin of such outliers, in some cases highlighting additional biological or technical factors within the experiment. Further details can be downloaded from the project website: http://imlspenticton.uzh.ch/robinson_lab/edgeR_robust/.

Breath alcohol of anesthesiologists using alcohol hand gel and the "five moments for hand hygiene" in routine practice.

PURPOSE: Appropriate hand hygiene reduces hospital-acquired infections. Anesthesiologists work in environments with numerous hand hygiene opportunities (HHOs). In a prospective observational study, we investigated the potential for an anesthesiologist to return a positive alcohol breath test during routine practice when using alcohol hand gel. METHODS: We observed ten volunteer anesthesiologists over four hours while they implemented the World Health Organization (WHO) "five moments for hand hygiene" using our hospital's adopted standard 70% ethanol hand gel. We measured the expired alcohol concentration at shift start and every fifteen minutes thereafter with a fuel cell breathalyzer calibrated to measure the percentage of blood alcohol concentration (BAC). Blood alcohol specimens (analyzed with gas chromatography) were collected at shift start and, when possible, immediately after a participant's first positive breathalyzer test. RESULTS: Of the 130 breathalyzer tests obtained, there were eight (6.2%) positive breath alcohol results from six of the ten participants, all within two minutes of a HHO. The highest value breathalyzer BAC recorded was 0.064%, with an overall mean (SD) of 0.023 (0.017)%. Five (62.5%) of the positive breathalyzer tests returned to zero in less than seven minutes. All of three blood specimens obtained immediately after a positive breathalyzer reading tested negative for alcohol. CONCLUSION: Anesthesia practitioners using alcohol hand gel in a manner that conforms with recommended hand hygiene can test positive for alcohol on a breathalyzer assay. Positive tests probably arose from inhalation of alcohol vapour into the respiratory dead space following gel application. If workplace breath testing for alcohol is implemented, it should be completed more than 15 min after applying alcohol hand gel. Positive results should be verified with a BAC test.

Robust diagnostic genetic testing using solution capture enrichment and a novel variant-filtering interface.

Targeted hybridization enrichment prior to next-generation sequencing is a widespread method for characterizing sequence variation in a research setting, and is being adopted by diagnostic laboratories. However, the number of variants identified can overwhelm clinical laboratories with strict time constraints, the final interpretation of likely pathogenicity being a particular bottleneck. To address this, we have developed an approach in which, after automatic variant calling on a standard unix pipeline, subsequent variant filtering is performed interactively, using AgileExomeFilter and AgilePindelFilter (http://dna.leeds.ac.uk/agile), tools designed for clinical scientists with standard desktop computers. To demonstrate the method's diagnostic efficacy, we tested 128 patients using (1) a targeted capture of 36 cancer-predisposing genes or (2) whole-exome capture for diagnosis of the genetically heterogeneous disorder primary ciliary dyskinesia (PCD). In the cancer cohort, complete concordance with previous diagnostic data was achieved across 793 variant genotypes. A high yield (42%) was also achieved for exome-based PCD diagnosis, underscoring the scalability of our method. Simple adjustments to the variant filtering parameters further allowed the identification of a homozygous truncating mutation in a presumptive new PCD gene, DNAH8. These tools should allow diagnostic laboratories to expand their testing portfolios flexibly, using a standard set of reagents and techniques.

Estimates of the effect of natural selection on protein-coding content.

Analysis of natural selection is key to understanding many core biological processes, including the emergence of competition, cooperation, and complexity, and has important applications in the targeted development of vaccines. Selection is hard to observe directly but can be inferred from molecular sequence variation. For protein-coding nucleotide sequences, the ratio of nonsynonymous to synonymous substitutions (omega) distinguishes neutrally evolving sequences (omega = 1) from those subjected to purifying (omega < 1) or positive Darwinian (omega > 1) selection. We show that current models used to estimate omega are substantially biased by naturally occurring sequence compositions. We present a novel model that weights substitutions by conditional nucleotide frequencies and which escapes these artifacts. Applying it to the genomes of pathogens causing malaria, leprosy, tuberculosis, and Lyme disease gave significant discrepancies in estimates with approximately 10-30% of genes affected. Our work has substantial implications for how vaccine targets are chosen and for studying the molecular basis of adaptive evolution.

General practitioner and nurse practitioner attitudes towards electronic reminders in primary care: a qualitative analysis.

