Anti-CRISPR prediction using deep learning reveals an inhibitor of Cas13b nucleases.

As part of the ongoing bacterial-phage arms race, CRISPR-Cas systems in bacteria clear invading phages whereas anti-CRISPR proteins (Acrs) in phages inhibit CRISPR defenses. Known Acrs have proven extremely diverse, complicating their identification. Here, we report a deep learning algorithm for Acr identification that revealed an Acr against type VI-B CRISPR-Cas systems. The algorithm predicted numerous putative Acrs spanning almost all CRISPR-Cas types and subtypes, including over 7,000 putative type IV and VI Acrs not predicted by other algorithms. By performing a cell-free screen for Acr hits against type VI-B systems, we identified a potent inhibitor of Cas13b nucleases we named AcrVIB1. AcrVIB1 blocks Cas13b-mediated defense against a targeted plasmid and lytic phage, and its inhibitory function principally occurs upstream of ribonucleoprotein complex formation. Overall, our work helps expand the known Acr universe, aiding our understanding of the bacteria-phage arms race and the use of Acrs to control CRISPR technologies.

Interrogating two extensively self-targeting Type I CRISPR-Cas systems in Xanthomonas albilineans reveals distinct anti-CRISPR proteins that block DNA degradation.

CRISPR-Cas systems store fragments of invader DNA as spacers to recognize and clear those same invaders in the future. Spacers can also be acquired from the host's genomic DNA, leading to lethal self-targeting. While self-targeting can be circumvented through different mechanisms, natural examples remain poorly explored. Here, we investigate extensive self-targeting by two CRISPR-Cas systems encoding 24 self-targeting spacers in the plant pathogen Xanthomonas albilineans. We show that the native I-C and I-F1 systems are actively expressed and that CRISPR RNAs are properly processed. When expressed in Escherichia coli, each Cascade complex binds its PAM-flanked DNA target to block transcription, while the addition of Cas3 paired with genome targeting induces cell killing. While exploring how X. albilineans survives self-targeting, we predicted putative anti-CRISPR proteins (Acrs) encoded within the bacterium's genome. Screening of identified candidates with cell-free transcription-translation systems and in E. coli revealed two Acrs, which we named AcrIC11 and AcrIF12Xal, that inhibit the activity of Cas3 but not Cascade of the respective system. While AcrF12Xal is homologous to AcrIF12, AcrIC11 shares sequence and structural homology with the anti-restriction protein KlcA. These findings help explain tolerance of self-targeting through two CRISPR-Cas systems and expand the known suite of DNA degradation-inhibiting Acrs.

RiboReport - benchmarking tools for ribosome profiling-based identification of open reading frames in bacteria.

Small proteins encoded by short open reading frames (ORFs) with 50 codons or fewer are emerging as an important class of cellular macromolecules in diverse organisms. However, they often evade detection by proteomics or in silico methods. Ribosome profiling (Ribo-seq) has revealed widespread translation in genomic regions previously thought to be non-coding, driving the development of ORF detection tools using Ribo-seq data. However, only a handful of tools have been designed for bacteria, and these have not yet been systematically compared. Here, we aimed to identify tools that use Ribo-seq data to correctly determine the translational status of annotated bacterial ORFs and also discover novel translated regions with high sensitivity. To this end, we generated a large set of annotated ORFs from four diverse bacterial organisms, manually labeled for their translation status based on Ribo-seq data, which are available for future benchmarking studies. This set was used to investigate the predictive performance of seven Ribo-seq-based ORF detection tools (REPARATION_blast, DeepRibo, Ribo-TISH, PRICE, smORFer, ribotricer and SPECtre), as well as IRSOM, which uses coding potential and RNA-seq coverage only. DeepRibo and REPARATION_blast robustly predicted translated ORFs, including sORFs, with no significant difference for ORFs in close proximity to other genes versus stand-alone genes. However, no tool predicted a set of novel, experimentally verified sORFs with high sensitivity. Start codon predictions with smORFer show the value of initiation site profiling data to further improve the sensitivity of ORF prediction tools in bacteria. Overall, we find that bacterial tools perform well for sORF detection, although there is potential for improving their performance, applicability, usability and reproducibility.

