Establishing Monoclonal Gammopathy of Undetermined Significance as an Independent Pre-Disease State of Multiple Myeloma Using Raman Spectroscopy, Dynamical Network Biomarker Theory, and Energy Landscape Analysis.

Multiple myeloma (MM) is a cancer of plasma cells. Normal (NL) cells are considered to pass through a precancerous state, such as monoclonal gammopathy of undetermined significance (MGUS), before transitioning to MM. In the present study, we acquired Raman spectra at three stages-834 NL, 711 MGUS, and 970 MM spectra-and applied the dynamical network biomarker (DNB) theory to these spectra. The DNB analysis identified MGUS as the unstable pre-disease state of MM and extracted Raman shifts at 1149 and 1527-1530 cm-1 as DNB variables. The distribution of DNB scores for each patient showed a significant difference between the mean values for MGUS and MM patients. Furthermore, an energy landscape (EL) analysis showed that the NL and MM stages were likely to become stable states. Raman spectroscopy, the DNB theory, and, complementarily, the EL analysis will be applicable to the identification of the pre-disease state in clinical samples.

Practices, Potential, and Perspectives for Detecting Predisease Using Raman Spectroscopy.

Raman spectroscopy shows great potential for practical clinical applications. By analyzing the structure and composition of molecules through real-time, non-destructive measurements of the scattered light from living cells and tissues, it offers valuable insights. The Raman spectral data directly link to the molecular composition of the cells and tissues and provides a "molecular fingerprint" for various disease states. This review focuses on the practical and clinical applications of Raman spectroscopy, especially in the early detection of human diseases. Identifying predisease, which marks the transition from a healthy to a disease state, is crucial for effective interventions to prevent disease onset. Raman spectroscopy can reveal biological processes occurring during the transition states and may eventually detect the molecular dynamics in predisease conditions.

Efficacy of Vitrectomy With Tamponade Versus No Tamponade for Myopic Traction Maculopathy: A Multicenter Study (SCHISIS Report No.1).

PURPOSE: To evaluate the influence of tamponade on the visual and anatomic outcomes of pars plana vitrectomy for myopic traction maculopathy (MTM). DESIGN: Multicenter, retrospective clinical cohort study. METHODS: Consecutive eyes that underwent vitrectomy for advanced MTM with tamponade of air, sulfur hexafluoride (SF6), or perfluoropropane (C3F8) or without tamponade with a minimum follow-up of 12 months were included. Main outcome measures included postoperative visual acuity (VA) at 12 months in eyes with vs without tamponade. RESULTS: We included a total of 193 eyes (193 patients) in this study; 136 eyes (70%) treated with tamponade were compared with 57 eyes (30%) treated without tamponade. Baseline characteristics did not differ significantly between the groups. Both groups showed significant visual improvement at 12 months (both P < .001). However, postoperative visual acuity and visual improvement at 12 months were significantly better (P = .003 and P = .028, respectively) in eyes without tamponade, although the MTM in these eyes without tamponade took longer to resolve (P = .039). Retinal thickness and the ellipsoid zone were more preserved in eyes without tamponade (P < .001 and P = .001, respectively). Complications such as macular holes did not differ between the groups. A novel imaging finding of "schisis bending (accordioning)" was identified during MTM resolution. CONCLUSIONS: Vitrectomy either with or without tamponade for MTM was effective in improving vision in this study. However, eyes without tamponade experienced even better visual improvement and preserved retinal anatomy, despite a longer schisis resolution time. Surgery without tamponade may achieve better visual outcomes.

Risk Factors and Outcomes of Postoperative Macular Hole Formation after Vitrectomy for Myopic Traction Maculopathy: SCHISIS Report No. 2.

