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Study Design: The study included 2 fresh-frozen cadavers., Objective: To elucidate the positional relationship between surgical instruments and nerve roots during full endoscopic facet-sparing (FE fs-TLIF) and full endoscopic facet-resecting (FE fr-TLIF) transforaminal lumbar interbody fusion and propose safe instrumentation insertion procedures and recommend cage glider designs aimed at protecting nerve roots., Background: Endoscopic surgical techniques are increasingly used for minimally invasive lumbar fusion surgery, with FE fr-TLIF and FE fs-TLIF being common approaches. However, the risk of nerve root injury remains a significant concern during these procedures., Patients and Methods: Eight experienced endoscopic spine surgeons performed uniportal FE fr-TLIF and FE fs-TLIF on cadaveric lumbar spines, totaling 16 surgeries. Postoperation, soft tissues were removed to assess the positional relationship between the cage entry point and nerve roots. Distances between the cage entry point, traversing nerve root, and exiting nerve root were measured. Safe instrumentation design and insertion procedures were determined., Results: In FE fr-TLIF, the mean distance between the cage entry point and traversing nerve root was significantly shorter compared with FE fs-TLIF (3.30 ± 1.35 vs . 8.58 ± 2.47 mm, respectively; P < 0.0001). Conversely, the mean distance between the cage entry point and the exiting nerve root was significantly shorter in FE fs-TLIF compared with FE fr-TLIF (3.73 ± 1.97 vs . 6.90 ± 1.36 mm, respectively; P < 0.0001). For FE fr-TLIF, prioritizing the protection of the traversing root using a 2-bevel tip cage glider was crucial. In contrast, for FE fs-TLIF, a single-bevel tip cage glider placed in the caudal location was recommended., Conclusion: This study elucidates the anatomic relationship between cage entry points and nerve roots in uniportal endoscopic lumbar fusion surgery. Protection strategies should prioritize the traversing root in FE fr-TLIF and the exiting root in FE fs-TLIF, with corresponding variations in surgical techniques., Level of Evidence: Level V., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

High-temperature-requirement protein A (HtrA) family proteins play important roles in controlling protein quality and are recognized as virulence factors in numerous animal and human bacterial pathogens. The role of HtrA family proteins in plant pathogens remains largely unexplored. Here, we investigated the HtrA family protein, DegQ, in the crucifer black rot pathogen Xanthomonas campestris pathovar campestris (Xcc). DegQ is essential for bacterial attachment and full virulence of Xcc. Moreover, the degQ mutant strain showed increased sensitivity to heat treatment and sodium dodecyl sulfate. Expressing the intact degQ gene in trans in the degQ mutant could reverse the observed phenotypic changes. In addition, we demonstrated that the DegQ protein exhibited chaperone-like activity. Transcriptional analysis displayed that degQ expression was induced under heat treatment. Our results contribute to understanding the function and expression of DegQ of Xcc for the first time and provide a novel perspective about HtrA family proteins in plant pathogen., (© 2024 Wiley‐VCH GmbH.)

As an essential nutrient element, phosphorus (P) is primarily acquired and translocated as inorganic phosphate (Pi) by plant roots. Pi is often sequestered in the soil and becomes limited for plant growth. Plants have developed a sophisticated array of adaptive responses, termed P starvation responses, to cope with P deficiency by improving its external acquisition and internal utilization. Over the past 2 to 3 decades, remarkable progress has been made toward understanding how plants sense and respond to changing environmental P. This review provides an overview of the molecular mechanisms that regulate or coordinate P starvation responses, emphasizing P transport, sensing, and signaling. We present the major players and regulators responsible for Pi uptake and translocation. We then introduce how P is perceived at the root tip, how systemic P signaling is operated, and the mechanisms by which the intracellular P status is sensed and conveyed. Additionally, the recent exciting findings about the influence of P on plant-microbe interactions are highlighted. Finally, the challenges and prospects concerning the interplay between P and other nutrients and strategies to enhance P utilization efficiency are discussed. Insights obtained from this knowledge may guide future research endeavors in sustainable agriculture., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Background: Cervical spondylotic myelopathy (CSM) may seriously affect quality of life. In the literature, there is scarce evidence of the pros and cons of full endoscopic spine surgery in the treatment of CSM. The main purpose of this study was to conduct a systematic review to elucidate the efficacy of full endoscopic spine surgery in the management of patients with CSM., Methods: This systematic review was conducted in accordance with the PRISMA guidelines. A systematic search of Web of Science, PubMed MEDLINE, Embase, and Cochrane Library was conducted from the database inception to February 1, 2023., Results: The study included 183 patients and their age was 56.78 ± 7.87 years. The average surgical time calculated was 96.34 ± 33.58 minutes. Intraoperative blood loss ranged from a minimal amount to 51 mL. The average duration of hospital stay was 3.56 ± 1.6 days. The average span for follow-up was on an interval of 18.7 ± 6.76 months. Significant improvements were noted in all aspects of functional outcomes and image results after full endoscopic cervical spine surgery, with no major complications., Conclusions: The current study found that both anterior transcorporeal and posterior surgical approaches could be used for the treatment of CSM with a full endoscopic technique. Indications of full endoscopic cervical spine surgery for CSM included cervical disc herniation, central canal stenosis, calcified ligamentum flavum, and ossification of the posterior longitudinal ligament. Improved postoperative outcomes with acceptable surgical complications were noted in this systematic review., (Copyright © 2023 Elsevier Inc. All rights reserved.)

SsrA and SmpB are known to play important roles in translational quality control and are essential for virulence in many human and animal pathogenic bacteria. The physiological roles and contribution of SsrA and SmpB to plant pathogen are unclear. Here, we present evidence to show that ssrA and smpB are involved in pathogenesis of Xanthomonas campestris pathovar campestris, the cause of black rot diseases in crucifers. The ssrA and smpB mutants exhibited defects in bacterial attachment, cell motility, and extracellular enzyme activity. The mutation of ssrA and smpB also resulted in a reduction in temperature tolerance. These altered phenotypes of the ssrA and smpB mutants could be complemented to wild-type levels by the intact ssrA and smpB genes. This is the first demonstration of the roles of SsrA and SmpB in phytopathogen., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

