Potential Candidate Genes for Therapeutic Targeting in Chronic Myeloid Leukemia: A Pilot Study.

BACKGROUND: Chronic myeloid leukemia (CML) is a prevalent hematological malignancy known for the presence of the Philadelphia chromosome and activation of the BCR-Abl kinase activity. Although tyrosine kinase inhibitors are widely used as the standard treatment, resistance remains a concern among certain patients. This study aimed to investigate the gene expression profile of a group of CML patients in comparison to a control group in order to identify novel candidate genes associated with the disease. METHODS: Whole transcriptome sequencing was performed, and gene expression levels were validated using quantitative real-time PCR. Additionally, single nucleotide and insertion/deletion variants were analyzed in the selected candidate genes among 10 CML patients and 4 healthy control subjects. RESULTS: Analysis revealed a set of differentially expressed genes, whose up- or downregulation was further confirmed by qRT-PCR. Among the upregulated genes in the patient group were ribosomal protein like (RPL) members, specifically RPL9, RPL34, RPL36A, and RPL39, while downregulation was observed in CCDC170, LDB1, and SBF1 compared to the healthy subjects. Furthermore, gene variant studies identified novel genetic changes in these candidate genes, suggesting potential clinical significance in CML. CONCLUSIONS: This study highlights RPL9, RPL34, RPL36A, RPL39, CCDC170, LDB1, and SBF1 as potential targets in CML. Additionally, it underscores the importance of investigating these genes and their variants in larger cohort studies to assess their clinical significance in CML patients.

Whole-transcriptome bioinformatics revealed HTRA3, KRT8, KRT17, and RHEX as novel targets in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is characterised by heterogeneous genomic signatures that vary among different patient groups. Hence, the current study aims to conduct a whole transcriptome analysis of a female patient with AML and a family history of the disease at the time of diagnosis. Genetic profiling has a useful impact on clinical management and treatment success of the disease as the complex genetic landscape of AML and differential responses to treatment might indicate inadequate therapeutic targeting. A 37 year old female patient with AML was admitted to the hospital complaining of general fatigue arthralgia and chest pain. AML diagnosis was confirmed by complete blood count and blood smears before being confirmed by cytogenetic analysis. Herein, we conducted whole-transcriptome sequencing analysis to assess differential gene expression profiles in patients and healthy control subjects. In addition, single nucleotide polymorphism/insertion-deletion analyses (SNP/INDEL) were performed to investigate gene variants in the present case. The results revealed a remarkable differential gene expression profile in AML compared to the corresponding control at the time of diagnosis, indicating that HTRA3, KRT8, KRT17, and RHEX are potential novel therapeutic targets. Additionally, a number of novel gene variants were also reported in the current study, as concluded from the SNP/INDEL analysis, which might be associated with disease risk assessment and probably affect prognosis. These genes and their new variants might be worth reporting to the scientific community for further exploration of AML.

Oral Intragastric DMBA Administration Induces Acute Lymphocytic Leukemia and Other Tumors in Male Wistar Rats.

BACKGROUND: Animal models of blood cancer are important tools to study these malignancies and also screen for novel therapeutic agents. Evidence from past research on the carcinogenic properties of 7,12-dimethylbenz[a]-anthracene (DMBA) was provided by a handful of studies. However, recent literature on DMBA carcinogenic activity and the underlying mechanisms is scarce. OBJECTIVE: The aim of this study was to develop a chemical model of leukemia using DMBA. Male Wistar rats (6 weeks old) were administered 1.5 mg of DMBA dissolved in sesame oil in biweekly doses using oral intragastric intubation. MATERIALS AND METHODS: Frequent complete blood counts and blood smear morphology assessment were used to assess the development of leukemia, while gross pathology and histopathology staining were used to evaluate malignancy development. RESULTS: The results showed that only 4% of rats developed acute lymphocytic leukemia. Interestingly, 36% of the rats developed tumors (parotid tumors [24%] and fibrosarcomas [12%]). CONCLUSION: These results suggest the pleiotropic potential of DMBA in the induction of multiple types of malignancies, including leukemia. This could be used as a model to validate therapeutic targets for leukemia and other induced malignancies.