PURPOSE: To evaluate the incidence, pathogenesis, risk factors, and treatment outcomes of postoperative macular hole (MH) after pars plana vitrectomy (PPV) for myopic traction maculopathy (MTM). DESIGN: Multicenter, interventional, retrospective case series. SUBJECTS: Consecutive eyes that underwent PPV for MTM with a minimum 6-month follow-up. METHODS: We investigated the characteristics and treatment outcomes of postoperative MH after MTM surgery. MAIN OUTCOME MEASURES: Incidence, risk factors, and anatomic and visual outcomes of postoperative MH. RESULTS: We included 207 eyes (207 patients) with a mean follow-up of 25.9 months. During follow-up, 24 (11.6%) eyes developed MH (10 with concurrent MH retinal detachment); 15 eyes within 30 days (early), 4 eyes between 31 and 180 days (intermediate), and 5 eyes after 180 days (late). Logistic regression analysis revealed male gender (odds ratio [OR], 2.917; 95% confidence interval [CI], 1.198-7.100; P = 0.018), thinner preoperative choroidal thickness (OR, 0.988; 95% CI, 0.976-1.000; P = 0.048), and use of indocyanine green for internal limiting membrane peeling (OR, 2.960; 95% CI, 1.172-7.476; P = 0.022) as significant risk factors for postoperative MH. Internal limiting membrane peeling with a fovea-sparing technique tended to protect against postoperative MH, but it was not statistically significant (P = 0.096), because 1 eye still developed MH. Postoperative MHs were treated by observation (6 eyes), in-office octafluoropropane (C3F8) gas injection (7 eyes), or PPV (11 eyes). Macular hole closure was achieved in 20 eyes (83%). The hole closure rate was 67% (4/6 eyes) after observation, 71% (5/7 eyes) after C3F8 gas injection, and 91% (10/11 eyes) after PPV. However, visual outcomes were significantly worse for eyes with postoperative MH than those without (0.38 ± 0.43 vs. 0.68 ± 0.46; P = 0.002). CONCLUSIONS: Postoperative MH may occur in 11.6% of patients with MTM at any time after surgery. Retreatment resulted in relatively favorable anatomic closure but unfavorable visual outcomes. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

27-GAUGE PARS PLANA/PLICATA VITRECTOMY FOR PEDIATRIC VITREORETINAL SURGERY.

PURPOSE: To report on the feasibility of 27-gauge (G) vitrectomy for pediatric patients. METHODS: This study is an international, multicenter, retrospective, interventional case series. Participants were patients 17 years or younger who underwent 27-G vitrectomy for various indications. RESULTS: The records of 56 eyes from 47 patients were reviewed. Mean age was 5.7 ± 5.2 years. Diagnoses included retinopathy of prematurity (Stages 3 with vitreous hemorrhage, 4A, 4B, and 5), Terson's syndrome, traumatic macular hole, posterior capsular opacification, endophthalmitis, and others. Instruments used were the 27-G infusion, 27-G vitreous cutter, 27-G light pipe, and 27-G internal limiting membrane forceps. Instrument bending was noted in one (1.8%) case. There were no cases with intraoperative complications, infusion issues, or postoperative endophthalmitis. There were 67/145 (46%) sclerotomies that required suturing, of which most (51/145) were sutured out of precaution. There were four cases (7.1%) that required conversion to a larger gauge and three cases (5.3%) that developed postoperative hypotony. Mean visual acuity improved from logarithm of the minimum angle of resolution 1.32 (20/420) to 0.72 (20/105), after a mean follow-up of 125.1 days (P = 0.01). Anatomic success was achieved in 96.4% of eyes after a single surgery. CONCLUSION: Twenty-seven-gauge vitrectomy was safe and feasible in selected pediatric vitreoretinopathies. Further studies are warranted to examine indications and outcomes.

Efficacy of cooling therapy and α-lipoic acid derivative against chemotherapy-induced alopecia in an animal model.

Chemotherapy-induced alopecia is frequently induced by various regimens of chemotherapy and has a significant impact on mental health and quality of life. However, the effect of available current treatment for chemotherapy-induced alopecia is not sufficient. This study aimed to clarify the therapeutic effects and mechanism of skin cooling and the antioxidant α-lipoic acid derivative on chemotherapy-induced alopecia. We developed a chemotherapy-induced alopecia model of cyclophosphamide (120 µg/g) using Institute of Cancer Research mice. We used cooling therapy and α-lipoic acid derivative application as the treatments. We compared the alopecia score, hair bulb diameter, insulin-like growth factor-1 level, vascular permeability, and apoptosis between the control and treatment groups. The alopecia score significantly improved in each treatment group compared with that in the cyclophosphamide group. Hair bulb diameter significantly improved in the cyclophosphamide + cooling group compared with that in the cyclophosphamide group. The insulin-like growth factor-1 level and vascular permeability level was significantly retained and suppressed, respectively, in each treatment group compared with that in the cyclophosphamide group. The number of apoptotic cells in the vascular endothelium significantly decreased in the cyclophosphamide + α-lipoic acid derivative group compared with that in the cyclophosphamide group. In conclusion, cooling therapy and α-lipoic acid derivative facilitated recovery from chemotherapy-induced alopecia caused by cyclophosphamide through decreasing vascular permeability.

