OBJECTIVE: To assess whether the relationship between abnormal fasting plasma glucose (FPG) levels and patient outcomes holds for both older men and older women with acute myocardial infarction (AMI).
PATIENTS AND METHODS: From April 1, 2004, to October 31, 2006, a total of 2016 consecutive older patients (age ≥65 years) presenting with AMI were screened. Of these patients, 1854 were consecutively enrolled in the study. Patients were categorized into 4 groups: the hypoglycemic group (FPG, ≤90.0 mg/dL [to convert to mmol/L, multiply by 0.0555]; n=443, 23.9%), the euglycemic group (FPG, 90.1-126.0 mg/dL; n=812, 43.8%), the mildly hyperglycemic group (FPG, 126.1-162.0 mg/dL; n=308, 16.6%), and the severely hyperglycemic group (FPG, ≥162.1 mg/dL; n=291, 15.7%). The primary outcomes were rates of in-hospital and 3-year mortality.
RESULTS: Female patients were older and had a higher incidence of diabetes mellitus but lower rates of smoking and use of invasive therapy. Men tended to have a higher frequency of hypoglycemia, whereas women tended to have a higher frequency of hyperglycemia. No significant difference was found in in-hospital (10.9% vs 9.1%; P=.36) or 3-year (24.5% vs 24.5%; P=.99) mortality between male and female patients, and FPG-associated mortality did not vary significantly by sex.
CONCLUSION: An increased FPG level was associated with a relatively higher risk of in-hospital mortality in men but not in women. Nonetheless, increased and decreased FPG levels at admission could predict higher mortality rates regardless of sex. There was a striking U-shaped relationship between FPG levels and in-hospital and 3-year mortality. The effect of abnormal FPG level on outcomes among older patients with AMI did not vary significantly by sex.
An increased fasting plasma glucose level was associated with a relatively higher risk of in-hospital mortality in men but not in women. Nonetheless, increased and decreased fasting plasma glucose levels at admission could predict higher mortality rates regardless of sex.
A highly divergent human papillomavirus (HPV) strain, HPV-L55, was identified in fecal samples from children hospitalized with diarrhea in China. The L1 gene of HPV-L55 shares <75% identity with previously reported HPVs, indicating that this virus represents a novel type of HPV. Phylogenetic analysis classified this virus as a member of the gammapapillomaviruses.
Inflammation leads to transcriptional downregulation of many hepatic genes, particulary those activated by RXRα-heterodimers. Inflammation-mediated reduction of nuclear RXRα levels is a main factor in reduced nuclear receptor (NR)–regulated hepatic gene expression, eventually leading to cholestasis and liver damage.
To investigate roles for RXRα in hepatic gene expression during inflammation, using two complementary mouse models: ligand–activation of RXRα, and in mice expressing hepatocyte-specific expression of RXRα missing its DNA-binding-domain (DBD; hs-RxrαΔex4−/−)
To activate RXRα, mice were gavage-fed with LG268 or vehicle for 5 days. To inhibit RXRα function, hs-RxrαΔex4−/− were used. All mice were IP-injected with LPS or saline for 16 hrs prior to analysis of hepatic RNA, protein and NR-DNA binding.
LG268-treatment attenuated the LPS-mediated reductions of several RXRα-regulated genes, coinciding with maintained RXRα occupancy in both Bsep and Ostβ promoters. Lacking full hepatocyte-RXRα function (hs-RxrαΔex4−/− mice) led to enhancement of LPS-mediated changes in gene expression, but surprisingly, maintenance of RNA levels of some RXRα-regulated genes. Investigations revealed that Hs-Rxrα−/− hepatocytes expressed an internally-truncated, ~44 kDa, RXRα-form. DNA-binding capacity of NR-heterodimers was equivalent in wt and hs-RxrαΔex4−/− livers, but reduced by LPS in both. ChIP-QPCR revealed reduced RXRα occupancy to the Bsep RXRα:FXR site was reduced, but not absent, in hs-RxrαΔex4−/− livers.
