Scalable Generation of 3D Pancreatic Islet Organoids from Human Pluripotent Stem Cells in Suspension Bioreactors.

We describe a scalable method for the robust generation of 3D pancreatic islet-like organoids from human pluripotent stem cells using suspension bioreactors. Our protocol involves a 6-stage, 20-day directed differentiation process, resulting in the production of 104-105 organoids. These organoids comprise α- and ß-like cells that exhibit glucose-responsive insulin and glucagon secretion. We detail methods for culturing, passaging, and cryopreserving stem cells as suspended clusters and for differentiating them through specific growth media and exogenous factors added in a stepwise manner. Additionally, we address quality control measures, troubleshooting strategies, and functional assays for research applications.

Structure-Guided Discovery of Potent and Selective CLK2 Inhibitors for the Treatment of Knee Osteoarthritis.

Osteoarthritis is the most common joint disorder. However, there are no disease-modifying drugs approved for OA treatment. CDC2-like kinase 2 (CLK2) could modulate Wnt signaling via alternative splicing of Wnt target genes and further affect bone differentiation, chondrocyte function, and inflammation, making CLK2 an attractive target for OA therapy. In this study, we designed and synthesized a series of highly potent CLK2 inhibitors based on Indazole 1. Among them, compound LQ23 showed more elevated inhibitory activity against CLK2 than the lead compound (IC50, 1.4 nM) with high CLK2/CLK3 selectivity (>70-fold). Furthermore, LQ23 showed outstanding antiosteoarthritis effects in vitro and in vivo, with the roles specific in decreased inflammatory cytokines, downregulated cartilage degradative enzymes, and increased joint cartilage via suppressing CLK2/Wnt signaling pathway. Overall, these data support LQ23 as a potential candidate for intra-articular knee OA therapy, leveraging its unique mechanism of action for targeted treatment.

Expansion of human hematopoietic stem cells by inhibiting translation.

Hematopoietic stem cell (HSC) transplantation using umbilical cord blood (UCB) is a potentially life-saving treatment for leukemia and bone marrow failure but is limited by the low number of HSCs in UCB. The loss of HSCs after ex vivo manipulation is also a major obstacle to gene editing for inherited blood disorders. HSCs require a low rate of translation to maintain their capacity for self-renewal, but hematopoietic cytokines used to expand HSCs stimulate protein synthesis and impair long-term self-renewal. We previously described cytokine-free conditions that maintain but do not expand human and mouse HSCs ex vivo. Here we performed a high throughput screen and identified translation inhibitors that allow ex vivo expansion of human HSCs while minimizing cytokine exposure. Transplantation assays show a ~5-fold expansion of long-term HSCs from UCB after one week of culture in low cytokine conditions. Single cell transcriptomic analysis demonstrates maintenance of HSCs expressing mediators of the unfolded protein stress response, further supporting the importance of regulated proteostasis in HSC maintenance and expansion. This expansion method maintains and expands human HSCs after CRISPR/Cas9 editing of the BCL11A+58 enhancer, overcoming a major obstacle to ex vivo gene correction for human hemoglobinopathies.

Scalable generation of 3D pancreatic islet organoids from human pluripotent stem cells in suspension bioreactors.

Here, we present a protocol for producing 3D pancreatic-like organoids from human pluripotent stem cells in suspension bioreactors. We describe scalable techniques for generating 10,000-100,000 organoids that further mature in 4-5 weeks into α- and ß-like cells with glucose-responsive insulin and glucagon release. We detail procedures for culturing, passaging, and cryopreserving stem cells as suspended clusters and specify growth media and differentiation factors for differentiation. Finally, we discuss functional assays for research applications. For complete details on the use and execution of this protocol, please refer to Alvarez-Dominguez et al.1.

An adult clock component links circadian rhythms to pancreatic ß-cell maturation.