Application of the Dynamical Network Biomarker Theory to Raman Spectra.

The dynamical network biomarker (DNB) theory detects the early warning signals of state transitions utilizing fluctuations in and correlations between variables in complex systems. Although the DNB theory has been applied to gene expression in several diseases, destructive testing by microarrays is a critical issue. Therefore, other biological information obtained by non-destructive testing is desirable; one such piece of information is Raman spectra measured by Raman spectroscopy. Raman spectroscopy is a powerful tool in life sciences and many other fields that enable the label-free non-invasive imaging of live cells and tissues along with detailed molecular fingerprints. Naïve and activated T cells have recently been successfully distinguished from each other using Raman spectroscopy without labeling. In the present study, we applied the DNB theory to Raman spectra of T cell activation as a model case. The dataset consisted of Raman spectra of the T cell activation process observed at 0 (naïve T cells), 2, 6, 12, 24 and 48 h (fully activated T cells). In the DNB analysis, the F-test and hierarchical clustering were used to detect the transition state and identify DNB Raman shifts. We successfully detected the transition state at 6 h and related DNB Raman shifts during the T cell activation process. The present results suggest novel applications of the DNB theory to Raman spectra ranging from fundamental research on cellular mechanisms to clinical examinations.

Prognostic potential and pathological validation of a diagnostic application using Raman spectroscopy in the characterization of degenerative changes in the cartilage of the humeral head.

Significance: Raman spectroscopy is a well-established analytical method in the fields of chemistry, industry, biology, pharmaceutics, and medicine. Previous studies have investigated optical imaging and Raman spectroscopy for osteoarthritis (OA) diagnosis in weight-bearing joints such as hip and knee joints. However, to realize early diagnosis or a curable treatment, it is still challenging to understand the correlations with intrinsic factors or patients' background. Aim: To elucidate the correlation between the Raman spectral features and pathological variations of human shoulder joint cartilage. Approach: Osteoarthritic cartilage specimens excised from the humeral heads of 14 patients who underwent shoulder arthroplasty were assessed by a confocal Raman microscope and histological staining. The Raman spectroscopic dataset of degenerative cartilage was further analyzed by principal component analysis and hierarchical cluster analysis. Results: Multivariate association of the Raman spectral data generated three major clusters. The first cluster of patients shows a relatively high Raman intensity of collagen. The second cluster displays relatively low Raman intensities of proteoglycans (PGs) and glycosaminoglycans (GAGs), whereas the third cluster shows relatively high Raman intensities of PGs and GAGs. The reduced PGs and GAGs are typical changes in OA cartilage, which have been confirmed by safranin­O staining. In contrast, the increased Raman intensities of collagen, PGs, and GAGs may reflect the instability of the cartilage matrix structure in OA patients. Conclusions: The results obtained confirm the correlation between the Raman spectral features and pathological variations of human shoulder joint cartilage. Unsupervised machine learning methods successfully yielded a clinically meaningful classification between the shoulder OA patients. This approach not only has potential to confirm severity of cartilage defects but also to determine the origin of an individual's OA by evaluating the cartilage quality.

Synthesis and Application of AgBiS2 and Ag2S Nanoinks for the Production of IR Photodetectors.

Nanoinks composed of quantum dots (QDs) are applied in light-receiving devices and light-emitting devices such as solar cells and displays. However, since the most widely used QDs, PbS and CdS, are toxic and environmentally concerning, alternative materials need to be developed. We synthesized and analyzed Ag chalcogenide nanoparticles, including AgBiS2 and Ag2S nanoparticles, which are eco-friendly materials. AgBiS2 and Ag2S QD films were prepared by spin-coating nanoparticle solutions and subsequent heat treatment. The effects of the heat treatment on residual ligands and photoluminescence were determined by surface analysis. The photocurrent response of the AgBiS2 and Ag2S QD films was measured in the near-infrared region, and the effect of the heat treatment temperature was investigated. The results indicate that AgBiS2 and Ag2S are prospective materials for near-infrared photodetectors.

