Direct construction of an open-sandwich enzyme immunoassay for one-step noncompetitive detection of thyroid hormone T4

To establish a sensitive noncompetitive immunoassay for thyroxine (T4), we attempted to isolate anti-T4 antibodies from a phage display library based on a phagemid pDong1 ( Dong et al. Anal. Biochem.2009, 36, 386 ), which was designed to enable open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) after selection on immobilized antigen. After the Fab-displaying phage library made from the splenocytes of T4-KLH immunized mice was subjected to biopanning on T4-BSA, two T4-specific clones were obtained. When they were assayed by indirect competitive ELISA, both clones showed low IC(50) (5-13 ng/mL), indicating their high affinity to T4. When they were used for OS-ELISA that detects antigen-dependency of the interaction between variable domains V(H) and V(L), a clone successfully detected 1 ng/mL of T4 with a working range superior to that of competitive IA. OS-ELISA was also performed with maltose binding protein (MBP)-fused V(H)/V(L) of this clone, which showed a detection limit less than 0.1 ng/mL T4. Moreover, the assay showed cross-reactivity with T3 similar to that of competitive ELISA, and also gave a reasonable total serum T4 concentration (90 ng/mL) from ethanol-extracted sample serum using the recombinant proteins. This is the first direct construction of an OS-ELISA system bypassing hybridoma, which will be applicable to the detection of many other small molecule antigens.     

Distribution of the sulfite resistance gene SSU1-R and the variation in its promoter region in wine yeasts

The SSU1-R gene provides high sulfite resistance to the wine yeast Y9-16B. In this study, we examined the distribution of this gene in 61 wine yeasts and 4 laboratory yeasts. We also analyzed the number of repeats of a 76-bp promoter sequence and its relationship to sulfite resistance. We found that the SSU1-R gene was present in 31 of the 61 wine yeasts. Furthermore, we found that the number of repeats in the promoter region of SSU1-R varied from two to six. Using RsaI, which cuts only once in the repeat, we suggested that the repeats all consisted of the 76-bp sequence. Finally, we found that there was a complex relationship between the number of repeats and sulfite resistance.     

Detection of short tandem repeat (STR) polymorphisms by microchip electrophoresis for individual identification of cattle

A simple and rapid detection of short tandem repeat (STR) markers was studied as a screening test for individual identification of cattle. DNAs were extracted from eight commercial beef samples by a proteinase K-boil method followed by purification with 2-propanol precipitation. Five STR markers, known to be highly polymorphic, were amplified by PCR and analyzed both by a conventional sequencing analysis (SEQ) and by a proposed microchip electrophoresis (MEP). Every marker revealed high polymorphism, such as 5-9 alleles in SEQ analysis, and 4-6 alleles in MEP analysis. This simple and rapid MEP analysis is expected to be an effective screening tool with use of confirmatory SEQ analysis.     

Effect of a dietary supplement containing glucosamine hydrochloride, chondroitin sulfate and quercetin glycosides on symptomatic knee osteoarthritis: a randomized, double-blind, placebo-controlled study

BACKGROUND: Oral glucosamine and chondroitin sulfate, alone and in combination, have been used worldwide for the treatment of osteoarthritis (OA), but their efficacy is controversial. This clinical study was aimed at investigating the potential of a dietary supplement containing glucosamine and chondroitin sulfate in combination with derivatives of quercetin, a naturally occurring flavonoid, (GCQ supplement) for knee OA care. RESULTS: A randomized, double‐blind, placebo‐controlled study was conducted in 40 Japanese subjects with symptomatic knee OA. Subjects were randomly assigned to GCQ supplement (1200 mg glucosamine hydrochloride, 60 mg chondroitin sulfate and 45 mg quercetin glycosides per day) or placebo and the treatment and follow‐up were continued for 16 weeks. The results of symptomatic efficacy assessment based on Japanese Orthopaedic Association criteria showed that scores for two of the four symptom/function subscales, as well as the aggregate scores, were significantly improved at week 16 or earlier in the GCQ group compared to the placebo group. Moreover, analyses of cartilage metabolism biomarkers showed a trend of improvement in type II collagen synthesis/degradation balance in the GCQ group during follow‐up. CONCLUSION: GCQ supplement was thought to be more effective than placebo in decreasing the intensity of knee OA‐associated clinical symptoms. Copyright © 2011 Society of Chemical Industry     