How ubiquitous circadian clocks orchestrate tissue-specific outputs is not well understood. Pancreatic ß cell-autonomous clocks attune insulin secretion to daily energy cycles, and desynchrony from genetic or behavioral disruptions raises type 2 diabetes risk. We show that the transcription factor DEC1, a clock component induced in adult ß cells, coordinates their glucose responsiveness by synchronizing energy metabolism and secretory gene oscillations. Dec1-ablated mice develop lifelong hypo-insulinemic diabetes, despite normal islet formation and intact circadian Clock and Bmal1 activators. DEC1, but not CLOCK/BMAL1, binds maturity-linked genes that mediate respiratory metabolism and insulin exocytosis, and Dec1 loss disrupts their transcription synchrony. Accordingly, ß-cell Dec1 ablation causes hypo-insulinemia due to immature glucose responsiveness, dampening insulin rhythms. Thus, Dec1 links circadian clockwork to the ß-cell maturation process, aligning metabolism to diurnal energy cycles.

Prognostic value of CD247 in patients with head and neck squamous cell carcinoma: bioinformatic analysis of TCGA database.

Background: Head and neck squamous cell carcinoma (HNSC) is the 7th most common type of cancer in the world. Through the advantages of The Cancer Genome Atlas (TCGA) large-scale sequencing-based genome analysis technology, we can explore the potential molecular mechanisms that can improve the prognosis of HNSC patients. Methods: The HNSC transcriptome and clinical data were downloaded from TCGA database. A univariate survival analysis and differential expression analysis were conducted to obtain the intersection gene set. A protein-protein interaction (PPI) analysis, modular analysis, and Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis were then conducted to identify the hub genes. Clinical correlation analysis, univariate and multivariate Cox regression analyses were performed on the identified hub genes to determine the prognostic impact of hub genes on HNSC patients. Results: In total, 601 intersecting gene sets were obtained. A modular analysis was conducted, and the highest scoring module was 19.304. Based on the GO/KEGG enrichment analysis results, CD247 molecule (CD247) was ultimately selected as the gene for this study. The CD247 were divided into a high-expression group and a low-expression group. The Kaplan-Meier survival curve analysis showed that there was a significant difference between the 2 groups (P<0.0001). The median survival time of the low-expression CD247 group was 30.9 months, and the 5-year survival rate was 36.4%. While the median survival time of the high-expression CD247 group was 68.8 months, and the 5-year survival rate was 52.3%. The clinical correlation analysis showed that CD247 was significantly negatively correlated with pathological tumor stage (pT) and pathological nodal extracapsular spread. Gene Set Enrichment Analysis (GSEA) showed that CD247 activating KEGG pathway hsa04650 and hsa04660. Conclusions: CD247 is an independent protective factor in the prognosis of HNSC patients. By activating the hsa04650 and hsa04660 pathways, the expression of interferon gamma, interleukin (IL)-2, and IL-10 is promoted, which in turn improves the tumor immune monitoring ability of the body, induces tumor cell apoptosis, and inhibits tumor cell growth. CD247 is a potential target for improving the clinical treatment effect of HNSC and the prognosis of patients.

An integrated microfluidics platform with high-throughput single-cell cloning array and concentration gradient generator for efficient cancer drug effect screening.