There are differential regulatory roles for hepatic RXRα, both in basal and inflammatory states, suggesting new and complex multi-domain roles for RXRα in regulating hepatic gene expression. Moreover, there is an unexpected non-obligate role for the DBD of RXRα.
liver; inflammation; nuclear receptor; RXRa; mouse; LG268; rexinoid; gene expression; DNA-binding
Primary liver cancer and liver metastases are among the most frequent malignancies worldwide, with an increasing number of new cases and deaths every year. Traditional surgery is only suitable for a limited proportion of patients and imaging-guided percutaneous thermal ablation has achieved optimistic results for management of hepatic malignancy. This synopsis outlines the first clinical practice guidelines for ultrasound-guided percutaneous microwave ablation therapy for hepatic malignancy, which was created by a joint task force of the Society of Chinese Interventional Ultrasound. The guidelines aim at standardizing the microwave ablation procedure and therapeutic efficacy assessment, as well as proposing the criteria for the treatment candidates.
Practice guidelines; Microwave radiation; Catheter ablation; Liver cancer; Ultrasound
Electrical impedance is one of the most frequently used parameters for characterizing material properties. The resistive and capacitive characteristics of tissue may be revealed by electrical impedance spectroscopy (EIS) as electrical biopsy. This technique could be used to monitor the sequelae after irradiation. In this study, rat intestinal tissues after irradiation were assessed by EIS system based on commercially available integrated circuits. The EIS results were fitted to a resistor-capacitor circuit model to determine the electrical properties of the tissue. The variations in the electrical characteristics of the tissue were compared to radiation injury score (RIS) by morphological and histological findings. The electrical properties, based on receiver operation curve (ROC) analysis, strongly reflected the histological changes with excellent diagnosis performance. The results of this study suggest that electrical biopsy reflects histological changes after irradiation. This approach may significantly augment the evaluation of tissue after irradiation. It could provide rapid results for decision making in monitoring radiation sequelae prospectively.
To evaluate neurotrophin (NT) expression in the endometrium of women with and without endometriosis
Prospective, cross-sectional, translational study
Thirty-three reproductive age women undergoing laparoscopy for infertility, pelvic pain, intramural fibroids, or tubal ligation
Endometrial biopsies, protein microarrays, RT-PCR, ELISAs and Western blotting
Main Outcome Measures
Neurotrophin proteins and mRNAs in eutopic endometrial biopsies
Among seven neurotrophic proteins detected on the antibody microarrays, RT-PCR analysis confirmed nerve growth factor (NGF), NT-4/5, and brain-derived neurotrophic factor (BDNF) mRNAs in endometrial tissue. Quantitative ELISAs revealed that NT-4/5 (806 ± 701 vs. 256 ± 190 pg/100 mg protein, P=0.04) and BDNF (121 ± 97 vs. 14 ± 11 ng/100 mg protein, P<0.01) concentrations were significantly higher in women with endometriosis. NGF (100 ± 74 vs. 93 ± 83 pg/100 mg protein) levels did not differ between cases and controls (P=0.83).
Neurotrophins are synthesized in situ within the endometrium. NT-4/5 and BDNF proteins were more concentrated in biopsies from endometriosis cases than controls, whereas NGF levels were similar. We hypothesize that the local production of NTs induces sensory innervation of endometrium of women with endometriosis. These NTs represent novel targets for the diagnosis and treatment of endometriosis.
nerves; endometriosis; proteomics; endometrium; pro-neurotrophins
The NEDD8 activating enzyme (NAE) is upstream of the 20S proteasome in the ubiquitin/proteasome pathway and catalyzes the first step in the neddylation pathway. NEDD8 modification of cullins is required for ubiquitination of cullin-ring ligases (CRLs), which regulate degradation of a distinct subset of proteins. The more targeted impact of NAE on protein degradation prompted us to study MLN4924, an investigational NAE inhibitor, in preclinical multiple myeloma (MM) models. In vitro treatment with MLN4924 led to dose-dependent decrease of viability (EC50=25–150nM) in a panel of human MM cell lines. MLN4924 was similarly active against a bortezomib-resistant ANBL-6 subline and its bortezomib-sensitive parental cells. MLN4924 had sub-μM activity (EC50 values <500nM) against primary CD138+ MM patient cells and exhibited at least additive effect when combined with dexamethasone, doxorubicin and bortezomib against MM.1S cells. The bortezomib-induced compensatory up-regulation of transcripts for ubiquitin/proteasome was not observed with MLN4924 treatment, suggesting distinct functional roles of NAE vs 20S proteasome. MLN4924 was well tolerated at doses up to 60mg/kg 2x daily and significantly reduced tumor burden in both a subcutaneous and an orthotopic mouse model of MM. These studies provide the framework for the clinical investigation of MLN4924 in MM.
