Enhancement of thermal stability of chondroitinase ABC I by site-directed mutagenesis: An insight from Ramachandran plot

The application of chondroitinase ABC I (cABC I) in damaged nervous tissue is believed to prune glycosaminoglycan chains of proteoglycans, thereby facilitates axon regeneration. However, the utilization of cABC I as therapeutics is notably restricted due to its thermal instability. In the present study, we have explored the possibility of thermostabilization of cABC I through release of its conformational strain using Ramachandran plot information. In this regard, Gln140 with non-optimal φ and ψ values were replaced with Gly, Ala and Asn. The results indicated that Q140G and Q140A mutants were able to improve both activity and thermal stability of the enzyme while Q140N variant reduced the enzyme activity and destabilized it. Moreover, the two former variants displayed a remarkable resistance to trypsin degradation. Structural analysis of all mutants showed an increase in intrinsic fluorescence intensity and secondary structure content of Q140G and Q140A compared to the wild type which indicated more compact structure upon mutation. This investigation demonstrated that relief of conformational tension can be considered as a possible approach to increase the stability of the protein.     

Reduced striatal ecto-nucleotidase activity in schizophrenia patients supports the “adenosine hypothesis”

Schizophrenia (SZ) is a major chronic neuropsychiatric disorder characterized by a hyperdopaminergic state. The hypoadenosinergic hypothesis proposes that reduced extracellular adenosine levels contribute to dopamine D₂ receptor hyperactivity. ATP, through the action of ecto-nucleotidases, constitutes a main source of extracellular adenosine. In the present study, we examined the activity of ecto-nucleotidases (NTPDases, ecto-5′-nucleotidase, and alkaline phosphatase) in the postmortem putamen of SZ patients (n = 13) compared with aged-matched controls (n = 10). We firstly demonstrated, by means of artificial postmortem delay experiments, that ecto-nucleotidase activity in human brains was stable up to 24 h, indicating the reliability of this tissue for these enzyme determinations. Remarkably, NTPDase-attributable activity (both ATPase and ADPase) was found to be reduced in SZ patients, while ecto-5′-nucleotidase and alkaline phosphatase activity remained unchanged. In the present study, we also describe the localization of these ecto-enzymes in human putamen control samples, showing differential expression in blood vessels, neurons, and glial cells. In conclusion, reduced striatal NTPDase activity may contribute to the pathophysiology of SZ, and it represents a potential mechanism of adenosine signalling impairment in this illness.     

CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

The P2X7 receptor is a trimeric ATP-gated cation channel important in health and disease. We have observed that the specific phospholipase D (PLD)1 antagonist, CAY10593 impairs P2X7-induced shedding of the ‘low affinity’ IgE receptor, CD23. The current study investigated the mode of action of this compound on P2X7 activation. Measurements of ATP-induced ethidium⁺ uptake revealed that CAY10593 impaired P2X7-induced pore formation in human RPMI 8226 B cells, P2X7-transfected HEK-293 cells and peripheral blood mononuclear cells. Concentration response curves demonstrated that CAY10593 impaired P2X7-induced pore formation in RPMI 8226 cells more potently than the PLD2 antagonist CAY10594 and the non-specific PLD antagonist halopemide. Electrophysiology measurements demonstrated that CAY10593 also inhibited P2X7-induced inward currents. Notably, RT-PCR demonstrated that PLD1 was absent in RPMI 8226 cells, while choline-Cl medium or 1-butanol, which block PLD stimulation and signalling respectively did not impair P2X7 activation in these cells. This data indicates that CAY10593 impairs human P2X7 independently of PLD1 stimulation and highlights the importance of ensuring that compounds used in signalling studies downstream of P2X7 activation do not affect the receptor itself.     