ATP-dependent proteases (FtsH, Lon, and Clp family proteins) are ubiquitous in bacteria and play essential roles in numerous regulatory cell processes. Xanthomonas campestris pv. campestris is a Gram-negative pathogen that can cause black rot diseases in crucifers. The genome of X. campestris pv. campestris has several clp genes, namely, clpS, clpA, clpX, clpP, clpQ, and clpY. Among these genes, only clpX and clpP is known to be required for pathogenicity. Here, we focused on two uncharacterized clp genes (clpS and clpA) that encode the adaptor (ClpS) and ATPase subunit (ClpA) of the ClpAP protease complex. Transcriptional analysis revealed that the expression of clpS and clpA was growth phase-dependent and affected by the growth temperature. The inactivation of clpA, but not of clpS, resulted in susceptibility to high temperature and attenuated virulence in the host plant. The altered phenotypes of the clpA mutant could be complemented in trans. Site-directed mutagenesis revealed that K223 and K504 were the amino acid residues critical for ClpA function in heat tolerance. The protein expression profile shown by the clpA mutant in response to heat stress was different from that exhibited by the wild type. In summary, we characterized two clp genes (clpS and clpA) by examining their expression profiles and functions in different processes, including stress tolerance and pathogenicity. We demonstrated that clpS and clpA were expressed in a temperature-dependent manner and that clpA was required for the survival at high temperature and full virulence of X. campestris pv. campestris. This work represents the first time that clpS and clpA were characterized in Xanthomonas., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Objective: Anterior cervical discectomy and fusion (ACDF) has long been regarded as a gold standard in the treatment of cervical myelopathy. Subsequently, cervical artificial disc replacement (c-ADR) was developed and provides the advantage of motion preservation at the level of the intervertebral disc surgical site, which may also reduce stress at adjacent levels. The goal of this study was to compare clinical and functional outcomes in patients undergoing ACDF with those in patients undergoing c-ADR for cervical spondylotic myelopathy (CSM)., Methods: A systematic literature review and meta-analysis were performed using the Embase, PubMed, and Cochrane Central Register of Controlled Trials databases from database inception to November 21, 2021. The authors compared Neck Disability Index (NDI), SF-36, and Japanese Orthopaedic Association (JOA) scores; complication rates; and reoperation rates for these two surgical procedures in CSM patients. The Mantel-Haenszel method and variance-weighted means were used to analyze outcomes after identifying articles that met study inclusion criteria., Results: More surgical time was consumed in the c-ADR surgery (p = 0.04). Shorter hospital stays were noted in patients who had undergone c-ADR (p = 0.04). Patients who had undergone c-ADR tended to have better NDI scores (p = 0.02) and SF-36 scores (p = 0.001). Comparable outcomes in terms of JOA scores (p = 0.24) and neurological success rate (p = 0.12) were noted after the surgery. There was no significant between-group difference in the overall complication rates (c-ADR: 18% vs ACDF: 25%, p = 0.17). However, patients in the ACDF group had a higher reoperation rate than patients in the c-ADR group (4.6% vs 1.5%, p = 0.02)., Conclusions: At the midterm follow-up after treatment of CSM, better functional outcomes as reflected by NDI and SF-36 scores were noted in the c-ADR group than those in the ACDF group. c-ADR had the advantage of retaining range of motion at the level of the intervertebral disc surgical site without causing more complications. A large sample size with long-term follow-up studies may be required to confirm these findings in the future.

Background: Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium that can cause black rot disease in crucifers. The lipoprotein outer membrane localization (Lol) system is involved in the lipoprotein sorting to the outer membrane. Although Xcc has a set of annotated lol genes, there is still little known about the physiological role in this phytopathogen. In this study, we aimed to characterize the role of LolB of Xcc in bacterial attachment, stress tolerance, and virulence., Results: To characterize the role of LolB, lolB mutant was constructed and phenotypic evaluation was performed. The lolB mutant revealed reductions in bacterial attachment, extracellular enzyme production, and virulence. Mutation of lolB also resulted in reduced tolerance to a myriad of stresses, including heat and a range of membrane-perturbing agents. Trans-complementation of lolB mutant with intact lolB gene reverted these altered phenotypes to the wild-type levels. From subsequent reporter assay and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis, the expression of genes that encode the major extracellular enzymes and the stress-related proteins was reduced after lolB mutation., Conclusions: The results in this work contribute to the functional understanding of lolB in Xanthomonas for the first time, and provide new insights into the function of lolB in bacteria., (© 2022. The Author(s).)

Blood supply interruption induces hypoxia and reduces serum provision to cause ischemia-induced osteonecrosis, including avascular osteonecrosis of the femoral head (ONFH). Oxygen deficiency (hypoxia) is known to induce different expression patterns in osteoblasts and osteoclasts, which have been extensively studied. However, the effects of serum insufficiency in nutrients, growth factors, and hormones on osteoblast and osteoclast activity in the damaged area and nearby regions remain poorly understood. In this study, the expression of osteoblast and osteoclast marker proteins was elucidated through in vitro and ex vivo studies. The results indicate that serum insufficiency accelerates the formation of monocyte-derived osteoclasts. The combined effect of serum insufficiency and hypoxia (mimicking ischemia) suppressed the activity of alkaline phosphatase and calcification in osteoblasts after the stimulation of osteogenic growth factors. Serum insufficiency increased the activity of tartrate-resistant acid phosphatase, expression of phosphorylated extracellular signal-regulated kinases, and production of reactive oxygen species in monocyte-derived osteoclasts in the absence of receptor activator of nuclear factor kappa-Β ligand stimulation. The findings indicate that changes in the expression of osteoblast and osteoclast markers in necrotic bone extracts were similar to those observed during an in vitro study. These results also suggest that serum insufficiency may be involved in the regulation of osteoclast formation in patients with ONFH.

Background: Platelet transfusion is required to treat haemo-oncology or trauma patients. Platelet apheresis (PA) performed with apheresis equipment has increased rapidly in recent years. Leucocyte-reduced platelet apheresis (LRPA) can reduce the risk of platelet refractoriness and febrile nonhemolytic transfusion reactions (FNHTRs) for transfusion. Accordingly, this study aimed to investigate and compare the platelet metabolic and functional responses between PA performed with Haemonetics and LRPA performed with Trima Accel cell separator., Methods: The qualities of platelets collected through PA and LRPA were evaluated in terms of visual appearance, morphology, platelet-aggregation changes, metabolic activities, and bacterium-screening test during 5-day storage. Statistical analyses included two-sample t-test and generalised estimating equation(GEE) method., Results: During 5-day storage in LRPA, residual leucocytes were all <1.0×10 6 , and the parameters of platelet function were as follows: platelet aggregated to agonists such as adenosine 5'-diphosphate (ADP) and collagen, and the extent of shape change and pO 2 showed no statistically significant difference between PA and LRPA. The hypotonic shock reaction (HSR) on days 0, 1, and 3 were significantly higher in LRPA than in PA (71.78±6.92 vs. 64.10±7.42; P=0.002; 71.53±8.98 vs. 62.96±9.84; P=0.007; 68.05±7.28 vs. 57.76±6.80; P<0.0001, respectively). Values of mean platelet volume (MPV) were statistically larger in PA than in LRPA on days 0, 1, and 3. On day 5, the swirling score was higher in LRPA than in PA. The mean lactate levels had no statistically significant difference between PA and LRPA. Moreover, no growth was observed through bacterium-screening test conducted on 40 samples., Conclusion: Comparison of LRPA and PA products collected from the Trima Accel and Haemonetics automated blood-collection systems, respectively, revealed that both products possessed good platelet qualities even though additional processes are needed to reduce leucocytes. Furthermore, investigating the outcomes of other apheresis instruments with focus on the safety of donors, products, and recipients is necessary., (Copyright © 2020 Société française de transfusion sanguine (SFTS). Published by Elsevier Masson SAS. All rights reserved.)

The caseinolytic protease (Clp) system is essential for survival under stress conditions and for virulence in several pathogenic bacteria. Xanthomonas campestris pv. campestris (Xcc) is a plant pathogen which causes black rot disease in crucifers. In this study, the Xcc clpP gene which is annotated to encode the proteolytic core of Clp was characterized. Mutation of clpP resulted in susceptibility to high temperature and puromycin stresses. Site-directed mutagenesis revealed that S105, H130, and D179 are critical amino acid residues for ClpP function in puromycin tolerance. Inactivation of clpP also revealed an attenuation of virulence on the host plant and a reduction in the production of extracellular cellulase, mannanase, pectinase, and protease. The affected phenotypes of the clpP mutant could be complemented to wild-type levels by the intact clpP gene. Transcriptional analysis revealed that expression of clpP is induced under heat shock condition.

