Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR-Cas9 gene editing

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. The 10th most common mutation, c.3178-2477C>T (3849+10kb C>T), involves a cryptic, intronic splice site. This mutation was corrected in CF primary cells homozygous for this mutation by delivering pairs of guide RNAs (gRNAs) with Cas9 protein in ribonucleoprotein (RNP) complexes that introduce double-strand breaks to flanking sites to excise the 3849+10kb C>T mutation, followed by DNA repair by the non-homologous end-joining pathway, which functions in all cells of the airway epithelium. RNP complexes were delivered to CF basal epithelial cell by a non-viral, receptor-targeted nanocomplex comprising a formulation of targeting peptides and lipids. Canonical CFTR mRNA splicing was, thus, restored leading to the restoration of CFTR protein expression with concomitant restoration of electrophysiological function in airway epithelial air-liquid interface cultures. Off-target editing was not detected by Sanger sequencing of in silico-selected genomic sites with the highest sequence similarities to the gRNAs, although more sensitive unbiased whole genome sequencing methods would be required for possible translational developments. This approach could potentially be used to correct aberrant splicing signals in several other CF mutations and other genetic disorders where deep-intronic mutations are pathogenic

The Grizzly, January 28, 1991

Policies and Statistics, A Security Concern: New Quad Regulations; Crime Report Released • Sonia Sanchez: Poet for Peace • Gulf Dialogue Continues • Independence Dogs • Financial Aid Month • You Asked for it: You Got It!! • New Quad Policy • Presidency Symposium • When is Dr. Martin Luther King, Jr.\u27s Birthday? • Who\u27s Who Announced • Mission Conference Held • Back to the Basics • The Innocent • Swimmers on Win Streak • Women Hope for Good Things to Come • A Roller Coaster Season for the Hoopsters • Women Rounding-out Season • The Dream Lives On • Letter: Bundle Up! • Researchers Psyched Out • The Technology of War • A Wasted Solution?https://digitalcommons.ursinus.edu/grizzlynews/1268/thumbnail.jp

The transport and fate of microplastic fibres in the Antarctic: The role of multiple global processes

Understanding the transport and accumulation of microplastics is useful to determine the relative risk they pose to global biodiversity. The exact contribution of microplastic sources is hard to elucidate; therefore, investigating the Antarctic Weddell Sea, an area known for its remoteness and little human presence (i.e. limited pollution sources), will help us to better understand microplastic transportation. Here, we investigate the presence of microplastics in a range of Antarctic sample media including air, seawater, and sediment. We hypothesised that multiple transportation processes including atmospheric and oceanic vectors determine the presence of microplastics in the Antarctic. Using techniques including Polarised Light Microscopy and Raman Spectrometry, we identified mostly fibres and categorised them based on their optical and chemical properties. A total of 47 individual microplastic categories (45 of which were fibres) were identified in the air, seawater, and sediment samples. The majority of categories did not overlap multiple media (42/47); however, four fibre categories were present in both air and water samples, and another fibre category was found in all three media (category 27). We suggest that the large variety of fibres identified and the overlap of fibre categories among media indicates that the pollution may result from multiple diffuse sources and transportation pathways. Additionally, our Air Mass Back Trajectory analyses demonstrates that microplastic fibres are being transported by air masses or wind, and strongly suggests that they are transported to the Antarctic from southern South America. We also propose that fibres may be transported into the Antarctic in subsurface waters, and as pollution was identified in our sediment and additional sea ice samples, we suggest that the coastal and Antarctic deep sea may be a sink for microplastic fibres. The results shown here from a remote, near-pristine system, further highlight the need for a global response to the plastic pollution crisis

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension

Randomized controlled trial of molnupiravir SARS-CoV-2 viral and antibody response in at-risk adult outpatients

Viral clearance, antibody response and the mutagenic effect of molnupiravir has not 77 been elucidated in at-risk populations. Non-hospitalised participants within 5 days of 78 SARS-CoV-2 symptoms randomised to receive molnupiravir (n=253) or Usual Care 79 (n=324) were recruited to study viral and antibody dynamics and the effect of molnupiravir on viral whole genome sequence from 1437 viral genomes. Molnupiravir accelerates viral load decline, but virus is detectable by Day 5 in most cases. At Day 14 (9 days post-treatment), molnupiravir is associated with significantly higher viral persistence and significantly lower anti-SARS-CoV-2 spike antibody titres compared to Usual Care. Serial sequencing reveals increased mutagenesis with molnupiravir treatment. Persistence of detectable viral RNA at Day 14 in the molnupiravir group is associated with higher transition mutations following treatment cessation. Viral viability at Day 14 is similar in both groups with post-molnupiravir treated samples cultured up to 9 days post cessation of treatment. The current 5-day molnupiravir course is too short. Longer courses should be tested to reduce the risk of potentially transmissible molnupiravir-mutated variants being generated