Assessment of South Asian Pediatric Acute Kidney Injury: Epidemiology and Risk Factors (ASPIRE)-a prospective study on "severe dialysis dependent pediatric AKI".

BACKGROUND: Pediatric acute kidney injury (AKI) is a global health concern with an associated mortality risk disproportionately pronounced in resource-limited settings. There is a pertinent need to understand the epidemiology of pediatric AKI in vulnerable populations. Here, we proposed a prospective study to investigate the epidemiology and associated risk factors of "severe dialysis dependent AKI" in children among South Asian nations which would be the first and largest of its kind. METHODS: The ASPIRE study (part of PCRRT-ICONIC Foundation initiative) is a multi-center, prospective observational study conducted in South Asian countries. All children and adolescents ≤ 18 years of age who required dialysis for AKI in any of the collaborating medical centers were enrolled. Data collection was performed until one of the following endpoints was observed: (1) discharge, (2) death, and (3) discharge against medical advice. RESULTS: From 2019 to 2022, a total of 308 children with severe AKI were enrolled. The mean age was 6.17 years (63% males). Secondary AKI was more prevalent than primary AKI (67.2%), which predominantly occurred due to infections, dehydration, and nephrotoxins. Common causes of primary AKI were glomerulonephritis, hemolytic uremic syndrome, lupus nephritis, and obstructive uropathy. Shock, need for ventilation, and coagulopathy were commonly seen in children with severe AKI who needed dialysis. The foremost kidney replacement therapy used was peritoneal dialysis (60.7%). The mortality rate was 32.1%. CONCLUSIONS: Common causes of AKI in children in South Asia are preventable. Mortality is high among these children suffering from "severe dialysis dependent AKI." Targeted interventions to prevent and identify AKI early and initiate supportive care in less-resourced nations are needed.

Effect of Vitamin D Deficiency on Periprosthetic Joint Infection and Complications After Primary Total Joint Arthroplasty.

BACKGROUND: Vitamin D deficiency is a global problem, and 13 to 75% of patients undergoing total joint arthroplasty (TJA) have vitamin D deficiency. Several studies have shown that low preoperative vitamin D levels may increase the risk of postoperative complications, including periprosthetic joint infection (PJI), in patients undergoing primary TJA. Most of the studies are underpowered. This study aimed to investigate the relationship between vitamin D deficiency and surgical and medical complications after primary TJA, with a specific focus on PJI. METHODS: Prospectively collected institutional multicenter arthroplasty databases were reviewed to identify patients who underwent primary total knee and hip arthroplasty. The study group was defined as patients whose vitamin D level is < 30 ng/dL and who received a single oral dose of 7.5 mg (300,000 IU) D3 within two weeks before index surgery (n = 488; mean age 63 years). Patients in the control group were those whose preoperative vitamin D levels were unknown and who did not receive vitamin D supplementation (n = 592, mean age 66). The groups were compared regarding 90-day medical and surgical complications, including PJI, mortality, and readmission rates. RESULTS: The total number of complications (8.6 and 4.3%; respectively; P = .005), superficial wound infection (2.5 and 0.2%, respectively; P < .001), and postoperative cellulitis (2.2 and 0% respectively; P < .001) were statistically significantly higher in the patient group who did not receive vitamin D supplementation. However, 90-day mortality (P = .524), PJI (P = .23), and readmission rate (P = .683) were similar between the groups. CONCLUSIONS: This study demonstrated that preoperative optimization of vitamin D levels may be beneficial in reducing postoperative complications, including superficial wound infection and postoperative cellulitis. Administering an oral 300,000 U single-dose vitamin D regimen to correct vitamin D deficiency can positively impact outcomes following primary TJA.

Unseen Threefold Mortality After the First Ten Days in Hemodialysis Patients Following Joint Arthroplasty: A Nationwide Retrospective Cohort Study of 1,287 Arthroplasty Patients on Hemodialysis.

