Objective: An imbalance in CD4+CD25+ regulatory T (Treg) cells and Th17 cells has been found to correlate to occurrence of acute coronary syndrome [ACS, including unstable angina (UA) and acute myocardial infarction (AMI)]. However, the mechanisms of Th17/Treg imbalance in ACS patients are still unclear. The purpose of this study is to investigate the possibility of differences in sensitivity of Th17 and Tregs to Fas-mediated apoptosis which could lead to Th17/Treg imbalance in ACS patients. Methods: We examined the apoptosis of Th17 and Treg cells, apoptosis-related Fas/Fas ligand(FasL) pathway, and inflammatory markers in patients with AMI, UA, stable angina (SA) and controls by Flow cytometry and ELISA. Then we analysed the correlation of inflammatory markers and sFasL to Treg apoptosis, and the effect of anti-FasL antibody on Treg apoptois in vitro. Results: Our study demonstrated that apoptotic Tregs, Fas and FasL expression, Caspase-3 activity of Tregs were significantly higher in ACS patients than those in NCA and SA patients (all P < 0.05). The percentage of apoptotic Tregs is positively correlated with the levels of inflammatory markers and sFasL. In vitro incubation of peripheral blood mononuclear cells from ACS patients with anti-FasL antibody resulted in a markedly reduction of apoptotic Treg cells. However, there were no significant differences in apoptotic Th17 cells and in Fas and FasL expression for Th17 cells between the four groups (all P >0.05). Conclusions: Tregs, but not Th17 cells, become apoptotic through Fas/FasL pathway, which contributed to reduction of Tregs leading to an imbalance between Th17 and Treg cells. This could be the mechanism underlying Th17/Treg imbalance and occurrence of ACS.
Apoptosis; Fas; Fas ligand; T helper 17; regulatory T cells; acute coronary syndrome
The design, synthesis, and biological evaluation of novel C3-substituted cyclopentyltetrahydrofuranyl (Cp-THF)-derived HIV-1 protease inhibitors are described. Various C3-functional groups on the Cp-THF ligand were investigated in order to maximize the ligand-binding site interactions in the flap region of the protease. Inhibitors 3c and 3d have displayed the most potent enzyme inhibitory and antiviral activity. Both inhibitors have maintained impressive activity against a panel of multidrug resistant HIV-1 variants. A high-resolution X-ray crystal structure of 3c-bound HIV-1 protease revealed a number of important molecular insights into the ligand-binding site interactions.
HIV-1 protease inhibitors; P2 ligand; Drug resistance; Design and synthesis; X-ray crystal structure
We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34+ cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: β-globin promoter (βp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the β-globin locus control region (HS2-βbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-βp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34+ cells in vitro. Transgene expression from the B19p6 or the HS2-βp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-βp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-βp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as β-thalassemia and sickle cell disease.
Afferent input regulates neuronal dendritic patterning locally and globally through distinct mechanisms. To begin to understand these mechanisms, we differentially manipulate afferent input in vivo and assess effects on dendritic patterning of individual neurons in chicken nucleus laminaris (NL). Dendrites of NL neurons segregate into dorsal and ventral domains, receiving excitatory input from the ipsilateral and contralateral ears, respectively, via nucleus magnocellularis (NM). Blocking action potentials from one ear, by either cochlea removal or temporary treatment with tetrodotoxin (TTX), leads to rapid and significant retraction of affected NL dendrites (dorsal ipsilaterally and ventral contralaterally) within 8h as compared to the other dendrites of the same neurons. The degree of retraction is comparable to that induced by direct deafferentation resulting from transection of NM axons. Importantly, when inner ear activity is allowed to recover from TTX treatments, retracted NL dendrites regrow to their normal length within 48h. The retraction and growth involve elimination of terminal branches and addition of new branches. Examination of changes in NL dendrites at 96h following unilateral cochlear removal, a manipulation that induces cell loss in NM and persistent blockage of afferent excitatory action potentials, reveals a significant correlation between cell death in the ipsilateral NM and the degree of dendritic retraction in NL. These results demonstrate that presynaptic action potentials rapidly and reversibly regulate dendritic patterning of postsynaptic neurons in a compartment specific manner, while long-term dendritic maintenance may be regulated in a way that is correlated with the presence of silent presynaptic appositions.
