EPM2A or EPM2B, leading to tissue accumulation of polyglucosan aggregates termed Lafora bodies (LBs). This study aimed to characterize the retinal phenotype in −/−Epm2a mice by examining knockout  and control (WT) littermates at two time points . In vivo exams included electroretinogram (ERG) testing, optical coherence tomography (OCT) and retinal photography. Ex vivo retinal testing included Periodic acid Schiff Diastase (PASD) staining, followed by imaging to assess and quantify LB deposition. There was no significant difference in any dark-adapted or light-adapted ERG parameters between KO and WT mice. The total retinal thickness was comparable between the groups and the retinal appearance was normal in both groups. On PASD staining, LBs were observed in KO mice within the inner and outer plexiform layers and in the inner nuclear layer. The average number of LBs within the inner plexiform layer in KO mice were 1743 ± 533 and 2615 ± 915 per mm2, at 10 and 14 months, respectively. This is the first study to characterize the retinal phenotype in an −/−Epm2a mouse model, demonstrating significant LB deposition in the bipolar cell nuclear layer and its synapses. This finding may be used to monitor the efficacy of experimental treatments in mouse models.Lafora disease (LD) is a progressive neurologic disorder caused by biallelic pathogenic variants in  EPM2A or EPM2B [Lafora Disease (LD), first described in 1911 [or EPM2B ,4. The dor EPM2B . This isor EPM2B ,7. Neuroor EPM2B ,6,7. Theor EPM2B ,6,7,8. Ior EPM2B ,11,12. Aor EPM2B ,11,12. Aor EPM2B ,14, retior EPM2B ,14,15. For EPM2B ,14; addior EPM2B . Opticalor EPM2B , but regNHLRC1 (EPM2B) [−/−Epm2a and −/−Epm2b have been generated [−/−Epm2a mice developed behavioral abnormalities at 4 months of age, motor coordination and memory deficits between 4 and 8 months, and myoclonus by 9 months [−/−Epm2a mice showed spike and slow-wave formation on EEG; notably, the regular and well-formed diffuse 3–6 Hz spike-wave complexes found in human LD are absent in this mouse model even at 1 year of age [−/−Epm2a mice as young as 3 months [−/−Epm2b mice developed impaired motor coordination, memory deficits and epileptiform activity including tonic-clonic seizures between 3 and 8 months of age that progressively worsened [−/−Epm2b showed no behavioural, cognitive or learning abnormalities up to 11 months of age, suggesting variability between genetic strains [Lafora Disease has been reported in multiple dog breeds ,18,19,20 (EPM2B) ,22, with (EPM2B) ,24. The  (EPM2B) . The EEG (EPM2B) ,26. Impaenerated ,28,29. T9 months ,30; thesr of age . Howeverworsened ; howeverEpm2a and Epm2b knock-out (KO) mice, LBs were identified in the various regions of the brain, footpads, as well as skeletal and cardiac muscles [A pathological hallmark of LD is the progressive deposition of polyglucosan aggregates in tissues, termed Lafora Bodies (LBs) . In huma muscles ,28.−/−Epm2a) by performing in-depth in vivo and ex vivo assessments.Currently, multiple therapeutic options are being pursued for LD . Since t−/−Epm2a KO mice were a gift from Drs. Subramaniam Ganesh and Antonio Delgado–Escueta. Briefly, the mice were generated by deleting the dual-specificity phosphatase domain (DPSD) coding region (exon 4) of the Epm2a gene and replacing it with a neomycin resistance gene via homologous recombination [The bination . The absbination . Mice we−/−Epm2a) and control (WT) littermates were examined at two time points . After overnight dark adaptation (DA), pupils were dilated using tropicamide 1% and phenylephrine 2.5% eye drops. Subsequently, mice were injected with 0.01 mL/g body weight of anesthetic solution (combination of 100 mg/mL Ketamine and 20 mg/mL Xylazine). Binocular electroretinogram (ERG) testing was then performed using the Lab Cradle (Diagnosys LLC) system. A DA (scotopic) intensity series ERG was performed  followed by a 10-min light adaptation . Subsequently, LA ERGs were performed using a 5 cd·s·m−2 stimulus flash to four stimulus frequencies . For analysis, the ERG from the eye with superior technical quality was chosen. If responses from either eye were similar, the right eye was chosen. Both a-wave and b-wave amplitudes were measured for the steps of the DA ERG. The a-wave amplitude was measured from the baseline to the trough of the first negative waveform, whilst the b-wave amplitude was measured from the trough of the a-wave to the peak of the subsequent positive waveform. The a-wave amplitudes were measured for 7 light intensities (DA 0.01 cd·s·m−2 to DA 10 cd·s·m−2) and the b-wave amplitudes were measured for all steps of the DA ERG. For the LA ERG, the trough-to-peak amplitude of the responses was calculated for all four stimulus frequencies.Five knockouts  were performed centered at the optic disc and at regions dorsal and ventral to the disc in each eye. The OCT allows for the detection of changes to the retinal thickness and lamination; also, the OCT can detect abnormal retinal deposition or any disruption to the retinal layers ,41,42,43After the ERG and OCT testing, animals were sacrificed by cervical dislocation. Eyes were removed and immediately fixed in 10% buffered formalin followed by paraffin embedding. Paraffin-embedded retinal tissue was sectioned and stained with the Periodic acid Schiff-Diastase (PASD) at The Centre for Phenogenomics Pathology Services, Toronto. PASD is a sensitive histochemical method to assess for glycogen; diastase application depolymerises glycogen to maltose and glucose, which are washed out of the slide . The LBsFull-field ERG was performed to assess the effects of LBs deposition on retinal function. p = 0.29) and 14 months (p = 0.17).Both KO and WT mice had a normal appearance on retinal photography at both 10 and 14 months. Overall, on OCT imaging, the retinal lamination appeared preserved and no localized changes in reflectivity were observed in KO mice. The average retinal thickness in KO mice was 198.53 ± 0.97 µm and 199.86 ± 0.67 µm at 10 and 14 months, respectively D. The av2 at 10 and 14 months, respectively  and 14 months (p < 0.0001). There was a significant increase in LB deposition over time in KO mice (p = 0.035). Further, the average diameter of the LB in KO mice was 0.45 ± 0.11 µm (range: 0.31–0.60 µm) and 0.47 ± 0.14 µm (range: 0.30–0.71 µm) at 10 and 14 months, respectively.Periodic acid Schiff-Diastase staining was executed to label LBs in the retinal tissue. The LBs were only observed in the retinas of KO mice and not in the WT littermates  also noted thinning of the retinal nerve fiber layer [−/−Epm2a mice in the current study did not have the capability to measure regional retinal thickness, limiting our analysis. Whilst the LBs were seen on PASD staining in the KO mice, they were not visualised on in vivo OCT scanning. This is likely due to the average diameter of LB in KO mice measuring ~0.45 and 0.47 µm at 10 and 14 months, respectively, which is beyond the axial resolution capability of the OCT system (~3 µm). Upon review of the literature, the size of LB noted in the hippocampus in −/−Epm2a mice at 9 months appeared to be ≤2 µm [−/−Epm2a mice, one might need higher resolution OCT or AOSLO technology (<2 µm) [The retinal lamination was preserved in KO mice at both time points. This finding is similar to human LD, where also, the retinal lamination is preserved ,14. The er layer , althougbe ≤2 µm  and thosbe ≤2 µm . Hence,  (<2 µm) . It is n (<2 µm) . Further−/−Epm2a mice, we did not ascertain LB deposition in the brain, liver or skeletal muscle in these mice. In retrospect, this is a missed opportunity as we could have compared the increase in LBs in KO mice retina over time to any changes in LB accumulation over time in other tissues. It is noted that an increase in LB deposition in the brain over time (4 to 17 months of age) has previously been reported by others in the same −/−Epm2a mouse model [Since the primary aim of our study was to characterize the eye phenotype in se model ,30.Epm2a, widely used as a human disease model for LD. Whilst these −/−Epm2a mice had deposition of LBs, a pathognomonic feature of LD, within the middle and inner retinal layers, these bodies did not lead to any structural and functional retinal defects that were identifiable in vivo. The degree of retinal involvement in this mouse model is milder than in the patients affected with EPM2A-related LD, as most human subjects showed ERG changes indicative of a functional defect. Nonetheless, as several treatment options are being explored for LD, the extent of LB deposition in the retina may be monitored ex vivo in the −/−Epm2a mice to ascertain treatment success for eye involvement. Further, in vivo monitoring of the neurological and behavioural deficits, as well as the EEG abnormalities in −/−Epm2a mice [To summarize, we have characterised the retinal phenotype in a KO mouse model of −/− mice ,30 could


