Postoperative outcomes and identification of risk factors for complications after emergency intestinal stoma surgery - a multicentre retrospective study.

AIM: Approximately 4000 patients in the UK have an emergency intestinal stoma formed each year. Stoma-related complications (SRCs) are heterogeneous but have previously been subcategorized into early or late SRCs, with early SRCs generally occurring within 30 days postoperatively. Early SRCs include skin excoriation, stoma necrosis and high output, while late SRCs include parastomal hernia, retraction and prolapse. There is a paucity of research on specific risk factors within the emergency cohort for development of SRCs. This paper aims to describe the incidence of SRCs after emergency intestinal surgery and to identify potential risk factors for SRCs within this cohort. METHOD: Consecutive patients undergoing emergency formation of an intestinal stoma (colostomy, ileostomy or jejunostomy) were identified prospectively from across three acute hospital sites over a 3-year period from the ELLSA (Emergency Laparotomy and Laparoscopic Scottish Audit) database. All patients were followed up for a minimum of 1 year. A multivariate logistic regression model was used to identify risk factors for early and late SRCs. RESULTS: A total of 455 patients were included (median follow-up 19 months, median age 64 years, male:female 0.52, 56.7% ileostomies). Early SRCs were experienced by 54.1% of patients, while 51% experienced late SRCs. A total of 219 patients (48.1%) had their stoma sited preoperatively. Risk factors for early SRCs included end ileostomy formation [OR 3.51 (2.24-5.49), p < 0.001], while preoperative stoma siting was found to be protective [OR 0.53 (0.35-0.83), p = 0.005]. Patient obesity [OR 3.11 (1.92-5.03), p < 0.001] and reoperation for complications following elective surgery [OR 4.18 (2.01-8.69), p < 0.001] were risk factors for late SRCs. CONCLUSION: Stoma-related complications after emergency surgery are common. Preoperative stoma siting is the only truly modifiable risk factor to reduce SRCs, and further research should be aimed at methods of improving the frequency and accuracy of this in the emergency setting.

Decoding the complexity of on-target integration: characterizing DNA insertions at the CRISPR-Cas9 targeted locus using nanopore sequencing.

BACKGROUND: CRISPR-Cas9 technology has advanced in vivo gene therapy for disorders like hemophilia A, notably through the successful targeted incorporation of the F8 gene into the Alb locus in hepatocytes, effectively curing this disorder in mice. However, thoroughly evaluating the safety and specificity of this therapy is essential. Our study introduces a novel methodology to analyze complex insertion sequences at the on-target edited locus, utilizing barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, magnetic bead-based long amplicon enrichment, and nanopore sequencing. RESULTS: We identified the expected F8 insertions and various fragment combinations resulting from the in vivo linearization of the double-cut plasmid donor. Notably, our research is the first to document insertions exceeding ten kbp. We also found that a small proportion of these insertions were derived from sources other than donor plasmids, including Cas9-sgRNA plasmids, genomic DNA fragments, and LINE-1 elements. CONCLUSIONS: Our study presents a robust method for analyzing the complexity of on-target editing, particularly for in vivo long insertions, where donor template integration can be challenging. This work offers a new tool for quality control in gene editing outcomes and underscores the importance of detailed characterization of edited genomic sequences. Our findings have significant implications for enhancing the safety and effectiveness of CRISPR-Cas9 gene therapy in treating various disorders, including hemophilia A.

First report of prevalence and assemblage analysis of Giardia duodenalis in pigs from Guangxi Zhuang Autonomous Region, southern China.