ADAMTS14, ARHGAP22, and EPDR1 as potential novel targets in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a blood cancer with a heterogeneous genomic landscape. This study aimed to mine bioinformatics data generated by RNA sequencing to unveil an AML case transcriptome profile and identify novel therapeutic targets and markers. In this study, we have determined the transcriptomic profile and analysed gene variants of an AML patient at the time of diagnosis and validated some genes by quantitative reverse transcriptase polymerase chain reaction. ADAMTS14, ARHGAP22, and ependymin-related protein 1 (EPDR1) were markedly upregulated compared to the corresponding control. In addition, novel exonic single-nucleotide and insertion/deletion variants were identified in these genes. Hence, ADAMTS14, ARHGAP22, and EPDR1 can be proposed as potential novel targets in AML, and their exact roles should be further explored.

RNA sequencing identified novel target genes for Adansonia digitata in breast and colon cancer cells.

Adansonia digitata exhibits numerous beneficial effects. In the current study, we investigated the anti-cancer effects of four different extracts of A. digitata (polar and non-polar extracts of fruit powder and fibers) on the proliferation of human colon cancer (HCT116), human breast cancer (MCF-7), and human ovarian cancer (OVCAR-3 and OVCAR-4) cell lines. RNA sequencing revealed the influence of the effective A. digitata fraction on the gene expression profiles of responsive cells. The results indicated that only the polar extract of the A. digitata fibers exhibited anti-proliferative activities against HCT116 and MCF-7 cells, but not ovarian cancer cells. Moreover, the polar extract of the fibers resulted in the modulation of the expression of multiple genes in HCT116 and MCF-7 cells. We propose that casein kinase 2 alpha 3 (CSNK2A3) is a novel casein kinase 2 (CSNK2) isoform in HCT116 cells and report, for the first time, the potential involvement of FYVE, RhoGEF, and PH domain-containing 3 (FGD3) in colon cancer. Together, these findings provide evidence supporting the anti-cancer potential of the polar extract of A. digitata fibers in this experimental model of breast and colon cancers.

Effect of novel leukemia mutations (K75E & E222K) on interferon regulatory factor 1 and its interaction with DNA: insights from molecular dynamics simulations and docking studies.

Interferon regulatory factor 1 (IRF-1) plays a vital role in cell proliferation and cell differentiation by acting as a tumor suppressor gene and its role is linked to various types of cancers, including leukemia and pre-leukemia myelodysplasia. Mutations in the coding region of the IRF-1 are likely to influence the IRF-1 and its DNA binding affinity. The molecular mechanism of the DNA recognition with the IRF-1 protein upon mutations is still unknown. In this study, we have elucidated the structural and functional behavior of the wild-type and mutant (K75E and E222K) IRF-1 proteins and their corresponding molecular mechanisms with DNA recognition at the molecular level, using molecular dynamics simulations. Furthermore, we also applied the docking approach to examine the binding between the IRF-1 protein and DNA upon mutations. This study evidently explains that, due to mutations, the IRF-1 structure loses its stability and becomes more flexible than the wild-type protein. This structural loss might affect IRF-1-DNA interaction and lead to the inhibition of cancer suppression. Identifying the effects of IRF-1 at the molecular level will be beneficial for designing drugs for IRF-1 associated cancers. These drugs should be designed so that they can help reactivate the IRF-1 function, by increasing the transcriptional activity, to treat leukemia.

RNA sequencing-based identification of potential targets in acute myeloid leukemia: A case report.

Acute myeloid leukemia (AML) refers to heterogenous types of blood cancer which possess a complicated genomic landscape, and multiple novel mutational alterations are frequently being reported. Herein, a case report of a 37-year old AML patient is presented, who was diagnosed following laboratory investigation after admission. The patient had thrombocytopenia, and three consecutive blast counts of 40, 30 and 41%, respectively. A blood sample was collected for whole-genome RNA sequencing to understand the transcriptomic profile at the time of diagnosis and compared with a matched female control. Gene expression was quantified using the RSEM software package. Bioinformatics analysis revealed a significant number of differentially expressed genes in the patient, suggesting a marked change in the transcriptomic landscape in this patient. By mining the bioinformatics data and screening the highly expressed genes with ≥80% probability of gene expression, four novel genes were highlighted that may serve as potential future targets in AML patients; Rh associated glycoprotein, succinate receptor 1, transmembrane-4 L-six family member-1 and ADGRA3, although further validation of their value is required.

