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1.  Antifungal Activity of C-27 Steroidal Saponins 
As part of our search for new antifungal agents from natural resources, 22 C-27 steroidal saponins and 6 steroidal sapogenins isolated from several monocotyledonous plants were tested for their antifungal activity against the opportunistic pathogens Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, and Aspergillus fumigatus. The results showed that the antifungal activity of the steroidal saponins was associated with their aglycone moieties and the number and structure of monosaccharide units in their sugar chains. Within the 10 active saponins, four tigogenin saponins (compounds 1 to 4) with a sugar moiety of four or five monosaccharide units exhibited significant activity against C. neoformans and A. fumigatus, comparable to the positive control amphotericin B. The antifungal potency of these compounds was not associated with cytotoxicity to mammalian cells. This suggests that the C-27 steroidal saponins may be considered potential antifungal leads for further preclinical study.
doi:10.1128/AAC.50.5.1710-1714.2006
PMCID: PMC1472193  PMID: 16641439
2.  Complete Genome Sequence of the Nitrogen-Fixing and Solvent-Producing Clostridium pasteurianum DSM 525 
Genome Announcements  2015;3(1):e01591-14.
Here, we report on the closed genome sequence of Clostridium pasteurianum DSM 525, which is an anaerobic, Gram-positive and endospore-forming organism. C. pasteurianum can fix N2 and produce solvents such as butanol and 1,3-propanediol from carbohydrates. The genome consists of a single 4,350,673-bp replicon.
doi:10.1128/genomeA.01591-14
PMCID: PMC4335339
3.  Association of the Genetic Polymorphisms in Pre-MicroRNAs with Risk of Ischemic Stroke in a Chinese Population 
PLoS ONE  2015;10(2):e0117007.
microRNA (miRNA) plays a role in the pathogenesis of ischemic stroke, and single nucleotide polymorphisms in miRNA genes may contribute to disease susceptibility. However, the effect of miR-146a, miR-196a2, and miR-499 polymorphisms on ischemic stroke susceptibility has been rarely reported. Using the TaqMan assay, we evaluated the association of hsa-miR-146a/rs2910164, hsa-miR-196a2/rs11614913, and hsa-miR-499/rs3746444 polymorphisms with the risk of ischemic stroke in a Chinese population with 531 ischemic stroke patients and 531 control subjects. Rs2910164 C/G genotypes were significantly associated with increased risk of ischemic stroke in different genetic model (homozygote comparison: OR = 2.00, 95% CI, 1.29–3.12, P = 0.002; additive model: OR = 1.35, 95% CI, 1.10–1.65, P = 0.004;dominant model: OR = 1.33, 95% CI, 1.00–1.75, P = 0.049; recessive model: OR = 1.82, 95% CI, 1.20–2.74, P = 0.004). Subjects with allele G of hsa-miR-146a/ rs2910164 also showed increased risk of ischemic stroke (OR = 1.33, 95% CI, 1.09–1.62, P = 0.005). Stratification analysis showed that the association between rs2910164 and the risk of ischemic stroke was more pronounced in subjects over 60 years old, females, non-drinkers, subjects without hypertension or diabetes mellitus. There were significant combined effects between miR-146a/rs2910164 and fasting glucose/low-density lipoprotein cholesterol levels on ischemic stroke susceptibility. However, we failed to find any association between the alleles/genotypes of rs11614913 T/C and ischemic stroke, respectively (P> 0.05). In summary, this study provides evidence that miR-146a/rs2910164 might be associated with a significantly increased risk of ischemic stroke in a Chinese population, and the combined effects between miRNA polymorphism and fasting glucose /blood lipid levels may contribute to stroke pathogenesis.
doi:10.1371/journal.pone.0117007
PMCID: PMC4319971  PMID: 25658319
4.  Deep Proteome Mapping of Mouse Kidney Based on OFFGel Prefractionation Reveals Remarkable Protein Post- Translational Modifications 
Journal of Proteome Research  2014;13(3):1636-1646.
Performing a comprehensive nonbiased proteome analysis is an extraordinary challenge due to sample complexity and wide dynamic range, especially in eukaryotic tissues. Thus, prefractionation steps conducted prior to mass spectrometric analysis are critically important to reduce complex biological matrices and allow in-depth analysis. Here we demonstrated the use of OFFGel prefractionation to identify more low abundant and hydrophobic proteins than in a nonfractionated sample. Moreover, OFFGel prefractionation of a kidney protein sample was able to unveil protein functional relevance by detecting PTMs, especially when prefractionation was augmented with a targeted enrichment strategy such as TiO2 phospho-enrichment. The OFFGel-TiO2 combination used in this study was comparable to other global phosphoproteomics approaches (SCX-TiO2, ERLIC-TiO2, or HILIC-TiO2). The detailed mouse kidney proteome with the phosphopeptide enrichment presented here serves as a useful platform for a better understanding of how the renal protein modification machinery works and, ultimately, will contribute to our understanding of pathological processes as well as normal physiological renal functions.
doi:10.1021/pr401122m
PMCID: PMC3993965  PMID: 24495006
OFFGel; kidney; proteomics; fractionation; phospho-enrichment
5.  Correlation between Serum Levels of High Mobility Group Box-1 Protein and Pancreatitis: A Meta-Analysis 
BioMed Research International  2015;2015:430185.