BACKGROUND: The study addresses the growing number of hemodialysis (HD) patients undergoing joint arthroplasty, who are at higher risk of complications and mortality. Previous research has often overlooked deaths after discharge. This study aimed to examine early outcomes in a large nationwide cohort of patients who underwent arthroplasty for elective and fracture-related reasons. METHODS: Between 2016 and 2022, a study was conducted using the e-Nabiz database of the Türkiye Ministry of Health, focusing on patients aged 18 years and above who underwent elective or fracture-related arthroplasty. This study included 1,287 patients reliant on dialysis who underwent total hip arthroplasty, total knee arthroplasty, or hemiarthroplasty (HA), with 7.7% of them receiving dialysis for the first time. Propensity score matching was used to create an equally sized group of non-dialysis-dependent patients, ensuring demographic balance in terms of age, sex, a comorbidity index, and surgery type. The primary objective was to compare mortality rates 10, 30, and 90 days after arthroplasty. RESULTS: The first-time dialysis patients who underwent HA had significantly higher 30- and 90-day mortality rates compared to the chronic dialysis group (P = .040 and P < .001, respectively). Also, the HD patients consistently exhibited higher 90-day mortality rates across all surgery types. With total knee arthroplasty, HD patients had a mortality rate of 8.7%, in stark contrast to 0% among non-HD patients (P < .001). Similarly, with total hip arthroplasty, HD patients had a 12% mortality rate, while non-HD patients had a markedly lower rate of 2.7% (P = .008). In the case of HA, HD patients had a significantly elevated 90-day mortality rate of 31.9%, in contrast to 17.1% among non-HD patients (P < .001). CONCLUSIONS: Joint arthroplasty has higher rates of mortality and complications among HD patients. Surgical decisions must be based on patients' overall health, necessitating collaboration among specialists. These patients should be closely monitored.

Gene Expression-Based Cancer Classification for Handling the Class Imbalance Problem and Curse of Dimensionality.

Cancer is a leading cause of death globally. The majority of cancer cases are only diagnosed in the late stages of cancer due to the use of conventional methods. This reduces the chance of survival for cancer patients. Therefore, early detection consequently followed by early diagnoses are important tasks in cancer research. Gene expression microarray technology has been applied to detect and diagnose most types of cancers in their early stages and has gained encouraging results. In this paper, we address the problem of classifying cancer based on gene expression for handling the class imbalance problem and the curse of dimensionality. The oversampling technique is utilized to overcome this problem by adding synthetic samples. Another common issue related to the gene expression dataset addressed in this paper is the curse of dimensionality. This problem is addressed by applying chi-square and information gain feature selection techniques. After applying these techniques individually, we proposed a method to select the most significant genes by combining those two techniques (CHiS and IG). We investigated the effect of these techniques individually and in combination. Four benchmarking biomedical datasets (Leukemia-subtypes, Leukemia-ALLAML, Colon, and CuMiDa) were used. The experimental results reveal that the oversampling techniques improve the results in most cases. Additionally, the performance of the proposed feature selection technique outperforms individual techniques in nearly all cases. In addition, this study provides an empirical study for evaluating several oversampling techniques along with ensemble-based learning. The experimental results also reveal that SVM-SMOTE, along with the random forests classifier, achieved the highest results, with a reporting accuracy of 100%. The obtained results surpass the findings in the existing literature as well.

Angiographic measurement of the superficial temporal artery for potential use in cerebral bypass surgery: a combined radiological and cadaveric study.