Giardia duodenalis is a common intestinal protozoan that can cause diarrhea and intestinal disease in animals and in humans. However, the prevalence and assemblages of G. duodenalis in pigs from Guangxi Zhuang Autonomous Region have not been reported. In this study, a total of 724 fecal samples (201 from nursery pigs, 183 from piglets, 175 from breeding pigs, and 165 from fattening pigs) were obtained in four areas of the region (Nanning, Yulin, Hezhou, and Guigang). The gene of the small subunit ribosomal RNA (SSU rRNA) of G. duodenalis was amplified by nested PCR. The results show that the prevalence of G. duodenalis in pigs was 3.59% (26/724), of which 14 samples belonged to assemblage A (53.85%) and 12 samples belonged to assemblage E (46.15%). The infection rates of G. duodenalis in Hezhou, Yulin, Nanning, and Guigang were 0%, 0.7%, 10.8% and 1.1%, respectively (χ2 = 45.616, p < 0.01); whereas 5.1% of breeding pigs, 6.0% of piglets, 2.4% of fattening pigs, and 1.0% of nursery pigs were infected with G. duodenalis (χ2 = 8.874, p < 0.05). The SSU rRNA-positive samples were amplified by PCR based on the ß-giardin (bg), glutamate dehydrogenase (gdh), and triphosphate isomerase (tpi) genes. Ten, eight and seven positive samples were detected, respectively. Based on phylogenetic analysis of the three genetic loci sequences, a multilocus genotyping A1 was found. The findings of this study provide basic data for the development of prevention and control of G. duodenalis infections in pigs and humans in the Guangxi Zhuang Autonomous Region.

Title: Premier rapport sur la prévalence et l'analyse des assemblages de Giardia duodenalis chez les porcs de la région autonome Zhuang du Guangxi, dans le sud de la Chine. Abstract: Giardia duodenalis est un protozoaire intestinal commun qui peut provoquer des diarrhées et des maladies intestinales chez les animaux et les humains. Cependant, la prévalence et les assemblages de G. duodenalis chez les porcs de la région autonome Zhuang du Guangxi n'ont pas été rapportés. Dans cette étude, un total de 724 échantillons fécaux (201 provenant de jeunes porcelets, 183 de porcelets, 175 de porcs reproducteurs et 165 de porcs à l'engrais) ont été obtenus dans quatre zones de la région (Nanning, Yulin, Hezhou et Guigang). Le gène de la petite sous-unité de l'ARN ribosomal (ARNr SSU) de G. duodenalis a été amplifié par PCR nichée. Les résultats ont montré que la prévalence de G. duodenalis chez les porcs était de 3,59 % (26/724), dont 14 échantillons appartenaient à l'assemblages A (53,85 %) et 12 échantillons à l'assemblage E (46,15 %). Les taux d'infection par G. duodenalis à Hezhou, Yulin, Nanning et Guigang étaient respectivement de 0, 0,7 %, 10,8 % et 1,1 % (χ2 = 45,616, p < 0,01), alors que 5,1 % des porcs reproducteurs, 6,0 % des porcelets, 2,4 % de porcs à l'engrais et 1,0 % des jeunes porcelets étaient infectés par G. duodenalis (χ2 = 8,874, p < 0,05). Les échantillons positifs pour l'ARNr SSU ont été amplifiés par PCR basée sur les gènes de la ß-giardine (bg), de la glutamate déshydrogénase (gdh) et de la triphosphate isomérase (tpi), et dix, huit et sept échantillons positifs ont été détectés, respectivement. Sur la base de l'analyse phylogénétique des trois séquences de loci génétiques, un génotypage multilocus A1 a été trouvé. Les résultats de cette étude fournissent des données de base pour le développement de la prévention et du contrôle des infections à G. duodenalis chez les porcs et les humains dans la région autonome Zhuang du Guangxi.

First Molecular Detection and Genotype Identification of Toxoplasma gondii in Chickens from Farmers' Markets in Fujian Province, Southeastern China.