Background. Aberrant expression of high mobility group box-1 protein (HMGB1) contributes to the progression of various inflammatory diseases. This meta-analysis focused on the clinical significance of serum HMGB1 levels in pancreatitis patients, with the goal of building a novel diagnostic score model. Method. We conducted a meta-analysis by searching in the PubMed, Embase, Web of Science, Cochrane Library, CISCOM, CINAHL, Google Scholar, China BioMedicine (CBM), and China National Knowledge Infrastructure (CNKI) databases without any language restrictions. Studies were pooled and standard mean difference (SMD) and its corresponding 95% confidence intervals (95% CIs) were calculated. Version 12.0 STATA software was used for statistical analysis. Results. We performed a final analysis of 841 subjects from 12 clinical case-control studies. The meta-analysis results showed a positive association between serum HMGB1 levels and the progression of pancreatitis. In the subgroup analysis by country, high serum level of HMGB1 may be related to pancreatitis progression in China, Korea, Hungary, and Japan populations (all P < 0.05). Conclusion. The present meta-analysis indicated that serum HMGB1 level was statistically elevated in patients with pancreatitis, and thus serum levels of HMGB1 could be determined to be a useful biomarker for pancreatitis patients.
doi:10.1155/2015/430185
PMCID: PMC4324490
6.  Enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA on human brain metastatic breast cancer 
Scientific Reports  2015;5:8029.
Novel molecularly targeted agents that block the development and metastasis of human brain metastatic breast cancer hold great promise for their translational value. In this study, we constructed a novel targeting composite peptide BRBP1-TAT-KLA comprising of three elements: a brain metastatic breast carcinoma cell (231-BR)-binding peptide BRBP1, a cell penetrating peptide TAT, and a proapoptotic peptide KLA. This composite peptide efficiently internalized in 231-BR cells and consequently induced mitochondrial damage and cellular apoptosis. Exposure of 231-BR cells to BRBP1-TAT-KLA significantly decreased cell viability and increased apoptosis compared with the cells treated with the control peptides. In vivo relevance of these findings was further corroborated in the 231-BR tumor-bearing mice that demonstrated significantly delayed tumor development and metastasis following administration of BRBP1-TAT-KLA compared with those treated with TAT-KLA alone. Interestingly, BRBP1-TAT-KLA inhibited the formation of both large and micro-metastases, while TAT-KLA alone failed to significantly reduce micro-metastases in the breast cancer brain metastasis mice. BRBP1-TAT-KLA selectively homed to the tumors in vivo where it induced cellular apoptosis without significant toxicity on non-tumor tissues. Our findings therefore demonstrated the enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA, providing insights toward development of a potential therapeutic strategy for brain metastatic breast cancer.
doi:10.1038/srep08029
PMCID: PMC4306141  PMID: 25619721
7.  Short Leukocyte Telomere Length Predicts Risk of Diabetes in American Indians: the Strong Heart Family Study 
Diabetes  2013;63(1):354-362.
Telomeres play a central role in cellular aging, and shorter telomere length has been associated with age-related disorders including diabetes. However, a causal link between telomere shortening and diabetes risk has not been established. In a well-characterized longitudinal cohort of American Indians participating in the Strong Heart Family Study, we examined whether leukocyte telomere length (LTL) at baseline predicts incident diabetes independent of known diabetes risk factors. Among 2,328 participants free of diabetes at baseline, 292 subjects developed diabetes during an average 5.5 years of follow-up. Compared with subjects in the highest quartile (longest) of LTL, those in the lowest quartile (shortest) had an almost twofold increased risk of incident diabetes (hazard ratio [HR] 1.83 [95% CI 1.26–2.66]), whereas the risk for those in the second (HR 0.87 [95% CI 0.59–1.29]) and the third (HR 0.95 [95% CI 0.65–1.38]) quartiles was statistically nonsignificant. These findings suggest a nonlinear association between LTL and incident diabetes and indicate that LTL could serve as a predictive marker for diabetes development in American Indians, who suffer from disproportionately high rates of diabetes.
doi:10.2337/db13-0744
PMCID: PMC3868043  PMID: 23949319
8.  The risk factors of neurologic deficits of one-stage posterior vertebral column resection for patients with severe and rigid spinal deformities 
European Spine Journal  2013;23(1):149-156.
Purpose
To determine the risk factors of neurologic deficits during PVCR correction, so as to help improve safety during and after surgery.
