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.

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.

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.

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.

Mutational Signature Analysis Reveals NTHL1 Deficiency to Cause a Multi-tumor Phenotype.

Biallelic germline mutations affecting NTHL1 predispose carriers to adenomatous polyposis and colorectal cancer, but the complete phenotype is unknown. We describe 29 individuals carrying biallelic germline NTHL1 mutations from 17 families, of which 26 developed one (n = 10) or multiple (n = 16) malignancies in 14 different tissues. An unexpected high breast cancer incidence was observed in female carriers (60%). Mutational signature analysis of 14 tumors from 7 organs revealed that NTHL1 deficiency underlies the main mutational process in all but one of the tumors (93%). These results reveal NTHL1 as a multi-tumor predisposition gene with a high lifetime risk for extracolonic cancers and a typical mutational signature observed across tumor types, which can assist in the recognition of this syndrome.

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.

Treatment of a metabolic liver disease by in vivo genome base editing in adult mice.

CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)1, using recently developed CRISPR-Cas-associated base editors2-4. These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pahenu2 gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pahenu2 mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.

Cancer modeling by Transgene Electroporation in Adult Zebrafish (TEAZ).

Transgenic animals are invaluable for modeling cancer genomics, but often require complex crosses of multiple germline alleles to obtain the desired combinations. Zebrafish models have advantages in that transgenes can be rapidly tested by mosaic expression, but typically lack spatial and temporal control of tumor onset, which limits their utility for the study of tumor progression and metastasis. To overcome these limitations, we have developed a method referred to as Transgene Electroporation in Adult Zebrafish (TEAZ). TEAZ can deliver DNA constructs with promoter elements of interest to drive fluorophores, oncogenes or CRISPR-Cas9-based mutagenic cassettes in specific cell types. Using TEAZ, we created a highly aggressive melanoma model via Cas9-mediated inactivation of Rb1 in the context of BRAFV600E in spatially constrained melanocytes. Unlike prior models that take ∼4 months to develop, we found that TEAZ leads to tumor onset in ∼7 weeks, and these tumors develop in fully immunocompetent animals. As the resulting tumors initiated at highly defined locations, we could track their progression via fluorescence, and documented deep invasion into tissues and metastatic deposits. TEAZ can be deployed to other tissues and cell types, such as the heart, with the use of suitable transgenic promoters. The versatility of TEAZ makes it widely accessible for rapid modeling of somatic gene alterations and cancer progression at a scale not achievable in other in vivo systems.

Genomic Subtypes of Non-invasive Bladder Cancer with Distinct Metabolic Profile and Female Gender Bias in KDM6A Mutation Frequency.

Bladder cancer incurs a higher lifetime treatment cost than other cancers due to frequent recurrence of non-invasive disease. Improved prognostic biomarkers and localized therapy are needed for this large patient group. We defined two major genomic subtypes of primary stage Ta tumors. One of these was characterized by loss of 9q including TSC1, increased KI67 labeling index, upregulated glycolysis, DNA repair, mTORC1 signaling, features of the unfolded protein response, and altered cholesterol homeostasis. Comparison with muscle-invasive bladder cancer mutation profiles revealed lower overall mutation rates and more frequent mutations in RHOB and chromatin modifier genes. More mutations in the histone lysine demethylase KDM6A were present in non-invasive tumors from females than males.

Neuroprotection during aortic surgery.

Neurological injury is a major limitation of aortic surgery, whether it involves spinal cord injury following intervention to the thoracoabdominal aorta, or stroke following surgery on the arch and ascending aorta. Despite an extensive body of literature and various proposals, a completely effective strategy to prevent or treat neurological injury remains elusive. In this article, we summarise the evidence for established and emerging strategies, and review current concepts in pathophysiology and risk assessment as they relate to neurological injury.

Statistical methods for detecting differentially methylated loci and regions.

DNA methylation, the reversible addition of methyl groups at CpG dinucleotides, represents an important regulatory layer associated with gene expression. Changed methylation status has been noted across diverse pathological states, including cancer. The rapid development and uptake of microarrays and large scale DNA sequencing has prompted an explosion of data analytic methods for processing and discovering changes in DNA methylation across varied data types. In this mini-review, we present a compact and accessible discussion of many of the salient challenges, such as experimental design, statistical methods for differential methylation detection, critical considerations such as cell type composition and the potential confounding that can arise from batch effects. From a statistical perspective, our main interests include the use of empirical Bayes or hierarchical models, which have proved immensely powerful in genomics, and the procedures by which false discovery control is achieved.

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.

Mechanisms that determine the internal environment of the developing brain: a transcriptomic, functional and ultrastructural approach.

We provide comprehensive identification of embryonic (E15) and adult rat lateral ventricular choroid plexus transcriptome, with focus on junction-associated proteins, ionic influx transporters and channels. Additionally, these data are related to new structural and previously published permeability studies. Results reveal that most genes associated with intercellular junctions are expressed at similar levels at both ages. In total, 32 molecules known to be associated with brain barrier interfaces were identified. Nine claudins showed unaltered expression, while two claudins (6 and 8) were expressed at higher levels in the embryo. Expression levels for most cytoplasmic/regulatory adaptors (10 of 12) were similar at the two ages. A few junctional genes displayed lower expression in embryos, including 5 claudins, occludin and one junctional adhesion molecule. Three gap junction genes were enriched in the embryo. The functional effectiveness of these junctions was assessed using blood-delivered water-soluble tracers at both the light and electron microscopic level: embryo and adult junctions halted movement of both 286Da and 3kDa molecules into the cerebrospinal fluid (CSF). The molecular identities of many ion channel and transporter genes previously reported as important for CSF formation and secretion in the adult were demonstrated in the embryonic choroid plexus (and validated with immunohistochemistry of protein products), but with some major age-related differences in expression. In addition, a large number of previously unidentified ion channel and transporter genes were identified for the first time in plexus epithelium. These results, in addition to data obtained from electron microscopical and physiological permeability experiments in immature brains, indicate that exchange between blood and CSF is mainly transcellular, as well-formed tight junctions restrict movement of small water-soluble molecules from early in development. These data strongly indicate the brain develops within a well-protected internal environment and the exchange between the blood, brain and CSF is transcellular and not through incomplete barriers.

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.

PyCogent: a toolkit for making sense from sequence.

We have implemented in Python the COmparative GENomic Toolkit, a fully integrated and thoroughly tested framework for novel probabilistic analyses of biological sequences, devising workflows, and generating publication quality graphics. PyCogent includes connectors to remote databases, built-in generalized probabilistic techniques for working with biological sequences, and controllers for third-party applications. The toolkit takes advantage of parallel architectures and runs on a range of hardware and operating systems, and is available under the general public license from http://sourceforge.net/projects/pycogent.