Employing CRISPR-Cas9 to Generate CD133 Synthetic Lethal Melanoma Stem Cells.

Malignant melanoma is a lethal skin cancer containing melanoma-initiating cells (MIC) implicated in tumorigenesis, invasion, and drug resistance, and is characterized by the elevated expression of stem cell markers, including CD133. The siRNA knockdown of CD133 enhances apoptosis induced by the MEK inhibitor trametinib in melanoma cells. This study investigates the underlying mechanisms of CD133's anti-apoptotic activity in patient-derived BAKP and POT cells, harboring difficult-to-treat NRASQ61K and NRASQ61R drivers, after CRISPR-Cas9 CD133 knockout or Dox-inducible expression of CD133. MACS-sorted CD133(+) BAKP cells were conditionally reprogrammed to derive BAKR cells with sustained CD133 expression and MIC features. Compared to BAKP, CD133(+) BAKR exhibit increased cell survival and reduced apoptosis in response to trametinib or the chemotherapeutic dacarbazine (DTIC). CRISPR-Cas9-mediated CD133 knockout in BAKR cells (BAKR-KO) re-sensitized cells to trametinib. CD133 knockout in BAKP and POT cells increased trametinib-induced apoptosis by reducing anti-apoptotic BCL-xL, p-AKT, and p-BAD and increasing pro-apoptotic BAX. Conversely, Dox-induced CD133 expression diminished apoptosis in both trametinib-treated cell lines, coincident with elevated p-AKT, p-BAD, BCL-2, and BCL-xL and decreased activation of BAX and caspases-3 and -9. AKT1/2 siRNA knockdown or inhibition of BCL-2 family members with navitoclax (ABT-263) in BAKP-KO cells further enhanced caspase-mediated apoptotic PARP cleavage. CD133 may therefore activate a survival pathway where (1) increased AKT phosphorylation and activation induces (2) BAD phosphorylation and inactivation, (3) decreases BAX activation, and (4) reduces caspases-3 and -9 activity and caspase-mediated PARP cleavage, leading to apoptosis suppression and drug resistance in melanoma. Targeting nodes of the CD133, AKT, or BCL-2 survival pathways with trametinib highlights the potential for combination therapies for NRAS-mutant melanoma stem cells for the development of more effective treatments for patients with high-risk melanoma.

Glial cell line-derived neurotrophic factor-induced mice liver defatting: A novel strategy to enable transplantation of steatotic livers.

Moderate macrovesicular steatosis (>30%), which is present in almost 50% of livers considered for transplantation, increases the risk of primary graft dysfunction. Our previously published data showed that glial cell line-derived neurotrophic factor (GDNF) is protective against high-fat diet (HFD)-induced hepatic steatosis in mice. Hence, we hypothesized that perfusion of steatotic livers with GDNF may reduce liver fat content before transplantation. Livers from 8 weeks of regular diet (RD) and of HFD-fed mice were perfused ex vivo for 4 hours with either vehicle, GDNF, or a previously described defatting cocktail. The liver's residual fat was quantified colorimetrically using a triglyceride (TG) assay kit and by Oil Red O (ORO) and Nile red/Hoechst staining. Liver tissue injury was assessed by using a lactate dehydrogenase (LDH) activity assay. In vitro induction of lipolysis in HepG2 cells was assessed by measuring glycerol and free fatty acid release. ORO staining showed significantly more steatosis in livers from HFD-fed mice compared with RD-fed mice (P < 0.001). HFD livers perfused with GDNF had significantly less steatosis than those not perfused (P = 0.001) or perfused with vehicle (P < 0.05). GDNF is equally effective in steatotic liver defatting compared to the defatting cocktail; however, GDNF induces less liver damage than the defatting cocktail. These observations were consistent with data obtained from assessment of liver TG content. Assessment of liver injury revealed significant hepatocyte injury in livers perfused with the control defatting cocktail but no evidence of injury in livers perfused with either GDNF or vehicle. In vitro, GDNF reduced TG accumulation in HepG2 cells and stimulated increased TG lipolysis. In conclusion, GDNF can decrease mice liver fat content to an acceptable range and could be a potential defatting agent before liver transplantation.

Loin pain hematuria syndrome.

Loin pain hematuria syndrome is a rare disease with a prevalence of ∼0.012%. The most prominent clinical features include periods of severe intermittent or persistent unilateral or bilateral loin pain accompanied by either microscopic or gross hematuria. Patients with loin pain hematuria syndrome initially present with hematuria, flank pain, or most often both hematuria and flank pain. Kidney biopsies from patients with loin pain hematuria typically reveal only minor pathologic abnormalities. Further, loin pain hematuria syndrome is not associated with loss of kidney function or urinary tract infections. Loin pain hematuria syndrome-associated hematuria and pain are postulated to be linked to vascular disease of the kidney, coagulopathy, renal vasospasm with microinfarction, hypersensitivity, complement activation on arterioles, venocalyceal fistula, abnormal ureteral peristalsis, and intratubular deposition of calcium or uric acid microcrystals. Many patients with loin pain hematuria syndrome also meet criteria for a somatoform disorder, and analgesic medications, including narcotics, commonly are used to treat loin pain hematuria syndrome-associated pain. Interventional treatments include renal denervation, kidney autotransplantation, and nephrectomy; however, these methods should be used only as a last resort when less invasive measures have been tried unsuccessfully. In this review article, we discuss and critique current clinical practices related to loin pain hematuria syndrome pathophysiology, diagnosis, treatment, and prognosis.

CRISPR-Cas9 Knockdown and Induced Expression of CD133 Reveal Essential Roles in Melanoma Invasion and Metastasis.

