CRASH2p: Closed-loop Two Photon Imaging in Freely Moving Animals.

Direct measurement of neural activity in freely moving animals is essential for understanding how the brain controls and represents behaviors. Genetically encoded calcium indicators report neural activity as changes in fluorescence intensity, but brain motion confounds quantitative measurement of fluorescence. Translation, rotation, and deformation of the brain and the movements of intervening scattering or auto-fluorescent tissue all alter the amount of fluorescent light captured by a microscope. Compared to single-photon approaches, two photon microscopy is less sensitive to scattering and off-target fluorescence, but more sensitive to motion, and two photon imaging has always required anchoring the microscope to the brain. We developed a closed-loop resonant axial-scanning high-speed two photon (CRASH2p) microscope for real-time 3D motion correction in unrestrained animals, without implantation of reference markers. We complemented CRASH2p with a novel scanning strategy and a multistage registration pipeline. We performed volumetric ratiometrically corrected functional imaging in the CNS of freely moving Drosophila larvae and discovered previously unknown neural correlates of behavior.

Multiple asynchronous recurrence as a predictive factor for refractoriness against locoregional and surgical therapy in patients with intermediate-stage hepatocellular carcinoma.

Development of subclassification of intermediate-stage hepatocellular carcinoma (HCC) by treatment suitability is in demand. We aimed to identify predictors that define treatment refractoriness against locoregional(transarterial chemoembolization(TACE) or thermal ablation) and surgical therapy. This multicenter retrospective study enrolled 1167 HCC patients between 2015 and 2021. Of those, 209 patients were initially diagnosed with intermediate-stage HCC. Treatment refractoriness was defined as clinical settings that meets the following untreatable progressive conditions by TACE (1) 25% increase of intrahepatic tumor, (2) transient deterioration to Child-Pugh class C, (3) macrovascular invasion or extrahepatic spread, within one year. We then analyzed factors contributing to treatment refractoriness. The Child-Pugh score/class, number of tumors, infiltrative radiological type, and recurrence were significant factors. Focusing on recurrence as a predictor, median time to untreatable progression (TTUP) was 17.2 months in the recurrence subgroup whereas 35.5 months in the initial occurrence subgroup (HR, 2.06; 95% CI, 1.44-2.96; P = 0.001). Median TTUP decreased in cases with more later times of recurrence (3-5 recurrences, 17.3 months; ≥ 6 recurrences, 7.7 months). Recurrence, even more at later times, leads to increased treatment refractoriness. Early introduction of multidisciplinary treatment should be considered against HCC patients after multiple recurrent episodes.

Design strategy for blends of biodegradable polyester and thermoplastic starch based on a molecular dynamics study of the phase-separated interface.

Molecular insight into the phase-separated interface formed when biodegradable polyesters and thermoplastic starch (TPS) are melt-blended is valuable for the design of composites. In this study, eight different interfaces combining four major biodegradable polyesters (PLA, PBS, PHB and PBAT) and two TPSs [unmodified TPS (nTPS) and citrate-modified TPS (cTPS)] were investigated by using molecular dynamics (MD) simulations. According to the MD simulation results, PBS, PHB and PBAT diffuse readily into the TPS and form compatible interfaces, whereas PLA is less compatible with the TPS. The results of tensile simulations show that PBS and PBAT adhere well to TPS; in particular, PBS/cTPS and PBAT/cTPS exhibit high interfacial-fracture energy (G). Both PLA and PHB blended with TPS exhibit low G because PLA is less compatible with TPS and PHB and TPS have low electrostatic interaction. The reason for the high G of PBS/cTPS and PBAT/cTPS is thought to be a combination of three factors: (i) formation of a deep compatible interface, (ii) suppression of void growth by electrostatic interactions and (iii) absorption of strain energy by a change in the conformation of the molecular chains. These three interfacial adhesion mechanisms should be considered when designing biodegradable polyester/TPS blends with good mechanical properties.

Antifungal activities of vineyard-habitat wild yeast for grape gray-mold disease and its effects on spontaneous winemaking.