Methods
A consecutive series of 76 patients with severe and rigid spinal deformities who were treated with PVCR at a single institution between October 2004 and July 2011 were included in our study. Of the 76 patients, 37 were male and 39 female, with an average age of 17.5 years (range 10–48 years). There were 52 adolescent patients (with an age <18 years) and 24 adult patients (with an age ≥18 years). Preoperatively, postoperatively and 6 months after surgery, we performed systemically neurologic function evaluations of each patients through meticulous physical examination. Any new abnormality or deterioration in evaluation of neurologic function than preoperative is reckoned postoperative neurologic deficits. Ten variables that might affect the safety of neurologic deficits during PVCR procedures, including imaging factors, clinical factors and operational factors, were analyzed using univariate analysis. Then the variables with statistical difference were analyzed by using multi-factor unconditional logistic regression analysis.
Results
No patient in this series had permanent paraplegia and nerve root injury due to operation. Change of neurologic status was found in six patients after surgery. Results of single-factor comparison demonstrated that the following seven variables were statistically different (P < 0.05): location of apex at main curve (X3), Cobb angle at the main curve at the coronal plane (X4), scoliosis associated with thoracic hyperkyphosis (X5), level of vertebral column resected (X6), number of segmental vessels ligated (X7), preexisting neurologic dysfunction (X8), and associated with intraspinal and brain stem anomalies (X9). The multi-factor unconditional logistic regression analysis revealed that X8 (OR = 49.322), X9 (OR = 18.423), X5 (OR = 11.883), and X6 (OR = 8.769) were independent and positively correlated with the neurologic deficit.
Conclusions
Preexisting neurologic dysfunction, associated with intraspinal and brain stem anomalies, scoliosis associated with thoracic hyperkyphosis and level of vertebral column resected are independent risk factors for neurologic deficits during PVCR procedure.
doi:10.1007/s00586-013-2793-6
PMCID: PMC3897813  PMID: 23619771
Spinal deformity; Vertebral column resection; Surgical procedures; Neurologic deficits; Risk factor
9.  Molecular Variation and Horizontal Gene Transfer of the Homocysteine Methyltransferase Gene mmuM and its Distribution in Clinical Pathogens 
The homocysteine methyltransferase encoded by mmuM is widely distributed among microbial organisms. It is the key enzyme that catalyzes the last step in methionine biosynthesis and plays an important role in the metabolism process. It also enables the microbial organisms to tolerate high concentrations of selenium in the environment. In this research, 533 mmuM gene sequences covering 70 genera of the bacteria were selected from GenBank database. The distribution frequency of mmuM is different in the investigated genera of bacteria. The mapping results of 160 mmuM reference sequences showed that the mmuM genes were found in 7 species of pathogen genomes sequenced in this work. The polymerase chain reaction products of one mmuM genotype (NC_013951 as the reference) were sequenced and the sequencing results confirmed the mapping results. Furthermore, 144 representative sequences were chosen for phylogenetic analysis and some mmuM genes from totally different genera (such as the genes between Escherichia and Klebsiella and between Enterobacter and Kosakonia) shared closer phylogenetic relationship than those from the same genus. Comparative genomic analysis of the mmuM encoding regions on plasmids and bacterial chromosomes showed that pKF3-140 and pIP1206 plasmids shared a 21 kb homology region and a 4.9 kb fragment in this region was in fact originated from the Escherichia coli chromosome. These results further suggested that mmuM gene did go through the gene horizontal transfer among different species or genera of bacteria. High-throughput sequencing combined with comparative genomics analysis would explore distribution and dissemination of the mmuM gene among bacteria and its evolution at a molecular level.
doi:10.7150/ijbs.10320
PMCID: PMC4278250  PMID: 25552925
comparative genomics; homocysteine methyltransferase gene; horizontal gene transfer; molecular variation
10.  CEP2 Attenuates Myoblast Differentiation But Does Not Affect Proliferation 
CEP2 (CDC42EP2) is a member of the CDC42 subfamily that belongs to the Rho family. The Rho family plays an important role in a variety of cellular processes including skeletal myogenesis. Here, we find the expression of CEP2 increased significantly during C2C12 myogenesis. Overexpression of CEP2 could attenuate myoblast differentiation, while knockdown of CEP2 by siRNA results in enhancing myogenesis. Furthermore, we demonstrate for the first time that CEP2 attenuates myoblast differentiation via suppression of muscle regulatory factors (MRFs) rather than influencing myoblast proliferation. These results indicate that CEP2 acts as a repressor during myogenesis, which provides new insights into the role of CEP2 in muscle development.
doi:10.7150/ijbs.8621
PMCID: PMC4278259  PMID: 25552934
CEP2; myogenesis; skeletal muscle; myoblast proliferation.
11.  Fetal Growth Is Associated with Maternal Fasting Plasma Glucose at First Prenatal Visit 
PLoS ONE  2014;9(12):e116352.