Chronic kidney disease (CKD) is a major cause of death and morbidity in Australia and worldwide. DNA vaccination has been used for targeting foreign antigens to induce immune responses and prevent autoimmune disease, viral infection and cancer. However, the use of DNA vaccination has been restricted by a limited ability to induce strong immune responses, especially against self-antigens which are limited by mechanisms of self-tolerance. Furthermore, there have been few studies on the potential of DNA vaccination in chronic inflammatory diseases, including CKD. We have established strategies of DNA vaccination targeting specific self-antigens in the immune system including co-stimulatory pathways, T cell receptors and chemokine molecules, which have been effective in protecting against the development of CKD in a variety of animal models. In particular, we find that the efficacy of DNA vaccination is improved by dendritic cell (DC) targeting and can protect against animal models of autoimmune nephritis mimicking human membranous nephropathy. In this review, we summarize several approaches that have been tested to improve the efficacy of DNA vaccination in CKD models, including enhanced DNA vaccine delivery methods, DNA vaccine modifications and new molecular targets for DNA vaccination. Finally, we discuss the specific application of DNA vaccination for preventing and treating CKD.
DNA vaccination; dendritic cell; DEC205; CD40; cytokine; costimulatory molecular; active Heymann nephritis (HN); adriamycin nephropathy (AN)
Hemoglobinopathies are the most common inherited diseases in southern China. However, there have been only a few epidemiological studies of hemoglobinopathies in Guangdong province.
Materials and Methods
Peripheral blood samples were collected from 15299 “healthy” unrelated subjects of dominantly ethnic Hakka in the Meizhou region, on which hemoglobin electrophoresis and routine blood tests were performed. Suspected cases with hemoglobin variants and hereditary persistence of fetal hemoglobin (HPFH) were further characterized by PCR, DNA sequencing, reverse dot blot (RDB) or multiplex ligation-dependent probe amplification (MLPA). In addition, 1743 samples were randomly selected from the 15299 subjects for thalassemia screening, and suspected thalassemia carriers were identified by PCR and RDB.
The gene frequency of hemoglobin variants was 0.477% (73/15299). The five main subgroups of the ten hemoglobin variants were Hb E, Hb G-Chinese, Hb Q-Tahiland, Hb New York and Hb J-Bangkok. 277 cases (15.89%, 277/1743) of suspected thalassemia carriers with microcytosis (MCV<82 fl) were found by thalassemia screening, and were tested by a RDB gene chip to reveal a total of 196 mutant chromosomes: including 124 α-thalassemia mutant chromosomes and 72 β-thalassemia mutant chromosomes. These results give a heterozygote frequency of 11.24% for common α and β thalassemia in the Hakka population in the Meizhou region. 3 cases of HPFH/δβ-thalassemia were found, including 2 cases of Vietnamese HPFH (FPFH-7) and a rare Belgian Gγ(Aγδβ)0–thalassemia identified in Chinese.
Our results provide a detailed prevalence and molecular characterization of hemoglobinopathies in Hakka people of the Meizhou region. The estimated numbers of pregnancies each year in the Meizhou region, in which the fetus would be at risk for β thalassemia major or intermedia, Bart’s hydrops fetalis, and Hb H disease, are 25 (95% CI, 15 to 38), 40 (95% CI, 26 to 57), and 15 (95% CI, 8 to 23), respectively.
