[Therapeutic effect of gene silencing peptidyl arginine deaminase 4 on pulmonary interstitial lesions induced by collagen-induced arthritis mice].

OBJECTIVE: To investigate the therapeutic effect of gene silencing peptidyl arginine deaminase 4 (PAD4) on pulmonary interstitial lesions induced by collagen-induced arthritis (CIA) mice, and possible mechanisms. METHODS: A CIA mouse model was established in DBA/1 mice, followed by a tail vein injection of the virus solution prepared by the PAD4-siRNA expression vector once a week for 8 times. The mice were sacrificed at the end of the experiment. The expression of PAD4 mRNA in lungs was detected by real-time quantitative PCR (qRT-PCR). The expression of PAD4 protein was detected by tissue immunohistochemistry. Cell culture was performed by spleen tissue. Flow cytometry changes in the ratio of Tfh cells to Tfr cells were examined; lung staining was performed in the lungs to observe changes in lung pathology. RESULTS: (1) Compared with the blank group, the expression of PAD4 mRNA in the lung tissue of the model group increased, the difference was statistically significant (P < 0.05). PAD4 mRNA in the lung tissue of the CIA mice after PAD4-siRNA treatment. The expression level was significantly lower than that of the model group and the negative control group, and the difference was statistically significant (P < 0.05). (2) Red fluorescence was less in the lung tissue of the blank group, while more red fluorescence was observed in the inflammatory cell infiltration area and trachea around the lung tissue of the model group and the negative control group, and the red fluorescence of the three groups after PAD4-siRNA treatment was significantly reduced; (3) Compared with the blank group, the proportion of Tfh cells in the model group increased, the difference was statistically significant (P < 0.05), the proportion of Tfh cells in spleen cells of the CIA mice after PAD4-siRNA treatment was significantly lower than that of the model group and the negative control group, the difference was statistically significant (P < 0.05); compared with the blank group, in the mouse spleen cells in the model group the proportion of Tfr cells was slightly decreased, but the difference was not statistically signifi-cant. The proportion of Tfr cells in the spleen cells of the mice increased after PAD4-siRNA treatment, but the difference was statistically significant only in the PAD4-siRNA2 group compared with the model group and the negative control group (P < 0.05); (4) The proportion of Tfh/Tfr in the spleen cells of the model group was increased, compared with the blank group, the difference was statistically significant (P < 0.05); the ratio of Tfh/Tfr in the three groups after PAD4-siRNA treatment all decreased, the difference was statistically significant (P < 0.05); (5) Compared with the blank group, the alveolar wall of the lung tissue of the model group was thickened, the inflammatory cell infiltration was increased, and the lung tissue destruction and inflammatory infiltration of the CIA mice were decreased after PAD4-siRNA treatment. The degree of reduction was reduced. CONCLUSION: Gene silencing of PAD4 can reduce the proportion of Tfh cells, increase the proportion of Tfr cells, reverse the proportion of Tfh/Tfr, and reduce the degree of interstitial lesions and inflammatory infiltration of lung tissue.

"Pauci-Hemosiderotic" Fibrolipomatous Tumor: A Mimicker of Various Lipomatous Lesions.

Hemosiderotic fibrolipomatous tumor is a rare soft tissue tumor that preferentially affects the dorsum of foot, shows recurrent t(1;10) translocation targeting TGFBR3 and OGA (MGEA5) genes, and has a high recurrence potential. Hemosiderin deposits, mature adipocytes, and interspersed spindle cells are the 3 cardinal morphologic features of this tumor. We describe a "pauci-hemosiderotic" example involving the left wrist of a 45-year-old female, posing a diagnostic pitfall. The tumor comprised mature adipose tissue traversed by variably thick fibrous septa containing short fascicles of spindle cells. Prominent small- to medium-sized blood vessels were present, often with perivascular fibrosis or aggregates of foamy histiocytes, sometimes associated with red cell extravasation. Hemosiderin was not conspicuous, but fine deposits could be found focally on careful search and with the aid of Perls stain. The diagnosis was further confirmed by diffuse expression of CD34 and presence of OGA translocation by fluorescence in situ hybridization. Pathologists should be aware that hemosiderin deposition can be scanty and focal in hemosiderotic fibrolipomatous, but the rich vasculature with a "damaged" appearance is a useful diagnostic clue.