BACKGROUND: Tumor cell heterogeneity mediated drug resistance has been recognized as the stumbling block of cancer treatment. Elucidating the cytotoxicity of anticancer drugs at single-cell level in a high-throughput way is thus of great value for developing precision therapy. However, current techniques suffer from limitations in dynamically characterizing the responses of thousands of single cells or cell clones presented to multiple drug conditions. METHODS: We developed a new microfluidics-based "SMART" platform that is Simple to operate, able to generate a Massive single-cell array and Multiplex drug concentrations, capable of keeping cells Alive, Retainable and Trackable in the microchambers. These features are achieved by integrating a Microfluidic chamber Array (4320 units) and a six-Concentration gradient generator (MAC), which enables highly efficient analysis of leukemia drug effects on single cells and cell clones in a high-throughput way. RESULTS: A simple procedure produces 6 on-chip drug gradients to treat more than 3000 single cells or single-cell derived clones and thus allows an efficient and precise analysis of cell heterogeneity. The statistic results reveal that Imatinib (Ima) and Resveratrol (Res) combination treatment on single cells or clones is much more efficient than Ima or Res single drug treatment, indicated by the markedly reduced half maximal inhibitory concentration (IC50). Additionally, single-cell derived clones demonstrate a higher IC50 in each drug treatment compared to single cells. Moreover, primary cells isolated from two leukemia patients are also found with apparent heterogeneity upon drug treatment on MAC. CONCLUSION: This microfluidics-based "SMART" platform allows high-throughput single-cell capture and culture, dynamic drug-gradient treatment and cell response monitoring, which represents a new approach to efficiently investigate anticancer drug effects and should benefit drug discovery for leukemia and other cancers.

Correlation of structural extracellular polymeric substances in the mesh biofilms with solids retention time and biofilm hydraulic resistance in dynamic membrane bioreactors.

Dynamic membrane bioreactor (DMBR), which mainly relied on the in-situ formed biofilms on support materials with large aperture (e.g., nylon mesh) to separate fine particles in wastewater, has attracted a lot of attentions due to low cost. The filtration performance of DMBR is mainly determined by the structure and hydraulic resistance of biofilms formed on the mesh. Therefore, understanding the correlation of operation conditions with mesh biofilm compositions and permeability are critically important for optimizing DMBR operation. In present study, how structural extracellular polymeric substances, including alginate-like extracellular polysaccharide (ALE) and amyloid-like protein (AP), in mesh biofilms correlate to solids retention time (SRT) and biofilm structures was explored in DMBRs. At 5d-SRT, compact and gel-like mesh biofilms were formed with a high specific filtration resistance (SFR) of 459 × 109 m/g, while at 40d-SRT porous mesh biofilms were developed with a low SFR of 24 × 109 m/g. Consequently, the 5d-SRT MBR experienced more rapid rise in transmembrane pressure. Further studies found that the 5d-SRT mesh biofilms had a higher AP content, which was positively correlated to biofilm hydraulic resistance. On the contrary, the 40d-SRT mesh biofilms contained a higher content of ALE, suggesting that ALE was negatively correlated to biofilm hydraulic resistance. Therefore, AP instead of ALE likely played a more important role in the formation of compact and gel-like mesh biofilms.

Inactivation of Polymicrobial Biofilms of Foodborne Pathogens Using Epsilon Poly-L-Lysin Conjugated Chitosan Nanoparticles.

A mixed culture (polymicrobial) biofilm provides a favorable environment for pathogens to persist in the food processing environment and to contaminate food products. Inactivation and eradication of such biofilms from food processing environments are achieved by using harsh disinfectants, but their toxicity and environmentally hostile characteristics are unsustainable. This study aims to use food-grade natural nanoparticulated antimicrobials to control mixed-culture biofilms. Chitosan, a natural broad-spectrum antimicrobial biopolymer (polysaccharide) from crustaceans, was derivatized to produce chitosan nanoparticles (ChNP) as a carrier for another broad-spectrum antimicrobial agent, ε-poly-L-lysine (PL), to synthesize ChNP-PL conjugate. The antimicrobial activity of ChNP and ChNP-PL was tested against mixed-culture biofilms. ChNP-PL (~100 nm) exhibited a synergistic antimicrobial and anti-biofilm effect against mono or mixed-culture biofilms of five foodborne pathogens, including Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica serovar Enteritidis, Escherichia coli O157:H7, and Pseudomonas aeruginosa. ChNP-PL treatment prevented biofilm formation by mono or mixed cultures of L. monocytogenes, P. aeruginosa, and E. coli O157:H7, and bacterial counts were either below the detection limit or caused 3.5-5 log reduction. ChNP-PL also inactivated preformed biofilms. In monoculture biofilm, ChNP-PL treatment reduced L. monocytogenes counts by 4.5 logs, S. Enteritidis by 2 logs, E. coli by 2 logs, and S. aureus by 0.5 logs, while ChNP-PL had no inhibitory effect on P. aeruginosa. In vitro mammalian cell-based cytotoxicity analysis confirmed ChNP-PL to have no deleterious effect on intestinal HCT-8 cell line. In conclusion, our results show ChNP-PL has strong potential to prevent the formation or inactivation of preformed polymicrobial biofilms of foodborne pathogens.