Experimental data suggest a protective effect of vitamin D on breast cancer; however, epidemiologic results remain inclusive. With a Chinese population-based case-control study and meta-analysis of the observational studies, we here systematically evaluated the association of blood 25(OH)D level and breast cancer risk. With 593 breast cancer cases and 580 cancer-free controls from Shanghai, China, we found that 80% of the normal women had severe vitamin D deficiency (less than 20 ng/mL) and 15.2% had mild deficiency (20 to 30 ng/mL) and only 4.8% of women had sufficient vitamin D level (>30 ng/mL) while the proportion was 96.1%, 3.2% and 0.7% respectively for the breast cancer patients. Compared to those with the lowest quartile of plasma 25(OH)D level, women with highest quartile 25(OH)D level showed a significant decreased breast cancer risk (Q4 vs.Q1: OR = 0.10, 95% CI = 0.06–0.15) and every 1 ng/ml increment of plasma 25(OH)D level led to a 16% lower odds of breast cancer (OR = 0.84, 95% CI = 0.81–0.87; P<0.001). From the meta-analysis of the observational studies, we found that women with highest quantile of blood 25(OH)D level was associated with a significantly reduced breast cancer risk compared to those with lowest quantile of blood 25(OH)D level for the 11 nested case-control and retrospective studies (pooled OR = 0.86, 95% CI = 0.75–1.00) and 10 case-control studies (7 population based, OR = 0.35, 95% CI = 0.24–0.52; 3 hospital based, OR = 0.08, 95% CI = 0.02–0.33). These results suggest that vitamin D may have a chemo-preventive effect against breast cancer.
Commercial production of acarbose is exclusively via done microbial fermentation with strains from the genera of Actinoplanes. The addition of C7N-aminocyclitols for enhanced production of acarbose and concurrently reduced formation of impurity C by cultivation of A. utahensis ZJB-08196 in 500-mL shake flasks was investigated, and validamine was found to be the most effective strategy. Under the optimal conditions of validamine addition, acarbose titer was increased from 3560 ± 128 mg/L to 4950 ± 156 mg/L, and impurity C concentration was concurrently decreased from 289 ± 24 mg/L to 107 ± 29 mg/L in batch fermentation after 168 h of cultivation. A further fed-batch experiment coupled with the addition of validamine (20 mg/L) in the fermentation medium prior to inoculation was designed to enhance the production of acarbose. When twice feedings of a mixture of 6 g/L glucose, 14 g/L maltose, and 9 g/L soybean flour were performed at 72 h and 96 h, acarbose titer reached 6606 ± 103 mg/L and impurity C concentration was only 212 ± 12 mg/L at 168 h of cultivation. Acarbose titer and proportion of acarbose/impurity C increased by 85.6% and 152.9% when compared with control experiments. This work demonstrates for the first time that validamine addition is a simple and effective strategy for increasing acarbose production and reducing impurity C formation.
Angiotensin II (AngII) induces cardiac hypertrophy and increases the expression of TR3. To determine whether TR3 is involved in the regulation of the pathological cardiac hypertrophy induced by AngII, we established mouse and rat hypertrophy models using chronic AngII administration. Our results reveal that a deficiency of TR3 in mice or the knockdown of TR3 in the left ventricle of rats attenuated AngII-induced cardiac hypertrophy compared with the respective controls. A mechanistic analysis demonstrates that the TR3-mediated activation of mTORC1 is associated with AngII-induced cardiac hypertrophy. TR3 was shown to form a trimer with the TSC1/TSC2 complex that specifically promoted TSC2 degradation via a proteasome/ubiquitination pathway. As a result, mTORC1, but not mTORC2, was activated; this was accompanied by increased protein synthesis, enhanced production of reactive oxygen species and enlarged cell size, thereby resulting in cardiac hypertrophy. This study demonstrates that TR3 positively regulates cardiac hypertrophy by influencing the effect of AngII on the mTOR pathway. The elimination or reduction of TR3 may reduce cardiac hypertrophy; therefore, TR3 is a potential target for clinical therapy.
