More than 80% of schistosomiasis patients in China live in the lake and marshland regions. The purpose of our study is to assess the effect of a comprehensive strategy to control transmission of Schistosoma japonicum in marshland regions.
In a cluster randomized controlled trial, we implemented an integrated control strategy in twelve villages from 2009 through 2011 in Gong'an County, Hubei Province. The routine interventions included praziquantel chemotherapy and controlling snails, and were implemented in all villages. New interventions, mainly consisting of building fences to limit the grazing area for bovines, building safe pastures for grazing, improving the residents' health conditions and facilities, were only implemented in six intervention villages. Results showed that the rate of S. japonicum infection in humans, bovines, snails, cow dung and mice in the intervention group decreased from 3.41% in 2008 to 0.81% in 2011, 3.3% to none, 11 of 6,219 to none, 3.9% to none and 31.7% to 1.7%, respectively (P<0.001 for all comparisons). In contrast, there were no statistically significant reductions of S. japonicum infection in humans, bovines and snails from 2008 to 2011 in the control group (P>0.05 for all comparisons). Moreover, a generalized linear model showed that there was a higher infection risk in humans in the control group than in the intervention group (OR = 1.250, P = 0.001) and an overall significant downward trend in infection risk during the study period.
The integrated control strategy, designed to reduce the role of bovines and humans as sources of S. japonicum infection, was highly effective in controlling the transmission of S. japonicum in marshland regions in China.
Chinese Clinical Trial Registry ChiCTR-PRC-12002405.
More than 80% of schistosomiasis patients in China live in the lake and marshland regions. Hence, how to control transmission of Schistosoma japonicum in these regions is especially important. From 2009 through 2011, we implemented an integrated control strategy, designed to reduce the role of bovines and humans as sources of S. japonicum infection, in twelve villages Gong'an County of Hubei Province, which is located in typical marshland. After three years, the rate of S. japonicum infection in humans, bovines and snails significantly declined in the six intervention villages. In contrast, there was no significant decline in these indexes in the six control villages. Moreover, there was a higher infection risk in humans in the control group than the intervention group. Our study showed that the integrated control strategy was highly effective in controlling the transmission of S. japonicum in marshland regions of China.
Previous studies have shown that the JAK2/STAT3 signaling pathway plays a regulatory role in cellular oxidative stress injury (OSI). In this study, we explored the role of the JAK2/STAT3 signaling pathway in hydrogen peroxide (H2O2)-induced OSI and the protective effect of melatonin against (H2O2)-induced injury in human umbilical vein endothelial cells (HUVECs). AG490 (a specific inhibitor of the JAK2/STAT3 signaling pathway) and JAK2 siRNA were used to manipulate JAK2/STAT3 activity, and the results showed that AG490 and JAK2 siRNA inhibited OSI and the levels of p-JAK2 and p-STAT3. HUVECs were then subjected to H2O2 in the absence or presence of melatonin, the main secretory product of the pineal gland. Melatonin conferred a protective effect against H2O2, which was evidenced by improvements in cell viability, adhesive ability and migratory ability, decreases in the apoptotic index and reactive oxygen species (ROS) production and several biochemical parameters in HUVECs. Immunofluorescence and Western blotting showed that H2O2 treatment increased the levels of p-JAK2, p-STAT3, Cytochrome c, Bax and Caspase3 and decreased the levels of Bcl2, whereas melatonin treatment partially reversed these effects. We, for the first time, demonstrate that the inhibition of the JAK2/STAT3 signaling pathway results in a protective effect against endothelial OSI. The protective effects of melatonin against OSI, at least partially, depend upon JAK2/STAT3 inhibition.
Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle and may have implications for human health. Establishment of chronic infection by M. avium subsp. paratuberculosis depends on its subversion of host immune responses. This includes blocking the ability of infected macrophages to be activated by gamma interferon (IFN-γ) for clearance of this intracellular pathogen. To define the mechanism by which M. avium subsp. paratuberculosis subverts this critical host cell function, patterns of signal transduction to IFN-γ stimulation of uninfected and M. avium subsp. paratuberculosis-infected bovine monocytes were determined through bovine-specific peptide arrays for kinome analysis. Pathway analysis of the kinome data indicated activation of the JAK-STAT pathway, a hallmark of IFN-γ signaling, in uninfected monocytes. In contrast, IFN-γ stimulation of M. avium subsp. paratuberculosis-infected monocytes failed to induce patterns of peptide phosphorylation consistent with JAK-STAT activation. The inability of IFN-γ to induce differential phosphorylation of peptides corresponding to early JAK-STAT intermediates in infected monocytes indicates that M. avium subsp. paratuberculosis blocks responsiveness at, or near, the IFN-γ receptor. Consistent with this hypothesis, increased expression of negative regulators of the IFN-γ receptors SOCS1 and SOCS3 as well as decreased expression of IFN-γ receptor chains 1 and 2 is observed in M. avium subsp. paratuberculosis-infected monocytes. These patterns of expression are functionally consistent with the kinome data and offer a mechanistic explanation for this critical M. avium subsp. paratuberculosis behavior. Understanding this mechanism may contribute to the rational design of more effective vaccines and/or therapeutics for Johne's disease.
The gene encoding the GroEL chaperonin is duplicated in nearly 30% of bacterial genomes; and although duplicated groEL genes have been comprehensively determined to have distinct physiological functions in different species, the mechanisms involved have not been characterized to date. Myxococcus xanthus DK1622 has two copies of the groEL gene, each of which can be deleted without affecting cell viability; however, the deletion of either gene does result in distinct defects in the cellular heat-shock response, predation, and development. In this study, we show that, from the expression levels of different groELs, the distinct functions of groEL1 and groEL2 in predation and development are probably the result of the substrate selectivity of the paralogous GroEL chaperonins, whereas the lethal effect of heat shock due to the deletion of groEL1 is caused by a decrease in the total groEL expression level. Following a bioinformatics analysis of the composition characteristics of GroELs from different bacteria, we performed region-swapping assays in M. xanthus, demonstrating that the differences in the apical and the C-terminal equatorial regions determine the substrate specificity of the two GroELs. Site-directed mutagenesis experiments indicated that the GGM repeat sequence at the C-terminus of GroEL1 plays an important role in functional divergence. Divergent functions of duplicated GroELs, which have similar patterns of variation in different bacterial species, have thus evolved mainly via alteration of the apical and the C-terminal equatorial regions. We identified the specific substrates of strain DK1622's GroEL1 and GroEL2 using immunoprecipitation and mass spectrometry techniques. Although 68 proteins bound to both GroEL1 and GroEL2, 83 and 46 proteins bound exclusively to GroEL1 or GroEL2, respectively. The GroEL-specific substrates exhibited distinct molecular sizes and secondary structures, providing an encouraging indication for GroEL evolution for functional divergence.
GroEL is a type I chaperonin, involved in protein folding, assembly, and transport. It is a major group of heat-shock proteins that are over-expressed at high temperatures and has fundamental roles in growth and survival at non-permissive temperatures. Because of its importance in many cellular processes, the groEL gene is ubiquitously distributed in bacteria. Most bacterial species possess a single groEL gene, while others (close to 30% of sequenced bacterial genomes) have two or more groEL copies. Many studies have described the functional divergence of duplicated groEL genes in different bacterial species, but the involved mechanisms have not yet been characterized. Myxobacteria are characterized by their unique multicellular behaviors. Myxococcus xanthus DK1622, the model strain of myxobacteria, possesses a large genome (9.14 Mb), containing many gene duplications, including two copies of the groEL gene. Gene duplications and their functional divergence are suggested for complex cellular behaviors, which, however, have not yet been testified. In this paper, using combined proteomic and genetic approaches, we explored how the duplicated groEL genes of M. xanthus DK1622 evolved to fit the functional divergence for social behaviors.