Antibiotic resistance and molecular characterization of enteroaggregative Escherichia coli isolated from patients with diarrhea in the Eastern Province of Saudi Arabia.

AIM: To investigate presence of Enteroaggregative Escherichia coli (EAEC) in patients suffering with diarrhea by targeting the pCVD432 (pAA) gene using PCR. METHODS: There were 63 non-duplicate isolates of E. coli isolated from diarrheal cases in teaching hospital in Eastern Province of Saudi Arabia between May 2013 to July 2014. All E. coli strains were examined for antibiotic susceptibility testing and polymerase chain reaction (PCR) for detection of virulence gene markers for EAEC. RESULTS: Of the 63 E coli strains that were reported with diarrheal cases, 35 (55.6%) EAEC were tested positive for pCVD432 gene and aggR gene was present in 19 (54.3%) strains. All strains tested positive for pCVD432 and aggR genes were classified as typical EAEC (tEAEC). EAEC revealed resistance to tetracycline, ampicillin, nalidixic acid, trimethoprim sulfamethoxazole, ciprofloxacin, streptomycin, noroxin, and piperacillin. CONCLUSION: EAEC was detected for the first time, among Saudi patients with diarrhea in this region of Saudi Arabia. The reported antibiotic resistance in this study is considered high among isolated EAEC strains to routinely prescribed antibiotics in our area.

Regulation of the Expression of DAPK1 by SUMO Pathway.

Death Associated Protein Kinase 1 (DAPK1) is an important signaling kinase mediating the biological effect of multiple natural biomolecules such as IFN-γ, TNF-α, curcumin, etc. DAPK1 is degraded through both ubiquitin-proteasomal and lysosomal degradation pathways. To investigate the crosstalk between these two DAPK1 degradation pathways, we carried out a screen using a set of ubiquitin E2 siRNAs at the presence of Tuberous Sclerous 2 (TSC2) and identified that the small ubiquitin-like molecule (SUMO) pathway is able to regulate the protein levels of DAPK1. Inhibition of the SUMO pathway enhanced DAPK1 protein levels and the minimum domain of DAPK1 protein required for this regulation is the kinase domain, suggesting that the SUMO pathway regulates DAPK1 protein levels independent of TSC2. Suppression of the SUMO pathway did not enhance DAPK1 protein stability. In addition, mutation of the potential SUMO conjugation sites on DAPK1 kinase domain did not alter its protein stability or response to SUMO pathway inhibition. These data suggested that the SUMO pathway does not regulate DAPK1 protein degradation. The exact molecular mechanism underlying this regulation is yet to be discovered.

Developmental lead (Pb)-induced deficits in hippocampal protein translation at the synapses are ameliorated by ascorbate supplementation.

BACKGROUND: Lead (Pb) is a persistent environmental neurotoxin and its exposure even in minute quantities has been known to induce neuronal defects. The immature brain is singularly sensitive to Pb neurotoxicity, and its exposure during development has permanent detrimental effects on the brain developmental trajectory and neuronal signaling and plasticity, culminating into compromises in the cognitive and behavioral attributes which persists even later in adulthood. Several molecular pathways have been implicated in the Pb-mediated disruption of neuronal signaling, including elevated oxidative stress, alterations in neurotransmitter biology, and mitochondrial dysfunction. Nevertheless, the neuronal targets and biochemical pathways underlying these Pb-mediated alterations in synaptic development and function have not been completely deduced. In this respect, recent studies have shown that synaptic signaling and its maintenance and plasticity are critically dependent on localized de novo protein translation at the synaptic terminals. MATERIALS AND METHODS: The present study hence aimed to assess the alterations in the synapse-specific translation induced by developmental Pb exposure. To this end, in vitro protein translation rate was analyzed in the hippocampal synaptoneurosomal fractions of rat pups pre- and postnatally exposed to Pb using a puromycin incorporation assay. Moreover, we evaluated the therapeutic effects of ascorbic acid supplementation against Pb-induced deficits in synapse-localized protein translation. RESULTS: We observed a significant loss in the rates of de novo protein translation in synaptoneurosomes of Pb-exposed pups compared to age-matched control pups. Interestingly, ascorbate supplementation lead to an appreciable recovery in Pb-induced translational deficits. Moreover, the deficit in activity-dependent synaptic protein translation was found to correlate significantly with the increase in the blood Pb levels. CONCLUSION: Dysregulation of synapse-localized de novo protein translation is a potentially critical determinant of Pb-induced synaptic dysfunction and the consequent deficits in behavioral, social, and psychological attributes of the organisms. In addition, our study establishes ascorbate supplementation as a key ameliorative agent against Pb-induced neurotoxicity.