angiotensin II; cardiac hypertrophy; mammalian target of rapamycin; orphan receptor TR3; tuberous sclerosis complex
dimerization inhibition; HIV-1 protease inhibitor; multidrug-resistant HIV-1 strains; oxatricyclic ligands; X-ray structure
RNA binding motif 5 (RBM5) is a tumor suppressor gene that regulates cell proliferation, differentiation and apoptosis through pre-mRNA splicing of related genes. This study aimed to detect RBM5 and KRAS expression in pancreatic ductal adenocarcinoma and their association with clinicopathological features. Detection of RBM5 and KRAS expression by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting was performed at mRNA and protein levels, respectively, in pancreatic cancer and non-tumor tissues. In addition, the association of RBM5 and KRAS expression with clinicopathological parameters and tumor recurrence was analyzed. The expression of RBM5 was significantly downregulated in pancreatic cancer tissues compared to peritumoral tissues at the mRNA and protein levels. Contrastingly, KRAS was significantly overexpressed in pancreatic cancerous tissues compared to peritumoral tissues. Analysis revealed that RBM5 expression was negatively correlated with KRAS expression in pancreatic cancer. Furthermore, reduced RBM5 expression has a close association with lymph node metastasis, distant metastasis, Union for International Cancer Control (UICC) stage and nerve and venous invasion, while overexpression of KRAS proteins was significantly correlated with tumor size, lymph node metastasis, UICC stage and nerve and venous invasion of pancreatic cancer. Significant RBM5 underexpression and KRAS overexpression were observed in pancreatic cancer compared to non-tumor tissues. There is a close association of differential RBM5 and KRAS with poor clinicopathological features, suggesting their potential roles in the progression and metastasis of pancreatic cancer.
pancreatic cancer; RNA binding motif 5; KRAS; clinicopathological features
In the recent years, China's auto industry develops rapidly, thus bringing a series of burdens to society and environment. This paper uses Logistic model to simulate the future trend of China's vehicle population and finds that China's auto industry would come into high speed development time during 2020–2050. Moreover, this paper predicts vehicles' fuel consumption and exhaust emissions (CO, HC, NOx, and PM) and quantificationally evaluates related industry policies. It can be concluded that (1) by 2020, China should develop at least 47 million medium/heavy hybrid cars to prevent the growth of vehicle fuel consumption; (2) China should take the more stringent vehicle emission standard V over 2017–2021 to hold back the growth of exhaust emissions; (3) developing new energy vehicles is the most effective measure to ease the pressure brought by auto industry.
Neuropathic pain is detrimental to human health; however, its pathogenesis still remains largely unknown. Overexpression of pain-associated genes and increased nociceptive somato-sensitivity are well observed in neuropathic pain. The importance of epigenetic mechanisms in regulating the expression of pro- or anti-nociceptive genes has been revealed by studies recently, and we hypothesize that the transcriptional coactivator and the histone acetyltransferase E1A binding protein p300 (p300), as a part of the epigenetic mechanisms of gene regulation, may be involved in the pathogenesis of neuropathic pain induced by chronic constriction injury (CCI). To test this hypothesis, two different approaches were used in this study: (I) down-regulating p300 with specific small hairpin RNA (shRNA) and (II) chemical inhibition of p300 acetyltransferase activity by a small molecule inhibitor, C646.
Using the CCI rat model, we found that the p300 expression was increased in the lumbar spinal cord on day 14 after CCI. The treatment with intrathecal p300 shRNA reversed CCI-induced mechanical allodynia and thermal hyperalgesia, and suppressed the expression of cyclooxygenase-2 (COX-2), a neuropathic pain-associated factor. Furthermore, C646, an inhibitor of p300 acetyltransferase, also attenuated mechanical allodynia and thermal hyperalgesia, accompanied by a suppressed COX-2 expression, in the spinal cord.
The results suggest that, through its acetyltransferase activity in the spinal cord after CCI, p300 epigenetically plays an important role in neuropathic pain. Inhibiting p300, using interfering RNA or C646, may be a promising approach to the development of new neuropathic pain therapies.
