Mazin A. Zamzami

The role of Gut Microbiota in the development of obesity and Diabetes

Obesity and its associated complications like type 2 diabetes (T2D) are reaching epidemic stages. Increased food intake and lack of exercise are two main contributing factors. Recent work has been highlighting an increasingly more important role of gut microbiota in metabolic disorders. It’s well known that gut microbiota plays a major role in the development of food absorption and low grade inflammation, two key processes in obesity and diabetes. This review summarizes key discoveries during the past decade that established the role of gut microbiota in the development of obesity and diabetes. It will look at the role of key metabolites mainly the short chain fatty acids (SCFA) that are produced by gut microbiota and how they impact key metabolic pathways such as insulin signalling, incretin production as well as inflammation. It will further look at the possible ways to harness the beneficial aspects of the gut microbiota to combat these metabolic disorders and reduce their impact.

Aqueous normal phase retention of nucleotides on silica hydride‐based columns: Method development strategies for analytes revelant in clinical analysis

An aqueous normal phase HPLC method coupled with UV or ESI/MS detection was used for the determination of a wide variety of nucleotides, essential in metabolomics studies. Fifteen nucleotides were tested in clinically relevant mixtures at levels of 100 microg/mL for UV detection and 1 microg/mL for ESI-MS detection. Analysis times for all protocols developed were less than 20 min. The chromatographic conditions were changed to achieve optimized retention and separation of the nucleotides studied. The aqueous normal phase-HPLC methods were developed utilizing two columns, one having a minimally modified hydride surface another having an undecanoic acid moiety on a hydride surface. Volatile, low ionic strength mobile phases were used. Negative ion mode ESI-MS at near neutral pH mobile phase, combined with a TOF detector provided a highly sensitive and specific method, which is equally suitable for quadrupole and ion trap instruments.

Acoustic and hybrid 3D-printed electrochemical biosensors for the real-time immunodetection of liver cancer cells (HepG2)

This study presents an efficient acoustic and hybrid three-dimensional (3D)-printed electrochemical biosensors for the detection of liver cancer cells. The biosensors function by recognizing the highly expressed tumor marker CD133, which is located on the surface of liver cancer cells. Detection was achieved by recrystallizing a recombinant S-layer fusion protein (rSbpA/ZZ) on the surface of the sensors. The fused ZZ-domain enables immobilization of the anti-CD133 antibody in a defined manner. These highly accessible anti-CD133 antibodies were employed as a sensing layer, thereby enabling the efficient detection of liver cancer cells (HepG2). The recognition of HepG2 cells was investigated in situ using a quartz crystal microbalance with dissipation monitoring (QCM-D), which enabled the label-free, real-time detection of living cells on the modified sensor surface under controlled conditions. Furthermore, the hybrid 3D additive printing strategy for biosensors facilitates both rapid development and small-scale manufacturing. The hybrid strategy of combining 3D-printed parts and more traditionally fabricated parts enables the use of optimal materials: a ceramic substrate with noble metals for the sensing element and 3D-printed capillary channels to guide and constrain the clinical sample. Cyclic voltammetry (CV) measurements confirmed the efficiency of the fabricated sensors. Most importantly, these sensors offer low-cost and disposable detection platforms for real-world applications. Thus, as demonstrated in this study, both fabricated acoustic and electrochemical sensing platforms can detect cancer cells and therefore may have further potential in other clinical applications and drug-screening studies.

miRNAs as Circulating Biomarkers for Alzheimer's Disease and Parkinson's Disease.

Detection of biomarkers for neurodegenerative disorders (NDDs) within brain tissues of Alzheimers disease (AD) and Parkinsons disease (PD) patients has always been hampered by our inability to access and biopsy tissue of key brain regions implicated in disease occurrence and progression. Currently, diagnosis of NDDs is principally based on clinical observations of symptoms that present at later stages of disease progression, followed by neuroimaging and, possibly, CSF evaluation. One way to potentially detect and diagnose NDDs at a far earlier stage is to screen for abnormal levels of specific disease markers within the peripheral circulation of patients with NDDs. Increasing evidence suggests that there is dysregulation of microRNAs (miRNAs) in NDDs. Peripheral blood mononuclear cells, as well as biofluids, such as plasma, serum, urine and cerebrospinal fluid, contain miRNAs that can be identified and quantified. Circulating miRNAs within blood and other biofluids may thus be characterized and used as non-invasive, diagnostic biomarkers that facilitate the early detection of disease and potentially the continual monitoring of disease progression for NDDs such as AD and PD. Plainly, such a screen is only possible with a clear understanding of which miRNAs change with disease, and when these changes occur during the progression of AD and PD. Such information is becoming increasingly available and, in the near future, may not only support disease diagnosis, but provide the opportunity to evaluate therapeutic interventions earlier in the disease process.

