Bartonella species in fleas from Palestinian territories: Prevalence and genetic diversity

Bartonellosis is an infectious bacterial disease. The prevalence and genetic characteristics of Bartonella spp. in fleas of wild and domestic animals from Palestinian territories are described. Flea samples (n=289) were collected from 121 cats, 135 dogs, 26 hyraxes and seven rats from northern (n=165), central (n=113), and southern Palestinian territories (n=11). The prevalent flea species were: Ctenocephalides felis (n=119/289; 41.2%), Ctenocephalides canis (n=159/289; 55%), and Xenopsylla sp. (n=7/289; 2.4%). Targeting the Intergenic Transcribed Spacer (ITS) locus, DNA of Bartonella was detected in 22% (64/289) of all fleas. Fifty percent of the C. felis and 57% of the Xenopsylla sp. contained Bartonella DNA. DNA sequencing showed the presence of Bartonella clarridgeiae (50%), Bartonella henselae (27%), and Bartonella koehlerae (3%) in C. felis. Xenopsylla sp. collected from Rattus rattus rats were infected with Bartonella tribocorum, Bartonella elizabethae, and Bartonella rochalimae. Phylogenetic sequence analysis using the 16S ribosomal RNA gene obtained four genetic clusters, B. henselae and B. koehlerae as subcluster 1, B. clarridgeiae as cluster 2, while the rat Bartonella species (B. tribocorum and B. elizabethae) were an outgroup cluster. These findings showed the important role of cat and rat fleas as vectors of zoonotic Bartonella species in Palestinian territories. It is hoped that this publication will raise awareness among physicians, veterinarians, and other health workers of the high prevalence of Bartonella spp. in fleas in Palestinian territories and the potential risk of these pathogens to humans and animals in this region.     

Plant polyphenols mobilize endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for anticancer properties

Plant polyphenols are important components of human diet and a number of them are considered to possess chemopreventive and therapeutic properties against cancer. They are recognized as naturally occurring antioxidants but also act as prooxidants catalyzing DNA degradation in the presence of transition metal ions such as copper. Using human peripheral lymphocytes and Comet assay we have previously confirmed that resveratrol-Cu(II) is indeed capable of causing DNA degradation in cells. In this paper we show that the polyphenols alone (in the absence of added copper) are also capable of causing DNA breakage in cells. Incubation of lymphocytes with neocuproine inhibited the DNA degradation confirming that Cu(I) is an intermediate in the DNA cleavage reaction. Further, we have also shown that polyphenols generate oxidative stress in lymphocytes which is inhibited by scavengers of reactive oxygen species and neocuproine. These results are in further support of our hypothesis that anticancer mechanism of plant polyphenols involves mobilization of endogenous copper, possibly chromatin bound copper, and the consequent prooxidant action.     

Microbial lipases: at the interface of aqueous and non-aqueous media. A review

In recent times, biotechnological applications of microbial lipases in synthesis of many organic molecules have rapidly increased in non-aqueous media. Microbial lipases are the 'working horses' in biocatalysis and have been extensively studied when their exceptionally high stability in non-aqueous media has been discovered. Stability of lipases in organic solvents makes them commercially feasibile in the enzymatic esterification reactions. Their stability is affected by temperature, reaction medium, water concentration and by the biocatalyst's preparation. An optimization process for ester synthesis from pilot scale to industrial scale in the reaction medium is discussed. The water released during the esterification process can be controlled over a wide range and has a profound effect on the activity of the lipases. Approaches to lipase catalysis like protein engineering, directed evolution and metagenome approach were studied. This review reports the recent development in the field ofnon-aqueous microbial lipase catalysis and factors controlling the esterification/transesterification processes in organic media.     

ESCRT-III family members stimulate Vps4 ATPase activity directly or via Vta1

The AAA-ATPase Vps4 is critical for function of the MVB sorting pathway, which in turn impacts cellular phenomena ranging from receptor downregulation to viral budding to cytokinesis. Vps4 dissociates ESCRTs from endosomal membranes during MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. Vta1 potentiates Vps4 activity and interacts with ESCRT-III family members. We have investigated the impact of Vta1 and ESCRT-III family members on Vps4 ATPase activity. Two distinct mechanisms of Vps4 stimulation are described: Vps2 can directly stimulate Vps4 via its MIT domain, whereas Vps60 stimulates via Vta1. Moreover, Did2 can stimulate Vps4 by both mechanisms in distinct contexts. Recent structural determination of the ESCRT-III-binding region of Vta1 unexpectedly revealed a MIT-like region. These data support a model wherein a network of MIT and MIT-like domain interactions with ESCRT-III subunits contributes to the regulation of Vps4 activity during MVB sorting.     