Neuropathic pain; p300; COX-2; Acetyltransferase activity; CCI
CyberKnife (CK), hypofractionated stereotactic radiosurgery, is a preferred option for the treatment of advanced refractory lung cancer which is usually inoperable. Cytokine-induced killer (CIK) cell immunotherapy has a marked radiosensitization effect which aids the elimination of residual tumor cells in distant areas. The main purpose of the present study was to evaluate the clinical efficacy of CK alone and combined with CIK cell therapy for advanced refractory lung cancer. In one year, 22 patients with advanced lung cancer underwent CK therapy at a CyberKnife Center. Of these patients, 11 received CIK cell therapy before or after the CK therapy course. The median prescribed dose in the combined CK and CIK group was 35 Gy (mean, 33.8±5.0 Gy) with a median number of fractions of 5. The median dose for patients who underwent CK alone was 35 Gy (mean, 35.2±6.0 Gy). CIK cell therapy was administered according to the condition of each patient, generally 2 continuous therapeutic sessions in 2 months. The median follow-up period was 3 months. The preliminary curative efficiency rate was 81.82% for patients who underwent CK/CIK and 72.73% for those who received CK alone, according to radiographic re-examination (P>0.05). The median improvement in the Karnofsky scores of the CK/CIK group was 20 (18±10.51) compared with 10 (8.6±11.85) for those who underwent CK alone (P<0.05). The median expression of carcinoembryonic antigen (CEA) before and after treatment was 40.81 and 12.21 ng/ml, respectively, for the CK/CIK group compared with 39.04 and 26.36 ng/ml for CK alone. The median percentage of phenotype expression of the CIK cells (CD3+/CD8+ and CD3+/CD56+) in the patients who underwent CK/CIK was recorded as 64.35% (57.08±16.94%) and 15.27% (18.80±7.00%), respectively, prior to transfusion. The preliminary results of the present study suggest that CK combined with CIK cell immunotherapy improved the short-term outcomes of patients for curative efficacy, Karnofsky scores, tumor marker levels and immune status compared with alternative CK treatments, although further studies are required.
CyberKnife; stereotactic radiosurgery; cytokine-induced killer; immunotherapy; clinical efficacy
Erlin1 and erlin2 are highly homologous, ~ 40kDa, endoplasmic reticulum membrane proteins that assemble into a ring-shaped complex with a mass of ~2MDa. How this complex is formed is not understood, but appears to involve multiple interactions, including a coiled-coil region that mediates lower-order erlin assembly, and a short hydrophobic region, termed the “assembly domain”, that mediates higher-order assembly into ~2MDa complexes. Here we have used molecular modeling, mutagenesis and cross-linking to examine the role of the assembly domain in higher-order assembly. We find (i) that the assembly domains of erlin1 and erlin2 are amphipathic helices, (ii) that erlin1 alone and erlin2 alone can assemble into ~2MDa complexes, (iii) that higher-order assembly is strongly inhibited by point mutations to the assembly domain, (iv) that three interacting hydrophobic residues in the assembly domain and aromaticity are essential for higher-order assembly, and (iv) that while erlins1 and 2 are equally capable of forming lower-order homo- and hetero-oligomers, hetero-oligomers are the most prevalent form when erlin1 and erlin2 are co-expressed. Overall, we conclude that the ~2MDa erlin1/2 complex is composed of an assemblage of lower-order hetero-oligomers, probably heterotrimers, linked together by assembly domain hydrophobic residues.
erlin1; erlin2; amphipathic helix; assembly; crosslinking
Elevated blood glucose is generally regarded as one of the risk factors that lead to coronary heart disease in patients with type 2 diabetes. However, our studies show that after inducing short-term damage, high blood glucose subsequently provides paradoxical protection for vessel function of animals with high blood pressure. Vessels can adapt to sustained high blood glucose and produce different stress proteins to counteract, to some extent, the damage brought about by hypertension. The results help us understand part of the basis for vessel adaptation in diabetes. The implication for treatment of diabetes is that if the patients have long-standing diabetes and established cardiovascular disease, the target of blood glucose lowering should be less stringent and reached gradually to avoid abrupt cancellation of the pre-existing adaptations.
