The natural tannins oligomeric proanthocyanidins and punicalagin are potent inhibitors of infection by SARS-CoV-2.

The Coronavirus Disease 2019 (COVID-19) pandemic continues to infect people worldwide. While the vaccinated population has been increasing, the rising breakthrough infection persists in the vaccinated population. For living with the virus, the dietary guidelines to prevent virus infection are worthy of and timely to develop further. Tannic acid has been demonstrated to be an effective inhibitor of coronavirus and is under clinical trial. Here we found that two other members of the tannins family, oligomeric proanthocyanidins (OPCs) and punicalagin, are also potent inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection with different mechanisms. OPCs and punicalagin showed inhibitory activity against omicron variants of SARS-CoV-2 infection. The water extractant of the grape seed was rich in OPCs and also exhibited the strongest inhibitory activities for viral entry of wild-type and other variants in vitro. Moreover, we evaluated the inhibitory activity of grape seed extractants (GSE) supplementation against SARS-CoV-2 viral entry in vivo and observed that serum samples from the healthy human subjects had suppressive activity against different variants of SARS-CoV-2 Vpp infection after taking GSE capsules. Our results suggest that natural tannins acted as potent inhibitors against SARS-CoV-2 infection, and GSE supplementation could serve as healthy food for infection prevention.

Since it first surfaced in late 2019, the COVID-19 pandemic has had a significant impact on people's lives. While several vaccines have been created, infections have not disappeared. This is largely due to new variants of the virus responsible for the disease (SARS-CoV-2) emerging, which current vaccines do not work as well against. Indeed, several reports suggest that protection from the omicron variant wanes as shortly as four to six months after vaccination. Therefore, other strategies are needed to reduce the risk of SARS-CoV-2 infections. In 2022, researchers discovered that tannic acid blocked two proteins that SARS-CoV-2 needs to enter and replicate inside human cells. Tannic acid is part of the tannin family, which includes natural molecules found in plant-based meals and beverages. Here, Chen et al. ­ including some of the researchers involved in the 2022 studies ­ set out to find whether two other tannins found in nature (OPCs and punicalagin) could also inhibit SARS-CoV-2. Chen et al. administered tannic acid, OPCs and punicalagin to human cells cultured in a laboratory that had been infected with SARS-CoV-2. This revealed that all three tannins suppress the activity of the same proteins required for viral entry and replication, but to varying degrees suggesting that they block SARS-CoV-2 infections via different mechanisms. The compounds were also able to inhibit different variants of the virus, including omicron, from infecting the lab-grown cells. Further experiments revealed that water extracted from seeded grapes, which contains high levels of OPCs, could also block SARS-CoV-2 entry in the cell culture system. To test this further, Chen et al. gave 18 healthy individuals capsules containing different concentrations of grape seed extract and collected samples of their serum. The serum samples suppressed entry of different variants of SARS-CoV-2 in the cell culture system, with serums from subjects that received the higher dose having the greatest effect. These findings suggest that naturally occurring tannins can suppress multiple variants of SARS-CoV-2 from entering and replicating in cells. Consuming supplements of grape seed extract could potentially reduce the risk of SARS-CoV-2 infections. However, further experiments, including clinical trials, are needed to test this possibility.

Development and Optimization of Black Rice (Oryza sativa L.) Sourdough Fermented by Levilactobacillus brevis LUC 247 for Physicochemical Characteristics and Antioxidant Capacity.

This study used Levilactobacillus brevis LUC 247 to ferment black rice sourdough, made into Type III black rice sourdough powder to produce black rice sourdough bread. The composition analysis, antioxidant capacity, and storage stability of the black rice sourdough bread with different proportions of black rice powder (0-60%) and fermented for different lengths of time (0-48 h) were discussed. The results showed that the black rice sourdough had the maximum lactic acid bacteria count (9 Log CFU/g) during 12 to 36 h of fermentation. The titratable acid, lactic acid, and acetic acid yields increased with the fermentation time and the proportion of black rice powder. The total anthocyanin content and antioxidant capacity increased with the fermentation time. The hardness and chewiness of the black rice sourdough bread were positively correlated with the black rice sourdough powder content and increased with storage time. In addition, the growth of fungi was significantly slowed as the additional level of black rice sourdough powder increased.