PURPOSE: This study aims to investigate the microsurgical anatomy of the superficial temporal artery (STA), explore the relationship between STA length and lumen diameter, and develop a reliable radiologic method for selecting STA segments for bypass surgery. METHODS: This study used 10 cadaveric dissections (20 STAs, both sides) and 20 retrospective radiological examinations (40 STAs, both sides), employing curved multiplanar reformation and flow color lookup table (CLUT) DICOM processing. Measurements included vessel lumen diameters and luminal cross-sectional thicknesses 3 mm proximal to the STA bifurcation, 3 mm distal to the frontal branch, 5 cm distal to the frontal branch, 3 mm distal to the parietal branch, and 5 cm distal to the parietal branch. The distance between the STA bifurcation and the superior zygomatic border (SZB) was also measured. In our analysis, descriptive statistics encompassed mean, standard deviation (SD), standard error, minimum and maximum values, and distributions. Comparative statistics were performed using Student's t-test, with statistical significance set at p < 0.05. RESULTS: There were no statistically significant differences between STA measurements of bifurcation distances (p = 0.88) and lumen diameters (p = 0.46) between cadavers and radiological measures. However, lumen thicknesses were larger in frontal branches than parietal branches at the seventh and eighth centimeter (p = 0.012, p = 0.039). Branches became thinner distally from the zygoma in both cadavers and radiological image measurements. CONCLUSION: The CLUT DICOM processing radiological measures provided the high-precision required to enable pre-surgical vessel selection for extracranial-intracranial bypass. The results show that STA vessel luminal diameters are sufficient (> 1 mm) for bypass surgery in the first 9 cm but gradually decrease after that. Also shown is that the choice of frontal versus parietal branches depends on individual anatomical features; therefore, careful preoperative radiological examination is critical.

CRISPRtracrRNA: robust approach for CRISPR tracrRNA detection.

MOTIVATION: The CRISPR-Cas9 system is a Type II CRISPR system that has rapidly become the most versatile and widespread tool for genome engineering. It consists of two components, the Cas9 effector protein, and a single guide RNA that combines the spacer (for identifying the target) with the tracrRNA, a trans-activating small RNA required for both crRNA maturation and interference. While there are well-established methods for screening Cas effector proteins and CRISPR arrays, the detection of tracrRNA remains the bottleneck in detecting Class 2 CRISPR systems. RESULTS: We introduce a new pipeline CRISPRtracrRNA for screening and evaluation of tracrRNA candidates in genomes. This pipeline combines evidence from different components of the Cas9-sgRNA complex. The core is a newly developed structural model via covariance models from a sequence-structure alignment of experimentally validated tracrRNAs. As additional evidence, we determine the terminator signal (required for the tracrRNA transcription) and the RNA-RNA interaction between the CRISPR array repeat and the 5'-part of the tracrRNA. Repeats are detected via an ML-based approach (CRISPRidenify). Providing further evidence, we detect the cassette containing the Cas9 (Type II CRISPR systems) and Cas12 (Type V CRISPR systems) effector protein. Our tool is the first for detecting tracrRNA for Type V systems. AVAILABILITY AND IMPLEMENTATION: The implementation of the CRISPRtracrRNA is available on GitHub upon requesting the access permission, (https://github.com/BackofenLab/CRISPRtracrRNA). Data generated in this study can be obtained upon request to the corresponding person: Rolf Backofen (backofen@informatik.uni-freiburg.de). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

CRISPRidentify: identification of CRISPR arrays using machine learning approach.

CRISPR-Cas are adaptive immune systems that degrade foreign genetic elements in archaea and bacteria. In carrying out their immune functions, CRISPR-Cas systems heavily rely on RNA components. These CRISPR (cr) RNAs are repeat-spacer units that are produced by processing of pre-crRNA, the transcript of CRISPR arrays, and guide Cas protein(s) to the cognate invading nucleic acids, enabling their destruction. Several bioinformatics tools have been developed to detect CRISPR arrays based solely on DNA sequences, but all these tools employ the same strategy of looking for repetitive patterns, which might correspond to CRISPR array repeats. The identified patterns are evaluated using a fixed, built-in scoring function, and arrays exceeding a cut-off value are reported. Here, we instead introduce a data-driven approach that uses machine learning to detect and differentiate true CRISPR arrays from false ones based on several features. Our CRISPR detection tool, CRISPRidentify, performs three steps: detection, feature extraction and classification based on manually curated sets of positive and negative examples of CRISPR arrays. The identified CRISPR arrays are then reported to the user accompanied by detailed annotation. We demonstrate that our approach identifies not only previously detected CRISPR arrays, but also CRISPR array candidates not detected by other tools. Compared to other methods, our tool has a drastically reduced false positive rate. In contrast to the existing tools, our approach not only provides the user with the basic statistics on the identified CRISPR arrays but also produces a certainty score as a practical measure of the likelihood that a given genomic region is a CRISPR array.