Toxoplasma gondii is an opportunistic pathogenic protozoan that can infect all nucleated cells in almost all warm-blooded animals, including humans. T. gondii infection has been reported in many food animals worldwide. However, the prevalence and genotypes of T. gondii in chickens from farmers' markets in Fujian province in southeastern China remain unreported. In the present study, four tissue samples from each of the 577 chickens (namely, the heart, liver, lungs, and muscles) were collected from farmers' markets in five regions of Fujian province (Zhangzhou, Sanming, Quanzhou, Fuzhou, and Longyan). We first analyzed the prevalence and genotypes of T. gondii using PCR targeting of the B1 gene of T. gondii. Of the 577 chickens, thirty-two (5.5%) tested positive for the B1 gene. Among the five regions, Sanming had the highest infection rate (16.8%, 16/95), followed by Quanzhou (8.0%, 8/100), Longyan (5.0%, 5/100), Zhangzhou (1.1%, 2/182), and Fuzhou (1.0%, 1/100). Among these thirty-two T. gondii-positive chickens, the infection rates of the lungs, heart, liver, and muscles were 68.8% (22/32), 34.4% (11/32), 28.1% (9/32), and 9.4% (3/32), respectively. Significant differences in prevalence were found among the different regions (χ2 = 35.164, p < 0.05) and tissues (χ2 = 25.874, p < 0.05). A total of 128 tissue and organ samples of the thirty-two T. gondii-positive chickens from the different regions were analyzed using PCR-restriction fragment length polymorphism (PCR-RFLP) on the basis of 10 genetic markers. Seven tissue samples (lung samples from five chickens, heart samples from one chicken, and liver samples from one chicken) underwent successful amplification at all the genetic markers, and all the T. gondii genotypes were identified as genotype I (ToxoDB #10). These findings serve as a foundation for evaluating the risk of T. gondii contamination in chicken products intended for human consumption and offer insight into preventing the transmission of the parasite from chickens to humans.

Outcomes of Fluorescence-Guided vs White Light Resection of Glioblastoma in a Single Institution.

Background Glioblastoma (GBM) is the most common malignant primary brain tumour and confers a very poor prognosis. Maximal safe resection of tumour is the goal of neurosurgical intervention and may be more easily achieved through the use of surgical adjuncts such as fluorescence-guided surgery (FGS). 5-Aminolevulinic acid (5-ALA) accumulates in GBM tissue and fluoresce red, distinguishing tumour cells from the surrounding tissue and therefore making resection easier. 5-ALA-guided resection in GBM has been shown to increase resection rates and prolong progression-free survival without impacting post-operative morbidity. Radiotherapy and concomitant chemotherapy also improve survival in GBM. Other factors such as patient age and molecular status of the tumour also impact prognosis. Aims The aim of this study was to compare the outcomes of 5-ALA vs white light-guided resection for glioblastoma in the west of Scotland. Methods  This was a retrospective analysis of baseline characteristics (age, sex, tumour molecular markers, radiotherapy, chemotherapy, anatomical location of tumour and treatment group) and outcomes (mortality, survival, degree of resection and performance status) of 239 patients who underwent primary resection of glioblastoma over a four-year period (2017-2020). A variety of statistical methods were used to analyse the relationship between each variable and surgical technique; multivariate Cox regression and the Kaplan-Meier method were used in survival analysis. Results  5-ALA-guided resection substantially improved resection rates (74.0% vs 40.2%). Mortality at 15 months was 5.1% lower in the 5-ALA group (52.0% vs 57.1%, p = 0.53), and patients lived an average of 68 days longer compared to the white light group (444 days vs 376 days, p = 0.21). There were negligible differences between treatment groups in terms of post-operative performance status (PS) and post-operative complications. In our multivariate Cox regression model, six factors were statistically significant at a level of p ≤ 0.05: age, radiotherapy, chemotherapy, O(6)-methylguanine-DNA methyltransferase (MGMT) methylation, anatomical location and >90% resection. Receiving chemotherapy and radiotherapy, MGMT methylation and undergoing >90% resection conferred a survival benefit at 15 months. Older age and multi-focal disease were related to a worsened mortality rate. Undergoing radiotherapy and maximal resection were the two greatest predictors of improved survival, reducing mortality risk by 58% and 51%, respectively. Conclusion 5-ALA-guided resection improved resection rates without impacting post-operative morbidity. 5-ALA-guided resection was associated with improved survival and lower mortality rate, but this was not statistically significant. Receiving chemoradiotherapy, MGMT methylation and undergoing maximal resection conferred a survival benefit, whilst older age and multi-focal disease were associated with a poorer prognosis.

Detection of Rickettsia spp. and Anaplasma ovis in Melophagus ovinus from southern Xinjiang, China.

