Treatment of lichen sclerosus with hydroxychloroquine: a Mayo Clinic experience.

BACKGROUND: There is a dearth of studies investigating the efficacy of hydroxychloroquine in the treatment of either anogenital lichen sclerosus or extragenital lichen sclerosus, a condition that, if left untreated, could lead to a greater degree of scarring and malignant transformation. OBJECTIVE: This study aims to analyze the demographic characteristics, clinicopathological features, treatment response, and outcomes of patients diagnosed with either anogenital or extragenital lichen sclerosus who received hydroxychloroquine therapy. METHODS: A retrospective analysis was conducted involving 70 patients diagnosed with lichen sclerosus who underwent treatment with hydroxychloroquine at our institution between 2018 and 2023. RESULTS: Among the cohort, 67 patients were female, and 3 were male. Extragenital lichen sclerosus was diagnosed in 23 patients, with 16 exhibiting concomitant morphea overlap. Itching was the predominant clinical presentation (67%). A notable proportion of patients (36%) had a connective tissue disorder, prompting hydroxychloroquine therapy. Among the 30 patients treated solely for lichen sclerosus, 21 demonstrated response and 9 had no response. From a broader comparison of response to hydroxychloroquine, the overall anogenital response rate was 84.6% as opposed to 50% in extragenital lichen sclerosus. The median time to initial response was 4 months. Adverse effects, predominantly mild, were observed in 10 (14.3%) patients. LIMITATION: This study is constrained by its retrospective nature and reliance on data from a single center, resulting in a limited sample size. CONCLUSION: Hydroxychloroquine demonstrates promise as a therapeutic option for anogenital lichen sclerosus because of its favorable response rates and low incidence of adverse effects. However, further investigations, including larger-scale or prospective studies, are imperative to ascertain its definitive efficacy.

Tissue of origin detection for cancer tumor using low-depth cfDNA samples through combination of tumor-specific methylation atlas and genome-wide methylation density in graph convolutional neural networks.

BACKGROUND: Cell free DNA (cfDNA)-based assays hold great potential in detecting early cancer signals yet determining the tissue-of-origin (TOO) for cancer signals remains a challenging task. Here, we investigated the contribution of a methylation atlas to TOO detection in low depth cfDNA samples. METHODS: We constructed a tumor-specific methylation atlas (TSMA) using whole-genome bisulfite sequencing (WGBS) data from five types of tumor tissues (breast, colorectal, gastric, liver and lung cancer) and paired white blood cells (WBC). TSMA was used with a non-negative least square matrix factorization (NNLS) deconvolution algorithm to identify the abundance of tumor tissue types in a WGBS sample. We showed that TSMA worked well with tumor tissue but struggled with cfDNA samples due to the overwhelming amount of WBC-derived DNA. To construct a model for TOO, we adopted the multi-modal strategy and used as inputs the combination of deconvolution scores from TSMA with other features of cfDNA. RESULTS: Our final model comprised of a graph convolutional neural network using deconvolution scores and genome-wide methylation density features, which achieved an accuracy of 69% in a held-out validation dataset of 239 low-depth cfDNA samples. CONCLUSIONS: In conclusion, we have demonstrated that our TSMA in combination with other cfDNA features can improve TOO detection in low-depth cfDNA samples.

Exploring the Therapeutic Potential of Camellia longii Orel & Luu Leaf Extracts for Memory Loss in Alzheimer's Disease: Novel Findings and Functional Food Applications.

Novel research on the chemical compositions and biochemical activities of Camellia longii Orel and Luu leaf extracts revealed valuable resources with potential applications in Alzheimer's disease treatment. Qualitative phytochemicals detected various compound groups, including polyphenols, saponins, tannins, flavonoids, alkaloids, amino acids, coumarins, and polysaccharides. HPLC-MS identified 23 compounds in C. longii leaves with compounds found at significant levels, including epicatechin gallate (17.12%), tryptophan (13.73%), isovitexin (12.91%), gallic acid (3.06%), and quercetin (3.06%). Interestingly, the ethanol extract (CLL-Ew) exhibited the highest extraction yield (26.6%) and potent antioxidant and acetylcholinesterase (AChE) inhibitory effects in vitro. In the Drosophila melanogaster model, CLL-Ew improved longevity, movement, and memory by reducing malondialdehyde and increasing glutathione levels. Docking simulations suggested that the above compounds bind tightly to AChE's active site, potentially contributing to memory enhancement. Interestingly, observations of male and female mice after administration of a dose of 5000 mg/kg C. longii leaf extract were recorded normally throughout the 14 day experiment. These findings highlight the potential of C. longii leaf extracts in functional foods and therapeutic interventions for memory impairment prevention and treatment.