Microorganisms, including native yeasts, are abundant in vineyard fields. Herein, we studied the possibility of using vineyard-derived wild yeast as a microbial pesticide against Botrytis cinerea, a pathogen that causes grape gray mold disease, to boost the initial alcohol production of spontaneously fermented wine. We identified the Saccharomyces cerevisiae strain KONDO170908, which showed the most effective antifungal activity in an ex vivo yeast dripping experiment on grape berries. This strain was utilized in an in vivo spray test on grape bunches in vineyard fields and was proven to significantly suppress gray mold disease on the grape berries in test plot #16 when the yeast was sprayed during both the flowering and ripening periods (morbidity 11.2% against 15.3% of the control plot, χ2 test, p < 0.0001). However, in test plot #17, spraying the yeast during only the ripening period had no effect (morbidity 16.3%). The grapes from each test plot were also submitted for spontaneous wine fermentation. Alcoholic fermentation of the grapes from test plot #16 provided the most active bubbling of CO2 gas and the highest ethanol production and colony counts over seven days of fermentation. Unique changes in the different strains of S. cerevisiae among the plots were observed throughout the early fermentation stage. Thus, yeast spraying during the flowering period might trigger modification of the entire microbiota and could ultimately contribute to promoting alcohol production in the spontaneously fermented wine, although it decreased the grape yield by 20%.

Meningeal carcinomatosis with decreased visual acuity: a case report.

Meningeal carcinomatosis is a condition in which cancer cells diffusely metastasize to the cerebral pia mater in the cerebrospinal membrane or cerebrospinal cavity. It causes a wide array of symptoms according to the site of metastasis. The prognosis is poor, especially in metastasis from solid tumors. This study reports a case of meningeal carcinomatosis caused by advanced gastric cancer, manifested by headache and vision loss. The patient was a 69-year-old man who underwent head computed tomography (CT) and magnetic resonance imaging (MRI) for persistent headaches. No abnormal findings were found; however, his vision declined, convulsions occurred, and cerebrospinal fluid (CSF) cytology showed poorly differentiated adenocarcinoma. Therefore, meningeal carcinomatosis was diagnosed. The patient died after receiving FOLFOX therapy to relieve symptoms and prolong his life. An autopsy showed no invasion of the optic nerve or surrounding tissues. As the frequency of complications of meningeal carcinomatosis in solid cancers is rare, it is crucial to actively suspect and make an early diagnosis.

MELAS-Derived Neurons Functionally Improve by Mitochondrial Transfer from Highly Purified Mesenchymal Stem Cells (REC).

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS) syndrome, caused by a single base substitution in mitochondrial DNA (m.3243A>G), is one of the most common maternally inherited mitochondrial diseases accompanied by neuronal damage due to defects in the oxidative phosphorylation system. There is no established treatment. Our previous study reported a superior restoration of mitochondrial function and bioenergetics in mitochondria-deficient cells using highly purified mesenchymal stem cells (RECs). However, whether such exogenous mitochondrial donation occurs in mitochondrial disease models and whether it plays a role in the recovery of pathological neuronal functions is unknown. Here, utilizing induced pluripotent stem cells (iPSC), we differentiated neurons with impaired mitochondrial function from patients with MELAS. MELAS neurons and RECs/mesenchymal stem cells (MSCs) were cultured under contact or non-contact conditions. Both RECs and MSCs can donate mitochondria to MELAS neurons, but RECs are more excellent than MSCs for mitochondrial transfer in both systems. In addition, REC-mediated mitochondrial transfer significantly restored mitochondrial function, including mitochondrial membrane potential, ATP/ROS production, intracellular calcium storage, and oxygen consumption rate. Moreover, mitochondrial function was maintained for at least three weeks. Thus, REC-donated exogenous mitochondria might offer a potential therapeutic strategy for treating neurological dysfunction in MELAS.

Multi-neuronal recording in unrestrained animals with all acousto-optic random-access line-scanning two-photon microscopy.