CRISPRloci: comprehensive and accurate annotation of CRISPR-Cas systems.

CRISPR-Cas systems are adaptive immune systems in prokaryotes, providing resistance against invading viruses and plasmids. The identification of CRISPR loci is currently a non-standardized, ambiguous process, requiring the manual combination of multiple tools, where existing tools detect only parts of the CRISPR-systems, and lack quality control, annotation and assessment capabilities of the detected CRISPR loci. Our CRISPRloci server provides the first resource for the prediction and assessment of all possible CRISPR loci. The server integrates a series of advanced Machine Learning tools within a seamless web interface featuring: (i) prediction of all CRISPR arrays in the correct orientation; (ii) definition of CRISPR leaders for each locus; and (iii) annotation of cas genes and their unambiguous classification. As a result, CRISPRloci is able to accurately determine the CRISPR array and associated information, such as: the Cas subtypes; cassette boundaries; accuracy of the repeat structure, orientation and leader sequence; virus-host interactions; self-targeting; as well as the annotation of cas genes, all of which have been missing from existing tools. This annotation is presented in an interactive interface, making it easy for scientists to gain an overview of the CRISPR system in their organism of interest. Predictions are also rendered in GFF format, enabling in-depth genome browser inspection. In summary, CRISPRloci constitutes a full suite for CRISPR-Cas system characterization that offers annotation quality previously available only after manual inspection.

Current Bioinformatics Tools to Optimize CRISPR/Cas9 Experiments to Reduce Off-Target Effects.

The CRISPR-Cas system has evolved into a cutting-edge technology that has transformed the field of biological sciences through precise genetic manipulation. CRISPR/Cas9 nuclease is evolving into a revolutionizing method to edit any gene of any species with desirable outcomes. The swift advancement of CRISPR-Cas technology is reflected in an ever-expanding ecosystem of bioinformatics tools designed to make CRISPR/Cas9 experiments easier. To assist researchers with efficient guide RNA designs with fewer off-target effects, nuclease target site selection, and experimental validation, bioinformaticians have built and developed a comprehensive set of tools. In this article, we will review the various computational tools available for the assessment of off-target effects, as well as the quantification of nuclease activity and specificity, including web-based search tools and experimental methods, and we will describe how these tools can be optimized for gene knock-out (KO) and gene knock-in (KI) for model organisms. We also discuss future directions in precision genome editing and its applications, as well as challenges in target selection, particularly in predicting off-target effects.

Evaluation of Forced Expiratory Volume in One Second and Forced Vital Capacity from Age and Height for Pulmonary Function Test.

BACKGROUND: Lung function tests (LFTs) are a collection of clinical examinations used to assess lung function and monitor potential declines in the lungs, respiratory muscles, and chest wall's mechanical performance. This cross-sectional study aimed to identify the relation of age and height to lung function. MATERIAL AND METHODS: The study was conducted at AlHussein Medical City, 70 adult male subjects were enrolled in the study. All subjects were screened physically to ensure that they were normal and there were no respiratory disorders that could affect the lung function. Age and height were taken for these subjects, forced vital capacity (FVC), FEV1 (Forced expiratory volume in first second) as well as FEV1 /FVC ratio were measured. RESULTS: The results of the study showed that the average values of FVC and FEV1 were 4.75 and 3.88 respectively. There was a significant negative correlation observed between age and FVC (r=0.48), FEV1 (r= 0.6). Also there was a significant positive correlation noticed between Height and FVC (r = 0.62), FEV1 (r =0.69). There was a very high correlation evidenced between FEV1 and FVC, the relation between FEV1 and FVC is practically height and age-independent. CONCLUSION: Our study highlights a great interest in the study of the relation between age, height, and lung function. The study also creates simple and convenience equations that can be used for reference standards in clinical practice to give reasonable theoretical values for a large sector of the population.