Lipoteichoic acid (LTA) is a cell wall component of Gram-positive bacteria. Limited data suggest that LTA is beneficial for bone regeneration in vitro. Thus, we used a mouse model of femoral defects to explore the effects of LTA on bone healing in vivo. Micro-computed tomography analysis and double-fluorochrome labeling were utilized to examine whether LTA can accelerate dynamic bone formation in vivo. The effects of LTA on osteoblastogenesis and osteoclastogenesis were also studied in vitro. LTA treatment induced prompt bone bridge formation, rapid endochondral ossification, and accelerated healing of fractures in mice with femoral bone defects. In vitro, LTA directly enhanced indicators of osteogenic factor-induced MC3T3-E1 cell differentiation, including alkaline phosphatase activity, calcium deposition and osteopontin expression. LTA also inhibited osteoclast activation induced by receptor activator of nuclear factor-kappa B ligand. We identified six molecules that may be associated with LTA-accelerated bone healing: monocyte chemoattractant protein 1, chemokine (C-X-C motif) ligand 1, cystatin C, growth/differentiation factor 15, endostatin and neutrophil gelatinase-associated lipocalin. Finally, double-fluorochrome, dynamic-labeling data indicated that LTA significantly enhanced bone-formation rates in vivo. In conclusion, our findings suggest that LTA has promising bone-regeneration properties.

Background: Although native to North America, the invasion of the aphid-like grape phylloxera Daktulosphaira vitifoliae across the globe altered the course of grape cultivation. For the past 150 years, viticulture relied on grafting-resistant North American Vitis species as rootstocks, thereby limiting genetic stocks tolerant to other stressors such as pathogens and climate change. Limited understanding of the insect genetics resulted in successive outbreaks across the globe when rootstocks failed. Here we report the 294-Mb genome of D. vitifoliae as a basic tool to understand host plant manipulation, nutritional endosymbiosis, and enhance global viticulture., Results: Using a combination of genome, RNA, and population resequencing, we found grape phylloxera showed high duplication rates since its common ancestor with aphids, but similarity in most metabolic genes, despite lacking obligate nutritional symbioses and feeding from parenchyma. Similarly, no enrichment occurred in development genes in relation to viviparity. However, phylloxera evolved > 2700 unique genes that resemble putative effectors and are active during feeding. Population sequencing revealed the global invasion began from the upper Mississippi River in North America, spread to Europe and from there to the rest of the world., Conclusions: The grape phylloxera genome reveals genetic architecture relative to the evolution of nutritional endosymbiosis, viviparity, and herbivory. The extraordinary expansion in effector genes also suggests novel adaptations to plant feeding and how insects induce complex plant phenotypes, for instance galls. Finally, our understanding of the origin of this invasive species and its genome provide genetics resources to alleviate rootstock bottlenecks restricting the advancement of viticulture.

Because of lipopolysaccharide (LPS)-mediated effects on osteoclast differentiation and bone loss, periprosthetic joint infection (PJI) caused by Gram-negative bacteria increases the risk of aseptic loosening after reimplantation. Synovial fluid interleukin-16 (IL-16) expression was higher in patients with PJI than in patients without joint infection. Thus, we explored the effects of IL-16 on bone. We investigated whether IL-16 modulates osteoclast or osteoblast differentiation in vitro. An LPS-induced bone loss mice model was used to explore the possible advantages of IL-16 inhibition for the prevention of bone loss. IL-16 directly activated p38 and c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) signaling and increased osteoclast activation markers, including tartrate-resistant acid phosphatase (TRAP), cathepsin K, and nuclear factor of activated T cells 1 (NFATc1). IL-16 directly caused monocytes to differentiate into TRAP-positive osteoclast-like cells through NFATc1 activation dependent on JNK/MAPK signaling. Moreover, IL-16 did not alter alkaline phosphatase activity or calcium deposition during osteoblastic differentiation. Finally, IL-16 inhibition prevented LPS-induced trabecular bone loss and osteoclast activation in vivo. IL-16 directly increased osteoclast activation through the JNK/NFATc1 pathway. IL-16 inhibition could represent a new strategy for treating infection-associated bone loss.

Xanthomonas campestris pv. campestris is a bacterial pathogen and the causal agent of black rot in crucifers. In this study, a clpX mutant was obtained by EZ-Tn5 transposon mutagenesis of the X. campestris pv. campestris. The clpX gene was annotated to encode ClpX, the ATP-binding subunit of ATP-dependent Clp protease. The clpX mutant exhibited reduced bacterial attachment, extracellular enzyme production and virulence. Mutation of clpX also resulted in increased sensitivity to a myriad of stresses, including heat, puromycin, and sodium dodecyl sulfate. These altered phenotypes of the clpX mutant could be restored to wild-type levels by in trans expression of the intact clpX gene. Proteomic analysis revealed that the expression of 211 proteins differed not less than twofold between the wild-type and mutant strains. Cluster of orthologous group analysis revealed that these proteins are mainly involved in metabolism, cell wall biogenesis, chaperone, and signal transduction. The reverse transcription quantitative real-time polymerase chain reaction analysis demonstrated that the expression of genes encoding attachment-related proteins, extracellular enzymes, and virulence-associated proteins was reduced after clpX mutation. The results in this study contribute to the functional understanding of the role of clpX in Xanthomonas for the first time, and extend new insights into the function of clpX in bacteria.

Background: The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae., Results: The 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins. The discovery of intron gain and turnover specific to the Hemiptera also prompted the evaluation of lineage and genome size as predictors of gene structure evolution. Furthermore, we identify enzymatic gains and losses that correlate with feeding biology, particularly for reductions associated with derived, fluid nutrition feeding., Conclusions: With the milkweed bug, we now have a critical mass of sequenced species for a hemimetabolous insect order and close outgroup to the Holometabola, substantially improving the diversity of insect genomics. We thereby define commonalities among the Hemiptera and delve into how hemipteran genomes reflect distinct feeding ecologies. Given Oncopeltus's strength as an experimental model, these new sequence resources bolster the foundation for molecular research and highlight technical considerations for the analysis of medium-sized invertebrate genomes.

Background: The gram-negative Xanthomonas campestris pv. campestris is the pathogenic bacterium that causes black rot disease in crucifers. The virulence determinants of this bacterium include extracellular enzymes, exopolysaccharides, and biofilm formation. Here, one transposon mutant of X. campestris pv. campestris strain 17 that affects biofilm formation was isolated, and subsequent analyses led to the identification of the lolA gene, which encodes an outer membrane lipoprotein chaperone., Results: The lolA mutant exhibited significant reductions in bacterial attachment, extracellular enzyme production, virulence, and tolerance in the presence of myriad membrane-perturbing agents. These phenotypic changes of the mutant could be complemented to the wild-type level through the intact lolA gene. Proteomic analysis revealed that 109 proteins were differentially expressed after lolA mutation. These differentially expressed proteins were categorized in various functional groups and were mainly associated with the membrane component, were involved in transport, and contained receptor activity. Through reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis, deletion of lolA was determined to have caused significantly reduced expression of genes that encode the major extracellular enzymes, the biofilm-related proteins, and the virulence-related proteins. The RT-qPCR analysis also indicated that the expression of several genes that encode putative outer membrane lipoproteins and TonB-dependent receptors was reduced after lolA mutation., Conclusions: This is the first report to define the lolA gene as a virulence factor and to contribute to the functional understanding of, and provide new information concerning, the role of lolA in Xanthomonas. Furthermore, the results of this study provide and extend new insights into the function of lolA in bacteria.

In animals, differentiation of germline from soma usually takes place during embryogenesis. Genes and their products that are preferentially expressed in the embryonic germ cells are regarded as candidates for maintaining germline fate or promoting germline identity. In Drosophila, for example, the protein encoded by the germline gene vasa is specifically restricted to the germ cells, while products of the gap gene hunchback (hb), a somatic gene, are preferentially expressed in the neuroblasts. In this study, we report the expression of both messenger RNA and protein encoded by Aphb, an hb orthologue in the asexual viviparous pea aphid Acyrthosiphon pisum, in germ cells as well as in neuroblasts. We infer that expression of Aphb messenger RNA in the germ cells during the formation of germaria is required for the anterior localization of Aphb in the protruding oocytes. Germarial expression and anterior localization of ApKrüppel was also identified but, unlike Aphb, its expression was not detected in the migrating germ cells. Very similar patterns of hb expression were also identified in the green peach aphid Myzus persicae, suggesting that germline expression of hb is conserved within the Aphididae. To date, this pattern of hb germline expression has not been reported in other insects., (© 2018 The Royal Entomological Society.)

