Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during post-natal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors, including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feed-forward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals.
PIWI-interacting RNA; piRNA; MIWI; MILI; A-MYB; MYBL1
Trans-splicing, a process involving the cleavage and joining of two separate transcripts, can expand the transcriptome and proteome in eukaryotes. Chimeric RNAs generated by trans-splicing are increasingly described in literatures. The widespread presence of antibiotic resistance genes in natural environments and human intestines is becoming an important challenge for public health. Certain antibiotic resistance genes, such as ampicillin resistance gene (Ampr), are frequently used in recombinant plasmids. Until now, trans-splicing involving recombinant plasmid-derived exogenous transcripts and endogenous cellular RNAs has not been reported. Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) is a key enzyme involved in cellular cholesterol homeostasis. The 4.3-kb human ACAT1 chimeric mRNA can produce 50-kDa and 56-kDa isoforms with different enzymatic activities. Here, we show that human ACAT1 56-kDa isoform is produced from an mRNA species generated through the trans-splicing of an exogenous transcript encoded by the antisense strand of Ampr (asAmp) present in common Ampr-plasmids and the 4.3-kb endogenous ACAT1 chimeric mRNA, which is presumably processed through a prior event of interchromosomal trans-splicing. Strikingly, DNA fragments containing the asAmp with an upstream recombined cryptic promoter and the corresponding exogenous asAmp transcripts have been detected in human cells. Our findings shed lights on the mechanism of human ACAT1 56-kDa isoform production, reveal an exogenous-endogenous trans-splicing system, in which recombinant plasmid-derived exogenous transcripts are linked with endogenous cellular RNAs in human cells, and suggest that exogenous DNA might affect human gene expression at both DNA and RNA levels.
trans-splicing; chimeric RNA; ampicillin resistance gene; recombinant plasmid; ACAT1
To evaluate the therapeutic benefit of 3D-image-guided high-dose-rate intracavitary brachytherapy (3D-image-guided HDR-BT) used as a salvage treatment of intensity modulated radiation therapy (IMRT) in patients with locally persistent nasopharyngeal carcinoma (NPC).
Thirty-two patients with locally persistent NPC after full dose of IMRT were evaluated retrospectively. 3D-image-guided HDR-BT treatment plan was performed on a 3D treatment planning system (PLATO BPS 14.2). The median dose of 16 Gy was delivered to the 100% isodose line of the Gross Tumor Volume.
The whole procedure was well tolerated under local anesthesia. The actuarial 5-y local control rate for 3D-image-guided HDR-BT was 93.8%, patients with early-T stage at initial diagnosis had 100% local control rate. The 5-y actuarial progression-free survival and distant metastasis-free survival rate were 78.1%, 87.5%. One patient developed and died of lung metastases. The 5-y actuarial overall survival rate was 96.9%.
Our results showed that 3D-image-guided HDR-BT would provide excellent local control as a salvage therapeutic modality to IMRT for patients with locally persistent disease at initial diagnosis of early-T stage NPC.
Nasopharyngeal carcinoma; Intensity-modulated radiotherapy; Persistent disease; 3D-image-guided HDR Brachytherapy; Local tumor control
Microsatellite instability (MSI) has been found in a range of human tumors, and little is known of the links between MSI and herpesvirus. In order to investigate the relationship between MSI and Gallid herpesvirus 2 (GaHV-2)-induced lymphoma, fifteen Marek’s disease (MD) lymphomas were analyzed through using 46 microsatellite markers, which were amplified by PCR from DNA specimens of lymphoma and normal muscular tissues from the same chicken. PCR products were evaluated by denaturing polyacrylamide gel electrophoresis for MSI analysis. MSI was proved in all lymphomas, at least in one locus. Thirty of the 46 microsatellite markers had microsatellite alterations. These results suggested that GaHV-2-induced lymphoma in chickens is related to MSI, and this is the first report to demonstrate that MSI is associated with the GaHV-2 induced lymphoma in chicken.
The causal genes for congenital cataract are good candidates for the genetic susceptibility for age-related cataract (ARC). The aim of this study was to investigate association between the polymorphisms in the causal genes for congenital cataract and ARC in a Chinese population. Meanwhile, we performed the replication study for previous identified risk genes for ARC.
We recruited 212 sporadic Han Chinese patients with age-related cataracts (ARC) and 172 normal controls in this study. We analyzed 31 SNPs from 13 genes which mostly possible contributes the progress of ARC in a Chinese population, comprising 212 cataract patients and 172 controls. Polymorphism-spanning fragments were amplified by using the multiplex polymerase chain reaction (PCR) and genotyped using primer extension method in MassARRAY platform. Allelic and haplotypic difference in the frequencies were estimated using the SHEsis software platform. P-value was adjusted by the Bonferroni correction.