Loss of RhoE Function in Dermatofibroma Promotes Disorganized Dermal Fibroblast Extracellular Matrix and Increased Integrin Activation.

Dermatofibromas (DFs) are common, benign fibrous skin tumors that can occur at any skin site. In most cases, DFs are solitary and sporadic, but a few are multiple and familial, and the mechanisms leading to these lesions are currently unclear. Using exome sequencing, we have identified a heterozygous variant in a pedigree with autosomal dominant multiple familial DF within RND3 (c.692C>T,p.T231M) that encodes for the small GTPase RhoE, a regulator of the actin cytoskeleton. Expression of T231M-RhoE or RhoE depletion using CRISPR in human dermal fibroblasts increased proliferation and adhesion to extracellular matrix through enhanced ß1 integrin activation and more disorganized matrix. The enzyme PLOD2 was identified as a binding partner for RhoE, and the formation of this complex was disrupted by T231M-RhoE. PLOD2 promotes collagen cross-linking and activation of ß1 integrins, and depleting PLOD2 in T231M-RhoE-expressing cells reduced T231M-RhoE-mediated ß1 integrin activation and led to increased matrix alignment. Immunohistochemical analysis revealed reduced expression of RhoE but increased expression of PLOD2 in the dermis of DF skin samples compared with that of the controls. Our data show that loss of RhoE function leads to increased PLOD2 activation, enhancing integrin activation and leading to a disorganized extracellular matrix, contributing to DF.

A possible role for second-hit postzygotic GJB2 mutation in porokeratotic eccrine ostial and dermal duct nevus.

Porokeratotic eccrine ostial and dermal duct nevus (PEODDN) is a rare type of epidermal nevus involving the eccrine acrosyringia. It typically presents as asymptomatic linear keratotic papules and plaques along the lines of Blaschko and predominantly affects the extremities. This disease has recently been linked to somatic mutations within the GJB2 locus. Only four GJB2 mutations have been previously documented for PEODDN, and the underlying genetic basis remains inconclusive. Herein, we report an 18-year-old female with a hyperkeratotic plaque on the dorsa of the proximal interphalangeal joint of her right ring finger, as well as multiple small hyperkeratotic papules linearly distributed on the lateral sides of her fingers occurring since birth. Histopathological results revealed prominent parakeratotic cornoid lamella-like tiers at the opening of the eccrine secretory ducts. Whole-exome sequencing of the affected skin tissue revealed a heterozygous germline mutation and a postzygotic somatic mutation in GJB2. In summary, this study presents a case of PEODDN with compound heterozygous mutations in GJB2, which broadens the genetic spectrum of this disease entity and implies a possible role for second-hit mutations in the pathogenesis of PEODDN.

Mutational analysis of epidermolysis bullosa in Taiwan by whole-exome sequencing complemented by RNA sequencing: a series of 77 patients.

BACKGROUND: Epidermolysis bullosa (EB) is a heterogeneous group of hereditary skin diseases characterized by skin fragility. Primary data on Taiwanese population remain scarce. METHODS: We gathered clinical information from EB patients at National Cheng Kung University Hospital from January, 2012, to June, 2021. Diagnostic tests including transmission electron microscopy, immunofluorescence studies, and whole-exome sequencing (WES) were performed. The pathogenicity of novel splice-site mutations was determined through reverse transcriptase-PCR of skin mRNA followed by Sanger and/or RNA sequencing. RESULTS: Seventy-seven EB patients from 45 families were included: 19 EB simplex, six junctional EB, and 52 dystrophic EB. Pathogenic variants were identified in 37 of 38 families (97.4%), in which WES was used as a first-line tool for mutational analysis; RNA sequencing determined pathogenic variants in the remaining one family. A total of 60 mutations in EB-related genes were identified, including 22 novel mutations. The mutations involved KRT5, KRT14, PLEC, COL17A1, LAMB3, LAMA3, ITGB4, and COL7A1. Over one-quarter of DEB patients had EB pruriginosa. CONCLUSIONS: The distinct clinical presentation and molecular pathology of EB in Taiwan expand our understanding of this disorder. WES was an effective first-line diagnostic tool for identifying EB-associated variants. RNA sequencing complemented WES when multiple potentially pathogenic splice-site mutations were found.