myeloma; cancer; NEDD8; NAE; microenvironment
Chaihu-Shugan-San (CSS) is a traditional Chinese herbal formula that is widely used for treating perimenopausal symptoms in China; however, its mechanisms remain unknown. The present study was designed to investigate potential CSS mechanisms in rats with unpredicted chronic mild stress (UCMS) and normally aging rats (52 weeks of age). We performed the sucrose consumption test along with the forced swimming test to confirm depression-like behavior and the open field test (OFT) to confirm anxiety-like behavior in the animals. In addition, we used an enzyme-linked immunosorbent assay to measure serum and hippocampal estradiol (E2) levels and a quantitative real-time polymerase chain reaction to assess hippocampal mRNA levels of estrogen receptors (ERs) α and β as well as G protein-coupled receptor 30 (GPR30). We found that CSS administration resulted in a significant increase in the ratio of hippocampal ERα and ERβ mRNA (ERα/ERβ ratio) in UCMS rats (p<0.001). However, no significant changes were observed in E2 levels, ERα mRNA expression, and GPR30 mRNA expression. In contrast, changes in ERα/ERβ mRNA ratio were sensitively associated with changes in mood states in the animal models. These findings suggest that enhancement of ERα/ERβ ratio may play a role in the pharmacological mechanisms of CSS. Furthermore, this ratio can be employed as a potential index for evaluating mood states in animal models and can be considered as a therapeutic target for perimenopausal anxiety and depression in the future.
In this study, we describe a novel porcine parechovirus-like virus (tentatively named PLV-CHN) from healthy piglets in China using 454 high-throughput sequencing. The complete genome of the virus comprises 6832 bp, encoding a predicted polyprotein of 2132 amino acids that is most similar to Ljungan virus (32% identity). A similar virus that belongs to a novel Picornaviridae genus, named swine pasivirus 1 (SPaV-1), was reported during the preparation of this paper. Sequence analysis revealed that PLV-CHN and SPaV1 shared 82% nucleotide identity and 89% amino acid identity. Further genomic and phylogenetic analyses suggested that both SPaV1 and PLV-CHN shared similar genomic characteristics and belong to the same novel Picornaviridae genus. A total of 36 (20.0%) fecal samples from 180 healthy piglets were positive for PLV-CHN by RT-PCR, while no fecal samples from 100 healthy children and 100 children with diarrhea, and no cerebrospinal fluid samples from 196 children with suspected viral encephalitis, was positive for the virus. However, Western blot and enzyme-linked immunosorbent assays using recombinant PLV-CHN VP1 polypeptide as an antigen showed a high seroprevalence of 63.5% in the healthy population. When grouped by age, the antibody-positivity rates showed that the majority of children under 12 years of age have been infected by the virus. It was suggested that PLV-CHN, SPaV1, or an as-yet-uncharacterized virus can infect humans early in life. Thus, investigation of the role of this novel virus is vital.
Clinical trials on Traditional Chinese Medicine (TCM) should be registered in a publicly accessible international trial register and report on all outcomes. We systematically assessed and evaluated TCM trials in registries with their subsequent publications.
To describe the characteristics of TCM trials, estimate bias risk and outcome-reporting bias in clinical trials.
Data sources and study selection
Fifteen trial registries were searched from their inception to July 2012 to identify randomised trials on TCM including Chinese herbs, acupuncture and/or moxibustion, cupping, tuina, qigong, etc.
We extracted data including TCM specialty and treated disease/conditions from the registries and searched for subsequent publications in PubMed and Chinese databases. We compared information in the registries of completed trials with any publications focusing on study design, sample size, randomisation, bias risk including reporting bias from the register protocol.
1096 registered randomised trials were identified evaluating TCM, of which 505 were completed studies (46.1%). The most frequent conditions were pain (13.3%), musculoskeletal (11.7%), nervous (8.7%), digestive (7.1%), circulatory (6.5%), respiratory (6.3%), mental and behavioural disorders (6.2%) and cancer (6.0%). The trial register data identified parallel, phase II/III randomised trials with sample size estimations and blinding, but limited information about randomisation (sequence generation and allocation concealment). Comparing trial registration data of 115 completed trials (22.8%) with their subsequent 136 publications, inconsistencies were identified in one or more of the following: sample size (11%), outcome assessor blinding (37.5%), primary outcomes (29%) and safety (28%) reporting.