We used Medicare administrative data to examine trends in primary and revision THA utilization and hospital volume. Between 1991 and 2005 primary and revision THA utilization increased by 40.9% and 16.8% respectively. The percentage of primary THA procedures performed in high volume hospitals (those in the highest quintile of volume) increased slightly from 58.0% of all procedures in 1991 to 58.7% in 2005 (P-value < 0.01). The percentage of revisions performed in high volume hospitals increased from 60.9% to 62.4% (P-value < 0.01). The percentage of primary THA procedures performed by low volume hospitals remained relatively stable (P-value = 0.36) while the percentage of revision THA performed by low volume hospitals declined (P-value < 0.001). In aggregate these results suggest minimal evidence that regionalization of THA is occurring.
Right ventricular failure (RVF) is the main cause of death in patients with pulmonary artery hypertension (PAH). Sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, was recently approved for treatment of PAH patients. However, the mechanisms underlying RV contractile malfunction and the benefits of sildenafil on RV function are not well understood. We aimed to investigate 1) the ultrastructural and excitation-contraction coupling alterations underlying PAH-induced RVF; 2) whether the ultrastructural changes are reversible; 3) the mechanisms underlying the therapeutic benefits of sildenafil in PAH-RVF. We used a single injection of monocrotaline (MCT) in Wistar rats to induce pulmonary vascular proliferation, which led to PAH and RVF. RV myocytes displayed severe T-tubule loss and disorganization as well as blunted and dys-synchronous SR Ca2+ release. Sildenafil prevented and reversed the MCT-induced PAH and LV filling impairment. Early intervention with sildenafil prevented RV hypertrophy and the development of RVF, T-tubule remodeling and Ca2+ handling dysfunction. While late treatment with sildenafil did not reverse RV hypertrophy in animals with established RVF, RV systolic function was improved. Furthermore, late intervention partially reversed both the impairment of myocyte T-tubule integrity and Ca2+ handling protein and SR Ca2+ release function in MCT-treated rats. In conclusion, PAH-induced increase in RV afterload causes severe T-tubule remodeling and Ca2+ handling dysfunction in RV myocytes, leading to RV contractile failure. Sildenafil prevents and partially reverses ultrastructural, molecular and functional remodeling of failing RV myocytes. Reversal of pathological T-tubule remodeling, although incomplete, is achievable without the regression of RV hypertrophy.
right ventricle failure; pulmonary artery hypertension; PDE5 inhibitor; calcium; t-tubule; excitation-contraction coupling
GH15 is a polyvalent phage that shows activity against a wide range of Staphylococcus aureus strains. In this work, the complete genome sequence of GH15 was determined. With a genome size of 139,806 bp (double-stranded DNA), GH15 is the largest staphylococcal phage sequenced to date. The complete genome encodes 214 open reading frames (ORFs) and 4 tRNAs. The closest relatives are the class III staphylococcal myobacteriophages, including K, A5W, ISP, Sb-1, and G1. Interestingly, although corresponding gene sequences demonstrate very high similarity, all the introns and inteins present in the phages listed above are absent in GH15. As such, GH15 can be considered phylogenetically unique among the staphylococcal myobacteriophages, indicating the diversity of this family.
Our previous studies showed that glioma-associated oncogene (Gli)2 plays an important role in the proliferation and apoptosis resistance of hepatocellular carcinoma (HCC) cells. The aim of this study was to explore the clinical significance of Gli2 expression in HCC.
Expression of Gli2 protein was detected in samples from 68 paired HCC samples, the corresponding paraneoplastic liver tissues, and 20 normal liver tissues using immunohistochemistry. Correlation of the immunohistochemistry results with clinicopathologic parameters, prognosis, and the expression of E-cadherin, N-cadherin, and vimentin were analyzed.
Immunohistochemical staining showed high levels of Gli2 protein expression in HCC, compared with paraneoplastic and normal liver tissues (P < 0.05). This high expression level of Gli2 was significantly associated with tumor differentiation, encapsulation, vascular invasion, early recurrence, and intra-hepatic metastasis (P < 0.05). There was a significantly negative correlation between Gli2 and E-cadherin expression (r = −0.302, P < 0.05) and a significantly positive correlation between expression of Gli2 and expression of vimentin (r = −0.468, P < 0.05) and N-cadherin (r = −0.505, P < 0.05). Kaplan-Meier analysis showed that patients with overexpressed Gli2 had significantly shorter overall survival and disease-free survival times (P < 0.05). Multivariate analysis suggested that the level of Gli2 expression was an independent prognostic factor for HCC.