Background: Hepatitis C virus (HCV) is a globally prevalent pathogen and a leading cause of death and morbidity. Traditional therapy with pegylated interferon- and ribavirin has had only limited success, with some adverse effects. Direct-acting antivirals (DAAs) are effective in suppressing HCV replication, but are expensive., Purpose: Hypericin has been reported to be a good antiviral agent for inhibiting HCV replication, however, little is known about its mechanisms of action. The aim of this study is to elucidate the mode of action of hypericin in Ava5 human hepatoma cell line (Huh7 derivative) harboring HCV subgenomic replicon RNA., Methods: To determine the non-structure protein 5A (NS5A) mRNA and NS3 protein expression levels, real-time PCR and Western blot analysis were performed, respectively. To investigate how hypericin inhibits HCV replication, 5-aza-2'-deoxycytidine (5-Aza-dC) and chidamide were used for determining histone modification. Furthermore, shRNA was applied to confirm the role of heme oxygenase (HO-1) in HCV repression., Results: Hypericin in experiment were tested and showed no cytotoxicity. Hypericin reduced HO-1 and NS5A in a time- and dose- dependent manner. Chidamide, but not 5-Aza-dc, restored hypericin-induced reduction in HCV NS3 expression and reversed HO-1 expression in Ava5 cells. LY294002 inhibited HCV replication via HO-1 down-regulation. Constitutive expressed p-AKT was not involved in hypericin-induced reduction in HCV replication. In addition, shHO-1 inhibited HCV replication., Conclusion: In conclusion, hypericin inhibits HCV replication via down-regulation of HO-1 expression and deacetylation., (Copyright © 2017. Published by Elsevier GmbH.)

Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers. Here, one EZ-Tn5 transposon mutant of Xcc, altered in bacterial attachment, was isolated. Further analysis revealed that the transposon was inserted in the wxcX gene (encodes a hypothetical protein) of the transposon mutant. Sequence analysis revealed that WxcX is highly conserved in Xanthomonas, but none has been characterized. In this study, it was indicated that mutation of wxcX resulted in enhanced bacterial attachment, reduced virulence on the host cabbage, and increased sensitivity to sodium dodecyl sulfate. The affected phenotypes of the wxcX mutant could be complemented to wild-type levels by the intact wxcX gene. Site-directed mutagenesis revealed that E408 and E411 are critical amino acid residues for WxcX function in bacterial attachment. Taken together, our results demonstrate the roles of wxcX in attachment, virulence, and tolerance to sodium dodecyl sulfate in Xanthomonas for the first time., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Xanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot disease in cruciferous plants. The XCC2366 gene product is annotated as a protein belonging to the TetR family of transcriptional regulators. In this study, we evaluated the function and expression of the XCC2366 gene. Mutational analysis demonstrated that XCC2366 is involved in the resistance to acriflavin and is necessary for virulence in Xcc. In addition, the XCC2366 transcription initiation site was mapped at nucleotide A, 63 nucleotide upstream of the XCC2366 translation start codon. Furthermore, transcriptional analysis revealed that the expression of XCC2366 is induced in the presence of acriflavin. Reporter assay also showed that XCC2366 regulates its own expression under acriflavin-supplemented condition. To the best of our knowledge, acriflavin resistance-related gene in the crucifer pathogen Xcc was characterized for the first time.

Nickel compounds have been classified as carcinogens and shown to be associated with induction of epithelial-mesenchymal transition (EMT) in fibrogenesis and tumorigenesis, as well as the crucial role of microRNAs (miRNAs) and their related genes in controlling EMT and cancer metastasis. Thus, the mechanisms involved in the regulation of EMT in nickel-treated cells are of potential interest in understanding lung fibrosis and tumor progression. We investigated the miRNA-dependent mechanisms involved in nickel-induced EMT in lung epithelial cells. Nickel increased miR-4417 expression and decreased its target gene TAB2 expression. Treatment of cells with TGF-β inhibitor SB525334 significantly blocked NiCl 2 and TGF-β-induced EMT. The expression of miR-4417 was abolished by SB525334 in TGF-β-treated cells, but not in nickel-treated cells. Both overexpression of miR-4417 and silencing of TAB2 induced fibronectin expression, but did not reduce E-cadherin expression. Moreover, oral administration of nickel promoted lung tumor growth in nude mice that had received BEAS-2B transformed cells by intravenous injection. The induction of EMT by nickel is mediated through multiple pathways. Induction of abundant miR-4417 and reduction of TAB2 expression following nickel exposure and may be involved in nickel-induced fibronectin. These findings provide novel insight into the roles of nickel in fibrogenesis and tumor progression.

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate. In the genome of Xanthomonas campestris pv. campestris, the phytopathogen that causes black rot in cruciferous plants, two putative IDH genes, icd1 and icd2, have been annotated. Their physiological roles in X. campestris pv. campestris are unclear. In this study, the icd2 gene from X. campestris pv. campestris was characterized in detail. We demonstrated genetically that icd2 gene encodes a functional IDH, and is involved in virulence as well as bacterial attachment. Furthermore, the icd2 transcription initiation site was mapped at nucleotide G, 127 nucleotide upstream of the icd2 translation start codon. In addition, promoter analysis revealed that icd2 expression exhibits a distinct expression profile under different culture conditions, is subjected to catabolite repression, and is affected by acetate. This is the first time that the function and transcription of icd2 have been characterized in the crucifer pathogen X. campestris pv. campestris.

Gram-negative phytopathogenic Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in crucifers. The ability of Xcc to incite this disease in plants depends on a number of factors, including exopolysaccharides, extracellular enzymes and biofilm production. In this study, transposon mutagenesis led to identification of the prc gene, encoding a tail-specific protease, which plays a role in Xcc pathogenesis. Mutation of prc resulted in decreased virulence, extracellular protease production and bacterial attachment, with restoration to the levels of wild type by the intact prc gene. From subsequent quantitative RT-PCR analysis and reporter assay, the major extracellular protease gene prt1, biofilm-related gene galE encoding a UDP-galactose 4-epimerase and two putative adhesin genes (yapH and XC&#95;4290 encoding autotransporter-like protein H and hemagglutinin, respectively) were found to be reduced in the prc mutant. Results of transcriptome profiling of Xcc wild type and prc mutant by RNA sequencing (RNA-Seq) showed that mutation of prc in Xcc leads to alteration in the transcriptional levels (more than twofold) of 91 genes. These differentially expressed genes were associated with a wide range of biological functions such as carbohydrate transport and metabolism, cell wall/membrane biogenesis, posttranslational modification, protein turnover and chaperones, inorganic ion transport and metabolism and signal transduction mechanisms. The results of this study facilitate the functional understanding of and provide new information about the regulatory role of prc., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Stressful experiences during early life (ELS) can affect brain development, thereby exerting a profound and long-lasting influence on mental development and psychological health. The stress inoculation hypothesis presupposes that individuals who have early experienced an attenuated form of stressors may gain immunity to its more virulent forms later in life. Increasing evidence demonstrates that ELS may promote the development of subsequent stress resistance, but the mechanisms underlying such adaptive changes are not fully understood. The present study evaluated the impact of fragmented dam-pup interactions by limiting the bedding and nesting material in the cage during postnatal days 2-9, a naturalistic animal model of chronic ELS, on the physiological and behavioral responses to different stressors in adolescent mice and characterized the possible underlying mechanisms. We found that ELS mice showed less social interaction deficits after chronic social defeat stress and acute restraint-tailshock stress-induced impaired long-term potentiation (LTP) and enhanced long-term depression (LTD) in hippocampal CA1 region compared with control mice. The effects of ELS on LTP and LTD were rescued by adrenalectomy. While ELS did not cause alterations in basal emotional behaviors, it significantly enhanced stress coping behaviors in both the tail suspension and the forced swimming tests. ELS mice exhibited a significant decrease in corticosterone response and trafficking of glucocorticoid receptors to the nucleus in response to acute restraint stress. Altogether, our data support the hypothesis that stress inoculation training, via early exposure to manageable stress, may enhance resistance to other unrelated extreme stressors in adolescence., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The Gram-negative plant pathogen Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers. The production of Xcc virulence factors is regulated by Clp and RpfF. HD-related output domain (HDOD) is a protein domain of unknown biochemical function. The genome of Xcc encodes three proteins (GsmR, HdpA, and HdpB) with an HDOD. The GsmR has been reported to play a role in the general stress response and cell motility and its expression is positively regulated by Clp. Here, the function and transcription of hdpA and hdpB were characterized. Mutation of hdpA resulted in enhanced bacterial attachment. In addition, the expression of hdpA was positively regulated by RpfF but not by Clp, subject to catabolite repression and affected by several stress conditions. However, mutational analysis and reporter assay showed that hdpB had no effect on the production of a range of virulence factors and its expression was independent of Clp and RpfF. The results shown here not only extend the previous work on RpfF regulation to show that it influences the expression of hdpA in Xcc, but also expand knowledge of the function of the HDOD containing proteins in bacteria.

