The production of a dextran binding antibody by phage display library and its applications to sugar processing

A phage collection (M1114), which is a mixture of two collections, M710-3R and Silica 426 each derived from a human synthetic phage antibody library (Fab 2lox), was used further enriched for dextran binding using enzyme-linked immunosorbent assays (ELISA) screening. The effects of dextran concentration on phage binding affinity were tested using indirect sandwich ELISA on phage collections, M1114-m74 and AE-M1114-m74-2R. Most of the phage bound to dextran (T2000) coated on a sandwich ELISA. The combination of ELISA screening and a sephadex columns enriched dextran binding 7 fold over enrichment by a single ELISA screen. Phage collection (M1114-m74) produced by combination screening showed the greatest binding on ELISA. The color intensity produced by phage collection (AE-M1114-m74-2R) obtained after the 2nd round of selection was 3.5 fold higher than that of phage collections, AE-M1114-m74-1R after the 1st round. Dextran binding by phage collection (AE-M1114-m74-2R) was illustrated using image analysis of transmission electron micrographs. Sephadex bead agarose electrophoresis (SBAE) screening produced phage collections (AE-M1114-m74-1R and 2R) which were used in a paper-dip assay. A dip stick assay using a protein blocked paper with adsorbed high molecular size dextran (T10,000, 107) produced the most color (59 ±5) using anti-dextran phage enzyme linked assays. Low molecular size dextran (T40, 4x104) produced significantly lower color (15 ±1). Phage collection (AE-M1114-m74-2R) was tested for specificity against dextran (T2000), corn starch, sucrose, dextrose, and chitin. Dextran produced up to 18 fold the normalized intensity of the other carbohydrates. The presence of Fab inserts in the phage collections was confirmed using PCR, and the presence of the same insert in the host E.coli was checked using a â-galactosidase linked assay. DNA sequencing of phage collection (AE-M1114-m74-2R) confirmed that human origin antibody was present. The PCR products of ë, ê light chains and heavy chain from phage collection (AE-M1114-m74-2R) were approximately 420 bp, 550 bp and 600 bp. This research used various selection methods to isolate anti-dextran phages from a library. These were used to develop a paper-dip stick method for dextran detection used for routine screening of sugar juices

Effects of Alginate Oligosaccharide on Testosterone-Induced Benign Prostatic Hyperplasia in Orchiectomized Rats

Benign prostatic hyperplasia (BPH) is an age-related disease of the urinary system that affects elderly men. Current treatments for BPH are associated with several adverse effects, thus highlighting the need for alternative agents. Alginate oligosaccharide (AOS), a water-soluble functional oligomer derived from brown algae, inhibits prostate cancer cell proliferation. However, the effects of AOS on BPH and the underlying molecular mechanisms remain unclear. Therefore, here, we aimed to investigate the therapeutic potential of AOS in BPH by using human benign prostatic epithelial cells (BPH-1) and a rat model of testosterone-induced BPH. Treatment with AOS inhibited in vitro and in vivo proliferation of prostatic epithelial cells and the testosterone-induced expression of androgen receptor (AR) and androgen-associated genes, such as those encoding 5α-reductase type 2 and prostate-specific antigen. Oral administration of AOS remarkably reduced the serum levels of dihydrotestosterone (DHT) and testosterone as well as the expression of proliferating cell nuclear antigen, inflammatory cytokines, and enzymes, which showed increased levels in prostatic tissues of rats with testosterone-induced BPH. Taken together, these data demonstrate that AOS suppresses testosterone-induced BPH in rats by downregulating AR and the expression of androgen-associated genes, supporting the hypothesis that AOS might be of potential use for the treatment of BPH

Sirtuin 6 Overexpression Improves Rotator Cuff Tendon-to-Bone Healing in the Aged

Aging is an independent risk factor for recurrent tearing after surgical repair of rotator cuff ruptures around the tendon-to-bone area. However, aging signature factors and related mechanisms involved in the healing of the rotator cuff are still unknown. We hypothesized that differences in proteins involved in the rotator cuff according to age may affect tendon-to-bone healing. The proteome analysis performed to identify the signature aging proteins of the rotator cuff confirmed the sirtuin signal as an age-specific protein. In particular, the expression of SIRT6 was markedly down-regulated with age. Ingenuity pathway analysis of omics data from age-dependent rat rotator cuffs and linear regression from human rotator cuffs showed SIRT6 to be closely related to the Wnt/β-catenin signal. We confirmed that overexpression of SIRT6 in the rotator cuff and primary tenocyte regulated canonical Wnt signaling by inhibiting the transcriptional expression of sclerostin, a Wnt antagonist. Finally, SIRT6 overexpression promoted tendon-to-bone healing after tenotomy with reconstruction in elderly rats. This approach is considered an effective treatment method for recovery from recurrent rotator cuff tears, which frequently occur in the elderly

Development of Lectin-Linked Immunomagnetic Separation for the Detection of Hepatitis A Virus

The accuracy and sensitivity of PCR-based methods for detection of hepatitis A virus (HAV) are dependent on the methods used to separate and concentrate the HAV from the infected cells. The pH and ionic strength affect the binding affinity of the virus to cells. In this study, we initially investigated the effects of pH (4.0–10.0) and metal ions (Fe2+, Co2+, Cu2+, Mg2+, K+, and Ca2+) on the binding of HAV to oyster digestive cells. The lowest relative binding (RB) of HAV to the cells was found at pH 4.0 and in FeSO4 solution (64.6% and 68.1%, respectively). To develop an alternative to antibody-dependent immunomagnetic separation prior to detection of HAV using RT-PCR, the binding of HAV to five lectins, peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Ulex europaeus agglutinin (UEA-1) and soybean agglutinin (SBA), was evaluated using ELISAs. SBA showed significantly higher RB to HAV than the other lectins tested. In addition, HAV could be concentrated within 30 min using SBA-linked magnetic bead separation (SMS) prior to the RT-PCR assay. Our findings demonstrate the feasibility of using SMS combined with RT-PCR to detect HAV at dilutions ranging from 10−1–10−4 of a HAV stock (titer: 104 TCID50/mL)

Effectiveness of Periodic Treatment of Quercetin against Influenza A Virus H1N1 through Modulation of Protein Expression

Kimchi, a traditional fermented food regularly consumed in Korea, contains various types of antimicrobial compounds. Among the tested compounds present in common spices used in Kimchi, quercetin showed the highest selectivity index against influenza A virus (IAV) H1N1. In this study, the effect of pretreatment and periodic treatment with quercetin against IAV in Madin–Darby canine kidney cells was observed. Compared to pretreatment, periodic treatment resulted in significantly higher cell viability but lower relative expression of the IAV PA gene and total apoptosis and cell death. To explain the mechanisms underlying the antiviral effects of quercetin treatment, a comparative proteomic analysis was performed in four samples (mock, quercetin-treated, IAV-infected, and quercetin-treated IAV-infected). Among the 220 proteins, 56 proteins were classified nonhierarchically into three clusters and were differentially modulated by quercetin treatment in IAV-infected cells. Post-translational modifications were identified in 68 proteins. In conclusion, periodic treatment with quercetin is effective in reducing IAV infection, and differentially regulates the expression of key proteins, including heat shock proteins, fibronectin 1, and prohibitin to reduce IAV replication

Author’s Reply to “Concerns regarding Validity of the Use of Bean Extract-Based Gargle for COVID-19 Diagnosis”

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