Fatty acid composition of the methylotrophic yeast Komagataella phaffii grown under low- and high-methanol conditions.

In this study, we analysed the intracellular fatty acid profiles of Komagataella phaffii during methylotrophic growth. K. phaffii grown on methanol had significantly lower total fatty acid contents in the cells compared with glucose-grown cells. C18 and C16 fatty acids were the predominant fatty acids in K. phaffii, although the contents of odd-chain fatty acids such as C17 fatty acids were also relatively high. Moreover, the intracellular fatty acid composition of K. phaffii changed in response to not only carbon sources but also methanol concentrations: C17 fatty acids and C18:2 content increased significantly as methanol concentration increased, whereas C18:1 and C18:3 contents were significantly lower in methanol-grown cells. The intracellular content of unidentified compounds (Cn H2n O4 ), on the other hand, was significantly greater in cells grown on methanol. As the intracellular contents of these Cn H2n O4 compounds were significantly higher in a gene-disrupted strain for glutathione peroxidase (gpx1Δ) than in the wild-type strain, we presume that the Cn H2n O4 compounds are fatty acid peroxides. These results indicate that K. phaffii can coordinate intracellular fatty acid composition during methylotrophic growth in order to adapt to high-methanol conditions and that certain fatty acid species such as C17:0, C17:1, C17:2 and C18:2 may be related to the physiological functions by which K. phaffii adapts to high-methanol conditions.

Label-free detection of human enteric nerve system using Raman spectroscopy: A pilot study for diagnosis of Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR) is characterized by the absence of an enteric nerve system (ENS). To remove aganglionosis, bowel reconstruction is only a curative treatment. It is mandatory to identify the extent of aganglionosis during surgery. Raman spectroscopy is a nondestructive chemical analysis technique that provides detailed information regarding molecular vibrations. The purpose of this study is to detect the ENS using Raman spectroscopy in the human intestine for diagnosis of HSCR. METHODS: The Raman spectra of each layer of the gastrointestinal wall were collected from surgical specimens of the human rectum. Based on collected spectral data, principal component analysis was performed to determine the ENS. Subsequently, the Raman spectra of HSCR sections were analyzed. RESULTS: Molecular structures of the gastrointestinal wall were characterized by Raman spectroscopy. Raman spectroscopy could discriminate between ganglion and muscle layers, and the spectra of the border between muscle layers in the aganglionosis were collagen-associated peaks. Either absence on presence of ENS was also confirmed in HSCR material. CONCLUSIONS: Label-free detection of the ENS was successfully demonstrated using Raman spectroscopy. Since this is a preliminary study, the strategy which may contribute to differentiate between ganglionic and aganglionic segments using noninvasive techniques in HSCR should be evaluated by prospective studies in near future.

Role of increased vascular permeability in chemotherapy-induced alopecia: In vivo imaging of the hair follicular microenvironment in mice.

Chemotherapy-induced alopecia is one of the most difficult adverse events of cancer treatment for patients. However, it is still unknown why anticancer drugs cause hair loss. We aimed to clarify the mechanism of chemotherapy-induced alopecia in mice using an in vivo imaging technique with a two-photon microscope, which enables observation of the deep reaction in the living body in real time. In this study, ICR mice were injected intraperitoneally with cyclophosphamide (120 µg/g). Changes in the hair bulb morphology, subcutaneous vessel permeability, and vessel density were evaluated by two-photon microscopy and conventional methods. In order to determine whether there is a causal relationship between vascular permeability and hair loss, we combined cyclophosphamide (50 µg/g) with subcutaneous histamine. Using two­photon microscopy and conventional examination, we confirmed that the hair bulbs became smaller, blood vessels around the hair follicle decreased, and vascular permeability increased at 24 hours after cyclophosphamide injection [corrected]. Apoptosis occurred in vascular endothelial cells around the hair follicle. Additionally, hair loss was exacerbated by temporarily enhancing vascular permeability with histamine. In conclusion, cyclophosphamide caused a decrease in vascular density and an increase in vascular permeability, therefore increased vascular permeability might be one of the causes of chemotherapy-induced alopecia.