Expression of Gli2 is high in HCC tissue, and is associated with poor prognosis in patients with HCC after hepatectomy.
Gli2; Hepatocellular carcinoma; Prognosis; Epithelial-to-mesenchymal transition
Epigenetic mechanisms, including DNA methylation, histone modification, and microRNAs, play pivotal roles in stem cell biology. Methyl-CpG binding protein 1 (MBD1), an important epigenetic regulator of adult neurogenesis, controls the proliferation and differentiation of adult neural stem/progenitor cells (aNSCs). We recently demonstrated that MBD1 deficiency in aNSCs leads to altered expression of several noncoding microRNAs (miRNAs).
Here we show that one of these miRNAs, miR-195, and MBD1 form a negative feedback loop. While MBD1 directly represses the expression of miR-195 in aNSCs, high levels of miR-195 in turn repress the expression of MBD1. Both gain-of-function and loss-of-function investigations show that alterations of the MBD1–miR-195 feedback loop tip the balance between aNSC proliferation and differentiation.
Therefore the regulatory loop formed by MBD1 and miR-195 is an important component of the epigenetic network that controls aNSC fate.
Yeast cytosine deaminase catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, NMR, and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in kcat and a dramatic increase in Km, indicating Glu64 is important for both binding and catalysis in the activation of 5FC. 19F-NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. 1H and 15N NMR analysis revealed trans-hydrogen-bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has higher activation energy as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by 1H and 15N NMR analysis. To explore the functional role of Glu64 in catalysis, deamination of cytosine catalyzed by the E64A mutant was investigated by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and after the formation of the tetrahedral reaction intermediate become partially rate-limiting steps. The results of the experimental and computational studies together indicate that Glu64 plays a critical role in both binding and chemical transformation in the conversion of the prodrug 5FC to the anticancer drug 5-fluorouracil.
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthetic pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin. The enzyme is essential for microorganisms, is absent from humans, and is not the target for any existing antibiotics. Therefore, HPPK is an attractive target for developing novel antimicrobial agents. Previously, we characterized the reaction trajectory of HPPK-catalyzed pyrophosphoryl transfer and synthesized a series of bisubstrate analog inhibitors of the enzyme by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups. Here, we report a new generation of bisubstrate analog inhibitors. To improve protein binding and linker properties of such inhibitors, we have replaced the pterin moiety with 7,7-dimethyl-7,8-dihydropterin and the phosphate bridge with a piperidine linked thioether. We have synthesized the new inhibitors, measured their Kd and IC50 values, determined their crystal structures in complex with HPPK, and established their structure-activity relationship. 6-Carboxylic acid ethyl ester-7,7-dimethyl-7,8-dihydropterin, a novel intermediate that we developed recently for easy derivatization at position 6 of 7,7-dimethyl-7,8-dihydropterin, offers a much high yield for the synthesis of bisubstrate analogs than that of previously established procedure.
Antibacterial; Bisubstrate; Folate; HPPK; Pterin
The mechanisms involved in diabetic neuropathic pain are complex and involve peripheral and central pathophysiological phenomena. Proinflammatory tumour necrosis factor α (TNF-α) and TNF-α receptor 1, which are markers of inflammation, contribute to neuropathic pain. The purpose of this experimental study was to evaluate the effect of curcumin on diabetic pain in rats. We tested 24 rats with diabetes induced by a single intraperitoneal injection of streptozotocin and 24 healthy control rats. Twelve rats in each group received 60 mg/kg oral curcumin daily for 28 days, and the other 12 received vehicle. On days 7, 14, 21, and 28, we tested mechanical allodynia with von Frey hairs and thermal hyperalgesia with radiant heat. Markers of inflammation in the spinal cord dorsal horn on day 28 were estimated with a commercial assay and Western blot analysis. Compared to control rats, diabetic rats exhibited increased mean plasma glucose concentration, decreased mean body weight, and significant pain hypersensitivity, as evidenced by decreased paw withdrawal threshold to von Frey hairs and decreased paw withdrawal latency to heat. Curcumin significantly attenuated the diabetes-induced allodynia and hyperalgesia and reduced the expression of both TNF-α and TNF-α receptor 1. Curcumin seems to relieve diabetic hyperalgesia, possibly through an inhibitory action on TNF-α and TNF-α receptor 1.
diabetic neuropathic pain; hyperalgesia; curcumin; tumour necrosis factor α; tumour necrosis factor α receptor 1.