To understand how neural activity encodes and coordinates behavior, it is desirable to record multi-neuronal activity in freely behaving animals. Imaging in unrestrained animals is challenging, especially for those, like larval Drosophila melanogaster, whose brains are deformed by body motion. A previously demonstrated two-photon tracking microscope recorded from individual neurons in freely crawling Drosophila larvae but faced limits in multi-neuronal recording. Here we demonstrate a new tracking microscope using acousto-optic deflectors (AODs) and an acoustic GRIN lens (TAG lens) to achieve axially resonant 2D random access scanning, sampling along arbitrarily located axial lines at a line rate of 70 kHz. With a tracking latency of 0.1 ms, this microscope recorded activities of various neurons in moving larval Drosophila CNS and VNC including premotor neurons, bilateral visual interneurons, and descending command neurons. This technique can be applied to the existing two-photon microscope to allow for fast 3D tracking and scanning.

Molecular insight into toughening induced by core-shell structure formation in starch-blended bioplastic composites.

Binary and ternary blends with poly(lactic acid) (PLA), poly(butylene succinate) (PBS), and thermoplastic starch (TPS) were prepared by a melt process to produce biodegradable biomass plastics with both economical and good mechanical properties. The mechanical and structural properties of each blend were evaluated. Molecular dynamics (MD) simulations were also conducted to examine the mechanisms underlying the mechanical and structural properties. PLA/PBS/TPS blends showed improved mechanical properties compared with PLA/TPS blends. The PLA/PBS/TPS blends with a TPS ratio of 25-40 wt% showed higher impact strength than PLA/PBS blends. Morphology observations showed that in the PLA/PBS/TPS blends, a structure similar to that of core-shell particles with TPS as the embedding phase and PBS as the coating phase was formed, and that the trends in morphology and impact strength changes were consistent. The MD simulations suggested that PBS and TPS tightly adhered to each other in a stable structure at a specific intermolecular distance. From these results, it is clear that PLA/PBS/TPS blends are toughened by the formation of a core-shell structure in which the TPS core and the PBS shell adhered well together and stress concentration and energy absorption occurred in the vicinity of the core-shell structure.

A Comparative Study on Fungal Diversity in Organic and Conventionally Cultivated Lemons During Accelerated Storage.

There has been a growing interest in organic farming as a countermeasure to the environmental burden caused by chemical pesticides. We analyzed and compared the fungal diversity of lemon fruits from organic and conventional cultivation by automated rRNA intergenic spacer analysis (ARISA), accompanied by isolation of cultured colonies and metagenomic analysis. Lemon peels were cut out and subjected to the analyses at purchase and after accelerated storage at 28 °C. The organic lemons did not decay even after 14 weeks, while most of the conventional lemons did decay. The fungal colony counts were not significantly different, although the number of fungal species together with the Shannon index, considering the abundance of each species, clearly showed more diversity in organic lemons than in conventional lemons (p = 0.011). Fusarium sp. (putative F. solani) accounted for as much as 90% of the relative abundance in the decayed conventional lemons. Metagenomic analysis also supported the lack of fungal diversity in conventional lemons. These results may suggest that organic cultivation maintains the diversity of native fungal flora in lemon fruit and could contribute to preventing decay during ambient storage.

[A Case of Curative Resection of the Local Advanced Gastric Cancer after SOX plus Nivolumab Chemotherapy, and the Pathological Diagnosis Was Complete Response].

A-78-year-old woman presented with difficulty swallowing. She was diagnosed as having advanced upper gastric cancer with invasion to the esophagus and the diaphragm. Biopsy examination of the tumor showed MSI-high and HER2-negative. The patient received 4 courses of SOX plus nivolumab in combination. After the chemotherapy, the size of the tumor decreased remarkably. We performed total gastrectomy with D2 lymph node dissection. Pathological examination revealed no residual cancer at the primary tumor location and the regional lymph nodes. The patient has shown no recurrence for 9 months without adjuvant chemotherapy. We conclude that SOX plus nivolumab in combination as first-line chemotherapy is an effective strategy against advanced gastric cancer.

[A Strategy of Treatment for Perforated Gastric Cancer].

In past reports, the incidence of gastric perforation accounts for 0.08 to 3.6% of all gastric cancers, and the proportion of perforated gastric cancer(PGC)in gastric perforations is 26 to 32%. In the treatment of PGC, critical care for peritonitis, diagnosis of gastric cancer and curability for gastric cancer are required simultaneously, so it is not easy to decide the treatment strategies. Therefore, for the purpose to consider treatment strategies for PGC, we conducted a clinicopathological study on PGC in our hospital for the past 12 years. There were 22 cases of PGC, and we analyzed clinicopathologically 19 cases excluding perforation during endoscopic resection and perforation during chemotherapy. The R0 surgery group tended to have a good prognosis even in PGC cases, and there was surgery-related death in the one-stage gastrectomy group. So it was considered desirable to perform radical surgery after the general condition was stable by the treatment of peritonitis was given priority in the PGC.