A simplified colorimetric method for rapid detection of cell viability and toxicity in adherent cell culture systems.

PURPOSE: Evaluation of cell viability and toxicity in adherent culture systems is of critical relevance for a wide range of disciplines of biomedical sciences research, including cancer research, toxicology, pharmacology, cell biology, neurology and nanomedicine. Several well-established cytotoxicity assays are widely used by researchers, including the most-preferred MTT assay. Nevertheless, there are problems associated with them, for example; in terms of the time-factor and solubilization of the formzan crystals before its spectroscopic quantification. In this study, we propose a simple, fast and cost-effective colorimetric assay that is free of these issues. METHODS: Our assay was based upon reductive splitting of blue-green colored supravital safranin derivative dye Janus green B (JG-B) to pink colored diethyl safranin by oxidoreductases of the electron transport chain (ETC) of actively respiring mitochondria. Because this conversion can be easily and reliably followed spectroscopically, measure of diethyl safranin formed from extraneously added JG-B provides a proficient indicator of cellular health and viability. RESULTS: Using MCF-7, a breast cancer cell line, we provide a proof of concept for the suggested assay and compare it with the MTT assay. CONCLUSION: Unlike the MTT assay, our JG-B assay does not require a solubilization/extraction step, and hence follows a much simpler and time-efficient protocol suitable for high-throughput analysis of cell viability in anchorage-dependent cell culture models. Additionally, the JG-B cell viability assay reported here can be suitably applied either independently or in complementation with other assays for the analysis of cellular viability and toxicity in both analytic and therapeutic aspects of research.

Staurosporine alleviates cisplatin chemoresistance in human cancer cell models by suppressing the induction of SQSTM1/p62.

Cancer is one of the leading causes of mortality worldwide. Platinum­based chemotherapeutic agents such as cisplatin are the first line of treatment for many types of cancers. However, the development of cisplatin resistance after prolonged treatment is a common cause of cancer recurrence. In the present study, we investigated an approach designed to overcome resistance to cisplatin involving co­treatment with a second chemotherapeutic agent, staurosporine, and examined the role of sequestosome 1 (SQSTM1/p62) in enhancing cellular sensitivity to cisplatin. We utilized experimental models of three different cancers comprising cell lines derived from colon, breast, and ovarian tumors and investigated cell proliferation, morphology and p62 levels after treatment with cisplatin, staurosporine, or a combination of the two. Western blot analysis showed that cisplatin treatment resulted in elevation of p62 levels when compared to the corresponding control cells. Conversely, treatment with staurosporine resulted in a marked reduction in p62 levels in all three cell types and abrogated the cisplatin­induced upregulation of p62. These results suggest that staurosporine could sensitize cancer cells to cisplatin via a mechanism involving downregulation of p62.

Simple, reliable, and time-efficient colorimetric method for the assessment of mitochondrial function and toxicity.

