Kinetic analysis of E. coli inactivation by high hydrostatic pressure with salts

Inactivation of E. coli by high hydrostatic pressure (250 to 400 MPa) with salts was investigated based on kinetic analysis. At concentrations from 0.074 to 0.145 M and from 0.240 to 0.290 M, both the absolute activation volumes and the preexponential factors were similar in KCl, NaCl, and LiCl solutions, suggesting that pressure inactivation is not salt-specific. On the other hand, in the intermediate salt-concentration range of 0.145 to 0.240 M, inactivation kinetics in the presence of the Na(+) and K(+) differed significantly from those in the presence of Li(+) (P < 0.05). In this concentration range, effect of salt stress and osmotic stress differed significantly from those in concentrations below 0.145 M or above 0.240 M. The cellular response to pressure varies with salt type and salt concentration. These novel findings provide important clues to distinguish between salt stress and osmotic stress in the inactivation of E. coli.     

Immunoassay based on monoclonal antibody for aflatoxin detection in poultry feed

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on an anti-aflatoxin B₁ monoclonal antibody was standardised and validated for aflatoxin screening in poultry feed samples and its performance was compared to high-performance liquid chromatography (HPLC). The ic-ELISA showed good linearity (r² = 0.994) and detection limits of 1.25 ng g⁻¹ for broiler feed and 1.41 ng g⁻¹ for laying hen feed. Mean aflatoxin recovery rates by ic-ELISA were 102% (laying hen feed) and 98% (broiler feed). Aflatoxins were detected in 88.2% of the 34 broiler feed samples by ic-ELISA and HPLC at means of 10.48 ng g⁻¹ and 8.41 ng g⁻¹, respectively, while 92% of laying hen feed samples (n = 36) showed aflatoxin contamination at means of 20.83 and 19.75 ng g⁻¹. The standardised ic-ELISA showed reliability and a high correlation with HPLC of 0.97 (broiler feed) and 0.98 (laying hen feed) indicating its potential for aflatoxin screening in poultry feed samples.     

Production of Fab fragment corresponding to surface protein antigen of Streptococcus mutans serotype c-derived peptide by Escherichia coli and cultured tobacco cells

The cDNA of a mouse Fab fragment was cloned from a hybridoma cell line that produces a mouse monoclonal antibody, KH5, that reacts with the peptide fragment of the surface protein antigen of Streptococcus mutans serotype c (PAc). After transfection with cDNA, recombinant Fab fragments were produced by Escherichia coli (T15 Fab) and cultured tobacco cells (X253 and X262 Fabs). The antipeptide activities of T15 and X253 were similar to that of KH5. X253 was secreted into the culture media, which had a specific affinity for the PAc peptide.     

Inhibitory effects of acidic xylooligosaccharide on stress-induced gastric inflammation in mice

The preventive effects of acidic xylooligosaccharide prepared from xylan of corncobs and related sugars on stress-induced gastric inflammation in mice were investigated. Oral administration of acidic xylooligosaccharide and hydrocortisone at doses of 100 and 200 mg/kg body weight significantly reduced the number of bleeding points in the gastric mucosa of mice loaded with cold-restraint stress. Acidic xylooligosaccharide showed concentration-dependent superoxide anion radical-scavenging activity at concentrations of 3.3-4.3 mg/mL and its IC50 was 3.5 mg/mL, although this value is approximately six times that of quercetin. The antioxidant activity of acidic xylooligosaccharide could contribute, in part, to its suppressive activities on stress-induced mouse gastritis. Xylose, xylobiose, xylan, and glucuronic acid showed no significant suppressive activities on mouse gastric inflammation at a dose of 100 mg/kg body weight. These results suggest that an appropriate degree of polymerization of xylan (larger than trimer) is necessary for the activities of acidic xylooligosaccharide.     

