Solid-state modifications of poly(γ-ethyl-L-glutamate)

Several modification of the arrangements of α-helical molecules were found in the solid films of poly (γ-ethyl-L -glutamate), depending on the casting solvent and the temperature. The helical conformation is somewhat looser than the normal 18-residue, 5-turn α-helix. Using x-ray diffraction, the types of molecular arrangements were classified into tetragonal, pseudohexagonal, and hexagonal ones. Tetragonal packing was observed in the filmm (form T) prepared by casting the solution in trifluorethanol or dichlorethane. The sample obtained from chloroform solution is a well-ordered, pseudohexagonal modification (form I). Forms I and T change into a poorly crystalline form III by annealing at temperatures above 130° C. It is particularly noteworthy that the less-ordered form III exhibits a thermoreversible transition around 110°C into a well-ordered form H with the hexagonal molecular packing.     

Iodine distribution in the thyroid follicles of the hagfish,Eptatretus burgeri and lamprey,Lampetra japonica: Electron-probe X-ray microanalysis

Summary In the thyroid follicles of species of cyclostomes, a hagfish and a lamprey, the distribution of stable iodine was examined by electron-probe X-ray microanalysis. A high concentration of stable iodine, heterogeneously distributed, was observed in the follicular cells of hagfish thyroid follicles. In the lamprey a low concentration of iodine was seen in the follicular lumina. The relative values for stable iodine determined in this way corresponded to values obtained by a chemical analytical method.     

Hepatic stellate cells function as regulatory bystanders

Regulatory T cells (Tregs) contribute significantly to the tolerogenic nature of the liver. The mechanisms, however, underlying liver-associated Treg induction are still elusive. We recently identified the vitamin A metabolite, retinoic acid (RA), as a key controller that promotes TGF-β-dependent Foxp3(+) Treg induction but inhibits TGF-β-driven Th17 differentiation. To investigate whether the RA producing hepatic stellate cells (HSC) are part of the liver tolerance mechanism, we investigated the ability of HSC to function as regulatory APC. Different from previous reports, we found that highly purified HSC did not express costimulatory molecules and only upregulated MHC class II after in vitro culture in the presence of exogenous IFN-γ. Consistent with an insufficient APC function, HSC failed to stimulate naive OT-II TCR transgenic CD4(+) T cells and only moderately stimulated α-galactosylceramide-primed invariant NKT cells. In contrast, HSC functioned as regulatory bystanders and promoted enhanced Foxp3 induction by OT-II TCR transgenic T cells primed by spleen dendritic cells, whereas they greatly inhibited the Th17 differentiation. Furthermore, the regulatory bystander capacity of the HSC was completely dependent on their ability to produce RA. Our data thus suggest that HSC can function as regulatory bystanders, and therefore, by promoting Tregs and suppressing Th17 differentiation, they might represent key players in the mechanism that drives liver-induced tolerance.     

Effects of acorn masting on population dynamics of three forest-dwelling rodent species in Hokkaido,Japan

The effects of the abundance of acorns of the oak, Quercus crispula, on the population dynamics of three rodent species (Apodemus speciosus, A. argenteus, and Clethrionomys rufocanus) were analyzed using time series data (1992–2006). The data were obtained in a forest in northern Hokkaido, Japan, by live trapping rodents and directly counting acorns on the ground. Apodemus speciosus generally increased in abundance following acorn masting. However, the clear effect of acorn abundance was not detected for the other two rodent species. Acorns of Q. crispula contain tannins, which potentially have detrimental effects on herbivores. Apodemus speciosus may reduce the damage caused by acorn tannins with tannin-binding salivary proteins and tannase-producing bacteria, whereas such physiological tolerance to tannins is not known in the other two rodent species. The differences in the effects of acorns between the three species may be due to differences in their physiological tolerance to tannins.     

Cytotoxic enhancement of a bispecific diabody by format conversion to tandem single-chain variable fragment (taFv): the case of the hEx3 diabody

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.     

Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling

MyD88 is a Toll/IL-1 receptor (TIR) domain-containing adapter common to signaling pathways via Toll-like receptor (TLR) family. However, accumulating evidence demonstrates the existence of a MyD88-independent pathway, which may explain unique biological responses of individual TLRs, particularly TLR3 and TLR4. TIR domain-containing adapter protein (TIRAP)/MyD88 adapter-like, a second adapter harboring the TIR domain, is essential for MyD88-dependent TLR2 and TLR4 signaling pathways, but not for MyD88-independent pathways. Here, we identified a novel TIR domain-containing molecule, named TIR domain-containing adapter inducing IFN-beta (TRIF). As is the case in MyD88 and TIRAP, overexpression of TRIF activated the NF-kappaB-dependent promoter. A dominant-negative form of TRIF inhibited TLR2-, TLR4-, and TLR7-dependent NF-kappaB activation. Furthermore, TRIF, but neither MyD88 nor TIRAP, activated the IFN-beta promoter. Dominant-negative TRIF inhibited TLR3-dependent activation of both the NF-kappaB-dependent and IFN-beta promoters. TRIF associated with TLR3 and IFN regulatory factor 3. These findings suggest that TRIF is involved in the TLR signaling, particularly in the MyD88-independent pathway.     

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.     

Identification of 2-phenylethanol with a rose-like odor from anal sac secretions of the small Indian mongoose (Herpestes auropunctatus)

The small Indian mongoose (Herpestes auropunctatus) is an invasive species in Okinawa and Amami-Oshima, Japan. Major strategies for their eradication have been the use of baited traps, which suffer from decreasing efficiency with declining populations and the bycatch of native animals. To address these concerns, mongoose-specific lures are required. In this study, we aimed to identify species- and/or sex-specific compounds from anal sac secretions of small Indian mongooses. Volatile compounds emitted from male and female mongoose anal sac secretions were analyzed by thermal desorption-gas chromatography-mass spectrometry. In addition to several fatty acids, 2-phenylethanol was identified as a minor compound, which is uncommon in mammalian secretions but a dominant odorant in roses. Female samples emitted higher levels of 2-phenylethanol than male samples did. These findings indicate that 2-phenylethanol is a female-specific volatile compound of anal sac secretions in small Indian mongooses, and it may be useful as an ingredient of mongoose-specific scent lures.     

Flexible Ion‐Conducting Composite Membranes for Lithium Batteries

The use of metallic lithium anodes enables higher energy density and higher specific capacity Li‐based batteries. However, it is essential to suppress lithium dendrite growth during electrodeposition. Li‐ion‐conducting ceramics (LICC) can mechanically suppress dendritic growth but are too fragile and also have low Li‐ion conductivity. Here, a simple, versatile, and scalable procedure for fabricating flexible Li‐ion‐conducting composite membranes composed of a single layer of LICC particles firmly embedded in a polymer matrix with their top and bottom surfaces exposed to allow for ionic transport is described. The membranes are thin (<100 μm) and possess high Li‐ion conductance at thicknesses where LICC disks are mechanically unstable. It is demonstrated that these membranes suppress Li dendrite growth even when the shear modulus of the matrix is lower than that of lithium. It is anticipated that these membranes enable the use of metallic lithium anodes in conventional and solid‐state Li‐ion batteries as well as in future Li? S and Li? O₂ batteries.     

Overexpression of glutathione peroxidase attenuates myocardial remodeling and preserves diastolic function in diabetic heart

Oxidative stress plays an important role in the structural and functional abnormalities of diabetic heart. Glutathione peroxidase (GSHPx) is a critical antioxidant enzyme that removes H(2)O(2) in both the cytosol and mitochondia. We hypothesized that the overexpression of GSHPx gene could attenuate left ventricular (LV) remodeling in diabetes mellitus (DM). We induced DM by injection of streptozotocin (160 mg/kg ip) in male GSHPx transgenic mice (TG+DM) and nontransgenic wildtype littermates (WT+DM). GSHPx activity was higher in the hearts of TG mice compared with WT mice, with no significant changes in other antioxidant enzymes. LV thiobarbituric acid-reactive substances measured in TG+DM at 8 wk were significantly lower than those in WT+DM (58 +/- 3 vs. 71 +/- 5 nmol/g, P < 0.05). Heart rate and aortic blood pressure were comparable between groups. Systolic function was preserved normal in WT+DM and TG+DM mice. In contrast, diastolic function was impaired in WT+DM and was improved in TG+DM as assessed by the deceleration time of peak velocity of transmitral diastolic flow and the time needed for relaxation of 50% maximal LV pressure to baseline value (tau; 13.5 +/- 1.2 vs. 8.9 +/- 0.7 ms, P < 0.01). The TG+DM values were comparable with those of WT+Control (tau; 7.8 +/- 0.2 ms). Improvement of LV diastolic function was accompanied by the attenuation of myocyte hypertrophy, interstitial fibrosis, and apoptosis. Overexpression of GSHPx gene ameliorated LV remodeling and diastolic dysfunction in DM. Therapies designed to interfere with oxidative stress might be beneficial to prevent cardiac abnormalities in DM.