Understanding the quality and safety of food production through the lens of The Microbiome of The Built Environment.

The Microbiome of the Built Environment (MoBE) is profoundly implicated in various sectors, including food science. The balance between beneficial and pathogenic microbes in these facilities directly influences product quality and public health. Maintaining a careful check on MoBE and external microbes is vital to the food industry to ensure quality control. There is also a risk of contamination in the meat processing facility as well. However, over-sanitization can increase drug-resistant microbes, highlighting the importance of balanced microbial management. Additionally, facility design, influenced by understanding MoBE, can optimize the growth of beneficial microbes and inhibit pathogenic microbes. Microbial mapping, an emerging practice, offers insights into microbial hotspots within facilities, resulting in targeted interventions. As the food industry evolves, the intricate understanding and management of MoBE will be pivotal to ensuring optimal food quality, safety, and innovation.

The Similarities in Microbial and Chemical Patterns of Fermentation in Two Open Environments were Promoted by Using 150-Year-Old Nukadoko as Starters.

Nukadoko, a fermented rice bran employed in traditional Japanese pickling, uses lactic acid bacteria to ferment vegetables. Here, we report the microbial and chemical data of a mixture of matured 150-year-old nukadoko and commercially available rice bran placed in two open environments over 29 days. Across the two environments, Loigolactobacillus was identified as the dominant microbial genera in the later stages of fermentation in nukadoko. The period of increase in the relative abundance of Loigolactobacillus correlated with a decrease in pH and Oxidation-Reduction Potential (ORP) values. While the two environments showed a difference in the rate of change in microbial diversity, they shared the common process through which Loigolactobacillus outcompeted adventitious bacteria in nukadoko, as indicated by the alpha and beta diversity index. Thus, the similarities in microbial and chemical data across two open environments during fermentation using starters indicate that starters contribute to the stability of fermentation in open environments.

Endocrinological evaluation of dawn phenomenon in patients with diabetes and comparison of insulin glargine U-100 biosimilar (Insulin Glargine BS Injection "Lilly") and glargine U-300 (Lantus XR): a randomized controlled study.

We investigated the pathophysiology of the dawn phenomenon by examining the effects of changes in blood glucose levels from late night to early morning on various hormones in a group taking glargine BS and a group taking Lantus XR, with the goal of achieving better glycemic control. Patients with types 1 and 2 diabetes scheduled for inpatient education were divided into BS and XR groups. Blood glucose levels were tracked from 0:00 to 7:00, while blood samples were extracted at 3:00 and 7:00 to measure glucose levels and hormones related to the dawn phenomenon. Overall, we analyzed blood sample and intermittently scanned Continuous Glucose Monitoring data of 43 and 40 patients, respectively. From 0:00 to 7:00, the mean blood glucose was significantly lower in the BS group, although the fluctuation was similar (p < 0.0001). The BS group also exhibited significantly higher ∆ACTH (p = 0.0215) and ∆ cortisol (p = 0.0430) than the XR group. In the BS group, ∆Glu exhibited a significant negative correlation with ∆ACTH and ∆cortisol (p = 0.0491). Similar findings were not observed in the XR group. These results suggest that XR may be a better choice for long-acting insulin since it is less likely to induce cortisol secretion. Further, analysis of the dawn phenomenon and non-dawn phenomenon groups showed the mean CPR levels at 3:00 and 7:00 were significantly higher in the latter (p = 0.0135). This supports the conventional belief that appropriate basal insulin replacement therapy is a beneficial treatment for the dawn phenomenon.

In Vivo Gene Expression Profile of Human Intestinal Epithelial Cells: From the Viewpoint of Drug Metabolism and Pharmacokinetics.