Aβ42 is believed to play a causative role in Alzheimer’s disease (AD) pathogenesis. γ-Secretase modulators (GSMs) are actively being pursued as potential AD therapeutics because they selectively alter the cleavage site of the amyloid precursor protein (APP) to reduce the formation of Aβ42. However, the binding partner of acid based GSMs was unresolved until now. We have developed clickable photoaffinity probes based on piperidine acetic acid GSM-1 and identified PS1 as the target within the γ-secretase complex. Furthermore, we provide evidence that allosteric interaction of GSMs with PS1 results in a conformational change in the active site of the γ-secretase complex leading to the observed modulation of γ-secretase activity.
Aβ42 is believed to play a causative role in Alzheimer’s
disease (AD) pathogenesis. γ-Secretase modulators (GSMs) are
actively being pursued as potential AD therapeutics because they selectively
alter the cleavage site of the amyloid precursor protein (APP) to
reduce the formation of Aβ42. However, the binding partner of
acid based GSMs was unresolved until now. We have developed clickable
photoaffinity probes based on piperidine acetic acid GSM-1 and identified
PS1 as the target within the γ-secretase complex. Furthermore,
we provide evidence that allosteric interaction of GSMs with PS1 results
in a conformational change in the active site of the γ-secretase
complex leading to the observed modulation of γ-secretase activity.
Alzheimer's disease, presenilin, γ-secretase modulator,
GSM-1, click chemistry, photoaffinity labeling
Nursing homes certified by the Medicare and/or Medicaid program are subject to federally mandated and state-enforced quality and safety standards. We examined the relationship between state quality enforcement and nursing home terminations from the two programs.
Using data from a survey of state licensure and certification agencies and other secondary databases, we performed bivariate and multivariate analyses on the strength of state quality regulation in 2005, and nursing home voluntary terminations (decisions made by the facility) or involuntary terminations (imposed by the state) in 2006–2007.
Involuntary terminations were rarely imposed by state regulators, while voluntary terminations were relatively more common (2.16 percent in 2006–2007) and varied considerably across states. After controlling for facility, market, and state covariates, nursing homes in states implementing stronger quality enforcement were more likely to voluntarily terminate from the Medicare and Medicaid programs (odds ratio=1.53, p=.018).
Although involuntary nursing home terminations occurred rarely in most states, nursing homes in states with stronger quality regulations tend to voluntarily exit the publicly financed market. Because of the consequences of voluntary terminations on patient care and access, state regulators need to consider the effects of increased enforcement on both enhanced quality and the costs of termination.
Nursing home regulation; Medicare and Medicaid; termination; state variation
The increasing prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C infection worldwide calls for efforts to develop a relevant animal model for evaluating AIDS candidate vaccines. In China, the prevalent HIV strains comprise a circulating recombinant form, BC (CRF07_BC), in which the envelope belongs to subtype C. To evaluate potential AIDS vaccines targeting Chinese viral strains in nonhuman primate models, we constructed a simian-human immunodeficiency virus (SHIV) carrying most of the envelope sequence of a primary HIV-1 clade C strain isolated from an HIV-positive intravenous drug user from YunNan province in China. Infection of six Chinese rhesus macaques with SHIV-CN97001 resulted in a low level of viremia and no significant alteration in CD4+ T-lymphocyte counts. To determine whether in vivo adaptation would enhance the infectivity of SHIV-CN97001, the parental infectious strain was serially passaged through eight Chinese rhesus macaques. The hallmarks of SHIV infectivity developed gradually, as shown by the increasingly elevated peak viremia with each passage. These findings establish that the R5-tropic SHIV-CN97001/Chinese rhesus macaque model should be very useful for evaluation of HIV-1 subtype C vaccines in China.