Although both diabetes and hypertension are risk factors for cardiovascular disease, the role of hyperglycaemia per se in endothelial dysfunction is controversial. This study was designed to examine whether hyperglycaemia, or streptozotocin-induced diabetes, could aggravate endothelial dysfunction in stroke-prone spontaneously hypertensive rats (SHRSP). Hyperglycaemia was induced by streptozotocin in 2-month-old SHRSP and age-matched normotensive Wistar–Kyoto (WKY) rats. The aorta was isolated 8 weeks after induction of hyperglycaemia to record its function and to examine its morphology with transmission electron microscopy. Endothelial/inducible nitric oxide synthase (eNOS/iNOS) and inducible/constitutive haem oxygenase (HO-1/HO-2) levels were determined with Western blotting. Aortic endothelial function and production of reactive oxygen species and nitric oxide were assayed after incubation in vitro in hyperglycaemic, hyperosmolar solution. Streptozotocin-induced diabetes of 8 weeks duration did not result in endothelial dysfunction in normotensive WKY rats. In contrast, hyperglycaemic WKY rats showed significantly enhanced endothelium-dependent vasodilatation, which was abrogated by simultaneous blocking of NOS and HO. The enhanced vasodilatation was associated with elevation of vascular eNOS and HO-1. Significant endothelial dysfunction and massive macrophage–monocyte infiltration were found in SHRSP aorta (the ratio of the number of macrophages to endothelial cells in the intima, expressed as a percentage, was 20.9 ± 2.8% in SHRSP versus 1.9 ± 0.5% in WKY rats, P < 0.01), which was attenuated significantly in hyperglycaemic SHRSP (11.3 ± 1.6%, P < 0.01 versus SHRSP). Acute hyperglycaemia (10 min) aggravated endothelial dysfunction in SHRSP, with a marked increase in intracellular reactive oxygen species and NO production. Sustained in vitro incubation in hyperglycaemic/hyperosmolar conditions (addition of an extra 50 mmol L−1 of glucose or mannitol to the usual buffer, to produce a final osmolarity of 350 mosmol L−1) for 5 h enhanced endothelium-dependent vasodilatation, with elevated vessel NO production and upregulation of eNOS/HO-1 proteins. Sustained hyperglycaemia does not aggravate endothelial dysfunction and macrophage infiltration in SHRSP. Hyperglycaemia/hyperosmolarity-induced upregulation of eNOS and HO-1 may play a role in this paradoxical adaptation of endothelial function.
Internal medicine includes several subspecialties. This study aimed to describe change trend of impact factors in different subspecialties of internal medicine during the past 12 years, as well as the developmental differences among each subspecialty, and the possible influencing factors behind these changes and differences.
Nine subspecialties of internal medicine were chosen for comparison. All data were collected from the Science Citation Index Expanded and Journal Citation Reports database.
(1) Journal numbers in nine subspecialties increased significantly from 1998 to 2010, with an average increment of 80.23%, in which cardiac and cardiovascular system diseases increased 131.2% rank the first; hematology increased 45% rank the least. (2) Impact Factor in subspecialties of infectious disease, cardiac and cardiovascular system diseases, gastroenterology and hepatology, hematology, endocrinology and metabolism increased significantly (p<0.05), in which gastroenterology and hepatology had the largest increase of 65.4%. (3) Journal impact factor of 0–2 had the largest proportion in all subspecialties. Among the journals with high impact factor (IF>6), hematology had the maximum proportion of 10%, nephrology and respiratory system disease had the minimum of 4%. Among the journal with low impact factor (IF<2), journal in nephrology and allergy had the most (60%), while endocrinology and metabolism had the least (40%). There were differences in median number of IF among the different subspecialties (p<0.05), in which endocrinology and metabolism had the highest, nephrology had the lowest. (4) The highest IF had a correlation with journal numbers and total paper numbers in each field.
The IF of internal medicine journals showed an increasingly positive trend, in which gastroenterology and hepatology increase the most. Hematology had more high IF journals. Endocrinology and metabolism had higher average IF. Nephrology remained the lowest position. Numbers of journals and total papers were associated with the highest IF.