Multiple calcium sources are required for intracellular calcium mobilization during gastric organoid epithelial repair.

Calcium (Ca2+ ) is a known accelerator for gastric wound repair. We have demonstrated in vivo and in vitro that intracellular Ca2+ increases in the gastric epithelial cells directly adjacent to a damaged cell, and that this Ca2+ rise is essential for the cellular migration that rapidly repairs the epithelium (restitution). While intracellular Ca2+ has been shown to be an important signaling factor during epithelial restitution, the source from which this intracellular Ca2+ originates remains unclear. Using gastric organoids derived from mice transgenic for a genetically encoded Ca2+ indicator, we sought to investigate the potential sources of intracellular Ca2+ mobilization. During confocal imaging, photodamage (PD) was induced to 1-2 gastric organoid epithelial cells and epithelial restitution measured simultaneously with changes in intracellular Ca2+ (measured as FRET/CFP ratio in migrating cells adjacent to the damaged area). Inhibition of voltage-gated Ca2+ channels (verapamil, 10 µM) or store-operated calcium entry (YM58483, 20 µM) resulted in delayed repair and dampened intracellular Ca2+ response. Furthermore, inhibition of phospholipase C (U73122, 10 µM) or inositol trisphosphate receptor (2-APB, 50 µM) likewise resulted in delayed repair and dampened Ca2+ response. Results suggest both extracellular and intracellular Ca2+ sources are essential for supplying the Ca2+ mobilization that stimulates repair.

Fucosyltransferase 8 plays a crucial role in the invasion and metastasis of pancreatic ductal adenocarcinoma.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. It is an aggressive malignancy associated with poor prognosis because of recurrence, metastasis, and treatment resistance. Aberrant glycosylation of cancer cells triggers their migration and invasion and is considered one of the most important prognostic cancer biomarkers. The current study aimed to identify glycan alterations and their relationship with the malignant potential of PDAC. METHODS: Using a lectin microarray, we evaluated glycan expression in 62 PDAC samples. Expression of fucosyltransferase 8 (FUT8), the only enzyme catalyzing core fucosylation, was investigated by immunohistochemistry. The role of FUT8 in PDAC invasion and metastasis was confirmed using an in vitro assay and a xenograft peritoneal metastasis mouse model. RESULTS: The microarray data demonstrated that core fucose-binding lectins were significantly higher in carcinoma than in normal pancreatic duct tissues. Similarly, FUT8 protein expression was significantly higher in carcinoma than in normal pancreatic duct tissues. High FUT8 protein expression was significantly associated with lymph-node metastases and relapse-free survival. FUT8 knockdown significantly reduced the invasion in PDAC cell lines and impaired peritoneal metastasis in the xenograft model. CONCLUSIONS: The findings of this study provide evidence that FUT8 plays a pivotal role in PDAC invasion and metastasis and might be a therapeutic target for this disease.

Pharmacokinetic and toxicodynamic evaluation of 5-fluorouracil administration after major hepatectomy in a rat model.

BACKGROUND: Chemotherapy after hepatectomy for colorectal liver metastasis has not been established, due to the toxic side effects, which are likely related to impaired drug clearance during liver regeneration. We investigated the pharmacokinetic and toxicodynamic evaluation of 5-fluorouracil (5-FU) during liver regeneration after major hepatectomy in a rat model. METHODS: Thirty-six male Wistar rats were divided into control (C), control with chemotherapy (CC), hepatectomy (H), and hepatectomy with chemotherapy (HC) groups. The CC and HC groups were administered 5-FU for 4 days. Plasma 5-FU, liver weight, and liver dihydropyrimidine dehydrogenase (DPD) were measured. The ileal villous height was measured to determine adverse effects. RESULTS: The area under the curve and maximum plasma concentration of 5-FU increased by up to 51% and 32%, respectively, in the HC group compared to the CC group. The liver regeneration rate was significantly lower in the HC group than in the H group (67.3 ± 7.4 vs 33.0 ± 5.7%, p < 0.001). The HC group had a significantly lower liver DPD than the CC group (4.4 ± 1.1 mg vs 6.9 ± 1.1 mg, p < 0.01). The HC group had a significantly lower ileal villous height than the CC group (253 ± 40 µm vs. 318 ± 36 µm, p < 0.05). CONCLUSIONS: Reduction of the total liver DPD following major hepatectomy caused increased plasma 5-FU levels and 5-FU-associated toxicity.

