Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a kind of tropical disease. Burkholderia thailandensis (Bt), with a high sequence similarity to Bp, is thought to be an avirulent organism. Since there are numerous similarities between Bp and Bt, their differences in pathogenesis of host response and related mechanism are still undermined. In recent years, microRNAs have been researched in many diseases, but seldom involved in bacterial infection, bacteria-host interaction or explaining the differences between virulent and avirulent species.
We found that Bp and Bt had similar phenotypes in terms of intracellular replication, dissemination (reflected by multinucleated giant cell formation), TNF-α release and apoptosis in RAW264.7 macrophages or TC-1 pulmonary cell but in different level. Especially, at the late infection phases (after 12 h post infection), Bp showed faster intracellular growth, stronger cytotoxicity, and higher TNF-α release. After microRNA array analysis, we found some microRNAs were significantly expressed in macrophages treated by Bp. miR-3473 was one of them specifically induced, but not significantly changed in Bt-treated macrophages. In addition, TargetScan suggested that miR-3473 possibly target TRAF3 (TNF receptor-associated factor 3), a well-known negative regulator of the NF-κB pathway, which was probably involved in the TNF-α induction and apoptosis in cells with Bp infection. In vivo, it was found that miR-3473 expression of total lungs cells from Bp-treated was higher than that from Bt-treated mice. And miR-3473 inhibitor was able to decrease the TNF-α release of mice and prolong the survival of mice with Bp infection.
In sum, miR-3473 plays an important role in the differential pathogenicity of Bp and Bt via miR-3473-TRAF3-TNF-α network, and regulates TNF-α release, cell apoptosis and animal survival after Bp treatment. In this study, we have found a specific microRNA is related to bacterial virulence and provide insight into the mechanism for host-bacteria interaction, which suggests that potential oligonucleotides should be applied against bacterial infection.
Burkholderia pseudomallei (Bp); Burkholderia thailandensis (Bt); TNF-α; Apoptosis; miR-3473; TRAF3; NF-κB
Fusobacterium nucleatum (F. nucleatum) plays a critical role in gastrointestinal inflammation. However, the exact mechanism by which F. nucleatum contributes to inflammation is unclear. In the present study, it was revealed that F. nucleatum could induce the production of proinflammatory cytokines (IL-8, IL-1β and TNF-α) and reactive oxygen species (ROS) in Caco-2 colorectal) adenocarcinoma cells. Furthermore, ROS scavengers (NAC or Tiron) could decrease the production of proinflammatory cytokines during F. nucleatum infection. In addition, we observed that autophagy is impaired in Caco-2 cells after F. nucleatum infection. The production of proinflammatory cytokines and ROS induced by F. nucleatum was enhanced with either autophagy pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (ATG5 or ATG12) in Caco-2 cells. Taken together, these results indicate that F. nucleatum-induced impairment of autophagic flux enhances the expression of proinflammatory cytokines via ROS in Caco-2 Cells.
Similar to its mammalian counterparts, teleost Toll-like receptor 9 (TLR9) recognizes unmethylated CpG DNA presented in the genome of bacteria or DNA viruses and initiates signaling pathway(s) for immune responses. We have previously shown that the TLR9 pathway in grouper, an economically important teleost, can be debilitated by an inhibitory gTLR9B isoform, whose production is mediated by RNA alternative splicing. However, how does grouper TLR9 (gTLR9) signaling impinge on the RNA splicing machinery to produce gTlr9B is unknown. Here we show that the gTlr9 alternative splicing is regulated through ligand-induced phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). We first observed that ligand-activated NF- κB pathway biased the production of the gTlr9B isoform. Because NF- κB is known to recruit p-TEFb kinase, which phosphorylates the Pol II CTD at Ser2 residues, we examined p-TEFb’s role in alternative splicing. We found that promoting p-TEFb kinase activity significantly favored the production of the gTlr9B isoform, whereas inhibiting p-TEFb yielded an opposite result. We further showed that p-TEFb-mediated production of the gTlr9B isoform down-regulates its own immune responses, suggesting a self-limiting mechanism. Taken together, our data indicate a feedback mechanism of the gTLR9 signaling pathway to regulate the alternative splicing machinery, which in turn produces an inhibitor to the pathway.
Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high mortality, which is prevalent in tropical regions of the world. A recent study shows that B. pseudomallei can survive inside mammalian cells because of its ability to actively evade cell autophagy. However, the underlying mechanisms remain unclear. In the present study, based on microarray screening, we found that ATG10 was downregulated following B. pseudomallei infection in A549 human lung epithelial cells. Forced expression of ATG10 accelerated the elimination of intracellular B. pseudomallei by enhancing the process of autophagy. Moreover, MIR4458, MIR4667-5p, and MIR4668-5p were found, by microarray screening, to be upregulated in response to B. pseudomallei infection. These 3 novel miRNAs, MIR4458, MIR4667-5p, and MIR4668-5p, targeted to the 3′-untranslated region of ATG10 in different time-course and spatial manners. Upregulation of these miRNAs reduced the level of ATG10 and inhibited autophagy, leading to increasing survival rate of intracellular B. pseudomallei. Furthermore, the increase of these miRNAs was correlated with the reduced promoter methylation status in A549 cells in response to B. pseudomallei infection. Our results reveal that 3 novel miRNAs regulate autophagy-mediated elimination of B. pseudomallei by targeting ATG10, and provide potential targets for clinical treatment.
ATG10; autophagy; Burkholderia pseudomallei; DNA methylation; MIR4458; MIR4667-5p; MIR4668-5p
Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A1, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.
autophagic cell death; autophagy; E. coli O157:H7; ER stress; Shiga toxins; 3-MA, 3-methyladenine; Δ, knockout; AO, acridine orange; ATF4, activating transcription factor 4; ATG, autophagy-related; Baf A1, bafilomycin A1; BECN1, Beclin 1, autophagy-related; CASP3, caspase 3, apoptosis-related cysteine peptidase; DDIT3, DNA-damage-inducible transcript 3; EHEC O157, Escherichia coli O157:H7; FACS, fluorescence activated cell sorting; MAP1LC3B, microtubule-associated protein 1 light chain 3 beta; MAPK, mitogen-activated protein kinase; MDC, monodansylcadaverine; NUPR1, nuclear protein, transcriptional regulator, 1; PBS, phosphate-buffered saline; PARP1, poly (ADP-ribose) polymerase 1; PI, propidium iodide; Stxs, Shiga toxins; Thap, thapsigargin; TEM, transmission electron microscopy; TRIB3, tribbles pseudokinase 3; WT, wild type; Z-VAD, Z-VAD-FMK
Association of a polymorphism rs3775291 in the toll-like receptor 3 (TLR3) gene with age-related macular degeneration (AMD) had been investigated intensively, with variable results across studies. Here we conducted a meta-analysis to verify the effect of rs3775291 on AMD. We searched for genetic association studies published in PubMed, EMBASE and Web of Science from start dates to March 10, 2015. Totally 235 reports were retrieved and 9 studies were included for meta-analysis, involving 7400 cases and 13579 controls. Summary odds ratios (ORs) with 95% confidence intervals (CIs) for alleles and genotypes were estimated. TLR3 rs3775291 was associated with both geographic atrophy (GA) and neovascular AMD (nAMD), with marginally significant pooled-P values. Stratification analysis by ethnicity indicated that rs3775291 was associated with all forms of AMD, GA and nAMD only in Caucasians (OR = 0.87, 0.78 and 0.77, respectively, for the TT genotype) but not in East Asians. However, the associations could not withstand Bonferroni correction. This meta-analysis has thus revealed suggestive evidence for TLR3 rs3775291 as an associated marker for AMD in Caucasians but not in Asians. This SNP may have only a small effect on AMD susceptibility. Further studies in larger samples are warranted to confirm its role.
