The impact of ß-glucan on the therapeutic outcome of experimental Trichinella spiralis infection.

Trichinellosis is a cosmopolitan zoonosis that is caused mainly by Trichinella spiralis infection. The human disease ranges from mild to severe and fatality may occur. The treatment of trichinellosis still presents a challenge for physicians. Anti-inflammatory drugs are usually added to antiparasitic agents to alleviate untoward immuno-inflammatory responses and possible tissue damage but they are not without adverse effects. Thus, there is a need for the discovery of safe and effective compounds with anti-inflammatory properties. This study aimed to evaluate the activity of ß-glucan during enteral and muscular phases of experimental T. spiralis infection as well as its therapeutic potential as an adjuvant to albendazole in treating trichinellosis. For this aim, mice were infected with T. spiralis and divided into the following groups: early and late ß-glucan treatment, albendazole treatment, and combined treatment groups. Infected mice were subjected to assessment of parasite burden, immunological markers, and histopathological changes in the small intestines and muscles. Immunohistochemical evaluation of NF-κB expression in small intestinal and muscle tissues was carried out in order to investigate the mechanism of action of ß-glucan. Interestingly, ß-glucan potentiated the efficacy of albendazole as noted by the significant reduction of counts of muscle larvae. The inflammatory responses in the small intestine and skeletal muscles were mitigated with some characteristic qualitative changes. ß-glucan also increased the expression of NF-κB in tissues which may account for some of its effects. In conclusion, ß-glucan showed a multifaceted beneficial impact on the therapeutic outcome of Trichinella infection and can be regarded as a promising adjuvant in the treatment of trichinellosis.

Curcumin attenuates cytoplasmic/endoplasmic reticulum stress, apoptosis and cholinergic dysfunction in diabetic rat hippocampus.

Diabetes mellitus (DM) is associated with the increased risk of the central nervous system complications as cerebrovascular disease, impaired cognition, dementia and neurodegeneration. Curcumin is a polyphenol with anti-oxidant, anti-inflammatory, anti-hyperlipidemic, and anti-cancer effects. Therefore, the present study was aimed to focus on the mechanistic insights of diabetes-induced hippocampal neurodegeneration in addition to shedding the light on the modulatory effect of curcumin. Twenty-eight male Wistar rats were randomly divided into four groups. Type I DM was induced by a single intra-peritoneal injection of streptozotocin (STZ) (65 mg/kg b.w.). Curcumin (100 mg/kg b.w.) was given to the diabetic group after the induction and for eight weeks. Hippocampal glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF-4), Bcl2 and choline acetyl transferase (ChAT) genes expression were assessed. Heat shock protein 70 (HSP70), Bcl-2-Associated X protein (Bax), Interferon-γ (INF-γ) and CCAAT-enhancer-binding protein homologous protein (CHOP) levels in the hippocampus were immunoassayed, in addition to the assessment of glycemic and redox status. Curcumin significantly improved blood glucose level, redox status, cellular stress, and decreased INF-γ and Bax levels, down-regulated GRP78 and ATF-4 expression, meanwhile, up-regulated Bcl2 and ChAT expression in hippocampus. Histological findings proved the biochemical and molecular findings. Our results support curcumin as a potential neuro-protective agent against diabetes induced hippocampal neurodegeneration.

Thrombomodulin, alarmin signaling, and copeptin: cross-talk between obesity and acute ischemic stroke initiation and severity in Egyptians.

Acute ischemic stroke (AIS) is followed by a strong inflammatory response contributing to brain damage and making early diagnosis and treatment inevitable. Hence, obesity is a state of chronic inflammation with amplified oxidative stress; this study aimed to assess the role played by thrombomodulin (TM)/alarmin signaling pathway and copeptin in AIS initiation and severity in addition to the implication of abnormal body weight. The study was conducted on 50 participants; 30 were patients with AIS (15 overweight/obese and 15 normal weight), 10 were overweight/obese, and 10 were normal weight. Plasma TM, copeptin, high mobility group box1 (HMGB1), and lipocalin 2 (LCN2) levels were immunoassayed. Toll-like receptor 4 (TLR4) mRNA expression was evaluated by real-time PCR, National Institutes of Health Stroke Scale (NIHSS), carotid intima media thickness; atherogenic index and glycemic status were also assessed. TM, copeptin, HMGB1, and LCN2 levels were significantly increased in overweight/obese AIS patients and in AIS patients with NIHSS score ≥ 7 when compared to other groups (p value=, ˂ 0.001*). Receiver operating characteristic (ROC) curve elaborated HMGB-1 and LCN2 as the best biomarker for diagnosis and prediction of AIS severity, respectively. Regression analysis avails LCN2 and TM as best biomarker for AIS severity predication. In conclusion, these results highlighted detrimental role of alarmin signaling with increased adaptive response to block this pathway through TM in addition to increased copeptin level as an acute damage marker and their tight relation to WC not to BMI in AIS which clarify the implication of central adiposity.