Orally administered drugs are absorbed and metabolized in the intestine. To accurately predict pharmacokinetics in the intestine, it is essential to understand the intestinal expression profiles of the genes related to drug absorption, distribution, metabolism, and excretion (ADME). However, in many previous studies, gene expression analysis in the intestine has been carried out using specimens from patients with cancer. In this study, to obtain more accurate gene expression profiles, biopsy samples were collected under endoscopic observation from the noninflammatory regions of 14 patients with inflammatory bowel disease, and RNA-seq analysis was performed. Gene expression analysis of drug-metabolizing enzymes (cytochromes P450), non-cytochrome P450 enzymes, nuclear receptors, drug-conjugating enzymes (UDP-glucuronosyltransferases and sulfotransferases), and apical and basolateral drug transporters was performed in biopsy samples from the duodenum, ileum, colon, and rectum. The proportions of the cytochromes P450 expressed in the ileum were 25% (CYP3A4), 19% (CYP2C18), and 14% (CYP3A5). CYP3A4 and CYP2C19 were highly expressed in the duodenum and ileum, but not in the colon and rectum. In the ileum, apical transporters such as P-gp, peptide transporter 1, breast cancer resistance protein, MRP2, and ASBT were strongly expressed, and the expression levels of P-gp and ASBT in the ileum were higher than those in other regions. In the ileum, basolateral transporters such as OSTα, OSTß, and MRP3 were strongly expressed. We succeeded in obtaining gene expression profiles of ADME-related genes in human intestinal epithelial cells in vivo. We expect that this information would be useful for accurate prediction of the pharmacokinetics of oral drugs. SIGNIFICANCE STATEMENT: To obtain gene expression profiles of ADME-related genes in human intestinal epithelial cells in vivo, biopsy samples were collected under endoscopic observation from the noninflammatory regions of 14 patients with inflammatory bowel disease, and RNA-seq analysis was performed. Gene expression profiles of drug-metabolizing enzymes (cytochromes P450), non-cytochrome P450 enzymes, nuclear receptors, drug-conjugating enzymes (UDP-glucuronosyltransferases and sulfotransferases), and apical and basolateral drug transporters in biopsy samples from the duodenum, ileum, colon, and rectum were obtained in this study.

Preventive Effects of Grape Extract on Ischemia/Reperfusion-Induced Acute Kidney Injury in Mice.

Since grape extract (GE) contains oligomeric proanthocyanidins and numerous polyphenols, dietary GE supplements may exert protective effects against various diseases. The present study investigated the pharmacological effects of GE derived from Chardonnay in vitro and in vivo. GE (100 µg/mL) completely inhibited tumor necrosis factor-α-induced endothelin-1, monocyte chemoattractant protein-1, interleukin-1ß, and intercellular adhesion molecule-1 gene expression in cultured endothelial cells. GE also strongly stimulated the phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) pathway. In the in vivo study, the effects of GE on ischemic acute kidney injury (AKI) were examined using male C57bl/6J wild-type and eNOS-/- mice. Right nephrectomized mice were exposed to 45 min of ischemia in the left kidney and this was followed by reperfusion. Although renal functional parameters in AKI mice significantly increased 48 h after reperfusion, the administration of GE (0.1 and 1 mg/kg, intravenous (i.v.)) 5 min before ischemia dose-dependently improved post-ischemic renal dysfunction in wild-type mice. Renal histopathological studies on AKI mice revealed tubular necrosis, proteinaceous casts in tubuli, and medullary congestion. The administration of GE ameliorated this damage in wild-type mice, but not in eNOS-/- mice. Furthermore, GE significantly restored decreases in the renal nitric oxide metabolite content due to ischemia in wild-type mice, but not in eNOS-/- mice. Thus, eNOS is closely involved in the renoprotective effects of GE, strongly suggesting that GE supplements are useful as a prophylactic treatment for the development of ischemic AKI.

T1-Weighted Magnetic Resonance Carotid Plaque Imaging: a Comparison between Conventional and Fast Spin-Echo Techniques.