The incidence of pancreatic carcinoma, a gastrointestinal malignancy, is on the increase and effective therapeutic strategies are therefore required. This study aimed to construct a recombinant plasmid pcDNA3.1(-) shCEACAM6-yCDglyTK from CEACAM6 targeting shRNA and the fusion suicide gene yCDglyTK for inhibition of SW1990 human pancreatic carcinoma cell growth and invasion. A plasmid containing hU6 promoter and CEACAM6 targeting short hairpin RNA (CEACAM6-shRNA) frame was constructed. It was subcloned to a CEA promoter-driven fusion suicide gene pcDNA3.1(-)yCDglyTK. The recombinant plasmid pcDNA3.1(-) shCEACAM6-yCDglyTK was identified by restriction endonuclease analysis and DNA sequencing. The recombinant plasmid was delivered into SW1990 human pancreatic carcinoma cells, the mRNA and protein expression of yCDglyTK and CEACAM6 was examined by RT-PCR, western blot analysis and immunofluorescence. SW1990 cells were treated with the prodrug 5-fluorocytosine (5-FC), and the cell viability was evaluated using the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The invasiveness and migration of SW1990 cells were evaluated by transwell migration assays. The restriction endonuclease analysis and DNA sequencing confirmed the construction of the recombinant plasmid pcDNA3.1(-) shCEACAM6-yCDglyTK. Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis outcomes showed that yCDglyTK was expressed in SW1990 cells and expression of CEACAM6 in SW1990 cells was significantly knocked down. MTT assay showed that the mean viability of SW1990 cells was significantly reduced after administration of the prodrug 5-FC in vitro. Transwell migration assays showed that invasion and migration action of SW1990 cells was significantly inhibited. In conclusion, recombinant plasmid pcDNA3.1(-) shCEACAM6-yCDglyTK was successfully constructed. The recombinant plasmid may therefore serve as a novel gene therapy approach for pancreatic carcinoma.
pancreatic carcinoma; RNA interference; suicide gene therapy; carcinoembryonic antigen-related cell adhesion molecule
We synthesized three 20mer caged circular antisense oligodeoxynucleotides (R20, R20B2 and R20B4) with a photocleavable linker and an amide bond linker between two 10mer oligodeoxynucleotides. With these caged circular antisense oligodeoxynucleotides, RNA-binding affinity and its digestion by ribonuclease H were readily photomodulated. RNA cleavage rates were upregulated ∼43-, 25- and 15-fold for R20, R20B2 and R20B4, respectively, upon light activation in vitro. R20B2 and R20B4 with 2- or 4-nt gaps in the target RNA lost their ability to bind the target RNA even though a small amount of RNA digestion was still observed. The loss of binding ability indicated promising gene photoregulation through a non-enzymatic strategy. To test this strategy, three caged circular antisense oligonucleotides (PS1, PS2 and PS3) with 2′-OMe RNA and phosphorothioate modifications were synthesized to target GFP expression. Upon light activation, photomodulation of target hybridization and GFP expression in cells was successfully achieved with PS1, PS2 and PS3. These caged circular antisense oligonucleotides show promising applications of photomodulating gene expression through both ribonuclease H and non-enzyme involved antisense strategies.
Morpholino oligomers (MOs) have been widely used to knock down specific genes in zebrafish, but their constitutive activities limit their experimental applications for studying a gene with multiple functions or within a gene network. We report herein a new design and synthesis of caged circular MOs (caged cMOs) with two ends linked by a photocleavable moiety. These caged cMOs were successfully used to photomodulate β-catenin-2 and no tail expression in zebrafish embryos.
The aim of this study was to investigate the correlation between breast cancer-specific gene 1 (BCSG1) and the effect of neoadjuvant chemotherapy (NAC) in patients with triple-negative breast cancer (TNBC). Real-time RT-PCR and immunohistochemistry were used to determine the expression of BCSG1 mRNA and protein levels of 32 TNBC patients before and after NAC. Tumor size was reduced significantly after NAC in all 32 TNBC patients. The expression of BCSG1 was also decreased after NAC at both mRNA and protein levels. There was a negative correlation between BCSG1 levels after NAC and the effect of NAC. BCSG1 may be a potential target for NAC in the treatment of TNBC.