Atorvastatin and metformin administration modulates experimental Trichinella spiralis infection.

The host-parasite interaction can be altered by the changes in the host environment that may be or may not be in favor of successful invasion by the nematode parasite Trichinella spiralis. Metformin and atorvastatin are applied on a wide scale, to the degree that they could be considered as part of the host biochemical environment that can affect the parasite. Therefore, this study aimed to investigate the impact of alteration of the host's biochemical environment by these commonly used drugs upon the course of T. spiralis infection. Mice were divided into three groups: (1) received atorvastatin, (2) received metformin, and (3) untreated, then after one week, animals were infected with T. spiralis. The treatment continued until the end of the experiment. From each group, small intestines and muscles were removed for histopathological, immunohistochemical, and biochemical analyses as well as total muscle larval counts. We found that the oxidative stress and the expression of vascular endothelial growth factor (VEGF) in the muscles were significantly reduced in both drug-receiving groups, while the total larval counts in muscles were only significantly reduced in atorvastatin-receiving group as compared to the infected control group. Moreover, marked reduction in the inflammatory cellular infiltration, cyclooxygenase-2 (COX-2) expression, and oxidative stress was noted in the small intestines of the treated groups as compared to the infected control group. In conclusion, this study provides many insights into the different biochemical changes in the host that the parasite has to face. Moreover, the anti-inflammatory and anti-angiogenic effects should be taken into consideration when treating infections in patients on therapy with atorvastatin or metformin.

Cloning and functional characterization of Ptpcd2 as a novel cell cycle related protein tyrosine phosphatase that regulates mitotic exit.

Faithful transmission of genetic information depends on accurate chromosome segregation as cells exit from mitosis, and errors in chromosomal segregation are catastrophic and may lead to aneuploidy which is the hallmark of cancer. In eukaryotes, an elaborate molecular control system ensures proper orchestration of events at mitotic exit. Phosphorylation of specific tyrosyl residues is a major control mechanism for cellular proliferation and the activities of protein tyrosine kinases and phosphatases must be integrated. Although mitotic kinases are well characterized, phosphatases involved in mitosis remain largely elusive. Here we identify a novel variant of mouse protein tyrosine phosphatase containing domain 1 (Ptpcd1), that we named Ptpcd2. Ptpcd1 is a Cdc14 related centrosomal phosphatase. Our newly identified Ptpcd2 shared a significant homology to yeast Cdc14p (34.1%) and other Cdc14 family of phosphatases. By subcellular fractionation Ptpcd2 was found to be enriched in the cytoplasm and nuclear pellets with catalytic phosphatase activity. By means of immunofluorescence, Ptpcd2 was spatiotemporally regulated in a cell cycle dependent manner with cytoplasmic abundance during mitosis, followed by nuclear localization during interphase. Overexpression of Ptpcd2 induced mitotic exit with decreased levels of some mitotic markers. Moreover, Ptpcd2 failed to colocalize with the centrosomal marker γ-tubulin, suggesting it as a non-centrosomal protein. Taken together, Ptpcd2 phosphatase appears a non-centrosomal variant of Ptpcd1 with probable mitotic functions. The identification of this new phosphatase suggests the existence of an interacting phosphatase network that controls mammalian mitosis and provides new drug targets for anticancer modalities.

Ptpcd-1 is a novel cell cycle related phosphatase that regulates centriole duplication and cytokinesis.