[Moxibustion on mental disorder in close contacts of coronavirus disease 2019: a randomized controlled trial].

OBJECTIVE: To observe the effect of moxibustion combined with Daiwenjiu plaster on mental disorder and self rating symptoms in close contacts of coronavirus disease 2019 (COVID-19) during quarantine. METHODS: A total of 240 close contacts of COVID-19 were randomized into an observation group (120 cases, 18 cases dropped off) and a control group (120 cases, 58 cases dropped off). Conventional observation was adopted in the control group. Moxibustion combined with Daiwenjiu plaster was given in the observation group, moxibustion was applied at Zusanli (ST 36), Hegu (LI 4) and Shenque (CV 8), 10 min each acupoint, once a day; Daiwenjiu plaster was applied at Dazhui (GV 14) and Feishu (BL 13) for 3 to 6 h each time, once every 2 days, the treatment was required for 2 weeks. Before and after treatment and in the follow-up of 2 weeks after treatment, self-reporting questionnaire 20 (SRQ-20), self-rating anxiety scale (SAS) scores and self rating symptoms were evaluated in the two groups. RESULTS: In the follow-up, SRQ-20 score was decreased compared before treatment (P<0.01), SAS score was decreased compared before and after treatment (P<0.01, P<0.05) in the observation group. After treatment and in the follow-up, SRQ-20 scores in the observation group were lower than those in the control group (P<0.05, P<0.01). After treatment, the case proportion of fatigue in the observation group was higher than that in the control group (P<0.05). In the follow-up, the case proportion of tension was lower than that in the control group (P<0.05). CONCLUSION: Moxibustion combined with Daiwenjiu plaster can effectively improve the mental disorder in close contacts of COVID-19.

[Analysis of theoretical basis and clinical application of dog-days moxibustion].

The present situation of the clinical application of dog-days moxibustion (moxibustion applied in the three periods of the hot season) is summarized so as to provide the guide for the theoretic study and clinical application of dog-days moxibustion. The intervention time of dog-days moxibustion is on the 1st day of each of the three periods of the hot season. Simultaneously, the geographic factors are considered. The disorders of lung system are mostly dominant among the indications of dog-days moxibustion, complicated with spleen and stomach disorders as well as cold and deficiency syndromes/patterns. The acupoints are mainly selected from the front-mu points on the chest and the back-shu points on the back, in combination with the differentiation of diseases, symptoms/patterns and the disorder stages. The duration of treatment is ranged from 1 to 3 years. The clinical therapeutic effect is improved constantly along with the increase of treatment periods by years.

[Significance and operation mode of moxibustion intervention for the group under quarantine after close contact with COVID-19].