OBJECTIVES: Reminders in primary care administrative systems aim to help clinicians provide evidence-based care, prescribe safely and save money. However, increased use of reminders can lead to alert fatigue. Our study aimed to assess general practitioners' (GPs) and nurse practitioners' (NPs) views on electronic reminders in primary care. DESIGN: A qualitative analysis using semistructured interviews. SETTING AND PARTICIPANTS: Fifteen GPs and NP based in general practices located in North-West London and Yorkshire, England. METHODS: We collected data on participants' views on: (1) perceptions of the value of information provided; (2) reminder-related behaviours and (3) how to improve reminders. We carried out a thematic analysis. RESULTS: Participants were familiar with reminders in their clinical systems and felt many were important to support their clinical work. However, participants reported, on average, 70% of reminders were ignored. Four major themes emerged: (1) reaction to a reminder, which was mixed and varied by situation. (2) Factors influencing the decision to act on reminders, often related to experience, consultation styles and interests of participants. Time constraints, alert design, inappropriate presentation and litigation were also factors. (3) Negative consequences of using reminders were increased workload or costs and compromising GP and NPs behaviour. (4) Factors relating to improving users' engagement with reminders were prevention of unnecessary reminders through data linkage across healthcare administrative systems or the development of more intelligent algorithms. Participants felt training was vital to effectively manage reminders. CONCLUSIONS: GPs and NPs believe reminders are useful in supporting the provision of good quality patient care. Improving GPs and NPs' engagement with reminders centres on further developing their relevance to their clinical practice, which is personalised, considers cognitive workflow and suppresses inappropriate presentation.

Genetic diagnosis of familial breast cancer using clonal sequencing.

Using conventional Sanger sequencing as a reference standard, we compared the sensitivity, specificity, and capacity of the Illumina GA II platform for the detection of TP53, BRCA1, and BRCA2 mutations in established tumor cell lines and DNA from patients with germline mutations. A total of 656 coding variants were identified in four cell lines and 65 patient DNAs. All of the known pathogenic mutations (including point mutations and insertions/deletions of up to 16 nucleotides) were identified, using a combination of the Illumina data analysis pipeline with custom and commercial sequence alignment software. In our configuration, clonal sequencing outperforms current diagnostic methods, providing a reduction in analysis times and in reagent costs compared with conventional sequencing. These improvements open the possibility of BRCA1/2 testing for a wider spectrum of at-risk women, and will allow the genetic classification of tumors prior to the use of novel PARP inhibitors to treat BRCA-deficient breast cancers.

Bioavailability of vitamin B12.

Vitamin B12 deficiency is common in people of all ages who consume a low intake of animal-source foods, including populations in developing countries. It is also prevalent among the elderly, even in wealthier countries, due to their malabsorption of B12 from food. Several methods have been applied to diagnose vitamin B12 malabsorption, including Schilling’s test, which is now used rarely, but these do not quantify percent bioavailability. Most of the information on B12 bioavailability from foods was collected 40 to 50 years ago, using radioactive isotopes of cobalt to label the corrinoid ring. The data are sparse, and the level of radioactivity required for in vivo labeling of animal tissues can be prohibitive. A newer method under development uses a low dose of radioactivity as (14)C-labeled B12, with measurement of the isotope excreted in urine and feces by accelerator mass spectrometry. This test has revealed that the unabsorbed vitamin is degraded in the intestine. The percent bioavailability is inversely proportional to the dose consumed due to saturation of the active absorption process, even within the range of usual intake from foods. This has important implications for the assessment and interpretation of bioavailability values, setting dietary requirements, and interpreting relationships between intake and status of the vitamin.

The dominant cold-sensitive Out-cold mutants of Drosophila melanogaster have novel missense mutations in the voltage-gated sodium channel gene paralytic.

Here we report the molecular characterization of Out-cold (Ocd) mutants of Drosophila melanogaster, which produce a dominant, X-linked, cold-sensitive paralytic phenotype. From its initial 1.5-Mb cytological location within 13F1-16A2, P-element and SNP mapping reduced the Ocd critical region to <100 kb and to six candidate genes: hangover, CG9947, CG4420, eIF2a, Rbp2, and paralytic (para). Complementation testing with para null mutations strongly suggests Ocd and para are allelic, as does gene rescue of Ocd semilethality with a wild-type para transgene. Pesticide resistance and electrophysiological phenotypes of Ocd mutants support this conclusion. The para gene encodes a voltage-gated sodium channel. Sequencing the Ocd lines revealed mutations within highly conserved regions of the para coding sequence, in the transmembrane segment S6 of domain III (I1545M and T1551I), and in the linker between domains III and IV (G1571R), the location of the channel inactivation gate. The G1571R mutation is of particular interest as mutations of the orthologous residue (G1306) in the human skeletal muscle sodium channel gene SCN4A are associated with cases of periodic paralysis and myotonia, including the human cold-sensitive disorder paramyotonia congenita. The mechanisms by which sodium channel mutations cause cold sensitivity are not well understood. Therefore, in the absence of suitable vertebrate models, Ocd provides a system in which genetic, molecular, physiological, and behavioral tools can be exploited to determine mechanisms underlying sodium channel periodic paralyses.