Case Report: A novel desmoplakin mutation in a taiwanese woman with familial dilated cardiomyopathy that necessitated heart transplantation.

Around one-third of patients diagnosed with idiopathic dilated cardiomyopathy (DCM) turn out to be familial cases, in only a few of which the identification of a pathogenic/likely pathogenic variant could be achieved. Cardiomyopathy caused by desmoplakin gene mutations represents a distinct form with a high prevalence of left ventricle involvement. We report a novel desmoplakin mutation carried by two individuals in a Taiwanese family, in which the proband recovered well after heart transplantation and under medical control, while her son had received an implantable cardioverter defibrillator and has been under guideline-directed medical therapy. The present study broadens the genetic spectrum of this disease entity and strengthens the notion that a detailed family history with genetic study contributes to the early detection and treatment of inherited diseases.

Cas9-guided haplotyping of three truncation variants in autosomal recessive disease.

An autosomal recessive disease is caused by biallelic loss-of-function mutations. However, when more than two disease-causing variants are found in a patient's gene, it is challenging to determine which two of the variants are responsible for the disease phenotype. Here, to decipher the pathogenic variants by precise haplotyping, we applied nanopore Cas9-targeted sequencing (nCATS) to three truncation COL7A1 variants detected in a patient with recessive dystrophic epidermolysis bullosa (EB). The distance between the most 5' and 3' variants was approximately 19 kb at the level of genomic DNA. nCATS successfully demonstrated that the most 5' and 3' variants were located in one allele while the variant in between was located in the other allele. Interestingly, the proband's mother, who was phenotypically intact, was heterozygous for the allele that harbored the two truncation variants, which could otherwise be misinterpreted as those of typical recessive dystrophic EB. Our study highlights the usefulness of nCATS as a tool to determine haplotypes of complicated genetic cases. Haplotyping of multiple variants in a gene can determine which variant should be therapeutically targeted when nucleotide-specific gene therapy is applied.

Genetic Diagnosis of Rubinstein-Taybi Syndrome With Multiplex Ligation-Dependent Probe Amplification (MLPA) and Whole-Exome Sequencing (WES): Case Series With a Novel CREBBP Variant.

Rubinstein-Taybi Syndrome (RSTS) is a rare congenital disease with distinctive facial features, broadening of the thumbs and halluces, and developmental delay. RSTS is caused by de novo genetic alterations in CREBBP and the homologous EP300 genes. In this study, we established a genetic diagnostic protocol by integrating multiplex ligation-dependent probe amplification (MLPA) and whole-exome sequencing (WES). Five patients clinically diagnosed with RSTS were enrolled for genetic testing. Germline DNA was extracted from the peripheral blood of the patients and their families. One patient (case 1) was identified as harboring a large heterozygous deletion in the 16p13.3 region, spanning the CREBBP gene. Three patients (Cases 2-4) harbored different CREBBP variants (c.2608C>T:p.Gln870Ter,c.4404_4405del:p.Thr1468fs,c.3649C>T:p.Gln1217Ter). No causative variants were identified for the fifth RSTS patient (case 5). Here, we propose a molecular diagnostic protocol that identified causative genetic alterations in 4/5 of the patients, yielding a molecular diagnostic rate of 80%. Given the rarity of RSTS, more research is needed to explore its pathogenesis and mechanism.

Effect of administering dexmedetomidine with or without atropine on cardiac troponin I level in isoflurane-anesthetized dogs.