SNCG protein; neoadjuvant therapy; adjuvant chemotherapy; breast neoplasms
We report the design, synthesis, biological evaluation, and the X-ray crystal structure of a novel inhibitor-bound HIV-1 protease. Various C3-functionalized cyclopentanyltetrahydrofurans (Cp-THF) were designed to interact with the flap Gly48 carbonyl or amide NH in the S2-subsite of the HIV-1 protease. We investigated the potential of those functionalized ligands in combination with hydroxyethyl sulfonamide isosteres. Inhibitor 26 containing a 3-(R)-hydroxyl group on the Cp-THF core, displayed the most potent enzyme inhibitory and antiviral activity. Our studies revealed a preference for the 3-(R)-configuration over the corresponding 3-(S)-derivative. Inhibitor 26 exhibited potent activity against a panel of multidrug-resistant HIV-1 variants. A high resolution X-ray structure of 26-bound HIV-1 protease revealed important molecular insight into the ligand-binding site interactions.
G1P rotaviruses are an important cause of diarrhea in humans in China. To date, there are no reports on the whole genomic analysis of the Chinese G1P rotaviruses. To determine the origin and overall genetic makeup of the recent Chinese G1P strains, the whole genomes of three strains, RVA/Human-wt/CHN/E1911/2009/G1P, RVA/Human-tc/CHN/R588/2005/G1P and RVA/Human-tc/CHN/Y128/2004/G1P, detected in an infant, a child and an adult, respectively, were analyzed. Strains E1911, R588 and Y128 exhibited a typical Wa-like genotype constellation. Except for the NSP3 gene of E1911, the whole genomes of strains E1911, R588 and Y128 were found to be more closely related to those of the recent Wa-like common human strains from different countries than those of the prototype G1P strain, or other old strains. On the other hand, the NSP3 gene of E1911 was genetically distinct from those of Y128, R588, or other Wa-like common human strains, and appeared to share a common origin with those of the porcine-like human G9 strains, providing evidence for intergenotype reassortment events. Comparisons of the amino acid residues defining the VP7 and VP4 antigenic domains revealed several mismatches between these Chinese G1P strains and the G1 and P strains contained in the currently licensed rotavirus vaccines RotarixTM and RotaTeqTM.
human G1P rotavirus; whole genomic analysis; China
Although mounting evidence indicates the involvement of galectin-3 in cancer progression and metastasis, the underlying molecular mechanisms remain largely unknown. In this study, we investigated the effect and possible mechanism of galectin-3 on the migration and invasion of B16F10, a metastatic melanoma cell line, in which galectin-3 and matrix metalloproteinase-1 (MMP-1) were both found to be highly expressed. Knockdown of galectin-3 with specific siRNA reduced migration and invasion, which was associated with reduced expression of MMP-1. To further investigate the underlying mechanism, we examined the effect of galectin-3 knockdown on the activity of AP-1, a transcriptional factor regulating MMP-1 expression. We found that galectin-3 directly interacted with AP-1 and facilitated the binding of this complex to the MMP-1 promoter that drives MMP-1 transcription. Moreover, silencing of galectin-3 inhibited binding of fra-1 and c-Jun to promoter sites of MMP-1 gene. Consistent with these in vitro findings, our in vivo study demonstrated that galectin-3 shRNA treatment significantly reduced the total number of mouse lung metastatic nodules. Taken together, galectin-3 facilitates cell migration and invasion in melanoma in vitro and can induce metastasis in vivo, in part through, regulating the transcription activity of AP-1 and thereby up-regulating MMP-1 expression.
galectin 3; matrix metalloproteinase 1; melanoma; neoplasm metastasis; RNA, small interfering; transcription factor AP-1
In the title compound, C25H22N4O2, the dihedral angles between the central pyrazole ring and the phenyl and benzene rings are 37.01 (3), 75.58 (7) and 49.67 (8)°. An intramolecular N—H⋯O hydrogen bond generates an S(6) motif. In the crystal, N—H⋯O hydrogen bonds link molecules into a zigzag chain extended along the b axis.