BACKGROUND: Magnetic resonance imaging is widely used to evaluate the intraplaque components of the cervical carotid artery. The non-gated T1-weighted spin-echo (SE) technique has been reported to have an excellent ability for discriminating stable and unstable plaques. However, the diagnostic performance of various SE-based techniques remains unclear. Hence, we compared plaque signals obtained by 3 kinds of SE-based methods with histological findings. METHODS: We prospectively examined 40 patients who underwent carotid endarterectomy by using 1.5-T scanners and obtained 2-dimensional (2D) conventional spin-echo (CSE), 2D fast spin-echo (FSE), and 3-dimensional (3D)-FSE images with identical repetition times. We calculated contrast ratios (CRs) of the plaques against adjacent muscles and compared these values with the pathological classification of the specimens. RESULTS: The CRs of type VII-VIII (calcific/fibrous), IV-V (lipid-rich/necrotic), and VI (complex/hemorrhagic) plaques were significantly different between all the methods (P <.001) and were discriminated from each other at sensitivities of 83%-100% and specificities of 94%-100%. The CRs of type IV-V plaques significantly differed between the methods (low to high, 2D-FSE, 2D-CSE, and 3D-FSE; P <.05); those of the type VI plaques were significantly lower with the 2D-FSE method than with the other methods (P <.01). CONCLUSIONS: The SE-based T1-weighted images can readily discriminate plaque characteristics with high sensitivities and specificities, although the signal intensity of unstable plaques was significantly high on the 3D-FSE images and significantly low on the 2D-FSE images.

Expression analysis of Baf60c during heart regeneration in axolotls and neonatal mice.

Some organisms, such as zebrafish, urodele amphibians, and newborn mice, have a capacity for heart regeneration following injury. However, adult mammals fail to regenerate their hearts. To know why newborn mice can regenerate their hearts, we focused on epigenetic factors, which are involved in cell differentiation in many tissues. Baf60c (BRG1/BRM-associated factor 60c), a component of ATP-dependent chromatin-remodeling complexes, has an essential role for cardiomyocyte differentiation at the early heart development. To address the function of Baf60c in postnatal heart homeostasis and regeneration, we examined the detailed expression/localization patterns of Baf60c in both mice and axolotls. In the mouse heart development, Baf60c was highly expressed in the entire heart at the early stages, but gradually downregulated at the postnatal stages. During heart regeneration in neonatal mice and axolotls, Baf60c expression was strongly upregulated after resection. Interestingly, the timing of Baf60c upregulation after resection was consistent with the temporal dynamics of cardiomyocyte proliferation. Moreover, knockdown of Baf60c downregulated proliferation of neonatal mouse cardiomyocytes. These data suggested that Baf60c plays an important role in cardiomyocyte proliferation in heart development and regeneration. This is the first study indicating that Baf60c contributes to the heart regeneration in vertebrates.

Intracranial Plaque Characterization in Patients with Acute Ischemic Stroke Using Pre- and Post-Contrast Three-Dimensional Magnetic Resonance Vessel Wall Imaging.

BACKGROUND: Magnetic resonance vessel wall imaging (VWI) techniques have been developed to assess atherosclerotic plaques in intracranial arteries, which are a cardinal cause of ischemic stroke. However, the clinical roles of plaque-related vulnerability and inflammation remain unclear. Hence, we evaluated plaque characteristics using VWI of the proximal middle cerebral artery (M1) in patients with acute ischemic stroke. METHODS: We prospectively examined 30 consecutive patients with acute noncardioembolic stroke in the M1 territory using pre-/postcontrast T1-weighted (T1W) three-dimensional (3D) VWI with a 3-Tesla scanner. The contrast ratio (CR) and contrast enhancement of the plaques were measured bilaterally at M1. RESULTS: Plaques were identified in the bilateral M1s of all patients, and no substantial stenosis existed. The M1 plaque CRs ipsilateral to the infarct (46.7%-67.9%) were significantly higher than the plaque CRs on the contralateral side (34.3%-69.4%), particularly in patients with lacunar infarcts (P <.01). In contrast, the occurrence of plaque enhancement was not different between the ipsilateral (20.0%) and contralateral (16.7%) sides. Further, the CRs in the nonlacunar group were significantly higher than the CRs in the lacunar group (P <.05), whereas enhanced plaques tended to be more frequent in the nonlacunar group, but this difference was not significant (P = .09). CONCLUSIONS: T1W 3D-VWI revealed that the signal intensity of M1 plaques was significantly higher in the affected side and in nonlacunar-type infarcts of patients with acute stroke, suggesting that unstable plaques in the M1 can cause stroke events presumably due to atherothrombotic mechanisms.