Review Processing and mechanical properties of fine-grained magnesium alloys

Magnesium alloys are promising light structural materials. The present paper focuses on fine-grained magnesium-based materials. Grain refinement is attained by hot working without additional treatments. Also, a very small grain size of less than 1 m is obtained by equal channel angular extrusion. A good combination of high strength and high ductility at room temperature is attained by grain refinement. Furthermore, fine-grained magnesium-based materials exhibit superplastic behavior at high stain rates (10^–1 s^–1) or low temperatures (473 K). These point out the importance of grain refinement to process magnesium-based materials with excellent mechanical properties.     

In Vitro Tumor Cell-Binding Assay to Select High-Binding Antibody and Predict Therapy Response for Personalized 64Cu-Intraperitoneal Radioimmunotherapy against Peritoneal Dissemination of Pancreatic Cancer: A Feasibility Study

Peritoneal dissemination of pancreatic cancer has a poor prognosis. We have reported that intraperitoneal radioimmunotherapy using a ⁶⁴Cu-labeled antibody (⁶⁴Cu-ipRIT) is a promising adjuvant therapy option to prevent this complication. To achieve personalized ⁶⁴Cu-ipRIT, we developed a new in vitro tumor cell-binding assay (⁶⁴Cu-TuBA) system with a panel containing nine candidate ⁶⁴Cu-labeled antibodies targeting seven antigens (EGFR, HER2, HER3, TfR, EpCAM, LAT1, and CD98), which are reportedly overexpressed in patients with pancreatic cancer. We investigated the feasibility of ⁶⁴Cu-TuBA to select the highest-binding antibody for individual cancer cell lines and predict the treatment response in vivo for ⁶⁴Cu-ipRIT. ⁶⁴Cu-TuBA was performed using six human pancreatic cancer cell lines. For three cell lines, an in vivo treatment study was performed with ⁶⁴Cu-ipRIT using high-, middle-, or low-binding antibodies in each peritoneal dissemination mouse model. The high-binding antibodies significantly prolonged survival in each mouse model, while low-and middle-binding antibodies were ineffective. There was a correlation between in vitro cell binding and in vivo therapeutic efficacy. Our findings suggest that ⁶⁴Cu-TuBA can be used for patient selection to enable personalized ⁶⁴Cu-ipRIT. Tumor cells isolated from surgically resected tumor tissues would be suitable for analysis with the ⁶⁴Cu-TuBA system in future clinical studies.     

NLRP3 Inflammasome Negatively Regulates RANKL-Induced Osteoclastogenesis of Mouse Bone Marrow Macrophages but Positively Regulates It in the Presence of Lipopolysaccharides

In inflammatory bone diseases such as periodontitis, the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3) inflammasome accelerates bone resorption by promoting proinflammatory cytokine IL-1β production. However, the role of the NLRP3 inflammasome in physiological bone remodeling remains unclear. Here, we investigated its role in osteoclastogenesis in the presence and absence of lipopolysaccharide (LPS), a Gram-negative bacterial component. When bone marrow macrophages (BMMs) were treated with receptor activator of nuclear factor-κB ligand (RANKL) in the presence of NLRP3 inflammasome inhibitors, osteoclast formation was promoted in the absence of LPS but attenuated in its presence. BMMs treated with RANKL and LPS produced IL-1β, and IL-1 receptor antagonist inhibited osteoclastogenesis, indicating IL-1β involvement. BMMs treated with RANKL alone produced no IL-1β but increased reactive oxygen species (ROS) production. A ROS inhibitor suppressed apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) speck formation and NLRP3 inflammasome inhibitors abrogated cytotoxicity in BMMs treated with RANKL, indicating that RANKL induces pyroptotic cell death in BMMs by activating the NLRP3 inflammasome via ROS. This suggests that the NLRP3 inflammasome promotes osteoclastogenesis via IL-1β production under infectious conditions, but suppresses osteoclastogenesis by inducing pyroptosis in osteoclast precursors under physiological conditions.     