Melophagus ovinus is a hematophagous insect that is distributed worldwide and plays a crucial role in transmitting disease-causing pathogens. From June 2021 to March 2022, a total of 370 M. ovinus were collected from 11 sampling points in southern Xinjiang, China. The specimens were identified using morphological and molecular analyses. Rickettsia spp. and Anaplasma ovis were detected from all the samples using seven Rickettsia-specific genetic markers and the msp-4 gene of A. ovis. Approximately 11% of the M. ovinus specimens were positive for Rickettsia spp., and Candidatus Rickettsia barbariae was the most predominant species (35/41; 85.4%), while R. massiliae was least prevalent (6/41; 14.6%). Approximately 10.5% (39/370) of the M. ovinus specimens were positive for A. ovis of genotype III, which was co-detected with Candidatus R. barbariae in M. ovinus (3/370; 0.8%). To the best of our knowledge, this is the first report of the detection of R. massiliae and Candidatus R. barbariae in M. ovinus globally. The detection and control of insect-borne diseases originating from M. ovinus should be strengthened in southern Xinjiang, an area important to animal husbandry and production.

The impact of intestinal stoma formation on patient quality of life after emergency surgery-A systematic review.

AIM: Elective stoma formation has a negative effect on patient quality of life (QoL), with a potential detrimental impact on body image, confidence and social functioning being shown previously. However, the impact of emergency stoma formation on QoL has been explored less frequently. This systematic review aims to synthesize all available literature exploring QoL via patient-reported outcome measures. METHODS: A search strategy was implemented on 24 November 2022 across Embase, MEDLINE, PsycInfo and the Cochrane Library database after registration on PROSPERO (CRD42022370606). Studies were included if they used a standardized patient-reported outcome measure, had more than five emergency stoma patients, age > 18 years and were fully published in English. Two of three independent researchers screened articles, extracted data and performed quality assessment using the Newcastle-Ottawa Scale and the Cochrane risk of bias tool. RESULTS: In all, 1775 articles were screened, with 16 included in the systematic review. This included 1868 emergency stoma patients (men:women 0.53; median age 64.6 years) followed up for a median of 12 months. Patients who had a Hartmann's procedure for perforated diverticulitis had poorer QoL than those who underwent primary anastomosis. There was a negligible difference in QoL between those who had a colonic stent for obstructing colorectal cancer compared with those who underwent emergency stoma formation. Female sex, end stoma formation and ileostomy formation were all identified as risk factors for poorer QoL. CONCLUSION: Patients undergoing emergency stoma surgery have marginally poorer QoL compared with those undergoing similar procedures without stoma formation. Further work is required to identify risk factors associated with this and also to compare QoL after stoma reversal.

Detection of Babesia Spp., Theileria Spp., and Anaplasma Ovis in Ornithodoros Lahorensis from Southern Xinjiang, China.

Introduction: Ticks are blood-sucking arthropods that have negative economic impacts and can spread a variety of diseases through their bites. There are few reports on soft ticks (Acari: Argasidae) and tick-borne pathogens in southern Xinjiang, China. This investigation supplements the available information for this region and is concerned with an argasid tick, apicomplexan parasites of the Babesia and Theileria genera and a bacterium of the Anaplasma genus. Material and Methods: In this study, 330 soft ticks were collected from nine sampling sites in southern Xinjiang between 2020 and 2021. The ticks were identified according to their morphological characteristics and confirmed as Ornithodoros lahorensis using mitochondrial 16S rDNA sequences. Babesia and Theileria were identified at the species level based on two fragments of the 18S rRNA gene, and one set of primers targeting the 16S rRNA gene was used to identify the Anaplasma genus. Results: Among the 330 samples, one Babesia species (Babesia sp.), two Theileria species (T. ovis and T. annulata), and one Anaplasma (A. ovis) species were detected. Conclusion: This study provides fundamental evidence for the occurrence of Babesia, Theileria and Anaplasma spp. in soft ticks. To the best of our knowledge, this is the first report of the detection of Babesia sp. and T. annulata in O. lahorensis. Therefore, the potential threat of soft ticks to livestock and humans should not be ignored.

Distribution and Molecular Epidemiology of Anaplasma ovis in Melophagus ovinus and Small Ruminants in Border Regions of South Xinjiang, China.