Adaptive mutation related to cellulose producibility in Komagataeibacter medellinensis (Gluconacetobacter xylinus) NBRC 3288.

Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.

Differential reparative phenotypes between zebrafish and medaka after cardiac injury.

BACKGROUND: Zebrafish have the ability for heart regeneration. However, another teleost animal model, the medaka, had not yet been investigated for this capacity. RESULTS: Compared with zebrafish, the medaka heart responded differently to an injury: An excessive fibrotic response occurred in the medaka heart, and existing cardiomyocytes or cardiac progenitor cells remained dormant, resulting in no numerical difference between the uncut and injured heart with respect to the number of EdU-incorporated cardiomyocytes. The results obtained from the analysis of the medaka raldh2-GFP transgenic line showed a lack of raldh2 expression in the endocardium. Regarding periostin expression, the localization of medaka periostin-b, a marker of fibrillogenesis, in the medaka heart remained at the wound site at 30 dpa; whereas zebrafish periostin-b was no longer localized at the wound but was detected in the epicardium at that time. CONCLUSIONS: Compared with zebrafish heart regeneration, the medaka heart phenotypes suggest the possibility that the medaka could hardly regenerate its heart tissue or that these phenotypes for heart regeneration showed a delay.

Effect of dulaglutide and long-acting insulin combination therapy in patients with type 2 diabetes: a retrospective observational study.

Objective: The study aimed to evaluate the long-term effects of combination therapy comprising dulaglutide and long-acting insulin, on glycemic control in patients with type 2 diabetes. Methods: This retrospective observational study included 20 patients with type 2 diabetes who underwent blood glucose management with intensive insulin therapy for a limited period. All patients were switched from intensive insulin therapy to combination therapy comprising dulaglutide and long-acting insulin. Hemoglobin A1c was evaluated before and 4, 12, and 24 weeks after starting combination therapy. Continuous glucose monitoring was conducted before and 1 and 24 weeks after starting combination therapy. Results: Hemoglobin A1c levels were significantly reduced after 4, 12, and 24 weeks of combination therapy (- 2.2% ± 0.4%, P < 0.0001; - 3.7% ± 0.8%, P = 0.0003; and - 3.6% ± 0.8%, P = 0.0005, respectively). Glycemic variability (% coefficient of variation) was significantly decreased after 1 and 24 weeks of combination therapy (- 5.7% ± 2.1%, P = 0.011; and - 8.7% ± 2.4%, P = 0.003, respectively) and the percentage of readings and time > 250 mg/dL at 24 weeks was significantly improved (- 2.2% ± 0.8%, P = 0.019). Conclusion: Combination therapy with dulaglutide and long-acting insulin resulted in better blood glucose control than intensive insulin therapy, which persisted for 24 weeks. Combination therapy also reduced blood glucose fluctuations and the number of self-injections needed. Supplementary Information: The online version contains supplementary material available at 10.1007/s13340-022-00592-z.

Direct contact of fermented rice bran beds promotes food-to-hand transmission of lactic acid bacteria.

The skin microbiome, which varies widely between individuals, plays a crucial role in human health. It also interacts with the environment in various ways, including during the preparation of fermented food. Nukadoko is a pickle and traditional fermented food in Japan that utilizes lactic acid bacteria to ferment vegetables. When preparing or maintaining Nukadoko, it is mixed with bare hands. Despite the known interaction between Nukadoko and human skin, no studies have explored its impact on Nukadoko quality or skin microbiome changes. This study examines these effects during Nukadoko maintenance. Three participants were asked to stir commercially available late-stage Nukadoko for 14 days and not stir it for the remaining 14 days to examine microbial settlement and shedding. Microbiome analysis was performed on human skin and Nukadoko. We found that microorganisms from rice bran beds can temporarily settle on human skin but are shed quickly. Stirring rice bran beds by hand may have short-term effects on the skin microbiome. This study provides insights into the communication between human and food microbiomes in traditional Japanese fermented foods.