S-adenosylmethionine decarboxylase degradation by the 26 S proteasome is accelerated by substrate-mediated transamination

The short-lived enzyme S-adenosylmethionine decarboxylase uses a covalently bound pyruvoyl cofactor to catalyze the formation of decarboxylated S-adenosylmethionine, which then donates an aminopropyl group for polyamine biosynthesis. Here we demonstrate that S-adenosylmethionine decarboxylase is ubiquitinated and degraded by the 26 S proteasome in vivo, a process that is accelerated by inactivation of S-adenosylmethionine decarboxylase by substrate-mediated transamination of its pyruvoyl cofactor. Proteasome inhibition in COS-7 cells prevents the degradation of S-adenosylmethionine decarboxylase antigen; however, even brief inhibition of the 26 S proteasome caused substantial losses of S-adenosylmethionine decarboxylase activity despite accumulation of S-adenosylmethionine decarboxylase antigen. Levels of the enzyme's substrate (S-adenosylmethionine) increased rapidly after 26 S proteasome inhibition, and this increase in substrate level is consistent with the observed loss of activity arising from an increased rate of inactivation by substrate-mediated transamination. Evidence is also presented that this substrate-mediated transamination accelerates normal degradation of S-adenosylmethionine decarboxylase, as the rate of degradation of the enzyme was increased in the presence of AbeAdo (5'-([(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine) (a substrate analogue that transaminates the enzyme); conversely, when the intracellular substrate level was reduced by methionine deprivation, the rate of degradation of the enzyme was decreased. Ubiquitination of S-adenosylmethionine decarboxylase is demonstrated by isolation of His-tagged AdoMetDC (S-adenosylmethionine decarboxylase) from COS-7 cells co-transfected with hemagglutinin-tagged ubiquitin and showing bands that were immunoreactive to both anti-AdoMetDC antibody and anti-hemagglutinin antibody. This is the first study to demonstrate that AdoMetDC is ubiquitinated and degraded by the 26 S proteasome, and substrate-mediated acceleration of degradation is a unique finding.     

Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle

The mitotic inducer gene from Schizosaccharomyces pombe, Spcdc25, was used as a tool to investigate regulation of G2/M in higher plants using the BY-2 (Nicotiana tabacum) cell line as a model. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size through a shortening of the G2 phase. The cells often formed isodiametric double files both in BY-2 cells and in cell suspensions derived from 35S::Spcdc25 tobacco plants. In Spcdc25-expressing cells, the tobacco cyclin-dependent kinase, NtCDKB1, showed high activity in early S phase, S/G2 and early M phase, whereas in empty vector cells CDKB1 activity was transiently high in early S phase but thereafter remained lower. Spcdc25-expressing cells also bypassed a block on G2/M imposed by the cytokinin biosynthetic inhibitor lovastatin (LVS). Surprisingly, cytokinins were at remarkably low levels in Spcdc25-expressing cells compared with the empty vector, explaining why these cells retained mitotic competence despite the presence of LVS. In conclusion, synchronised Spcdc25-expressing BY-2 cells divided prematurely at a small cell size, and they exhibited premature, but sustained, CDKB1 activity even though endogenous cytokinins were virtually undetectable.     

Cancer-Associated Fibroblasts Promote Proliferation of Endometrial Cancer Cells

Endometrial cancer is the most commonly diagnosed gynecologic malignancy worldwide; yet the tumor microenvironment, especially the fibroblast cells surrounding the cancer cells, is poorly understood. We established four primary cultures of fibroblasts from human endometrial cancer tissues (cancer-associated fibroblasts, CAFs) using antibody-conjugated magnetic bead isolation. These relatively homogenous fibroblast cultures expressed fibroblast markers (CD90, vimentin and alpha-smooth muscle actin) and hormonal (estrogen and progesterone) receptors. Conditioned media collected from CAFs induced a dose-dependent proliferation of both primary cultures and cell lines of endometrial cancer in vitro (175%) when compared to non-treated cells, in contrast to those from normal endometrial fibroblast cell line (51%) (P<0.0001). These effects were not observed in fibroblast culture derived from benign endometrial hyperplasia tissues, indicating the specificity of CAFs in affecting endometrial cancer cell proliferation. To determine the mechanism underlying the differential fibroblast effects, we compared the activation of PI3K/Akt and MAPK/Erk pathways in endometrial cancer cells following treatment with normal fibroblasts- and CAFs-conditioned media. Western blot analysis showed that the expression of both phosphorylated forms of Akt and Erk were significantly down-regulated in normal fibroblasts-treated cells, but were up-regulated/maintained in CAFs-treated cells. Treatment with specific inhibitors {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and U0126 reversed the CAFs-mediated cell proliferation (P<0.0001), suggesting for a role of these pathways in modulating endometrial cancer cell proliferation. Rapamycin, which targets a downstream molecule in PI3K pathway (mTOR), also suppressed CAFs-induced cell proliferation by inducing apoptosis. Cytokine profiling analysis revealed that CAFs secrete higher levels of macrophage chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, RANTES and vascular endothelial growth factor (VEGF) than normal fibroblasts. Our data suggests that in contrast to normal fibroblasts, CAFs may exhibit a pro-tumorigenic effect in the progression of endometrial cancer, and PI3K/Akt and MAPK/Erk signaling may represent critical regulators in how endometrial cancer cells respond to their microenvironment.     