Local Cutaneous Scrotal Involvement of Paratesticular Mesothelioma.

ABSTRACT: Paratesticular mesothelioma (malignant mesothelioma arising from the tunica vaginalis of the testis) represents a small proportion of mesothelial neoplasms, and cutaneous involvement by paratesticular mesothelioma is very rare. Cutaneous involvement can manifest as scrotal subcutaneous nodules from regional spread, distant metastasis, or direct extension through surgical scars. Mesothelioma has 3 histopathologic classifications that include epithelioid, biphasic, and sarcomatoid, which is rarely seen in paratesticular mesothelioma. Given the rarity of this condition, cutaneous mesothelioma may be misdiagnosed as histologic mimics, such as metastatic adenocarcinoma or adnexal neoplasms; thus, appropriate immunohistochemical workup and clinical correlation are required to make an accurate diagnosis. In this case, a 75-year-old man with a history of paratesticular mesothelioma, status postorchiectomy, presented with right-sided scrotal swelling, erythema, and subcutaneous nodules. These nodules were identified as local recurrence with cutaneous involvement by paratesticular mesothelioma on histopathologic examination. This case highlights the clinical and histopathologic features of this diagnosis and underscores the importance of dermatopathologists being aware of this condition to ensure accurate diagnosis.

Effectiveness of community-based diabetes and hypertension prevention and management programmes in Indonesia and Viet Nam: a quasi-experimental study.

INTRODUCTION: Non-communicable diseases (NCDs) have surpassed infectious diseases as the leading global cause of death, with the Southeast Asian region experiencing a significant rise in NCD prevalence over the past decades. Despite the escalating burden, screening for NCDs remains at very low levels, resulting in undetected cases, premature mortality and high public healthcare costs. We investigate whether community-based NCD prevention and management programmes are an effective solution. METHODS: In Indonesia, we compare participants in the community-based NCD screening and management programme Pos Pembinaan Terpadu-Penyakit Tidak Menular with matched non-participants with respect to their uptake of screening activities, health-related behaviour and knowledge and metabolic risk factors. We use statistical matching to redress a possible selection bias (n=1669). In Viet Nam, we compare members of Intergenerational Self-Help Clubs, which were offered similar NCD health services, with members of other community groups, where such services were not offered. We can rely on two waves of data and use a double-difference approach to redress a possible selection bias and to measure the impacts of participation (n=1710). We discuss strengths and weaknesses of the two approaches in Indonesia and Viet Nam. RESULTS: In Indonesia, participants have significantly higher uptake of screening for hypertension and diabetes (+13% from a control mean of 88% (95% CI 9% to 17%); +93% from a control mean of 48% (95% CI 79% to 108%)). In both countries, participants show a higher knowledge about risk factors, symptoms and complications of NCDs (Indonesia: +0.29 SD (0.13-0.45), Viet Nam: +0.17 SD (0.03-0.30)). Yet, the improved knowledge is only partly reflected in improved health behaviour (Viet Nam: fruit consumption +0.33 SD (0.15-0.51), vegetable consumption +0.27 SD (0.04-0.50)), body mass index (BMI) (Viet Nam: BMI -0.07 SD (-0.13 to -0.00)) or metabolic risk factors (Indonesia: systolic blood pressure: -0.13 SD (-0.26 to -0.00)). CONCLUSION: Community-based NCD programmes are well suited to increase screening and to transmit health knowledge. Due to their extensive outreach within the community, they can serve as a valuable complement to the screening services provided at the primary healthcare level. Yet, limited coverage, insufficient resources and a high staff turnover remain a problem. TRIAL REGISTRATION NUMBER: NCT05239572.