Transplantation of adipose tissue-derived stem cells ameliorates autoimmune pathogenesis in MRL/lpr mice : Modulation of the balance between Th17 and Treg.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells characterized by immunomodulatory properties and are therefore considered a promising tool for the treatment of autoimmune diseases. In this study, we aimed to investigate whether transplantation of adipose tissue-derived stem cells (ADSCs) affects the autoimmune pathogenesis in MRL/lpr mice. METHODS: Fifteen 12-week-old MRL/lpr mice were randomly divided into three groups: ADSC, cyclophosphamide (CTX), and control groups, with five mice in each group. ADSC and control groups were injected with 1â€¯× 106 ADSCs or PBS, respectively, via the tail vein, once a week for 8 weeks. The CTX group was injected with CTX at a dose of 15 mg/kg body weight, once a week for 2 weeks, and this was repeated after 2 weeks rest. Proteinuria, anti-double-stranded DNA (anti-dsDNA) antibody, and serum creatinine levels were then measured. The populations of Th17 and Treg cells in the spleen were detected by flow cytometry. All statistical analyses were performed using least square difference. RESULTS: Eight weeks after treatment, the 24 h proteinuria, anti-dsDNA antibody levels, and serum creatinine were decreased significantly with transplantation of mouse ADSCs. ADSCs markedly reduced the number of TH17 cells, increased Treg cells, and improved renal pathology. CONCLUSION: Our results indicate that transplantation of ADSCs could significantly inhibit autoimmune progression in MRL/lpr mice and the efficacy of ADSCs was comparable to that of CTX.

[Adipose tissue derived stem cells'treatment effects in MRL/lpr mice and its effects on the imbalance of Th17/Treg cells].

OBJECTIVE: Preliminary study on therapeutic effects of adipose tissue derived stem cells (ADSCs) on MRL/lpr mice and the effect on imbalance of Th17/Treg. METHODS: Fifteen 12-week-old MRL/lpr mice were randomly divided into 3 groups by using random number table, including ADSCs group, control group and cyclophosphamide (CTX) group, with 5 in each group. ADSCs group and control group were injected with 1×106 ADSCs or phosphate buffered solution (PBS) via tail vein respectively, once a week, a total of eight times. CTX group was injected CTX at a dose of 15 mg/kg body weight, once a week for 2 weeks, and then repeated after 2 weeks'rest, a total of four times. The 24-hour proteinuria was measured before and after treatment. All the mice were sacrificed after treatment for 8 weeks. Th17 cells and Treg cells in splenic were examined by flow cytometry. RESULTS: (1)The 24-hour proteinuria in the three groups had no significant difference before treatment (P>0.05). After therapy for 4 weeks, the 24-hour proteinuria in the ADSCs and CTX groups was much lower than those in control group, and the difference was significant [(5.02±1.61) g/L vs. (7.10±1.63) g/L, (4.90±0.71) g/L vs. (7.10±1.63) g/L, P<0.05], and the longer the duration of treatment (8 weeks), the more obvious effect [(2.24±0.73) g/L vs. (10.36±1.64) g/L, (3.80±1.45) g/L vs. (10.36±1.64) g/L, P<0.01]. There was no significant difference in 24-hour proteinuria between ADSCs group and CTX group (P>0.05). (2) Percentage of Treg cells/CD4+T cells in the spleen lymphocytes: The percentages in ADSCs and CTX groups were higher than that in control group. The levels were 13.62%±1.87%, 14.14%±1.29%, 10.71%±1.23%, respectively, but there was no significant difference (P>0.05). (3) Percentage of Th17 cells/CD4+T cells in the spleen lymphocytes: The percentages in ADSCs and CTX groups were significantly lower than that in control group. The levels were 1.43%±0.20%, 1.63%±0.65%, 6.37%±1.64%, respectively, with statistical significance (P<0.01). CONCLUSION: Transplantation of ADSCs can reduce the 24-hour proteinuria in MRL/lpr mice. To prolong the time of treatment, the effect is more significant. Transplantation of ADSCs can up-regulate Treg cells and down-regulate Th17 cells. ADSCs have the ability to regulate the immune balance of Th17/Treg in MRL/lpr mice, suggesting that ADSCs play the role of anti-inflammatory and immune regulation by regulating the Treg and Th17 cells.

