Variable Behavior of iPSCs Derived from CML Patients for Response to TKI and Hematopoietic Differentiation

Chronic myeloid leukemia disease (CML) found effective therapy by treating patients with tyrosine kinase inhibitors (TKI), which suppress the BCR-ABL1 oncogene activity. However, the majority of patients achieving remission with TKI still have molecular evidences of disease persistence. Various mechanisms have been proposed to explain the disease persistence and recurrence. One of the hypotheses is that the primitive leukemic stem cells (LSCs) can survive in the presence of TKI. Understanding the mechanisms leading to TKI resistance of the LSCs in CML is a critical issue but is limited by availability of cells from patients. We generated induced pluripotent stem cells (iPSCs) derived from CD34⁺ blood cells isolated from CML patients (CML-iPSCs) as a model for studying LSCs survival in the presence of TKI and the mechanisms supporting TKI resistance. Interestingly, CML-iPSCs resisted to TKI treatment and their survival did not depend on BCR-ABL1, as for primitive LSCs. Induction of hematopoietic differentiation of CML-iPSC clones was reduced compared to normal clones. Hematopoietic progenitors obtained from iPSCs partially recovered TKI sensitivity. Notably, different CML-iPSCs obtained from the same CML patients were heterogeneous, in terms of BCR-ABL1 level and proliferation. Thus, several clones of CML-iPSCs are a powerful model to decipher all the mechanisms leading to LSC survival following TKI therapy and are a promising tool for testing new therapeutic agents.     

Evidence of an increased incidence of day 3 parasitaemia in Suriname: an indicator of the emerging resistance of Plasmodium falciparum to artemether

The emerging resistance to artemisinin derivatives that has been reported in South-East Asia led us to assess the efficacy of artemether-lumefantrine as the first line therapy for uncomplicated Plasmodium falciparum infections in Suriname. This drug assessment was performed according to the recommendations of the World Health Organization in 2011. The decreasing number of malaria cases in Suriname, which are currently limited to migrating populations and gold miners, precludes any conclusions on artemether efficacy because adequate numbers of patients with 28-day follow-up data are difficult to obtain. Therefore, a comparison of day 3 parasitaemia in a 2011 study and in a 2005/2006 study was used to detect the emergence of resistance to artemether. The prevalence of day 3 parasitaemia was assessed in a study in 2011 and was compared to that in a study in 2005/2006. The same protocol was used in both studies and artemether-lumefantrine was the study drug. Of 48 evaluable patients in 2011, 15 (31%) still had parasitaemia on day 3 compared to one (2%) out of 45 evaluable patients in 2005/2006. Overall, 11 evaluable patients in the 2011 study who were followed up until day 28 had negative slides and similar findings were obtained in all 38 evaluable patients in the 2005/2006 study. The significantly increased incidence of parasite persistence on day 3 may be an indication of emerging resistance to artemether.     

In vitro and in silico molecular interaction of multiphase nanoparticles containing inositol hexaphosphate and jacalin: Therapeutic potential against colon cancer cells (HCT-15)

Inositol hexaphosphate (IP6) is a natural constituent found in almost all cereals and legumes. It is known to cause numerous antiangiogenic manifestations. Notwithstanding its great potential, it is underutilized due to the chelation and rapid excretion from the body. Jacalin is another natural constituent obtained from seeds of jackfruit and can target disaccharides overexpressed in tumor cells. The current study was in-quested to develop and evaluate a surface-modified gold nanoparticulate system containing IP6 and jacalin which may maximize the apoptotic effect of IP6 against HCT-15 cell lines. IP6 loaded jacalin-pectin-gold nanoparticles (IJP-GNPs) were developed through reduction followed by incubation method. The developed formulation was tested for various in vitro and in silico studies to investigate its potential. HCT-15 cells when exposed to IJP-GNP resulted in significant apoptotic effects in dose as well as time-dependent manner, as measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, micronucleus, and reactive oxygen species assay. IJP-GNP displayed cell cycle arrest at the G0/G1 phase. To further explore the mechanism of chemoprevention, in silico studies were performed. The docking results revealed that the interactive behavior of IP6, P-GNP, and jacalin could target and inhibit the tumor formation activity, supported by in vitro studies. Taken together, all the findings suggested that IP6 loaded nanoparticles may increase the hope of future drug delivery strategy for targeting colon cancer.     