Cisplatin-based therapy is common in the treatment of several types of cancers, including lung cancers. In our previous study, GMI, an immunomodulatory protein cloned from Ganoderma microsporum, induced a cytotoxic effect in lung cancer cells via autophagy. The aim of this study is to examine the role of GMI in enhancing cisplatin-mediated cell death. On the basis of MTT assay and Combination Index, GMI and cisplatin cotreatment induced a synergistic cytotoxic effect. GMI and cisplatin-induced apoptosis was determined by sub-G1, nuclear condensation, and annexin-V/propidium iodide analyses. On Western blot, expressions of γH2AX and cleaved forms of PARP, caspase-3, and caspase-7 were induced by combined treatment. Akt/mTOR pathway activity, LC3-II expression, and acidic vesicular organelle development demonstrated that cisplatin does not abolish GMI-mediated autophagy. Cyto-ID Green/hoechst 33342 double staining and time-dependent experiment indicated that GMI and cisplatin-treated A549 cells simultaneously express autophagosomes and apoptotic nuclei. To elucidate the role of autophagy in inducing apoptosis by GMI and cisplatin, chemical inhibitors and LC3 shRNA were used to inhibit autophagy. The results showed that 3-methyladenine decreases, while chloroquine increases GMI and cisplatin cotreatment-induced cleavage of caspase-7 and PARP. LC3 silencing abolished activation of apoptosis in A549 cells. Caspase inhibitors and caspase-7 silencing mitigated GMI and cisplatin-elicited cell viability inhibition and apoptosis. This is the first study to reveal the novel function of GMI in potentiating cisplatin-mediated apoptosis. GMI and cisplatin induce apoptosis via autophagy/caspase-7-dependent and survivin- and ERCC1-independent pathway. GMI may be a potential cisplatin adjuvant against lung cancer.

Benzo[a]pyrene (B[a]P) is a potent lung carcinogen derived from tobacco smoking and environmental contamination. This study aimed to investigate the signal transduction pathway responsible for B[a]P-induced non-small cell lung cancer (NSCLC) development. We exposed the human NSCLC cell lines Calu-1, CL3, H1299, CH27, H23, and H1355 to B[a]P and assessed cell cycle progression using flow cytometry. Expression of cell cycle mediators was measured using Western blot analyses and electrophoretic mobility shift assays (EMSAs). B[a]P exposure dramatically induced S-phase accumulation in H1355 cells. Phospho-p53 (Ser15 and Ser20), phospho-ERK, phospho-p38, and Bax were significantly increased in H1355 cells whereas phospho-Rb was decreased in these cells. In addition, B[a]P induced phosphorylation of checkpoint kinase-1 (Chk1) but not Chk2. EMSA experiments revealed a slower migrating band after c-Myc bound the E-box in response to B[a]P treatment, which was abolished upon the addition of the ERK inhibitor PD98059 in H1355 cells. Phospho-ERK inhibition and dominant negative mutant Chk1 expression reversed B[a]P-induced S phase accumulation and downregulated phospho-Chk1 and phospho-ERK expression. Taken together, these results suggest that activation of ERK and its downstream mediator Chk1 may contribute to B[a]P-induced S phase accumulation in H1355 cells. The results could help in the development of lung cancer treatments that target the Chk1 pathway through ERK., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Asthma is a major public health concern. Its greatest risk factor is house dust mite (HDM). Dermatophagoides microceras (Der m) is a type of HDM, and in central Taiwan, there is approximately 80% prevalence of sensitization to Der m. FIP-fve is a fungal immunomodulatory protein (FIP) isolated from the fungus Flammulina velutipes, and exhibits anti-inflammatory properties. To investigate whether FIP-fve affects Der m-induced asthma and inflammation, we evaluated hyper-responsiveness (AHR), pathological changes, and cytokines in mice. We demonstrated that oral FIP-fve decreased Der m-induced airway AHR, airway inflammation, cell infiltration, and expression of cytokines in the bronchoalveolar lavage fluid of Balb/c mice. The results of this study suggest that FIP-fve suppresses asthma, inflammation, and respiratory pathogenesis stimulated by Der m. FIP-fve is able to maintain immunomodulatory activity even in simulated gastric fluid and intestinal fluid. FIP-fve could be a safe and stable agent for suppression of allergic asthma.

Uridine triphosphate (UTP)-glucose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.9) is an enzyme that catalyzes the formation of uridine diphosphate (UDP)-glucose from UTP and glucose-1-phosphate. GalU is involved in virulence in a number of animal-pathogenic bacteria since its product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharide and exopolysaccharide. However, its function in Xanthomonas campestris pv. campestris, the phytopathogen that causes black rot in cruciferous plants, is unclear. Here, we characterized a galU mutant of X. campestris pv. campestris and showed that the X. campestris pv. campestris galU mutant resulted in a reduction in virulence on the host cabbage. We also demonstrated that galU is involved in bacterial attachment, cell motility, and polysaccharide synthesis. Furthermore, the galU mutant showed increased sensitivity to various stress conditions including copper sulfate, hydrogen peroxide, and sodium dodecyl sulfate. In addition, mutation of galU impairs the expression of the flagellin gene fliC as well as the attachment-related genes xadA, fhaC, and yapH. In conclusion, our results indicate involvement of galU in the virulence factor production and pathogenicity in X. campestris pv. campestris, and a role for galU in stress tolerance of this crucifer pathogen.