The 3-year overall survival (OS) rate, the 3-year progression-free survival (PFS) rate and the 3-year local control (LC) rates were 60.5, 53.2 and 72.7%, respectively. For each clinical stage, the 3-year OS rates were I, 84.8%; II, 60.3% and III, 42.9%; the 3-year PFS rates were I, 71.9%; II, 58.3% and III, 37.0% and the 3-year LC were I, 78.4%; II, 79.8% and III, 65.2%, respectively. Notably, four patients (2.3%) with ≥Grade 3 cardiopulmonary toxicities were observed . Our study showed that particle therapy for esophageal cancer has lower rates of adverse cardiopulmonary events than X-ray radiotherapy.This study aimed to evaluate the efficacy and safety of particle therapy (proton beam therapy and carbon-ion radiotherapy) for esophageal cancer by analyzing prospective nationwide registry data from particle therapy facilities throughout Japan. Patients diagnosed with esophageal cancer who received particle therapy between May 2016 and June 2018 were recruited from the registries of 12 particle therapy centers in Japan. Eventually, we enrolled 174 patients who met the inclusion criteria. Of the 174 patients, 137 (78.7%) were male, with a median age of 69 years (range: 41–88 years). Clinical stages included I ( According to the International Agency for Research on Cancer, esophageal cancer is the sixth leading cause of cancer-related deaths worldwide . The RegPresently, surgery remains the first choice of treatment for operable esophageal cancer in Japan, with 61.2% of the affected patients undergoing treatment . In partProton beam therapy (PBT) and carbon-ion radiation therapy (CIRT) are types of the particle therapy. Particle therapy delivers concentrated doses of protons or carbon-ion beams to target tissues with minimal harm to surrounding normal tissues, compared with conventional X-ray radiotherapy or intensity-modulated radiotherapy (IMRT) .Therefore, this study aimed to evaluate the efficacy and safety of particle therapy. It is worth noting that an increasing number of facilities have recently become available for particle therapy. Moreover, this Japanese Nationwide Registry Study on particle therapy for esophageal cancer was initiated in 2016, and all institutions that performed particle therapy for patients with esophageal cancer than in Japan were also included in this study.This study was a multicenter, prospective observational registry conducted by Proton-Net (a Japanese PBT network) and the Japan Carbon-ion Radiation Oncology Study Group (J-CROS). Proton-Net is a study group that includes all 19 PBT institutions, and J-CROS is a study group that includes all 7 CIRT institutions currently in Japan. About 12 facilities  participated in this multi-institutional study on esophageal cancer. This study was approved by the Esophageal Cancer Working Group of the Particle Beam Therapy Committee and Subcommittee of the Japanese Society for Radiation Oncology (JASTRO). Written informed consent was obtained from the patients.Between May 2016 and June 2018, patients diagnosed with esophageal cancer and treated with particle therapy, with or without chemotherapy, absence of metastases to distant organs and no other sites with cancer were recruited from the prospective databases of particle therapy facilities in Japan. The clinical stage was evaluated using the TNM Classification of Malignant Tumors, seventh edition .10 = total dose × (1 + dose per fraction/10), these values range from 67.2 to 84.0 (Gy (RBE) BED10). Gross tumor volume (GTV) was defined as the primary tumor and lymph node metastasis based on endoscopy and imaging. Typically, the clinical target volume (CTV) was defined as GTV obtained by adding 2–5 cm margins to both cranial and caudal edges and 0.5–2 cm margins from other directions, the planning target volume was defined as the CTV plus 0.5–1 cm. The elective nodal irradiation (ENI) field usually depends on the location of the primary esophageal cancer and the institution’s treatment decisions. For example, ENI can be performed either from the bilateral supraclavicular lymph node to the celiac artery or from the aortic arch to the peri-gastric region, etc. Moreover, due to the narrow field of particle therapy, the use of X-ray radiotherapy in ENI is acceptable. Importantly, in the unified treatment policy of PBT .#2 “cancer”[TIAB] “cancers”[TIAB] OR “neoplasm”[TIAB] “neoplasms”[TIAB] OR “neoplasms”[MH] OR “tumor”[TIAB] OR “tumors”[TIAB] OR “tumor”[TIAB] OR “carcinoma”[TIAB] OR “carcinomas”[TIAB] .#3 “proton therapy”[Title/Abstract] OR “proton radiotherapy” [Title/Abstract] OR “proton beam therapy”[Title/Abstract] OR “proton beam radiotherapy”[Title/Abstract] OR “carbon ion therapy” [Title/Abstract] OR “carbon ion radiotherapy”[Title/Abstract] OR “carbon ion beam therapy”[Title/Abstract] OR “carbon ion beam radiotherapy”[Title/Abstract] OR “heavy ion radiotherapy”[Title/Abstract] OR “heavy ion radiotherapy”[MeSH Terms] OR “hadron therapy”[Title/Abstract] OR “hadrontherapy”[Title/Abstract].n = 55; 31.6%), II , III , IV  and unknown . Notably, 91 (52.3%) patients had lymph node metastases, 160 (92.0%) had squamous cell carcinoma and only 5 (2.9%) had adenocarcinoma. In addition, 169 (97.1%) patients were with PS (performance status) 0 or PS 1, and no patients were with PS 3 or higher.One hundred and seventy-four patients fulfilled the inclusion criteria. The baseline characteristics of the cohort are summarized in 10 was 79.2 Gy (RBE) (range: 57.6–91.2 Gy [RBE]). ENI was performed in 95 (54.6%) patients, and 118 (67.8%) patients underwent concurrent chemotherapy at 8 PBT centers. In particular, the most commonly used chemotherapy regimens were cisplatin and 5-fluorouracil (101/118).Scheduled particle therapy was performed in all except two patients, and their median total BEDThe median follow-up time was 908 days (range: 76–1669 days). From the data collected, we found that 67 patients died; among them, 52 died from esophageal cancer and 15 from other causes. In addition, recurrence was observed in 71 patients, and the sites of recurrence were local (irradiated site) in 36 cases, lymph nodes in 14 cases and distant metastasis in 25 cases, including duplication.The 3-year OS rate was 60.5%  , the 3-yZ-test based on a double-logarithmic transformation  for esophageal cancer worldwide.In a previous retrospective analysis of PBT at four centers in Japan from 2009 to 2013 , the 3-yParticle therapy delivers concentrated doses of proton beams or carbon-ion beams to target tissues with minimal harm to surrounding normal tissues than conventional X-ray radiotherapy or IMRT . Among tZ-test . In addition, Wang et al. described that the frequency of adverse events for PBT was significantly lower than that for X-ray radiotherapy in patients with cardiac dysfunction  [In patients with esophageal cancer, cardiopulmonary toxicity is one of the biggest problems post-radiotherapy . Among tet al. : 23 vs 1i et al. : 7.1 vs g et al. : 21 vs 1= 0.018) .Cardiopulmonary adverse events have been suggested to be significantly reduced in patients with cervical esophageal cancer. From our results, we found that there were three cases 1.7%) and one case (0.6%) of cardiac and pulmonary adverse events of Grade 3 or higher. Our particle therapy cohort included 22 cases (12.6%) of cervical esophageal cancer cases, and when cervical esophageal cases were excluded, there were two cases (1.3%) and one case (0.7%) of cardiac and pulmonary adverse events of Grade 3 or higher. Comparing these with the results of the Japan Clinical Oncology Group (JCOG) study and Japanese Radiation Oncology Study Group (JROSG), regarding stage I esophageal cancer, and the frequency of Grade 3 or higher adverse events  in JCOG9708 % and one, and theThis study has a limitation. We compared the survival rate of X-ray radiotherapy and that of particle therapy; therefore, we considered that the results might reflect the differences in background factors  rather than the superiority of the treatment methods.In conclusion, particle therapy for esophageal cancer may reduce cardiopulmonary toxicity compared with X-ray radiotherapy. In our future study, we will reexamine survival in a reanalysis scheduled for 2023.The datasets analyzed during the current study are available from the corresponding author on reasonable request.


