Cleaving Folded RNA by Multifunctional DNAzyme Nanomachines.

Both tight and specific binding of folded biological mRNA is required for gene silencing by oligonucleotide gene therapy agents. However, this is fundamentally impossible using the conventional oligonucleotide probes according to the affinity/specificity dilemma. This study addresses this problem for cleaving folded RNA by using multicomponent agents (dubbed 'DNA nanomachine' or DNM). DNMs bind RNA by four short RNA binding arms, which ensure tight and highly selective RNA binding. Along with the improved affinity, DNM maintain the high sequence selectivity of the conventional DNAzymes. DNM enabled up to 3-fold improvement in DNAzymes catalytic efficiency (kcat/Km) by facilitating both RNA substrate binding and product release steps of the catalytic cycle. This study demonstrates that multicomponent probes organized in sophisticated structures can help to achieve the balance between affinity and selectivity in recognizing folded RNA and thus creates a foundation for applying complex DNA nanostructures derived by DNA nanotechnology in gene therapy.

RNase H-dependent DNA thresholder modulated by cancer marker concentration.

Threshold antisense oligonucleotide constructs were designed to cleave mRNA within different biomarker concentrations. The mRNA cleavage is activated by 2.6, 7.5 or 39.5 nM of biomarker depending on the construct design. The constructs can be used to differentiate cancer from normal cells by the level of oncogene expression followed by silencing of a targeted gene.

Cleaving Folded RNA with DNAzyme Agents.

Cleavage of biological mRNA by DNAzymes (Dz) has been proposed as a variation of oligonucleotide gene therapy (OGT). The design of Dz-based OGT agents includes computational prediction of two RNA-binding arms with low affinity (melting temperatures (Tm ) close to the reaction temperature of 37 °C) to avoid product inhibition and maintain high specificity. However, RNA cleavage might be limited by the RNA binding step especially if the RNA is folded in secondary structures. This calls for the need for two high-affinity RNA-binding arms. In this study, we optimized 10-23 Dz-based OGT agents for cleavage of three RNA targets with different folding energies under multiple turnover conditions in 2 mM Mg2+ at 37 °C. Unexpectedly, one optimized Dz had each RNA-binding arm with a Tm ≥60 °C, without suffering from product inhibition or low selectivity. This phenomenon was explained by the folding of the RNA cleavage products into stable secondary structures. This result suggests that Dz with long (high affinity) RNA-binding arms should not be excluded from the candidate pool for OGT agents. Rather, analysis of the cleavage products' folding should be included in Dz selection algorithms. The Dz optimization workflow should include testing with folded rather than linear RNA substrates.

Effect of Ramadan fasting on chronic inflammation markers and gut bacterial endotoxins among Egyptian hemodialysis patients.

BACKGROUND: Chronic kidney disease (CKD) is associated with a state of chronic inflammation. This study aimed to investigate effects of Ramadan fasting on the markers of chronic inflammation and gut bacterial endotoxin levels in maintenance hemodialysis. METHOD: A prospective self-controlled observational study included 45 patients. Serum levels of High Sensitive CRP (hsCRP), indoxyl sulfate, and trimethylamine-n-levels were measured within a week before and a week after Ramadan fasting. RESULTS: Twenty-seven patients have fasted more than 15 days (29 ± 2.2 days). The levels of high sensitive C-reactive protein (hsCRP) (median of 62 mg/L vs. 91 mg/L), trimethylamine-n-oxide (TMAO) (median of 4.5 µmoL/L vs. 17 µmoL/L), platelet-to-lymphocyte ratio (PLR) (mean of 98.9 mg/L vs. 111.8 mg/L) and neutrophil-to-lymphocyte ratio (NLR) (median of 1.56 vs. 1.59) were significantly lower after Ramadan fasting with p < 0.001, p < 0.001, p < 0.001, and p = 0.04, respectively. CONCLUSION: A beneficial effect of Ramadan fasting on levels of bacterial endotoxins and markers of chronic inflammation in hemodialysis patients was observed.

Thresholding DNA Nanomachine as a Highly Cooperative and Efficient Enzyme-Like System for Controlled RNA Cleavage.