Bone morphogenetic proteins (BMPs) play positive roles in cartilage development, but they can barely be detected in healthy articular cartilage. However, recent evidence has indicated that BMPs could be detected in osteoarthritic and damaged cartilage and their precise roles have not been well defined. Extremely high amounts of leptin have been reported in obese individuals, which can be associated with osteoarthritis (OA) development. The aim of this study was to investigate whether BMPs could be induced in human primary chondrocytes during leptin-stimulated OA development and the underlying mechanism. We found that expression of BMP-2 mRNA, but not BMP-4, BMP-6, or BMP-7 mRNA, could be increased in human primary chondrocytes under leptin stimulation. Moreover, this BMP-2 induction was mediated through transcription factor-signal transducer and activator of transcription (STAT) 3 activation via JAK2-ERK1/2-induced Ser727-phosphorylation. Of note, histone deacetylases (HDACs) 3 and 4 were both involved in modulating leptin-induced BMP-2 mRNA expression through different pathways: HDAC3, but not HDAC4, associated with STAT3 to form a complex. Our results further demonstrated that the role of BMP-2 induction under leptin stimulation is to increase collagen II expression. The findings in this study provide new insights into the regulatory mechanism of BMP-2 induction in leptin-stimulated chondrocytes and suggest that BMP-2 may play a reparative role in regulating leptin-induced OA development.
Novel composites were synthesized from keratin (KER), cellulose (CEL) and chitosan (CS). The method is recyclable because majority (>88%) of [BMIm+Cl−], an ionic liquid (IL), used as the sole solvent, was recovered for reuse. Experimentally, it was confirmed that unique properties of each component remain intact in the composites, namely bactericide (from KER and CS) and anti-inflammatory property (from KER). Specifically, the composites were examined for their anti-inflammatory influence on macrophages. The cells were imaged and immunophenotyped to determine the quantity using the macrophage marker CD11b. The 75:25 [KER+CS] composite was found to have the least amount of CD11b macrophages compared to other composites. Bactericidal assays indicated that all composites, except the 25:75 [KER+CS], substantially reduce the growth of organisms such as vancomycin resistant Enterococcus (VRE) and Eschericia coli. The results clearly indicate that the composites possess all properties needed for effective use as a wound dressing.
Toll-like receptor 9 (TLR9) recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length) and gTLR9B (with a truncated Cʹ-terminal signal transducing domain), whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides), whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN), gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish.
Many cell therapies currently being tested are based on mesenchymal stromal cells (MSCs). However, MSCs start to enter the senescent state upon long-term expansion. The role of retinoblastoma (RB) protein in regulating MSC properties is not well studied. Here, we show that RB levels are higher in early-passage MSCs compared with late-passage MSCs. RB knockdown induces premature senescence and reduced differentiation potentials in early-passage MSCs. RB overexpression inhibits senescence and increases differentiation potentials in late-passage MSCs. Expression of DNMT1, but not DNMT3A or DNMT3B, is also higher in early-passage MSCs than in late-passage MSCs. Furthermore, DNMT1 knockdown in early-passage MSCs induces senescence and reduces differentiation potentials, whereas DNMT1 overexpression in late-passage MSCs has the opposite effect. These results demonstrate that RB expressed in early-passage MSCs upregulates DNMT1 expression and inhibits senescence in MSCs. Therefore, genetic modification of RB could be a way to improve the efficiency of MSCs in clinical use.
•RB levels are higher in early-passage MSCs than in late-passage MSCs•RB knockdown induces a premature senescence and reduces differentiation potentials•RB overexpression reverses senescence and increases differentiation potentials•RB upregulates DNMT1 to inhibit senescence and promote differentiation potentials
In this article, Hung, Kao, and colleagues show that retinoblastoma (RB) levels are higher in early-passage mesenchymal stromal cells (MSCs) than in late-passage MSCs, which serves to regulate senescence and differentiation potentials. Expression of DNMT1 is also higher in early-passage MSCs than in late-passage MSCs. This study demonstrates that RB expressed in early-passage MSCs upregulates DNMT1 expression and inhibits senescence in MSCs.