The Gram-negative plant pathogen Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers, a disease that causes tremendous agricultural loss. In this study, the Xcc galE gene was characterized. Sequence and mutational analysis demonstrated that the Xcc galE encodes a UDP-galactose 4-epimerase (EC 5.1.3.2), which catalyzes the interconversion of UDP-galactose and UDP-glucose. Alanine substitution of the putative catalytic residues (Ser124, Tyr147, and Lys151) of GalE caused loss of epimerase activity. Further study showed that the Xcc galE mutant had reduced biofilm formation ability. Furthermore, reporter assays revealed that galE transcription exhibits a distinct expression profile under different culture conditions, is subject to catabolite repression, and is positively regulated by Clp and RpfF. In addition, the galE transcription initiation site was mapped. This is the first time that UDP-galactose 4-epimerase has been characterized in the crucifer pathogen Xcc., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

FIP-fve is an immunomodulatory protein isolated from Flammulina velutipes that possesses anti-inflammatory and immunomodulatory activities. However, little is known about its anticancer effects. It is suppressed cell proliferation of A549 lung cancer cells on MTT assay following 48 h treatment of FIP-fve. FIP-fve treatment also resulted in cell cycle arrest but not apoptosis on flow cytometry. This immunomodulatory protein was observed to increase p53 expression, as well as the expression of its downstream gene p21, on Western blot. FIP-fve inhibited migration of A549 cells on wound healing assay and decreased filopodia fiber formation on labeling with Texas Red-X phalloidin. To confirm the effect of FIP-fve on the role of Rac1 in filopodia formation, we investigated the activity of Rac1 in A549 cells following FIP-fve treatment. FIP-fve inhibited EGF-induced activation of Rac1. We demonstrated that FIP-fve decreases RACGAP1 mRNA and protein levels on RT-PCR and Western blot. In addition, the reporter activity of RACGAP1 was reduced by FIP-fve on RacGAP1 promoter assay. Silencing of RacGAP1 decreased cell migration, and overexpression of RacGAP1 increased cell migration in A549 cells. In conclusion, FIP-fve inhibits lung cancer cell migration via RacGAP1 and suppresses the proliferation of A549 via p53 activation pathway.

Previously we identified anterior localization of hunchback (Aphb) mRNA in oocytes and early embryos of the parthenogenetic and viviparous pea aphid Acyrthosiphon pisum, suggesting that the breaking of anterior asymmetry in the oocytes leads to the formation of the anterior axis in embryos. In order to study posterior development in the asexual pea aphid, we cloned and analysed the developmental expression of caudal (Apcad), a posterior gene highly conserved in many animal phyla. We found that transcripts of Apcad were not detected in germaria, oocytes and embryos prior to the formation of the blastoderm in the asexual (viviparous) pea aphid. This unusual expression pattern differs from that of the existing insect models, including long- and short-germ insects, where maternal cad mRNA is passed to the early embryos and forms a posterior-anterior gradient. The first detectable Apcad expression occurred in the newly formed primordial germ cells and their adjacent blastodermal cells during late blastulation. From gastrulation onward, and as in other insects, Apcad mRNA is restricted to the posteriormost region of the germ band. Similarly, in the sexual (oviparous) oocytes we were able to identify anterior localization of Aphb mRNA but posterior localization of Apcad was not detected. This suggests that cad-driven posterior development is not conserved during early embryogenesis in asexual and sexual pea aphids., (© 2013 Royal Entomological Society.)

In prokaryotes, two-component signal transduction systems, consisting of a histidine kinase and a response regulator, play a critical role in regulating a range of cellular functions. A recent study suggests that XCC3315, a response regulator with a CheY-like receiver domain attached to an uncharacterized HD-related output domain (HDOD domain), plays a role in the general stress response of the Gram-negative bacterium Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in cruciferous plants. Here, we demonstrated genetically that XCC3315, designated as gsmR (general stress and motility regulator), is involved in the expression of genes responsible for flagellum synthesis, including rpoN2, flhF, flhB, and fliC. Site-directed mutagenesis revealed that Glu9 and Arg100 in the receiver domain and Gly205, Asp263, His287, Trp298 and His311 in the HDOD are critical amino acids for GsmR function in cell motility regulation. The gsmR transcription initiation site was mapped. Promoter analysis and gel retardation assay revealed that the expression of gsmR is positively controlled by the global transcriptional regulator Clp in a direct manner, and is subject to catabolite repression. Our findings not only extend the previous work on Clp regulation to show that it influences the expression of gsmR in Xcc, but are also the first to characterize the expression of this response regulator gene in this phytopathogen. Furthermore, GsmR is the first HDOD-containing protein of bacteria in which key amino acids have been experimentally identified and characterized., (© 2012 The Authors Journal compilation © 2012 FEBS.)

The nucleotide cyclic di-GMP is a second messenger in bacteria that regulates a range of cellular functions including the virulence of pathogens. GGDEF is a protein domain involved in the synthesis of cyclic di-GMP. The genome of the crucifer pathogen Xanthomonas campestris pv. campestris (Xcc) encodes 21 proteins with a GGDEF domain. Clp, a homolog of the model transcription factor Crp of Escherichia coli, is a global regulator in Xcc. The aim of this study is to identify genes encoding GGDEF domain proteins whose expression is regulated by Clp. Results of reporter assay and RT-PCR analysis suggested that Clp regulates the expression of a set of genes encoding proteins harboring GGDEF domain. The transcription initiation site of XCC1294, one of the Clp regulated gene encoding a GGDEF domain protein, was mapped. Promoter analysis and gel retardation assay indicated that the transcription of XCC1294 is positively and directly regulated by Clp. Furthermore, transcription of XCC1294 was subject to catabolite repression and affected by several stress conditions. We also showed that mutation of XCC1294 results in enhanced surface attachment. In addition, transcription of three putative adhesin genes (xadA, fhaC, and yapH) was increased in the XCC1294 mutant. Taken together, the data presented here indicate that Clp positively regulates expression of XCC1294, and that XCC1294 serves a regulator of bacterial attachment and regulates different adhesin genes expression.

In Xanthomonas campestris pv. campestris (Xcc), which is the causative agent of black rot in crucifers, the virulence factor level is substantially decreased in the mutant deficient in RpfG, a phosphodiesterase that degrades the second messenger cyclic di-GMP. The rpfG mutant also grew in an aggregated state. It is indicated that expression of Pseudomonas GGDEF domain protein WspR (a diguanylate cyclase that synthesizes cyclic di-GMP) in wild-type Xcc can produce a phenocopy of the rpfG mutant. In this study, we showed that over-expression of GGDEF domain protein XCC2731 in wild-type Xcc caused (i) aggregation of cells, (ii) reduction in motility, and (iii) decrease in production of virulence factor extracellular enzymes and exopolysaccharides. Site-directed mutagenesis of the conserved G, G, and E residues of the GGDEF domain in XCC2731 abolished its function. The XCC2731 mutant has attenuated virulence. Furthermore, XCC2731 mutant was affected in surface attachment. Using the 5' RACE method, the XCC2731 transcription initiation site was mapped at nucleotide G, 15nt upstream of the XCC2731 start codon. Transcriptional fusion assay and gel retardation analysis indicated that Clp (cAMP receptor protein-like protein) positively regulates XCC2731 transcription in a direct manner. Reporter analysis also revealed that XCC2731 transcription is subject to catabolite repression, and reduced under conditions of oxygen limitation and high osmolarity. Our findings not only extend previous work on Clp regulation to show that it influences the expression of XCC2731 in Xcc, but also are the first to characterize the GGDEF domain protein gene expression in this phytopathogen., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Vorinostat (suberoylanilide hydroxamic acid), a class of histone deacetylase inhibitors, represents an emerging class of anticancer agents currently progressing in clinical trials. It causes cell growth inhibition, differentiation, and apoptosis of many tumor types in vitro and in vivo. Recently, it was reported that hTERT is one of the targets for cancer therapy in cancer cells. Telomerase repeat amplification protocol assay was used to analyze the expression of hTERT after vorinostat treatment in the A549 lung cancer cells. Vorinostat inhibited telomerase activity by reducing the expression of human telomerase reverse transcriptase (hTERT) in A549 human lung cancer cells. The epigenetic regulation mechanism is responsible for the repression of hTERT by vorinostat, analyzed through the methylation-specific PCR and bisulfite sequencing of the hTERT promoter. Vorinostat induced the demethylation of site-specific CpGs on the promoter region of hTERT, which was caused by the down-regulation of DNA methyltransferases. DNA methyltransferases (DNMT1 and DNMT3b) were also decreased in vorinostat-treated A549 cancer cells. Furthermore, chromatin immunoprecipitation analysis of the hTERT promoter revealed that vorinostat decreased the level of inactive chromatin markers dimethyl-H3K9, and the declined binding of DNMT1 and DNMT3b were associated. The novel insights showed that vorinostat down-regulated telomerase via epigenetic alteration in lung cancer to vorinostat-mediated cancer-specific therapies., (Copyright © 2011 Wiley-Liss, Inc.)

