Background Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).Methodology/Principal FindingsRobust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200–320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.Conclusions/SignificanceIn spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection.

Background and objectiveThe coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents multiple, diverse challenges to providing appropriate medical care, especially in terms of medication and treatment adherence for chronic diseases such as type 2 diabetes mellitus (T2DM). The COVID-19 pandemic has exacerbated these barriers by potentially forcing physicians to modify their treatment plans due to limitations on in-person visits and changes to patients' financial and social support systems. It remains uncertain whether physicians believe they can provide the same standard of care using telehealth technology or other means to their patients during the pandemic. The goal of this study was to explore physician perceptions about their ability to provide care to patients with T2DM during the COVID-19 pandemic. MethodologyThis cross-sectional study collected data between January 25, 2021, and February 2, 2021, using an anonymous, self-administered online survey involving DO and MD physicians including residents treating patients with T2DM. The survey was administered via REDCap and collected data on participant demographics, attitudes, perceptions, knowledge, and prior and current (COVID-19-era) experience with care for T2DM patients. Physicians registered with the Florida Department of Health with publicly available emails were invited to participate. ResultsThe survey showed that during the COVID-19 pandemic, 57.9% of physicians (n=48) believed that their patients have a weaker social support system; 68.7% (n=57) modified their patient care plans due to patients' financial difficulties; 78.4% (n=65) believed a regular physical exam is necessary to properly treat patients; 48.2% (n=40) did not believe they had a more complete picture of the case with remote consultations; 47.0% (n=39) were not as satisfied with remote consultations as with face-to-face patient visits; 68.7% (n=57) believed telehealth is necessary to adequately treat patients; 38.5% (n=32) have been less likely to refer their patients to other providers or specialists; 45.8% (n=38) reported concerns over admitting their patients to the hospital for acute medical care; 61.5% (n=51) reported having more patients delay scheduling their routine follow-up care; 61.5% (n=51) believed their patients have been less compliant with the healthcare plans recommended to them. ConclusionsThe study showed that COVID-19 has significantly impacted physicians' perceptions and abilities to provide care for patients with T2DM. COVID-19 has negatively impacted several crucial aspects of diabetes management, including consistent in-person examinations, social support, and referral to other required services, which could result in long-term consequences for these patients. Furthermore, our study suggests that physicians may not be as satisfied with the care they are able to provide via remote consultations as they are with in-person visits, which has significant implications as we move toward a more teleheal...

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