Valve Interstitial Cell-Specific Cyclooxygenase-1 Associated With Calcification of Aortic Valves.

BACKGROUND: The molecular mechanisms underlying aortic valve calcification are poorly understood. Here, we aimed to identify the master regulators of calcification by comparison of genes in valve interstitial cells (VICs) with calcified and noncalcified aortic valves. METHODS: Calcified aortic valves were surgically excised from patients with aortic valve stenosis who required aortic valve replacements. Noncalcified and calcified sections were obtained from aortic valve leaflets. Collagenase-digested tissues were seeded into dishes, and VICs adhering to the dishes were cultured for 3 weeks, followed by comprehensive gene expression analysis. Functional analyses of identified proteins were performed by in vitro calcification assays. Tissue localization was determined by immunohistochemical staining for normal (n = 11) and stenotic valves (n = 30). RESULTS: We found 87 genes showing greater than a twofold change in calcified tissues. Among these genes, 68 were downregulated and 19 were upregulated. Cyclooxygenase-1 (COX1) messenger RNA and protein levels were upregulated in VICs from calcified tissues. The COX1 messenger RNA and protein levels in VICs were also strongly increased by stimulation with osteoblast differentiation medium. These were VIC-specific phenotypes and were not observed in other cell types. Immunohistochemical staining revealed that COX1-positive VICs were specifically localized in the calcified area of aortic valve tissues. CONCLUSIONS: The VIC-specific COX1 overexpression played a crucial role in calcification by promoting osteoblast differentiation in aortic valve tissues.

Multicenter survey of sutureless 27-gauge vitrectomy for primary rhegmatogenous retinal detachment: a consecutive series of 410 cases.

PURPOSE: To evaluate the surgical outcomes of the 27-gauge (G) vitrectomy system for the treatment of primary rhegmatogenous retinal detachment (RRD). METHODS: This retrospective consecutive series multicenter study involved a total of 410 eyes of 406 patients who underwent 3-port transconjunctival 27G pars plana vitrectomy (PPV) for RRD between November 2014 and December 2016 and who were followed for a minimum of 3 months postoperative. The main outcome measure was primary reattachment, with the secondary outcome measures being final reattachment, improvement of visual acuity (VA), intraocular pressure (IOP), intraoperative and postoperative complications, and surgery time. RESULTS: Of the 410 treated eyes, primary reattachment was achieved in 392 (95.6%) and final reattachment was achieved in 410 (100%). In 226 eyes (55.1%) with macula-on RRD, the mean logarithm of the minimum angle of resolution (logMAR) VA improved from 0.16 ± 0.51 pre-surgery to 0.02 ± 0.14 post-surgery (P = 0.11). In 184 eyes (44.9%) with macula-off RRD, logMAR VA improved from 1.06 ± 0.77 pre-surgery to 0.26 ± 0.35 post-surgery (P < 0.001). Following surgery, the mean IOP was highest at 1 day (15.7 ± 7.0 mmHg) postoperative. In all eyes, surgery was concluded without the use of sutures or the need of conversion to a larger-gauge instrument. Although hypotony was observed in 14 (3.4%) of the 410 treated eyes at 1 day postoperative, it spontaneously resolved within 1 week without additional surgical intervention. No postoperative complications such as infectious endophthalmitis were observed throughout the follow-up period. CONCLUSION: Our findings show that 27G PPV is both safe and effective for the treatment of primary RRD.

Trefoil factor 2 activation of CXCR4 requires calcium mobilization to drive epithelial repair in gastric organoids.