Increasing numbers of clinical trials investigating a variety of TCM interventions have been registered in international trial registries. The study design of registered TCM trials has improved in estimating sample size, use of blinding and placebos. However, selective outcome reporting is widespread and similar to conventional medicine and therefore study conclusions should be interpreted with caution.
Cardiac lipomatous metaplasia (LM) occurs in patients with chronic ischemic heart disease and heart failure with unclear mechanisms. We studied coronary occlusion/reperfusion-induced myocardial infarction (MI) in rabbits during a 9-months follow-up using 3.0 T magnetic resonance scanner, and confirmed the presence of MI in acute phase and LM in chronic phase using histopathology.
MI was surgically induced in 10 rabbits by 90-min coronary artery occlusion and reperfusion. Forty-eight hours later, multiparametric cardiac magnetic resonance imaging (cMRI) was performed at a 3.0 T clinical scanner for MI diagnosis and cardiac function analysis. Afterwards, seven rabbits were scarified for histochemical staining with triphenyltetrazolium chloride (TTC), and hematoxylin-eosin (HE), and 3 were scanned with cMRI at 2 days, 2 weeks, 2 months and 9 months for longitudinal observations of morphological and functional changes, and the fate of the animals. Post-mortem TTC, HE and Masson's trichrome (MTC) were studied for chronic stage of MI.
The size of acute MI correlated well between cMRI and TTC staining (r2=0.83). Global cardiac morphology-function analysis showed significant correlation between increasing acute MI size and decreasing ejection fraction (p<0.001). During 9 months, cMRI documented evolving morphological and functional changes from acute MI to chronic scar transformation and fat deposition with a definite diagnosis of LM established by histopathology.
Acute MI and chronic LM were induced in rabbits and monitored with 3.0 T MRI. Studies on this platform may help investigate the mechanisms and therapeutic interventions for LM.
Acute myocardial infarction; Animal model; Chronic ischemia; Cardiovascular magnetic resonance imaging; Lipomatous metaplasia
Reduction of glutamine synthetase (GS) function is closely related to established epilepsy, but little is known regarding its role in epileptogenesis. The present study aimed to elucidate the functional changes of GS in the brain and its involvement in epileptogenesis using the amygdala kindling model of epilepsy induced by daily electrical stimulation of basolateral amygdala in rats. Both expression and activity of GS in the ipsilateral dentate gyrus (DG) were upregulated when kindled seizures progressed to stage 4. A single dose of L-methionine sulfoximine (MSO, in 2 µl), a selective GS inhibitor, was administered into the ipsilateral DG on the third day following the first stage 3 seizure (just before GS was upregulated). It was found that low doses of MSO (5 or 10 µg) significantly and dose-dependently reduced the severity of and susceptibility to evoked seizures, whereas MSO at a high dose (20 µg) aggravated kindled seizures. In animals that seizure acquisition had been successfully suppressed with 10 µg MSO, GS upregulation reoccurred when seizures re-progressed to stage 4 and re-administration of 10 µg MSO consistently reduced the seizures. GLN at a dose of 1.5 µg abolished the alleviative effect of 10 µg MSO and deleterious effect of 20 µg MSO on kindled seizures. Moreover, appropriate artificial microRNA interference (1 and 1.5×106 TU/2 µl) of GS expression in the ipsilateral DG also inhibited seizure progression. In addition, a transient increase of GS expression and activity in the cortex was also observed during epileptogenesis evoked by pentylenetetrazole kindling. These results strongly suggest that a transient and region-specific upregulation of GS function occurs when epilepsy develops into a certain stage and eventually promotes the process of epileptogenesis. Inhibition of GS to an adequate degree and at an appropriate timing may be a potential therapeutic approach to interrupting epileptogenesis.