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

Background: Many patients with COVID-19 have been treated with plasma containing anti-SARS-CoV-2 antibodies. We aimed to evaluate the safety and efficacy of convalescent plasma therapy in patients admitted to hospital with COVID-19. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]) is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. The trial is underway at 177 NHS hospitals from across the UK. Eligible and consenting patients were randomly assigned (1:1) to receive either usual care alone (usual care group) or usual care plus high-titre convalescent plasma (convalescent plasma group). The primary outcome was 28-day mortality, analysed on an intention-to-treat basis. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936. Findings: Between May 28, 2020, and Jan 15, 2021, 11558 (71%) of 16287 patients enrolled in RECOVERY were eligible to receive convalescent plasma and were assigned to either the convalescent plasma group or the usual care group. There was no significant difference in 28-day mortality between the two groups: 1399 (24%) of 5795 patients in the convalescent plasma group and 1408 (24%) of 5763 patients in the usual care group died within 28 days (rate ratio 1·00, 95% CI 0·93–1·07; p=0·95). The 28-day mortality rate ratio was similar in all prespecified subgroups of patients, including in those patients without detectable SARS-CoV-2 antibodies at randomisation. Allocation to convalescent plasma had no significant effect on the proportion of patients discharged from hospital within 28 days (3832 [66%] patients in the convalescent plasma group vs 3822 [66%] patients in the usual care group; rate ratio 0·99, 95% CI 0·94–1·03; p=0·57). Among those not on invasive mechanical ventilation at randomisation, there was no significant difference in the proportion of patients meeting the composite endpoint of progression to invasive mechanical ventilation or death (1568 [29%] of 5493 patients in the convalescent plasma group vs 1568 [29%] of 5448 patients in the usual care group; rate ratio 0·99, 95% CI 0·93–1·05; p=0·79). Interpretation: In patients hospitalised with COVID-19, high-titre convalescent plasma did not improve survival or other prespecified clinical outcomes. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research

Disparities in Mortality Trends for Infants of Teenagers: 1996 to 2019.

BACKGROUND AND OBJECTIVES: Although mortality rates are highest for infants of teens aged 15 to 19, no studies have examined the long-term trends by race and ethnicity, urbanicity, or maternal age. The objectives of this study were to examine trends and differences in mortality for infants of teens by race and ethnicity and urbanicity from 1996 to 2019 and estimate the contribution of changes in the maternal age distribution and maternal age-specific (infant) mortality rates (ASMRs) to differences in infant deaths in 1996 and 2019. METHODS: We used 1996 to 2019 period-linked birth and infant death data from the United States to assess biennial mortality rates per 1000 live births. Pairwise comparisons of rates were conducted using z test statistics and Joinpoint Regression was used to examine trends. Kitagawa decomposition analysis was used to estimate the proportion of change in infant deaths because of changes in the maternal age distribution and ASMRs. RESULTS: From 1996 to 2019, the mortality rate for infants of teens declined 16.7%, from 10.30 deaths per 1000 live births to 8.58. The decline was significant across racial and ethnic and urbanization subgroups; however, within rural counties, mortality rates did not change significantly for infants of Black or Hispanic teens. Changes in ASMRs accounted for 93.3% of the difference between 1996 and 2019 infant mortality rates, whereas changes in the maternal age distribution accounted for 6.7%. CONCLUSIONS: Additional research into the contextual factors in rural counties that are driving the lack of progress for infants of Black and Hispanic teens may help inform efforts to advance health equity

When a tree falls: Controls on wood decay predict standing dead tree fall and new risks in changing forests.

When standing dead trees (snags) fall, they have major impacts on forest ecosystems. Snag fall can redistribute wildlife habitat and impact public safety, while governing important carbon (C) cycle consequences of tree mortality because ground contact accelerates C emissions during deadwood decay. Managing the consequences of altered snag dynamics in changing forests requires predicting when snags fall as wood decay erodes mechanical resistance to breaking forces. Previous studies have pointed to common predictors, such as stem size, degree of decay and species identity, but few have assessed the relative strength of underlying mechanisms driving snag fall across biomes. Here, we analyze nearly 100,000 repeated snag observations from boreal to subtropical forests across the eastern United States to show that wood decay controls snag fall in ways that could generate previously unrecognized forest-climate feedback. Warmer locations where wood decays quickly had much faster rates of snag fall. The effect of temperature on snag fall was so strong that in a simple forest C model, anticipated warming by mid-century reduced snag C by 22%. Furthermore, species-level differences in wood decay resistance (durability) accurately predicted the timing of snag fall. Differences in half-life for standing dead trees were similar to expected differences in the service lifetimes of wooden structures built from their timber. Strong effects of temperature and wood durability imply future forests where dying trees fall and decay faster than at present, reducing terrestrial C storage and snag-dependent wildlife habitat. These results can improve the representation of forest C cycling and assist forest managers by helping predict when a dead tree may fall

Estimates of the reproduction numbers of Spanish influenza using morbidity data.

BACKGROUND: There have been several studies of the transmissibility of the 1918 (Spanish) influenza virus, which has attributed to >20 million deaths. Many of the analyses to date have involved fitting predictions from a transmission model to the observed epidemic curves from different settings. METHODS: Using morbidity data from cities in Europe and America and from confined settings during the 1918 influenza pandemic, we contrast the use of several different methods based on the growth rate and final size of the epidemic, which do not rely on transmission models, to estimate the effective and basic reproduction numbers. RESULTS: The effective reproduction number (the average number of secondary infectious cases produced by a typical infectious case in a given population) for the 1918 influenza virus was in the range 1.2-3.0 and 2.1-7.5 for community-based and confined settings, respectively. CONCLUSIONS: Assuming further that 30 and 50% of individuals were immune to Spanish influenza after the wave in April 1918 and the first subsequent wave, respectively, these findings imply that, in a totally susceptible population, an infectious case could have led to 2.4-4.3 and 2.6-10.6 cases in community-based and confined settings, respectively. These findings for community-based populations confirm the relatively low transmissibility of the 1918 (Spanish) influenza virus, which has been found by other studies using alternative data sources and methods