The gram-negative plant pathogenic Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers, a disease causing tremendous loss in agriculture. Copper-containing bactericides have been widely used to control this disease for many years, possibly leading to the development of copper resistance in Xcc. Homologues of copper resistance genes copLAB are present in the Xcc genome, but none has been characterized. In this study, mutations in copL, copA, and copB decreased Xcc copper tolerance. Among them, the copA mutant displayed the most significant reduction. The copA mutant also resulted in a reduction in virulence on the host cabbage. Sequence and mutational analysis demonstrated that copA encodes a multicopper oxidase and that CopA is able to catalyze the oxidation of 2,6-dimethoxyphenol. Alanine substitutions in each of the putative copper binding residues (H538, H583, C584, and H585) of CopA caused a loss of function including copper tolerance and oxidase activity. Furthermore, reporter assays showed that copA transcription is inducible in the presence of copper, subject to catabolite repression, and repressed under conditions of high osmolarity, nitrogen starvation, or oxygen limitation. This is the first time that multicopper oxidase has been characterized in the crucifer pathogen Xcc.

Exopolysaccharides and several extracellular enzymes of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, are virulence determinants. It is known that Clp (cAMP receptor protein-like protein) and RpfF (an enoyl-CoA hydratase homologue required for the synthesis of the diffusible signal factor, DSF) regulate production of these factors. In this study, plate assay revealed that Xcc possesses pectin methylesterase activity and that its expression is controlled by Clp and RpfF. Mutational analysis has demonstrated that pmeA encodes a pectin methylesterase. Using the 5' RACE method, the pmeA transcription initiation site was mapped. Transcriptional fusion assays showed that pmeA transcription is positively regulated by Clp and RpfF, subject to catabolite repression which is independent of Clp or RpfF, and repressed under conditions of high osmolarity or oxygen limitation. This study not only extends previous work on Clp and RpfF regulation by showing that they both influence the expression of pmeA in Xcc, but also, for the first time, characterizes pectin methylesterase gene expression in Xanthomonas., (Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

Matrix metalloproteinase 9 (MMP-9) has been implicated in airway injury in chronic obstructive pulmonary disease (COPD), lung inflammation, and lung cancer and plays a major role in tumor necrosis factor-α (TNF-α)-stimulated tumor invasion and lung inflammation. MMP-9 activity is promoted by the pro-inflammatory cytokine TNF-α. GMI, cloned from Ganoderma microsporum and purified, is one of the recombinant fungal immunomodulatory proteins. To understand the molecular mechanisms involved in the suppression of TNF-α-mediated tumor invasion and inflammation, GMI modulation of this pathway was investigated in human alveolar epithelial A549 cells in this study. GMI exhibited an inhibitory effect on TNF-α-induced invasion, with GMI treatment and TNF-α exposure presenting the most anti-invasive properties on Boyden chamber assay. GMI reduced TNF-α-induced MMP-9 activities on gelatin zymography assay through inhibition of MMP-9 transcriptional activity. RT-PCR and MMP-9 promoter luciferase analysis revealed that GMI inhibits the transcription of MMP-9 mRNA. Moreover, in vitro and in vivo binding experiments, an electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation assay (ChIP) demonstrated that GMI suppresses DNA binding of nuclear factor (NF)-κB transcription factors to MMP-9 promoter. Western blot analysis indicated that GMI blocks the phosphorylation and degradation of IκBα, which in turn leads to suppression of the phosphorylation and nuclear translocation of p65. Thus, overall, our results indicated that GMI mediates antitumor invasion and anti-inflammatory effects through modulation of NF-κB/MMP-9 pathways.

Biosynthesis of xanthan polysaccharide, a virulence factor of phytopathogenic Xanthomonas campestris pv. campestris (Xcc), involves the gum operon and the cyclic AMP receptor protein (CRP) homologue Clp. Clp was shown to have the same DNA binding specificity as the CRP at positions 5, 6, and 7 (GTG motif) of the left arm. Therefore, Clp binding sites (CBSs) have typically been identified by pattern searching of the Xcc genome using the consensus CRP binding sequence. Here, results of a reporter assay and electrophoretic mobility shift assay suggest that Clp upregulates the gum operon by binding to two non-consensus sites, in which a more conserved right arm may compensate for the lack of conservation in the left arm, a high GC content in the central region (6 bp) may be important for binding, and binding may be enhanced if the GC-rich central region is palindromic. These suggest that atypical CBSs exist in Xcc promoters and that Clp, while retaining the capacity to bind typical CBSs, has evolved to bind atypical CBS because: 1) Clp shares only moderate homology with the CRP and is modulated by cyclic di-GMP; and 2) Xcc has a higher GC content (65%) than Escherichia coli (50%)., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

Background: Pemetrexed disodium (Alimta), LY231514, is an antifolate that is able to simultaneously inhibit the synthesis of purines and pyrimidines. Pemetrexed has been approved for first- and second-line treatment in patients with non-small cell lung cancer (NSCLC). However, there is still a lack of clinical biomarkers for predicting the therapeutic response to pemetrexed. The aim of this study is to establish new biomarkers for pemetrexed treatment in NSCLC., Methods: Human NSCLC cell lines were exposed to pemetrexed. The antitumor effect was measured by growth inhibition with MTT assay and expression of cell cycle mediators with immunoblots. Using the Superarray cancer pathway gene array, 482 genes were screened for differential expression in A549 cells that were untreated or treated with pemetrexed., Results: A549 cells exhibited sensitivity but H1355 cells showed resistance to pemetrexed. To investigate the mechanisms of responsiveness and nonresponsiveness to pemetrexed in these cell lines, we measured the expression levels of thymidylate synthase (TS), dihydrofolate reductase (DHFR), reduced folate carrier, and folylpoly-gamma-glutamate synthetase genes. TS, DHFR, and reduced folate carrier gene expressions were significantly reduced in A549 and H1355 cells. Pemetrexed caused cell cycle arrest in the G1 phase and S phase in H1355 and A549 cells, respectively. Significantly higher expressions of many genes, especially lipocalin-2 (Lcn-2) and nm23-H1 proteins, were noted in A549 cells treated with pemetrexed in comparison with untreated cells. Furthermore, reverse transcriptase polymerase chain reaction and Western blot showed that Lcn-2 and nm23-H1 expressions increase in response to pemetrexed treatment in a dose-responsive manner in pemetrexed-sensitive A549 cells but not in resistant H1355 cells., Conclusions: Our results indicated that downregulation of TS and DHFR genes and upregulation of p21, p27, Lcn-2, and nm23-H1 genes may serve as new biomarkers for predicting responsiveness to pemetrexed.

Exopolysaccharide and several extracellular enzymes of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, are important virulence determinants. It is known that Clp (cAMP receptor protein-like protein) and RpfF (an enoyl-CoA hydratase homologue required for the synthesis of diffusible signal factor, DSF) regulate the production of these determinants. Addition of DSF or Xcc extracellular protein containing partially purified mannanase (EC 3.2.1.78, encoded by manA) can disperse the cell aggregates formed by rpfF mutant. In this study, nucleotide G 64 nt upstream of the manA translation start codon was determined as the transcription initiation site by the 5' RACE technique. Transcriptional fusion assays showed that manA transcription is positively regulated by Clp and RpfF and induced by locust bean gum. The manA coding region was cloned and expressed in E. coli as recombinant ManA (rManA). The rManA was purified by affinity chromatography, and its biochemical properties were characterized. The rManA had a pH optimum at 7.0 (0.1 M Hepes) and a temperature optimum at about 37 degrees C. Sequence and mutational analyses demonstrated that Xcc manA encodes the major mannanase, a member of family 5 of glycosyl hydrolases. This study not only extends previous work on Clp and RpfF regulation by showing that they both influence the expression of manA in Xcc, but it also for the first time characterizes Xanthomonas mannanase at the protein level.

