Increased mutation efficiency of CRISPR/Cas9 genome editing in banana by optimized construct

The CRISPR/Cas9-mediated genome editing system has been used extensively to engineer targeted mutations in a wide variety of species. Its application in banana, however, has been hindered because of the species’ triploid nature and low genome editing efficiency. This has delayed the development of a DNA-free genome editing approach. In this study, we reported that the endogenous U6 promoter and banana codon-optimized Cas9 apparently increased mutation frequency in banana, and we generated a method to validate the mutation efficiency of the CRISPR/Cas9-mediated genome editing system based on transient expression in protoplasts. The activity of the MaU6c promoter was approximately four times higher than that of the OsU6a promoter in banana protoplasts. The application of this promoter and banana codon-optimized Cas9 in CRISPR/Cas9 cassette resulted in a fourfold increase in mutation efficiency compared with the previous CRISPR/Cas9 cassette for banana. Our results indicated that the optimized CRISPR/Cas9 system was effective for mutating targeted genes in banana and thus will improve the applications for basic functional genomics. These findings are relevant to future germplasm improvement and provide a foundation for developing DNA-free genome editing technology in banana.

Cancer Registry of Population in the City Ozyorsk: Structure of Malignant Neoplasms First Diagnosed in Period From 1948 to 2016

Purpose: The analysis of the structure of malignant neoplasms (MN) incidence among the population in the city Ozyorsk, located near Mayak Production Association, based on the information from the territorial cancer registry. Materials and methods: There were 14681 first diagnosed cases of malignant neoplasms at the period from 1948 to 2016 in the territorial cancer registry of Ozyorsk. The diagnoses were verified by all available medical documents. The structure of MN incidence, vital status of diseased people, age in a year of diagnostics, rank distribution of the basic localizations were studied. Results: For 70 years period of follow-up there were 7676 cases from 14681 of MN incidence cases during the last 17 years (from 2000 to 2017) – as much as for the previous 51 years of follow-up from 1948 to 1999 (7005 cases). In the MN structure solid cancer constitutes 94.5 %, hemoblastoses – 5.5 %. As at December 31, 2016 23.3 % of the diseased people were alive, 75.2 % died from all causes, 1.5 % lost to follow-up. The cause of 80,6 % deaths was MN. Age in the year of diagnostics constitutes 61,7 years. 75.8 % diagnoses have morphological confirmation. During the entire period of follow-up first three places belong to lung cancer, gastric cancer and prostate cancer for males; breast cancer, gastric cancer and colon cancer for females in the MN structure. Conclusions: During the period from 1948 to 2016 the growth of the first diagnosed MN cases among the population of Ozyorsk was a result of the population doubling and a fourfold increase of the citizens over the age of 50. Developed and supported cancer registry allows to analyze and partially control oncological situation in the closed cities of Rosatom.

When Innovation Goes Wrong: Technological Regress and the Opioid Epidemic

The fourfold increase in opioid deaths between 2000 and 2017 rivals even the COVID-19 pandemic as a health crisis for America. Why did it happen? Measures of demand for pain relief – physical pain and despair – are high and in many cases rising, but their increase was nowhere near as large as the increase in deaths. The primary shift is in supply, primarily of new forms of allegedly safer narcotics. These new pain relievers flowed in greater volume to areas with more physical pain and mental health impairment, but since their apparent safety was an illusion, opioid deaths followed. By the end of the 2000s, restrictions on legal opioids led to further supply-side innovations, which created the burgeoning illegal market that accounts for the bulk of opioid deaths today. Because opioid use is easier to start than end, America's opioid epidemicis likely to persist for some time.

The Respiratory Resistance Sensitivity Task: An Automated Method for Quantifying Respiratory Interoception and Metacognition

The ability to sense, monitor, and control respiration - e.g., respiratory interoception (henceforth, respiroception) is a core homeostatic ability. Beyond the mere regulation of gas exchange, enhanced awareness of respiratory sensations is directly related to psychiatric symptoms such as panic and anxiety. Indeed, chronic breathlessness (dyspnea) is associated with a fourfold increase in the risk of developing depression and anxiety, and the regulation of the breath is a key aspect of many mindfulness-based approaches to the treatment of mental illness. Physiologically speaking, the ability to accurately monitor respiratory sensations is important for optimizing cardiorespiratory function during athletic exertion, and can be a key indicator of illness. Given the important role of respiroception in mental and physical health, it is unsurprising that there is increased interest in the quantification of respiratory psychophysiology across different perceptual and metacognitive levels of the psychological hierarchy. Compared to other more popular modalities of interoception, such as in the cardiac domain, there are relatively few methods available for measuring aspects of respiroception. Existing inspiratory loading tasks are difficult to administer and frequently require expensive medical equipment, or offer poor granularity in their quantification of respiratory-related perceptual ability. To facilitate the study of respiroception, we here present a new, fully automated and computer-controlled apparatus and psychophysiological method, which can flexibly and easily measure respiratory-related interoceptive sensitivity, bias and metacognition, in as little as 30 minutes of testing, using easy to make 3D printable parts.