[Clinical application of intraoperative CT in management of maxillofacial fracture].

Nine patients with maxillofacial fracture that received intraoperative CT examination in Lanzhou General Hospital of Lanzhou Military Command from January 2017 to March 2017 were retrospectively studied. The procedure of intraoperative CT was introduced. The value of this technique was preliminarily discussed in order to provide a new method for the accurate implementation of maxillofacial fracture surgery.

[Therapeutic effect of adipose tissue-derived stem cells on bleomycin-induced mice of scleroderma].

OBJECTIVE: To investigate the effects and mechanisms of adipose-derived stem cells (ADSCs) on bleomycin-induced mice of scleroderma. METHODS: In the study, 24 C57BL/6J female mice were randomly divided into control group, bleomycin(BLM)group, ADSCs (hypodermic injection) group and ADSCs (intravenous injection) group . BLM [2 mg/(kg×d)] was injected into the mice to establish the model of scleroderma. There were 6 mice in each group .The control group mice were injected with normal saline 2 mL/(kg×d) by subcutaneously. The rest of the three groups were injected with BLM. ADSCs groups were injected with ADSCs (2×105) subcutaneously and intravenously, respectively. T-helper 17 (Th17) and regulatory T cell (Treg cell) of spleen cells were detected by flow cytometry. The levels of cytokines in the lung tissue and in the serum were detected by real-time fluorescence quantification. Real-time polymerase chain reaction(PCR) and enzyme-linked immuno sorbent assay(ELISA). The pathology change of skin and lung tissue was observed by hematoxylin eosin (HE) staining. RESULTS: The proportion of Th17 and Treg increased in BLM group than in control group(15.30%±1.29% vs.4.32%±0.79%; 9.90%±1.95% vs.5.18%±1.35%, P<0.05), the expression of Th17 significantly decreased (5.02%±0.83%, 6.00%±0.82% vs.15.30%±1.29%, P<0.05) and the expression of Treg increased after the ADSCs therapy (14.32%±1.59%, 11.09%±4.31% vs. 9.90%±1.95%, P<0.05). The expression levels of IL-17,IL-6,tumor necrosis factor-α (TNF-α)mRNA in the lung tissue and IL-6 in the serum increased in BLM group than in control group [3.54±0.30, 10.65±0.66, 5.37±0.52 vs. 1.00±0.00; (21.2±1.74) ng/L vs. (16.87±1.09) ng/L, P<0.05]. The expression of these cytokines significant decreased after the ADSCs therapy [1.63±0.45,1.50±0.29 vs.3.54±0.30; 3.11±0.85, 2.98±0.76 vs.10.65±0.66;1.45±0.47, 1.59±0.41 vs. 5.37±0.52; (17.87±1.45) ng/L, (17.61±1.16) ng/L vs. (21.2±1.74) ng/L, P<0.05]. But there was no obvious difference between ADSCs (hypodermic injection) group and ADSCs (intravenous injection) group(P>0.05). The expression of TGF-ß in the serum increased in BLM group than in control group[(33.95±2.49) ng/L vs. (28.8± 2.29) ng/L, P<0.05], however, the expression of TGF-ß mRNA had no significant differences than that of control group (1.17±0.11 vs.1.00±0.00, P>0.05). The expression of TGF-ß mRNA and protein had no significant differences than that of BLM group [1.25±0.11,1.26±0.12 vs.1.17±0.11; (31.84±2.04) ng/L, (31.25±2.36) ng/L vs. (33.95±2.49) ng/L, P>0.05]. HE staining showed that the inflammation of lung tissue was relieved and the dermal thickness and collagen deposition were decreased after the ADSCs therapy. CONCLUSION: ADSCs could effectively alleviate inflammation of the lungs and fibrosis of skin; the effects of anti-inflammatory and anti-fibrosis were associated with immune regulating function.

[Effect of type 1 sphingosine-1-phosphate receptor siRNA on human salivary gland cells].