Therapeutic nucleic acid agents (TNA) can be activated by a marker RNA sequence followed by initiation of targeted RNA cleavage. This property can be used in conditional cell suppression, e. g., cancer marker-dependent cell death. However, healthy cells often express lower levels of cancer markers, thus jeopardizing TNA activation exclusively in cancer cells. Earlier, we developed a conditionally activated split deoxyribozyme construct (DNA thresholder or DTh) that can be activated by high but not by low concentrations of cancer markers. It's activity, however, was suppressed by very high marker concentrations. Herein, we combine the DTh functional units in a single DNA association (Thresholding DNA nanomachine or Th-DNM). Th-DNM maintains a high level of RNA cleavage activity in the presence of marker concentrations above the threshold level. Th-DNM differentiated fully complementary miR17 markers sequence from double base mismatched miR-20. Th-DNM can become a building block of DNA nanorobots for cancer treatment.

Binary Antisense Oligonucleotide Agent for Cancer Marker-Dependent Degradation of Targeted RNA.

Antisense oligonucleotide technology is one of the most successful gene therapy (GT) approaches. However, low selectivity of antisense agents limits their application as anticancer drugs. To achieve activation of antisense agent selectively in cancer cells, herein, we propose the concept of binary antisense oligonucleotide (biASO) agent. biASO recognizes an RNA sequence of a gene associated with cancer development (marker) and then activates RNase H-dependent cleavage of a targeted messenger RNA. biASO was optimized to produce only the background cleavage of the targeted RNA in the absence of the activator. The approach lays the foundation for the development of highly selective and efficient GT agents.

RNA-Cleaving DNA Thresholder Controlled by Concentrations of miRNA Cancer Marker.

Oligonucleotide gene therapy (OGT) agents suppress specific mRNAs in cells and thus reduce the expression of targeted genes. The ability to unambiguously distinguish cancer from healthy cells can solve the low selectivity problem of OGT agents. Cancer RNA markers are expressed in both healthy and cancer cells with a higher expression level in cancer cells. We have designed a DNA-based construct, named DNA thresholder (DTh) that cleaves targeted RNA only at high concentrations of cancer marker RNA and demonstrates low cleavage activity at low marker concentrations. The RNA-cleaving activity can be adjusted within one order of magnitude of the cancer marker RNA concentration by simply redesigning DTh. Importantly, DTh recognizes cancer marker RNA, while cleaving targeted RNA; this offers a possibility to suppress vital genes exclusively in cancer cells, thus triggering their death. DTh is a prototype of computation-inspired molecular device for controlling gene expression and cancer treatment.

Effect of Ramadan fasting on arterial stiffness parameters among Egyptian hypertensive patients with and without chronic kidney disease.

Arterial stiffness (AS) increases progressively in patients with chronic kidney disease (CKD). It is a strong predictor of cardiovascular and all-cause mortality. This study aims at evaluate of the effect of Ramadan fasting on AS parameters, augmentation index (AIx), and pulse wave velocity (PWV) in hypertensive patients with and without CKD. A cohort of 71 patients (mean age = 57.14 ± 14.5 years, 42 females and 29 males) were enrolled in this study; 34 with CKD and 37 without CKD. All patients had hypertension, while 25 patients had diabetes mellitus. Serum creatinine (Cr), serum urea, estimated glomerular filtration rate (eGFR) by CKD-EPI formula, brachial and central systolic blood pressure (BSP and CSP respectively), brachial and central diastolic blood pressure (BDP, CDP, respectively), AIx and PWV (measured by cuff based oscillometric method) were assessed before and after Ramadan fasting. In patients without CKD BSP, BDP, CSP, and CDP significantly decreased (P = 0.0001, 0.0001, 0.0001, and 0.0001, respectively). In patients with CKD BSP and CSP significantly decreased (P = 0.005 and 0.005), while BDP and CDP decreased, but the change was not statistically significant. AIx significantly decreased in patients without CKD (P = 0.0001, mean 36.24 before and 26.22 after Ramadan fasting), but did not significantly change in patients with CKD (P 0.381 mean 25.94 before and 25 after Ramadan fasting). PWV decreased in both groups, but the change was not significant. Serum Cr significantly increased (P = 0.03 mean 1.06 mg/dL before and 1.11 mg/dL after Ramadan fasting), while eGFR did not significantly decrease (P = 0.072, mean 69.73 mL/ min/1.73 m2 before and 67.3 mL/min/1.73 m2 after Ramadan fasting) in patients without CKD. Serum Cr significantly decreased (P 0.028 mean 1.93 mg/dL before and 1.87 mg/dL after Ramadan fasting) and eGFR significantly increased (P 0.006 mean 32.65 mL/min/1.73 m2 before and 34.68 mL/min/1.73 m2 after RF) in patients with CKD. Ramadan fasting is associated with improved peripheral and central blood pressure control in hypertensive patients with and without CKD. It is also associated with improved arterial compliance (decreased AIx) in hypertensive patients without CKD.