Proper progression of mitosis requires spatio-temporal regulation of protein phosphorylation by orchestrated activities of kinases and phosphatases. Although many kinases, such as Aurora kinases, polo-like kinases (Plks), and cyclin B-Cdk1 are relatively well characterized in the context of their physiological functions at mitosis and regulation of their enzymatic activities during mitotic progression, phosphatases involved are largely unknown. Here we identified a novel protein tyrosine phosphatase containing domain 1 (Ptpcd 1) as a mitotic phosphatase, which shares sequence homology to Cdc14. Immunofluorescence studies revealed that Ptpcd1 partially colocalized with gamma-tubulin, an archetypical centrosomal marker. Overexpression of this phosphatase prevented unscheduled centrosomal amplification in hydroxyurea arrested U2OS cells. Intriguingly, Ptpcd 1-associated and colocalized with polo-like kinase 1(Plk1). Hence, overexpression of Ptpcd1 rescued prometaphase arrest of Plk-1 depleted cells, but resulted in aberrant cytokinesis as did as Plk1 overexpression. These results suggested that Ptpcd1 is involved in centrosomal duplication and cytokinesis.

Cyclin A-Cdk1 regulates the origin firing program in mammalian cells.

Somatic mammalian cells possess well-established S-phase programs with specific regions of the genome replicated at precise times. The ATR-Chk1 pathway plays a central role in these programs, but the mechanism for how Chk1 regulates origin firing remains unknown. We demonstrate here the essential role of cyclin A2-Cdk1 in the regulation of late origin firing. Activity of cyclin A2-Cdk1 was hardly detected at the onset of S phase, but it was obvious at middle to late S phase under unperturbed condition. Chk1 depletion resulted in increased expression of Cdc25A, subsequent hyperactivation of cyclin A2-Cdk1, and abnormal replication at early S phase. Hence, the ectopic expression of cyclin A2-Cdk1AF (constitutively active mutant) fusion constructs resulted in abnormal origin firing, causing the premature appearance of DNA replication at late origins at early S phase. Intriguingly, inactivation of Cdk1 in temperature-sensitive Cdk1 mutant cell lines (FT210) resulted in a prolonged S phase and inefficient activation of late origin firing even at late S phase. Our results thus suggest that cyclin A2-Cdk1 is a key regulator of S-phase programs.

Essential role of Chk1 in S phase progression through regulation of RNR2 expression.

Chk1 is an essential kinase for maintaining genome integrity and cell cycle checkpoints through phosphorylating several downstream targets. Recently, we demonstrated that Chk1 is also required for cell proliferation in somatic cells under unperturbed condition through regulating transcription of several genes. Here, we show that Chk1 is required for S phase progression and RNR2 is a critical downstream target of genes transcriptionally regulated by Chk1. Hence, although RNR2 expression reached maximum at S phase in the presence of Chk1, Chk1 depletion arrested the cell cycle at S phase and reduced RNR2 expression at both mRNA and protein levels. Ectopic expression of RNR2 failed to rescue the S phase arrest observed in Chk1 depleted cells, suggesting the presence of an additional Chk1-target(s) for completion of S phase other than RNR2. Therefore, our results suggest that Chk1 is required for DNA replication at least through regulating RNR2 gene transcription.

Chk1 is a histone H3 threonine 11 kinase that regulates DNA damage-induced transcriptional repression.

DNA damage results in activation or suppression of transcription of a large number of genes. Transcriptional activation has been well characterized in the context of sequence-specific DNA-bound activators, whereas mechanisms of transcriptional suppression are largely unexplored. We show here that DNA damage rapidly reduces histone H3 Threonine 11 (T11) phosphorylation. This correlates with repression of genes, including cyclin B1 and cdk1. H3-T11 phosphorylation occurs throughout the cell cycle and is Chk1 dependent in vivo. Following DNA damage, Chk1 undergoes rapid chromatin dissociation, concomitant with reduced H3-T11 phosphorylation. Furthermore, we find that loss of H3-T11 phosphorylation correlates with reduced binding of the histone acetyltransferase GCN5 at cyclin B1 and cdk1 promoters and reduced H3-K9 acetylation. We propose a mechanism for Chk1 as a histone kinase, responsible for DNA-damage-induced transcriptional repression by loss of histone acetylation.