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Genomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish, Amphiprion ocellaris . Microinjection of zygotes was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene ( RH2B ) involved in vision, and Tyrosinase-producing gene ( tyr ) involved in the production of melanin. Analysis of the sequenced target gene regions in A . ocellaris embryos showed that uptake was as high as 73.3% of injected embryos. Further analysis of the subcloned mutant gene sequences combined with amplicon shotgun sequencing revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in F0 A . ocellaris embryos. Moreover, we clearly show a loss-of-function in tyr mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful starting point to further explore the potential application of CRISPR/Cas9 in A . ocellaris , as a platform for studying gene function in anemonefishes and other reef fishes.

The coconut rhinoceros beetle (CRB) is an important pest of palms in the Asia/Pacific regions. A natural pathogen of CRB, Oryctes rhinoceros nudivirus (OrNV) has successfully been introduced into virus-free, invasive CRB populations in many Pacific islands leading to pest suppression. While PCR is currently used for virus monitoring, a more portable, simplified in-field method of virus detection would be helpful. Here, we developed a Recombinase Polymerase Amplification (RPA)-based assay to detect the partial gp54 gene of OrNV (OrNV-RPA) using fluorescence-based chemistry. The assay has a high degree of specificity with no cross-reactivity to the genomic DNA of various insects, soil, and bacterial species commonly found in the environment. Additionally, the assay has a high level of sensitivity with the ability to detect approximately 1610 copies of the gp54 gene (15 min reaction). OrNV-RPA assay results showed high consistency with the current standard PCR-based assay, whether using purified DNA (95.4%) or crude lysate (93.5%) from CRB midgut tissue, validating the ability of the OrNV-RPA assay to detect virus-free and virus-infected CRB specimens. The newly developed RPA-based assay provides an option for rapid in-field detection of OrNV to help improve CRB management in the Pacific Islands and other regions.

Lipid nanoparticles (LNPs) are the preeminent non-viral drug delivery vehicle for mRNA-based therapies. Immense effort has been placed on optimizing the ionizable lipid (IL) structure, which contains an amine core conjugated to lipid tails, as small molecular adjustments can result in substantial changes in the overall efficacy of the resulting LNPs. However, despite some advancements, a major barrier for LNP delivery is endosomal escape. Here, we develop a platform for synthesizing a class of branched ILs that improve endosomal escape. These compounds incorporate terminally branched groups that increase hepatic mRNA and ribonucleoprotein complex delivery and gene editing efficiency as well as T cell transfection compared to non-branched lipids. Through an array of complementary experiments, we determine that our lipid architecture induces greater endosomal penetration and disruption. This work provides a scheme to generate a class of ILs for both mRNA and protein delivery. Lipid nanoparticles (LNPs) are the preeminent drug delivery vehicle for mRNA therapies, partially due to the ionizable lipid (IL) components that facilitate endosomal escape. Here, authors devise terminally branched ILs that enhance endosome escape, resulting in increased liver and T cell delivery.

This study evaluates the efficacy of capecitabine using data from a large, well-characterised population of patients with metastatic colorectal cancer (mCRC) treated in two identically designed phase III studies. A total of 1207 patients with previously untreated mCRC were randomised to either oral capecitabine (1250 mg m −2 twice daily, days 1−14 every 21 days; n =603) or intravenous (i.v.) bolus 5-fluorouracil/leucovorin (5-FU/LV; Mayo Clinic regimen; n =604). Capecitabine demonstrated a statistically significant superior response rate compared with 5-FU/LV (26 vs 17%; P <0.0002). Subgroup analysis demonstrated that capecitabine consistently resulted in superior response rates ( P <0.05), even in patient subgroups with poor prognostic indicators. The median time to response and duration of response were similar and time to progression (TTP) was equivalent in the two arms (hazard ratio (HR) 0.997, 95% confidence interval (CI) 0.885–1.123, P =0.95; median 4.6 vs 4.7 months with capecitabine and 5-FU/LV, respectively). Multivariate Cox regression analysis identified younger age, liver metastases, multiple metastases and poor Karnofsky Performance Status as independent prognostic indicators for poor TTP. Overall survival was equivalent in the two arms (HR 0.95, 95% CI 0.84–1.06, P =0.48; median 12.9 vs 12.8 months, respectively). Capecitabine results in superior response rate, equivalent TTP and overall survival, an improved safety profile and improved convenience compared with i.v. 5-FU/LV as first-line treatment for MCRC. For patients in whom fluoropyrimidine monotherapy is indicated, capecitabine should be strongly considered. Following encouraging results from phase I and II trials, randomised trials are evaluating capecitabine in combination with irinotecan, oxaliplatin and radiotherapy. Capecitabine is a suitable replacement for i.v. 5-FU as the backbone of colorectal cancer therapy.

[This corrects the article DOI: 10.1371/journal.pone.0261331.].

A randomized trial evaluating spinal as compared with general anesthesia for hip-fracture surgery in adults 50 years of age or older did not show superiority of spinal anesthesia with respect to a composite of death or an inability to walk unassisted at 60 days. Postoperative delirium occurred in similar percentages of patients in the two groups.

Camera trapping for detecting wildlife is increasingly used as a primary method of non‐invasive wildlife monitoring. Yet understanding among researchers and conservationists on how camera trap make, and model affect detection rates is limited. Published studies often fail to make clear why a given camera trap model was chosen or what specifications or parameters were used to capture target species within a given study area, prohibiting replicability. Here we present a comparison of predator and herbivore detection efficacy using three makes and models of camera trap at differing price ranges, year of release (hereafter vintages) and specifications. We used a passive monitoring survey design at six sites in open field conditions across the Flow Country, Northern Scotland. Detection efficacy varied substantially between grades and vintages of camera traps and depended on species captured. Older models of camera with lower trigger speed and night vision range performed particularly poorly for nocturnal predatory mammal detection. This has implications for how researchers, conservationists, developers and other users approach experimental design and analyses, but also on the conclusions that may be drawn from studies. We caution against using the results of one or more camera trap studies using different makes and models of cameras to inform experimental design or policy interventions. Top left: proportion of deer of total deer images taken in light and darkness for each camera trap type. Top right and bottom images: The same deer event as taken by each camera. Notice differences in FOV and image quality/range.

