Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.

A High-Sensitivity Analysis Method Of Furanocoumarins In Radix Angelicae Dahuricae By Reverse-Phase High-Performance Liquid Chromatography Coupled With Integrated Pulsed Amperometric Detection

Radix Angelicae Dahuricae is a traditional Chinese medicine. We developed a high-sensitivity method for detection of furanocoumarins in Radix Angelicae Dahuricae and Gumiganghwal-tang (GMGHT). The six furanocoumarins of Radix Angelicae Dahuricae were sonication-extracted from 50% ethanol for 60 min. Six furanocoumarins were separated through a gradient elution system. The limits of detection of the components were 0.002–0.3 ng (0.2–30 ng/mL). The coefficients of determination were 0.9995–1.0000, all inter-day and intra-day precision values were < 4.9%, and the mean recoveries and relative standard deviations were 96.4%–104.5% and 0.5%–4.8% for Radix Angelicae Dahuricae extract, respectively. Our method does not require any pretreatment steps and exhibits good reproducibility, selectivity, and sensitivity. Therefore, our method will contribute to a Radix Angelicae Dahuricae quality control measure.

Both microbiological surveillance and audit of procedures improve reprocessing of flexible bronchoscopes and patient safety

Background: Microbiological surveillance of bronchoscopes and automatic endoscope reprocessors (AERs)/washer disinfectors as a quality control measure is controversial. Experts also are divided on the infection risks associated with bronchoscopic procedures. Objective: We evaluated the impact of routine microbiological surveillance and audits of cleaning/disinfection practices on contamination rates of reprocessed bronchoscopes. Design: Audits were conducted of reprocessing procedures and microbiological surveillance on all flexible bronchoscopes used from January 2007 to June 2020 at a teaching hospital in France. Contamination rates per year were calculated and analyzed using a Poisson regression model. The risk factors for microbiological contamination were analyzed using a multivariable logistical regression model. Results: In total, 478 microbiological tests were conducted on 91 different bronchoscopes and 57 on AERs. The rate of bronchoscope contamination significantly decreased between 2007 and 2020, varying from 30.2 to 0% (P < .0001). Multivariate analysis confirmed that retesting after a previous contaminated test was significantly associated with higher risk of bronchoscope contamination (OR, 2.58; P = .015). This finding was explained by the persistence of microorganisms in bronchoscopes despite repeated disinfections. However, the risk of persistent contamination was not associated with the age of the bronchoscope. Conclusions: Our results confirm that bronchoscopes can remain contaminated despite repeated reprocessing. Routine microbial testing of bronchoscopes for quality assurance and audit of decontamination and disinfection procedures can improve the reprocessing of bronchoscopes and minimize the rate of persistent contamination.

Papanicolaou-Stained Cytosmear Preparations in the Evaluation of Leucocytospermia: A Tertiary Centre Experience and Assessment of Utility

Objectives Leucocytospermia is a rare cause of infertility with a variable incidence in infertile men. In many andrology laboratories, semen analysis is primarily centered on analyzing basic parameters (sperm count, motility, and viability). We examined the role of cytomorphological analysis on Papanicolaou (PAP)-stained smears in the work up of male infertility, with special reference to leukocytospermia and assessed the morphological features of sperms in these cases. Materials and Methods All cases signed out as “leukocytospermia” between November 2017 and January 2018 were evaluated. Cases showing pus cells (≥ 1/5hpf) on cytosmear evaluation were also analyzed. Parameters obtained on modified Neubauer’s chamber, wet mount preparations, and autoanalyzer SQA-IIC-P (Medical Electronic Systems, Israel) were tabulated. PAP-stained smears were examined to assess morphological defects and other findings, if any. Results Out of 348 semen samples, 6 (1.72%) were diagnosed as leukocytospermia, all displaying pus cells on cytomorphological evaluation. Five cases having white blood cells < 1 × 10 9/L were analyzed, which displayed the presence of pus cells (≥ 1/5 hpf) on cytosmear preparations (1.43%). Nine cases (81.8%) showed reduced motile sperm concentration, functional sperm concentration, and sperm motility index. Morphological evaluation of the PAP-stained smears showed mid-piece defects in 18.1% cases. Conclusion Diligent cytomorphological evaluation of semen is essential to diagnose leukocytospermia, assess morphological defects, and serve as a quality control measure.