Coronin1C Is a GDP-Specific Rab44 Effector That Controls Osteoclast Formation by Regulating Cell Motility in Macrophages

Osteoclasts are multinucleated bone-resorbing cells that are formed by the fusion of macrophages. Recently, we identified Rab44, a large Rab GTPase, as an upregulated gene during osteoclast differentiation that negatively regulates osteoclast differentiation. However, the molecular mechanisms by which Rab44 negatively regulates osteoclast differentiation remain unknown. Here, we found that the GDP form of Rab44 interacted with the actin-binding protein, Coronin1C, in murine macrophages. Immunoprecipitation experiments revealed that the interaction of Rab44 and Coronin1C occurred in wild-type and a dominant-negative (DN) mutant of Rab44, but not in a constitutively active (CA) mutant of Rab44. Consistent with these findings, the expression of the CA mutant inhibited osteoclast differentiation, whereas that of the DN mutant enhanced this differentiation. Using a phase-contrast microscope, Coronin1C-knockdown osteoclasts apparently impaired multinuclear formation. Moreover, Coronin1C knockdown impaired the migration and chemotaxis of RAW-D macrophages. An in vivo experimental system demonstrated that Coronin1C knockdown suppresses osteoclastogenesis. Therefore, the decreased cell formation and fusion of Coronin1C-depleted osteoclasts might be due to the decreased migration of Coronin1C-knockdown macrophages. These results indicate that Coronin1C is a GDP-specific Rab44 effector that controls osteoclast formation by regulating cell motility in macrophages.     

Effects of Two Distinct Psychoactive Microbes,Lacticaseibacillus rhamnosus JB-1 and Limosilactobacillus reuteri 6475, on Circulating and Hippocampal mRNA in Male Mice

Discovery of the microbiota-gut–brain axis has led to proposed microbe-based therapeutic strategies in mental health, including the use of mood-altering bacterial species, termed psychobiotics. However, we still have limited understanding of the key signaling pathways engaged by specific organisms in modulating brain function, and evidence suggests that bacteria with broadly similar neuroactive and immunomodulatory actions can drive different behavioral outcomes. We sought to identify pathways distinguishing two psychoactive bacterial strains that seemingly engage similar gut–brain signaling pathways but have distinct effects on behaviour. We used RNAseq to identify mRNAs differentially expressed in the blood and hippocampus of mice following Lacticaseibacillus rhamnosus JB-1, and Limosilactobacillus reuteri 6475 treatment and performed Gene Set Enrichment Analysis (GSEA) to identify enrichment in pathway activity. L. rhamnosus, but not L. reuteri treatment altered several pathways in the blood and hippocampus, and the rhamnosus could be clearly distinguished based on mRNA profile. In particular, L. rhamnosus treatment modulated the activity of interferon signaling, JAK/STAT, and TNF-alpha via NF-KB pathways. Our results highlight that psychobiotics can induce complex changes in host gene expression, andin understanding these changes, we may help fine-tune selection of psychobiotics for treating mood disorders.     

Analysis of Selenoprotein F Binding to UDP-Glucose:Glycoprotein Glucosyltransferase (UGGT) by a Photoreactive Crosslinker

In the endoplasmic reticulum glycoprotein quality control system, UDP-glucose : glycoprotein glucosyltransferase (UGGT) functions as a folding sensor. Although it is known to form a heterodimer with selenoprotein F (SelenoF), the details of the complex formation remain obscure. A pulldown assay using co-transfected SelenoF and truncated mutants of human UGGT1 (HUGT1) revealed that SelenoF binds to the TRXL2 domain of HUGT1. Additionally, a newly developed photoaffinity crosslinker was selectively introduced into cysteine residues of recombinant SelenoF to determine the spatial orientation of SelenoF to HUGT1. The crosslinking experiments showed that SelenoF formed a covalent bond with amino acids in the TRXL3 region and the interdomain between βS2 and GT24 of HUGT1 via the synthetic crosslinker. SelenoF might play a role in assessing and refining the disulfide bonds of misfolded glycoproteins in the hydrophobic cavity of HUGT1 as it binds to the highly flexible region of HUGT1 to reach its long hydrophobic cavity. Clarification of the SelenoF-binding domain of UGGT and its relative position will help predict and reveal the function of SelenoF from a structural perspective.