Clade C; Passage; Rhesus macaques; SHIV
To quantify age-dependent iron deposition changes in healthy subjects using Susceptibility Weighted Imaging (SWI).
Materials and Methods
In total, 143 healthy volunteers were enrolled. All underwent conventional MR and SWI sequences. Subjects were divided into eight groups according to age. Using phase images to quantify iron deposition in the head of the caudate nucleus and the lenticular nucleus, the angle radian value was calculated and compared between groups. ANOVA/Pearson correlation coefficient linear regression analysis and polynomial fitting were performed to analyze the relationship between iron deposition in the head of the caudate nucleus and lenticular nucleus with age.
Iron deposition in the lenticular nucleus increased in individuals aged up to 40 years, but did not change in those aged over 40 years once a peak had been reached. In the head of the caudate nucleus, iron deposition peaked at 60 years (p<0.05). The correlation coefficients for iron deposition in the L-head of the caudate nucleus, R-head of the caudate nucleus, L-lenticular nucleus and R-lenticular nucleus with age were 0.67691, 0.48585, 0.5228 and 0.5228 (p<0.001, respectively). Linear regression analyses showed a significant correlation between iron deposition levels in with age groups.
Iron deposition in the lenticular nucleus was found to increase with age, reaching a plateau at 40 years. Iron deposition in the head of the caudate nucleus also increased with age, reaching a plateau at 60 years.
Toxicarioside A is a cardenolide isolated mainly from plants and animals. Emerging evidence demonstrate that cardenolides not only have cardiac effects but also anticancer effects. In this study, we used in vivo models to investigate the antitumor activities of toxicarioside A and the potential mechanisms behind them. Murine colorectal carcinoma (CT26) and Lewis lung carcinoma (LL/2) models were established in syngeneic BALB/c and C57BL/6 mice, respectively. We found that the optimum effective dose of toxicarioside A treatment significantly suppressed tumor growth and angiogenesis in CT and LL/2 tumor models in vivo. Northern and Western blot analysis showed significant inhibition of endoglin expression in toxicarioside A-treated human umbilical vein endothelial cells (HUVECs) in vitro and tumor tissues in vivo. Toxicarioside A treatment significantly inhibited cell proliferation, migration and invasion, but did not cause significant cell apoptosis and affected other membrane protein (such as CD31 and MHC I) expression. In addition, TGF-β expression was also significantly inhibited in CT26 and LL/2 tumor cells treated with toxicarioside A. Western blot analysis indicated that Smad1 and phosphorylated Smad1 but not Smad2/3 and phosphorylated Smad2/3 were attenuated in HUVECs treated with toxicarioside A. Smad1 and Smad2/3 signaling remained unchanged in CT26 and LL/2 tumor cells treated with toxicarioside A. Endoglin knockout by small interfering RNA against endoglin induced alternations in Smad1 and Smad2/3 signaling in HUVECs. Our results indicate that toxicarioside A suppresses tumor growth through inhibition of endoglin-related tumor angiogenesis, which involves in the endoglin/TGF-β signal pathway.
Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF21 via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native FGF21. Two specific point mutations were introduced into FGF21. The leucine to arginine substitution at position 98 (L98R) suppressed FGF21 aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF21 analog with a favorable pharmaceutical property for potential clinical development.
Radiofrequency catheter ablation (RFCA) has been used for the ablation of premature ventricular contractions (PVCs) or ventricular tachycardia (VT). To date, the mapping and catheter ablation of the arrhythmias originating from the left ventricular outflow tract (LVOT) has not been specified. This study investigates the electrocardiogram (ECG) feature of PVCs or VT originating from the LVOT. Moreover, the treatment outcome of RFCA is analyzed.
Mapping and ablation were performed on the supravalvular or subvalvular aorta in 52 cases with PVCs/VT originating from the LVOT. The data were compared with those from 104 patients with PVCs/VT originating from the right ventricular outflow tract (RVOT). A differential procedure was prepared based on the comparison of the ECG features of PVCs/VT originating from the RVOT, LVOT, and their different parts.