Artificial intelligence for the detection of polyps or cancer with colon capsule endoscopy

Colorectal cancer is common and can be devastating, with long-term survival rates vastly improved by early diagnosis. Colon capsule endoscopy (CCE) is increasingly recognised as a reliable option for colonic surveillance, but widespread adoption has been slow for several reasons, including the time-consuming reading process of the CCE recording. Automated image recognition and artificial intelligence (AI) are appealing solutions in CCE. Through a review of the currently available and developmental technologies, we discuss how AI is poised to deliver at the forefront of CCE in the coming years. Current practice for CCE reporting often involves a two-step approach, with a ‘pre-reader’ and ‘validator’. This requires skilled and experienced readers with a significant time commitment. Therefore, CCE is well-positioned to reap the benefits of the ongoing digital innovation. This is likely to initially involve an automated AI check of finished CCE evaluations as a quality control measure. Once felt reliable, AI could be used in conjunction with a ‘pre-reader’, before adopting more of this role by sending provisional results and abnormal frames to the validator. With time, AI would be able to evaluate the findings more thoroughly and reduce the input required from human readers and ultimately autogenerate a highly accurate report and recommendation of therapy, if required, for any pathology identified. As with many medical fields reliant on image recognition, AI will be a welcome aid in CCE. Initially, this will be as an adjunct to ‘double-check’ that nothing has been missed, but with time will hopefully lead to a faster, more convenient diagnostic service for the screening population.

Infra Red Spectra of Different Species of Cultivated Oyster Mushrooms Possible Tool for Identifying Bioactive Compounds and Establishing Taxonomic Linkage

Mushrooms are macrofungi that serve as a vegetarian source of protein along with various bio-active molecules of primary health importance. The activity of the bio-active molecules range from antioxidant, immunomodulation, hepatoprotection. Cultivated oyster mushrooms are also rich in these components which may be estimated quantitatively by skill intensive ‘destructive’ chemical techniques. Infra Red spectroscopy provides a non-destructive user friendly technique to quickly assess the presence of bio-active compounds in mushroom species to be used as a quality control measure as this non-destructive tool can be used to segregate mushroom harvest according to availability of bioactive compounds. IR spectra based strain classification and taxa delimitation of mushroom samples are also attempted vis a vis DNA sequence based phylogeny analysis of the same, but no correlation is observed between the two types of phylogeny analysis.

Rapid Detection of Botulinum Neurotoxins—A Review

A toxin is a poisonous substance produced within living cells or organisms. One of the most potent groups of toxins currently known are the Botulinum Neurotoxins (BoNTs). These are so deadly that as little as 62 ng could kill an average human; to put this into context that is approximately 200,000 × less than the weight of a grain of sand. The extreme toxicity of BoNTs leads to the need for methods of determining their concentration at very low levels of sensitivity. Currently the mouse bioassay is the most widely used detection method monitoring the activity of the toxin; however, this assay is not only lengthy, it also has both cost and ethical issues due to the use of live animals. This review focuses on detection methods both existing and emerging that remove the need for the use of animals and will look at three areas; speed of detection, sensitivity of detection and finally cost. The assays will have wide reaching interest, ranging from the pharmaceutical/clinical industry for production quality management or as a point of care sensor in suspected cases of botulism, the food industry as a quality control measure, to the military, detecting BoNT that has been potentially used as a bio warfare agent.

FastQ Screen: A tool for multi-genome mapping and quality control

DNA sequencing analysis typically involves mapping reads to just one reference genome. Mapping against multiple genomes is necessary, however, when the genome of origin requires confirmation. Mapping against multiple genomes is also advisable for detecting contamination or for identifying sample swaps which, if left undetected, may lead to incorrect experimental conclusions. Consequently, we present FastQ Screen, a tool to validate the origin of DNA samples by quantifying the proportion of reads that map to a panel of reference genomes. FastQ Screen is intended to be used routinely as a quality control measure and for analysing samples in which the origin of the DNA is uncertain or has multiple sources.