Background: Anaplasma ovis are obligate intracellular bacteria that can endanger human and animal health, and they can be transmitted by arthropod vectors, such as Melophagus ovinus and ticks. Materials and Methods: In this study, 433 specimens, including 370 M. ovinus and 63 sheep blood samples, were collected from nine districts of South Xinjiang to investigate the distribution and molecular epidemiology of A. ovis in M. ovinus and small ruminant. Results: DNA of A. ovis was detected in 109 (25.2%, 109/433) of the 433 samples using PCR and sequencing. The analysis of A. ovis msp4 sequences revealed four different genotypes, including genotype III (47.7%; 52/109), GB3 (34.0%; 37/109), AoGOv3 (15.6%; 17/109), and XJ9 (2.8%; 3/109). Conclusions: To the best of our knowledge, A. ovis genotypes GB3, AoGOv3, and XJ9 detected in this study are the first to be reported in M. ovinus, and our data indicate that XJ9 is a novel A. ovis genotype presented herein for the first time. These findings provide important references for the new understanding and prevention of A. ovis in border counties in China.

Superior Fidelity and Distinct Editing Outcomes of SaCas9 Compared to SpCas9 in Genome Editing.

A series of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) systems have been engineered for genome editing. The most widely used Cas9 is SpCas9 from Streptococcus pyogenes and SaCas9 from Staphylococcus aureus. However, a comparison of their detailed gene editing outcomes is still lacking. By characterizing the editing outcomes of 11 sites in human induced pluripotent stem cells (iPSCs) and K562 cells, we found that SaCas9 could edit the genome with greater efficiency than SpCas9. We also compared the spacer lengths of single-guide RNA (sgRNA, 18-21 nt for SpCas9 and 19-23 nt for SaCas9) and found that the optimal spacer lengths were 20 nt and 21 nt for SpCas9 and SaCas9, respectively. However, the optimal spacer size for a particular guide RNA ranged from 18-21 nt or 21-22 nt for SpCas9 and SaCas9, respectively. Furthermore, SpCas9 exhibited a more substantial bias than SaCas9 for nonhomologous end-joining (NHEJ) +1 insertion at the fourth nucleotide upstream of the protospacer adjacent motif (PAM), characteristic of a staggered cut. Accordingly, editing with SaCas9 led to higher knock-in efficiencies of NHEJ-mediated double-stranded oligodeoxynucleotide (dsODN) insertion or adeno-associated virus serotype 6 (AAV6) donor-mediated homology-directed repair (HDR). Finally, GUIDE-seq analysis revealed that SaCas9 exhibited significantly reduced off-target effects compared with SpCas9. Our work indicates the superior performance of SaCas9 to SpCas9 in transgene integration-based therapeutic gene editing and the necessity to identify the optimal spacer length to achieve desired editing results.

Sputter-Deposited High Entropy Alloy Thin Film Electrocatalyst for Enhanced Oxygen Evolution Reaction Performance.

Thin film catalysts, giving a different morphology, provide a significant advantage over catalyst particles for the gas evolution reaction. Taking the advantages of sputter deposition, a high entropy alloy (HEA) thin film electrocatalyst is hereby reported for the oxygen evolution reaction (OER). The catalyst characteristics are investigated not only in its as-deposited state, but also during and after the OER. For comparison, unary, binary, ternary, and quaternary thin film catalysts are prepared and characterized. The surface electronic structure modification due to the addition of a metal is studied experimentally and theoretically using density functional theory calculation. It is demonstrated that sputtered FeNiMoCrAl HEA thin film exhibits OER performance superior to all the reported HEA catalysts with robust electrocatalytic activity having a low overpotential of 220 mV at 10 mA cm-2 , and excellent electrochemical stability at different constant current densities of 10 and 100 mA cm-2 for 50 h. Furthermore, the microstructure transformation is investigated during the OER, which is important for the understanding of the OER mechanism provided by HEA electrocatalyst. Such a finding will contribute to future catalyst design.

Antithrombotic therapy to prevent cognitive decline in people with small vessel disease on neuroimaging but without dementia.