Prunus salicina Lindl.) is an important stone fruit crop in Sichuan that is increasingly in demand by consumers owing to its flavor and outstanding nutraceutical properties. The physicochemical characteristics, antioxidant capacity, and volatile profiles of five traditional and new plum cultivars in Sichuan were determined using high-performance liquid chromatography and gas chromatography time-of-flight mass spectrometry. The results showed that all plums exhibited an appropriate quality profile for fresh consumption; the new cultivar ‘ZH’ exhibited the highest soluble solids content, sugar–acid ratio, total phenolic content, total flavonoid content, and antioxidant capacity. High sugar–low acid properties were observed in five plum cultivars. Sucrose was the main sugar, while quinic acid and malic acid were the main organic acids. The plums were rich in volatile compounds and had specific volatile characteristics. A total of 737 volatiles were identified in the plum fruit, and orthogonal partial least-squares discriminant analysis was employed to screen 40 differential volatiles as markers for cultivar distinction. These findings offer comprehensive information on the physicochemical characteristics, antioxidant capacity, and volatile profiles of plums.Plum ( Prunus (family Rosaceae). Through decades of domestication and the ongoing selection of wild plums, there are more than 40 species of plums distributed worldwide [Prunus salicina) is considered one of the most important fruit crops commercially grown in China, Spain, and the USA [Plums are popular stone fruits belonging to the subgenus orldwide . The Jap the USA . Around  the USA . SichuanFruit flavor and nutrition are important factors in fruit quality. As delicious table fruits, plums are characterized by a pleasant taste and aroma. The composition and content of sugars and organic acids are critical contributors affecting fruit taste, and these characteristics are strongly influenced by the cultivar and fruit maturity . In addiIn addition to their juiciness and tastiness, mature plums contain various bioactive components. Since the 1990s, there has been a sharp rise in interest in research involving plums due to their high concentrations of phenolic compounds, particularly flavonoids and a subclass of anthocyanins ,9. PhenoThe local market demand for safe and high-quality fruits is increasing because consumers are aware of their characteristic taste and nutritional value . In this2, then stored at −80 °C.Five plum cultivars were collected from Deyang : xiangli (XL), xiangtian (XT), ailisi (AL), shengxuepo (SX), and zihuang (ZH) . A total1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and the ferric reducing antioxidant power (FRAP) assay were purchased from Sigma-Aldrich . Standards for sucrose, glucose, fructose, sorbitol, and quinic, malic, citric, and succinic acids were obtained from Yuanye Biotechnology Co., Ltd. . All of the other chemicals used were of analytical grade.Fresh juice was produced using a juicer  for use in the determination of SSC and TA. SSC was analyzed using a sugar refractometer  . TA was Sugars and organic acids were extracted according to the method described by Kodagoda et al. , and the2+ column (100 × 7.8 mm × 8 mum) at 80 °C with an injection volume of 10 μL. The chromatographic separation of sugars was performed using twice-distilled water as the mobile phase, a 0.3 mL/min flow rate, and a 40 min run time. Organic acids  were detected using a 2487 UV detector  set at 210 nm after separation with an Aminex HPX-87H column (300 mm × 7.8 mm × 9 mm) kept at 60 °C. A dilute sulfuric acid solution (0.05 mol/L) was used as the mobile phase, with a 0.5 mL/min flow rate and a 35 min run time.Sugars  were detected using an RID-10A refractive index detector  and RCM-Monosaccharide Cav/v) and ultrasound three times at 35 °C for 30 min each time and then centrifuged at 10,000 rpm for 8 min. After extraction, the TPC was determined using the Folin–Ciocalteu method [v/v) reagent was mixed with 1 mL of polyphenol extract and left for 5 min and then neutralized with 4 mL sodium carbonate (7.5% w/w) and reacted at 25 °C for 90 min. The absorbance of the sample was measured at 760 nm, and the results were expressed as milligram equivalents of gallic acid per 100 g of fresh weight (mg GAE/100 g).The TPC was extracted by the following method as reported previously . Brieflyu method . First, 2 (5% m/v) for 5 min at room temperature (24 °C). Subsequently, 0.3 mL of AlCl3 (10% v/v) and 2 mL of NaOH (1 mol/L) were added. The absorbance of the mixture was measured at 510 nm and compared with that of the catechin standard. The results were expressed as milligrams of catechin equivalent (CE) per 100 g of fresh weight (mg CE/100 g).TFC was measured using a colorimetric assay . In detaTAC was determined using a Plant ANTH ELISA Kit  . The pluv/v, 80%) was added to the powder (1.0 g) at room temperature (24 °C), extracted using ultrasonication , and centrifuged at 8000 rpm for 15 min. Finally, the supernatant was stored at −20 °C for later experimental use.The extract was prepared as described by Yu et al. . BrieflyThe antioxidant capacity of the samples was studied using ABTS and DPPH assays  and the FRAP method (for assessing the reducing power) . The absVolatiles in the plums were isolated according to the solid-phase microextraction (SPME) method previously described by Özdemir et al. . Briefly® 4D GC × GC-TOF mass spectrometer  and a DB-Heavy Wax column (30 m × 250 μm × 0.5 μm). The injection-port temperature was 260 °C and high-purity helium (>99.999%) was used as the carrier gas at a rate of 1.0 mL/min. The initial temperature of the GC was 40 °C for 3 min, then increased to 100 °C at a ramp rate of 4 °C/min for 2 min, 3 °C/min up to 180 °C for 2 min, followed by a ramp rate of 10 °C/min to 250 °C for 6 min. The ion source used for the mass spectrometer was 250 °C and the electron energy was 70 eV. Mass spectra were obtained at a scan rate of 2.84 scans/s in the m/z 35–550 range.GC-TOF-MS analysis was performed using an Agilent 8890A GC equipped with a LECO Pegasusp < 0.05) among the samples were determined using ANOVA with Tukey’s post hoc test using SPSS 26.0 . Bar charts, heat maps, and radar charts were generated using GraphPad Prism 9.0 , and orthogonal partial least-squares discriminant analysis (OPLS-DA) was performed using SIMCA 14.1 .All data are presented as the mean values ± standard deviation. Significant differences   a, and thp < 0.05) among the selected fresh plums. Maietti et al. [Polyphenols with flavonoid structures combine with aluminum chloride to produce a yellow solution that immediately turns red under alkaline conditions . In thisi et al.  explaineAnthocyanins are an important class of phenolic compounds that belong to the flavonoid class of plant secondary metabolites. Many studies have confirmed that anthocyanins are responsible for the color of many fruits and are economically important . In thisAntioxidants can form stable products by donating electrons to free radicals, thus preventing the chain reactions of free radicals and exerting antioxidant effects . Taking p < 0.05) (p > 0.05). Research suggests that the highest TPC corresponds to the strongest anti-free radical potential of the relevant fruit extracts, which is similar to our results [The DPPH assay is based on the hydrogen donor-mediated reduction of the stable radical DPPH to the yellow-colored diphenyl picrylhydrazine . The fin < 0.05) d. The ne results ,48.+-reducing capability of the five plums differed significantly (p < 0.05) between 17.17 and 54.34 µmol TEs/g , followed by finger lime (46.16 ± 3.74 µmol Fe2+/g DW) and native pepperberry (48.48 ± 3.23 µmol Fe2+/g DW).Unlike DPPH, the FRAP assay is based on the ability of a sample to donate electrons to reduce the ferric tripyridyltriazine complex to the ferrous tripyridyltriazine under low-pH conditions . The FRAVolatile organic compounds (VOCs) are key components of the fruit metabolome and provide a deeper understanding of the differences in aroma composition and their impacts on flavor perception . An untap < 0.05 [Prunus salicina Lindl. and contribute to the overall green odor and fruity aroma of plums [To better define the most representative volatile compounds among the different plum cultivars, OPLS-DA, a multivariate statistical analysis, was employed to identify potential volatile markers for which the criteria were set to satisfy the conditions of VIP > 1 and p < 0.05 . Accordip < 0.05 . Ethyl ep < 0.05 . Ethyl 2p < 0.05 . Aldehydof plums ,56,57. Iof plums . In addiA comparative analysis of the research data on the physicochemical indices, functional characteristics, and volatile flavor components of different plum cultivars from Sichuan was carried out to reveal their potential commercial value. These results suggest that all the investigated plums were characterized by a good taste, mainly due to their low acid–high sugar characteristics. The total sugar content of the five plums varied between 82.19 mg/g FW and 141.38 mg/g FW, and the total organic acid content ranged from 13.32 mg/g FW to 30.35 mg/g FW. The sweetness was mainly determined by sucrose (29.22–52.04%), and malic acid (33.21–46.78%) and quinic acid (40.59–52.44%) were the dominant contributors to the acidity of the five plums. All of the plums exhibited high levels of phenolic compounds, total flavonoids, and strong antioxidant properties. The radical scavenging capacity of the five plums measured using ABTS and DPPH assays ranged from 17.17 to 54.34 µmol TEs/g and 27.16 to 21.71 µmol TEs/g, respectively, and the FRAP reducing capacity of the plums varied between 7.75 and 32.19 µmol TEs/g. Among the five plum cultivars, the new cultivar ‘ZH’ had the highest TPC (271.74 mg of GAE/100 g) and TFC (169.84 mg of GAE/100 g) as well as strong antioxidant capacity. Furthermore, the characteristics of the volatile compounds in the different plum cultivars were investigated. The VOCs of the plums consisted of 31 acids, 106 alcohols, 48 aldehydes, 75 alkanes, 93 alkenes, 111 aromatic compounds, 76 ketones, 108 esters, 9 furans, and 80 other compounds. Among them, alcohols and esters were the major components in the plums. A total of 44 potential volatile markers related to the five plum cultivars were identified using our model. The major sweet fruit aroma contributors included ethyl acetate, isobutyl acetate, ethyl propionate, ethyl 3-methylbutanoate, and isoamyl acetate, which were identified in ‘AL’, and hexenyl acetate, butyl acetate, and hexyl acetate in ‘SX’. Aldehydes and alcohols were associated with green odors, such as 2-hexenal, 2-octenal, 2,4-heptadienal, nonanal, pentenal, and (Z)-2-decenal in ‘XT’; nonanol, hexanal, and (Z)-3-hexenol in ‘ZH’; and 2-phenylethanol, 1-octanol, 2-ethyl-1-hexanol, and nonanol in ‘XL’. This study demonstrates the quality characteristics of the important commercial cultivars of plum fruit in Sichuan and contributes to their wider use in breeding practice. Among the five plums, the new cultivar ‘ZH’ could be used as a source of new properties when improving the quality properties of plums due to its outstanding antioxidant activity and good taste. Furthermore, this study is an initial analysis of the volatile profile of plums from Sichuan; in future research, we will clarify the sensory contribution of volatiles to the overall aroma of plums and identify new markers based on different varietal genotypes.