CONTEXTE: Les tests de fonction pulmonaire (TFP) regroupent une série d'examens cliniques utilisés pour évaluer la fonction pulmonaire et surveiller d'éventuelles réductions des performances mécaniques des poumons, des muscles respiratoires et de la paroi thoracique. Cette étude transversale visait à déterminer la relation entre l'âge et la taille et la fonction pulmonaire. MATÉRIEL ET MÉTHODES: L'étude a été menée à la ville médicale Al-Hussein. Soixante-dix sujets masculins adultes ont été inscrits à l'étude. Tous les sujets ont été soumis à un examen physique pour s'assurer qu'ils étaient en bonne santé et ne présentaient pas de troubles respiratoires susceptibles d'affecter la fonction pulmonaire. L'âge et la taille de ces sujets ont été relevés, et la capacité vitale forcée (CVF), le VEMS (volume expiratoire maximal en une seconde) ainsi que le rapport VEMS/CVF ont été mesurés. RÉSULTATS: Les résultats de l'étude ont montré que les valeurs moyennes de la CVF et du VEMS étaient respectivement de 4,75 et 3,88. Une corrélation négative significative a été observée entre l'âge et la CVF (r = 0,48) ainsi qu'entre l'âge et le VEMS (r = 0,6). De plus, une corrélation positive significative a été remarquée entre la taille et la CVF (r = 0,62) ainsi qu'entre la taille et le VEMS (r = 0,69). Une corrélation très élevée a été mise en évidence entre le VEMS et la CVF, la relation entre le VEMS et la CVF est pratiquement indépendante de la taille et de l'âge. CONCLUSION: Notre étude met en évidence un intérêt particulier pour l'étude de la relation entre l'âge, la taille et la fonction pulmonaire. L'étude crée également des équations simples et pratiques qui peuvent être utilisées comme référence dans la pratique clinique pour fournir des valeurs théoriques raisonnables pour une grande partie de la population. Mots-clés: VEMS, CVF, VEMS/CVF, Spirométrie.

Assessing the feasibility of a web-based outcome measurement system in child and adolescent mental health services - myHealthE a randomised controlled feasibility pilot study.

BACKGROUND: Interest in internet-based patient reported outcome measure (PROM) collection is increasing. The NHS myHealthE (MHE) web-based monitoring system was developed to address the limitations of paper-based PROM completion. MHE provides a simple and secure way for families accessing Child and Adolescent Mental Health Services to report clinical information and track their child's progress. This study aimed to assess whether MHE improves the completion of the Strengths and Difficulties Questionnaire (SDQ) compared with paper collection. Secondary objectives were to explore caregiver satisfaction and application acceptability. METHODS: A 12-week single-blinded randomised controlled feasibility pilot trial of MHE was conducted with 196 families accessing neurodevelopmental services in south London to examine whether electronic questionnaires are completed more readily than paper-based questionnaires over a 3-month period. Follow up process evaluation phone calls with a subset (n = 8) of caregivers explored system satisfaction and usability. RESULTS: MHE group assignment was significantly associated with an increased probability of completing an SDQ-P in the study period (adjusted hazard ratio (HR) 12.1, 95% CI 4.7-31.0; p = <.001). Of those caregivers' who received the MHE invitation (n = 68) 69.1% completed an SDQ using the platform compared to 8.8% in the control group (n = 68). The system was well received by caregivers, who cited numerous benefits of using MHE, for example, real-time feedback and ease of completion. CONCLUSIONS: MHE holds promise for improving PROM completion rates. Research is needed to refine MHE, evaluate large-scale MHE implementation, cost effectiveness and explore factors associated with differences in electronic questionnaire uptake.

Casboundary: automated definition of integral Cas cassettes.