BACKGROUND: Cerebral small vessel disease is a progressive disease of the brain's deep perforating blood vessels. It is usually diagnosed based on lesions seen on brain imaging. Cerebral small vessel disease is a common cause of stroke but can also cause a progressive cognitive decline. As antithrombotic therapy is an established treatment for stroke prevention, we sought to determine whether antithrombotic therapy might also be effective in preventing cognitive decline in people with small vessel disease. OBJECTIVES: To assess the effects of antithrombotic therapy for prevention of cognitive decline in people with small vessel disease on neuroimaging but without dementia. SEARCH METHODS: We searched ALOIS, the Cochrane Dementia and Cognitive Improvement Review Group's Specialised Register, and the Cochrane Stroke Group's Specialised Register; the most recent search was on 21 July 2021. We also searched MEDLINE, Embase, four other databases and two trials registries. We searched the reference lists of the articles retrieved from these searches. As trials with a stroke focus may include relevant subgroup data, we complemented these searches with a focussed search of all antithrombotic titles in the Cochrane Stroke Group database.  SELECTION CRITERIA: We included randomised controlled trials (RCT) of people with neuroimaging evidence of at least mild cerebral small vessel disease (defined here as white matter hyperintensities, lacunes of presumed vascular origin and subcortical infarcts) but with no evidence of dementia. The trials had to compare antithrombotic therapy of minimum 24 weeks' duration to no antithrombotic therapy (either placebo or treatment as usual), or compare different antithrombotic treatment regimens. Antithrombotic therapy could include antiplatelet agents (as monotherapy or combination therapy), anticoagulants or a combination. DATA COLLECTION AND ANALYSIS: Two review authors independently screened all the titles identified by the searches. We assessed full texts for eligibility for inclusion according to our prespecified selection criteria, extracted data to a proforma and assessed risk of bias using the Cochrane tool for RCTs. We evaluated the certainty of evidence using GRADE. Due to heterogeneity across included participants, interventions and outcomes of eligible trials, it was not possible to perform meta-analyses. MAIN RESULTS: We included three RCTs (3384 participants). One study investigated the effect of antithrombotic therapy in participants not yet on antithrombotic therapy; two studies investigated the effect of additional antithrombotic therapy, one in a population already taking a single antithrombotic agent and one in a mixed population (participants on an antithrombotic drug and antithrombotic-naive participants). Intervention and follow-up durations varied from 24 weeks to four years. Jia 2016 was a placebo-controlled trial assessing 24 weeks of treatment with DL-3-n-butylphthalide (a compound with multimodal actions, including a putative antiplatelet effect) in 280 Chinese participants with vascular cognitive impairment caused by subcortical ischaemic small vessel disease, but without dementia. There was very low-certainty evidence for a small difference in cognitive test scores favouring treatment with DL-3-n-butylphthalide, as measured by the 12-item Alzheimer's Disease Assessment Scale-Cognitive subscale (adjusted mean difference -1.07, 95% confidence interval (CI) -2.02 to -0.12), but this difference may not be clinically relevant. There was also very low-certainty evidence for greater proportional improvement measured with the Clinician Interview-Based Impression of Change-Plus Caregiver Input (57% with DL-3-n-butylphthalide versus 42% with placebo; P = 0.01), but there was no difference in other measures of cognition (Mini-Mental State Examination and Clinical Dementia Rating) or function. There was no evidence of a difference in adverse events between treatment groups. The SILENCE RCT compared antithrombotic therapy (aspirin) and placebo during four years of treatment in 83 participants with 'silent brain infarcts' who were on no prior antithrombotic therapy. There was very low-certainty evidence for no difference between groups across various measures of cognition and function, rates of stroke or adverse events. The Secondary Prevention of Subcortical Stroke Study (SPS3) compared dual antiplatelet therapy (clopidogrel plus aspirin) to aspirin alone in 3020 participants with recent lacunar stroke. There was low-certainty evidence of no effect on cognitive outcomes as measured by the Cognitive Abilities Screening Instruments (CASI) assessed annually over five years. There was also low-certainty evidence of no difference in the annual incidence of mild cognitive decline between the two treatment groups (9.7% with dual antiplatelet therapy versus 9.9% with aspirin), or the annual stroke recurrence rate (2.5% with dual antiplatelet therapy versus 2.7% with aspirin). Bleeding risk may be higher with dual antiplatelet therapy (hazard ratio (HR) 2.15, 95% CI 1.49 to 3.11; low certainty evidence), but there may be no significant increase in intracerebral bleeding risk (HR 1.52, 95% CI 0.79 to 2.93; low-certainty evidence). None of the included trials assessed the incidence of new dementia. AUTHORS' CONCLUSIONS: We found no convincing evidence to suggest any clinically relevant cognitive benefit of using antithrombotic therapy in addition to standard treatment in people with cerebral small vessel disease but without dementia, but there may be an increased bleeding risk with this approach. There was marked heterogeneity across the trials and the certainty of the evidence was generally poor.