Telocytes express PDGFRα in the human gastrointestinal tract

Telocytes (TC), a cell population located in the connective tissue of many organs of humans and laboratory mammals, are characterized by a small cell body and extremely long and thin processes. Different TC subpopulations share unique ultrastructural features, but express different markers. In the gastrointestinal (GI) tract, cells with features of TC were seen to be CD34‐positive/c‐kit‐negative and several roles have been proposed for them. Other interstitial cell types with regulatory roles described in the gut are the c‐kit‐positive/CD34‐negative/platelet‐derived growth factor receptor α (PDGFRα)‐negative interstitial cells of Cajal (ICC) and the PDGFRα‐positive/c‐kit‐negative fibroblast‐like cells (FLC). As TC display the same features and locations of the PDGFRα‐positive cells, we investigated whether TC and PDGFRα‐positive cells could be the same cell type. PDGFRα/CD34, PDGFRα/c‐kit and CD34/c‐kit double immunolabelling was performed in full‐thickness specimens from human oesophagus, stomach and small and large intestines. All TC in the mucosa, submucosa and muscle coat were PDGFRα/CD34‐positive. TC formed a three‐dimensional network in the submucosa and in the interstitium between muscle layers, and an almost continuous layer at the submucosal borders of muscularis mucosae and circular muscle layer. Moreover, TC encircled muscle bundles, nerve structures, blood vessels, funds of gastric glands and intestinal crypts. Some TC were located within the muscle bundles, displaying the same location of ICC and running intermingled with them. ICC were c‐kit‐positive and CD34/PDGFRα‐negative. In conclusion, in the human GI tract the TC are PDGFRα‐positive and, therefore, might correspond to the FLC. We also hypothesize that in human gut, there are different TC subpopulations probably playing region‐specific roles.     

Transforming growth factor-β promotes the function of HIV-specific CXCR5+CD8 T cells

HIV replication can be inhibited by CXCR5⁺CD8 T cells (follicular cytotoxic T cell [TFC]) which transfer into B-cell follicles where latent HIV infection persists. However, how cytokines affect TFC remain unclear. Understanding which cytokines show the ability to affect TFC could be a key strategy toward curing HIV. Similar mechanisms could be used for the growth and transfer of TFCs and follicular helper T (TFH) cells; as a result, we hypothesized that cytokines IL-6, IL-21, and transforming growth factor-β (TGF-β), which are necessary for the differentiation of TFH cells, could also dictate the development of TFCs. In this work, lymph node mononuclear cells and peripheral blood mononuclear cells from HIV-infected individuals were cocultured with IL-6, IL-21, and TGF-β. We then carried out T-cell receptor (TCR) repertoire analysis to compare the differences between CXCR5^– and CXCR5⁺CD8 T cells. Our results showed that the percentage and function of TFC can be enhanced by stimulation with TGF-β. Besides, TGF-β stimulation enhanced the diversity of TCR and complementarity-determining region 3 sequences. HIV DNA showed a negative correlation with TFC. The use of TGF-β to promote the expression of CXCR5⁺CD8 T cells could become a new treatment approach for curing HIV.     

The molecular markers of cancer stem cells in head and neck tumors

Head and neck cancer (HNC) is the six most common malignancy worldwide leading to more than 350,000 deaths annually. Despite recent advances in treatment modalities for these patients, there has been only a slight improvement of prognosis. As cancer stem cells (CSCs) have been implicated in tumor cell survival, progression, and response to therapy, the identification of this tumor subpopulation would have important therapeutic and prognostic implications. In this structured appraisal of the literature, Embase, PubMed, and Ovid were searched for publications that investigated CSC markers of HNC in humans. The search was conducted under the PRISMA guidelines with clear inclusion and exclusion criteria for articles published in the last two decades. The review process resulted in the identification of some key CSC-associated molecules such as CD44, ALDH1, CD133, Oct3/4, Nanog, and Sox2, although a single common CSC sorting marker could not be found. These biomarkers were identified in a range of HNCs but the most common one was squamous cell carcinoma (SCC), predominantly oral SCC. Patient cohorts were of variable size (3–195 individuals) and the most common technique used for detection was immunohistochemistry. Some of the molecules were associated with poor prognosis and may be able to inform the choice of appropriate treatment for these patients.     

In silico investigation of novel biological pathways: The role of CD200 in regulation of T cell priming in experimental autoimmune encephalomyelitis

The use of simulation to investigate biological domains will inevitably lead to the need to extend existing simulations as new areas of these domains become more fully understood. Such simulation extensions can entail the incorporation of additional cell types, molecules or molecular pathways, all of which can exert a profound influence on the simulation behaviour. Where the biological domain is not well characterised, a structured development methodology must be employed to ensure that the extended simulation is well aligned with its predecessor. We develop and discuss such a methodology, relying on iterative simulation development and sensitivity analysis. The utility of this methodology is demonstrated using a case study simulation of experimental autoimmune encephalomyelitis (EAE), a murine T cell-mediated autoimmune disease model of multiple sclerosis, where it is used to investigate the activity of an additional regulatory pathway. We discuss how application of this methodology guards against creating inappropriate simulation representations of the biology when investigating poorly characterised biological mechanisms.