[A Patient with Gastric Adenosquamous Cell Carcinoma].

A 73-year-old female was referred from a local clinic with abdominal pain. A diagnosis of gastric cancer(cT3, cN0, M0, cStage ⅡB)and acute cholecystitis was made. Distal gastrectomy, D2, and cholecystectomy were performed. Postoperative pathological examination led to a diagnosis of adenosquamous cell carcinoma(pT3, pN2, M0, pStage ⅢA). SOX therapy was administered as postoperative adjuvant chemotherapy. However, multiple liver metastases were detected. XP and DTX therapies were administered; however, there was a reduction in performance status. The patient died 10 months after surgery. Gastric adenosquamous cell carcinoma is classified as a specific type according to the Japanese Classification of Gastric Carcinoma(15th edition). This carcinoma accounts for 0.3 to 0.5% of patients undergoing gastric cancer surgery and is relatively rare. Its malignancy level is higher than that of gastric adenocarcinoma, and its prognosis is poorer.

Methods to Induce Small-Scale Differentiation of iPS Cells into Dopaminergic Neurons and to Detect Disease Phenotypes.

Disease-specific induced pluripotent stem (iPS) cells are useful tools to analyze the pathology of neurodegenerative diseases. In this chapter, we describe a procedure to efficiently induce small-scale differentiation of patient iPS cells into midbrain dopaminergic neurons to detect cell death and mitochondrial clearance by using immunostaining. A combination of our method described here and an image analysis system, such as the IN Cell Analyzer, will enable the quantitative assessment of cell vulnerability and mitochondrial quality control abnormalities in cells derived from patients with Parkinson's disease; this set-up might be used to perform drug screening.

Hepatic Adenosine Triphosphate Reduction Through the Short-Chain Fatty Acids-Peroxisome Proliferator-Activated Receptor γ-Uncoupling Protein 2 Axis Alleviates Immune-Mediated Acute Hepatitis in Inulin-Supplemented Mice.

How liver tolerance is disrupted in immune-mediated liver injury is currently unclear. There is also insufficient information available regarding susceptibility, precipitation, escalation, and perpetuation of autoimmune hepatitis. To explore how dietary fiber influences hepatic damage, we applied the concanavalin A (ConA)-induced acute immune-mediated liver injury model in mice fed a diet supplemented with 6.8% inulin, a water-soluble fermentable fiber. Twelve hours after ConA administration, inulin-supplemented diet-fed mice demonstrated significantly alleviated hepatic damage histologically and serologically, with down-regulation of hepatic interferon-γ and tumor necrosis factor and reduced myeloperoxidase (MPO)-producing neutrophil infiltration. Preconditioning with an inulin-supplemented diet for 2 weeks significantly reduced hepatic adenosine triphosphate (ATP) content; suramin, a purinergic P2 receptor antagonist, abolished the protective effect. Of note, the portal plasma derived from mice fed the inulin-supplemented diet significantly alleviated ConA-induced immune-mediated liver injury. Mechanistically, increased portal short-chain fatty acid (SCFA) levels, such as those of acetate and butyrate, by inulin supplementation leads to up-regulation of hepatic γ-type peroxisome proliferator-activated receptor (Pparg) and uncoupling protein 2 (Ucp2), which uncouples mitochondrial ATP synthesis downstream of PPARγ. Pparg down-regulating small interfering RNA cancelled the protective effect of inulin supplementation against MPO-producing neutrophil infiltration and the subsequent immune-mediated liver injury, suggesting that the SCFA-PPARγ-UCP2 axis plays a key role in the protective effect by inulin supplementation. Moreover, significant changes in the gut microbiota, including increased operational taxonomic units in genera Akkermansia and Allobaculum, also characterized the protective effect of the inulin-supplemented diet. Conclusion: There is a possible unraveled etiopathophysiological link between the maintenance of liver tolerance and dietary fiber. The SCFA-PPARγ-UCP2 axis may provide therapeutic targets for immune-mediated liver injury in the future.