OBJECTIVE: To construct sphingosine 1-phosphate receptor-1 (S1P1)-small interfering RNA (siRNA) lentiviral vectors and infect human salivary gland cells (HSG), and to investigate its possible therapy on Sjogren's syndrome. METHODS: HSG cells were divided into blank group, empty vector group, scramble-siRNA group and S1P1-siRNA group. The lentiviral vectors expressing siRNA against S1P1 and the pLL3.7 were respectively transfected into 293T cells with pMD2.G, pMDL g/p RRE, pRSV-REV to produce virus, and then infect HSG cells. The efficiency was observed by flow cytometry after the transfection for 48 h. The expression levels of S1P1 mRNA of HSG were detected by real-time RT-PCR and the expression of S1P1 protein was detected by immunohistochemistry method. The expression levels of interferon-γ (IFN-γ) and interleukin (IL)-17 in the supernatant of the cells were detected by ELISA method. RESULTS: (1) The scramble-siRNA, S1P1-siRNA lentiviral vector was successfully constructed, and the lentivirus titer was about 3.5×108 TU/mL. (2) The level of S1P1 mRNA was lower in S1P1-siRNA group than those in the blank group, empty vector group, and scramble-siRNA group 48 h after infection, there were significant differences between them (P<0.05). (3) The expression of S1P1 protein was lower in S1P1-siRNA group than those in blank group, empty vector group, and scramble-siRNA group 48 h after transfection, there were significant differences between them (P<0.05). (4) The levels of IL-17 were lower in S1P1-siRNA group than those in blank group, empty vector group, and scramble-siRNA group 48 h after transfection, there were significant differences between them (P<0.05). (5) The levels of IFN-γ in S1P1-siRNA group were lower than those in blank group, empty vector group, and scramble-siRNA group 48 h after transfection, there were significant differences between them (P<0.05). CONCLUSION: The lentiviral vector targeting S1P1 was successfully constructed. S1P1 siRNA could suppress the levels of S1P1 mRNA and protein, and decrease the expression of IL-17 and IFN-γ. S1P1 siRNA could infect HSG cells stably and inhibit the expression of S1P1 gene specifically and efficiently, and reduce the levels of inflammatory cytokines.

[Effect of CD40 siRNA on inflammatory response of MRL/Lpr mice].

OBJECTIVE: To observe the effect of CD40 siRNA on expression of IFN-γ, IL-17, IL-4 and anti-dsDNA antibody of systemic lupus erythematosus (SLE) animal model MRL/Lpr mice and to discuss its therapy on MRL/Lpr mice. METHODS: In the study, 16 female MRL/Lpr mice were randomly divided into control group (n=4), empty vector group (n=4), CD40-siRNA1 group (n=4) and CD40-siRNA2 group (n=4). The vectors expressing siRNA against CD40 were injected by tail veil into MRL/Lpr mice, while MRL/Lpr mice in control group and empty vector group were injected with the same dose of PBS and pGFP-V-RS vector respectively. The injection was given six times and every one day. The mice were sacrificed 14 d after injection, and the spleen tissue was weighed. The pGFP-V-RS was labeled by green fluorescent protein (GFP) and the tissue sections were observed whether siRNA expressed in the spleen. The expression levels of IFN-γ, IL-17, IL-4 and anti-dsDNA antibody in the sera were detected by ELISA method on the 1st day before the first time and the 2nd, 5th, 8th, 11th, and 14th days after last injection, and the expression levels of CD40 mRNA in spleen tissue of MRL/Lpr mice were detected by RT-PCR and the expression levels of CD40 protein in spleen tissue of MRL/Lpr mice were detected by immunohistochemistry method. RESULTS: The expression vector of CD40-siRNA could express in the spleen of MRL/Lpr. The spleens in CD40-siRNA1 group [(78.85±5.61) mg] and CD40-siRNA2 group [(80.25±4.07) mg] were lower than those in control [(141.88±7.81) mg] and empty vector group [(153.10±7.60) mg]. The levels of IL-17, IFN-γ and anti-dsDNA antibody were lower and the levels of IL-4 was higher in CD40-siRNA1 group and CD40-siRNA2 group on the 2nd, 5th and 8th days after last injection than on the 1st day before the first time (P<0.05). The levels of IFN-γ in CD40-siRNA1 group were (118.74±10.32) ng/L, (115.24±8.26) ng/L and (113.71±5.02) ng/L in turn, the levels of IFN-γ in CD40-siRNA2 group were (117.83±6.83) ng/L, (114.07±0.97) ng/L and (112.67±9.66) ng/L in turn. The levels of IL-17 in CD40-siRNA1 group were (7.05±0.41) ng/L, (6.34±0.76) ng/L and (5.83±0.43) ng/L in turn, the levels of IL-17 in CD40-siRNA2 group were (7.07±0.22) ng/L, (6.35±0.49) ng/L and (6.12±0.80) ng/L in turn. The levels of anti-dsDNA antibody in CD40-siRNA1 group were (7.51±0.29) ng/L, (6.74±0.45) ng/L and (6.32±0.39) ng/L in turn, the levels of anti-dsDNA antibody in CD40-siRNA2 group were (8.19±0.38) ng/L, (7.14±0.50) ng/L and (6.48±0.29) ng/L in turn. The levels of IL-4 in CD40-siRNA1 group were (26.51±1.81)ng/L (27.80±1.72) ng/L and (28.08±2.21) ng/L in turn, the level of IL-4 in CD40-siRNA2 group were (26.28±2.03) ng/L, (28.15±2.95) ng/L and (28.37±1.71) ng/L in turn. The expression levels of IL-17 and IFN-γ antibody increased gradually and the levels of IL-4 decreased gradually in CD40-siRNA1 group and CD40-siRNA2 group on the 11th and 14th days after last injection, then reached to the levels of control group and empty vector group (P>0.05). Though the levels of anti-dsDNA antibody in CD40-siRNA1 group and CD40-siRNA2 group on the 11th day was higher than on the 8th day, there was more significance than those in control group and empty vector group (P<0.05). There was no significance between the 4 groups on the 14th day. The levels of CD40 mRNA and protein were lower in CD40-siRNA1 group and CD40-siRNA2 group than in control group and empty vector group on the 14th day after last injection (P<0.05). CONCLUSION: CD40-siRNA can reduce the concentration of IL-17, IFN-γ and of anti-dsDNA antibody in serum, and at the same time, it can elevate the concentration of IL-4 and suppress CD40 mRNA and protein of spleen in MRL/Lpr. Meanwhile after suppressing CD40 mRNA and protein, it can reduce inflammatory response of the mice and the disease activity of MRL/Lpr, suggesting that CD40-siRNA has therapy effect on SLE.