A dark matter in sake brewing: Origin of microbes producing a Kimoto-style fermentation starter.

Introduction: In Kimoto-style fermentation, a fermentation starter is produced before the primary brewing process to stabilize fermentation. Nitrate-reducing bacteria, mainly derived from brewing water, produce nitrite, and lactic acid bacteria such as Leuconostoc can proliferate because of their tolerance toward low temperature and their low nutritional requirements. Later, Lactobacillus becomes the dominant genus, leading to weakly acidic conditions that contribute to control yeasts and undesired bacterial contaminants. However, the sources of these microorganisms that play a pivotal role in Sake brewing have not yet been revealed. Thus, comprehensive elucidation of the microbiome is necessary. Methods: In this study, we performed 16S rRNA amplicon sequencing analysis after sampling from floor, equipment surfaces, and raw materials for making fermentation starters, including koji, and water in Tsuchida Sake brewery, Gunma, Japan. Results: Amplicon sequence variants (ASVs) between the external environments and the fermentation starter were compared, and it was verified that the microorganisms in the external environments, such as built environments, equipment surfaces, and raw materials in the sake brewery, were introduced into the fermentation starter. Furthermore, various adventitious microbes present in the fermentation starter of early days and from the external environments were detected in a nonnegligible proportion in the starter, which may impact the taste and flavor. Discussion: These findings illuminate the uncharacterized microbial dark matter of sake brewing, the sources of microbes in Kimoto-style fermentation.

Impact of P-glycoprotein on intracellular drug concentration in peripheral blood mononuclear cells and K562 cells.

P-glycoprotein (P-gp) expression in lymphocytes is variable and 2-fold higher in rheumatoid arthritis (RA) patients with treatment resistance than in healthy subjects. To date the information on P-gp-mediated drug interaction in lymphocyte is limited. We analyzed the importance on P-gp in lymphocytes using peripheral blood mononuclear cells (PBMCs) together with K562, K562/Adr, and K562/Vin cells, which have various P-gp levels, as cell models, and dexamethasone, nintedanib and apafant as weak to good P-gp substrates. P-gp levels in K562, K562/Adr, and K562/Vin cells were 0.3-, 20-, and 106-fold of healthy PBMCs, respectively. While cell accumulation of apafant and nintedanib decreased in all cells with increasing P-gp levels, dexamethasone accumulation in K562/Adr was comparable to that in healthy PBMCs and K562 cells. Cell accumulations of substrates in cells with low P-gp expression were not significantly changed by the P-gp inhibitors at therapeutic concentrations. However, accumulation increased to 1.4-fold at highest in K562/Adr cells with higher P-gp expression than in PBMCs of the RA patients. These results suggest P-gp controls the cellular concentration of P-gp substrates in PBMCs or K562 cells but cellular concentration of a weak P-gp substrate would not be apparently affected even in cells with a sufficient P-gp expression.

A unique case in which Kimoto-style fermentation was completed with Leuconostoc as the dominant genus without transitioning to Lactobacillus.

The Kimoto-style fermentation starter is a traditional preparation method of sake brewing. In this process, specific microbial transition patterns have been observed within nitrate-reducing bacteria and lactic acid bacteria during the production process of the fermentation starter. We have characterized phylogenetic compositions and diversity of the bacterial community in a sake brewery performing the Kimoto-style fermentation. Comparing the time-series changes with other sake breweries previously reported, we found a novel type of Kimoto-style fermentation in which the microbial transition differed significantly from other breweries during the fermentation step. Specifically, the lactic acid bacteria, Leuconostoc spp. was a predominant species in the late stage in the preparation process of fermentation starter, on the other hand, Lactobacillus spp., which plays a pivotal role in other breweries, was not detected in this analysis. The discovery of this new variation of microbiome transition in Kimoto-style fermentation has further deepened our understanding of the diversity of sake brewing.