Cutaneous Collagenous Vasculopathy: A Rare Entity Treated With Pulsed Dye Laser.

Cutaneous collagenous vasculopathy (CCV) is a rare idiopathic dermal microangiopathy. Clinically, it presents as diffuse cutaneous telangiectasias that are indistinguishable from other benign vascular entities, thereby posing a diagnostic challenge. We present a case of CCV successfully treated with pulsed dye laser (PDL). A 27-year-old male presented with generalized erythematous macules, diagnosed as CCV via histopathology. After a successful test spot, PDL treatment resulted in significant improvement. The pathogenesis of CCV involves altered dermal microvasculature and veil cell activation. Epidemiologically, it primarily affects Caucasians, most often in the middle-aged adult population. A negative family history of similar lesions can help narrow down the differential diagnosis. Diagnosis requires biopsy, with histopathological examination demonstrating vessel ectasia and collagenous vessel wall thickening. Given its rarity, CCV presents diagnostic and management challenges though PDL emerges as a promising treatment modality for this condition.

Five Dimensions of Needs for Help: The Efficacy of a Technology-Based Intervention Among Asian American Breast Cancer Survivors.

Cancer survivors including Asian American breast cancer survivors have reported their high needs for help during their survivorship process. With the COVID-19 pandemic, the necessity of technology-based programs to address their needs for help without face-to-face interactions has been highlighted. The purpose of this randomized intervention study was to determine the efficacy of a technology-based program in reducing various types of needs for help among this specific population. This was a randomized clinical trial with repeated measures. A total of 199 participants were included in the data analysis. The recruitment settings included both online and offline communities/groups for Asian Americans. The needs for help were assessed using the Support Care Needs Survey-34 Short Form (SCNS) subscales measuring psychological, information, physical, support, and communication needs. Data analysis was conducted through an intent-to-treat approach. In the mixed effect models, psychological needs, information needs, physical needs, and communication needs decreased over time (P < .001). However, there were no significant group * time effects. Social support significantly mediated the effects of a technology-based intervention on psychological, information, and support needs at the pre-test and the post-1 month. This study supported significant decreases in the needs for help of Asian American breast cancer survivors by a technology-based intervention. Further studies are needed with other racial/ethnic groups of cancer survivors to confirm the efficacy of a technology-based intervention in reducing cancer survivors' needs for help during their survivorship process.

Spinal cord watershed infarction after surgery.

Spinal cord watershed infarction is a rare phenomenon due to the rich collateral blood supply to the organ. It often occurs in elderly patients with preexisting atherosclerotic disease in the setting of global hypoperfusion, such as thoracoabdominal surgery, dissection, coagulopathies, or idiopathic. We present a case of spinal cord infarction (SCI) in both longitudinal and transverse watershed areas as a complication of postoperative blood loss. In addition, we demonstrate the imaging evolution of spinal cord infarct in the subacute phase with peripheral enhancement due to the breakdown of the blood-spinal cord barrier.

Gut Microbiome of Patients With Breast Cancer in Vietnam.

PURPOSE: Gut microbiota play an important role in human health, including cancer. Cancer and its treatment, in turn, may alter the gut microbiome. To understand this complex relationship, we profiled the gut microbiome of 356 Vietnamese patients with breast cancer. MATERIALS AND METHODS: Stool samples were collected before chemotherapy, with 162 pre- and 194 postsurgery. The gut microbiome was measured by shotgun metagenomic sequencing. Associations of gut microbial diversity, taxa abundance, and gut microbiome health index (GMHI) with sociodemographic, clinical factors, and tumor characteristics were evaluated. RESULTS: Postsurgery samples were associated with significantly lower α- and ß-diversities (P < .001) and showed significant differences in the abundance of 15% of 2,864 investigated taxa (false discovery rate [FDR] <0.1) compared with presurgery samples. An unhealthy gut microbiome was prevalent among patients with breast cancer, with a mean GMHI of -0.79 and -2.81 in pre- and postsurgery stool samples, respectively. In an analysis of 162 presurgery stool samples, diagnosis delay was significantly associated with lower α-diversity, variation in ß-diversity, an increased abundance of species Enorma massiliensis, and a decreased abundance of Faecalicoccus pleomorphus. High intake of fiber was significantly associated with lower α-diversity and a higher abundance of species belonging to Bifidobacterium, Prevotella, and Bacteroides gena (FDR < 0.1). We did not find that cancer stage and subtype, menopausal status, comorbidity, antibiotic use during 3 months before stool collection, or physical activity was significantly associated with α- and ß-diversities or GMHI although a few significant differences were observed in taxa abundance. CONCLUSION: Our study revealed that diagnosis delay, high fiber intake, and breast cancer surgery, which is always followed by antibiotic prophylaxis in Vietnam, led to a less diverse and unhealthy gut microbiome among patients with breast cancer.