Acetaldehyde exposure underlies functional defects in monocytes induced by excessive alcohol consumption.

Increased intestinal permeability and hepatic macrophage activation by endotoxins are involved in alcohol-induced liver injury pathogenesis. Long-term alcohol exposure conversely induces endotoxin immune tolerance; however, the precise mechanism and reversibility are unclear. Seventy-two alcohol-dependent patients with alcohol dehydrogenase-1B (ADH1B, rs1229984) and aldehyde dehydrogenase-2 (ALDH2, rs671) gene polymorphisms admitted for alcohol abstinence were enrolled. Blood and fecal samples were collected on admission and 4 weeks after alcohol cessation and were sequentially analyzed. Wild-type and ALDH2*2 transgenic mice were used to examine the effect of acetaldehyde exposure on liver immune responses. The productivity of inflammatory cytokines of peripheral CD14+ monocytes in response to LPS stimulation was significantly suppressed in alcohol dependent patients on admission relative to that in healthy controls, which was partially restored by alcohol abstinence with little impact on the gut microbiota composition. Notably, immune suppression was associated with ALDH2/ADH1B gene polymorphisms, and patients with a combination of ALDH2*1/*2 and ADH1B*2 genotypes, the most acetaldehyde-exposed group, demonstrated a deeply suppressed phenotype, suggesting a direct role of acetaldehyde. In vitro LPS and malondialdehyde-acetaldehyde adducted protein stimulation induced direct cytotoxicity on monocytes derived from healthy controls, and a second LPS stimulation suppressed the inflammatory cytokines production. Consistently, hepatic macrophages of ethanol-administered ALDH2*2 transgenic mice exhibited suppressed inflammatory cytokines production in response to LPS compared to that in wild-type mice, reinforcing the contribution of acetaldehyde to liver macrophage function. These results collectively provide new perspectives on the systemic influence of excessive alcohol consumption based on alcohol-metabolizing enzyme genetic polymorphisms.

Compact and adjustable compensator for AOD spatial and temporal dispersion using off-the-shelf components.

Random access multiphoton microscopy using two orthogonal acousto-optic deflectors (AODs) allows sampling only particular regions of interest within a plane, greatly speeding up the sampling rate. AODs introduce spatial and temporal dispersions, which distort the point spread function and decrease the peak intensity of the pulse. Both of these effects can be compensated for with a single dispersive element placed a distance before the AODs. An additional acousto-optic modulator, a custom cut prism, and a standard prism used with additional cylindrical optics have been demonstrated. All of these introduce additional cost or complexity and require an extended path length to achieve the needed negative group delay dispersion (GDD). By introducing a telescope between a transmission grating and the AODs, we correct for spatial and temporal dispersions in a compact design using only off-the-shelf components, and we show that the GDD can be tuned by translation of the telescope without adjustment of any other elements.

[Clinicopathological Examination of 81 Cases Added Gastrectomy for Early Gastric Cancer after ESD].

As a general rule, our department has performed additional gastrectomy with lymph node dissection(radical surgery: RS) for non-curative endoscopic submucosal dissection(ESD)cases. This time, we performed a clinicopathological study on 81 patients who underwent RS after ESD for 10 years from May 2009 to April 2019. Lymph node metastasis(LNM)was observed in 5 cases and local cancer residue(LCR)was observed in 8 cases. Examination of the presence or absence of LNM and LCR by clinicopathological factors(histopathological type, tumor size, lymphatic invasion[ly], venous invasion[v], horizontal margin[HM], vertical margin[VM], submucosal invasion, ulceration[scar])revealed no significant risk factor for LNM, however, tumor size and HM were significant risk factors for LCR. The relationship between the eCura system and the case rate associated with LNM in our hospital was similar to that in the original report. Regarding the prognosis, there was one local recurrence and no death from the primary disease.

C-C motif chemokine receptor 9 regulates obesity-induced insulin resistance via inflammation of the small intestine in mice.