GLP1 81±9.6 vs 37±6.9 pM×min, P=0.002) and insulin . GPR119 activation also significantly increased glucagon levels in both groups (P<0.01) with, however, no difference between the groups. By contrast, GPR119 activation did not affect islet hormone secretion from isolated islets. Glucose elimination after meal ingestion was significantly increased by GPR119 activation in HFD mice  but not in control mice. Islet GLP1R protein levels was higher in HFD vs CD mice . In conclusion, insulin-resistant mice display increased islet GLP1R protein levels and augmented meal-induced GLP1 and insulin responses to GPR119 activation, which results in increased glucose elimination. We suggest that the increased islet GLP1R protein levels together with the increased GLP1 release may contribute to the upregulated β-cell function in insulin resistance.We previously demonstrated that the overall incretin effect and the β-cell responsiveness to glucagon-like peptide-1 (GLP1) are increased in insulin-resistant mice and may contribute to the upregulated β-cell function. Now we examined whether this could, first, be explained by increased islet GLP1 receptor (GLP1R) protein levels and, secondly, be leveraged by G-protein-coupled receptor 119 (GPR119) activation, which stimulates GLP1 secretion. Female C57BL/6J mice, fed a control  or high-fat  diet for 8 weeks, were anesthetized and orally given a GPR119 receptor agonist  or vehicle, followed after 10 min with gavage with a liquid mixed meal . Blood was sampled for determination of glucose, insulin, intact GLP1, and glucagon, and islets were isolated for studies on insulin and glucagon secretion and GLP1R protein levels. In HFD vs CD mice, GPR119 activation augmented the meal-induced increase in the release of both GLP1 (AUC In fully compensated insulin resistance, there is a sufficient upregulation of insulin secretion whereas in glucose intolerance and type 2 diabetes this upregulation is inadequate The increased incretin effect in insulin resistance raises the question of whether GLP1 secretion from the intestine is increased and may thus be therapeutically leveraged. One approach to test this hypothesis would be to activate G-protein-coupled receptor 119 (GPR119). This receptor is expressed in gut enteroendocrine cells In this study, we explored, first, whether the increased β-cell responsiveness to GLP1 in insulin-resistant mice is associated with increased islet GLP1 receptor (GLP1R) protein levels. We then examined the potential to improve islet function and glucose tolerance in glucose-intolerant insulin-resistant mice through GPR119 activation by administering a specific GPR119 receptor agonist. As the effects of GPR119 activation on islet α-cells are still unknown, glucagon release was also investigated in view of the importance of glucagon for glycemic dysregulation and reduction of glucagon in therapy of type 2 diabetes 50=4 nM against the human GPR119 receptor. GSK2041706 (GSK706) is a potent and selective agonists at the rodent and human GPR119 receptor that have been discovered by GlaxoSmithKline in vivo studies, the compound was formulated in a vehicle consisting of 0.5% methocel K15M premium EP hydroxypropyl methylcellulose  and 0.1% Tween 80 (Fluka/Sigma–Aldrich) in water. For the in vitro experiments, the compound was dissolved in 1% DMSO (Sigma–Aldrich).In this study, we used a novel small-molecule GPR119 receptor agonist -1-(1--4-piperidinyl)ethyl)oxy]-5-[4-(methylsulfonyl)phenyl]pyrazine) (GlaxoSmithKline) ad libitum. The animals were housed in groups of eight per cage in a temperature-controlled (22 °C) room with artificial lighting maintained on a 12 h light:12 h darkness cycle. All experimental procedures were performed in agreement with the Animal Ethics Committee in Lund, Sweden.Female C57BL/6J mice (average 22 g) were obtained from Taconic . After 1 week of acclimatization, the mice were divided into two groups and fed either a control diet  or a high-fat diet  for 8 weeks. Body weight and food intake were monitored once a week. Food and water was provided Fasted (5 h) mice were anesthetized with an i.p. injection of midazolam  and a combination of fluanisone (0.9 mg/mouse) and fentanyl . The mice were orally gavaged (0.25 ml) with vehicle or GSK706A (10 mg/kg) 10 min before a new gavage with the previously described mixed meal 2, 1.2 mmol/l MgCl2, 25 mmol/l HEPES (pH 7.4), 5.6 mmol/l glucose, and 0.1% fatty acid free BSA (Boehringer Mannheim) at 37 °C during 60 min. Thereafter, islets in groups of three were incubated in 200 μl of the above-described buffer with varying concentrations of glucose. In the first experiment, the direct effect of GSK706A on insulin secretion was determined by incubating isolated islets from mice fed the CD and HFD in different concentrations of glucose , with or without addition of GSK706A (1 nmol/l). In a second experiment, the effect of non-glucose stimuli on GPR119 activation was determined by incubating isolated islets from mice fed the CD and HFD in different concentrations of glucose  with or without arginine (10 mmol/l) and GSK706A (1 nmol/l). The islets were incubated for 60 min at 37 °C, where after aliquots of 25 or 50 μl buffer in duplicate were collected and stored at −20 °C until analysis of insulin and glucagon respectively.Mouse islets were isolated from the pancreata of normal and HFD-fed mice by collagenase digestion and handpicked under the microscope. Batches of freshly isolated islets were preincubated in HEPES balanced salt solution containing 125 mmol/l NaCl, 5.9 mmol/l KCl, 1.28 mmol/l CaClThe protein levels of the GLP1R in isolated islets of CD and HFD-fed mice were analyzed by western blot. The islets were homogenized in a buffer containing 150 mmol/l NaCl, 2 mmol/l EDTA, 20 mmol/l Tris–HCl, pH 7.5, 1% Triton X-100, and 0.2% protease inhibitor cocktail (Sigma–Aldrich). The total amount of proteins in each sample was measured using a BCA Protein assay reagent kit . Aliquots of tissue homogenates containing equal amounts of protein (30 μg) were separated on SDS–PAGE and blotted onto nitrocellulose membranes (Bio-Rad). The membranes were probed with primary antibodies against the GLP1R (53 kDa) (Abcam) and actin (42 kDa) (Abcam). The secondary antibody was a HRP-conjugated goat anti-rabbit IgG antibody (Amersham Pharmacia Biotech). The blots were developed by enhanced chemiluminescence  and the proteins were detected and quantified using a ChemiDoc XRS+ system with image lab software (Bio-Rad).Plasma glucose was measured with the glucose oxidase method using 2,2′-azino-bis as a substrate with the absorbance measured at 420 nm on a microtiter plate reader . Insulin was analyzed with sandwich immunoassay technique (ELISA) using double MABs against insulin . Glucagon was measured by RIA . Levels of intact GLP1 were determined by sandwich immunoassay technique (ELISA) using MABs specific for the active form of GLP1, whereby electrochemiluminescent labeling enables detection of voltage-mediated chemiluminescence in the SECTOR Imager plate reader .s.e.m. Statistical significances were assessed by using Student's t-test. Suprabasal areas under the curve (AUC) were calculated by the trapezoidal rule for glucose and insulin data during time interval 0–60 min and for glucagon and intact GLP1 during 0–20 min. The glucose elimination constant (KG) was determined as the glucose elimination rate (% per min) between 15 and 60 min after oral challenge. β-Cell function (0–60 min) was determined as the ratio between AUC insulin and glucose.Data are reported as mean values ±P<0.001) and elevated fasting glucose (P=0.0014) and insulin (P<0.001) levels in plasma (P=0.007) and reduced KG (P=0.009)  . Basal pP=0.009) .P=0.037) (P=0.0016)  (P=0.0004) (P=0.004) and levels stayed significantly elevated after 20 min  (P=0.0011) (P=0.03) (P=0.014)  (P=0.0017) and insulin-resistant mice , compared with vehicle but with no difference between the groups  or vehicle was given orally before (−10 min) an oral liquid test meal to CD and HFD-fed mice. In CD-fed mice, no significant difference in plasma glucose or insulin levels after meal ingestion was observed between gavage of vehicle vs GPR119 agonist A and B. P=0.037) . Further=0.0016) C. In insP=0.003) F, also s=0.0004) . The ins P=0.05) G. This w=0.0011) . We also(P=0.03) . The gluP=0.014) , althougP=0.014) E. β-CellP=0.002) . ConcernP=0.002) D and H ie groups .P=0.004) and  respectively. By incubating islets from CD and HFD-fed mice with GSK706A , we did not observe a significant increase in insulin release at 2.8, 5.6, 8.7, 11.1, or 16.7 mmol/l glucose (data not shown). In a second experiment, the islets were incubated with arginine (10 mmol/l), a potent stimulator of both islet insulin P<0.001) in both CD and HFD-fed mouse islets (P<0.001 and P=0.011 respectively) and 11.1 mmol/l glucose (P<0.001 and P=0.006 respectively) , moderate (8.3 mmol/l), and high glucose (16.7 mmol/l) compared with CD ,  B and D.P<0.05)  .Insulin resistance leads to a compensatory increase in insulin secretion, which enables sufficient control of blood glucose levels The GLP1R has been localized in several peripheral tissues Previous studies on humans report that individuals with an IGT were found to have an impaired insulinotropic effect of GLP1 et al. Gpr119−/− mice Studies on incretin hormone secretion in individuals with various degrees of glucose tolerance have reported conflicting results. Secretion of GLP1 was found to be impaired in obese subjects with both NGT in vitro experiments do not support such a notion. On the other hand, as these are static islet incubations, smaller effects of GSK706A such as variations in first-phase insulin release may be masked by the collective insulin secretion during the full incubation time. Thus, in the current study, GPR119 activation had no significant effect on islet insulin release even at very high doses. Therefore, although minor effects on insulin secretion may have been difficult to find in these studies in incubated islets, the marked insulin response to GPR119 activation in insulin-resistant mice is most likely mediated by GLP1.The increased GLP1 response to GPR119 activation in association with the increased β-cell levels of the GLP1R in the high-fat-fed mice explains the robust increase in insulin secretion. In pancreatic islets, GPR119 protein has been localized mainly in β-cells The elevated intact GLP1 and insulin levels after GPR119 activation in the insulin-resistant mice resulted in increased glucose elimination rate. However, the effect was small and the mechanisms behind the stimulated glucose elimination is not known, as it may be mediated not only by increased insulin levels but also by direct effects on hepatic glucose production and glucose kinetics, which were not studied in detail in the current study. Conversely, plasma glucose levels were not reduced by GSK706A at any of the individual time points and, similarly, the AUC for glucose was not lowered. Thus, no change in glucose tolerance but increased insulin levels occurred after GPR119 activation, which may suggest that insulin may be less effective, which also needs to be studied in more detail. Therefore, our overall and main conclusion based on these results is that GPR119 activation seems a weak tool to improve glycemia in the setting of insulin resistance.in vitro experiments do not indicate that GPR119 activation has any direct effect on islet glucagon release, and thus, not likely the main contributor to the increased in vivo plasma glucagon levels. As GPR119 protein levels is found not only in the gut L-cells, which produce GLP1, but also in other enteroendocrine cells, it is possible that receptor activation indirectly affects islet glucagon release via other gut hormones than GLP1. For example, GPR119 has been found in K-cells et al have showed that GIP infusion during the IIGI results in hypersecretion of glucagon The failure of GPR119 activation to lower glucose below baseline may be explained by the concomitant increase in plasma glucagon levels after GPR119 activation. The mechanism behind the glucagonotropic action of GPR119 activation remains to be established. As GLP1 and insulin are potent inhibitors of glucagon secretion In conclusion, we suggest that the normal adaptive response of the β-cell to insulin resistance involves an upregulation of the incretin system manifested as both increased islet GLP1R protein levels and enhanced GPR119-activated GLP1 secretion, resulting in improved β-cell insulin secretion. However, no major changes in glucose tolerance are observed. We also show that GPR119 activation stimulates glucagon secretion, which increases glucagon levels. Therefore, a further development of an anti-diabetic effect of GPR119 activation may require combination with glucagon antagonism. These results thus provide both a mechanistic basis for increased incretin effect and β-cell sensitivity to GLP1 in insulin-resistant mice and a rationale for improving islet function by GPR119 activation.L Ahlkvist contributed with the design of the study, undertaking of the experiments, analyses and statistics, interpretation and discussion of the data, and writing the manuscript. K Brown contributed with the design of the study and gave comments to the manuscript. B Ahrén contributed with design of the study, interpretation and discussion of the data, and writing the manuscript.