Insulin Resistance and Hepatitis C Virus-Associated Subclinical Inflammation Are Hidden Causes of Pruritus in Egyptian Hemodialysis Patients: A Multicenter Prospective Observational Study.

BACKGROUND: Pruritus is one of the most common and disturbing symptoms in hemodialysis (HD) patients. The pathogenesis of pruritus in HD patients is multifactorial; however, a little progress in understanding this pathogenesis has been achieved. OBJECTIVES: To evaluate the frequency and risk factors of pruritus among HD patients in Dakahlia, Egypt. METHODS: A total of 193 patients from 4 HD centers were included in this study. Pruritus intensity was assessed by visual analog scale. All patients were assessed by clinical and laboratory parameters, such as age, sex, duration of dialysis, dialysis adequacy, urea, calcium, phosphorus, parathyroid hormone, fasting insulin, fasting sugar, HOMA-IR, serum ferritin, high-sensitive C-reactive protein (hs-CRP) and hemoglobin. They were also investigated for hepatitis C virus (HCV) infection by HCV enzyme-linked immunosorbent assay, HCV-PCR for plasma and buffy coat for further detection of occult HCV infection. RESULTS: Male gender, dialysis duration, inadequate dialysis, anemia, high ferritin, hyperphosphatemia, hypocalcemia, hs-CRP, and insulin resistance were characteristic features in pruritic patients (p = 0.01, 0.006, 0.0001, 0.047, 0.01, 0.0001, 0.024, 0.000, and 0.0001, respectively). Significant positive correlations were found between pruritus score and each of age (p = 0.002, r = 0.222), duration of dialysis (p = 0.03, r = 0.151), serum ferritin (p = 0.001, r = 0.213), serum phosphorus (p = 0.0001, r = 0.59), fasting insulin (p = 0.001, r= 0.273), and HOMA-IR (p = 0.0001, r = 0.349), while there was a negative correlation with Kt/V (p= 0.0001, r = -0.459). Linear multivariate regression analysis showed that age, duration of dialysis, serum phosphorus, Kt/v, and hs-CRP were good predictors for pruritic score in HD patients. All HCV-infected patients (who were positive for both plasma and buffy coat HCV-PCR) had pruritus with -significantly higher pruritus score than non-infected patients (p = 0.009), they also showed significantly higher fasting insulin, HOMA-IR, and hs-CRP levels (p = 0.0001). CONCLUSIONS: Uremic pruritus (UP) is a serious problem in HD patients. hs-CRP, male gender, dialysis duration, insulin resistance, dialysis inadequacy, and hyperphosphatemia are positively correlated with the intensity of UP. HCV infection is associated with severe UP, insulin resistance, and inflammation.

Rifaximin in nonalcoholic fatty liver disease: hit multiple targets with a single shot.

BACKGROUND/AIMS: The pathogenesis of nonalcoholic fatty liver disease (NAFLD) may include increased insulin resistance, upregulation of proinflammatory cytokines, lipopolysaccharide, and BMI. Rifaximin is a minimally absorbable antibiotic that might act against a broad spectrum of gut bacteria. This study aimed to investigate the effects of rifaximin on NAFLD. PATIENTS AND METHODS: Fifty participants with biopsy-proven nonalcoholic steatohepatitis (NASH) were registered in this multicentric, double-blind, randomized, placebo-controlled study. BMI, alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, lipid profile, serum endotoxin, homeostatic model assessment, toll-like receptor-4, interleukin-10 (IL-10), IL-6, tumor necrosis factor-α, and cytokeratin-18 (CK-18) levels were evaluated at baseline and at 1, 3, and 6 months of rifaximin therapy (1100 mg/day). RESULTS: Patients were randomized into two groups (rifaximin group; n=25 and placebo group; n=25). After 6 months of rifaximin therapy, patients with NASH showed a significant reduction in homeostatic model assessment, alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, endotoxin, toll-like receptor-4, IL-6, tumor necrosis factor-α, CK-18, and NAFLD-liver fat score (all P<0.05), but no changes in the lipid profile; moreover, there was a mild nonstatistically significant reduction of BMI. However, in the placebo group, there was no significant difference in these variables at baseline and after therapy. CONCLUSION: Rifaximin therapy appears to be effective and safe in modifying NASH through reduction of serum endotoxin and improvement of insulin resistance, proinflammatory cytokines, CK-18, and NAFLD-liver fat score.