AIMS/HYPOTHESIS: Accumulation of adipose tissue macrophages is considered pivotal in the development of obesity-associated inflammation and insulin resistance. In addition, recent studies suggest an involvement of the intestine as the primary organ in inducing hyperglycaemia and insulin resistance. We have reported that the C-C motif chemokine receptor (CCR) CCR9 is associated with intestinal immunity and has a pathogenic role in various liver diseases. However, its contribution to type 2 diabetes is unknown. In the current study, we aimed to clarify the involvement of CCR9 in the pathology of type 2 diabetes and the potential underlying mechanisms. METHODS: To elucidate how CCR9 affects the development of metabolic phenotypes, we examined the impact of CCR9 deficiency on the pathogenesis of type 2 diabetes using male C57BL/6J (wild-type [WT]) and CCR9-deficient (CCR9 knockout [KO]) mice fed a 60% high-fat diet (HFD) for 12 weeks. RESULTS: WT and Ccr9KO mice fed an HFD exhibited a comparable weight gain; however, glucose tolerance and insulin resistance were significantly improved in Ccr9KO mice. Moreover, visceral adipose tissue (VAT) and the liver of Ccr9KO mice presented with less inflammation and increased expression of glucose metabolism-related genes than WT mice. Ccr9 and Ccl25 expression were specifically higher in the small intestine but was not altered by HFD feeding and type 2 diabetes development. Accumulation of IFN-γ-producing CD4+ T lymphocytes and increased intestinal permeability in the small intestine was observed in WT mice following HFD feeding, but these changes were suppressed in HFD-fed Ccr9KO mice. Adoptive transfer of gut-tropic CCR9-expressing T lymphocytes partially reversed the favourable glucose tolerance found in Ccr9KO mice via exacerbated inflammation in the small intestine and VAT. CONCLUSIONS/INTERPRETATION: CCR9 plays a central role in the pathogenesis of type 2 diabetes by inducing an inflammatory shift in the small intestine. Our findings support CCR9 as a new therapeutic target for type 2 diabetes via the gut-VAT-liver axis.

Role of CC chemokine receptor 9 in the progression of murine and human non-alcoholic steatohepatitis.

BACKGROUND & AIMS: The number of patients with non-alcoholic steatohepatitis (NASH) is increasing globally. Recently, specific chemokine receptors have garnered interest as therapeutic targets in NASH. This is the first report to examine the role of the C-C chemokine receptor 9 (CCR9)/C-C chemokine receptor ligand 25 (CCL25) axis, and to reveal its therapeutic potential in NASH. METHODS: Patients with biopsy-proven non-alcoholic liver disease (NAFLD) were recruited and their serum and hepatic chemokine expression was examined. Furthermore, wild-type (WT) and Ccr9-/- mice were fed a high-fat high-cholesterol (HFHC) diet for 24 weeks to establish NASH. RESULTS: Serum CCL25, and hepatic CCR9 and CCL25 expression levels were increased in patients with NASH compared to healthy volunteers. Furthermore, Ccr9-/- mice were protected from HFHC diet-induced NASH progression both serologically and histologically. Flow cytometry and immunohistochemistry analysis showed that CCR9+CD11b+ inflammatory macrophages accumulated in the inflamed livers of HFHC diet-fed mice, while the number was reduced in Ccr9-/- mice. Consistent with human NASH livers, CCR9 was also expressed on hepatic stellate cells (HSCs) in mice with NASH, while CCR9-deficient HSCs showed less fibrogenic potential in vitro. Administration of a CCR9 antagonist hampered further fibrosis progression in mice with NASH, supporting its potential clinical application. Finally, we showed that CCR9 blockade attenuated the development of NAFLD-related hepatocellular carcinoma in HF diet-fed mice injected with diethylnitrosamine. CONCLUSIONS: These results highlight the role of the CCR9/CCL25 axis on macrophage recruitment and fibrosis formation in a murine NASH model, providing new insights into therapeutic strategies for NASH. LAY SUMMARY: Herein, we show that a specific chemokine axis involving a receptor (CCR9) and its ligand (CCL25) contributes to the progression of non-alcoholic steatohepatitis and carcinogenesis in humans and mice. Furthermore, treatment with a CCR9 antagonist ameliorates the development of steatohepatitis and holds promise for the treatment of patients with non-alcoholic steatohepatitis.