On the base of the idea of traditional Chinese medicine as "disease prevention", the mode and the protocol of the moxibustion intervention for the group under quarantine after close contact with coronavirus disease 2019 (COVID-19) were explored. The group under quarantine after close contact with COVID-19 was taken as the subjects. By the non-contact physician-patient communication network platform co-developed by China Association of Acupuncture-Moxibustion, Hunan Provincial Association of Acupuncture-Moxibustion, Data Center of China Academy of Chinese Medical Sciences and Yuge Medicine Company, an exploratory randomized controlled trial was designed. A total of 100 cases were included and randomized into a moxibustion group and a conventional intervention group, 50 cases in each one. In the moxibustion group, moxibustion intervention was used. In the conventional intervention group, the conventional observation was adopted without moxibusiton intervention applied. The outcomes included the symptoms changes, e.g. anxiety, emotional disturbance, fatigue, headache and diarrhea, as well as whether quarantine release and the case confirmed or not, etc. The results were evaluated before intervention, in 14 days of intervention and 2 weeks after intervention separately. In this research, on the base of internet plus technology and with the internet communication platform adopted, through mobile phone WeChat App, it was to implement the subject screen, the random allocation and the instruction of moxibustion intervention as well as the quality control of patient's diary and data collection. It is anticipated that the significance and the implementation mode of moxibustion intervention can be assessed preliminarily for the group under quarantine after close contact with COVID-19.

Effect of Tai Chi Chuan in Breast Cancer Patients: A Systematic Review and Meta-Analysis.

Background: Tai Chi Chuan(TCC), as a mind-body exercise, may have a positive impact on physical function and psychological well-being in breast cancer patients. The latest systematic review and meta-analysis of TCC for breast cancer was made 4 years ago and some new clinical trials about it were published. We remade a systematic review and meta-analysis to evaluate the effect of TCC in breast cancer patients. Methods: In this systematic review and meta-analysis, we searched MEDLINE (via PubMed), EMBASE (via embase.com), CENTRAL, CNKI, COVIP, Wanfang, Chaoxing, CiNii, J-SSTAGE, DBpia, and ThaiJO with no language restrictions from inception to December 31, 2018 (updated on February 16, 2020), for randomized clinical trials comparing TCC with non-exercised therapy in breast cancer patients. The primary outcome was quality of life in patients with breast cancer and data pooled by a random-effects model. Subgroup analyses were conducted to estimate the effect of different durations of TCC for breast cancer patients. This study was registered in PROSPERO, number CRD 4201810326. Results: Fifteen articles involving a total of 885 breast cancer participants were included in this review. Compared with non-exercised therapy, TCC had a significant effect on quality of life in breast cancer patients (SMD = 0.37, 95% CI 0.15-0.59, p = 0.001), and subgroup analysis found that TCC showed beneficial effect in 12 weeks and 25 weeks (12 weeks: SMD = 0.40, 95% CI 0.19-0.62, p = 0.0003; 25 weeks: SMD = 0.38, 95% CI 0.15-0.62, p = 0.002). Meta-analyses of secondary outcomes showed that 3 weeks TCC increased shoulder function (SMD = 1.08, 95% CI 0.28-1.87, p = 0.008), 12 weeks TCC improved pain (SMD = 0.30, 95% CI 0.08-0.51, p = 0.007), shoulder function (SMD = 1.34, 95% CI 0.43-2.25, p = 0.004), strength of arm (SMD = 0.44, 95% CI 0.20-0.68, p = 0.0004), and anxiety (MD = -4.90, 95% CI -7.83 to -1.98, p = 0.001) in breast cancer patients compared with the control group. Conclusions: TCC appears to be effective on some physical and psychological symptoms and improves the quality of life in patients with breast cancer. Additional randomized controlled trials with a rigorous methodology and low risk of bias are needed to provide more reliable evidence.

Cancer stem-like cells with increased expression of NY-ESO-1 initiate breast cancer metastasis.