Age-related macular degeneration (AMD) is a major cause of severe vision loss in elderly people. Diabetes mellitus is a common endocrine disorder with serious consequences, and diabetic retinopathy (DR) is the main ophthalmic complication. DR and AMD are different diseases and we seek to explore the relationship between diabetes and AMD. MEDLINE, EMBASE, and the Cochrane Library were searched for potentially eligible studies. Studies based on longitudinal cohort, cross-sectional, and case-control associations, reporting evaluation data of diabetes as an independent factor for AMD were included. Reports of relative risks (RRs), hazard ratios (HRs), odds ratio (ORs), or evaluation data of diabetes as an independent factor for AMD were included. Review Manager and STATA were used for the meta-analysis. Twenty four articles involving 27 study populations were included for meta-analysis. In 7 cohort studies, diabetes was shown to be a risk factor for AMD (OR, 1.05; 95% CI, 1.00–1.14). Results of 9 cross-sectional studies revealed consistent association of diabetes with AMD (OR, 1.21; 95% CI, 1.00–1.45), especially for late AMD (OR, 1.48; 95% CI, 1.44–1.51). Similar association was also detected for AMD (OR, 1.29; 95% CI, 1.13–1.49) and late AMD (OR, 1.16; 95% CI, 1.11–1.21) in 11 case-control studies. The pooled ORs for risk of neovascular AMD (nAMD) were 1.10 (95% CI, 0.96–1.26), 1.48 (95% CI, 1.44–1.51), and 1.15 (95% CI, 1.11–1.21) from cohort, cross-sectional and case-control studies, respectively. No obvious divergence existed among different ethnic groups. Therefore, we find diabetes a risk factor for AMD, stronger for late AMD than earlier stages. However, most of the included studies only adjusted for age and sex; we thus cannot rule out confounding as a potential explanation for the association. More well-designed prospective cohort studies are still warranted to further examine the association.
The quality of cardiopulmonary resuscitation (CPR) is important to survival after cardiac arrest. Mechanical devices (MD) provide constant CPR, but their effectiveness may be affected by deployment timeliness.
To identify the timeliness of the overall and of each essential step in the deployment of a piston-type MD during emergency department (ED) resuscitation, and to identify factors associated with delayed MD deployment by video recordings.
Between December 2005 and December 2008, video clips from resuscitations with CPR sessions using a MD in the ED were reviewed using time-motion analyses. The overall deployment timeliness and the time spent on each essential step of deployment were measured.
There were 37 CPR recordings that used a MD. Deployment of MD took an average 122.6 ± 57.8 s. The 3 most time-consuming steps were: (1) setting the device (57.8 ± 38.3 s), (2) positioning the patient (33.4 ± 38.0 s), and (3) positioning the device (14.7 ± 9.5 s). Total no flow time was 89.1 ± 41.2 s (72.7% of total time) and associated with the 3 most time-consuming steps. There was no difference in the total timeliness, no-flow time, and no-flow ratio between different rescuer numbers, time of day of the resuscitation, or body size of patients.
Rescuers spent a significant amount of time on MD deployment, leading to long no-flow times. Lack of familiarity with the device and positioning strategy were associated with poor performance. Additional training in device deployment strategies are required to improve the benefits of mechanical CPR.
Mechanical devices; Cardiopulmonary resuscitation (CPR); Video-recording; Time-motion analysis; No-flow time Quality
Advances in next-generation sequencing technologies have enabled the identification of multiple rare single nucleotide polymorphisms involved in diseases or traits. Several strategies for identifying rare variants that contribute to disease susceptibility have recently been proposed. An important feature of many of these statistical methods is the pooling or collapsing of multiple rare single nucleotide variants to achieve a reasonably high frequency and effect. However, if the pooled rare variants are associated with the trait in different directions, then the pooling may weaken the signal, thereby reducing its statistical power. In the present paper, we propose a backward support vector machine (BSVM)-based variant selection procedure to identify informative disease-associated rare variants. In the selection procedure, the rare variants are weighted and collapsed according to their positive or negative associations with the disease, which may be associated with common variants and rare variants with protective, deleterious, or neutral effects. This nonparametric variant selection procedure is able to account for confounding factors and can also be adopted in other regression frameworks. The results of a simulation study and a data example show that the proposed BSVM approach is more powerful than four other approaches under the considered scenarios, while maintaining valid type I errors.
Melioidosis, caused by Burkholderia pseudomallei, is considered to be endemic to Northern Australia and Southeast Asia, with high mortality and relapse rates, regardless of powerful antibiotic therapy. Here we report the first genome sequence of Burkholderia pseudomallei strain BPC006, obtained from a melioidosis patient in Hainan, China. The genome sizes of the 2 chromosomes were determined to be 4,001,777 bp and 3,153,284 bp.