Among 52 cases with PVCs originating from the LVOT, 47 were successfully treated by RFCA, with a success rate of 90.38%. Several differences among the 12-lead ECG features were observed from the RVOT and LVOT in the left and right coronary sinus groups, as well as under the left coronary sinus group (left fibrous trigone): (1) If the precordial leads transition 0 are considered as the diagnostic parameters of PVCs/VT originating from the LVOT, then the sensitivity, specificity, as well as positive and negative predictive values are 94.12%, 93.00%, 87.27%, and 96.88%, respectively; (2) The analysis of different subgroups of the LVOT are as follows: (a) A mainly positive wave of r or m pattern was recorded in the lead I in 72.73% of patients in the right coronary sinus group, versus 12.90% of patients in the left coronary sinus group, and 0% in the under left coronary sinus group. (b) All patients in the right coronary sinus group presented waves of RII>RIII and QSaVR>QSaVL, whereas most patients in the other two groups showed waves of RIII>RII and QSaVL>QSaVR. (c) Most patients in the under left coronary sinus group in lead V1 had a mainly positive wave (R) (77.78%), whereas those in the right (81.82%) and left (62.50%) coronary sinus groups had mainly negative waves (rS).
RFCA is a safe and effective curative therapy for PVCs/VT originating from the LVOT. The 12-lead ECG features of the LVOT from different origins exhibit certain distinctions.
Electrophysiology; Ventricular arrhythmia; Left ventricular outflow; Catheter ablation; Radiofrequency current
Muscle growth depends on the fusion of proliferate satellite cells to existing myofibers. We reported previously that 0–14 day intermittent feeding led to persistent retardation in myofiber hypertrophy. However, how satellite cells respond to such nutritional insult has not been adequately elucidated.
One-day-old broiler chicks were allocated to control (Con, ad libitum feeding), intermittent feeding (IF, feed provided on alternate days) and re-feeding (RF, 2 days ad libitum feeding after 12 days of intermittent feeding) groups. Chickens were killed on Day 15 and satellite cells were isolated. When cultured, satellite cells from the IF group demonstrated significant retardation in proliferation and differentiation potential, while RF partly restored the proliferation rate and differentiation potential of the satellite cells. Significant up-regulation of insulin like growth factor I receptor (IGF-IR) (P<0.05) and thyroid hormone receptor α (TRα) (P<0.05), and down-regulation of growth hormone receptor (GHR) (P<0.01) and IGF-I (P<0.01) mRNA expression was observed in freshly isolated IF satellite cells when compared with Con cells. In RF cells, the mRNA expression of IGF-I was higher (P<0.05) and of TRα was lower (P<0.01) than in IF cells, suggesting that RF restored the mRNA expression of TRα and IGF-I, but not of GHR and IGF-IR. The Bax/Bcl-2 ratio tended to increase in the IF group, which was reversed in the RF group (P<0.05), indicating that RF reduced the pro-apoptotic influence of IF. Moreover, no significant effect of T3 was detected on cell survival in IF cells compared with Con (P<0.001) or RF (P<0.05) cells.
These data suggest that early-age feed restriction inhibits the proliferation and differentiation of satellite cells, induces changes in mRNA expression of the GH/IGF-I and thyroid hormone receptors in satellite cells, as well as blunted sensitivity of satellite cells to T3, and that RF partially reverses these effects. Thus, a moderate nutritional strategy for feed restriction should be chosen in early chick rearing systems.