Breast cancer stem-like cells (BCSLCs) with a CD44+/CD24-/low phenotype initiate the invasion and metastasis of breast cancer. The expression of New York oesophageal squamous cell carcinoma 1 (NY-ESO-1), one of the most immunogenic cancer-testicular antigens, is largely restricted to cancer and germ cells/placental trophoblasts, with little to no expression in normal adult somatic cells. Currently, few studies have reported the expression or function of NY-ESO-1 in BCSLCs. In the present study, immunohistochemistry indicated enhanced expression levels of NY-ESO-1/CD44 (P<0.01) and decreased expression levels of CD24 (P<0.01) in metastatic breast cancer tissues (MBCT) compared with non-MBCT. Additionally, the co-localization of CD44, CD24 and NY-ESO-1 in tissue samples was determined using immunofluorescence analysis. The results revealed that the expression of NY-ESO-1/CD44/CD24 was associated with breast cancer metastasis. Moreover, Spearman's rank correlation analysis indicated that CD44/CD24 expression was significantly correlated with that of NY-ESO-1. In the present study, mammosphere culture, a valuable method of BCSLC enrichment, was used to enrich MCF-7 and SK-BR-3 BCSLCs; immunofluorescence, western blotting and flow cytometry demonstrated increased expression levels of NY-ESO-1 and CD44, and low expression levels of CD24 in BCSLCs. Furthermore, the cell migration and invasion assays verified that BCSLCs with an increased NY-ESO-1 expression level exhibited greater invasive and migratory capacity compared with parental breast cancer cells. In addition to previously reported findings from the Oncomine database, it was ascertained that CD44+/CD24-/low BCSLCs with an increased level of NY-ESO-1 expression initiated the invasion and metastasis of breast cancer; therefore, NY-ESO-1 may serve as a novel target for metastatic breast cancer immunotherapy.

[Effects of herbal-cake-separated moxibustion on the repair of vascular endothelial and SDF-1 in rabbits with atherosclerosis].

OBJECTIVE: To observe the effects of herbal-cake-separated moxibustion on the repair of damaged vascular endothelium structure and the content of stromal cells derived factor 1 (SDF-1) in rabbits with atherosclerosis. METHODS: A total of 75 rabbits were randomly divided into a normal group, a model group, a direct moxibustion group, an atorvastatin calcium group and a herbal-cake-separated moxibustion group, 15 rabbits in each one. The rabbits in the normal group were fed with normal diet, and the remaining rabbits were fed with high-cholesterol diet for 12 weeks to prepare atherosclerotic model. Two groups of acupoints, one was "Juque" (CV 14), "Tianshu" (ST 25) and "Fenglong" (ST 40), the other one was "Xinshu" (BL 15), "Ganshu" (BL 18) and "Pishu" (BL 20), were applied in the direct moxibustion group and herbal-cake-separated moxibustion group; the two groups of acupoints were selected alternatively every other day. The moxibustion was given for 30 min per treatment, once a day for 4 weeks. The rabbits in the atorvastatin calcium group were treated with atorvastatin calcium tablets (1.96 mg•kg-1•d-1) which were crushed into powder and mixed into breakfast. After modeling, the rabbits in the normal group and model group received no treatment, and immobilized at the time when moxibustion was applied in other three groups. The levels of total cholesterol (TC) and triglyceride (TG) were measured by enzymic method; the low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured by colorimetric method; the morphological structure of aortic wall was observed under optical microscope; the serum level of SDF-1 was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: After treatment, compared with the normal group, the levels of TC, TG and LDL-C were significantly increased in the model group (all P<0.01), and the level of HDL-C was decreased (P<0.01). Compared with the model group, the levels of TC, TG and LDL-C were significantly decreased (all P<0.01), and the level of HDL-C was significantly increased in the direct moxibustion group, atorvastatin calcium group and herbal-cake-separated moxibustion group (P<0.01, P<0.05). Compared with the normal group, the morphological structure of aortic wall was significantly damaged in the model group. Compared with the model group, the vascular endothelial structure was improved in the atorvastatin calcium group and herbal-cake-separated moxibustion group, and the pathological change of aorta endothelial in the direct moxibustion group was relieved. After treatment, compared with the model group, the level of SDF-1 was increased in the direct moxibustion group, atorvastatin calcium group and herbal-cake-separated moxibustion group (P<0.05, P<0.01); the level of SDF-1 in the herbal-cake- separated moxibustion group was higher than that in the direct moxibustion group (P<0.05). CONCLUSION: The herbal- cake-separated moxibustion can promote the expression of SDF-1 in serum and repair the damaged aortic endothelial structure.

