A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy

CAMELLIA SINENSIS VAR. MADOENSIS (SECT. THEA, THEACEAE), A NEW TAXON FROM VIETNAM

Camellia sinensis var. madoensis is described and illustrated as a new variety of Camellia sinensis (section Thea, Theaceae) from Xuan Loc Commune, Song Cau District, Phu Yen Province. The new variety is easily distinguishable from C. sinensis var. sinensis by style free ½ to the base. The ITS sequence of this variety is also different from that of Camellia sinensis and its other varieties, while the matK gene sequences are nearly identical among Camellia taxa.Camellia sinensis var. madoensis được mô tả và minh họa với vai trò là một thứ mới của Camellia sinensis (section Thea, Theaceae) ghi nhận tại xã Xuân Lộc, huyện Sông Cầu, tỉnh Phú Yên. Thứ mới này có thể dễ dàng phân biệt với C. sinensis var. sinensis bởi vòi nhụy rời ½ tính từ đế. Trình tự ITS của thứ này cũng khác với Camellia sinensis và các thứ khác của nó

Quantitative antisense screening and optimization for exon 51 skipping in Duchenne muscular dystrophy

International audienceDuchenne muscular dystrophy (DMD), the most common lethal genetic disorder, is caused by mutations in the dystrophin (DMD) gene. Exon skipping is a therapeutic approach that uses antisense oligonucleotides (AOs) to modulate splicing and restore the reading frame, leading to truncated, yet functional protein expression. In 2016, the US Food and Drug Administration (FDA) conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based AO drug, eteplirsen, developed for DMD exon 51 skipping. Eteplirsen remains controversial with insufficient evidence of its therapeutic effect in patients. We recently developed an in silico tool to design antisense morpholino sequences for exon skipping. Here, we designed morpholino AOs targeting DMD exon 51 using the in silico tool and quantitatively evaluated the effects in immortalized DMD muscle cells in vitro. To our surprise, most of the newly designed morpholinos induced exon 51 skipping more efficiently compared with the eteplirsen sequence. The efficacy of exon 51 skipping and rescue of dystrophin protein expression were increased by up to more than 12-fold and 7-fold, respectively, compared with the eteplirsen sequence. Significant in vivo efficacy of the most effective morpholino, determined in vitro, was confirmed in mice carrying the human DMD gene. These findings underscore the importance of AO sequence optimization for exon skipping

CAMELLIA SINENSIS VAR. MADOENSIS (SECT. THEA, THEACEAE), A NEW TAXON FROM VIETNAM

Camellia sinensis var. madoensis is described and illustrated as a new variety of Camellia sinensis (section Thea, Theaceae) from Xuan Loc Commune, Song Cau District, Phu Yen Province. The new variety is easily distinguishable from C. sinensis var. sinensis by style free ½ to the base. The ITS sequence of this variety is also different from that of Camellia sinensis and its other varieties, while the matK gene sequences are nearly identical among Camellia taxa

Effectiveness of cervical pessary compared to cervical cerclage with or without vaginal progesterone for the prevention of preterm birth in women with twin pregnancies and a short cervix : Study protocol for a two-by-two factorial randomised clinical trial

Peer reviewedPublisher PD

Evaluation of awake prone positioning effectiveness in moderate to severe COVID-19

Evidence mainly from high income countries suggests that lying in the prone position may be beneficial in patients with COVID-19 even if they are not receiving invasive ventilation. Studies indicate that increased duration of prone position may be associated with improved outcomes, but achieving this requires additional staff time and resources. Our study aims to support prolonged (≥ 8hours/day) awake prone positioning in patients with moderate to severe COVID-19 disease in Vietnam. We use a specialist team to support prone positioning of patients and wearable devices to assist monitoring vital signs and prone position and an electronic data registry to capture routine clinical data

An open label randomized controlled trial of tamoxifen combined with amphotericin B and fluconazole for cryptococcal meningitis

Background: Cryptococcal meningitis has high mortality. Flucytosine is a key treatment but is expensive and rarely available. The anti-cancer agent tamoxifen has synergistic anti-cryptococcal activity with amphotericin in vitro. It is off-patent, cheap, and widely available. We performed a trial to determine its therapeutic potential. Methods:Open label randomized controlled trial. Participants received standard care - amphotericin combined with fluconazole for the first two weeks - or standard care plus tamoxifen 300mg/day. The primary end point was Early Fungicidal Activity (EFA) - the rate of yeast clearance from cerebrospinal fluid (CSF). Trial registration https://clinicaltrials.gov/ct2/show/NCT03112031 . Results: 50 patients were enrolled, (median age 34 years, 35 male). Tamoxifen had no effect on EFA (- 0.48log10 colony-forming units/mL/CSF control arm versus -0.49 tamoxifen arm, difference - 0.005log10CFU/ml/day, 95%CI: -0.16, 0.15, P=0.95). Tamoxifen caused QTc prolongation. Conclusion: High dose tamoxifen does not increase the clearance rate of Cryptococcus from CSF. Novel, affordable therapies are needed. Funding:The trial was funded through the Wellcome Trust Asia Programme Vietnam Core Grant 106680 and a Wellcome Trust Intermediate Fellowship to JND grant number WT097147MA

Distinct Peripheral Blood RNA Responses to Salmonella in Pigs Differing in Salmonella Shedding Levels: Intersection of IFNG, TLR and miRNA Pathways

Transcriptomic analysis of the response to bacterial pathogens has been reported for several species, yet few studies have investigated the transcriptional differences in whole blood in subjects that differ in their disease response phenotypes. Salmonella species infect many vertebrate species, and pigs colonized with Salmonella enterica serovar Typhimurium (ST) are usually asymptomatic, making detection of these Salmonella-carrier pigs difficult. The variable fecal shedding of Salmonella is an important cause of foodborne illness and zoonotic disease. To investigate gene pathways and biomarkers associated with the variance in Salmonella shedding following experimental inoculation, we initiated the first analysis of the whole blood transcriptional response induced by Salmonella. A population of pigs (n = 40) was inoculated with ST and peripheral blood and fecal Salmonella counts were collected between 2 and 20 days post-inoculation (dpi). Two groups of pigs with either low shedding (LS) or persistent shedding (PS) phenotypes were identified. Global transcriptional changes in response to ST inoculation were identified by Affymetrix Genechip® analysis of peripheral blood RNA at day 0 and 2 dpi. ST inoculation triggered substantial gene expression changes in the pigs and there was differential expression of many genes between LS and PS pigs. Analysis of the differential profiles of gene expression within and between PS and LS phenotypic classes identified distinct regulatory pathways mediated by IFN-γ, TNF, NF-κB, or one of several miRNAs. We confirmed the activation of two regulatory factors, SPI1 and CEBPB, and demonstrated that expression of miR-155 was decreased specifically in the PS animals. These data provide insight into specific pathways associated with extremes in Salmonella fecal shedding that can be targeted for further exploration on why some animals develop a carrier state. This knowledge can also be used to develop rational manipulations of genetics, pharmaceuticals, nutrition or husbandry methods to decrease Salmonella colonization, shedding and spread