Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l -lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.     

Solubilization of human placental tumor necrosis factor receptors as a complex with a guanine nucleotide-binding protein.

Human placental membranes exhibited high-affinity receptors for tumor necrosis factor (TNF) (Kd = 5.6 x 10(-10) M) with a density of 1.2-1.7 x 10(10) sites/mg protein. The receptors were solubilized from these membranes with 1% Nonidet P-40, and the solubilized receptor was adsorbed to Con A-Sepharose and wheat germ agglutinin agarose columns, indicating that the TNF receptor derived from human placenta contains carbohydrate chains recognized by these lectins. TNF binding activity was eluted from a column of Sephacryl S-300 as a single peak of Mr 300 kDa. The solubilized receptor was further purified by TNF-Sepharose prepared by coupling of TNF to tresyl-activated Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified sample resolved five major bands of Mr 90, 78, 41, 35, and 11 kDa, suggesting that these polypeptides constitute a multimeric complex with a molecular mass of 300 kDa, as observed in gel filtration study. Furthermore, the TNF-Sepharose-bound fraction demonstrated GTP gamma S binding and GTPase activity. Immunoblot analysis showed that the 41- and 35-kDa polypeptides were recognized by antisera against alpha subunits and beta subunit of GTP-binding proteins, respectively. These results suggest that the native TNF receptor couples to a guanine nucleotide-binding protein to form a large complex structure in human placental membranes.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

Lagrangian tracking measurements revealed the temporal dynamics of nitrogen and phosphorus spiralling in a large Japanese river

Limnology - The in-stream processing of nutrients plays an important role in the fluvial nutrient transport from lands to the ocean, but few empirical studies have addressed the temporal dynamics...     

Cleome hassleriana plants fully support the development and reproduction of Nesidiocoris tenuis

BioControl - Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) is a zoophytophagous predator that feeds on plants as well as prey. Several non-crop host plant species have been used to maintain...     

Rapid and accurate quantitative analysis of fermentation gases by Raman spectroscopy

Raman spectroscopy was employed for the precise quantitative analysis of a gaseous mixture. The band ratio between the Raman band of the target gas and that of the external standard was calculated and found to be proportional to the pressure of the target gas. The linearity of the calibration curves was very good (higher than 0.999). The mole fractions of atmospheric nitrogen and atmospheric oxygen were determined from their partial pressures. The average molar fraction of atmospheric nitrogen was calculated to be 0.790, similar to the literature value. Those of methane, hydrogen, and carbon dioxide, which are produced by fermentation, were also determined from the Raman spectrum. The values were identical to those obtained from the measured volume of each gas. The possibility of quantifying gas molecules by Raman spectroscopy is demonstrated.     

Anesthetic Effects of a Mixture of Medetomidine,Midazolam and Butorphanol in Two Strains of Mice

The combination of ketamine and xylazine is a widely used anesthetic for laboratory animals. However, due to an abuse problem in Japan, ketamine has been specified as a narcotic since 2007. Instead of using ketamine, Kawai et al. reported an injectable formula with an equivalent effect to the mixture of ketamine and xylazine [11]. The mixture of 0.3 mg/kg body weight (b.w.) medetomidine (Med.), 4.0 mg/kg b.w. midazoram (Mid.), and 5.0 mg/kg b.w. butorphanol (But.) produced an anesthetic duration of around 40 min in outbred ICR mice. However, the anesthetic effect of the mixture for inbred mice strains remains unknown. Therefore, we examined anesthetic effects of the mixture of Med., Mid., and But. in the BALB/c and C57BL/6J strains. After intraperitoneal injection into mice, right front paw, left hind paw, and tail pinch reflexes as well as corneal and righting reflexes were observed. Every 5 min, we scored each reflex category as 0 for reaction or 1 for no reaction. As long as the total score was at least 4 out of 5, we considered the mixture as putting a mouse in a surgical anesthetic state. The mixture produced an anesthetic duration of more than 45 min in both strains of mice. These results indicate that the mixture of Med., Mid., and But. can be a useful and effective anesthesia for the BALB/c and C57BL/6J strains of inbred mice as well as outbred ICR mice.     

Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (Ret^M919T, Ret^N396K and Ret^Y792F) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. Ret^M919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in Ret^M919T/- mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.     

Effects of providing pollen to Euseius sojaensis or Amblyseius eharai (Acari: Phytoseiidae) on populations of the pink citrus rust mite,Aculops pelekassi (Acari: Eriophyidae)

Applied Entomology and Zoology - The pink citrus rust mite, Aculops pelekassi (Keifer) (Acari: Eriophyidae), is a serious pest and is widely distributed in most citrus-growing areas of the world....