Aims
Fasting plasma glucose (FPG) concentration measured at the first prenatal visit is a predictor of gestational diabetes mellitus (GDM); however, whether this test is indicative of fetal growth has not been clarified. Thus, the purpose of this study was to determine whether birth weight and birth length were related to FPG levels at the first prenatal visit.
Materials and Methods
Research samples were collected from pregnant women who took an FPG test at their first prenatal visit (10–24 gestational weeks), received regular prenatal care, and delivered in our center. FPG value, maternal pre-gravid BMI, weight gain before FPG test, before and after Oral Glucose Tolerance Test (OGTT), neonatal birthweight, birth length, Ponderal Index and birthing method were recorded for analysis. Data were analyzed by independent sample t test, Pearson correlation, and Chi-square test, followed by partial correlation or logistic regression to confirm differences. Statistical significance level was α = 0.05.
Results
2284 pregnant women, including 462 GDM and 1822 with normal glucose tolerance (NGT) were recruited for the present study. FPG concentration at the first prenatal visit was associated with neonatal birth weight (partial correlation coefficient r′ = 0.089, P<0.001) and birth length (partial correlation coefficient r′ = 0.061, P = 0.005), but not with Ponderal Index or birthing method. Maternal pre-gravid BMI was associated with FPG value (partial correlation coefficient r′ = 0.113, P<0.001). FPG concentration at the first prenatal visit (OR = 2.945, P<0.001), weight gain before OGTT test (OR = 1.039, P = 0.010), and age (OR = 1.107, P<0.001) were independent related factors of GDM.
Conclusion
Fasting plasma glucose concentration at the first prenatal visit is associated with fetal growth. Maternal pre-gravid BMI and weight gain are related to glucose metabolism.
doi:10.1371/journal.pone.0116352
PMCID: PMC4281143  PMID: 25551824
12.  Influence of morphology and hemodynamic factors on rupture of multiple intracranial aneurysms: matched-pairs of ruptured-unruptured aneurysms located unilaterally on the anterior circulation 
BMC Neurology  2014;14(1):253.
Background
The authors evaluated the impact of morphological and hemodynamic factors on the rupture of matched-pairs of ruptured-unruptured intracranial aneurysms on one patient’s ipsilateral anterior circulation with 3D reconstruction model and computational fluid dynamic method simulation.
Methods
20 patients with intracranial aneurysms pairs on the same-side of anterior circulation but with different rupture status were retrospectively collected. Each pair was divided into ruptured-unruptured group. Patient-specific models based on their 3D-DSA images were constructed and analyzed. The relative locations, morphologic and hemodynamic factors of these two groups were compared.
Results
There was no significant difference in the relative bleeding location. The morphological factors analysis found that the ruptured aneurysms more often had irregular shape and had significantly higher maximum height and aspect ratio. The hemodynamic factors analysis found lower minimum wall shear stress (WSSmin) and more low-wall shear stress-area (LSA) in the ruptured aneurysms than that of the unruptured ones. The ruptured aneurysms more often had WSSmin on the dome.
Conclusions
Intracranial aneurysms pairs with different rupture status on unilateral side of anterior circulation may be a good disease model to investigate possible characteristics linked to rupture independent of patient characteristics. Irregular shape, larger size, higher aspect ratio, lower WSSmin and more LSA may indicate a higher risk for their rupture.
doi:10.1186/s12883-014-0253-5
PMCID: PMC4301794  PMID: 25551809
Intracranial aneurysms; Multiple aneurysm; Anterior circulation; Rupture; Computational fluid dynamics
13.  CCR7 Expressing Mesenchymal Stem Cells Potently Inhibit Graft-versus-Host Disease by Spoiling the Fourth Supplemental Billingham’s Tenet 
PLoS ONE  2014;9(12):e115720.
The clinical acute graft-versus-host disease (GvHD)-therapy of mesenchymal stem cells (MSCs) is not as satisfactory as expected. Secondary lymphoid organs (SLOs) are the major niches serve to initiate immune responses or induce tolerance. Our previous study showed that CCR7 guide murine MSC line C3H10T1/2 migrating to SLOs. In this study, CCR7 gene was engineered into murine MSCs by lentivirus transfection system (MSCs/CCR7). The immunomodulatory mechanism of MSCs/CCR7 was further investigated. Provoked by inflammatory cytokines, MSCs/CCR7 increased the secretion of nitric oxide and calmed down the T cell immune response in vitro. Immunofluorescent staining results showed that transfused MSCs/CCR7 can migrate to and relocate at the appropriate T cell-rich zones within SLOs in vivo. MSCs/CCR7 displayed enhanced effect in prolonging the survival and alleviating the clinical scores of the GvHD mice than normal MSCs. Owing to the critical relocation sites, MSCs/CCR7 co-infusion potently made the T cells in SLOs more naïve like, thus control T cells trafficking from SLOs to the target organs. Through spoiling the fourth supplemental Billingham’s tenet, MSCs/CCR7 potently inhibited the development of GvHD. The study here provides a novel therapeutic strategy of MSCs/CCR7 infusion at a low dosage to give potent immunomodulatory effect for clinical immune disease therapy.
doi:10.1371/journal.pone.0115720
PMCID: PMC4280136  PMID: 25549354
14.  Abnormal Pulmonary Function and Respiratory Muscle Strength Findings in Chinese Patients with Parkinson’s Disease and Multiple System Atrophy–Comparison with Normal Elderly 
PLoS ONE  2014;9(12):e116123.