MOTIVATION: CRISPR-Cas are important systems found in most archaeal and many bacterial genomes, providing adaptive immunity against mobile genetic elements in prokaryotes. The CRISPR-Cas systems are encoded by a set of consecutive cas genes, here termed cassette. The identification of cassette boundaries is key for finding cassettes in CRISPR research field. This is often carried out by using Hidden Markov Models and manual annotation. In this article, we propose the first method able to automatically define the cassette boundaries. In addition, we present a Cas-type predictive model used by the method to assign each gene located in the region defined by a cassette's boundaries a Cas label from a set of pre-defined Cas types. Furthermore, the proposed method can detect potentially new cas genes and decompose a cassette into its modules. RESULTS: We evaluate the predictive performance of our proposed method on data collected from the two most recent CRISPR classification studies. In our experiments, we obtain an average similarity of 0.86 between the predicted and expected cassettes. Besides, we achieve F-scores above 0.9 for the classification of cas genes of known types and 0.73 for the unknown ones. Finally, we conduct two additional study cases, where we investigate the occurrence of potentially new cas genes and the occurrence of module exchange between different genomes. AVAILABILITY AND IMPLEMENTATION: https://github.com/BackofenLab/Casboundary. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

ILC1 drive intestinal epithelial and matrix remodelling.

Organoids can shed light on the dynamic interplay between complex tissues and rare cell types within a controlled microenvironment. Here, we develop gut organoid cocultures with type-1 innate lymphoid cells (ILC1) to dissect the impact of their accumulation in inflamed intestines. We demonstrate that murine and human ILC1 secrete transforming growth factor ß1, driving expansion of CD44v6+ epithelial crypts. ILC1 additionally express MMP9 and drive gene signatures indicative of extracellular matrix remodelling. We therefore encapsulated human epithelial-mesenchymal intestinal organoids in MMP-sensitive, synthetic hydrogels designed to form efficient networks at low polymer concentrations. Harnessing this defined system, we demonstrate that ILC1 drive matrix softening and stiffening, which we suggest occurs through balanced matrix degradation and deposition. Our platform enabled us to elucidate previously undescribed interactions between ILC1 and their microenvironment, which suggest that they may exacerbate fibrosis and tumour growth when enriched in inflamed patient tissues.

Pandemic-related emergency psychiatric presentations for self-harm of children and adolescents in 10 countries (PREP-kids): a retrospective international cohort study.

To examine the differences in hospital emergency psychiatric presentations for self-harm of children and adolescents during the covid-19 lockdown in March and April 2020 compared with the same period in 2019. Retrospective cohort study. We used electronic patient records from 23 hospital emergency departments in ten countries grouped into 14 areas. We examined data on 2073 acute hospital presentations by 1795 unique children and adolescents through age 18. We examined the total number of emergency psychiatric hospital presentations and the proportion of children and adolescents presenting with severe self-harm as our two main outcome measures. In addition, we examined sociodemographic and clinical characteristics and clinical management variables for those presenting with self-harm. To compare the number of hospital presentations between 2020 and 2019 a negative binomial model was used. For other variables, individual participant data (IPD) meta-analyses were carried out. Emergency psychiatric hospital presentations decreased from 1239 in 2019 to 834 in 2020, incident rate ratio 0.67, 95% CI 0.62-0.73; p < 0.001. The proportion of children and adolescents presenting with self-harm increased from 50% in 2019 to 57% in 2020, odds ratio 1.33, 1.07-1.64; p = 0.009 but there was no difference in the proportion presenting with severe self-harm. Within the subpopulation presenting with self-harm the proportion of children and adolescents presenting with emotional disorders increased from 58 to 66%, odds ratio 1.58, 1.06-2.36; p = 0.025. The proportion of children and adolescents admitted to an observation ward also decreased from 13 to 9% in 2020, odds ratio 0.52, 0.28-0.96; p = 0.036. Service planners should consider that, during a lockdown, there are likely to be fewer emergency psychiatric presentations. Many children and adolescents with psychiatric emergencies might not receive any service. A focus on developing intensive community care services with outreach capabilities should be prioritised.

The Syringe External Fixator: Short- and Medium-Term Functional Outcomes From This Inexpensive and Customizable Treatment for Comminuted Intra-Articular Fractures of the Hand.