Functional integration of protein A binding ability to antibody fragments for convenient and tag-free purification.

Although the development of small therapeutic antibodies is important, the affinity tags used for their purification often result in heterogeneous production and immunogenicity. In this study, we integrated Staphylococcus aureus protein A (SpA) binding ability into antibody fragments for convenient and tag-free purification. SpA affinity chromatography is used as a global standard purification method for conventional antibodies owing to its high binding affinity to the Fc region. SpA also has a binding affinity for some variable heavy domains (VH) classified in the VH3 subfamily. Through mutagenesis based on alignment and structural modeling results using the SpA-VH3 cocrystal structure, we integrated the SpA-binding ability into the anti-CD3 single-chain Fv. Furthermore, we applied this mutagenesis approach to more complicated small bispecific antibodies and successfully purified the antibodies using SpA affinity chromatography. The antibodies retained their biological function after purification. Integration of SpA-binding ability into conventional antibody fragments simplifies the purification and monitoring of the production processes and, thus, is an ideal strategy for accelerating the development of small therapeutic antibodies. Furthermore, because of its immunoactivity, the anti-CD3 variable region with SpA-binding ability is an effective building block for developing engineered cancer therapeutic antibodies without the Fc region.

Comparison of the effects of empagliflozin and glimepiride on endothelial function in patients with type 2 diabetes: A randomized controlled study.

Patients with type 2 diabetes who have cardiovascular disease and are receiving empagliflozin have a lower rate of primary composite cardiovascular outcomes. In contrast, glimepiride increases cardiovascular hospitalization when combined with metformin. Here, we assessed the effects of empagliflozin and glimepiride on endothelial function using flow-mediated dilation (FMD). In this prospective, open-label, randomized, parallel-group study, 63 patients with type 2 diabetes received metformin and insulin glargine U100 for 12 weeks. This was followed by additional treatment with empagliflozin or glimepiride for 12 weeks. The primary outcome was the change in the FMD measurement (ΔFMDs) at 24 weeks of additional treatment. Secondary outcomes comprised changes in metabolic markers and body composition. The empagliflozin group (n = 33) and glimepiride group (n = 30) showed no significant differences in ΔFMDs (empagliflozin, -0.11 [95%CI: -1.02, 0.80]%; glimepiride, -0.34 [95%CI: -1.28, 0.60]%; P = 0.73). Additionally, changes in glycated hemoglobin were similar between the two groups. However, a significant difference in body weight change was observed (empagliflozin, -0.58 [95%CI: -1.60, 0.43] kg; glimepiride, 1.20 [95%CI: 0.15, 2.26] kg; P = 0.02). Moreover, a body composition analysis revealed that body fluid volume significantly decreased after empagliflozin treatment (baseline, 35.8 ± 6.8 L; after 12 weeks, -0.33 ± 0.72 L; P = 0.03). Hence, although empagliflozin did not improve endothelial function compared with glimepiride for patients with type 2 diabetes, it did decrease body fluid volumes. Thus, the coronary-protective effect of empagliflozin is not derived from endothelial function protection, but rather from heart failure risk reduction. Trial registration: This trial was registered on September 13, 2016; UMIN000024001.

Mutations in degP and spoT Genes Mediate Response to Fermentation Stress in Thermally Adapted Strains of Acetic Acid Bacterium Komagataeibacter medellinensis NBRC 3288.