Helicobacter pylori evade immune responses and achieve persistent colonization in the stomach. However, the mechanism by which H. pylori infections persist is not clear. In this study, we showed that MIR30B is upregulated during H. pylori infection of an AGS cell line and human gastric tissues. Upregulation of MIR30B benefited bacterial replication by compromising the process of autophagy during the H. pylori infection. As a potential mechanistic explanation for this observation, we demonstrate that MIR30B directly targets ATG12 and BECN1, which are important proteins involved in autophagy. These results suggest that compromise of autophagy by MIR30B allows intracellular H. pylori to evade autophagic clearance, thereby contributing to the persistence of H. pylori infections.
Helicobacter pylori; MIR30B; ATG12; BECN1; autophagy
The aim of this study was designed to assess the risk factors of lag-screw cutout in the treatment of intertrochanteric fracture with a dynamic hip screw (DHS). From 2003 to 2007, 1,150 patients who had acute unilateral intertrochanteric fractures of the femur were enrolled to the study. All fractures were managed by closed reduction and internal fixation with 135° DHS devices. Patient demographics, fracture patterns, reduction and fixation and perioperative course parameters were all recorded. The follow-up period was 38 months on average (range 16–60 months). Finally, 937 patients were available for evaluation of final results in which we focused on lag-screw cutout. Excluding complications not related to screw position, 64 patients (6.8%) with screw cutout were encountered, and the remaining 873 patients had uneventful union, with the average union time of 17.5 weeks (range15–24 weeks). Upon analysis with logistic regression, the tip−apex distance (TAD) was shown to be the most important predictive factor for cutout, followed by screw position, fracture pattern, reduction and patient age. In order to decrease the risk of lag-screw cutout, it is important to ensure good fracture reduction and to place the lag screw in either the middle/middle or inferior/middle position with appropriate TAD.
The result of treatment of infections involving antibiotic-resistant organisms in total knee arthroplasty (TKA) is often poor. We evaluated the efficacy of 2-stage revision in TKAs infected with resistant organisms and compared the clinical outcomes with articulating and conventional static spacers, in terms of both infection control and function.
In a prospective manner, from June 2003 to January 2007 selected patients with a TKA infected with resistant organisms were enrolled and treated with 2-stage re-implantation. The 45 patients were divided into 2 groups: group A (23 patients) implanted with the articulating spacers and group S (22 patients) implanted with static spacers. All patients followed the same antibiotic protocols and had the same re-implantation criteria. The efficacy of infection control was evaluated using re-implantation rate, recurrence rate, and overall success rate. The functional and radiographic results were interpreted with the Hospital of Special Surgery (HSS) knee score and the Insall-Salvati ratio.
With mean 40 (24–61) months of follow-up, 22 of 23 knees were re-implanted in group A and 21 of 22 were re-implanted in group S. Of these re-implanted prostheses, 1 re-infection occurred in group A and 2 occurred in group S. Range of motion after re-implantation, the final functional scores, and the satisfaction rate were better in group A. One third of the patients in group S, and none in group A, had a patella baja.
After 2-stage re-implantation of TKAs originally infected with resistant organisms, the clinical outcome was satisfactory—and similar to that reported after treatment of TKAs infected with low-virulence strains. Treatment with an articulating spacer resulted in better functional outcome and lower incidence of patella baja.
Muscle disability is a common sequel after fracture management. Previous research has shown divergent results concerning muscle-power recovery after bone healing. This study has investigated the muscle function of wrist extensors after lateral condylar fracture in children, as evaluated by a hand-held dynamometer and compared with sex- and age-matched children. From 1999 to 2004, 20 patients (13 boys and seven girls; mean age: 9 years and 4 months) with displaced lateral condylar fracture of the humerus were treated by open reduction and internal fixation with Kirschner wires (K-wire). The duration of K-wire fixation was 35 days and the mean follow-up time was 50 months. A total of 180 healthy age-, sex- and weight-matched children were used as control groups. A paired Student’s test was applied for the analysis of statistical significance. The range of motion of the elbow and radiographic findings were not significantly different between the injured limb and normal control groups. The maximum isometric power of wrist-extensor muscles after surgical treatment of lateral condylar fracture of the humerus in final follow-up was not statistically different from that in the normal control children. Muscle power therefore recovers to its normal status after the healing of lateral condylar fracture of the humerus in children.