We isolated human epidermis-derived mesenchymal stem cell-like pluripotent cells (hEMSCPCs) and demonstrate efficient harvesting, maintenance in vitro for at least 30 passages, reprogramming into multiple phenotypes in vivo, and integration into adult host tissues after injection into the mouse blastocyst to create chimeras. Cell phenotype was examined by karyotyping, immunostaining, immunofluorescence, and flow cytometry. A nested PCR protocol using primers specific for human SRY genes was designed to detect hEMSCPC-derived cells in female chimeric mice. FISH was used to validate the results of nested PCR. Results indicated that hEMSCPCs were derived from epidermis but were distinct from epidermal cells; they resembled mesenchymal stem cells (MSCs) morphologically and expressed the main markers of MSCs. About half of all female offspring of mice implanted with embryos injected with hEMSCPCs at the blastocyst stage harbored the human Y chromosome and tissue-specific human protein, thereby demonstrating the transdifferentiation of hEMSCPCs.
To evaluate the association of coronary artery endothelial function and plasma levels of low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) in patients with Type 2 Diabetes Mellitus (DM).
We investigated 90 participants from our institution between October 2007 to March 2010: non-DM (n = 60) and DM (n = 30). As an indicator of coronary endothelial dysfunction, we used non-invasive Doppler echocardiography to quantify coronary flow velocity reserve (CFVR) in the distal part of the left descending artery after rest and after intravenous adenosine administration.
Plasma level of LDL-C was significantly higher in patients with DM than in non-DM (3.21 ± 0.64 vs. 2.86 ± 0.72 mmo/L, P < 0.05), but HDL-C level did not differ between the groups (1.01 ± 0.17 vs. 1.05 ± 0.19 mmo/L). Furthermore, the CFVR value was lower in DM patients than non-diabetics (2.45 ± 0.62 vs. 2.98 ± 0.68, P < 0.001). Plasma levels of LDL-C were negatively correlated with CFVR in all subjects (r = −0.35, P < 0.001; 95% confidence interval (CI): −0.52 – −0.15) and in the non-DM (r = −0.29, P < 0.05; 95% CI: −0.51– −0.05), with an even stronger negative correlation in the DM group (r = −0.42, P < 0.05; 95% CI: −0.68 – −0.06). Age (β = −0.019, s = 0.007, sβ = −0.435, 95% CI: −0.033 – −0.005, P = 0.008), LDL-C (β = −0.217, s = 0.105, sβ = −0.282, 95% CI: −0.428 – −0.005, P = 0.045) remained independently correlated with CFVR in the DM group. However, we found no correlation between HDL-C level and CFVR in any group.
Diabetes may contribute to coronary artery disease (CAD) by inducing dysfunction of the coronary artery endothelium. Increased LDL-C level may adversely impair coronary endothelial function in DM. HDL-C may lose its endothelial-protective effects, in part as a result of pathological conditions, especially under abnormal glucose metabolism.
Coronary flow velocity reserve; Low-density lipoprotein cholesterol; Endothelial function; Diabetes mellitus; High-density lipoprotein cholesterol
The immunosuppressive agent cyclosporin A has been proven to reduce the rejection rate and prolong the survival time of transplanted hearts. But some reports showed that cyclosporine A did not completely suppress the rejection. We performed in vitro studies to model a time course to observe the effect of cyclosporin A. Methods: The experiment was divided into a control group (group I), an antigen group (group II), a cyclosporin A group (group III) and an antigen + cyclosporin A group (group IV). After transplantation, at 2 h, 6 h, 12 h, 24 h, 48 h and 72 h, leukocyte molecules were monitored. Results: The expression of IL-2R peaked at 12 h in group II and at 6 h in group III. There was a gradual decline in the expression of the P59 gene in group I, positive expression at 2 h and between 12 h and 24 h in group II, in group IV, there was a decrease at 48 h. The expression of the CD4 gene was lowest at 2 h in group I and at 6 h in group II. CD4 expression then quickly increased to a maximum at 48 h in group III, at 2 h in group IV. There was a minimal expression was reached at 12 h in group I and IV and at 6 h in group III in the expression of the CD8 gene. Conclusions: Alloantigen induced lymphocytes to release IL-2R and P59 and stimulated the induction of the CD4 gene’ transcription for 6 h. Cyclosporin A stimulated the release of IL-2R for 2 h. These results provide an in vitro basis for describing the time phases of rejection inhibited by cyclosporin A.