There was no difference in the frequencies of the genotype and allele of the all SNPs between the patients with ARC and the controls. In the haplotypic analysis, the haplotypes consisting of rs7154572, rs7150141 and rs12432994 in Kinesin Light Chain 1 Gene (KLC1) showed significant association with ARC (p = 0.000878). A rare haplotype CGT was more frequent in patients (p = 0.000106, and p = 0.00795 after corrected for 75 tests).
Our study provides evidence that the combined effect of three variants within the KLC1 gene may predispose to ARC, but the precise mechanism needs further investigating.
Our previous studies have shown that high-mobility group box 1 (HMGB1) could physically associate with the retinoblastoma (RB) protein via an LXCXE (leucine-X-cysteine-X-glutamic; X=any amino acid) motif. An identical LXCXE motif is present in the HMGB1–3 protein sequences, whereas a near-consensus LXCXD (leucine-X-cysteine-X-asparagine; X=any amino acid) motif is found in the HMGB4 protein. In this study, we have demonstrated that like HMGB1, HMGB2–3 also associated with the RB in vitro and in vivo, as evidenced by glutathione-s-transferase capture and immunoprecipitation–Western blot assays. A point mutation of the LXCXE or LXCXD motif led to disruption of RB:HMGB1–4 interactions. Enforced expression of HMGB1–3 or HMGB4 by adenoviral-vector-mediated gene transfer resulted in significant inhibition of breast cancer cell proliferation through an LXCXE- or LXCXD-dependent mechanism and an increased radiosensitivity through an LXCXE- or LXCXD-independent mechanism. These results suggest an important role of the LXCXE/D motif in RB:HMGB1–4 association and modulation of cancer cell growth, but not radiosensitivity.
breast cancer; biotherapy; cancer; gene therapy; irradiation
Female pattern hair loss (FPHL) is the most common hair loss disorder in women and it may impact on the psychological and social activities of patients, thereby reducing their quality of life (QoL). Topical minoxidil has been shown to be effective and safe in the treatment of patients with FPHL. The aim of this study was to assess the QoL of patients with FPHL and investigate whether topical minoxidil solution treatment improves the QoL of these patients. In this study, we enrolled 125 female patients aged 16–72 years to answer visual analog scale (VAS) and dermatology life quality index (DLQI) questionnaires. Of these patients, 31 were recruited for the follow-up study after 12 months of treatment with 2% minoxidil. Each index and the change in QoL prior to and following treatment were statistically analyzed. There was identified to be a correlation between clinical severity and the values of the indices in all patients. There was a statistically significant difference between the VAS and DLQI scores prior to and following treatment with 2% minoxidil. A comparison between the good responders (n=23) and the poor responders (n=8) revealed no significant difference in the improvement of VAS and DLQI scores. The QoL of the patients was severely impaired by FPHL. The DLQI and VAS used in this study were validated as useful indices for the evaluation of QoL due to their high reliability, sensitivity and simplicity. This evaluation is recommended for the management of FPHL treatment. The results of the study demonstrated that topical minoxidil improved the QoL of the patients.
China; dermatology life quality index; visual analog scale
Nucleosome organization at promoter regions plays an important role in regulating gene activity. Genome-wide studies in yeast, flies, worms, mammalian embryonic stem cells and transformed cell lines have found well-positioned nucleosomes flanking a nucleosome depleted region (NDR) at transcription start sites. This nucleosome arrangement depends on DNA sequence (cis-elements) as well as DNA binding factors and ATP-dependent chromatin modifiers (trans-factors). However, little is understood about how the nascent embryonic genome positions nucleosomes during development. This is particularly intriguing since the embryonic genome must undergo a broad reprogramming event upon fusion of sperm and oocyte. Using four stages of early embryonic zebrafish development, we map nucleosome positions at the promoter region of 37 zebrafish hox genes. We find that nucleosome arrangement at the hox promoters is a progressive process that takes place over several stages. At stages immediately after fertilization, nucleosomes appear to be largely disordered at hox promoter regions. At stages after activation of the embryonic genome, nucleosomes are detectable at hox promoters, with positions becoming more uniform and more highly occupied. Since the genomic sequence is invariant during embryogenesis, this progressive change in nucleosome arrangement suggests that trans-factors play an important role in organizing nucleosomes during embryogenesis. Separating hox genes into expressed and non-expressed groups shows that expressed promoters have better positioned and occupied nucleosomes, as well as distinct NDRs, than non-expressed promoters. Finally, by blocking the retinoic acid-signaling pathway, we disrupt early hox gene transcription, but observe no effect on nucleosome positions, suggesting that active hox transcription is not a driving force behind the arrangement of nucleosomes at the promoters of hox genes during early development.