Synonymous single nucleotide polymorphisms in the MC4R gene that are significantly associated with milk production traits in water buffaloes.

Melanocortin-4 receptor (MC4R) is associated with feed intake, growth, fatness, and carcass composition in many domestic animals. The aim of this study was to evaluate the association of single nucleotide polymorphisms (SNPs) in MC4R with milk production traits in water buffalo. Eight SNPs were identified by direct polymerase chain reaction sequencing of samples from 18 buffaloes. SNPs were then genotyped using the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) method in 332 buffaloes. Two of eight SNPs were not in Hardy-Weinberg equilibrium. Based on the SNP data, seven haplotypes were constructed. Three SNPs H1 (AGT), H2 (GAT), and H3 (GAC) accounted for 93.0% of the total individuals. Statistical analysis indicated that the SNP g.1104C>T was significantly associated with milk yield, protein, and fat percentage (P < 0.05). In conclusion, these results provide evidence that polymorphisms in the buffalo MC4R gene are associated with milk production traits and might be potential biomarkers for water buffalo breeding.

Associations between polymorphisms of the gene and milk production traits in water buffaloes.

Signal transducer and activator of transcription 1 () is an important regulator of mammary gland differentiation and cell survival that has been regarded as a candidate gene affecting milk production traits in mammals. Therefore, this study was conducted to evaluate significant associations between SNP of the gene and milk production traits in buffaloes. Here, 18 SNP were identified in the buffalo gene, including 15 intronic mutations and 3 exon mutations. All the identified SNP were then genotyped using matrix-assisted laser desorption/ionization time of flight mass spectrometry methods from 192 buffaloes. All the SNP were in Hardy-Weinberg equilibrium, and 2 haplotype blocks were successfully constructed based on these SNP data, which formed 5 and 3 major haplotypes in the population (>5%), respectively. The results of association analysis showed that only SNP13 located in exon 10 was significantly associated with the milk production traits in the population ( < 0.05). Single nucleotide polymorphism 2, SNP5, SNP8, and SNP9 were associated with protein percentage, and SNP4 and SNP10 were associated with 305-d milk yield ( < 0.05). Our results provide evidence that polymorphisms of the buffalo gene are associated with milk production traits and can be used as a candidate gene for marker-assisted selection in buffalo breeding.