Transplantation rejection model in vitro; early expression’ time phase; cyclosporin A
Corrinoids are cobalt-containing molecules that function as enzyme cofactors in a wide variety of organisms but are produced solely by a subset of prokaryotes. Specific corrinoids are identified by the structure of their axial ligands. The lower axial ligand of a corrinoid can be a benzimidazole, purine, or phenolic compound. Though it is known that many organisms obtain corrinoids from the environment, the variety of corrinoids that can serve as cofactors for any one organism is largely unstudied. Here, we examine the range of corrinoids that function as cofactors for corrinoid-dependent metabolism in Dehalococcoides mccartyi strain 195. Dehalococcoides bacteria play an important role in the bioremediation of chlorinated solvents in the environment because of their unique ability to convert the common groundwater contaminants perchloroethene and trichloroethene to the innocuous end product ethene. All isolated D. mccartyi strains require exogenous corrinoids such as vitamin B12 for growth. However, like many other corrinoid-dependent bacteria, none of the well-characterized D. mccartyi strains has been shown to be capable of synthesizing corrinoids de novo. In this study, we investigate the ability of D. mccartyi strain 195 to use specific corrinoids, as well as its ability to modify imported corrinoids to a functional form. We show that strain 195 can use only specific corrinoids containing benzimidazole lower ligands but is capable of remodeling other corrinoids by lower ligand replacement when provided a functional benzimidazole base. This study of corrinoid utilization and modification by D. mccartyi provides insight into the array of strategies that microorganisms employ in acquiring essential nutrients from the environment.
In the title complex, [Cu(C22H17Br2N2O2)2], the CuII ion is four-coordinated in a trans-CuN2O2 square-planar geometry by two phenolate O and two imino N atoms from two deprotonated N,O-bidentate ligands. In the crystal, the packing of the molecules is controlled by C—H⋯π and π–π interactions [centroid–centroid distances = 3.568 (3), 3.678 (2), 3.717 (3) and 3.799 (2) Å] and weak Br⋯Br halogen bonds [3.508 (4) Å], linking the molecules into an infinite three-dimensional supramolecular network.
It is well-established that lipid disorder is an important cardiovascular risk factor, and failure to reach optimal lipid levels significantly contributes to the residual cardiovascular risks. However, limited information is available on the management and the attainment of recommended cholesterol targets in real-world practice in China.
Methods and Results
A nationally representative sample of 12,040 patients with dyslipidemia from 19 provinces and 84 hospitals across China were consecutively enrolled in this survey. Risk stratification and individual cholesterol target was established for all participants. This survey identified a high-risk cohort, with over 50% of patients had hypertension, 37.5% had coronary artery disease, and more than 30% had peripheral artery disease. Thirty-nine percent of all participants received lipid lowering medications. And the majority of them (94.5%) had statins (42.5% with atorvastatin, 29.0% with simvastatin, and 15.2% with rosuvastatin). However, the overall attainment for low-density lipoprotein cholesterol (LDL-C) target is low (25.8%), especially, in female (22.2%), and in patients with increased body mass index (BMI) (38.3% for BMI<18.5, 28.1% for BMI 18.5–24.9, 26.0% for BMI 25.0–29.9, and 17.4% for BMI≥30, P<0.0001). Subgroup analysis also showed the attainment is significantly lower in patients who were stratified into high (19.9%) and very high (21.1%) risk category. In logistic regression analysis, eight factors (BMI, gender, coronary artery disease, systolic and diastolic blood pressure, hypertension, family history of premature coronary artery disease and current smoking) were identified as independent predictors of LDL-C attainment.
Despite the proven benefits of lipid-lowering therapies, current management of dyslipidemia continues to be unsatisfied. A considerable proportion of patients failed to achieve guideline-recommended targets in China, and this apparent treatment gap was more pronounced among patients with increased BMI, higher risk stratification and women.