We aimed to determine whether dexmedetomidine administration with or without atropine increases cardiac troponin I (cTnI) level in healthy dogs. We hypothesized that 10 µg/kg dexmedetomidine + atropine increases the cTnI level, whereas 5 µg/kg dexmedetomidine + atropine does not. Eighteen healthy, pet dogs that underwent an orthopedic surgery or ovariohysterectomy were included in this study. The dogs were randomly assigned to atropine (0.02 mg/kg)-dexmedetomidine (10 µg/kg), saline-dexmedetomidine (10 µg/kg), and atropine (0.02 mg/kg)-dexmedetomidine (5 µg/kg) groups. Each dog was premedicated with atropine or saline intramuscularly (IM). After 10 min, they were IM injected with dexmedetomidine (10 or 5 µg/kg)-morphine (0.5 mg/kg)-midazolam (0.2 mg/kg). Following this, anesthesia was induced after 10 min with propofol and maintained with isoflurane in 100% oxygen. The median plasma cTnI level at 6, 12 and 24 hr after premedication was significantly higher than that at baseline. The cTnI level in the atropine-dexmedetomidine (10 µg/kg) group was significantly higher than that in the saline-dexmedetomidine (10 µg/kg) and atropine-dexmedetomidine (5 µg/kg) groups at 6 and 12 hr after premedication. The cTnI level returned to normal within 72 hr after premedication in all groups. The administration of atropine in combination with 10 µg/kg dexmedetomidine increased the cTnI level, indicating subclinical myocardial damage.

Plectin Missense Mutation p.Leu319Pro in the Pathogenesis of Autosomal Recessive Epidermolysis Bullosa Simplex.

is missing (Short communication).

A Single High-Intensity Shock Wave Pulse With Microbubbles Opens the Blood-Brain Barrier in Rats.

Focused extracorporeal shockwave (FSW), one kind of focused high-intensity pulsed ultrasound, has been shown to induce blood-brain barrier (BBB) opening in targeted brain areas in rat animal models with minimal detrimental effects below threshold intensity levels or iterations. In the current study, we found that the thresholds could be further reduced by the addition of microbubbles (ultrasound contrast agents or UCA; SonoVue). FSW with 2 × 106 MBs/kg of UCA (20% of clinical dosage) at an intensity level of 0.1 (peak positive pressure 5.4 MPa; peak negative pressure -4.2 MPa; energy flux density 0.03 mJ/mm2) resulting in a 100% BBB opening rate without detectable hemorrhage or apoptosis in the brain. Significantly reduced free radical production was found compared with 0.5 MHz focused ultrasound at a peak negative pressure of 0.44 MPa (1% duty cycle and 4 × 107 MBs/kg of UCA). FSW devices offer advantages of commercial availability and high safety, and thus may facilitate future research and applications of focal BBB opening for oncological and pharmacological purposes.

Targeting Inflammation, PHA-767491 Shows a Broad Spectrum in Protein Aggregation Diseases.

Many protein aggregation diseases (PAD) affect the nervous system. Deposits of aggregated disease-specific proteins are found within or around the neuronal cells of neurodegenerative diseases. Although the main protein component is disease-specific, oligomeric aggregates are presumed to be the key agents causing the neurotoxicity. Evidence has shown that protein aggregates cause a chronic inflammatory reaction in the brain, resulting in neurodegeneration. Therefore, strategies targeting anti-inflammation could be beneficial to the therapeutics of PAD. PHA-767491 was originally identified as an inhibitor of CDC7/CDK9 and was found to reduce TDP-43 phosphorylation and prevent neurodegeneration in TDP-43 transgenic animals. We recently identified PHA-767491 as a GSK-3ß inhibitor. In this study, we established mouse hippocampal primary culture with tau-hyperphosphorylation through the activation of GSK-3ß using Wortmannin and GF109203X. We found that PHA-767491 significantly improved the neurite outgrowth of hippocampal primary neurons against the neurotoxicity induced by GSK-3ß. We further showed that PHA-767491 had neuroprotective ability in hippocampal primary culture under oligomeric Aß treatment. In addition, PHA-767491 attenuated the neuroinflammation in mouse cerebellar slice culture with human TBP-109Q agitation. Further study of SCA17 transgenic mice carrying human TBP-109Q showed that PHA-767491 ameliorated the gait ataxia and the inflammatory response both centrally and peripherally. Our findings suggest that PHA-767491 has a broad spectrum of activity in the treatment of different PAD and that this activity could be based on the anti-inflammation mechanism.