The antioxidant property of CACF was evaluated with DPPH, ORAC, and FRAP assays, which demonstrated significant antioxidant activity. The cytotoxicity of CACF was tested using the MTT assay; CACF effective inhibitory concentrations (IC50) for A549, PC-3, MCF-7, and WRL-68 cells were 31.42 ± 5.4, 22.61 ± 1.7, 8.1 ± 0.9, and 54.93 ± 8.3 μg/mL, respectively. CACF effectively and dose-dependently inhibited TNF-α release, in vitro and in vivo. CACF inhibited TNF-α secretion in stimulated RAW264.7 macrophage supernatants with an IC50 of 0.012 μg/mL, without affecting their viability; the highest dose tested reduced serum TNF-α by 61%. Acute toxicity testing in rats revealed that CACF was non-toxic at all doses tested. Matching the cytotoxic activity towards a mechanistic approach, CACF dose-dependently exhibited in vitro inhibitory effects against the activation of NF-κB translocation in MCF-7 cells. Preliminary phytochemical screening with GC/MS analysis detected 22 compounds in CACF, of which morpholinoethyl isothiocyanate was the most abundant (29.04%). The study reveals the potential of CACF in the treatment of breast cancer and in oxidative stress conditions with associated inflammatory responses.We investigated the antioxidant potential, cytotoxic effect, and TNF- Since time immemorial, traditional herbs have been used as remedies for several diseases . Even thCentratherum anthelminticum (L.) KUNTZE (family: Asteraceae) is an erect, pubescent annual herb found widely in the Indian subcontinent which is locally known as “Somraj,” and its seeds are known as “Kalijiri” in Hindi .  Murine macrophage cells RAW 264.7 were seeded in 96-well plates at 5 × 10ly as in . PercentWhereas percentage TNF inhibition was calculated as follows:n = 8 for each group). The rats were pretreated with the indicated solutions for 30 minutes before lipopolysaccharide (LPS) stimulation: group 1 was pretreated intraperitoneally (i. p.) with 1 mL of PBS alone, group 2 with dexamethasone (6 mg/kg BW) in 25% DMSO, group 3 with CACF (25 mg/kg BW) in 25% DMSO, group 4 with CACF (50 mg/kg BW) in 25% DMSO, and group 5 with CACF (100 mg/kg BW) in 25% DMSO. Groups 1–5 were treatment groups stimulated with LPS, while group 6 made up the untreated negative control in which rats were given a solution of 25% DMSO in PBS and no LPS. Subsequent to pretreatment, LPS (1 mg/kg) was then administered in 1 mL of pyrogen-free normal saline i. p for five treatment groups, and PBS was administered i. p. for the negative control group. Blood was withdrawn from the animals under ether anesthesia after 90 minutes of LPS or PBS administration. Serum was collected and stored at −80°C until analysis. Serum levels of TNF-α were determined using rat TNF-α ELISA kit according to the manufacturer's protocol .Healthy male rats were selected and divided into 6 groups (n = 6) and were orally fed with increasing doses of CACF: 10, 20, 100, and 1000 mg/kg body weight (BW). The rats were observed continuously for 2 hours for behavioral, neurological, and autonomic profiles and after 24 and 72 hours for any lethality. All procedures were according to the guidelines stated by OECD.Healthy adult rats of either sex were divided into 4 groups  at 6000 cells/well. After overnight, cells were treated with different concentrations of CACF for 1 hr, followed by treatment with 10 μm), with helium as the carrier gas at a flow rate of 1.0 mL/min. The injection volume was 1 μL with a split ratio of 10 : 1. The column temperature was initially held at 100°C for 3 min and then increased to 290°C at a rate of 10°C/min. The column temperature was then maintained at 290°C for 3 min. The temperatures of the injector and detector were 250°C and 280°C, respectively. Mass acquisition was performed in the range of 40–550 atomic mass units (a. m. u) using electron impact ionization at 70 eV. The major components in this sample were predicted by a spectral database matching against the library of National Institute of Standards and Technology (NIST21 and NIST Wiley).Gas chromatography mass spectrometry (GC-MS) analysis of the selected fraction was carried out on a Shimadzu GC-17 A network GC system coupled to a mass-selective detector: MS-QP50-50. Separation was conducted on an HP-5 MS column  of the number of experiments indicated in the legends. Statistical significance was assessed using one-way analysis of variance (ANOVA) followed by a multiple comparison test (Tukey's post-hoc test), were μg GAE/mg extract.P < 0.05), with an IC50 value of 22.56 ± 1.4 μg/mL  with a 1048.3 μmol/L for the fraction, while the positive control used in this study displayed a value of 6240 and 907.7 μmol/L for ascorbic acid and BHT, respectively  was calculated for oxygen radical absorbance capacity of CACF, trolox, and the positive control quercetin. ORAC results are demonstrated in 50 for the CACF towards different cell lines. CACF demonstrated dose-dependent cytotoxic effects with IC50 values of 31.42 ± 5.4, 22.61 ± 1.7, 8.1 ± 0.9, and 54.93 ± 8.3 μg/mL; in A549, PC-3, MCF-7 and WRL-68, respectively , prostate cancer (PC-3), breast cancer (MCF-7), and normal hepatic cells (WRL-68), respectively. After 24 hours, cell viability was determined by the MTT assay. CACF induced cell cytotoxicity in a concentration-dependent manner. These dose titration curves allowed determination of ICectively . In lineα production  seeds. The antioxidant assays performed using the DPPH, ORAC, and FRAP methodologies revealed the free radical-scavenging possibilities of this fraction. Antioxidants are substances that may protect cells from the damage caused by unstable molecules known as free radicals. Several factors can lead to the accumulation of free radicals in the cell; examples are certain chemicals, ultraviolet radiations, inflammatory cytokines, and bacterial lipopolysacharides, and other pesticides further generate oxidative stress conditions and extend their contribution in the progression of various ailments including neurodegenerative diseases, atherosclerosis, diabetes, inflammation, and carcinogenesis [The current study revealed the pleiotropic bioactivities of the chloroform fraction of ogenesis , 25–27. ogenesis , 29.50 value, nevertheless it was not cytotoxic to normal cell line, WRL-68. These results specify that cell lines differ in their sensitivity to the same CACF, which may be determined by multiple-cell type-specific signalling cascades and transcription factor activities [Having uncovered the antioxidant nature of CACF, we then turned to look at its effects in a living cell system. Cytotoxic screening models provide important preliminary data to select plant extracts or natural compounds with potential anticancer properties . In thistivities .in vitro and in vivo without affecting cell viability and animal survival, respectively. This also warrants the ethnomedical uses of this plant in the treatment of inflammatory conditions. The ability of CACF to inhibit TNF production may attribute to several factors. Previous studies on C. anthelminticum have shown the presence of several bioactive components [In addition to that, CACF exhibited inhibition of TNF production both mponents , for examponents . The abimponents .in vitro inhibitory effects on cell proliferation in MCF-7 cells, which moves our line of study towards carrying out additional experiments to get a better understanding in elucidating mechanisms of breast carcinogenesis with possible future strategies. It is well known that antioxidants are known for their ability to scavenge free radicals from the various stimuli, resulting in activation of transcription factors involved in the transcription of survival and inflammatory genes such as nuclear factor kappa B (NF-κB) [κB is a transcription factor involved in copious inflammatory and cancer-related ailments and has developed as a foremost target in drug discovery [κB transcription factors in constitutive activation includes increased survival signalling, cell proliferation, angiogenesis, and invasion, which are key features of the malignant phenotype [α in MCF-7 cells. TNF-α-induced transcript levels for the adhesion molecules, and this might interfer at an early stage of signaling event induced by TNF-α. This suggests that CACF may inhibit the expression of the cell adhesion molecules by interfering with the transcription of their respective genes and may inhibit either the initiation of transcription or the stability of the mRNAs encoding in these molecules. It is well understood that NF-κB is an important transcription factor involved in the gene regulation and contributing in immune and inflammatory responses, including genes encoding [κB is believed to play an important role in the regulation of inflammatory response associated with cancer therapy [Moreover, this fraction along with pleotropic bioactivities demonstrated the  (NF-κB) . NF-κB iiscovery . The conhenotype . Our stuencoding , 36. Mor therapy , 38.Analysis of the nonpolar extractable of plant material using GC/MS has been applied before . Our resCentratherum anthelminticum seed (CACF) displayed a range of interrelated in vitro activities, ranging between antioxidant, cytotoxic and in vitro and in vivo TNF-α inhibition while remaining safe. Furthermore, CACF is maintained concerning mechanistic approach linked with cytotoxic activity on targeted cell line which positively inhibited NF-kB translocation in MCF-7 cells. Therefore, these results merit further pharmacological investigations with detailed phytochemical analysis on C. anthelminticum.In summary, the chloroform fraction of