Chimeric antigen receptor (CAR) cell therapy represents a hallmark in cancer immunotherapy, with significant clinical results in the treatment of hematological tumors. However, current approved methods to engineer T cells to express CAR use viral vectors, which are integrative and have been associated with severe adverse effects due to constitutive expression of CAR. In this context, non-viral vectors such as ionizable lipid nanoparticles (LNPs) arise as an alternative to engineer CAR T cells with transient expression of CAR.IntroductionChimeric antigen receptor (CAR) cell therapy represents a hallmark in cancer immunotherapy, with significant clinical results in the treatment of hematological tumors. However, current approved methods to engineer T cells to express CAR use viral vectors, which are integrative and have been associated with severe adverse effects due to constitutive expression of CAR. In this context, non-viral vectors such as ionizable lipid nanoparticles (LNPs) arise as an alternative to engineer CAR T cells with transient expression of CAR.Here, we formulated a mini-library of LNPs to deliver pDNA to T cells by varying the molar ratios of excipient lipids in each formulation. LNPs were characterized and screened in vitro using a T cell line (Jurkat). The optimized formulation was used ex vivo to engineer T cells derived from human peripheral blood mononuclear cells (PBMCs) for the expression of an anti-CD19 CAR (CAR-CD19BBz). The effectiveness of these CAR T cells was assessed in vitro against Raji (CD19+) cells.MethodsHere, we formulated a mini-library of LNPs to deliver pDNA to T cells by varying the molar ratios of excipient lipids in each formulation. LNPs were characterized and screened in vitro using a T cell line (Jurkat). The optimized formulation was used ex vivo to engineer T cells derived from human peripheral blood mononuclear cells (PBMCs) for the expression of an anti-CD19 CAR (CAR-CD19BBz). The effectiveness of these CAR T cells was assessed in vitro against Raji (CD19+) cells.LNPs formulated with different molar ratios of excipient lipids efficiently delivered pDNA to Jurkat cells with low cytotoxicity compared to conventional transfection methods, such as electroporation and lipofectamine. We show that CAR-CD19BBz expression in T cells was transient after transfection with LNPs. Jurkat cells transfected with our top-performing LNPs underwent activation when exposed to CD19+ target cells. Using our top-performing LNP-9-CAR, we were able to engineer human primary T cells to express CAR-CD19BBz, which elicited significant specific killing of CD19+ target cells in vitro.ResultsLNPs formulated with different molar ratios of excipient lipids efficiently delivered pDNA to Jurkat cells with low cytotoxicity compared to conventional transfection methods, such as electroporation and lipofectamine. We show that CAR-CD19BBz expression in T cells was transient after transfection with LNPs. Jurkat cells transfected with our top-performing LNPs underwent activation when exposed to CD19+ target cells. Using our top-performing LNP-9-CAR, we were able to engineer human primary T cells to express CAR-CD19BBz, which elicited significant specific killing of CD19+ target cells in vitro.Collectively, our results show that LNP-mediated delivery of pDNA is a suitable method to engineer human T cells to express CAR, which holds promise for improving the production methods and broader application of this therapy in the future.ConclusionCollectively, our results show that LNP-mediated delivery of pDNA is a suitable method to engineer human T cells to express CAR, which holds promise for improving the production methods and broader application of this therapy in the future.

IntroductionPatients with stroke-like symptoms may be underutilising emergency medical services and avoiding hospitalisation during the COVID-19 pandemic. We investigated a decline in admissions for stroke and transient ischaemic attack (TIA) and emergency department (ED) stroke alert activations.MethodsWe retrospectively compiled total weekly hospital admissions for stroke and TIA between 31 December 2018 and 21 April 2019 versus 30 December 2019 and 19 April 2020 at five US tertiary academic comprehensive stroke centres in cities with early COVID-19 outbreaks in Boston, New York City, Providence and Seattle. We collected available data on ED stroke alerts, stroke severity using the National Institutes of Health Stroke Scale (NIHSS) and time from symptom onset to hospital arrival.ResultsCompared with 31 December 2018 to 21 April 2019, a decline in stroke/TIA admissions and ED stroke alerts occurred during 30 December 2019 to 19 April 2020 (p trend <0.001 for each). The declines coincided with state stay-at-home recommendations in late March. The greatest decline in hospital admissions was observed between 23 March and 19 April 2020, with a 31% decline compared with the corresponding weeks in 2019. Three of the five centres with 2019 and 2020 stroke alert data had a 46% decline in ED stroke alerts in late March and April 2020, compared with 2019. Median baseline NIHSS during these 4 weeks was 10 in 2020 and 7 in 2019. There was no difference in time from symptom onset to hospital arrival.ConclusionAt these five large academic US hospitals, admissions for stroke and TIA declined during the COVID-19 pandemic. There was a trend for fewer ED stroke alerts at three of the five centres with available 2019 and 2020 data. Acute stroke therapies are time-sensitive, so decreased healthcare access or utilisation may lead to more disabling or fatal strokes, or more severe non-neurological complications related to stroke. Our findings underscore the indirect effects of this pandemic. Public health officials, hospital systems and healthcare providers must continue to encourage patients with stroke to seek acute care during this crisis.