Flow-induced vibrations in closed side branch pipes and their attenuation methods

Some problems due to flow-induced vibrations related to closed side branch pipes have been observed in thermal and nuclear power plants. Fluctuating pressure generated in the main pipes was unusually, acoustically excited in closed side branch pipes, and intense vibrations were caused at pipes and components. For example, flow-excited acoustic resonance in closed side branches of stub pipes of safety relief valves caused the failure of steam dryers in the United States Quad City Unit 2 nuclear power plant. Furthermore, there was a possibility that residual air or gas in a closed side branch pipe unexpectedly caused severe vibrations of low frequency in the feed water piping system. We have investigated the root cause and influence of air on severe vibrations. Intense fluctuating pressure was often caused by water hammer due to valve closure and it became larger in the closed side branch pipes. We showed that an additional side branch with an orifice was very effective to suppress the flow-induced acoustic resonance. Design methods of the orifice to attenuate fluctuating pressure generated by water hammer were presented considering Mach number, the pressure loss coefficient of orifice and the intensity of particle velocity. Moreover, suitability of the characteristic curve method was confirmed for evaluation of the attenuation effect of an orifice on fluctuating pressure generated by water hammer. Finally, we considered some flow-induced vibration problems related to closed side branch pipes and their attenuation methods.     

Pathological Roles of Pulmonary Cells in Acute Lung Injury: Lessons from Clinical Practice

Interstitial lung diseases (ILD) are relatively rare and sometimes become life threatening. In particular, rapidly progressive ILD, which frequently presents as acute lung injury (ALI) on lung histopathology, shows poor prognosis if proper and immediate treatments are not initiated. These devastating conditions include acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF), clinically amyopathic dermatomyositis (CADM), epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-induced lung injury, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection named coronavirus disease 2019 (COVID-19). In this review, clinical information, physical findings, laboratory examinations, and findings on lung high-resolution computed tomography and lung histopathology are presented, focusing on majorly damaged cells in each disease. Furthermore, treatments that should be immediately initiated in clinical practice for each disease are illustrated to save patients with these diseases.     

Heart current in an anatomically realistic human model due to contact with a low-frequency energized source

Distributions of 60 Hz contact current around and inside the heart of the anatomically realistic human body are demonstrated under four scenarios: left hand to both feet, right hand to both feet, hand to hand, and foot to foot. The heart-current ratios and the heart-current factors are calculated. The new numerical method which is partly modified from the conventional scalar potential finite difference (SPFD) method is proposed. It is found that a part of ∼33–40% of the total contact current passes through the heart in three scenarios of one hand to either the other hand or feet, and these heart-current ratios are much larger than the ones reported by Freiberger. Furthermore, the heart-current factor is almost the same as the one in IEC 60479-1 for the right-hand-to-feet path, but is twice as much as the one in IEC 60479-1 for the hand-to-hand path. Copyright © 2009 Institute of Electrical Engineers of Japan. Published by John Wiley & Sons, Inc.     

Toward Engineered Biosynthesis of Drugs in Human Cells

Biosynthetic genes are not only responsible for the formation of bioactive substances but also suited for other applications including gene therapy. To test the feasibility of human cells producing antibiotics in situ when provided with a heterologous biosynthetic gene, we focused on cytochrome P450, the class of enzymes important in conferring bioactivity to natural product precursors. We selected Fma-P450 that plays a central role in the fumagillin antimicrobial biosynthesis in Aspergillus fumigatus to examine fungal metabolite production by HeLa cells that express fma-P450 heterologously. Here we show that HeLa cells harboring fma-P450 can biosynthesize 5-hydroxyl-β-trans-bergamoten and cytotoxic 5-epi-demethoxyfumagillol when supplemented with the nontoxic precursor β-trans-bergamotene. While the production level was insufficient to effect cell death, we demonstrate that programming human cells to autogenerate antibiotics by introducing a heterologous biosynthetic gene is feasible.