Background
There have been limited comparative data regarding the investigations on pulmonary and respiratory muscle function in the patients with different parkinsonism disorders such as Parkinson’s disease (PD) and multiple system atrophy (MSA) versus normal elderly. The present study is aiming to characterize the performance of pulmonary function and respiratory muscle strength in PD and MSA, and to investigate the association with severity of motor symptoms and disease duration.
Methods
Pulmonary function and respiratory muscle strength tests were performed in 30 patients with PD, 27 with MSA as well as in 20 age-, sex-, height-, weight-matched normal elderly controls. All the patients underwent United Parkinson’s disease rating scale (UPDRS) or united multiple system atrophy rating scale (UMSARS) separately as diagnosed.
Results
Vital capacity, forced expiratory volume in 1 second and forced vital capacity decreased, residual volume and ratio of residual volume to total lung capacity increased in both PD and MSA groups compared to controls (p<0.05). Diffusing capacity was decreased in the MSA group, compared with PD and normal elderly control groups (p<0.05). Respiratory muscle strength was lower in both PD and MSA groups than in controls (p<0.05). The values representing spirometry function and respiratory muscle strength were found to have a negative linear correlation with mean score of UPDRS-III in PD and mean score of UMSARS-I in MSA. Respiratory muscle strength showed a negative linear correlation with the mean score of UMSARS-II and disease duration in MSA patients.
Conclusions
These findings suggest that respiratory dysfunction is involved in PD and MSA. Respiratory muscle strength is remarkably reduced, and some of the parameters correlate with disease duration and illness severity. The compromised respiratory function in neurodegenerative disorders should be the focus of further researches.
doi:10.1371/journal.pone.0116123
PMCID: PMC4278864  PMID: 25546308
15.  Influenza Surveillance and Incidence in a Rural Area in China during the 2009/2010 Influenza Pandemic 
PLoS ONE  2014;9(12):e115347.
Background
Most influenza surveillance is based on data from urban sentinel hospitals; little is known about influenza activity in rural communities. We conducted influenza surveillance in a rural region of China with the aim of detecting influenza activity in the 2009/2010 influenza season.
Methods
The study was conducted from October 2009 to March 2010. Real-time polymerase chain reaction was used to confirm influenza cases. Over-the-counter (OTC) drug sales were daily collected in drugstores and hospitals/clinics. Space-time scan statistics were used to identify clusters of ILI in community. The incidence rate of ILI/influenza was estimated on the basis of the number of ILI/influenza cases detected by the hospitals/clinics.
Results
A total of 434 ILI cases (3.88% of all consultations) were reported; 64.71% of these cases were influenza A (H1N1) pdm09. The estimated incidence rate of ILI and influenza were 5.19/100 and 0.40/100, respectively. The numbers of ILI cases and OTC drug purchases in the previous 7 days were strongly correlated (Spearman rank correlation coefficient [r] = 0.620, P = 0.001). Four ILI outbreaks were detected by space-time permutation analysis.
Conclusions
This rural community surveillance detected influenza A (H1N1) pdm09 activity and outbreaks in the 2009/2010 influenza season and enabled estimation of the incidence rate of influenza. It also provides a scientific data for public health measures.
doi:10.1371/journal.pone.0115347
PMCID: PMC4277345  PMID: 25542003
16.  Detection of SRSF2-P95 Mutation by High-Resolution Melting Curve Analysis and Its Effect on Prognosis in Myelodysplastic Syndrome 
PLoS ONE  2014;9(12):e115693.