Drosophila Dicer-1 produces microRNAs (miRNAs) from pre-miRNA, whereas Dicer-2 generates small interfering RNAs (siRNAs) from long dsRNA. Alternative splicing of the loquacious (loqs) mRNA generates three distinct Dicer partner proteins. To understand the function of each, we constructed flies expressing Loqs-PA, Loqs-PB, or Loqs-PD. Loqs-PD promotes both endo- and exo-siRNA production by Dicer-2. Loqs-PA or Loqs-PB is required for viability, but the proteins are not fully redundant: a specific subset of miRNAs requires Loqs-PB. Surprisingly, Loqs-PB tunes where Dicer-1 cleaves pre-miR-307a, generating a longer miRNA isoform with a distinct seed sequence and target specificity. The longer form of miR-307a represses glycerol kinase and taranis mRNA expression. The mammalian Dicer-partner TRBP, a Loqs-PB homolog, similarly tunes where Dicer cleaves pre-miR-132. Thus, Dicer-binding partner proteins change the choice of cleavage site by Dicer, producing miRNAs with target specificities different from those made by Dicer alone or Dicer bound to alternative protein partners.
Epstein-Barr virus (EBV) infection has been associated with lymphoma development. EBV latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation and progression of different human cells, including lymphocytes. This meta-analysis investigated LMP1 expression with prognosis of patients with lymphoma.
The electronic databases of PubMed, Embase, and Chinese Biomedicine Databases were searched. There were 15 published studies available for a random effects model analysis. Quality assessment was performed using the Newcastle-Ottawa Quality Assessment Scale for cohort studies. A funnel plot was used to investigate publication bias, and sources of heterogeneity were identified by meta-regression analysis. The combined hazard ratios (HR) and their corresponding 95% confidence intervals of LMP1 expression were calculated by comparison to the overall survival.
Overall, there was no statistical significance found between LMP1 expression and survival of lymphoma patients (HR 1.25 [95% CI, 0.92–1.68]). In subgroup analyses, LMP1 expression was associated with survival in patients with non-Hodgkin lymphoma (NHL) (HR = 1.84, 95% CI: 1.02–3.34), but not with survival of patients with Hodgkin disease (HD) (HR = 1.03, 95% CI: 0.74–1.44). In addition, significant heterogeneity was present and the meta-regression revealed that the outcome of analysis was mainly influenced by the cutoff value.
This meta-analysis demonstrated that LMP1 expression appears to be an unfavorable prognostic factor for overall survival of NHL patients. The data suggested that EBV infection and LMP1 expression may be an important factor for NHL development or progression.
AIM: To compare the prognostic assessment of lymph node ratio and absolute number based staging system for gastric cancer after D2 resection.
METHODS: The clinical, pathologic, and long-term follow-up data of 427 patients with gastric cancer that underwent D2 curative gastrectomy were retrospectively analyzed. The relationships between the metastatic lymph node ratio (MLR), log odds of positive lymph nodes (LODDS), and positive lymph nodes (pN) staging methods and the long-term prognoses of the patients were compared. In addition, the survival curves, accuracy, and homogeneity were compared with stratification to evaluate the prognostic assessment of the 3 methods when the number of tested lymph nodes was insufficient (< 10 and 10-15).
RESULTS: MLR [hazard ratio (HR) = 1.401, P = 0.012], LODDS (HR = 1.012, P = 0.034), and pN (HR = 1.376, P = 0.005) were independent risk factors for gastric cancer patients. The receiver operating characteristic (ROC) curves showed that the prognostic accuracy of the 3 methods was comparable (P > 0.05). Spearman correlation analysis confirmed that MLR, LODDS, and pN were all positively correlated with the total number of tested lymph nodes. When the number of tested lymph node was < 10, the value of survival curves staged by MLR and LODDS was superior to those of pN staging. However, the difference in survival curves between adjacent stages was not significant. In addition, the survival rate of stage 4 patients using the MLR and LODDS staging methods was 26.7% and 27.3% with < 10 lymph node, respectively which were significantly higher than the survival rate of patients with > 15 tested lymph nodes (< 4%). The ROC curve showed that the accuracy of the prognostic assessment of MLR, LODDS, and pN staging methods was comparable (P > 0.05), and the area under the ROC curve of all 3 methods were increased progressively with the enhanced levels of examined lymph nodes. In addition, the homogeneity of the 3 methods in patients with ≤ 15 tested lymph nodes also showed no significant difference.