Using Carbonized Cotton Fabric Waste to Prepare Poly(ethylene glycol) Composite Phase Change Materials with Improved Thermal Conductivity and Solar-to-Thermal Conversion.

Poly(ethylene glycol) (PEG) boasts excellent thermal energy storage capabilities but lacks efficiency in thermal conductivity and solar absorption. Simultaneously, the escalating concern surrounding the substantial volume of discarded cotton fabric underscores environmental issues. In this study, we devised composite phase change materials (PCMs) by embedding PEG into a carbon cotton material (CCM), varying PEG content from 50 to 80%, and conducted a comprehensive analysis of their thermal properties and solar-to-thermal conversion. The CCM, crafted from a waste 100% cotton towel through carbonization, provided an optimal porous structure that accommodated a significant 70% PEG content without any leakage, thanks to interactions such as surface tension, capillary forces, and interfacial hydrogen bonds. These PEG/CCM composites exhibited impressive crystallization fractions (>92%), resulting in notable thermal energy storage capacities ranging from 85.3 to 143.2 J/g as the PEG content increased from 50 to 80%. Moreover, these composites showed high thermal stability and exceptional cycling durability even after 500 melting/crystallization cycles. Notably, their thermal conductivities markedly increased to a range of 0.46-0.85 W/m·K compared to pure PEG's modest 0.24 W/m·K. Furthermore, the PEG/CCM composites substantially augmented visible and near-infrared (VIS-NIR) light absorption. Evaluation of solar-to-thermal conversion illustrated the composite's ability to efficiently convert solar energy into thermal energy, storing and subsequently releasing it through melting and crystallization processes. This study introduces a novel class of composite PCMs characterized by cost-effectiveness, outstanding thermal performance, and impressive solar-to-thermal conversion capabilities. These composite PCMs hold significant promise for large-scale solar-to-thermal energy storage applications while contributing to the sustainability of cotton waste management.

Enzymatic Strategies for the Biosynthesis of N-Acyl Amino Acid Amides.

Amide bond-containing biomolecules are functionally significant and useful compounds with diverse applications. For example, N-acyl amino acids (NAAAs) are an important class of lipoamino acid amides with extensive use in food, cosmetic and pharmaceutical industries. Their conventional chemical synthesis involves the use of toxic chlorinating agents for carboxylic acid activation. Enzyme-catalyzed biotransformation for the green synthesis of these amides is therefore highly desirable. Here, we review a range of enzymes suitable for the synthesis of NAAA amides and their strategies adopted in carboxylic acid activation. Generally, ATP-dependent enzymes for NAAA biosynthesis are acyl-adenylating enzymes that couple the hydrolysis of phosphoanhydride bond in ATP with the formation of an acyl-adenylate intermediate. In contrast, ATP-independent enzymes involve hydrolases such as lipases or aminoacylases, which rely on the transient activation of the carboxylic acid. This occurs either through an acyl-enzyme intermediate or by favorable interactions with surrounding residues to anchor the acyl donor in a suitable orientation for the incoming amine nucleophile. Recently, the development of an alternative pathway involving ester-amide interconversion has unraveled another possible strategy for amide formation through esterification-aminolysis cascade reactions, potentially expanding the substrate scope for enzymes to catalyze the synthesis of a diverse range of NAAA amides.

Glass shards masquerading as calcifications in the breast: A case report.