Microsporidial stromal keratitis: characterisation of clinical features, ultrastructural study by electron microscopy and efficacy of different surgical modalities.

AIMS: To report the clinical manifestations, ultrastructure and evaluate the efficacy of therapeutic lamellar keratectomy (TLK) and penetrating keratoplasty (PK) for microsporidial stromal keratitis (MSK). METHODS: Fourteen MSK cases between 2009 and 2018 were recruited. Each patient's clinical presentation, light microscopy, histopathology, PCR and electron microscopy (EM) of corneal samples were reviewed. RESULTS: The patients were 70.0±4.7 years old (average follow-up, 4.5 years). Time from symptoms to presentation was 10.6±13.0 weeks. The corneal manifestations were highly variable. Corneal scrapings revealed Gram stain positivity in 12 cases (85.7%) and modified Ziehl-Neelsen stain positivity in 9 (64.3%). Histopathology revealed spores in all specimens, while sequencing of small subunit rRNA-based PCR products identified Vittaforma corneae in 82% of patients. EM demonstrated various forms of microsporidial sporoplasm in corneal keratocytes. All patients were treated with topical antimicrobial agents or combined with oral antiparasitic medications for >3 weeks. As all patients were refractory to medical therapy, they ultimately underwent surgical intervention (TLK in 7, PK in 6 and 1 received TLK first, followed by PK). Postoperatively, the infection was resolved in 78.6% of the patients. Nevertheless, a high recurrence rate (21.4%) was noted during 3-year follow-up, with only two patients retained a final visual acuity ≥20/100. CONCLUSION: MSK usually presents with a non-specific corneal infiltration refractory to antimicrobial therapy. The diagnosis relies on light microscopic examinations on corneal scrapings and histopathological analyses. Surgical intervention is warranted by limiting the infection; however, it was associated with an overall poor outcome.

Application of condensed molasses fermentation solubles and lactic acid bacteria in corn silage production.

BACKGROUND: The aim of this study is to investigate the application of two lactic acid bacteria and dry condensed molasses fermentation solubles (CMS) in the making and preservation of corn silage. Forage corn materials are divided into eight treatment groups as follows: (i) control, (ii) B2 (Lactobacillus plantarum B2, 1 × 109 cfu kg-1 ), (iii) LAS (Lactobacillus buchneri 40788, 3 × 108 cfu kg-1 ), (iv) B2 + LAS, (v) CMS (35 g kg-1 , fresh weight), (vi) B2 + CMS, (vii) LAS + CMS and (viii) B2 + LAS + CMS. The silage composition and aerobic stability are determined after ensiling for 90 days. Furthermore, the digestibility of the silage product and gas production are evaluated using a trotro digestion procedure. RESULTS: The assay results indicate that the CMS supplementation and B2 inoculation significantly increased lactic acid concentration (P < 0.01). However, they also reduced the content of acetic acid and silage pH at the initial fermentation stage. The CMS supplemented with B2 (B2 + CMS) showed an improvement in the quality of silage, but a significant decrease in aerobic stability (P < 0.01). The B2 + LAS + CMS treatment yielded an increase in acetic acid production during the late fermentation period and is able to extend the aerobic stability of corn silage. Furthermore, this study shows that CMS supplementation alone can significantly improve the digestibility of the in vitro dry matter (P < 0.01) and the microbial protein synthesis efficiency (P = 0.01). In addition, the CMS supplementation is beneficial for enhancing the aerobic stability of corn silage. CONCLUSIONS: These results suggest that the combination of CMS supplementation and a suitable inoculation lactic acid bacterial strain can be highly promising for enhancing the higher quality and stability of corn silage. © 2020 Society of Chemical Industry.

Two Cases of Interleukin-7-Deficient Generalized Verrucosis.

We report 2 generalized verrucosis (GV) patients homozygous for a novel mutation in the start codon of IL7. Unlike the previous report in which IL-7 deficiency accompanied CD4 T lymphocytopenia, circulating CD4 T cells were not depleted in one of our patients, suggesting a GV pathogenesis other than poor T-cell development.