Hypoxia Induced Aggressiveness of Prostate Cancer Cells Is Linked with Deregulated Expression of VEGF, IL-6 and miRNAs That Are Attenuated by CDF

Tumor hypoxia with deregulated expression of hypoxia inducing factor (HIF) and its biological consequence leads to poor prognosis of patients diagnosed with solid tumors, resulting in higher mortality, suggesting that understanding of the molecular relationship of hypoxia with other cellular features of tumor aggressiveness would be invaluable for developing newer targeted therapy for solid tumors. Emerging evidence also suggest that hypoxia and HIF signaling pathways contributes to the acquisition of epithelial-to-mesenchymal transition (EMT), maintenance of cancer stem cell (CSC) functions, and also maintains the vicious cycle of inflammation, all of which contribute to radiation therapy and chemotherapy resistance. However, the detailed mechanisms by which hypoxia/HIF drive these events are not fully understood. Here, we have shown that hypoxia leads to increased expression of VEGF, IL-6, and CSC marker genes such as Nanog, Oct4 and EZH2, and also increased the expression of miR-21, an oncogenic miRNA, in prostate cancer (PCa) cells (PC-3 and LNCaP). The treatment of PCa cells with CDF, a novel Curcumin-derived synthetic analogue previously showed anti-tumor activity in vivo, inhibited the productions of VEGF and IL-6, and down-regulated the expression of Nanog, Oct4, EZH2 mRNAs, as well as miR-21 under hypoxic condition. Moreover, CDF treatment of PCa cells led to decreased cell migration under hypoxic condition. Taken together, these results suggest that the anti-tumor effect of CDF is in part mediated through deregulation of tumor hypoxic pathways, and thus CDF could become useful for cancer therapy.     

Helicobacter pylori Eradication Causes Perturbation of the Human Gut Microbiome in Young Adults

Background

Accumulating evidence shows that Helicobacter pylori protects against some metabolic and immunological diseases in which the development of these diseases coincide with temporal or permanent dysbiosis. The aim of this study was to assess the effect of H. pylori eradication on the human gut microbiome.

Methods

As part of the currently on-going ESSAY (Eradication Study in Stable Adults/Youths) study, we collected stool samples from 17 H. pylori-positive young adult (18–30 years-old) volunteers. The same cohort was followed up 6, 12 and 18 months-post H. pylori eradication. The impact of H. pylori on the human gut microbiome pre- and post-eradication was investigated using high throughput 16S rRNA gene (V3-V4 region) sequencing using the Illumina Miseq followed by data analysis using Qiime pipeline.

Results

We compared the composition and diversity of bacterial communities in the fecal microbiome of the H. pylori-positive volunteers, before and after H. pylori eradication therapy. The 16S rRNA gene was sequenced at an average of 150,000–170,000 reads/sample. The microbial diversity were similar pre- and post-H. pylori eradication with no significant differences in richness and evenness of bacterial species. Despite that the general profile of the gut microbiome was similar pre- and post-eradication, some changes in the bacterial communities at the phylum and genus levels were notable, particularly the decrease in relative abundance of Bacterioidetes and corresponding increase in Firmicutes after H. pylori eradication. The significant increase of short-chain fatty acids (SCFA)-producing bacteria genera could also be associated with increased risk of metabolic disorders.

Conclusions

Our preliminary stool metagenomics study shows that eradication of H. pylori caused perturbation of the gut microbiome and may indirectly affect the health of human. Clinicians should be aware of the effect of broad spectrum antibiotics used in H. pylori eradication regimen and be cautious in the clinical management of H. pylori infection, particularly in immunocompromised patients.