Tanshinone IIA inhibits human prostate cancer cells growth by induction of endoplasmic reticulum stress in vitro and in vivo.

BACKGROUND: Tanshinone IIA (Tan-IIA) is one of the major lipophilic components isolated from the root of Salviae Miltiorrhizae Radix. We explored the mechanisms of cell death induced by Tan-IIA treatment in prostate cancer cells in vitro and in vivo. METHODS: Cells were treated with Tan-IIA and growth inhibition was assessed. Cell cycle profiles after Tan-IIA treatment were determined by flow cytometry. Expression levels of cell cycle regulatory proteins and apoptosis-related proteins were determined after Tan-IIA treatment. Expression levels of endoplasmic reticulum (ER) stress-regulated genes were determined to investigate their role in Tan-IIA-induced cell death. GADD153 expression was knocked down by small interfering RNA (siRNA) transfection. Rate of cell death and proliferation was obtained by 3-(4,5-dimethyl thizol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Antitumor activity of Tan-IIA was performed in LNCaP xenograft model. RESULTS: Our results showed that Tan-IIA caused prostate cancer cell death in a dose-dependent manner, and cell cycle arrest at G0/G1 phase was noted, in LNCaP cells. The G0/G1 phase arrest correlated with increase levels of CDK inhibitors (p16, p21 and p27) and decrease of the checkpoint proteins. Tan-IIA also induced ER stress in prostate cancer cells: activation and nuclear translocation of GADD153/CCAAT/enhancer-binding protein-homologous protein (CHOP) were identified, and increased expression of the downstream molecules GRP78/BiP, inositol-requiring protein-1α and GADD153/CHOP were evidenced. Blockage of GADD153/CHOP expression by siRNA reduced Tan-IIA-induced cell death in LNCaP cells. Tan-IIA also suppressed LNCaP xenograft tumor growth, causing 86.4% reduction in tumor volume after 13 days of treatment. CONCLUSIONS: Our findings suggest that Tan-IIA causes G0/G1 cell cycle arrest in LNCaP cells and its cytotoxicity is mediated at least partly by ER stress induction. These data provide evidence supporting Tan-IIA as a potential anticancer agent by inducing ER stress in prostate cancer.

Electrophysiological correlates of looking at paintings and its association with art expertise.

This study investigated the electrocortical correlates of art expertise, as defined by a newly developed, content-valid and internally consistent 23-item art expertise questionnaire in N=27 participants that varied in their degree of art expertise. Participants viewed each 50 paintings, filtering-distorted versions of these paintings and plain colour stimuli under free-viewing conditions whilst the EEG was recorded from 64 channels. Results revealed P3b-/LPC-like bilateral posterior event-related potentials (ERP) that were larger over the right hemisphere than over the left hemisphere. Art expertise correlated negatively with the amplitude of the ERP responses to paintings and control stimuli. We conclude that art expertise is associated with reduced ERP responses to visual stimuli in general that can be considered to reflect increased neural efficiency due to extensive practice in the contemplation of visual art.

Cloning and sequencing of the rDNA gene family of the water buffalo (Bubalus bubalis).

The rDNA genes coding for ribosomal RNA in animals are complicated repeat sequences with high GC content. We amplified water buffalo rDNA gene sequences with the long and accurate (LA) PCR method, using LA Taq DNA polymerase and GC buffer, based on bioinformatic analysis of related organisms. The rDNA genes were found to consist of 9016 nucleotides, including three rRNA genes and two internal transcribed spacers (ITS), which we named 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA. We tested and optimized conditions for cloning these complicated rDNA sequences, including specific rules of primer design, improvements in the reaction system, and selection of the DNA polymerase.

Optimization of cryopreservation of buffalo (Bubalus bubalis) blastocysts produced by in vitro fertilization and somatic cell nuclear transfer.