GREPore-seq: A robust workflow to detect changes after gene editing through long-range PCR and nanopore sequencing.

To achieve the enormous potential of gene-editing technology in clinical therapies, one needs to evaluate both the on-target efficiency and unintended editing consequences comprehensively. However, there is a lack of a pipelined, large-scale, and economical workflow for detecting genome editing outcomes, in particular insertion or deletion of a large fragment. Here, we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR/Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products. Recognizing the high error rates of Oxford nanopore sequencing, we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing (GREPore-seq). GREPore-seq can assess nonhomologous end-joining (NHEJ)-mediated double-stranded oligodeoxynucleotide (dsODN) insertions with comparable accuracy to Illumina next-generation sequencing (NGS). GREPore-seq also reveals a full spectrum of homology-directed repair (HDR)-mediated large gene knock-in, correlating well with the fluorescence-activated cell sorting (FACS) analysis results. Of note, we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting site. Therefore, we have established a practical workflow to evaluate various genetic changes, including quantifying insertions of short dsODNs, knock-ins of long pieces, plasmid insertions, and large fragment deletions after CRISPR editing. GREPore-seq is freely available at GitHub (https://github.com/lisiang/GREPore-seq) and the National Genomics Data Center (NGDC) BioCode (https://ngdc.cncb.ac.cn/biocode/tools/BT007293).

Improved and Flexible HDR Editing by Targeting Introns in iPSCs.

Highly efficient gene knockout (KO) editing of CRISPR-Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines.

Modulation of Immune Reaction in Hydrodynamic Gene Therapy for Hemophilia A.

Hemophilia A (HA) is a monogenic disease characterized by plasma clotting factor 8 (F8) deficiency due to F8 mutation. We have been attempting to cure HA permanently using a CRISPR-Cas9 gene-editing strategy. In this study, we induced targeted integration of BDDF8 (B-domain-deleted F8) gene into the albumin locus of HA mice by hydrodynamic tail vein injection of editing plasmid vectors. One week after treatment, a high F8 activity ranging from 70% to 280% of normal serum levels was observed in all treated HA mice but dropped to background levels 3-5 weeks later. We found that the humoral immune reaction targeting F8 is the predominant cause of the decreased F8 activity. We hypothesized that hydrodynamic injection-induced liver damage triggered the release of large quantities of inflammatory cytokines. However, coinjection of plasmids expressing a dozen immunomodulatory factors failed to curtail the immune reaction and stabilize F8 activity effectively. The spCas9 plasmid carrying a miR-142-3p target sequence alleviated the cellular immune response but could not deliver therapeutic efficacy. Strikingly, immunosuppressant cyclophosphamide virtually abolished the immune response, leading to a year-long stable F8 level. Our findings should have important implications in developing therapies in mouse models using the hydrodynamic gene delivery approach, highlighting the necessity of modulating the innate immune response triggered by liver damage.

Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion.

BACKGROUND: After repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences. RESULTS: We show that large deletions are ubiquitous but are dependent on editing sites and cell types. Human primary T cells display more significant deletions than hematopoietic stem and progenitor cells (HSPCs), whereas we observe low levels in induced pluripotent stem cells (iPSCs). We find that the homology-directed repair (HDR) with single-stranded oligodeoxynucleotides (ssODNs) carrying short homology reduces the deletion damage by almost half, while adeno-associated virus (AAV) donors with long homology reduce large deletions by approximately 80%. In the absence of HDR, the insertion of a short double-stranded ODN by NHEJ reduces deletion indexes by about 60%. CONCLUSIONS: Timely bridging of broken ends by HDR and NHEJ vastly decreases the unintended consequences of dsDNA cleavage. These strategies can be harnessed in gene editing applications to attenuate unintended outcomes.