Hotspot mutations of serine/arginine-rich splicing factor 2 (SRSF2) gene have been identified in a proportion of hematologic malignancies including myelodysplastic syndrome (MDS). The aim of the present study was to develop a new approach to screen SRSF2 mutation and analyze the clinical relevance of SRSF2 mutations in Chinese MDS. A protocol based on high-resolution melting analysis (HRMA) was established to screen SRSF2-P95 mutation in 108 MDS patients and was compared with Sanger sequencing. The clinical relevance of SRSF2 mutations was further evaluated. HRMA identified five (4.6%) cases with SRSF2 mutation, completely validated by Sanger sequencing without false positive or negative results. The sensitivities of HRMA and Sanger sequencing were 10% and 25% for the detection of SRSF2-P95H mutation, respectively, against the background of wild-type DNA. Patients with SRSF2 mutation had shorter overall survival time than those with wild-type SRSF2 in both the whole cohort of cases and those with normal karyotype (P = 0.069 and 0.023, respectively). Multivariate analysis confirmed SRSF2 mutation as an independent risk factor in both patient populations. We established a fast, high-throughput, and inexpensive HRMA-based method to screen SRSF2 mutation, which could be used in clinical diagnostic laboratories. SRSF2 mutations were significantly associated with mortality rate in the MDS affected Chinese.
doi:10.1371/journal.pone.0115693
PMCID: PMC4277410  PMID: 25541999
17.  Mechanisms of Pyrazinamide Action and Resistance 
Microbiology spectrum  2013;2(4):1-12.
PZA is a unique anti-tuberculosis drug that plays a key role in shortening the TB therapy. PZA kills non-replicating persisters that other TB drugs fail to kill, and thus making it an essential drug for inclusion in any drug combinations for treating drug susceptible and drug-resistant TB such as MDR-TB. PZA acts differently from common antibiotics by inhibiting multiple targets such as energy production, trans-translation and perhaps pantothenate /coenzyme A required for persister survival. Resistance to PZA is mostly caused by mutations in the pncA gene encoding pyrazinamidase involved in conversion of the prodrug PZA to the active form POA. Mutations in the drug target RpsA are also found in some PZA-resistant strains. The recent finding that panD mutations are found in some PZA-resistant strains without pncA or rpsA mutations may suggest a third PZA resistance gene and a potential new target of PZA. Current phenotype based PZA susceptibility testing is not reliable due to false resistance, and sequencing of the pncA gene represents a more rapid, cost-effective and more reliable molecular test for PZA susceptibility testing and should be used for guiding improved treatment of MDR/XDR-TB. Finally, the story of PZA has important implications for not only TB therapy but also chemotherapy in general. PZA serves as a model prototype persister drug and hopefully a ‘tipping point’ that inspires new efforts at developing a new type of antibiotics or drugs that target non-replicating persisters for improved treatment of not only TB but also other persistent bacterial infections.
doi:10.1128/microbiolspec.MGM2-0023-2013
PMCID: PMC4268777  PMID: 25530919
18.  A new class of inhibitors of peptide sorption and acylation in PLGA 
Acylation of peptides occurring within controlled-release depots prepared from copolymers of lactic and glycolic acid (PLGA) is a degradation reaction that may compromise product safety and efficacy. As peptide sorption to PLGA is believed to be a common precursor to peptide acylation, a new method to inhibit acylation is presented involving disruptors of peptide sorption, namely, inorganic divalent cations. Kinetics of sorption of a model peptide, octreotide acetate, to free-acid end-group PLGA was monitored in the presence and absence of water-soluble inorganic divalent cationic salts in HEPES buffer solution (pH 7.4, 37 °C). Sorption of cations and octreotide attained pseudo-equilibrium by 24 h. From 24-h sorption isotherms, all cations studied inhibited octreotide sorption to PLGA—the inhibiting effect of the cations increased in the order: Na+ < Mg2+ < Ca2+, Sr2+ < Ni2+ < Mn2+. Long-term inhibition of octreotide sorption in the presence of 15 mM CaCl2 and MnCl2 translated to decreased acylated octreotide present in solution by greater than 50% at 21 days incubation, i.e., from 32% in the cation-free control to 14 and 13% for CaCl2 and MnCl2, respectively. Over one month in vitro release, PLGA implants encapsulating octreotide acetate and CaCl2 or MnCl2 also showed substantial inhibition of acylation relative to no salt or NaCl controls, and similarly, strong inhibition of acylation upon divalent salt incorporation was observed during solvent extrusion of suspended peptide with polar organic carrier solvents. Hence, disrupting peptide sorption to PLGA with addition of inorganic divalent cations is a simple and viable strategy to inhibit acylation of peptides in PLGA delivery systems.
doi:10.1016/j.jconrel.2009.03.006
PMCID: PMC4269232  PMID: 19318114
acylation; octreotide acetate; sorption; divalent cations; PLGA
19.  Recycling Gene Carrier with High Efficiency and Low Toxicity Mediated by L-Cystine-Bridged Bis(β-cyclodextrin)s 
Scientific Reports  2014;4:7471.