Noniatrogenically acquired foreign bodies in the nipple-areola complex or breast skin are rare and can have variable imaging features, depending on the nature of the foreign material. We present the case of a 41-year-old female who had numerous apparent round and punctate calcifications in the right periareolar breast, predominantly within the skin. The biopsy showed multiple glass shards on a background of scar tissue. Further discussion with the patient confirmed that the glass shards were acquired during a motor vehicle accident several years earlier. We also review the types of foreign body material observed in the breast, the imaging appearance of glass foreign bodies in soft tissue, and methods of removal.

Multimodal analysis of methylomics and fragmentomics in plasma cell-free DNA for multi-cancer early detection and localization.

Despite their promise, circulating tumor DNA (ctDNA)-based assays for multi-cancer early detection face challenges in test performance, due mostly to the limited abundance of ctDNA and its inherent variability. To address these challenges, published assays to date demanded a very high-depth sequencing, resulting in an elevated price of test. Herein, we developed a multimodal assay called SPOT-MAS (screening for the presence of tumor by methylation and size) to simultaneously profile methylomics, fragmentomics, copy number, and end motifs in a single workflow using targeted and shallow genome-wide sequencing (~0.55×) of cell-free DNA. We applied SPOT-MAS to 738 non-metastatic patients with breast, colorectal, gastric, lung, and liver cancer, and 1550 healthy controls. We then employed machine learning to extract multiple cancer and tissue-specific signatures for detecting and locating cancer. SPOT-MAS successfully detected the five cancer types with a sensitivity of 72.4% at 97.0% specificity. The sensitivities for detecting early-stage cancers were 73.9% and 62.3% for stages I and II, respectively, increasing to 88.3% for non-metastatic stage IIIA. For tumor-of-origin, our assay achieved an accuracy of 0.7. Our study demonstrates comparable performance to other ctDNA-based assays while requiring significantly lower sequencing depth, making it economically feasible for population-wide screening.

A randomized controlled trial testing a virtual program for Asian American women breast cancer survivors.

A culturally tailored virtual program could meet the survivorship needs of Asian American women breast cancer survivors (AABC). This study aims to determine the efficacy of a culturally tailored virtual information and coaching/support program (TICAA) in improving AABC's survivorship experience. A randomized clinical trial (NCT02803593) was conducted from January 2017 to June 2020 among 199 AABC. The intervention group utilized TICAA and the American Cancer Society [ACS] website while the control group used only ACS website for 12 weeks. The outcomes were measured using the SCNS-34SF (needs; primary), the MSAS-SF (symptoms; secondary), and the FACT-B (quality of life; secondary). The data were analyzed using an intent-to-treat approach. The intervention group showed significant reductions in their needs from the baseline (T0) to post 4 weeks (T1) and to post 12 weeks (T2). Although the changes were not statistically significant, the intervention group had decreased symptoms from T0 to T2 while the control group had an increase in their symptoms. The intervention group had a significant increase in their quality of life from T0 to T2. A culturally tailored virtual program could therefore improve quality of life in AABC patients. Trial Registration: To Enhance Breast Cancer Survivorship of Asian Americans (TICAA), NCT02803593, https://clinicaltrials.gov/ct2/show/NCT02803593?titles=TICAA&draw=2&rank=1.

Enhanced podophyllotoxin production of endophyte Fusarium proliferatum TQN5T by host extract and phenylalanine.