Background. Laparoscopic Roux-en-Y gastric bypass (LRYGB) has been shown to be an effective treatment for type 2 diabetes mellitus (T2DM) in patients with morbid obesity. However, it is unclear just how effective the LRYGB procedure is on T2DM for patients with BMI less than 35 kg/m2. We report one obese patient with T2DM who did not meet the current NIH criteria for morbid obesity surgery. This patient underwent a laparoscopic truncal vagotomy, subtotal gastrectomy, and Roux-en-Y gastrojejunostomy for intractable gastric ulcers and subsequently had full resolution of her T2DM. Methods. A 48-year-old patient with a BMI of 34.6 kg/m2 underwent a laparoscopic truncal vagotomy, subtotal gastrectomy, and Roux-en-Y gastrojejunostomy for intractable gastric ulcers. The patient was seen 3 months preoperatively, followed for 24 months postoperatively, and evaluated for postoperative complications, weight loss, and improvement in comorbidities. Results. The patient had no postoperative surgical complications. Her BMI decreased from 34.6 kg/m2 to 22.3 kg/m2 by 24 months postoperatively. Significant improvements in her fasting blood glucose levels were seen 10 days postoperatively from a preoperative level of 147 mg/dl to 97 mg/dl. Conclusion. Patients with a BMI less than 35 kg/m2 and uncontrolled T2DM may benefit from a laparoscopic Roux-en-Y gastric bypass. In the United States, the development of T2DM has been strongly linked to obesity, with 50% of T2DM patients having a BMI >30 kg/m2 . T2DM is2 . BariatrThe role of surgical therapy as the primary management of T2DM is promising but controversial. The mechanism of action has been studied, but there remains an unclear explanation as to what postsurgical effects are the most important in alleviating the disease . Resolut2 or greater than 35 kg/m2 with life-threatening comorbidities such as T2DM [2. They achieved 80% remission of T2DM 1 month postoperatively and 100% euglycemia at 3-month followup [The role of RYGB as a long-lasting and effective procedure for controlling T2DM in the morbidly obese is well defined. Currently, under the NIH guidelines, bariatric surgery is indicated when a patient's body mass index (BMI) is greater than 40 kg/m as T2DM . Only a followup . We inve2. She had a Zollinger-Ellison syndrome workup, which was negative. Her T2DM medications included Lantus 30 U once a day, Januvia 100 mg once a day, and Metformin 1000 mg twice daily. Three months prior to surgery, her HbA1c was 7.9% and fasting glucose was 147 mg/dL. The patient is a 48-year-old Caucasian female who underwent laparoscopic truncal vagotomy, subtotal gastrectomy, and Roux-en-y gastrojejunostomy for gastric and duodenal ulcers unresponsive to medication. Past medical history was significant for intractable gastric and peptic ulcer disease for 3 years, type 2 diabetes mellitus (T2DM), hypertension, hypercholesterolemia, gastritis, esophagitis, gastroparesis, and obesity. Her BMI was 34.6 kg/m2, respectively. At her 2-month evaluation, her HbA1c had markedly improved to 6.5% and her BMI was 29.2 kg/m2. She remained off all insulin and hypoglycemic agents after her 10-day postoperative visit. Within 6 months, the patient's BMI had improved to 26.9 kg/m2 and she had a HbA1c of 6.1%  resolution or glucose control” in postgastrectomy patients. He reported 39 patients with a BMI less than 35 who underwent either a laparoscopic ileal interposition procedure associated with a sleeve gastrectomy or a laparoscopic ileal interposition procedure associated with a diverted sleeve gastrectomy. The inclusion criteria for the study specified T2DM patients whose disease had been diagnosed for at least 3 years; documentation of HbA1c exceeding 7.5% for at least 3 months; stable weight, defined as no significant change (>3%) over the 3 months before enrollment; evidence of stable treatment with oral hypoglycemic therapy or insulin for at least 12 months. All patients had a BMI less than 35 kg/mCaloric restriction, weight loss, and hormonal as well as anatomical changes are some of the major explanations offered as possible mechanisms for improvement in glucose metabolism following surgery. However, the rapid improvement of DM after RYGB counters the argument for weight loss and caloric restriction as these methods require time to achieve proper glycemic control . InsteadFollowing RYGB, the effects of GLP are enhanced leading to marked improvements in both glucose metabolism as well as insulin resistance . This co2 or greater. However, patients with uncontrolled T2DB with BMIs less than 35 should be considered for RYGB as definitive treatment of their T2DM. The NIH guidelines should be reevaluated to consider RYGB in patients with BMI <35 who have uncontrolled T2DM. More prospective studies for the use of RYGB in patients with BMI less than 35 kg/m2 with comorbidities should be conducted using larger sample sizes with longer follow-up evaluation to include 2 years.Currently, the role of RYGB as a weight loss procedure in patients with at least one comorbidity is reserved for patients with a BMI of 35 kg/m


There is increasing evidence to suggest that pericytes play a crucial role in regulating the remodeling state of blood vessels. As cerebral pericytes are embedded within the extracellular matrix (ECM) of the vascular basal lamina, it is important to understand how individual ECM components influence pericyte remodeling behavior, and how cytokines regulate these events.The influence of different vascular ECM substrates on cerebral pericyte behavior was examined in assays of cell adhesion, migration, and proliferation. Pericyte expression of integrin receptors was examined by flow cytometry. The influence of cytokines on pericyte functions and integrin expression was also examined, and the role of specific integrins in mediating these effects was defined by function-blocking antibodies. Expression of pericyte integrins within remodeling cerebral blood vessels was analyzed using dual immunofluorescence (IF) of brain sections derived from the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE).Fibronectin and collagen I promoted pericyte proliferation and migration, but heparan sulfate proteoglycan (HSPG) had an inhibitory influence on pericyte behavior. Flow cytometry showed that cerebral pericytes express high levels of α5 integrin, and lower levels of α1, α2, and α6 integrins. The pro-inflammatory cytokine tumor necrosis factor (TNF)-α strongly promoted pericyte proliferation and migration, and concomitantly induced a switch in pericyte integrins, from α1 to α2 integrin, the opposite to the switch seen when pericytes differentiated. Inhibition studies showed that α2 integrin mediates pericyte adhesion to collagens, and significantly, function blockade of α2 integrin abrogated the pro-modeling influence of TNF-α. Dual-IF on brain tissue with the pericyte marker NG2 showed that while α1 integrin was expressed by pericytes in both stable and remodeling vessels, pericyte expression of α2 integrin was strongly induced in remodeling vessels in EAE brain.Our results suggest a model in which ECM constituents exert an important influence on pericyte remodeling status. In this model, HSPG restricts pericyte remodeling in stable vessels, but during inflammation, TNF-α triggers a switch in pericyte integrins from α1 to α2, thereby stimulating pericyte proliferation and migration on collagen. These results thus define a fundamental molecular mechanism in which TNF-α stimulates pericyte remodeling in an α2 integrin-dependent manner. Pericytes are vascular mural cells that lie in close proximity to endothelial cells of capillaries, arterioles, and venules,2. PericThe basal lamina of cerebral blood vessels comprises a number of different ECM proteins and proteoglycans, the precise make-up of which varies with the vessel-maturation state. The basal lamina of mature vessels is comprised of three major constituents: collagen IV, laminins, and heparan sulfate proteoglycan (HSPG)-14. In aAs pericytes lie within the basal lamina ECM of cerebral microvessels, it seemThe studies described were reviewed and approved by the Scripps Research Institute (TSRI) Institutional Animal Care and Use Committee. All cell cultures were obtained from C57Bl/6 mice, which were maintained under pathogen-free conditions in the closed breeding colony of TSRI.33-35 peptide emulsified in complete Freund’s adjuvant (CFA) containing 2 mg/ml Mycobacterium tuberculosis by subcutaneous injection in both the base of the tail and upper back. In addition, on days 0 and 1, mice also received an intraperitoneal injection of 200 ng pertussis toxin. Control mice received CFA not containing the MOG peptide. This protocol leads to robust induction of clinical EAE on days 12 to 14 after immunization. Animals were monitored daily for clinical signs and scored as follows: 0, no symptoms; 1, flaccid tail; 2, paresis of hind limb; 3, paralysis of hind limbs; 4, quadriplegia; 5, death. At 21 days post-immunization, corresponding to the acute symptomatic stage of disease, mice were euthanized