CONCLUSION: Neither MLR or LODDS could reduce the staging bias. A sufficient number of tested lymph nodes is key to ensure an accurate prognosis for patients underwent D2 radical gastrectomy.
Gastric cancer; Metastatic lymph node ratio; Lymph node metastasis; Prognosis
Leishmania species belong to the family Trypanosomatidae and cause leishmaniasis, a geographically widespread disease that infects humans and other vertebrates. This disease remains endemic in China. Due to the large geographic area and complex ecological environment, the taxonomic position and phylogenetic relationship of Chinese Leishmania isolates remain uncertain. A recent internal transcribed spacer 1 and cytochrome oxidase II phylogeny of Chinese Leishmania isolates has challenged some aspects of their traditional taxonomy as well as cladistics hypotheses of their phylogeny. The current study was designed to provide further disease background and sequence analysis.
We systematically analyzed 50 cytochrome b (cyt b) gene sequences of 19 isolates (16 from China, 3 from other countries) sequenced after polymerase chain reaction (PCR) using a special primer for cyt b as well as 31 sequences downloaded from GenBank. After alignment, the data were analyzed using the maximum parsimony, Bayesian and netwok methods.
Sequences of six haplotypes representing 10 Chinese isolates formed a monophyletic group and clustered with Leishmania tarentolae. The isolates GS1, GS7, XJ771 of this study from China clustered with other isolates of Leishmania donovani complex. The isolate JS1 was a sister to Leishmania tropica, which represented an L. tropica complex instead of clustering with L. donovani complex or with the other 10 Chinese isolates. The isolates KXG-2 and GS-GER20 formed a monophyletic group with Leishmania turanica from central Asia. In the different phylogenetic trees, all of the Chinese isolates occurred in at least four groups regardless of geographic distribution.
The undescribed Leishmania species of China, which are clearly causative agents of canine leishmaniasis and human visceral leishmaniasis and are related to Sauroleishmania, may have evolved from a common ancestral parasite that came from the Americas and may have split off earlier than the other old world Leishmania. Our results also suggest the following: the isolates GS7, GS1 and XJ771 occur as part of the L. donovani complex; the JS1 isolate is L. tropica; and the isolate GS-GER20 identified as Leishmania gerbilli is close to KXG-2 which is L. turanica.
Leishmania; Phylogeny; cyt b; China; Sauroleishmania
Transposons evolve rapidly and can mobilize and trigger genetic instability. piRNAs silence these genome pathogens, but it is unclear how the piRNA pathway adapts to invasion of new transposons. In Drosophila, piRNAs are encoded by heterochromatic clusters and maternally deposited in the embryo. Paternally inherited P-element transposons thus escape silencing and trigger a hybrid sterility syndrome termed P-M hybrid dysgenesis. We show that P-M hybrid dysgenesis activates both P-elements and resident transposons, and disrupts the piRNA biogenesis machinery. As dysgenic hybrids age, however, fertility is restored, P-elements are silenced, and P-element piRNAs are produced de novo. In addition, the piRNA biogenesis machinery assembles and resident elements are silenced. Significantly, resident transposons insert into piRNA clusters, and these new insertions are transmitted to progeny, produce novel piRNAs, and are associated with reduced transposition. P-element invasion thus triggers heritable changes in genome structure that appear to enhance transposon silencing.
Latent membrane protein (LMP) 1 and LMP2A encoded by Epstein-Barr virus (EBV) are associated with the development of malignancies, but their expression in extranodal NK/T-cell lymphoma, nasal type (ENKTL) and the relationship with clinical characteristics of this disease remain poorly understood. In the present study, we examined the expression of LMP1 and LMP2A in ENKTL, and investigated the correlations between LMP1 and LMP2A expression with clinicopathological characteristics of ENKTL patients.
Paraffin sections of surgically removed samples from 16 ENKTL patients were analyzed by immunohistochemistry and the related clinicopathological data were collected and analyzed.
Elevated expression (immunohistochemistry score ≥ 4) of LMP1 and LMP2A was detected in the tumor cells of ENKTL. High LMP1 expression was associated with positive B symptoms (p = 0.012), while high LMP2A expression was related to gender (p = 0.029). The expression of both LMP1 and LMP2A showed significant correlations with patients’ overall survival (p = 0.049, p = 0.036).