The objective of this study was to optimize cryopreservation conditions for buffalo in vitro produced (IVP) embryos. The in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) blastocysts were vitrified with either 40% ethylene glycol (EG), 25% EG + 25% dimethylsulfoxide (DMSO), or 20% EG + 20% DMSO + 0.5 m sucrose, and the IVF blastocysts produced from abattoir-derived ovaries were also slow-frozen with either 10% EG or 0.05 m trehalose dehydrate + 1.8% EG + 0.4% BSA. Cryosurvival rates of blastocysts harvested on various days or at various developmental stages were also examined. In this study: (1) vitrification with 20% EG + 20% DMSO + 0.5 m sucrose had the best cryopreservation efficiency; (2) IVF and SCNT blastocysts had similar cryotolerance (P > 0.05); (3) after thawing, slow-frozen blastocysts reexpanded earlier than the vitrified blastocysts (P < 0.01); (4) cryosurvival rate of expanded blastocysts was higher than that of early blastocysts (P < 0.05); (5) cryosurvival rates of Days 5 to 7 blastocysts (Day 0 = day of IVF or SCNT) were higher than those of Days 8 to 9 blastocysts (P < 0.01); and (6) after embryo transfer, pregnancy rates for fresh and cryopreserved blastocysts were not different (P > 0.05). In conclusion, vitrification of Days 6 to 7 expanded blastocysts with 20% EG + 20% DMSO + 0.5 m sucrose was optimal for cryopreservation of buffalo IVP embryos.

Study on the inter-subspecies nuclear transfer of river buffalo somatic cell nuclei into swamp buffalo oocyte cytoplasm.

The objective of this study was to explore the feasibility of inter-subspecies somatic cell nuclear transfer (SCNT) of river buffalo (50 chromosomes) somatic cell nuclei into swamp buffalo (48 chromosomes) oocyte cytoplasm. The enucleated swamp buffalo oocytes were fused with four different types of river buffalo cells: freshly thawed ear fibroblasts, serum-starved ear fibroblasts, cumulus cells and ear fibroblasts from a cloned buffalo calf. As a result, the developmental competence of embryos reconstructed with freshly thawed ear fibroblasts was the poorest (P<0.01), while those of the other three types were not different from each other. Furthermore, the efficiency of swamp-swamp buffalo, swamp-river buffalo and bovine-buffalo SCNT were also compared. The results showed that the blastocyst rate of swamp-river reconstructed embryos was not different from swamp-swamp embryos, while significantly higher than that of bovine-buffalo embryos (P<0.01). A total of thirty cloned blastocysts derived from freshly thawed ear fibroblasts were transferred into thirteen recipient buffaloes, four recipients established pregnancy, while three of them aborted on Days 65, 75 and 90 of gestation, respectively. One cross-bred buffalo (Murrah x swamp, 49 chromosomes) receiving three embryos delivered a 39 kg female calf on Day 335 of gestation. These results indicate that the inter-subspecies SCNT is feasible to produce swamp-river buffalo embryos, and these can develop to full term and result in live buffalo calves.

An inter-subspecies cloned buffalo (Bubalus bubalis) obtained by transferring of cryopreserved embryos via somatic cell nuclear transfer.

The aim of this study was to explore the feasibility of cryopreservation of inter-subspecies cloned embryos in buffalo. In our experiment, river buffalo ear fibroblast nucleus was fused into swamp buffalo oocyte cytoplasm. The blastocyst formation rate for nuclear transfer of freshly thawed cells was not different from those of growing cells, confluent or serum-starved cells. A total of 122 cloned blastocysts derived from cryopreserved fibroblasts were cryopreserved and thawed, 37 were survived, the cryosurvival rate was 30.3%. The survived blastocysts were transferred into 15 recipient buffalos. Five of the recipients established pregnancy, but four of them aborted on day 53, 59, 145 and 179 of gestation respectively. One cross-bred buffalo (Murrah × Swamp buffalo (2n = 49) received three embryos delivered a 40.5 kg female calf by natural delivery on day 320 of gestation. Up to now (13-month old), the cloned calf has been growing well with no abnormity observed. These results demonstrated that cryopreservation of inter-subspecies cloned embryos is feasible to produce buffalo offspring.

In vitro embryo production in buffalo (Bubalus bubalis) using sexed sperm and oocytes from ovum pick up.