Constructing safe and effective gene delivery carriers is becoming highly desirable for gene therapy. Herein, a series of supramolecular crosslinking system were prepared through host-guest binding of adamantyl-modified low molecular weight of polyethyleneimine with L-cystine-bridged bis(β-cyclodextrin)s and characterized by 1H NMR titration, electron microscopy, zeta potential, dynamic light-scattering, gel electrophoresis, flow cytometry and confocal fluorescence microscopy. The results showed that these nanometersized supramolecular crosslinking systems exhibited higher DNA transfection efficiencies and lower cytotoxicity than the commercial DNA carrier gold standard (25 kDa bPEI) for both normal cells and cancer cells, giving a very high DNA transfection efficiency up to 54% for 293T cells. Significantly, this type of supramolecular crosslinking system possesses a number of enzyme-responsive disulfide bonds, which can be cleaved by reductive enzyme to promote the DNA release but recovered by oxidative enzyme to make the carrier renewable. These results demonstrate that these supramolecular crosslinking systems can be used as promising gene carriers.
doi:10.1038/srep07471
PMCID: PMC4265772  PMID: 25503268
20.  Minimizing acylation of peptides in PLGA microspheres 
The main objective of this study was to characterize and find mechanisms to prevent acylation of therapeutic peptides encapsulated in glucose-star poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres. The effect of addition of divalent cation salts CaCl2, MnCl2 as well as carboxymethyl chitosan (CMCS) on inhibition of acylation of octreotide (Oct), salmon calcitonin (sCT), and human parathyroid hormone (hPTH) was evaluated. Peptide content and integrity inside the degrading microspheres was monitored by reversed-phase high performance liquid chromatography (HPLC) and mass spectrometry during release incubation under physiological conditions. The extent of peptide acylation was strongly inhibited in the formulations containing divalent cations and/or CMCS as excipients, although specific effects were dependent on the specific peptide and excipient combinations. Both inorganic cations improved stability of Oct and hPTH but not sCT. Addition of CMCS alone was ineffective. Combining inorganic cations with CMCS improved stability of Oct and sCT but it had no effect on hPTH stability. The operative stabilization mechanisms are consistent with blocking peptide-PLGA interactions by a) directly competing for PLGA interactions with dications and/or b) increasing peptide affinity in the stabilizer phase within PLGA pores. Hence, inorganic multivalent cations are general stabilizers against peptide acylation, the effect of which may be augmented in certain instances with addition of CMCS.
doi:10.1016/j.jconrel.2012.04.022
PMCID: PMC4261194  PMID: 22546683
Acylation; Stability; Peptide; PLGA; Excipients; Sorption
21.  Inhibition of peptide acylation in PLGA microspheres with water-soluble divalent cationic salts 
Pharmaceutical research  2009;26(8):1986-1994.
Purpose
To test the potential of water-soluble divalent cationic salts to inhibit acylation of octreotide encapsulated in poly(D,L-lactic-co-glycolic acid)-star (PLGA) microspheres.
Methods
The divalent cationic salts, calcium chloride and manganese chloride, previously shown to disrupt peptide sorption, were introduced in PLGA microspheres prepared by the double emulsion-solvent evaporation method. Peptide stability was monitored by reversed-phase high performance liquid chromatography (RP-HPLC) and identified by liquid chromatography coupled with mass spectrometry (LC-MS) during microsphere degradation under physiological conditions for four weeks. Microsphere morphology and salt content were examined by scanning electron microscopy (SEM) and inductively coupled plasma-optical emission spectroscopy (ICP-OES), respectively.
Results
Addition of divalent cationic salts solely to the organic phase provided marginal acylation inhibition. However, upon addition of the salt inhibitors to both the primary emulsion and the outer water phase resulted in improved drug and salt encapsulation efficiency as well as significantly decreased salt leaching and octreotide acylation. After 28 days, the extent of acylation inhibition afforded by divalent cations was > 58% relative to 13 % for the NaCl control group.
Conclusions
Divalent cationic salts are suitable class of stabilizers of peptide acylation in PLGA microspheres and this study provides an important formulation approach to maximize stabilizer potency.
doi:10.1007/s11095-009-9914-2
PMCID: PMC4260456  PMID: 19533307
Peptide stability; Acylation; Sorption; Divalent cations; Controlled release
22.  Bias in Estimation of a Mixture of Normal Distributions 
Estimating parameters in a mixture of normal distributions dates back to the 19th century when Pearson originally considered data of crabs from the Bay of Naples. Since then, many real world applications of mixtures have led to various proposed methods for studying similar problems. Among them, maximum likelihood estimation (MLE) and the continuous empirical characteristic function (CECF) methods have drawn the most attention. However, the performance of these competing estimation methods has not been thoroughly studied in the literature and conclusions have not been consistent in published research. In this article, we review this classical problem with a focus on estimation bias. An extensive simulation study is conducted to compare the estimation bias between the MLE and CECF methods over a wide range of disparity values. We use the overlapping coefficient (OVL) to measure the amount of disparity, and provide a practical guideline for estimation quality in mixtures of normal distributions. Application to an ongoing multi-site Huntington disease study is illustrated for ascertaining cognitive biomarkers of disease progression.
doi:10.4172/2155-6180.1000179
PMCID: PMC4257062  PMID: 25485171
Biomarkers; Disparity index; EM algorithm; Mixtures
23.  RAN-Binding Protein 9 is Involved in Alternative Splicing and is Critical for Male Germ Cell Development and Male Fertility 
PLoS Genetics  2014;10(12):e1004825.