LMP1 and LMP2A may be prognostic indicators of survival in patients with ENKTL.
LMP1; LMP2A; EBV; ENKTL; Prognosis; Tumor marker
Genetic aberrations contribute to acute myeloid leukemia (AML). However, half of AML cases do not contain the well-known aberrations detectable mostly by cytogenetic analysis, and these cases are classified as normal karyotype AML. Different outcomes of normal karyotype AML suggest that this subgroup of AML could be genetically heterogeneous. But lack of genetic markers makes it difficult to further study this subgroup of AML. Using paired-end RNAseq method, we performed a transcriptome analysis in 45 AML cases including 29 normal karyotype AML, 8 abnormal karyotype AML and 8 AML without karyotype informaiton. Our study identified 134 fusion transcripts, all of which were formed between the partner genes adjacent in the same chromosome and distributed at different frequencies in the AML cases. Seven fusions are exclusively present in normal karyotype AML, and the rest fusions are shared between the normal karyotype AML and abnormal karyotype AML. CIITA, a master regulator of MHC class II gene expression and truncated in B-cell lymphoma and Hodgkin disease, is found to fuse with DEXI in 48% of normal karyotype AML cases. The fusion transcripts formed between adjacent genes highlight the possibility that certain such fusions could be involved in oncological process in AML, and provide a new source to identify genetic markers for normal karyotype AML.
β/γ-Crystallins, the major structural proteins in human lens, are highly conserved in their tertiary structures but distinct in the quaternary structures. The N- and C-terminal extensions have been proposed to play a crucial role in mediating the size of β-crystallin assembly. In this research, we investigated the molecular mechanism underlying the congenital hereditary cataract caused by the recently characterized A2V mutation in βB2-crystallin. Spectroscopic experiments indicated that the mutation did not affect the secondary and tertiary structures of βB2-crystallin. The mutation did not affect the formation of βB2/βA3-crystallin heteromer as well as the stability and folding of the heteromer, suggesting that the mutation might not interfere with the protein interacting network in the lens. However, the tetramerization of βB2-crystallin at high protein concentrations was retarded by the A2V mutation. The mutation slightly decreased the thermal stability and promoted the thermal aggregation of βB2-crystallin. Although it did not influence the stability of βB2-crystallin against denaturation induced by chemical denaturants and UV irradiation, the A2V mutant was more prone to be trapped in the off-pathway aggregation process during kinetic refolding. Our results suggested that the A2V mutation might lead to injury of lens optical properties by decreasing βB2-crystallin stability against heat treatment and by impairing βB2-crystallin assembly into high-order homo-oligomers.
This study aimed to identify novel GATA5 mutations that underlie familial atrial fibrillation.
A total of 110 unrelated patients with familial atrial fibrillation and 200 unrelated, ethnically matched healthy controls were recruited. The entire coding region of the GATA5 gene was sequenced in 110 atrial fibrillation probands. The available relatives of the mutation carriers and 200 controls were subsequently genotyped for the identified mutations. The functional effect of the mutated GATA5 was characterized using a luciferase reporter assay system.
Two novel heterozygous GATA5 mutations (p.Y138F and p.C210G) were identified in two of the 110 unrelated atrial fibrillation families. These missense mutations cosegregated with AF in the families and were absent in the 400 control chromosomes. A cross-species alignment of GATA5 protein sequence showed that the altered amino acids were completely conserved evolutionarily. A functional analysis revealed that the mutant GATA5 proteins were associated with significantly decreased transcriptional activation when compared with their wild-type counterpart.
The findings expand the spectrum of GATA5 mutations linked to AF and provide novel insights into the molecular mechanism involved in the pathogenesis of atrial fibrillation, suggesting potential implications for the early prophylaxis and personalized treatment of this common arrhythmia.
Atrial Fibrillation; Transcriptional Factor; GATA5; Genetics; Reporter Gene
Patients with os odontoideum always present instability in atlantoaxial joint and need atlantoaxial fixation. C2 pedicle or laminar screws fixation has proven to be efficient and reliable for atlantoaxial instability. However, os odontoideum is a congenital or developmental disease, featured with anomalous bony anatomies. The anatomic measurements and guidelines for C2 pedicle screw placement in general population tends to differ with those of os odontoideum patients, for whom C2 pedicle screws are often needed. The option and techniques of C2 fixation are still challenging and yet to be fully explored.
Material and methods
We recruited 29 adult patients with os odontoideum and measured the dimension of C2 pedicle and lamina for each patient to examine how well do they match with the screws anatomically. In order to access the intra-observer reliability and inter-observer repeatability of the measurements, the intraclass correlation coefficient (ICC) was also calculated.