Hyaluronic acid nanoparticle-encapsulated microRNA-125b repolarizes tumor-associated macrophages in pancreatic cancer

Aim: To investigate a novel strategy to target tumor-associated macrophages and reprogram them to an antitumor phenotype in pancreatic adenocarcinoma (PDAC). Methods: M2 peptides were conjugated to HA-PEG/HA-PEI polymer to form self-assembled nanoparticles with miR-125b. The efficacy of HA-PEI/PEG-M2peptide nanoparticles in pancreatic tumors from LSL-KrasG12D/+, LSL-Trp53R172H/+, Pdx1-Cre genetically engineered mice was evaluated. Results: In vitro M2 macrophage-specific delivery of targeted nanoformulations was demonstrated. Intraperitoneal administration of M2-targeted nanoparticles showed preferential accumulation in the pancreas of KPC-PDAC mice and an above fourfold increase in the M1-to-M2 macrophage ratio compared with transfection with scrambled miR. Conclusion: M2-targeted HA-PEI/PEG nanoparticles with miR-125b can transfect tumor-associated macrophages in pancreatic tissues and may have implications for PDAC immunotherapy.

An mRNA assay system demonstrates proteasomal-specific degradation contributes to cardiomyopathic phospholamban null mutation

Background The human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified. Previous studies have shown the implication between an impaired cellular degradation homeostasis and cardiomyopathy development. Therefore, uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics. Methods A panel of mutant and wild-type reporter tagged PLN modified mRNA (modRNA) constructs were transfected in human embryonic stem cell-derived cardiomyocytes. Lysosomal and proteasomal chemical inhibitors were used together with cell imaging and protein analysis tools in order to dissect degradation pathways associated with expressed PLN constructs. Transcriptional profiling of the cardiomyocytes transfected by wild-type or L39X mutant PLN modRNA was analysed with bulk RNA sequencing. Results Our modRNA assay system revealed that transfected L39X mRNA was stable and actively translated in vitro but with only trace amount of protein detectable. Proteasomal inhibition of cardiomyocytes transfected with L39X mutant PLN modRNA showed a fourfold increase in protein expression levels. Additionally, RNA sequencing analysis of protein degradational pathways showed a significant distinct transcriptomic signature between wild-type and L39X mutant PLN modRNA transfected cardiomyocytes. Conclusion Our results demonstrate that the cardiomyopathic PLN null mutant L39X is rapidly, actively and specifically degraded by proteasomal pathways. Herein, and to the best of our knowledge, we report for the first time the usage of modified mRNAs to screen for and illuminate alternative molecular pathways found in genes associated with inherited cardiomyopathies.

Increased biosynthesis of acetyl-CoA in the yeast Saccharomyces cerevisiae by overexpression of a deregulated pantothenate kinase gene and engineering of the coenzyme A biosynthetic pathway

Coenzyme A (CoA) and its derivatives such as acetyl-CoA are essential metabolites for several biosynthetic reactions. In the yeast S. cerevisiae, five enzymes (encoded by essential genes CAB1-CAB5; coenzyme A biosynthesis) are required to perform CoA biosynthesis from pantothenate, cysteine, and ATP. Similar to enzymes from other eukaryotes, yeast pantothenate kinase (PanK, encoded by CAB1) turned out to be inhibited by acetyl-CoA. By genetic selection of intragenic suppressors of a temperature-sensitive cab1 mutant combined with rationale mutagenesis of the presumed acetyl-CoA binding site within PanK, we were able to identify the variant CAB1 W331R, encoding a hyperactive PanK completely insensitive to inhibition by acetyl-CoA. Using a versatile gene integration cassette containing the TPI1 promoter, we constructed strains overexpressing CAB1 W331R in combination with additional genes of CoA biosynthesis (CAB2, CAB3, HAL3, CAB4, and CAB5). In these strains, the level of CoA nucleotides was 15-fold increased, compared to a reference strain without additional CAB genes. Overexpression of wild-type CAB1 instead of CAB1 W331R turned out as substantially less effective (fourfold increase of CoA nucleotides). Supplementation of overproducing strains with additional pantothenate could further elevate the level of CoA (2.3-fold). Minor increases were observed after overexpression of FEN2 (encoding a pantothenate permease) and deletion of PCD1 (CoA-specific phosphatase). We conclude that the strategy described in this work may improve the efficiency of biotechnological applications depending on acetyl-CoA.Key points• A gene encoding a hyperactive yeast pantothenate kinase (PanK) was constructed.• Overexpression of CoA biosynthetic genes elevated CoA nucleotides 15-fold.• Supplementation with pantothenate further increased the level of CoA nucleotides.