by intraperitoneal injection of sodium pentothal.Experimental autoimmune encephalomyelitis (EAE) was induced using a commercial protocol and materials . Briefly, C57Bl/6 female mice, 8 to 10 weeks old, were immunized with 100 μl of 1 mg/ml MOGPure cultures of mouse brain endothelial cells (BECs) or pericytes were prepared as previously described,25. BriePericytes were obtained using the same approach, except that the puromycin step was omitted. The pericyte cultures were grown in ECGM, with the medium changed every 3 days. On reaching confluency, cultures were harvested with trypsin and passaged. During the first two passages, pericyte cultures were grown in ECGM, but on the third passage, they were switched to pericyte medium  containing 2% FBS. In previous studies we found that, using this approach, cultures of pericytes become highly purified after the third passage, at which point these cultures are more than 99% pericytes as determined by expression of the pericyte marker NG2 and the PDGF-β receptor, and contain less than 1% of contaminating endothelial cells (CD31), astrocytes , or microglia (Mac-1), as determined by fluorescent immunocytochemistry. All stuTo investigate the influence of cytokines on pericyte behavior and expression of integrin subunits, pericytes were cultured on collagen I in the presence of 20 ng/ml basic fibroblast growth factor , 20 ng/ml platelet derived growth factor (PDGF-B) 2 ng/ml transforming growth factor (TGF)-β1 10 ng/ml tumor necrosis factor (TNF)-α, or 10 ng/ml vascular endothelial growth factor (VEGF) . These concentrations were selected based on the findings of previous studies,27. The Integrin expression by BECs and pericytes (treated with different cytokines for 2 days) was examined as described previously. BrieflyAdhesion assays were performed as described previously. BrieflyGlass coverslips were coated with the ECM substrates as described above, and pericytes plated out in serum-free N1 medium. Cytokines and/or integrin-blocking antibodies were included at the time of plating. The following morning, pericytes were incubated for 3 hours with bromodeoxyuridine , fixed in acid/alcohol, and analyzed by immunofluorescence for BrdU incorporation, in accordance with the manufacturer’s instructions. BrdU-positive cells were expressed as the percentage of total cells (Hoechst staining).Pericyte migration was quantified using the scratch assay. Pericytes were plated into ECM-coated wells of a 24-well plate, and cultured in PCM. Upon reaching confluence, vertical and horizontal scratches were made in the monolayer using a 1 ml pipette tip. The PCM and cell debris were removed, and replaced with serum-free N1 medium (cytokines and/or integrin-blocking antibodies were included at this time), and the scratch width was recorded for all samples. The new width of the scratch was recorded 16 hours later, and the distance of cell migration calculated. In each experiment, each condition was performed in duplicate.Immunohistochemistry was performed as described previously, on 10 μt-test was used to analyze the results of the proliferation assays and the immunohistochemistry, while the paired Student’s t-test was used to analyze the results for flow cytometry, and the cell-adhesion and migration assays. For all tests, P<0.05 was defined.All results represent the mean ± SEM of four experiments, except for immunohistochemistry, which was performed with three different animals per condition. The Student’s To investigate how pericyte behavior is influenced by the different ECM molecules present in the vascular basal lamina, pure populations of cerebral pericytes were cultured in serum-free N1 medium on glass coverslips coated with collagen I, collagen IV, fibronectin, HSPG, or laminin-1.P<0.01), with only 13 ± 7.2% cell attachment to laminin-1, and less than 1% of adhesion to HSPG. After 8 hours, fibronectin and collagen I and IV had all promoted greater than 90% adhesion of pericytes, whereas the pericyte adhesion to laminin and HSPG was significantly less (fibronectin 98.8 ± 3.8 versus laminin 51.3 ± 7.3 (P<0.01) and HSPG 29.2 ± 5.2 (P<0.001)). Thus, there was a clear hierarchy in the strength of adhesion of pericytes for different vascular ECM substrates in the order: fibronectin > collagens > laminin-1 > HSPG.First, the pericyte adhesion characteristics were investigated by performing adhesion assays at time-points of 1, 4, and 8 hours. A clear hierarchy among the ECM proteins quickly became evident . Collagen IV also supported pericyte proliferation (5.7 ± 2.2%). However, compared with collagen I (5.7 ± 0.8%), both laminin-1  and HSPG  significantly inhibited pericyte proliferation.Next, the influence of different ECM components on pericyte proliferation and migration was investigated. Pericyte proliferation was examined by BrdU incorporation. Pericytes were cultured in serum-free conditions on the different substrates, then BrdU was added to cells for 3-hours, followed by BrdU immunofluorescence (IF) detection. Pericyte proliferation was promoted most strongly by fibronectin Figure C, with aP<0.001), followed by 50% lower levels on fibronectin  and laminin-1   . Thus, TNF-α strongly promotes cerebral pericyte remodeling behavior, stimulating both pericyte proliferation and migration.Vascular remodeling is promoted by a number of different growth factors, including VEGF, bFGF, and PDGF-BB,30,31. CP<0.01), while strongly upregulating α2 integrin . Interestingly, this TNF-α-induced switch from α1 to α2 integrin was exactly the opposite to that which occurred when pericytes were induced to differentiate (indicated by increased expression of α-smooth muscle actin) by culture in DMEM containing 10% FBS (DF). In this system, DF increased α1 expression , but decreased α2 integrin . Thus, TNF-α and DF exert antagonistic effects on pericyte remodeling status; TNF-α promotes pericyte transition into a remodeling phenotype, which correlates with a switch from α1 to α2 integrins, whereas DF has the opposite effect. Second, several factors increased pericyte expression of the pro-angiogenic α5 integrin subunit. TGF-β1 was the most effective at promoting α5 integrin expression , and TNF-α also promoted this effect . Pericyte α6 integrin-expression levels were not significantly altered by any of the factors tested.It is now well established that growth factors and cytokines exert some of their effects on cell behavior by modulating ECM-integrin interactions,35. To aP<0.001) and anti-α2  antibodies, and pericyte adhesion to collagen IV was also inhibited by anti-β1  and anti-α2  antibodies, whereas the anti-α1 antibodies had no obvious effect  and α5  integrins, indicating that α5β1 is the major pericyte receptor responsible for adhesion to fibronectin. Similar experiments showed that pericyte adhesion to laminin-1 was almost totally blocked by anti-β1 antibody  and partially blocked by anti-α1  and anti-α6  antibodies, but was not significantly affected by the anti-α2 antibody, indicating that pericyte adhesion to laminin-1 is mediated by a combination of α1β1 and α6β1 integrins.Our studies reveal that TNF-α promotes transformation of pericytes into an active remodeling phenotype, and that this correlates with a switch in expression of collagen-binding integrins, from α1 to α2. So what is the functional significance of this switch in β1 integrins? One possibility is that upregulation of α2 integrin confers on pericytes additional adhesive or signaling properties, facilitating increased adhesion to basal lamina collagen and increasing migratory and mitotic capacity. In support of this hypothesis, previous studies have shown that α2 integrin has a much higher affinity than α1 integrin for the ECM substrate collagen I,37, raisP<0.02; proliferation reduced from 35.8 ± 4.7% to 17.6 ± 2.2%, P<0.02) or α2  integrin subunits, but were not significantly affected by the anti-α1 integrin antibody , and this strongly co-localized to NG2-positive pericytes TNF-α promotes cerebral pericyte remodeling Click here for file