The results for reliability of the CT measurements showed excellent intraobserver (ICC = 0.95 and 0.96) and interobserver correlation coefficient (ICC = 0.93). The diameter and length of C2 pedicle were found to be 6.06 ± 1.37 and 24.05 ± 2.54 mm, while the corresponding figures of C2 laminar were 6.95 ± 0.82 and 25.60 ± 2.18 mm, respectively. In the measurements, all 29 cases had suitable diameter (larger than 5.5 mm) for C2 laminar screw (the laminar diameters ranged from 5.52 to 8.82 mm). In C2 pedicle measurements, the diameters of the 29 cases were from 3.50 to 9.86 mm, while 20 pedicles (34.5%) in 14 cases were less than 5.5 mm in diameter. Six had bilateral small pedicles where the diameter was less than 5.5 mm.
Anatomically, we found laminar screw is a better match in comparison with pedicle screw for C2 fixation in os odontoideum. The options for C2 fixation should be made based on careful preoperative imaging and thorough consideration. Preoperative reconstructive CT scan can offer great assistance for the choice of fixation in os odontoideum by revealing the anatomy of the C2 pedicles in detail.
Os odontoideum pedicle screw; Laminar screw; C2 fixation
Hemorrhagic shock (HS) due to major trauma and surgery predisposes the host to the development of systemic inflammatory response syndrome (SIRS) including acute lung injury (ALI) through activating and exaggerating the innate immune response. IL-1β is a crucial pro-inflammatory cytokine that contributes to the development of SIRS and ALI. Lung endothelial cells (EC) are one important source of IL-1β, and the production of active IL-1β is controlled by the inflammasome. In this study, we addressed the mechanism underlying HS activation of the inflammasome in lung EC. We show that high mobility group box 1 (HMGB1) acting through TLR4, and a synergistic collaboration with TLR2 and RAGE signaling, mediates HS-induced activation of EC NAD(P)H oxidase. In turn, reactive oxygen species (ROS) derived from NAD(P)H oxidase promote the association of thioredoxin-interacting protein (TXNIP) with the Nod-like receptor protein NLRP3 and subsequently induce inflammasome activation and IL-1β secretion from the EC. We also show that neutrophil-derived ROS play a role in enhancing EC NAD(P)H oxidase activation, and therefore an amplified inflammasome activation in response to HS. The present study explores a novel mechanism underlying HS activation of EC inflammasome and, thus, presents a potential therapeutic target for SIRS and ALI induced after HS.
IL-1β; NAD(P)H oxidase; Reactive oxygen species; Toll-like receptors
Lactose repressor protein (LacI), a negative transcriptional regulator in Escherichia coli, relies on an allosteric conformational change for its function. The LacI effector isopropyl-β,D-thiogalactoside (IPTG) promotes this allosteric response and engages the side chains of residues N125 and D149 based on the crystallographic structure of LacI·IPTG. Targeted molecular dynamics (TMD) simulations have indicated involvement of these side chains during the protein structural changes in response to inducer binding. To examine this region further, we applied stochastic boundary molecular dynamics (SBMD) simulation and identified a transient interaction between residues N125 and D149. Based on these data, we introduced substitutions for either/both residues and analyzed their impact on protein function. The substitutions utilized were alanine to preclude hydrogen bonding or cysteine to allow disulfide bond formation, which was not observed for N125C/D149C. Minimal impacts were observed on operator affinity for all substitutions, but D149C, N125A/D149A and N125C/D149C bound to IPTG with 5-8 fold lower affinity than wild-type LacI and exhibited decreased allosteric amplitude (KRI/O/KR/O). Of interest, the double mutants did not exhibit an allosteric response to an alternate inducer, 2-phenylethyl-β,D-galactoside (PhEG), despite demonstration of PhEG binding. Further, the presence of the anti-inducer, o-nitrophenyl-β,D-fucoside (ONPF), enhanced operator affinity for wild-type LacI and all other mutant proteins examined, but behaved as an inducer for N125A/D149A, decreasing operator binding affinity. These results confirm the role of residues 125 and 149 in ligand binding and allosteric response and illustrate how readily the function of a regulatory protein can be altered.
