High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effect

CRISPR-Cas13 systems have recently been employed for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has imposed a major barrier for their in vivo applications. We designed a dual-fluorescent reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants (including Cas13d and Cas13X) exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. Importantly, high-fidelity Cas13 variants show comparable RNA knockdown activity with wild-type Cas13 but no detectable collateral damage in transgenic mice and adeno-associated virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effect are now available for targeted degradation of RNAs in basic research and therapeutic applications.

Changes in the gut microbiota diversity of brown frogs (Rana dybowskii) after an antibiotic bath

Background Captive amphibians frequently receive antibiotic baths to control bacterial diseases. The potential collateral effect of these antibiotics on the microbiota of frogs is largely unknown. To date, studies have mainly relied on oral administration to examine the effects of antibiotics on the gut microbiota; in contrast, little is known regarding the effects of bath-applied antibiotics on the gut microbiota. The gut microbiota compositions of the gentamicin, recovery, and control groups were compared by Illumina high-throughput sequencing, and the functional profiles were analysed using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Furthermore, the relationship between the structure and predicted functional composition of the gut microbiota was determined. Results The alpha diversity indices were significantly reduced by the gentamicin bath, illustrating that this treatment significantly changed the composition of the gut microbiota. After 7 days, the gut microbiota of the recovery group was not significantly different from that of the gentamicin group. Forty-four indicator taxa were selected at the genus level, comprising 42 indicators representing the control group and 2 indicators representing the gentamicin and recovery groups. Potential pathogenic bacteria of the genera Aeromonas, Citrobacter, and Chryseobacterium were significantly depleted after the gentamicin bath. There was no significant positive association between the community composition and functional composition of the gut microbiota in the gentamicin or control frogs, indicating that the functional redundancy of the gut bacterial community was high. Conclusions Gentamicin significantly changed the structure of the gut microbiota of R. dybowskii, and the gut microbiota exhibited weak resilience. However, the gentamicin bath did not change the functional composition of the gut microbiota of R. dybowskii, and there was no significant correlation between the structural composition and the functional composition of the gut microbiota.

Lactoferricins access the cytosol of Escherichia coli within few seconds

We report the real-time response of E. coli to lactoferricin-derived antimicrobial peptides (AMPs) on length-scales bridging microscopic cell-sizes to nanoscopic lipid packing using millisecond time-resolved synchrotron small-angle X-ray scattering. Coupling a multi-scale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly saturate the bacterial envelope and reach the cytosol within less than three seconds—much faster than previously considered. Final cytosolic AMP concentrations of ~ 100 mM suggest an efficient shut-down of metabolism as primary cause for bacterial killing. On the other hand, the damage of the cell envelope is a collateral effect of AMP activity that does not kill the bacteria. This implies that the impairment of the membrane barrier is a necessary but not sufficient condition for microbial killing by lactoferricins. The most efficient AMP studied exceeds others in both speed of reaching cytoplasm and lowest cytosolic peptide concentration.

Abstract 2: Acute Stroke Care During The Covid-19 Pandemic: The Society Of Vascular And Interventional Neurology Collaboration

Background: We sought to evaluate whether the coronavirus disease 2019 (COVID-19) pandemic may have contributed to delays in acute stroke management at Comprehensive Stroke Centers (CSCs). Methods: Pooled clinical data of consecutive adult stroke patients from 12 U.S. CSCs (1/1/2019-5/31/2020) were queried. The rate of thrombolysis for non-transferred patients within the Target: Stroke goal of 60min was compared between patients admitted 3/1/2019-5/31/2019 (pre-COVID-19) and 3/1/2020-5/31/2020 (COVID-19). The time from arrival to imaging and treatment with thrombolysis or thrombectomy, as continuous variables, were also assessed. Results: Of the 7906 patients included, 1319 were admitted pre-COVID-19 and 933 were admitted during COVID-19, 15% of whom underwent intravenous thrombolysis. There was no difference in the rate of thrombolysis within 60min during COVID-19 (OR 0.88, 95%CI 0.42-1.86, p=0.74), despite adjustment for variables associated with earlier treatment (adjusted OR 0.82, 95%CI 0.38-1.76, p=0.61). There was no significant overall delay to thrombolysis during the COVID-19 period vs. pe-COVID-19 (p=0.42), even after multivariable adjustment (p=0.63) or after comparison across months leading to COVID-19 (Figure). The only independent predictor of delayed treatment time between periods was the use of emergency medical services (adjusted β=-6.93, 95%CI -12.83 - -1.04, p=0.03). There was no significant delay from hospital arrival to imaging in all patients, or imaging to skin puncture in patients who underwent thrombectomy. Conclusions: There was no independent effect of the COVID-19 period on delays in acute care with respect to thrombolysis or thrombectomy in this multicenter observational cohort. Further studies are warranted to externally validate these findings, and determine if site volume or center accreditation may mediate a collateral effect of the pandemic on stroke care paradigms.