Optimization of α-ketoamide based p38 inhibitors through modifications to the region that binds to the allosteric site

We have optimized a novel series of potent p38 MAP kinase inhibitors based on an α-ketoamide scaffold through structure based design that due to their extended molecular architecture bind, in addition to the ATP site, to an allosteric pocket. In vitro ADME, in vivo PK and efficacy studies show these compounds to have drug-like characteristics and have resulted in the nomination of a development candidate which is currently in phase II clinical trials for the oral treatment of inflammatory conditions.     

Variation in Plasmodium falciparum Histidine-Rich Protein 2 (Pfhrp2) and Plasmodium falciparum Histidine-Rich Protein 3 (Pfhrp3) Gene Deletions in Guyana and Suriname

Guyana and Suriname have made important progress in reducing the burden of malaria. While both countries use microscopy as the primary tool for clinical diagnosis, malaria rapid diagnostic tests (RDTs) are useful in remote areas of the interior where laboratory support may be limited or unavailable. Recent reports indicate that histidine-rich protein 2 (PfHRP2)-based diagnostic tests specific for detection of P. falciparum may provide false negative results in some parts of South America due to the emergence of P. falciparum parasites that lack the pfhrp2 gene, and thus produce no PfHRP2 antigen. Pfhrp2 and pfhrp3 genes were amplified in parasite isolates collected from Guyana and Suriname to determine if there were circulating isolates with deletions in these genes. Pfhrp3 deletions were monitored because some monoclonal antibodies utilized in PfHRP2-based RDTs cross-react with the PfHRP3 protein. We found that all 97 isolates from Guyana that met the inclusion criteria were both pfhrp2- and pfhrp3-positive. In Suriname (N = 78), 14% of the samples tested were pfhrp2-negative while 4% were pfhrp3-negative. Furthermore, analysis of the genomic region proximal to pfhrp2 and pfhrp3 revealed that genomic deletions extended to the flanking genes. We also investigated the population substructure of the isolates collected to determine if the parasites that had deletions of pfhrp2 and pfhrp3 belonged to any genetic subtypes. Cluster analysis revealed that there was no predominant P. falciparum population substructure among the isolates from either country, an indication of genetic admixture among the parasite populations. Furthermore, the pfhrp2-deleted parasites from Suriname did not appear to share a single, unique genetic background.     

Catalytic Efficiency of Chitinase-D on Insoluble Chitinous Substrates Was Improved by Fusing Auxiliary Domains

Chitin is an abundant renewable polysaccharide, next only to cellulose. Chitinases are important for effective utilization of this biopolymer. Chitinase D from Serratia proteamaculans (SpChiD) is a single domain chitinase with both hydrolytic and transglycosylation (TG) activities. SpChiD had less of hydrolytic activity on insoluble polymeric chitin substrates due to the absence of auxiliary binding domains. We improved catalytic efficiency of SpChiD in degradation of insoluble chitin substrates by fusing with auxiliary domains like polycystic kidney disease (PKD) domain and chitin binding protein 21 (CBP21). Of the six different SpChiD fusion chimeras, two C-terminal fusions viz. ChiD+PKD and ChiD+CBP resulted in improved hydrolytic activity on α- and β-chitin, respectively. Time-course degradation of colloidal chitin also confirmed that these two C-terminal SpChiD fusion chimeras were more active than other chimeras. More TG products were produced for a longer duration by the fusion chimeras ChiD+PKD and PKD+ChiD+CBP.     