LacI allostery; sugar specificity; ligand binding; structure/function
In order to identify immunodominant antigens of Mycobacterium tuberculosis that may be used in the serodiagnosis of active tuberculosis (TB), we designed an M. tuberculosis fusion protein consisting of CFP-10 (10-kDa culture filtrate protein), ESAT-6 (6-kDa early secreted antigenic target), and the extracellular domain fragment of PPE68 (PPE68′). Then, the coding sequences of the three proteins were inserted into a prokaryotic expression vector, pET-32a(+). To enhance the immunological response, the proteins were linked together. The fusion proteins with a 6×His tag were successfully overexpressed in Escherichia coli BL21 and purified. The purified proteins were applied for detection of the total IgG titer by using an enzyme-linked immunosorbent assay (ELISA) with human sera from well-characterized TB cases and the control cases, and results were compared to those with purified protein derivative tuberculin (PPD). The ELISA results showed that among 140 cases of confirmed active TB and 70 control cases, CFP-10–ESAT-6–PPE68′ had a sensitivity of 73.3% and specificity of 94.3%, compared to a sensitivity of 66.7% and specificity of 74.3% for PPD and a sensitivity of 65% and specificity of 91.4% for CFP-10–ESAT-6. In addition, the fusion protein CFP-10–ESAT-6–PPE68′ stimulated a higher level of antigen-specific gamma interferon (IFN-γ) release for active-TB patients than PPD and CFP-10–ESAT-6. After immunization of C57BL/6 mice, the findings indicated that the total IgG titers and the concentrations of IFN-γ in mice immunized by CFP-10–ESAT-6–PPE68′ were high and induced strong, long-term humoral immunity compared to results with PPD and CFP-10–ESAT-6. Thus, our study indicates that the fusion protein CFP-10–ESAT-6–PPE68′ may be useful as an immunodominant antigen for the serodiagnosis of active TB.
The ubiquitously expressed 14-3-3 proteins regulate many pathways involved in transformation. Previously, we found that 14-3-3ζ overexpression increased Akt phosphorylation in human mammary epithelial cells. Here, we investigated the clinical relevance and molecular mechanism of 14-3-3ζ overexpression-mediated Akt phosphorylation and the potential impact on breast cancer progression. We found that 14-3-3ζ overexpression was significantly (P = 0.005) associated with increased Akt phosphorylation in human breast tumors. Additionally, 14-3-3ζ overexpression combined with strong Akt phosphorylation was significantly (P=0.01) associated with increased cancer recurrence in patients. In contrast, knockdown of 14-3-3ζ expression by siRNA in cancer cell lines and tumor xenografts reduced Akt phosphorylation. Furthermore, 14-3-3ζ enhanced Akt phosphorylation through activation of PI3K. Mechanistically, 14-3-3ζ bound to the p85 regulatory subunit of PI3K and increased PI3K translocation to the cell membrane. A single 14-3-3 binding motif encompassing serine 83 on p85 is largely responsible for 14-3-3ζ-mediated p85 binding and PI3K/Akt activation. Mutation of serine 83 to alanine on p85 inhibited 14-3-3ζ binding to the p85 subunit of PI3K, reduced PI3K membrane localization and activation, impeded anchorage independent growth and enhanced stress induced apoptosis. These findings revealed a novel mechanism by which 14-3-3ζ overexpression activates PI3K, a key node in the mitogenic signaling network known to promote malignancies in many cell types.
14-3-3ζ; breast cancer; PI3K; Akt
AIM: To investigate the role of anal cushions in hemorrhoidectomy and its effect on anal continence of the patients.
METHODS: Seventy-six consecutive patients (33 men and 43 women) with a mean age of 44 years were included. They underwent Milligan-Morgan hemorrhoidectomy because of symptomatic third- and fourth-degree hemorrhoids and failure in conservative treatment for years. Wexner score was recorded and liquid continence test was performed for each patient before and two months after operation using the techniques described in our previous work. The speed-constant rectal lavage apparatus was prepared in our laboratory. The device could output a pulsed and speed-constant saline stream with a high pressure, which is capable of overcoming any rectal resistance change. The patients were divided into three groups, group A (< 900 mL), group B (900-1200 mL) and group C (> 1200 mL) according to the results of the preoperative liquid continence test.