PURPOSE: Comminuted intra-articular fractures and fracture dislocations of the metacarpophalangeal (MCP) and interphalangeal joints are challenging. Dynamic external fixation, permitting early joint motion while still minimizing forces across the healing joint, can result in acceptable postoperative active range of motion (AROM). However, some fractures are not initially stable enough for early dynamic motion; further, many available dynamic external fixation systems are costly and cumbersome. We reviewed our experience using an external fixator made from a 1-mL syringe and K-wires and report outcomes using it as a static fixator, dynamic fixator, or configured as a static fixator and then converted to a dynamic fixator in the clinic. METHODS: Patients with intra-articular fractures and fracture dislocations of the MCP and proximal interphalangeal (PIP) joints treated between 2014 and 2020 using syringe external fixators were retrospectively reviewed. We reviewed demographics, mechanisms, treatment types and durations, and postoperative AROM, as well as complications including infection, pin loosening, nonunion, hardware failure, and need for further surgery. Patients were analyzed by the level of joint injury (MCP versus PIP) and by treatment pattern. RESULTS: After excluding 23 patients with 25 joint injuries who were lost to follow-up or had inadequate outcome data, 27 patients with 29 joint injuries were reviewed. The average follow-up was 171 days after surgery. The postoperative AROM at the MCP level averaged 55° for static fixation and 30° for static-to-dynamic fixation. The postoperative AROM at the PIP level averaged 64° for static fixation, 66° for static-to-dynamic fixation, and 80° for dynamic fixation. Three pin site infections and 2 loose pins were reported. CONCLUSIONS: The syringe external fixator is an inexpensive, effective, and customizable treatment for intra-articular MCP and interphalangeal fractures and fracture dislocations, and results in acceptable postoperative AROM outcomes and complication rates. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Adaptation induced by self-targeting in a type I-B CRISPR-Cas system.

Haloferax volcanii is, to our knowledge, the only prokaryote known to tolerate CRISPR-Cas-mediated damage to its genome in the WT background; the resulting cleavage of the genome is repaired by homologous recombination restoring the WT version. In mutant Haloferax strains with enhanced self-targeting, cell fitness decreases and microhomology-mediated end joining becomes active, generating deletions in the targeted gene. Here we use self-targeting to investigate adaptation in H. volcanii CRISPR-Cas type I-B. We show that self-targeting and genome breakage events that are induced by self-targeting, such as those catalyzed by active transposases, can generate DNA fragments that are used by the CRISPR-Cas adaptation machinery for integration into the CRISPR loci. Low cellular concentrations of self-targeting crRNAs resulted in acquisition of large numbers of spacers originating from the entire genomic DNA. In contrast, high concentrations of self-targeting crRNAs resulted in lower acquisition that was mostly centered on the targeting site. Furthermore, we observed naïve spacer acquisition at a low level in WT Haloferax cells and with higher efficiency upon overexpression of the Cas proteins Cas1, Cas2, and Cas4. Taken together, these findings indicate that naïve adaptation is a regulated process in H. volcanii that operates at low basal levels and is induced by DNA breaks.

Impact of alcohol on the progression of HCV-related liver disease: A systematic review and meta-analysis.

BACKGROUND & AIMS: Despite a marked reduction in new cases of cirrhosis caused by HCV infection, over 500,000 new cirrhosis cases in this category were estimated globally in 2019. This contribution quantifies the relationship between alcohol use and the progression of liver disease in people with HCV infections. METHODS: The causal impact of different levels of alcohol use on cirrhosis has previously been established. The quantification of this relationship was undertaken based on a systematic search of the literature and a meta-analysis. We limited our search to longitudinal and case-control studies with biologically verified outcomes. Different sensitivity analyses were conducted to check on key assumptions and on the generalizability of the relationship. RESULTS: Alcohol use has a dose-dependent relationship with incident cirrhosis, which is linear on the log-linear level, and thus exponential on the level of odds ratios or other risk indicators. Each standard drink of 12 grams of pure alcohol per day increases the risk by about 11%. The results were stable regardless of the statistical model used, level of adjustment, quality of the study, or outcome (i.e., cirrhosis, decompensated cirrhosis, liver-related death). CONCLUSIONS: Alcohol use has a marked impact on the progression of HCV infections to cirrhosis and more severe liver outcomes. LAY SUMMARY: Alcohol consumption has a significant impact on the progression of liver disease in people with HCV infections. Each alcoholic drink per day is associated with an increase in the risk of cirrhosis of 11%.