Mitochondria are organelles involved in the production of cellular energy, regulation of Ca2+ and redox signaling, and are critical for normal functioning of eukaryotic cells. The dysfunction of mitochondria has been implicated in a wide range of diseases, including metabolic and neurodegenerative disorders and different types of cancers. To better understand the role of mitochondria in healthy and disease states, the development of efficient and reliable tools for the assessment of mitochondrial function is particularly important. Janus green B (JG-B) is a supravital lipophilic cationic dye which, in its oxidized form, has a green-blue color. As JG-B is taken up and reduced by metabolically active mitochondria, the dye has been used for assessing the purity, integrity and metabolic activity of mitochondria with microscopy-based methods. Here we present a simple, time- and cost-efficient JG-B-based colorimetric assay for assessing mitochondrial function, activity and toxicity. The method is based upon reduction of JG-B by mitochondrial dehydrogenases to diethylsafranine, which is pink colored and has a maximum absorption at 550 nm. In this proof of principle study, using in vitro mitochondrial preparations isolated from rat brain, we provide evidence that monitoring JG-B conversion to diethylsafranine can be used as a reliable and robust indicator of mitochondrial activity and toxicity. Because of its simplicity and efficiency in terms of costs and time, this assay has a wide potential in analytical as well as therapeutic areas of biomedical research.

Neonatal maternal deprivation impairs localized de novo activity-induced protein translation at the synapse in the rat hippocampus.

Neonatal neuropsychiatric stress induces alterations in neurodevelopment that can lead to irreversible damage to neuronal physiology, and social, behavioral, and cognitive skills. In addition, this culminates to an elevated vulnerability to stress and anxiety later in life. Developmental deficits in hippocampal synaptic function and plasticity are among the primary contributors of detrimental alterations in brain function induced by early-life stress. However, the underlying molecular mechanisms are not completely understood. Localized protein translation, occurring at the synapse and triggered by neuronal activity, is critical for synapse function, maintenance, and plasticity. We used a rodent model of chronic maternal deprivation to characterize the effects of early-life neuropsychiatric stress on localized de novo protein translation at synaptic connections between neurons. Synaptoneurosomal preparations isolated biochemically from the hippocampi of rat pups that were subjected to maternal deprivation were deficient in depolarization-induced activity-dependent protein translation when compared with littermate controls. Conversely, basal unstimulated protein translation was not affected. Moreover, deficits in activity-driven synaptic protein translation were significantly correlated with a reduction in phosphorylated cell survival protein kinase protein B or Akt (p473 Ser and p308 Thr), but not phosphorylated extracellular signal-regulated kinase.

Interferon regulatory factor 1 inactivation in human cancer.

Interferon regulatory factors (IRFs) are a group of closely related proteins collectively referred to as the IRF family. Members of this family were originally recognized for their roles in inflammatory responses; however, recent research has suggested that they are also involved in tumor biology. This review focusses on current knowledge of the roles of IRF-1 and IRF-2 in human cancer, with particular attention paid to the impact of IRF-1 inactivation. The different mechanisms underlying IRF-1 inactivation and their implications for human cancers and the potential importance of IRF-1 in immunotherapy are also summarized.

ERIC-PCR Genotyping of Acinetobacter baumannii Isolated from Different Clinical Specimens.

BACKGROUND: Acinetobacter baumannii is a major cause of hospital care-acquired infections, and this bacterium poses a significant challenge to health care worldwide. At King Fahd Hospital of the University (KFHU), Al Khobar, Saudi Arabia, there had been a significant increase in the number of cases of A. baumannii infections. OBJECTIVE: The objective of this study was to determine the clonal relationship between A. baumannii collected from different specimens of patients admitted to KFHU using the enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) fingerprinting method. MATERIALS AND METHODS: A. baumannii strains were isolated from a total of 59 specimens from inpatients admitted to KFHU between January and September 2014. These specimens were mainly collected from wound, rectal and throat swabs and transtracheal aspiration. ERIC-PCR fingerprinting was used to determine the clonal relationship between the different isolated strains. RESULTS: Using ERIC-PCR fingerprinting genotype analysis, 51 strains of A. baumannii were clustered into seven groups, while the remaining 8 were single strains. The genetic relatedness of A. baumannii isolated from admitted patients was high, indicating cross-transmission within the hospitalized patients. CONCLUSION: This study found that the increase in the incidence of A. baumannii in patients at KFHU was likely due to the spread of seven epidemic clones, thereby highlighting the need for intensifying the infection control measures to prevent nosocomial transmission of A. baumannii. These results also demonstrate that ERIC-PCR is a reliable and rapid method for studying the clonal similarity between A. baumannii isolated from different clinical specimens.