Aster glehni (AG) is a Korean traditional herb that grows in Ulleungdo Island, Republic of Korea. None of the several reports on AG include a determination of the effect of AG on adipogenesis. The primary aim of this study was to determine whether AG attenuates adipogenesis in mouse 3T3-L1 cells and epididymal fat tissue. AG blocked the differentiation of 3T3-L1 preadipocytes in a concentration-dependent manner and suppressed the expression of adipogenesis-related genes such as γPPAR, αC/EBP, and SREBP1c, the master regulators of adipogenesis. Male C57BL/6J mice were divided randomly and equally into 4 diet groups: control diet (CON), high-fat diet (HFD), HFD with 1% AG extract added (AG1), and HFD with 5% AG extract added (AG5). The experimental animals were fed HFD and the 2 combinations for 10 weeks. Mice fed HFD with AG gained less body weight and visceral fat-pad weight than did the mice fed HFD alone. Moreover, AG inhibited the expression of important adipogenic genes such as γPPAR, αC/EBP, SREBP1c, LXR, and leptin in the epididymal adipose tissue of the mice treated with AG1 and AG5. These findings indicate antiadipogenic and antiobesity effects of AG and suggest its therapeutic potential in obesity and obesity-related diseases. Overweight and obesity are defined as abnormal or excessive fat accumulation that may impair health; globally, excessive body weight is the fifth leading risk factor for death. At least 2.8 million adults die each year as a result of obesity. Furthermore, 44% of the diabetes burden, 23% of the ischemic heart disease burden, and 7% to 41% of the burden of specific cancers are attributable to obesity [γ (PPARγ) and CCAAT/enhancer binding proteins (C/EBPα), which are crucial transcription factors in adipogenesis, activate the expression of other adipocyte markers [The process by which undifferentiated preadipocytes become adipocytes is termed adipogenesis. The differentiation to adipocyte is a complex developmental procedure that involves the coordinated interplay of numerous transcription factors . Peroxis markers . Sterol  markers . Lipopro markers , and liv markers ; leptin  markers .The common side effects of conventional pharmacological obesity treatments—hypertension, cardiac arrhythmia, constipation, headache, steatorrhea, and deficiencies of lipid-soluble vitamins and essential fatty acids , 10—have Aster glehni Franchet et Sckmidt (AG), which is common to Ulleungdo Island, Republic of Korea, is officially recognized as a regional specialty that grows only in this region. It has recently been named Aster pseudoglehni Lim, Hyun & Shin. In the republic of Korea, the leaves of AG are used in a dish called chwinamul, and AG has also been used to treat diabetes mellitus, hypercholesterolemia, insomnia, and cardiovascular disease [ disease . Accordi disease . Previou disease . Althoug disease , no studAG leaves from Ulleungdo Island, Republic of Korea, were dried and crushed for extraction. The dried material was refluxed with 70% EtOH for 6 h at 60°C. The extract was filtered, concentrated under reduced pressure, and then freeze-dried to obtain a solid extract powder.μg/mL streptomycin, and 100 units/mL penicillin at 37°C in 10% CO2. The cells were grown to confluency in 10% calf serum/DMEM. Two days after the cells had reached confluency (day 0), they were stimulated with a methylisobutylxanthine, dexamethasone, and insulin (MDI) induction medium , 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μm/mL dexamethasone, and 1 μg/mL of insulin). Two days after stimulation with MDI (day 2), the medium was changed to an insulin medium . Two days later (day 4), the medium was changed to 10% FBS/DMEM. The cells were fed with 10% FBS/DMEM every 2 days. Full differentiation was achieved by day 8.The 3T3-L1 mouse preadipocytes were cultured in Dulbecco's Modified Eagle's Medium  supplemented with 10% bovine calf serum, 100 μg/mL oil red O for 15 min. After rinsing with PBS, we captured representative photomicrographs using a microscope and measured fat accumulation.To determine both adipogenic potential and fat accumulation, we stained the cells with oil red O . On day 8, the cultured cells were washed thrice with phosphate-buffered saline (PBS) and then fixed with 4% formaldehyde for 30 min at room temperature. The cells were washed 3 times more with PBS and stained with 0.5 5 cells per well and set up to differentiate in the presence and absence of AG. On day 2 and day 8 of differentiation, the cells were exposed with AdipoRed , and intracellular triglyceride (TG) level was analyzed.3T3-L1 cells were cultured in 6-well plates at a density of 3 × 10For the periods indicated, 3T3-L1 cells were treated with AG or not. Conditioned media from the final 24 h of culture were collected. Leptin concentrations were determined using an enzyme-linked immunosorbent assay .Total RNA was isolated from the differentiated 3T3-L1 cells and epididymal adipose tissue by using Easy-Blue Reagent  according to the manufacturer's instructions. Then, total RNA qualification was performed with an Epoch micro-volume spectrophotometer system . An equal amount of total RNA was used to synthesize cDNA with a high-capacity cDNA reverse transcription kit . The program was set for 10 min of initiation at 25°C, followed by 90 min of incubation at 50°C and 5 min at 85°C. Primers were obtained from Bioneer . All primer sequences and annealing temperatures are shown in 2, 0.1 mM dithiothreitol, and protease inhibitor mixture). Determination of the protein was carried out on Tris-glycine SDS-polyacrylamide gels, followed by a transfer to PVDF membranes . Then, the membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% tween 20 (TBST) at room temperature for 1 h and incubated at 4°C overnight with 1 : 1000 dilutions of primary antibodies. The following day, the membranes were washed with TBST 3 times for 10 min each and made to react with 1 : 2500 dilutions of horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. After the reaction, the complexes were visualized with ECL plus detection reagents .The 3T3-L1 cells were lysed in a lysis buffer  were fed standard chow and those in group 2 (HFD) a HFD (40% fat). group 3 (AG1) consisted of mice fed with the same HFD, which included 1% AG extract (1% w/w in HFD), and group 4 (AG5) consisted of mice fed HFD with 5% added AG extract (5% w/w in HFD). Mice were fed experimental diet for 10 weeks. Body weight was recorded weekly. After an overnight fast at the end of the experimental period, tissue collection was performed. The animals were sacrificed by cervical spine dislocation and the livers, epididymal, retroperitoneal and total fat pads were dissected, weighed immediately, and frozen at −80°C.Male C57BL/6J mice, weighing 15–17 g at an age of 3-4 weeks, were purchased from Daehan Biolink . They were housed (5 mice/cage) under a 12 h light-dark cycle, at 23 ± 2°C, and a relative humidity of 55% ± 10%, under conditions that followed the NIH Guide for the Care and Use of Laboratory Animals; they were allowed diet and water μm were cut and stained with hematoxylin and eosin (H&E) for analysis of adipocyte diameter, surface area, and number. The stained slides were observed with a SZX10 microscope  and photographed.The liver and the epididymal adipose tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Sections of 4 and 8 μm particles  was used. The column eluent was monitored at UV 246 nm, following which all solvents were degassed with a micromembrane filter . Chromatography was performed at room temperature at a flow rate of 0.5 mL/min, and 10 μL was analyzed for 50 min. In brief, seven CQs and the four extracts dissolved in 80% MeOH were filtered using a syringe filter and injected for HPLC analysis. The two mobile phases of 0.05% phosphoric acid (solvent A) and MeOH (solvent B) were used for gradient elution at the rate of 1.00 mL/min: 0–10 min, 60% A : 40% B; 10–20 min, 50% A : 50% B; 20–30 min, 40% A : 60% B; 30–35 min, 60% A : 40% B. The two flavonoids, astragalin and kaempferol, were also used as the standard compounds for the analysis. Under these HPLC identical conditions, the retention times of astragalin and kaempferol were 14.4 min and 24.3 min, respectively. Plotting the peak area  versus concentration , regression equations of y = 27.10x + 50.50 (R2 = 0.998) and y = 54.85x + 287.01 (R2 = 0.999) for astragalin and kaempferol, respectively. Sample solutions were injected into the HPLC system at 1.000 mg/mL, and the contents were determined from the regression equation.The HPLC apparatus was a Gilson System equipped with a 234 autosampler, a UV/VIS-155 detector, and a 321 HPLC Pump . A Luna 4.60 × 250 mm C18 reversed-phase column with 5 P value of <0.05 was considered statistically significant.The results are presented as the mean ± S.E.M. of the 3 independent experiments. The data were analyzed by one-way analysis of variance (ANOVA); statistical analyses were performed using SPSS (Version 19.0). A To determine whether AG has an antiobesity effect, we carried out adipocyte differentiation using 3T3-L1 cells, both in the presence and the absence of AG. After 8 days, oil red O was added to the cultured cells, and fat accumulation and intracellular triglyceride levels were analyzed. Troglitazone, an antidiabetic and anti-inflammatory drug , was useγ, C/EBPα, and SREBP1c are key regulators of adipogenesis, and LXR is also involved. After cell differentiation for 8 days in the presence or absence of 100, 200, and 400 μg/mL of AG, the expression levels of adipogenic markers were determined by real-time PCR. The 8-day AG treatment of cells reduced the mRNA expression of PPARγ, C/EBPα, SREBP1c, and LXR . We usually use 10–20% doses for evaluating the antiobesity effect in animal models . HoweverAdipose tissue is considered a crucial factor in the regulation of many pathological processes, and it is linked to both the immune and the metabolic systems. For that reason, obesity is associated with both a chronic inflammatory response and immune functions . FurtherCaffeoylquinic acids (CQs) are esters of caffeic and quinic acids and are phenolic compounds. They exist naturally as chlorogenic acid (3-CQ), cryptochlorogenic acid (4-CQ), neochlorogenic acid (5-CQ), cynarin , isochlorogenic acid A , isochlorogenic acid B , and isochlorogenic acid C  . Their vWe have demonstrated that AG exerts antiobesity effects both in vitro and in vivo. Its effects on obesity are based on the downregulation of adipogenic-related transcription factors, suggesting that AG has a potential as a therapeutic agent for obesity and obesity-related diseases.