Clinical presentation and serological diagnosis of syphilis reinfection in men living with HIV: a retrospective cohort study

ObjectivesStudies on the characteristics of syphilis reinfection are scarce despite increasing numbers and proportions of cases. We aimed to gain insights into the clinical and serological presentation of reinfected men living with HIV and to evaluate diagnostic criteria for syphilis reinfection.MethodsWe conducted a retrospective cohort study of 259 HIV-positive men diagnosed with syphilis between January 1999 and September 2015 at the University Hospital Zurich. We compared patients with a single syphilis infection (n=109) to patients with reinfections (n=150).ResultsThe two groups matched in age, sexual orientation and numbers of other STIs. Reinfected patients more often presented with latent syphilis than patients with a single syphilis episode (41.9% vs 8.9%; p<0.001). Although generally high venereal diseases research laboratory (VDRL) or rapid plasma reagin (RPR) titres (median 1:32) were seen in reinfected patients, 19.4% had titres ≤1:8. Treponema pallidum passive particle agglutination (TPPA) titres were significantly higher (1:81 840 vs 1:10 240; p<0.001), while IgM values were significantly lower (1.27 vs 3.5; p<0.001) in syphilis reinfections than in first infections. The TPPA increased ≥fourfold in >92.3% of reinfected patients.ConclusionsOur data highlight the paramount importance of regularly screening patients at risk as syphilis reinfections in men living with HIV are more likely to be latent infections, that is, without symptoms. As non-treponemal tests might be biologically false-positive (up to a titre of 1:8) due to various conditions, a ≥fourfold increase of the TPPA might be considered as optional criterion for the diagnosis of syphilis reinfections. This could be especially valuable for diagnosing reinfected latent stage patients.

980 Technological Innovations in The Management of Pressure Ulcers: A Systematic Review and Discussion

Aim Pressure ulcers (PUs) affect over 700,000 patients per annum and are associated with an up to fourfold increase in mortality rate in older populations with complex pathologies. NICE guidelines for PU management involve a combination of low-grade technological interventions, including dressings and foam mattresses, while technology-centric therapies are not offered. The aim of this systematic review is to evaluate the effectiveness of any novel and high-grade technological PU interventions. Method The PubMed, Embase (via Ovid), Web of Science and CINAHL databases were electronically searched using free-text and MeSH search strings. Results evaluating the type of technological intervention, outcome measures and wound healing rates, duration of hospital stay, requirement for surgical debridement, infection and mortality rates were methodically assessed. Results An initial search identified 900 articles. Using the PRISMA guidance, 25 studies were included, encompassing 845 patients. 28 novel modalities of high-grade innovations were identified, with distinguished technologies including smart wireless bandages and 3D-printed wound repair scaffolds. Overall, 92.8% of devices demonstrated an improvement in wound healing (P &lt; 0.05). A novel framework was created from the different innovations identified to approach adopting innovation in PU management. Conclusions The majority of high-technology innovations identified in this systematic review significantly improve PU healing, thus offering an additional pathway to standard care. Given the annual cost of PUs (£531 million), it is vital innovative technologies are considered as the NHS transitions into a digital age. Therefore, further studies are essential and recommended to evaluate the potential benefit of the promising, innovative technologies identified.

940 Neck of Femur Fracture Management and Outcomes during COVID-19: Overview of the Results from a District General Hospital in Wales

Introduction The COVID-19 pandemic has delivered significant challenges to the Orthopaedic community. Our study aims to assess the impact of COVID-19 on the management and outcomes of patient’s presenting with Hip Fractures to our DGH. Method Retrospective data analysis was performed on a cohort of hip fracture patients who presented to our DGH before the COVID-19 pandemic (23/03/2019 to 05/05/2019) and were compared to those who presented during the COVID-19 pandemic (23/03/2020 to 05/05/2020). Minimum follow up was 30 days postoperatively. Results 38 patients with hip fractures presented to our unit in the pre-COVID-19 period compared to 27 patients in the COVID-19 period. Total time from presentation to discharge, during COVID-19, demonstrated a 70.23% decrease when compared to the pre-COVID cohort. 30-day Mortality rates were higher in the COVID-19 cohort. Conclusions During the COVID-19 pandemic our time to theatre and discharge of hip fractures has seen an improvement, with time to discharge decreasing by 70.23%. Comparison of our mortality rates has seen a fourfold increase likely influenced by the detrimental effects of COVID-19. Further work and larger numbers are required to see the true impact of COVID-19 on the management and outcomes of hip fractures in our DGH.