Inhibition of S. aureus alpha-hemolysin and B. anthracis lethal toxin by beta-cyclodextrin derivatives

Many pathogens utilize the formation of transmembrane pores in target cells in the process of infection. A great number of pore-forming proteins, both bacterial and viral, are considered to be important virulence factors, which makes them attractive targets for the discovery of new therapeutic agents. Our research is based on the idea that compounds designed to block the pores can inhibit the action of virulence factors, and that the chances to find high affinity blocking agents increase if they have the same symmetry as the target pore. Recently, we demonstrated that derivatives of beta-cyclodextrin inhibited anthrax lethal toxin (LeTx) action by blocking the transmembrane pore formed by the protective antigen (PA) subunit of the toxin. To test the broader applicability of this approach, we sought beta-cyclodextrin derivatives capable of inhibiting the activity of Staphylococcus aureus alpha-hemolysin (alpha-HL), which is regarded as a major virulence factor playing an important role in staphylococcal infection. We identified several amino acid derivatives of beta-cyclodextrin that inhibited the activity of alpha-HL and LeTx in cell-based assays at low micromolar concentrations. One of the compounds was tested for the ability to block ion conductance through the pores formed by alpha-HL and PA in artificial lipid membranes. We anticipate that this approach can serve as the basis for a structure-directed drug discovery program to find new and effective therapeutics against various pathogens that utilize pore-forming proteins as virulence factors.     

Fungal rDNA signatures in coronary atherosclerotic plaques

Bacterial DNA has been found in coronary plaques and it has therefore been concluded that bacteria may play a role as trigger factors in the chronic inflammatory process underlying coronary atherosclerosis. However, the microbial spectrum is complex and it is not known whether microorganisms other than bacteria are involved in coronary disease. Fungal 18S rDNA signatures were systematically investigated in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients and controls (unaffected coronary arteries) using clone libraries, denaturating gradient gel analysis (DGGE), in situ hybridization and fluorescence in situ hybridization (FISH). Fungal DNA was found in 35 of 38 (92.11%) coronary heart disease patients by either polymerase chain reaction (PCR) with universal primers or in situ hybridization analysis (n = 5), but not in any control sample. In a clone library with more than 350 sequenced clones from pooled patient DNA, an overall richness of 19 different fungal phylotypes could be observed. Fungal profiles of coronary heart disease patients obtained by DGGE analysis showed a median richness of fungal species of 5 (range from 2 to 9) with a high interindividual variability (mean similarity 18.83%). For the first time, the presence of fungal components in atherosclerotic plaques has been demonstrated. Coronary atheromatous plaques harbour diverse and variable fungal communities suggesting a polymicrobial contribution to the chronic inflammatory aetiology.     

Stress granules counteract senescence by sequestration of PAI‐1

Cellular senescence is a physiological response by which an organism halts the proliferation of potentially harmful and damaged cells. However, the accumulation of senescent cells over time can become deleterious leading to diseases and physiological decline. Our data reveal a novel interplay between senescence and the stress response that affects both the progression of senescence and the behavior of senescent cells. We show that constitutive exposure to stress induces the formation of stress granules (SGs) in proliferative and presenescent cells, but not in fully senescent cells. Stress granule assembly alone is sufficient to decrease the number of senescent cells without affecting the expression of bona fide senescence markers. SG‐mediated inhibition of senescence is associated with the recruitment of the plasminogen activator inhibitor‐1 (PAI‐1), a known promoter of senescence, to these entities. PAI‐1 localization to SGs increases the translocation of cyclin D1 to the nucleus, promotes RB phosphorylation, and maintains a proliferative, non‐senescent state. Together, our data indicate that SGs may be targets of intervention to modulate senescence in order to impair or prevent its deleterious effects.     

Characterisation of Pseudosuccinea columella and Radix natalensis (Gastropoda: Lymnaeidae) in Egypt using shell and molecular data

ABSTRACT

Pseudosuccinea columella and Radix natalensis live in the same habitat in Egypt and are important intermediate hosts of Fasciola hepatica and F. gigantica. Our study aimed to characterise both snail species using molecular analysis and shell measurements. The ranges of morphometric parameters overlapped in the two lymnaeids, indicating that they do not clearly differentiate the two species. PCR-sequence analysis of the nuclear ribosomal small subunit rRNA and the polymorphic mitochondrial cytochrome oxidase subunit 1 (CO1) genes were used to determine the genetic identity and the potential diversity of the snails. Little intrasequence variations were detected in the sequences of both gene loci, indicating the potential homogeneity of lymnaeid populations in Egypt. Generated sequences of the mitochondrial CO1 gene locus for R. natalensis showed obvious heterogeneity compared to other sequences in GenBank. Molecular characterisation of these lymnaeids might help to understand the snails’ biodiversity in a bid to control these populations and their related diseases.