Metabolic Mechanisms and a Rational Combinational Application of Carboxyamidotriazole in Fighting Pancreatic Cancer Progression after Chemotherapy.

The anticancer and anti-inflammatory effects of carboxyamidotriazole (CAI) have been demonstrated in several studies, but the underlying mechanisms remain to be elucidated. This study showed that CAI caused metabolic reprogramming of pancreatic cancer cells. The inhibition of mitochondrial oxidative metabolism by CAI led to increased glutamine-dependent reductive carboxylation and enhanced glycolytic metabolism. The presence of environmental substances that affect cellular metabolism, such as glutamine and pyruvate, attenuated the anticancer efficacy of CAI. Based on the action of CAI: 1) when glutamine was removed, the NAD+/NADH ratio was decreased, the synthesis of cellular aspartate was reduced, and autophagy flux was blocked; and 2) when glycolysis was pharmacologically inhibited, the ATP level was significantly decreased, the cell viability was greatly inhibited, and the compensatory rescue effect of glutamine was eliminated. When combined with chemotherapy, cotreatment with CAI and the glycolysis inhibitor 2-deoxyglucose (2-DG) inhibited the pancreatic cancer progression after chemotherapy. As the inhibition of mitochondrial oxidative metabolism can explain several anticancer activities of CAI reported previously, including inhibition of calcium entry and induction of reactive oxygen species, we demonstrate that inhibition of mitochondrial oxidative phosphorylation may be the fundamental mechanism of CAI. The combination of CAI and 2-DG causes energy depletion in cancer cells, eliminating the rescue effect of the metabolic environment. Inhibiting pancreatic cancer progression after chemotherapy is a rational application of this metabolism-disturbing combination strategy.

Characterization of the genome of a phylogenetically distinct tospovirus and its interactions with the local lesion-induced host Chenopodium quinoa by whole-transcriptome analyses.

Chenopodium quinoa is a natural local lesion host of numerous plant viruses, including tospoviruses (family Bunyaviridae). Groundnut chlorotic fan-spot tospovirus (GCFSV) has been shown to consistently induce local lesions on the leaves of C. quinoa 4 days post-inoculation (dpi). To reveal the whole genome of GCFSV and its interactions with C. quinoa, RNA-seq was performed to determine the transcriptome profiles of C. quinoa leaves. The high-throughput reads from infected C. quinoa leaves were used to identify the whole genome sequence of GCFSV and its single nucleotide polymorphisms. Our results indicated that GCFSV is a phylogenetically distinct tospovirus. Moreover, 27,170 coding and 29,563 non-coding sequences of C. quinoa were identified through de novo assembly, mixing reads from mock and infected samples. Several key genes involved in the modulation of hypersensitive response (HR) were identified. The expression levels of 4,893 deduced complete genes annotated using the Arabidopsis genome indicated that several HR-related orthologues of pathogenesis-related proteins, transcription factors, mitogen-activated protein kinases, and defense proteins were significantly expressed in leaves that formed local lesions. Here, we also provide new insights into the replication progression of a tospovirus and the molecular regulation of the C. quinoa response to virus infection.

A rapid process for fabricating gas sensors.

Zinc oxide (ZnO) is a low-toxicity and environmentally-friendly material applied on devices, sensors or actuators for "green" usage. A porous ZnO film deposited by a rapid process of aerosol deposition (AD) was employed as the gas-sensitive material in a CO gas sensor to reduce both manufacturing cost and time, and to further extend the AD application for a large-scale production. The relative resistance change (△R/R) of the ZnO gas sensor was used for gas measurement. The fabricated ZnO gas sensors were measured with operating temperatures ranging from 110 °C to 180 °C, and CO concentrations ranging from 100 ppm to 1000 ppm. The sensitivity and the response time presented good performance at increasing operating temperatures and CO concentrations. AD was successfully for applied for making ZnO gas sensors with great potential for achieving high deposition rates at low deposition temperatures, large-scale production and low cost.