Novel IRF-1 Mutations in a Small Cohort of Leukaemia Patients From Saudi Arabia

Involvement of the Interferon Regulatory Factor 1 (IRF-1) gene in regulation of cell differentiation and proliferation made it a potential target in cancer research. IRF-1 acts as a tumor suppressor gene, and is inactivated in chronic (CML) and non-chronic myelogenous leukemia (non-CML). In the light of numerous reports on genetic changes in the noncoding region of the IRF-1 gene, this study aimed to explore possible genomic changes in coding and non-coding regions of IRF-1 in a random sample of leukemic Saudi patients, in order to obtain insights into potential impact of genetic changes on clinicopathological characteristics. Patients were classified into two major leukemia subtypes: CML (8 cases; 36.4%) and non-CML (14 cases; 63.6%). Sequencing results revealed two novel mutations in the coding area of the IRF-1 gene likely to influence the IRF-1/DNA binding affinity. In addition, three mutational sites in the noncoding region between exon 5&6 (8985(T>G), 8,990(T>G) and 8995(A>G) were identified. In conclusion, a larger representative study might help provide better understanding of the possible contribution of the identified genetic changes in IRF-1 to disease prognosis and outcomes in leukemic patients.

First description of methyltransferases in extensively drug-resistant Klebsiella pneumoniae isolates from Saudi Arabia.

PURPOSE: The resistance determinants for carbapenems, fluoroquinolones and aminoglycosides were characterized in 16 extensively drug-resistant Klebsiella pneumoniae (XDRKPN) strains collected from Saudi hospitals during 2014. METHODOLOGY: PCR and sequencing were used to detect: blaKPC, blaNDM, blaVIM, blaIMP-1,blaOXA-48, blaCTX-M, blaTEM, blaSHV and ampC for ß-lactam resistance; qnrA, qnrB, qnrS, aac(6')-Ib-cr, qepA and mutations of gyrA and parC for fluoroquinolone resistance; and aacA4, aacC2, aadA1, aphA6, armA and rmtB for aminoglycoside resistance. Enterobacterial repetitive intergenic consensus sequence-based PCR was performed to detect the clonal relatedness. RESULTS: All isolates encoded blaCTX-M, aacC2 and aphA6, together with mutations in gyrA and parC. blaOXA-48, blaNDM-1, aadA1, aacA4, qnrB, aac(6')-Ib-cr, armA and/or rmtB were detected in different strains. At least 93.2 % clonal relatedness was detected among these strains. CONCLUSIONS: To our knowledge, this is the first report describing XDRKPN encoding at least seven resistance determinants and harbouring methyltransferases in Saudi Arabia.

Developmental Phases of Anticancer Screening Models.

The development of new screening models for cancer therapy is indispensable for the improvement of cancer treatment, and for the creation of alternative possibilities in the field of chemotherapy. Screening models are routinely used to reduce the cost and resources involved in anticancer drug development. The value of any screening model will ultimately be ascertained by its ability to reliably predict the clinical response. Traditional in vivo screening models have been replaced with cell-based screening assays, and these cell-based models are under constant development to better mimic in vivo conditions. In this review, the developmental phases of anticancer screening models are discussed and exemplified, including the two-dimensional, threedimensional, cancer stem cell, and non-mammalian screening models. In addition, the use of new virtual screening tools as a model for anticancer drug development is highlighted.

Chryseobacterium gleum pneumonia in an infant with nephrotic syndrome.

INTRODUCTION: Chryseobacterium gleum is commonly distributed in the environment. It can cause a wide variety of infections in immunocompromised patients in hospital setting. CASE PRESENTATION: A 6 month old infant with nephrotic syndrome was admitted to the emergency room for an acute onset of fever, difficulty breathing, cyanosis, and low oral intake. Cultures of endotracheal tube specimens were positive for Chryseobacterium gleum which was confirmed by ribosomal sequencing. The organism was susceptible to trimethoprim-sulfamethoxazole, minocycline, and levofloxacin. The patient clinically improved on levofloxacin treatment. CONCLUSION: To the best of our knowledge, this is the first case of pneumonia caused by Chryseobacterium gleum in an infant with nephrotic syndrome. It is also the first report of C. gleum causing respiratory tract infection in Saudi Arabia.