Fungal immunomodulatory protein, FIP-fve, has been isolated from Flammulina velutipes, and its immunomodulatory effects are believed to be associated with the enhanced activation of IFN-gamma-releasing Th1 cells. However, the mechanisms of FIP-fve-mediated signal transduction in the regulation of interferon-gamma (IFN-gamma) gene expression in human peripheral blood mononuclear cells (PBMCs) are still poorly understood. Using fluo-3 AM, we found that FIP-fve induces a rapid elevation in calcium concentration. ELISA, RT-PCR and Western blot assays demonstrated significant increases in the production and mRNA expression of IFN-gamma and protein kinase C-alpha (PKC-alpha) activation in activated PBMCs, which were abolished by EGTA, nifedipine and GO6976. In conclusion, Ca2+ release and PKC-alpha activation are required for IFN-gamma production induced by FIP-fve in PBMCs.

Exopolysaccharide and several extracellular enzymes of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, are virulence determinants. In this study, two Xcc annotated extracellular pectate lyase genes, pelA1 and pelA2, belonging to family 1 of the polysaccharide lyase, were characterized. Sequence and mutational analyses have demonstrated that pelA1 encodes the major pectate lyase, whereas pelA2 is not transcribed. Using the 5' RACE method, the pelA1 transcription initiation site was mapped at nucleotide G, 103 nt upstream of the pelA1 start codon. Promoter analysis demonstrated that polygalacturonic acid and CaCl(2) induce the expression of pelA1. Transcriptional fusion assays also indicated that Clp (cAMP receptor protein-like protein) and RpfF (an enoyl-CoA hydratase homologue that is required for the synthesis of cis-11-methyl-2-dodecenoic acid, a low molecular weight diffusible signal factor, DSF) positively regulate pelA1 transcription. Gel retardation assays showed that Clp exerts a positive control over expression of pelA1 by direct binding to the upstream Clp-binding site. In conclusion, the present research demonstrated that pelA1 codes for the major pectate lyase in Xcc strain Xc17 and that its expression is up-regulated by Clp and RpfF. This is the first study to characterize pectate lyase gene expression in Xcc.

Introduction: In locations that lack nearby cyclotron facilities for radionuclide production, generator-based (68)Ga radiopharmaceuticals might have clinical utility for positron emission tomography (PET) studies of myocardial perfusion and other physiological processes., Methods: The lipophilic and monocationic (67)Ga-labeled gallium chelates of five novel hexadentate bis(salicylaldimine) ligands the bis(salicylaldimine), bis(3-methoxysalicylaldimine), bis(4-methoxysalicylaldimine), bis(6-meth,oxysalicylaldimine), and bis(4,6-dimethoxysalicylaldimine) of N,N'-bis(3-aminopropyl)-N,N'-dimethylethylenediamine (BAPDMEN), were prepared. The structure of the unlabeled [Ga(4-MeOsal)(2)BAPDMEN](+)PF(6)(-) salt was determined by X-ray crystallography, and the biodistribution of each of the (67)Ga-labeled gallium chelates was determined in rats following intravenous administration and compared with the biodistribution of [(86)Rb]rubidium chloride., Results: The [Ga(4-MeOsal)(2)BAPDMEN](+)PF(6)(-) complex exhibited the expected pseudo-octahedral N(4)O(2)(2-) coordination sphere about the Ga(3+) center with a trans disposition of the phenolate oxygen atoms. All five (67)Ga radiopharmaceuticals were found to afford the desired myocardial retention of the radiogallium. The [(67/68)Ga][Ga(3-MeOsal)(2)BAPDMEN](1+) radiopharmaceutical appears to have the best properties for myocardial imaging, exhibiting 2% of the injected dose in the heart 1 min and 2 h postinjection and very high heart/nontarget ratios (heart/blood ratios of 7.6+/-1.0 and 54+/-10 at 1 and 120 min, respectively; heart/liver ratios of 1.8+/-0.4 and 39+/-3 at 1 and 120 min, respectively)., Conclusions: Most of these new agents, particularly [(67/68)Ga][Ga(3-MeOsal)(2)BAPDMEN](1+), would appear superior to previously reported bis(salicylaldimine) ligands of N,N'-bis(3-aminopropyl)ethylenediamine as candidates for PET imaging of the heart with (68)Ga.

Purified recombinant fungal immunomodulatory protein from Ganoderma tsugae (reFIP-gts) has anti-telomerase effects in human lung adenocarcinoma A549 cells. However, how reFIP-gts affects cancer cell fates remains unclear. Here, we demonstrated that reFIP-gts-treated lung cancer cells are arrested at G1 phase by flow cytometry and possess morphological phenotype consistent with cellular senescence. The senescent nature of these cells was supported by positive staining for senescence-associated beta-galactosidase activity and increased lysosomal content in A549 and CaLu-1 lung cancer cells. Arrest of cells at G1 appears to be the key means through which reFIP-gts induces premature cellular senescence in A549 cells. Finally, reFIP-gts- treated A549 cells grew more slowly and formed significantly fewer cell colonies in soft agar than untreated A549 cells. In an in vivo mouse model, A549 cells treated with reFIP-gts grew significantly slower than cells treated with PBS alone, confirming that lung tumor can be inhibited by reFIP-gts. The use of reFIP-gts may be a powerful new strategy for chemoprevention and antineoplastic therapy.

Exopolysaccharide and several extracellular enzymes of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, are virulence factors. In this study, sequence and mutational analysis has demonstrated that Xcc pehA encodes the major polygalacturonase, a member of family 28 of the glycosyl hydrolases. Using the 5' RACE (rapid amplification of cDNA ends) method, the pehA transcription initiation site was mapped at 102 nt downstream of a Clp (cAMP receptor protein-like protein)-binding site. Transcriptional fusion assays showed that pehA transcription is greatly induced by polygalacturonic acid, positively regulated by Clp and RpfF (an enoyl-CoA hydratase homologue which is required for the synthesis of cis-11-methyl-2-dodecenoic acid, a low-molecular-mass diffusible signal factor), subjected to catabolite repression, which is independent of Clp or RpfF, and repressed under conditions of oxygen limitation or nitrogen starvation. Our findings extend previous work on Clp and RpfF regulation to show that they both influence the expression of pehA in Xcc.

Recombinant fungal immunomodulatory protein, reFIP-gts, was cloned from Ganoderma tsugae and purified. In our previous study, it was shown that reFIP-gts has anti-telomerase effects in A549 cells. Here, we proved that reFIP-gts entry into the cell and localization in endoplasmic reticulum can result in ER stress, thereby increasing ER stress markers (CHOP/GADD153) and intracellular calcium release in A549 cells. The use of calcium chelator restores reFIP-gts-mediated reduction in telomerase activity. These results strongly suggest that ER stress induces intracellular calcium release and results in inhibition of telomerase activity. Although reFIP-gts decreased hTERT mRNA level in both A549 and H1299 cells, only the telomerase activity in A549 cells was inhibited. Surprisingly, we found that reFIP-gts induces translocation of hTERT from the nucleus into the cytosol in A549 cells but not in H1299 cells. Using leptomycin B, nuclear export inhibitor, we showed that hTERT is not transported. Using MG132, a proteasome inhibitor, reFIP-gts also prevents hTERT translocation from proteasome degradation. Taken together, these results indicate that reFIP-gts inhibits telomerase activity in lung cancer cells through nuclear export mechanisms, which might be mediated by ER stress-induced intracellular calcium level.