Signalling pathways in UHRF1-dependent regulation of tumor suppressor genes in cancer

Epigenetic silencing of tumor suppressor genes (TSGs) through DNA methylation and histone changes is a main hallmark of cancer. Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is a potent oncogene overexpressed in various solid and haematological tumors and its high expression levels are associated with decreased expression of several TSGs including p16INK4A, BRCA1, PPARG and KiSS1. Using its several functional domains, UHRF1 creates a strong coordinated dialogue between DNA methylation and histone post-translation modification changes causing the epigenetic silencing of TSGs which allows cancer cells to escape apoptosis. To ensure the silencing of TSGs during cell division, UHRF1 recruits several enzymes including histone deacetylase 1 (HDAC1), DNA methyltransferase 1 (DNMT1) and histone lysine methyltransferases G9a and Suv39H1 to the right place at the right moment. Several in vitro and in vivo works have reported the direct implication of the epigenetic player UHRF1 in tumorigenesis through the repression of TSGs expression and suggested UHRF1 as a promising target for cancer treatment. This review describes the molecular mechanisms underlying UHRF1 regulation in cancer and discusses its importance as a therapeutic target to induce the reactivation of TSGs and subsequent apoptosis.

Inosine Triphosphate Pyrophosphohydrolase (ITPA) polymorphic sequence variants in adult hematological malignancy patients and possible association with mitochondrial DNA defects

BackgroundInosine triphosphate pyrophosphohydrolase (ITPase) is a ‘house-cleaning’ enzyme that degrades non-canonical (‘rogue’) nucleotides. Complete deficiency is fatal in knockout mice, but a mutant polymorphism resulting in low enzyme activity with an accumulation of ITP and other non-canonical nucleotides, appears benign in humans. We hypothesised that reduced ITPase activity may cause acquired mitochondrial DNA (mtDNA) defects. Furthermore, we investigated whether accumulating mtDNA defects may then be a risk factor for cell transformation, in adult haematological malignancy (AHM).MethodsDNA was extracted from peripheral blood and bone marrow samples. Microarray-based sequencing of mtDNA was performed on 13 AHM patients confirmed as carrying the ITPA 94C>A mutation causing low ITPase activity, and 4 AHM patients with wildtype ITPA. The frequencies of ITPA 94C>A and IVS2+21A>C polymorphisms were studied from 85 available AHM patients.ResultsITPA 94C>A was associated with a significant increase in total heteroplasmic/homoplasmic mtDNA mutations (p<0.009) compared with wildtype ITPA, following exclusion of haplogroup variants. This suggested that low ITPase activity may induce mitochondrial abnormalities. Compared to the normal population, frequencies for the 94C>A and IVS2+21A>C mutant alleles among the AHM patients were higher for myelodyplastic syndrome (MDS) - but below significance; were approximately equivalent for chronic lymphoblastic leukemia; and were lower for acute myeloid leukemia.ConclusionsThis study invokes a new paradigm for the evolution of MDS, where nucleotide imbalances produced by defects in ‘house-cleaning’ genes may induce mitochondrial dysfunction, compromising cell integrity. It supports recent studies which point towards an important role for ITPase in cellular surveillance of rogue nucleotides.

Neuroprotective Mechanisms Mediated by CDK5 Inhibition.

Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase belonging to the family of cyclin-dependent kinases. In addition to maintaining the neuronal architecture, CDK5 plays an important role in the regulation of synaptic plasticity, neurotransmitter release, neuron migration and neurite outgrowth. Although various reports have shown links between neurodegeneration and deregulation of cyclin-dependent kinases, the specific role of CDK5 inhibition in causing neuroprotection in cases of neuronal insult or in neurodegenerative diseases is not wellunderstood. This article discusses current evidence for the involvement of CDK5 deregulation in neurodegenerative disorders and neurodegeneration associated with stroke through various mechanisms. These include upregulation of cyclin D1 and overactivation of CDK5 mediated neuronal cell death pathways, aberrant hyperphosphorylation of human tau proteins and/or neurofilament proteins, formation of neurofibrillary lesions, excitotoxicity, cytoskeletal disruption, motor neuron death (due to abnormally high levels of CDK5/p25) and colchicine- induced apoptosis in cerebellar granule neurons. A better understanding of the role of CDK5 inhibition in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious pharmacological inhibitors of CDK5 for therapeutic use against human neurodegenerative disorders, such as Alzheimers disease, amyotrophic lateral sclerosis and neuronal loss associated with stroke.

Computing disease-linked SOD1 mutations: deciphering protein stability and patient-phenotype relations

Protein stability is a requisite in the field of biotechnology, cell biology and drug design. To understand effects of amino acid substitutions, computational models are preferred to save time and expenses. As a systemically important, highly abundant, stable protein, the knowledge of Cu/Zn Superoxide dismutase1 (SOD1) is important, making it a suitable test case for genotype-phenotype correlation in understanding ALS. Here, we report performance of eight protein stability calculators (PoPMuSiC 3.1, I-Mutant 2.0, I-Mutant 3.0, CUPSAT, FoldX, mCSM, BeatMusic and ENCoM) against 54 experimental stability changes due to mutations of SOD1. Four different high-resolution structures were used to test structure sensitivity that may affect protein calculations. Bland-Altman plot was also used to assess agreement between stability analyses. Overall, PoPMuSiC and FoldX emerge as the best methods in this benchmark. The relative performance of all the eight methods was very much structure independent, and also displayed less structural sensitivity. We also analyzed patient’s data in relation to experimental and computed protein stabilities for mutations of human SOD1. Correlation between disease phenotypes and stability changes suggest that the changes in SOD1 stability correlate with ALS patient survival times. Thus, the results clearly demonstrate the importance of protein stability in SOD1 pathogenicity.