KEY POINTS: Determining the signalling cascade of epithelial repair, using murine gastric organoids, allows definition of regulatory processes intrinsic to epithelial cells, at the same time as validating and dissecting the signalling cascade with more precision than is possible in vivo Following single cell damage, intracellular calcium selectively increases within cells adjacent to the damage site and is essential for promoting repair. Trefoil factor 2 (TFF2) acts via chemokine C-X-C receptor 4 and epidermal growth factor receptor signalling, including extracellular signal-regulated kinase activation, to drive calcium mobilization and promote gastric repair. Sodium hydrogen exchanger 2, although essential for repair, acts downstream of TFF2 and calcium mobilization. ABSTRACT: The gastric mucosa of the stomach is continually exposed to environmental and physiological stress factors that can cause local epithelial damage. Although much is known about the complex nature of gastric wound repair, the stepwise process that characterizes epithelial restitution remains poorly defined. The present study aimed to determine the effectors that drive gastric epithelial repair using a reductionist culture model. To determine the role of trefoil factor 2 (TFF2) and intracellular calcium (Ca2+ ) mobilization in gastric restitution, gastric organoids were derived from TFF2 knockout (KO) mice and yellow Cameleon-Nano15 (fluorescent calcium reporter) transgenic mice, respectively. Inhibitors and recombinant protein were used to determine the upstream and downstream effectors of gastric restitution following photodamage (PD) to single cells within the gastric organoids. Single cell PD resulted in parallel events of dead cell exfoliation and migration of intact neighbouring cells to restore a continuous epithelium in the damage site. Under normal conditions following PD, Ca2+ levels increased within neighbour migrating cells, peaking at ∼1 min, suggesting localized Ca2+ mobilization at the site of cell protrusion/migration. TFF2 KO organoids exhibit delayed repair; however, this delay can be rescued by the addition of exogenous TFF2. Inhibition of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK)1/2 or a TFF2 receptor, chemokine C-X-C receptor 4 (CXCR4), resulted in significant delay and dampened Ca2+ mobilization. Inhibition of sodium hydrogen exchanger 2 (NHE2) caused significant delay but did not affect Ca2+ mobilization. A similar delay was observed in NHE2 KO organoids. In TFF2 KO gastric organoids, the addition of exogenous TFF2 in the presence of EGFR or CXCR4 inhibition was unable to rescue repair. The present study demonstrates that intracellular Ca2+ mobilization occurs within gastric epithelial cells adjacent to the damage site to promote repair by mechanisms that involve TFF2 signalling via CXCR4, as well as activation of EGFR and ERK1/2. Furthermore NHE2 is shown to be important for efficient repair and to operate via a mechanism either downstream or independent of calcium mobilization.

Raman Spectroscopic Analysis to Detect Reduced Bone Quality after Sciatic Neurectomy in Mice.

Bone mineral density (BMD) is a commonly used diagnostic indicator for bone fracture risk in osteoporosis. Along with low BMD, bone fragility accounts for reduced bone quality in addition to low BMD, but there is no diagnostic method to directly assess the bone quality. In this study, we investigated changes in bone quality using the Raman spectroscopic technique. Sciatic neurectomy (NX) was performed in male C57/BL6J mice (NX group) as a model of disuse osteoporosis, and sham surgery was used as an experimental control (Sham group). Eight months after surgery, we acquired Raman spectral data from the anterior cortical surface of the proximal tibia. We also performed a BMD measurement and micro-CT measurement to investigate the pathogenesis of osteoporosis. Quantitative analysis based on the Raman peak intensities showed that the carbonate/phosphate ratio and the mineral/matrix ratio were significantly higher in the NX group than in the Sham group. There was direct evidence of alterations in the mineral content associated with mechanical properties of bone. To fully understand the spectral changes, we performed principal component analysis of the spectral dataset, focusing on the matrix content. In conclusion, Raman spectroscopy provides reliable information on chemical changes in both mineral and matrix contents, and it also identifies possible mechanisms of disuse osteoporosis.

Determination of Formaldehyde in Water Samples by High-Performance Liquid Chromatography with Methyl Acetoacetate Derivatization.

A high-performance liquid chromatography method with methyl acetoacetate derivatization via the Hantzsch reaction was developed for the analysis of formaldehyde (HCHO) in several water samples. Under optimized conditions, HCHO was detected within 4 min and was not affected by excessive derivatization reagents. The calibration curve constructed from the peak height of HCHO was linear, with a correlation coefficient of 0.9998. The relative standard deviation of the peak height from ten replicates was 0.29%. The detection and quantitative limits were 0.96 µg/L and 3.16 µg/L, respectively. A recovery test of HCHO was performed to compare the developed method with the official analysis method (DNPH method). The developed method was used to determine the HCHO levels in several water samples (tap water, river water, and waste water).