Fermentation technology using endophytes is considered a potential alternative approach for producing pharmaceutical compounds like podophyllotoxin (PTOX). In this study, fungus TQN5T (VCCM 44284) was selected from endophytic fungi isolated from Dysosma versipellis in Vietnam for PTOX production through TLC. The presence of PTOX in TQN5T was further confirmed by HPLC. Molecular identification indicated TQN5T as Fusarium proliferatum with 99.43% identity. This result was asserted by morphological characteristics such as white cottony, filamentous colony, layer and branched mycelium, and clear hyphae septa. Cytotoxic assay indicated both biomass extract and culture filtrate of TQN5T presented strong cytotoxicity on LU-1 and HepG2 with IC50 of 0.11, 0.20, 0.041, and 0071, respectively, implying anti-cancer compounds were accumulated in the mycelium and secreted into the medium. Further, the production of PTOX in TQN5T was investigated in the fermentation condition supplemented with 10 µg/ml of host plant extract or phenylalanine as elicitors. The results revealed a significantly higher amount of PTOX in the PDB + PE and PDB + PA at all studied time points in comparison with PDB (control). Especially, after 168 h of culture, PTOX content in the PDB with plant extract reached the peak with 314 µg/g DW which is 10% higher than the best yield of PTOX in previous studies, denoting F. proliferatum TQN5T as a promising PTOX producer. This is the first study on enhancing the PTOX production in endophytic fungi by supplementing phenylalanine-a precursor for PTOX biosynthesis in plants into fermented media, suggesting a common PTOX biosynthetic pathway between host plant and endophytes. KEY POINTS: • Fusarium proliferatum TQN5T was proven for PTOX production. • Both mycelia extract and spent broth extract of Fusarium proliferatum TQN5T presented strong cytotoxicity on cancer cell lines LU-1 and HepG2. • The supplementation of 10 µg/ml host plant extract and phenylalanine into fermentation media of F. proliferatum TQN5T improved the yield of PTOX.

CTLA4 mRNA is downregulated by miR-155 in regulatory T cells, and reduced blood CTLA4 levels are associated with poor prognosis in metastatic melanoma patients.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is an immune checkpoint expressed in regulatory T (Treg) cells and activated T lymphocytes. Despite its potential as a treatment strategy for melanoma, CTLA-4 inhibition has limited efficacy. Using data from The Cancer Genome Atlas (TCGA) melanoma database and another dataset, we found that decreased CTLA4 mRNA was associated with a poorer prognosis in metastatic melanoma. To investigate further, we measured blood CTLA4 mRNA in 273 whole-blood samples from an Australian cohort and found that it was lower in metastatic melanoma than in healthy controls and associated with worse patient survival. We confirmed these findings using Cox proportional hazards model analysis and another cohort from the US. Fractionated blood analysis revealed that Treg cells were responsible for the downregulated CTLA4 in metastatic melanoma patients, which was confirmed by further analysis of published data showing downregulated CTLA-4 surface protein expression in Treg cells of metastatic melanoma compared to healthy donors. Mechanistically, we found that secretomes from human metastatic melanoma cells downregulate CTLA4 mRNA at the post-transcriptional level through miR-155 while upregulating FOXP3 expression in human Treg cells. Functionally, we demonstrated that CTLA4 expression inhibits the proliferation and suppressive function of human Treg cells. Finally, miR-155 was found to be upregulated in Treg cells from metastatic melanoma patients compared to healthy donors. Our study provides new insights into the underlying mechanisms of reduced CTLA4 expression observed in melanoma patients, demonstrating that post-transcriptional silencing of CTLA4 by miRNA-155 in Treg cells may play a critical role. Since CTLA-4 expression is downregulated in non-responder melanoma patients to anti-PD-1 immunotherapy, targeting miRNA-155 or other factors involved in regulating CTLA4 expression in Treg cells without affecting T cells could be a potential strategy to improve the efficacy of immunotherapy in melanoma. Further research is needed to understand the molecular mechanisms regulating CTLA4 expression in Treg cells and identify potential therapeutic targets for enhancing immune-based therapies.

Altered cleavage of human factor VIII at the B-domain and acidic region 3 interface enhances expression after gene therapy in hemophilia A mice.