[Effect of Herbal-cake-separated Moxibustion on Blood Lipid Levels and Expression of Hepatic PPARγ and SR-B 1 Proteins and Genes in Hyperlipidemia Atherosclerosis Rabbits].

OBJECTIVE: To observe the effect of herbal-cake-separated moxibustion on blood lipid levels and expression of peroxisome proliferator-activated receptor γ (PPARγ) and scavenger receptor B 1 (SR-B 1) proteins and genes in liver of hyperlipidemia atherosclerosis rabbits, so as to explore its mechanism underlying anti-atherosclerosis formation. METHODS: Forty male New Zealand white rabbits were randomly divided into normal control, model, moxibustion and Simvastatin groups (n＝10 rabbits in each group). The hyperlipidemia atherosclerosis model was established by high cholesterol diet and propylthiouracil for 12 weeks. Herbal-cake-separated moxibustion was applied to "Juque" (CV 14), and bilateral "Tianshu" (ST 25), "Fenglong" (ST 40) (point group 1), and bilateral "Xinshu" (BL 15), "Ganshu" (BL 18) and "Pishu" (BL 20) (point group 2). The two groups of points were used alternately. Simvastatin (1.96 mg•kg－1•d－1) mixed in the forage was given to rabbits of the Simva-statin group. Both moxibustion and medication treatments were given once daily for continuous 4 weeks. Total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) levels in plasma were detected by using an automatic biochemistry analyzer. The expression levels of PPARγ and SR-B 1 proteins and genes in the hepatic tissue were determined by Western blot and reverse transcription-polymerase chain reaction, separately. RESULTS: After modeling, plasma TC, TG and LDL-C levels in the model group were significantly increased (P<0.01), and the levels of plasma HDL-C and hepatic PPARγ and SR-B 1 protein and mRNA expression were obviously down-regulated relevant to the normal group (P<0.01). Compared with the model group, plasma TC, TG and LDL-C levels were significantly decreased (P<0.01), and plasma HDL-C and hepatic PPARγ and SR-B 1 protein and mRNA levels were significantly up-regulated in the two treatment groups (P<0.01, P<0.05). CONCLUSION: Herbal-cake-separated moxibustion can regulate blood lipid levels and suppress hyperlipidemia-induced decrease of expression of hepatic PPARγ and SR-B 1 proteins and genes in hyperlipidemia atherosclerosis rabbits, which maybe contribute to its action in anti-atherosclerosis through promoting reversal of cholesterol.

[Effects of Herbal-cake-separated Moxibustion on Blood Lipid Protein Levels and Expression of Toll-like Receptor and Nuclear Factor Genes in Atherosclerotic Plaques in Hyperglycemia Rabbits].