Bornyl gallate (BG) is a potential drug candidate synthesized by the reaction of two natural products, gallic acid and borneol. Previous studies have strongly suggested that BG is worthy of further investigation due to antioxidant, antiatherosclerosis activities, and obvious activity of stimulating intersegmental vessel growth in zebrafish. This work was designed to elucidate the metabolic profile of BG through analyzing its metabolites in vitro and in vivo by a chromatographic separation coupled with a mass spectrometry. The metabolites of BG were characterized from the rat liver microsome incubation solution, as well as rat urine and plasma after oral administration. Chromatographic separation was performed on an Agilent TC-C18 column (250 mm × 4.6 mm, 5 μm) with gradient elution using methanol and water containing 0.2% (V : V) formic acid as the mobile phase. Metabolites identification involved analyzing the retention behaviors, changes of molecular weights and MS/MS fragment patterns of BG and the metabolites. Five compounds were identified as isomers of hydroxylated BG metabolites in vitro. The major metabolites of BG in rat urine and plasma proved to be BG-O-glucuronide and O-methyl BG-O-glucuronide. The proposed method confirmed to be a reliable and sensitive alternative for characterizing metabolic pathways of BG.
Electrical biopsy illustrates a tissue’s electrical properties by electrical impedance spectroscopy. However, electrical biopsy parameters are different from conventional morphological-based examinations. The correlation between electrical biopsy and the morphological observation has not been checked. Considering the tissue responses to injury, extracellular resistance should be most sensitive with the accumulation of fluid in tissue, and it is expected to increase the ratio of optical low staining area on histological images. In this study, we calculated the ratio of optical low staining area of sampled histological images and compared with the results of electrical biopsy to verify the hypothesis of that the extracellular resistance of electrical biopsy most highly correlates with the ratio of optical low staining area on histological images.
The irradiated intestinal tissues of rats after different latent period were used for study. The sampled tissues were measured by electrical impedance spectroscopy for electrical biopsy and the microscopic images were acquired. The sampled histological images were transformed into the Hue-Saturation-Density (HSD) colour model to decouple the stain density. The ratio of optical low staining area on histological images was computed to quantify the morphological changes. The results were related to the parameters from electrical biopsy according to three element circuit model by Spearman’s rank correlation test.
The ratio of optical low staining area varied as well as the tissue’s electrical parameters. The extracellular resistance (Re) and intracellular resistance (Ri) by electrical biopsy tended to increase with the ratio of low staining area decreasing. The membrane capacitance (Cm) by electrical biopsy tended to increase with the ratio of optical low staining area increasing. The extracellular resistance (Re) of electrical biopsy was the parameter most highly correlated with the ratio of optical low staining area with a correlation coefficient of −0.757 (p < 0.001).
The results of this report confirm the hypothesis and support the idea that electrical biopsy results reflect the changes in tissues seen in conventional histological findings in a sense of conventional histological knowledge, and this approach may have a great potential for augmenting the pathological diagnosis of tissues.
Electrical impedance spectroscopy; Electrical biopsy; Radiation enteropathy; Hue-saturation-density (HSD) transformation; Ratio of low staining area; Extracellular resistance
The main keratinase (kerA) gene from the Bacillus licheniformis S90 was optimized by two codon optimization strategies and expressed in Pichia pastoris in order to improve the enzyme production compared to the preparations with the native kerA gene. The results showed that the corresponding mutations (synonymous codons) according to the codon bias in Pichia pastoris were successfully introduced into keratinase gene. The highest keratinase activity produced by P. pastoris pPICZαA-kerAwt, pPICZαA-kerAopti1 and pPICZαA-kerAopti2 was 195 U/ml, 324 U/ml and 293 U/ml respectively. In addition, there was no significant difference in biomass concentration, target gene copy numbers and relative mRNA expression levels of every positive strain. The molecular weight of keratinase secreted by recombinant P. pastori was approx. 39 kDa. It was optimally active at pH 7.5 and 50°C. The recombinant keratinase could efficiently degrade both α-keratin (keratin azure) and β-keratin (chicken feather meal). These properties make the P. pastoris pPICZαA-kerAopti1 a suitable candidate for industrial production of keratinases.
Fidelity and efficiency of pre-mRNA splicing are critical for generating functional mRNAs, but how such accuracy in 5′ splice site (SS) selection is attained is not fully clear. Through a series of yeast genetic screens, we isolated alleles of prp28 that improve splicing of suboptimal 5′SS substrates, demonstrating that WT-Prp28p proofreads, and consequently rejects, poor 5′SS. Prp28p is thought to facilitate the disruption of 5′SS–U1 snRNA pairing to allow for 5′SS–U6 snRNA pairing in the catalytic spliceosome; unexpectedly, 5′SS proofreading by Prp28p is dependent on competition with the stability of the 5′SS:U6 duplex, but not the 5′SS:U1 duplex. E404K, the strongest prp28 allele containing a mutation located in the linker region between adenosine triphosphatase (ATPase) subdomains, exhibited lower RNA-binding activity and enhanced splicing of suboptimal substrates before first-step catalysis, suggesting that decreased Prp28p activity allows longer time for suboptimal 5′SS substrates to pair with U6 snRNA and thereby reduces splicing fidelity. Residue E404 is critical for providing high splicing activity, demonstrated here in both yeast and Drosophila cells. Thus, the subdomain linker in Prp28p plays important roles both in splicing efficiency across species and in proofreading of 5′SS.