Comparative study of extrapolative factors linked with oxidative injury and anti-inflammatory status in chronic kidney disease patients experiencing cardiovascular distress

Background Chronic kidney disease (CKD) is a group of heterogeneous abnormalities affecting the function and structure of the kidney and mostly further proceeds to cardiovascular damage prior to end stage renal disease (ESRD). The oxidative insult and inflammatory mediators have some undefined role in CKD and cardiovascular complications. It is therefore, aimed at to pin point the predictive factors in the development of cardiovascular disorder in patients with chronic kidney disease. Methods Fifty patients of CKD experiencing cardiovascular distress and twenty normal individuals having same age and sex acted as control during these observations. Blood samples (Each 5 ml) were drawn and subjected to centrifugation for 10–15 minutes to separate the serum at 4000-5000rpm. The levels of MDA, GSH, SOD, CAT, VIT C, VIT E, IL-1, TNF-alpha, nitric oxide (NO) and advanced oxidation protein products (AOPPs) were estimated and analyzed. Results The nitric oxide levels in the CKD patients decreased significantly (13.26±1.25 ng/ml) compared to controls (42.15±5.26 ng/ml). The serum vitamin E and C levels in these patients recorded 2.15±0.25 μg/ml and 0.97±0.09 μg/ml respectively as against their assigned controls which read 6.35±1.22 μg/ml and 3.29±0.25 μg/ml. Furthermore, a significantly higher level of Malondialdehyde (MDA) as1.25±0.07 nmol/ml was observed in CKD patients viz-a-viz relevant control. However, the serum SOD, catalase (CAT) and GSH levels in the same patients registered a significant decline as evident from respective figures 0.07±0.002 μg/dl, 1.22±0.012 μmol/mol, and 3.25±1.05 μg/dl. The control for these was observed as0.99±0.06 μg/dl, 3.19±0.05 μmol/mol, and 8.64±0.03 μg/dL. On the other hand, the IL-1 levels in the CKD patients found quite higher (402.5±18.26 pg/ml). This clearly points to substantial increase in oxidative insult and reduced NO levels leading to the renal and cardiovascular damage. Conclusion Observations support the fact that the decrease in anti-oxidative capacity accompanied by higher inflammatory mediators in CKD is indicative of oxidative stress, consequently leading to CKD progression, in all probability to cardiovascular insult. The outcome reiterates that strategies be designed afresh to contain CKD progression to cardiovascular complications and ESRD. One way could be to focus on early detection of stress related to the disease. It requires analyzing the factors related to stress, such as the one reported here. Linking these factors with the symptoms could be a crucial step forward. And further, the disease could be monitored in a more disciplined manner.

The KIP/CIP family members p21^{Waf1/Cip1} and p57^{Kip2} as diagnostic markers for breast cancer

OBJECTIVE We examined the expression status of p21^{Waf1/Cip1} and p57^{Kip2} in breast cancer as well as their relationship with clinicopathological factors. Moreover, the diagnostic value of gene promoter methylation of p21^Waf1/Cip1 and p57^Kip2 was assessed in breast cancer patients. METHODS This study involved 85 patients diagnosed with breast cancer and 36 patients with benign breast lesions. The expression of p21^{Waf1/Cip1} and p57^{Kip2} in cell lysates was analyzed by ELISA and Western blot, respectively. The gene promoter methylation of p21^Waf1/Cip1 and p57^Kip2 was examined in cell lysates by methylation specific PCR. RESULTS p21^{Waf1/Cip1} expression was higher while p57^{Kip2} level was lower in breast cancer patients compared to patients with benign breast lesions. The combined use of p21^{Waf1/Cip1} and p57^{Kip2} provided sensitivity and specificity of 82.35% and 86.11%, respectively. None of the malignant and benign breast tumors were found to be hypermethylated at p21^Waf1/Cip1 gene promoter. However, aberrant methylation of p57^Kip2 gene promoter was detected in 49 of 85 (57.65%) of breast cancer tumors. High p21^{Waf1/Cip1} level was associated with high grade, late stages and lymph node involvement, whereas low p57^{Kip2} level was correlated with high grade and HER2 overexpressing breast cancer. Moreover, hypermethylated p57^Kip2 gene promoter was associated with high grade. CONCLUSION Our findings show that the overexpression of p21^{Waf1/Cip1}, down-expression of p57^{Kip2} and gene promoter methylation of p57^Kip2 could be considered as promising diagnostic markers for breast cancer.