Feed restriction; Satellite cells; Proliferation; Gene expression; Chicken
Interactions between cancer cells and their microenvironment are crucial for promoting tumor growth and invasiveness. In the tumor adaptive landscape model, hypoxic and acidic microenvironmental conditions reduce the fitness of cancer cells and significantly restrict their proliferation. This selects for enhanced motility as cancer cells may evolve an invasive phenotype if the consequent cell movement is rewarded by proliferation. Here, we used an integrative approach combining a mathematical tumor adaptive landscape model with experimental studies to examine the evolutionary dynamics that promote an invasive cancer phenotype. Computer simulation results hypothesized an explicit coupling of motility and proliferation in cancer cells. The mathematical modeling results were also experimentally examined by selecting Panc-1 cells with enhanced motility on a fibroblast-derived 3D matrix for cells that move away from the unfavorable metabolic constraints. After multiple rounds of selection, the cells that adapted through increased motility were characterized for their phenotypic properties compared to stationary cells. Microarray and gene depletion studies demonstrated the role of Rho-GDI2 in regulating both cell movement and proliferation. Together, this work illustrates the partnership between evolutionary mathematical modeling and experimental validation as a potentially useful approach to study the complex dynamics of the tumor microenvironment.
Adaptive landscape model; tumor microenvironment; cell motility; 3D matrix; Rho-GDI2
The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. Using single-molecule Fluorescence Resonance Energy Transfer (FRET) with a combined statistical data analysis approach, we have identified the intermittently appearing coherence of the enzymatic conformational state from the recorded single molecule intensity-time trajectories of enzyme 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in catalytic reaction. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. The coherence frequency, identified by statistical results of the correlation function analysis from single-molecule FRET trajectories, increases with the increasing substrate concentrations. The intermittent coherence in conformational state changes at the enzymatic reaction active site is likely to be common and exist in other conformation regulated enzymatic reactions. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.
Descemet's membrane detachment (DMD) can be a potentially serious complication of intraocular surgery or ocular trauma. The cause is not very clear. We are trying to remind an awareness of the spectrum of DMD resulting from trabeculectomy by presenting a case of extensive DMD after trabeculectomy which was successfully repaired.
Descemet membrane detachment; trabeculectomy; glaucoma; cornea
Migrating cells employ sophisticated signal transduction systems to respond to their environment and polarize towards attractant sources. Bacterial cells also regulate their polarity dynamically to reverse their direction of movement. In Myxococcus xanthus, a GTP-bound Ras-like G-protein, MglA, activates the motility machineries at the leading cell pole. Reversals are provoked by pole-to-pole switching of MglA, which is under the control of a chemosensory-like signal transduction cascade (Frz). It was previously known that the asymmetric localization of MglA at one cell pole is regulated by MglB, a GTPase Activating Protein (GAP). In this process, MglB specifically localizes at the opposite lagging cell pole and blocks MglA localization at that pole. However, how MglA is targeted to the leading pole and how Frz activity switches the localizations of MglA and MglB synchronously remained unknown. Here, we show that MglA requires RomR, a previously known response regulator protein, to localize to the leading cell pole efficiently. Specifically, RomR-MglA and RomR-MglB complexes are formed and act complementarily to establish the polarity axis, segregating MglA and MglB to opposite cell poles. Finally, we present evidence that Frz signaling may regulate MglA localization through RomR, suggesting that RomR constitutes a link between the Frz-signaling and MglAB polarity modules. Thus, in Myxococcus xanthus, a response regulator protein governs the localization of a small G-protein, adding further insight to the polarization mechanism and suggesting that motility regulation evolved by recruiting and combining existing signaling modules of diverse origins.
Migrating cells have evolved a molecular compass to rapidly respond to environmental signals. During chemotaxis, small G-proteins and their regulators are activated and determine a leading cell edge towards attractant molecules. Bacteria also move across surfaces in a directed manner. The rod-shaped bacterium Myxococcus xanthus can switch its direction of movement in a process where the cell poles exchange roles (reversal), allowing complex multicellular behaviors. In Myxococcus, a small G-protein, MglA, determines the leading cell pole. In this study, we investigated how MglA localizes to the pole and found that its localization depends on the dual complementary action of RomR and MglB. In this process, RomR targets MglA to the pole while, in turn, MglB prevents its accumulation at the back of the cell. Moreover, RomR potentially links MglA to the Frz signal transduction pathway, a chemosensory system controlling the reversal frequency. The results provide a new molecular basis to understand motility regulation in a bacterium, which may have arisen from co-optation and branching of prokaryotic and eukaryotic-like signaling modules.