As well known, both natural and synthetic steroidal compounds are powerful endocrine disrupting compounds (EDCs) which can cause reproductive toxicity and affect cellular development in mammals and thus are generally regarded as serious contributors to water pollution. Streptomyces virginiae IBL14 is an effective degradative strain for many steroidal compounds and can also catalyze the C25 hydroxylation of diosgenin, the first-ever biotransformation found on the F-ring of diosgenin.
To completely elucidate the hydroxylation function of cytochrome P450 genes (CYPs) found during biotransformation of steroids by S. virginiae IBL14, the whole genome sequencing of this strain was carried out via 454 Sequencing Systems. The analytical results of BLASTP showed that the strain IBL14 contains 33 CYPs, 7 ferredoxins and 3 ferredoxin reductases in its 8.0 Mb linear chromosome. CYPs from S. virginiae IBL14 are phylogenetically closed to those of Streptomyces sp. Mg1 and Streptomyces sp. C. One new subfamily was found as per the fact that the CYP Svu001 in S. virginiae IBL14 shares 66% identity only to that (ZP_05001937, protein identifer) from Streptomyces sp. Mg1. Further analysis showed that among all of the 33 CYPs in S. virginiae IBL14, three CYPs are clustered with ferredoxins, one with ferredoxin and ferredoxin reductase and three CYPs with ATP/GTP binding proteins, four CYPs arranged with transcriptional regulatory genes and one CYP located on the upstream of an ATP-binding protein and transcriptional regulators as well as four CYPs associated with other functional genes involved in secondary metabolism and degradation.
These characteristics found in CYPs from S. virginiae IBL14 show that the EXXR motif in the K-helix is not absolutely conserved in CYP157 family and I-helix not absolutely essential for the CYP structure, too. Experimental results showed that both CYP Svh01 and CYP Svu022 are two hydroxylases, capable of bioconverting diosgenone into isonuatigenone and β-estradiol into estriol, respectively.
Biotransformation; Cytochrome P450; Ferredoxin; Ferredoxin reductase; Gene sequencing; Secondary metabolism
OBJECTIVE: Differently located tumors of the same origin may exhibit diverse responses to the same therapeutics. To test this hypothesis, we compared the responses of rodent hepatic and subcutaneous engrafts of rhabdomyosarcoma-1 (R1) to a vascular disrupting agent Combretastatin A4 phosphate (CA4P). METHODS: Twelve WAG/Rij rats, each bearing three R1 implanted in the right and left hepatic lobes and subcutaneously in the thoracic region, received CA4P intravenously at 5 mg/kg (n = 6) or solvent (n = 6). Therapeutic responses were compared interindividually and intraindividually among tumors of different sites till 48 hours after injection using in vivo MRI, postmortem digital microangiography, and histopathology. RESULTS: MRI revealed that the subcutaneous tumors (STs) significantly increased in volume than hepatic tumors (HTs) 48 hours after CA4P (P < .05). Relative to vehicle controls and treated group at baseline, necrosis ratio, apparent diffusion coefficient, and enhancement ratio changed slightly with the STs but significantly with HTs (P < .05) after CA4P treatment. Vessel density derived from microangiography was significantly lower in STs compared to HTs without CA4P treatment. CA4P treatment resulted in decreased vessel density in HTs, while it did not affect vessel density in STs. MRI and microangiography outcomes were supported by histopathologic findings. CONCLUSIONS: MRI and microangiography allowed quantitative comparison of therapeutic responses to CA4P in rats with multifocal tumors. The discovered diverse effects of the same drug on tumors of the same origin but different locations emphasize the presence of cancer heterogeneity and the importance of individualization of drug delivery.