Analysing Touchscreen Gestures: A Study Based on Individuals with Down Syndrome Centred on Design for All

There has been a conscious shift towards developing increasingly inclusive applications. However, despite this fact, most research has focused on supporting those with visual or hearing impairments and less attention has been paid to cognitive impairments. The purpose of this study is to analyse touch gestures used for touchscreens and identify which gestures are suitable for individuals living with Down syndrome (DS) or other forms of physical or cognitive impairments. With this information, app developers can satisfy Design for All (DfA) requirements by selecting adequate gestures from existing lists of gesture sets. Twenty touch gestures were defined for this study and a sample group containing eighteen individuals with Down syndrome was used. A tool was developed to measure the performance of touch gestures and participants were asked to perform simple tasks that involved the repeated use of these twenty gestures. Three variables are analysed to establish whether they influence the success rates or completion times of gestures, as they could have a collateral effect on the skill with which gestures are performed. These variables are Gender, Type of Down syndrome, and Socioeconomic Status. Analysis reveals that significant difference is present when a pairwise comparison is performed, meaning individuals with DS cannot perform all gestures with the same ease. The variables Gender and Socioeconomic Status do not influence success rates or completion times, but Type of DS does.

Diagnostic utility of Sudoscan for detecting bortezomib-induced painful neuropathy: a study on 18 patients with multiple myeloma

IntroductionIn the past few years, treatment of multiple myeloma has undergone a deep change for the employment of novel treatment comprising proteasome inhibitors. Bortezomib is a first-line drug in therapy of multiple myeloma. The onset of peripheral neuropathy is a dose-limiting collateral effect of the drug. This neuropathy is a distal symmetric neuropathy that affects both large and small fibers. Nerve conduction study (NCS) can be used for the diagnosis of bortezomib neuropathy, but this technique demonstrates alterations of the large nerve fibers. Sudoscan is a novel technique utilized to offer an evaluation of sudomotor function. The main objective of this study was to compare the sensitivity and diagnostic specificity of Sudoscan with respect to the nerve conduction study after bortezomib treatment.Material and methodsA total of 18 multiple myeloma patients were studied, 10 (55.5%) men and 8 (44.5%) women. Patients were analyzed at baseline and after 6 months of treatment with bortezomib. Subjects were submitted to nerve conduction study and electrochemical skin conductance evaluation with the Sudoscan device. Patients were also submitted to a clinical measure of pain and neuropathy.ResultsAt baseline NCS showed that only the mean sural SAP amplitude was below the 2SD lower limit of normal in 3 (16.7%) patients, while at same time we found an alteration of Sudoscan profiles in 2 (11.1%) patients. After 6 months of treatment, the NCS profiles were altered in 13 (72.2%) patients, and the Sudoscan profiles were modified in 11 (61.1%) subjects.ConclusionsOur results suggest that Sudoscan can be considered for the diagnosis of bortezomib-induced neuropathy. It is objective, reproducible, and surely easier than the traditional nerve conduction study. Sudoscan may be a useful help to manage the therapeutic interventions in multiple myeloma.

A Rising Tide Drowns Unstable Boats: How Inequality Creates Homelessness

Is income inequality a driver of homelessness at the community level? We theorize that inequality affects homelessness both by crowding out low-income households from the rental market (what we call an “income channel”) and by causing home prices to rise (a “price channel”). We construct a dataset of information on inequality, homelessness, rent burden, and housing prices in 239 communities from 2007 to 2018 and use it to assess the income inequality–homelessness relationship. Our results suggest that income inequality is a significant driver of community homelessness and that the “income channel” is the more likely mechanism through which homelessness is created. We argue that broader policy efforts to reduce income inequality are likely to have the collateral effect of reducing homelessness, and we discuss the need for national and local policies to help low-income households afford housing.