OBJECTIVE: To observe the effect of herbal cake-separated moxibustion on blood lipid-apoprotein levels and the expression of Toll-like receptor 2 (TLR 2), TLR 4 and nuclear factor kappa B(NF-κB) mRNAs in atherosclerotic (AS) vulnerable plaques of hyperglycemia rabbits, so as to explore its mechanism underlying improvement of atherosclerosis. METHODS: Sixty New Zealand rabbits were randomly divided into 5 groups: control, model, direct moxibustion, herbal-cake-separated moxibustion and medication groups(n＝12 rabbits in each group). The AS vulnerable plaque model was established by high-fat forage feeding plus balloon-induced abdominal aorta injury and gene transfection of Ad 5-p 53 recombinant vector. Direct moxibustion or herbal-cake-separated moxibustion was applied to "Juque" (CV 14) and bilateral "Tianshu" (ST 25), "Fenglong" (ST 40), or bilateral "Xinshu" (BL 15), "Pishu" (BL 20) and "Ganshu" (BL 18) for 15－20 min every time. The medication group was treated by feeding Atorvastatin. All the treatments were conducted once daily for 8 weeks. Plasma total cholesterol(TC) and triglyceri-de(TG) contents were detected by enzyme method, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) contents detected by colorimetric determination, and plasma apolipoprotein A(Apo-A) and apolipoprotein B(Apo-B) levels determined by electrophoretic method. The pathological changes of vulnerable plaque and the aortic intima and media thickness were observed under light microscope after H．E. staining. The expression levels of TLR 2, TLR 4 and NF-κB mRNAs in AS plaques were determined by quantitative real-time PCR. RESULTS: After modeling, the levels of plasma TC, TG, LDL and Apo-B in the model group were remarkably increased (P<0.01), and Apo-A and HDL/LDL were significantly decreased in comparison with the control group (P<0.01). Additionally, the aortic intima and media thickness and the expression levels of TLR 2, TLR 4 and NF-κB mRNAs in AS plaques were significantly increased (P<0.01). After the treatment, the elevated levels of plasma TC, TG, LDL and Apo-B, the aortic intima thickness and media thickness, and the expression levels of TLR 2, TLR 4 and NF-κB mRNAs in the 3 treatment groups were significantly down-regulated in comparison with the model group (P<0.05, P<0.01), while the decreased levels of Apo-A and HDL/LDL were considerably increased (P<0.01). Comparison among the 3 treatment groups showed that the therapeutic effects of the herbal-cake-separated moxibustion and medication were significantly superior to those of the direct moxibustion in down-regulating the levels of TC, TG, LDL, Apo-B, TLR 2 mRNA, TLR 4 mRNA and NF-κB mRNA, and reducing the thickness of the aortic intima and media, as well as in up-regulating the levels of Apo-A and HDL/LDL (P<0.05, P<0.01). No significant differences were found between the herbal-cake-separated moxibustion and medication groups in the above-mentioned indexes (P>0.05). CONCLUSION: Herbal-cake-separated moxibustion has a positive role in stabilizing AS vulnerable plaque in hyperglycemia rabbits, which may be associated with its effects in regulating blood lipid-apolipoprotein levels and inhibiting the expression of TLR 2, TLR 4 and NF-κB mRNAs in vulnerable plaques.

Lys63-linked ubiquitin chain adopts multiple conformational states for specific target recognition.

A polyubiquitin comprises multiple covalently linked ubiquitins and recognizes myriad targets. Free or bound to ligands, polyubiquitins are found in different arrangements of ubiquitin subunits. To understand the structural basis for polyubiquitin quaternary plasticity and to explore the target recognition mechanism, we characterize the conformational space of Lys63-linked diubiquitin (K63-Ub2). Refining against inter-subunit paramagnetic NMR data, we show that free K63-Ub2 exists as a dynamic ensemble comprising multiple closed and open quaternary states. The quaternary dynamics enables K63-Ub2 to be specifically recognized in a variety of signaling pathways. When binding to a target protein, one of the preexisting quaternary states is selected and stabilized. A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands. This conformational selection mechanism at the quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition.

[Development of researches on acupuncture stimulation-induced regulatory effect under pathological state based on resting-state functional magnetic resonance technology].

Using keywords of "acupuncture, fMRI and resting state", authors of the present paper retrieved papers in both Chinese and English from CNKI and PubMed databases without time limitation. A total of 13 articles (11 for acupuncture and 2 for moxibustion) met the standards were subjected to analysis. Of the 13 papers, 6 involve acupuncture analgesia, 2 moxibustion analgesia, 3 mild cognitive impairment, one involves stroke, and the rest one is for depression. Research results of acupuncture analgesia revealed that the analgesic effect is closely related to its regulation on "pain matrix" brain regions and pain-related default mode network. In the studies, a single acupoint is often used, without acupoints-combined recipes. Therefore, it is highly recommended that researchers pay more attention to (OD studies on the underlying mechanism of moxibustion, and (improvement of design protocols targeting requirements of acupuncture clinical practice.