The objective was to explore the use of sexed sperm and OPU-derived oocytes in an IVP system to produce sex-preselected bubaline embryos. Oocytes were recovered from 20 fertile Murrah and Nili-Ravi buffalo cows by repeated (twice weekly) ultrasound-guided transvaginal ovum pick up (OPU), or by aspiration of abbatoir-derived bubaline ovaries, and subjected to IVF, using frozen-thawed sexed or unsexed bubaline semen. On average, 4.6 oocytes were retrieved per buffalo per session (70.9% were Grades A or B). Following IVF with sexed sperm, oocytes derived from OPU had similar developmental competence as those from abattoir-derived ovaries, in terms of cleavage rate (57.6 vs. 50.4%, P=0.357) and blastocyst development rate (16.0 vs. 23.9%, P=0.237). Furthermore, using frozen-thawed sexed versus unsexed semen did not affect rates of cleavage (50.5 vs. 50.9%, P=0.978) or blastocyst development (15.3 vs. 19.1%, P=0.291) after IVF using OPU-derived oocytes. Of the embryos produced in an OPU-IVP system, 9 of 34 sexed fresh embryos (26.5%) and 5 of 43 sexed frozen embryos (11.6%) transferred to recipients established pregnancies, whereas 7 of 26 unsexed fresh embryos (26.9%) and 6 out of 39 unsexed frozen embryos (15.4%) transferred to recipients established pregnancies. Eleven sex-preselected buffalo calves (10 females and one male) and 10 sexed buffalo calves (six females and four males) were born following embryo transfer. In the present study, OPU, sperm sexing technology, IVP, and embryo transfer, were used to produce sex-preselected buffalo calves. This study provided proof of concept for further research and wider field application of these technologies in buffalo.

Gene delivery to human sweat glands: a model for cystic fibrosis gene therapy.

Gene therapy vectors are mostly studied in cultured cells, rodents, and sometimes in non-human primates, but it is useful to test them in human tissue prior to clinical trials. In this study, we investigated the possibility of using human sweat glands as a model for testing cystic fibrosis (CF) gene therapy vectors. Human sweat glands are relatively easy to obtain from skin biopsy, and can be tested for CFTR function. Using patients' sweat glands could provide a safe model to study the efficacy of CF gene therapy. As the first step to explore using sweat glands as a model for CF gene therapy, we examined various ex vivo gene delivery methods for a helper-dependent adenovirus (HD-Ad) vector. Gene delivery to sweat glands in skin organ culture was studied by topical application, intradermal injection or submerged culture. We found that transduction efficiency can be enhanced by pretreating isolated sweat glands with dispase, which suggests that the basement membrane is a critical barrier to gene delivery by adenoviral vectors. Using this approach, we showed that Cftr could be efficiently delivered to and expressed by the epithelial cells of sweat glands with our helper-dependent adenoviral vector containing cytokeratin 18 regulatory elements. Based on this study we propose that sweat glands might be used as an alternative model to study CF gene therapy in humans.

Enhanced oxidative damage in human cells harboring A3243G mutation of mitochondrial DNA: implication of oxidative stress in the pathogenesis of mitochondrial diabetes.

Mitochondrial oxidative phosphorylation and the ATP production in pancreatic beta cells play significant roles in insulin secretion in response to glucose and other nutrients. An A to G mutation in the tRNA(Leu(UUR)) gene at nucleotide position (np) 3243 of mitochondrial DNA (mtDNA) has been observed in patients with MELAS syndrome and mitochondrial diabetes. Recently, some patients with mitochondrial diabetes associated with the A3243G mtDNA mutation were found to respond to coenzyme Q10 therapy. Thus, we investigated oxidative stress and peroxidative damage in a series of cybrids carrying either the wild-type adenine or the mutant-type guanine at np 3243 but having otherwise identical mtDNA sequence. The cybrids harboring >90% of the A3243G mutant mtDNA were found to have significantly lower oxygen consumption rate and electron transfer activities, and thereby had lower ATP/ADP ratios and declined energy charge. Importantly, the defective respiratory function elicited by the A3243G mtDNA mutation caused an increased oxidative stress as indicated by the decreased GSH/GSSG ratio and enhanced oxidative damage to lipids. Moreover, the cybrids harboring high proportions of the A3243G mtDNA mutation were found to be much more vulnerable to an exogenous oxidant, tert-butylhydroperoxide. We thus suggest that enhanced oxidative damage and elevated oxidative stress contribute to the decline of mitochondrial function and may be involved in the initiation and progression of the MELAS syndrome and mitochondrial diabetes.