BACKGROUND: Variants of human factor VIII (hFVIII) have been developed to further understand the structure and function of hFVIII and improve gene-based therapeutics. We have previously characterized several hFVIII variants of the furin cleavage site (1645-1648) with improved secretion. We have also identified a second cleavage site in the acidic region 3 (a3) (1657-1658) that becomes the primary hFVIII intracellular cleavage position in the absence of the furin site. We tested a hypothesis that modification of this site may confer additional functional advantages to hFVIII. OBJECTIVES: The aim of this study was to conduct the biochemical and functional characterization of hFVIII variants of the furin cleavage site, the a3 cleavage site, or in combination, both in vitro and in vivo after AAV mediated gene therapy. METHODS: Recombinant hFVIII variants of the furin cleavage site (hFVIII-Δ3), the a3 cleavage site (hFVIII-S1657P/D1658E [SP/DE]), or in combination (hFVIII-Δ3-SP/DE) were purified and characterized in vitro and in vivo. RESULTS: Recombinant hFVIII-Δ3, hFVIII-SP/DE, and hFVIII-Δ3-SP/DE variants all had comparable specific activity to B-domain deleted (BDD) hFVIII. Hemophilia A mice tolerant to hFVIII did not develop immune responses to hFVIII after protein challenge with these variants or after adeno-associated virus (AAV) delivery. Following AAV delivery, hFVIII-Δ3-SP/DE resulted in expression levels that were 2- to 5-fold higher than those with hFVIII-BDD in hemophilia A mice. CONCLUSION: The novel hFVIII-Δ3-SP/DE variant of the furin and a3 cleavage sites significantly improved secretion compared with hFVIII-BDD. This key feature of the Δ3-SP/DE variant provides a unique strategy that can be combined with other approaches to further improve factor VIII expression to achieve superior efficacy in AAV-based gene therapy for hemophilia A.

Isolation and Cytotoxic Potency of Endophytic Fungi Associated with Dysosma difformis, a Study for the Novel Resources of Podophyllotoxin.

Endophytic fungi are promising sources for the production of podophyllotoxin-an important anticancer compound, replacing depleted medical plants. In this study, the endophytes associated with Dysosma difformis-an ethnomedicinal plant species were isolated to explore novel sources of podophyllotoxin. Fifty-three endophytic fungi were isolated and identified by morphological observation and ITS-based rDNA sequencing, assigning them to 27 genera in 3 divisions. Fusarium was found the most prevalent genus with a colonization frequency of 11.11%, followed by Trametes (9.26%) and Penicillium (7.41%). Phylogenetic trees were constructed for the endophytic fungi community in two collection sites, Ha Giang and Lai Chau, revealing the adaptation of the species to the specific tissues and habitats. Cytotoxic activity of endophytic fungal extracts was investigated on cancer cell lines such as SK-LU-1, HL-60, and HepG2, demonstrating strong anti-cancer activity of six isolates belonging to Penicillium, Trametes, Purpureocillium, Aspergillus, and Ganoderma with IC50 value of lower than 10 µg/mL. The presence of podophyllotoxin was indicated in Penicillium, Trametes, Aspergillus and for the first time in Purpureocillium and Ganoderma via high-performance liquid chromatography, which implied them as a potential source of this anti-cancer compound.

Simultaneously induced mutations in eIF4E genes by CRISPR/Cas9 enhance PVY resistance in tobacco.

Tobacco is an important commercial crop and a rich source of alkaloids for pharmaceutical and agricultural applications. However, its yield can be reduced by up to 70% due to virus infections, especially by a potyvirus Potato virus Y (PVY). The replication of PVY relies on host factors, and eukaryotic translation initiation factor 4Es (eIF4Es) have already been identified as recessive resistance genes against potyviruses in many plant species. To investigate the molecular basis of PVY resistance in the widely cultivated allotetraploid tobacco variety K326, we developed a dual guide RNA CRISPR/Cas9 system for combinatorial gene editing of two clades, eIF4E1 (eIF4E1-S and eIF4E1-T) and eIF4E2 (eIF4E2-S and eIF4E2-T) in the eIF4E gene family comprising six members in tobacco. We screened for CRISPR/Cas9-induced mutations by heteroduplex analysis and Sanger sequencing, and monitored PVYO accumulation in virus challenged regenerated plants by DAS-ELISA both in T0 and T1 generations. We found that all T0 lines carrying targeted mutations in the eIF4E1-S gene displayed enhanced resistance to PVYO confirming previous reports. More importantly, our combinatorial approach revealed that eIF4E1-S is necessary but not sufficient for complete PVY resistance. Only the quadruple mutants harboring loss-of-function mutations in eIF4E1-S, eIF4E1-T, eIF4E2-S and eIF4E2-T showed heritable high-level resistance to PVYO in tobacco. Our work highlights the importance of understanding host factor redundancy in virus replication and provides a roadmap to generate virus resistance by combinatorial CRISPR/Cas9-mediated editing in non-model crop plants with complex genomes.