An acetic acid bacterium, Komagataeibacter medellinensis NBRC 3288, was adapted to higher growth temperatures through an experimental evolution approach in acetic acid fermentation conditions, in which the cells grew under high concentrations of ethanol and acetic acid. The thermally adapted strains were shown to exhibit significantly increased growth and fermentation ability, compared to the wild strain, at higher temperatures. Although the wild cells were largely elongated and exhibited a rough cell surface, the adapted strains repressed the elongation and exhibited a smaller cell size and a smoother cell surface than the wild strain. Among the adapted strains, the ITO-1 strain isolated during the initial rounds of adaptation was shown to have three indel mutations in the genes gyrB, degP, and spoT. Among these, two dispensable genes, degP and spoT, were further examined in this study. Rough cell surface morphology related to degP mutation suggested that membrane vesicle-like structures were increased on the cell surface of the wild-type strain but repressed in the ITO-1 strain under high-temperature acetic acid fermentation conditions. The ΔdegP strain could not grow at higher temperatures and accumulated a large amount of membrane vesicles in the culture supernatant when grown even at 30°C, suggesting that the degP mutation is involved in cell surface stability. As the spoT gene of ITO-1 lost a 3'-end of 424 bp, which includes one (Act-4) of the possible two regulatory domains (TGS and Act-4), two spoT mutant strains were created: one (ΔTGSAct) with a drug cassette in between the 5'-half catalytic domain and 3'-half regulatory domains of the gene, and the other (ΔAct-4) in between TGS and Act-4 domains of the regulatory domain. These spoT mutants exhibited different growth responses; ΔTGSAct grew better in both the fermentation and non-fermentation conditions, whereas ΔAct-4 did only under fermentation conditions, such as ITO-1 at higher temperatures. We suggest that cell elongation and/or cell size are largely related to these spoT mutations, which may be involved in fermentation stress and thermotolerance.

Development of a POCT type insulin sensor employing anti-insulin single chain variable fragment based on faradaic electrochemical impedance spectroscopy under single frequency measurement.

To improve glycemic control managed through insulin administration, recent studies have focused on developing hand-held point-of-care testing (POCT) electrochemical biosensors for insulin measurement. Amongst them, anti-insulin IgG-based sensors show promise in detecting insulin with high specificity and sensitivity. However, fabrication of electrochemical sensors with IgG antibodies can prove challenging because of their larger molecular size. To overcome these limitations, this study focuses on utilizing the anti-insulin single chain variable fragment (scFv) as a biosensing molecule with single-frequency faradaic electrochemical impedance spectroscopy (EIS). By comparing two different immobilization methods, covalent conjugation via succinimidyl ester and non-covalent poly-histidine chelation, we demonstrated effective modification of the electrode surface with anti-insulin scFv, while retaining its specific recognition toward insulin. Sensor performance was confirmed via the concentration-dependent faradaic electrochemical impedance change using potassium ferricyanide as a redox probe. The optimal frequency for measurement was determined to be the peak slope of the calculated impedance correlation with respect to frequency. Based on the identified optimized frequency, we performed single-frequency measurement of insulin within a concentration range of 10 pM-100 nM. This study can aid in developing a future point-of-care sensor which rapidly and sensitively measures insulin across a dynamic range of physiological concentrations, with label-free detection.

Autologous cell-enriched fat grafting for breast augmentation.

Autologous fat grafting for breast augmentation has faced some historical hurdles. However, in recent years it has been gaining acceptance from the medical community. This prospective, nonrandomized open-label study of 20 Japanese women supports the use of autologous fat grafting in breast augmentation and explores enhancement of fat graft tissue with autologous adipose-derived regenerative cells (ADRCs). After adipose harvesting using syringe liposuction, the tissue is processed in the Celution 800 System(®), which washes the graft and isolates ADRCs. The average cells per gram of harvested adipose tissue was 3.42 × 10(5), and the mean cell viability measured using an automated cell counting system before graft delivery was 85.3%. All patients demonstrated improvement in circumferential breast measurement (BRM) from their baseline state, and breast measurements were stable by 3 months after surgery. The mean BRM 9 months after surgery had increased 3.3 cm from preoperative measurements. Through 9 months, overall physician satisfaction was 69% and patient satisfaction was 75%. No serious or unexpected adverse events were reported, and the procedure was safe and well tolerated in all patients. Postoperative cyst formation was seen in two patients. These prospective results demonstrate that ADRC-enriched fat grafts processed with a closed automated system maintain high cell viability and that the procedure is safe and effective, with all patients showing improvement after a single treatment.