As a member of the large Ran-binding protein family, Ran-binding protein 9 (RANBP9) has been suggested to play a critical role in diverse cellular functions in somatic cell lineages in vitro, and this is further supported by the neonatal lethality phenotype in Ranbp9 global knockout mice. However, the exact molecular actions of RANBP9 remain largely unknown. By inactivation of Ranbp9 specifically in testicular somatic and spermatogenic cells, we discovered that Ranbp9 was dispensable for Sertoli cell development and functions, but critical for male germ cell development and male fertility. RIP-Seq and proteomic analyses revealed that RANBP9 was associated with multiple key splicing factors and directly targeted >2,300 mRNAs in spermatocytes and round spermatids. Many of the RANBP9 target and non-target mRNAs either displayed aberrant splicing patterns or were dysregulated in the absence of Ranbp9. Our data uncovered a novel role of Ranbp9 in regulating alternative splicing in spermatogenic cells, which is critical for normal spermatogenesis and male fertility.
Author Summary
Male fertility depends on successful production of functional sperm. Sperm are produced through spermatogenesis, a process of male germ cell proliferation and differentiation in the testis. Most of the genes involved in spermatogenesis are transcribed and processed into multiple isoforms, which are mainly achieved through alternative splicing. The testis-specific transcriptome, characterized by male germ cell-specific alternative splicing patterns, has been shown to be essential for successful spermatogenesis. However, how these male germ cells-specific alternative splicing events are regulated remains largely unknown. Here, we report that RANBP9 is involved in alternative splicing events that are critical for male germ cell development, and dysfunction of RANBP9 leads to disrupted spermatogenesis and compromised male fertility.
doi:10.1371/journal.pgen.1004825
PMCID: PMC4256260  PMID: 25474150
24.  Optimization of the Ethanol Recycling Reflux Extraction Process for Saponins Using a Design Space Approach 
PLoS ONE  2014;9(12):e114300.
A solvent recycling reflux extraction process for Panax notoginseng was optimized using a design space approach to improve the batch-to-batch consistency of the extract. Saponin yields, total saponin purity, and pigment yield were defined as the process critical quality attributes (CQAs). Ethanol content, extraction time, and the ratio of the recycling ethanol flow rate and initial solvent volume in the extraction tank (RES) were identified as the critical process parameters (CPPs) via quantitative risk assessment. Box-Behnken design experiments were performed. Quadratic models between CPPs and process CQAs were developed, with determination coefficients higher than 0.88. As the ethanol concentration decreases, saponin yields first increase and then decrease. A longer extraction time leads to higher yields of the ginsenosides Rb1 and Rd. The total saponin purity increases as the ethanol concentration increases. The pigment yield increases as the ethanol concentration decreases or extraction time increases. The design space was calculated using a Monte-Carlo simulation method with an acceptable probability of 0.90. Normal operation ranges to attain process CQA criteria with a probability of more than 0.914 are recommended as follows: ethanol content of 79–82%, extraction time of 6.1–7.1 h, and RES of 0.039–0.040 min−1. Most of the results of the verification experiments agreed well with the predictions. The verification experiment results showed that the selection of proper operating ethanol content, extraction time, and RES within the design space can ensure that the CQA criteria are met.
doi:10.1371/journal.pone.0114300
PMCID: PMC4255001  PMID: 25470598
25.  Salinomycin Suppresses PDGFRβ, MYC, and Notch Signaling in Human Medulloblastoma 
Austin journal of pharmacology and therapeutics  2014;2(3):www.austinpublishinggroup.com/pharmacology-therapeutics/fulltext/ajpt-v2-id1020.php.
Medulloblastoma (MB) is the most common childhood brain tumor. Despite improved therapy and management, approximately 30% of patients die of the disease. To search for a more effective therapeutic strategy, the effects of salinomycin were tested on cell proliferation, cell death, and cell cycle progression in human MB cell lines. The results demonstrated that salinomycin inhibits cell proliferation, induces cell death , and disrupts cell cycle progression in MB cells. Salinomycin was also tested on the expression levels of key genes involved in proliferation and survival signaling and revealed that salinomycin down-regulates the expression of PDGFRβ, MYC, p21 and Bcl-2 as well as up-regulates the expression of cyclin A. In addition, the results reveal that salinomycin suppresses the expression of Hes1 and Hes5 in MB cells. Our data shed light on the potential of using salinomycin as a novel therapeutic agent for patients with MB.
PMCID: PMC4251667  PMID: 25478603
Salinomycin; Medulloblastoma; PDGFRβ; MYC; Notch Signaling

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