RESULTS: All the patients completed the study. The average number of hemorrhoidal masses excised was 2.4. Most patients presented with hemorrhoidal symptoms for more than one year, including a mean duration of incontinence of 5.2 years. The most common symptoms before surgery were anal bleeding (n = 55), prolapsed lesion (n = 34), anal pain (n = 12) and constipation (n = 17). There were grade III hemorrhoids in 39 (51.3%) patients, and grade IV in 37 (48.7%) patients according to Goligher classification. Five patients had experienced hemorrhoid surgery at least once. Compared with postoperative results, the retained volume in the preoperative liquid continence test was higher in 40 patients, lower in 27 patients, and similar in the other 9 patients. The overall preoperative retained volume in the liquid continence test was 1130.61 ± 78.35 mL, and postoperative volume was slightly decreased (991.27 ± 42.77 mL), but there was no significant difference (P = 0.057). Difference was significant in the test value before and after hemorrhoidectomy in group A (858.24 ± 32.01 mL vs 574.18 ± 60.28 mL, P = 0.011), but no obvious difference was noted in group B or group C. There was no significant difference in Wexner score before and after operation (1.68 ± 0.13 vs 2.10 ± 0.17, P = 0.064). By further stratified analysis, there was significant difference before and 2 months after operation in group A (2.71 ± 0.30 vs 3.58 ± 0.40, P = 0.003). In contrast, there were no significant differences in group B or group C (1.89 ± 0.15 vs 2.11 ± 0.19, P = 0.179; 0.98 ± 0.11 vs 1.34 ± 0.19, P = 0.123).
CONCLUSION: There is no difference in the continence status of patients before and after Milligan-Morgan hemorrhoidectomy. However, patients with preoperative compromised continence may have further deterioration of their continence, hence Milligan-Morgan hemorrhoidectomy should be avoided in such patients.
Anal cushion; Anal incontinence; Liquids continence test; Wexner score; Hemorrhoidectomy
Berberine, a major pharmacological component of the Chinese herb Coptis chinensis, which was originally used to treat bacterial diarrhea, has recently been demonstrated to be clinically effective in alleviating type 2 diabetes. In this study, we revealed that berberine effectively prevented the development of obesity and insulin resistance in high-fat diet (HFD)-fed rats, which showed decreased food intake. Increases in the levels of serum lipopolysaccharide-binding protein, monocyte chemoattractant protein-1, and leptin and decrease in the serum level of adiponectin corrected for body fat in HFD-fed rats were also significantly retarded by the co-administration of berberine at 100 mg/kg body weight. Bar-coded pyrosequencing of the V3 region of 16S rRNA genes revealed a significant reduction in the gut microbiota diversity of berberine-treated rats. UniFrac principal coordinates analysis revealed a marked shift of the gut microbiota structure in berberine-treated rats away from that of the controls. Redundancy analysis identified 268 berberine-responding operational taxonomic units (OTUs), most of which were essentially eliminated, whereas a few putative short-chain fatty acid (SCFA)-producing bacteria, including Blautia and Allobaculum, were selectively enriched, along with elevations of fecal SCFA concentrations. Partial least square regression models based on these 268 OTUs were established (Q2>0.6) for predicting the adiposity index, body weight, leptin and adiponectin corrected for body fat, indicating that these discrete phylotypes might have a close association with the host metabolic phenotypes. Taken together, our findings suggest that the prevention of obesity and insulin resistance by berberine in HFD-fed rats is at least partially mediated by structural modulation of the gut microbiota, which may help to alleviate inflammation by reducing the exogenous antigen load in the host and elevating SCFA levels in the intestine.
Imaging biomarkers are useful outcome measures in treatment trials. We compared sample size estimates for future treatment trials performed over 6 or 12-months in progressive supranuclear palsy using both imaging and clinical measures. We recruited 16 probable progressive supranuclear palsy patients that underwent baseline, 6 and 12 month brain scans, and 16 age-matched controls with serial scans. Disease severity was measured at each time-point using the progressive supranuclear palsy rating scale. Rates of ventricular expansion and rates of atrophy of the whole brain, superior frontal lobe, thalamus, caudate and midbrain were calculated. Rates of atrophy and clinical decline were used to calculate sample sizes required to power placebo-controlled treatment trials over 6 and 12-months. Rates of whole brain, thalamus and midbrain atrophy, and ventricular expansion, were increased over 6 and 12-months in progressive supranuclear palsy compared to controls. The progressive supranuclear palsy rating scale increased by 9 points over 6-months, and 18 points over 12-months. The smallest sample size estimates for treatment trials over 6-months were achieved using rate of midbrain atrophy, followed by rate of whole brain atrophy and ventricular expansion. Sample size estimates were further reduced over 12-month intervals. Sample size estimates for the progressive supranuclear palsy rating scale were worse than imaging measures over 6-months, but comparable over 12-months. Atrophy and clinical decline can be detected over 6-months in progressive supranuclear palsy. Sample size estimates suggest that treatment trials could be performed over this interval, with rate of midbrain atrophy providing the best outcome measure.
Progressive supranuclear palsy; atrophy; midbrain; power calculations; short interval