Homocysteine: a new diagnostic marker in spontaneous bacterial peritonitis.

BACKGROUND AND AIMS: The diagnosis of spontaneous bacterial peritonitis (SBP) depends primarily on a polymorphonuclear leukocyte cell count more than 250/mm. This method is invasive, and not diagnostic in all variants of SBP; we aimed to assess serum homocysteine as a precise indicative marker for the diagnosis of all variants of SBP. PATIENTS AND METHODS: A total 323 consecutive ascitic patients were registered in this prospective work. Serum and ascitic fluid of homocysteine were evaluated utilizing an enzyme-linked immunosorbent assay. RESULTS: Participants were classified into a non-SBP group, including 262 participants and 61 patients with SBP. Serum and ascitic homocysteine were considerably elevated in the SBP group than in the non-SBP group (17.94±7.57 vs. 11.75±5.68 µmol/l; P<0.001 and 14.70±5.45 vs. 9.75±4.55 µmol/l; P<0.001). At a cutoff value of 17.79 µmol/l, serum homocysteine had 89.3% specificity and 95.1% sensitivity for distinguishing SBP (area under the curve: 0.932) and, at a cutoff value of 16.1 µmol/l, ascitic homocysteine had 84.4% specificity and 92.7% sensitivity for distinguishing SBP (area under the curve: 0.901). Both were positively correlated with the polymorphonuclear count, C-reactive protein, Child-Pugh score, and Model For End-Stage Liver Disease score as well as negatively correlated with the protein content in the ascitic fluid and estimated glomerular filtration rate. After SBP therapy, there was a marked reduction in serum and ascitic homocysteine levels. CONCLUSION: This study demonstrates that serum and ascitic homocysteine are considerably higher in SBP participants versus non-SBP patients. Serum homocysteine may provide a reliable and noninvasive diagnostic marker for all variants of SBP.

New predictive factors of poor response to therapy in autoimmune hepatitis: role of mean platelet volume.

BACKGROUND AND OBJECTIVES: The response to immunosuppressive therapy in autoimmune hepatitis (AIH) is a matter of debate. The aim of this work is to identify the histological, biochemical, and clinical predictive factors of incomplete response/treatment failure to the standard treatment (prednisone with or without azathioprine) in a well-characterized series of AIH Egyptian patients. PATIENTS AND METHODS: Of 49 AIH patients, only 36 patients completed this retrospective cohort study. The immunological, biochemical, histopathological, and clinical characteristics of patients were evaluated at diagnosis and during follow-up. RESULTS: Patients were classified into two groups; group A showed a complete response to therapy (n=22; 61%) and group B showed partial response/treatment failure (n=14; 39%). In a multivariate analysis, we observed that age at diagnosis up to 22 years [odds ratio (OR): 23.22; confidence interval (CI): 3.978-135.549; P<0.001], serum albumin up to 3.2 g/dl (OR: 5.36; CI: 1.237-23.209; P=0.025), mean platelet volume (MPV) of at least 10.75 fl (OR: 16.5; CI: 3.093-88.037; P<0.001), and presence of cirrhosis at diagnosis (OR: 8.44; CI: 1.682-42.392; P=0.001) were independent variables that can predict partial response/treatment failure. MPV correlated positively with stages of fibrosis/cirrhosis and grades of activity in liver biopsy at diagnosis and correlated inversely with serum albumin and age at presentation. During therapy, group B showed a fluctuation in MPV levels, however, group A showed a progressive decline until the end point. CONCLUSION: Our study confirmed that younger age, hypoalbuminemia, increased MPV, and cirrhosis at diagnosis were all independent predictors of incomplete response/treatment failure in AIH patients. MPV may reflect the response to therapy.