CD133, known as prominin1, is a penta-span transmembrane glycoprotein presumably a cancer stem cell marker for carcinomas, glioblastomas, and melanomas. We showed that CD133(+) 'melanoma-initiating cells' are associated with chemoresistance, contributing to poor patient outcome. The current study investigates the role(s) of CD133 in invasion and metastasis. Magnetic-activated cell sorting of a melanoma cell line (BAKP) followed by transwell invasion assays revealed that CD133(+) cells are significantly more invasive than CD133(-) cells. Conditional reprogramming of BAKP CD133(+) cells maintained stable CD133 overexpression (BAK-R), and induced cancer stem cell markers, melanosphere formation, and chemoresistance to kinase inhibitors. BAK-R cells showed upregulated CD133 expression, and consequently were more invasive and metastatic than BAK-P cells in transwell and zebrafish assays. CD133 knockdown by siRNA or CRISPR-Cas9 (BAK-R-T3) in BAK-R cells reduced invasion and levels of matrix metalloproteinases MMP2/MMP9. BAK-R-SC cells, but not BAK-R-T3, were metastatic in zebrafish. While CD133 knockdown by siRNA or CRISPR-Cas9 in BAK-P cells attenuated invasion and diminished MMP2/MMP9 levels, doxycycline-induced CD133 expression in BAK-P cells enhanced invasion and MMP2/MMP9 concentrations. CD133 may therefore play an essential role in invasion and metastasis via upregulation of MMP2/MMP9, leading to tumor progression, and represents an attractive target for intervention in melanoma.

CD133 Is Associated with Increased Melanoma Cell Survival after Multikinase Inhibition.

FDA-approved kinase inhibitors are now used for melanoma, including combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib for BRAFV600 mutations. NRAS-mutated cell lines are also sensitive to MEK inhibition in vitro, and NRAS-mutated tumors have also shown partial response to MEK inhibitors. However, melanoma still has high recurrence rates due to subpopulations, sometimes described as "melanoma initiating cells," resistant to treatment. Since CD133 is a putative cancer stem cell marker for different cancers, associated with decreased survival, we examined resistance of patient-derived CD133(+) and CD133(-) melanoma cells to MAPK inhibitors. Human melanoma cells were exposed to increasing concentrations of trametinib and/or dabrafenib, either before or after separation into CD133(+) and CD133(-) subpopulations. In parental CD133-mixed lines, the percentages of CD133(+) cells increased significantly (p<0.05) after high-dose drug treatment. Presorted CD133(+) cells also exhibited significantly greater (p<0.05) IC50s for single and combination MAPKI treatment. siRNA knockdown revealed a causal relationship between CD133 and drug resistance. Microarray and qRT-PCR analyses revealed that ten of 18 ABC transporter genes were significantly (P<0.05) upregulated in the CD133(+) subpopulation, while inhibition of ABC activity increased sensitivity, suggesting a mechanism for increased drug resistance of CD133(+) cells.

The Relation between Helicobacter pylori Infection and Acute Bacterial Diarrhea in Children.

Background. H. pylori infection leads to chronic gastritis in both children and adults. But recently, there are arising theories of its protective effect in diarrheal diseases. Aim. To explore the prevalence of H. pylori infection in children with bacterial diarrhea and compare it with healthy controls. Patients and Methods. Two matched groups consisted of 122 consecutive children, aged 24-72 months old, with acute bacterial diarrhea, who had Shigellosis (N = 68) and Salmonellosis (N = 54) as patients group and 204 healthy asymptomatic children as control group enrolled in this study. Results. The prevalence of H. pylori infection in healthy control children was significantly higher than in patients group, (odds ratio = 3.6, 95% CI: 1.33-9.5, P = 0.007). In our study, only 2/54 Salmonella infected patients and 3/68 of Shigellosis had evidence of H. pylori infection, while normal control children had 27/204 infected individuals. Conclusion. H. pylori infection may play a protective role against bacterial diarrhea in children. So it is important to consider all of the positive and negative aspects of H. pylori infection before its eradication.

Correction of Diabetic Hyperglycemia and Amelioration of Metabolic Anomalies by Minicircle DNA Mediated Glucose-Dependent Hepatic Insulin Production.

Type 1 diabetes mellitus (T1DM) is caused by immune destruction of insulin-producing pancreatic ß-cells. Commonly used insulin injection therapy does not provide a dynamic blood glucose control to prevent long-term systemic T1DM-associated damages. Donor shortage and the limited long-term success of islet transplants have stimulated the development of novel therapies for T1DM. Gene therapy-based glucose-regulated hepatic insulin production is a promising strategy to treat T1DM. We have developed gene constructs which cause glucose-concentration-dependent human insulin production in liver cells. A novel set of human insulin expression constructs containing a combination of elements to improve gene transcription, mRNA processing, and translation efficiency were generated as minicircle DNA preparations that lack bacterial and viral DNA. Hepatocytes transduced with the new constructs, ex vivo, produced large amounts of glucose-inducible human insulin. In vivo, insulin minicircle DNA (TA1m) treated streptozotocin (STZ)-diabetic rats demonstrated euglycemia when fasted or fed, ad libitum. Weight loss due to uncontrolled hyperglycemia was reversed in insulin gene treated diabetic rats to normal rate of weight gain, lasting ∼1 month. Intraperitoneal glucose tolerance test (IPGT) demonstrated in vivo glucose-responsive changes in insulin levels to correct hyperglycemia within 45 minutes. A single TA1m treatment raised serum albumin levels in diabetic rats to normal and significantly reduced hypertriglyceridemia and hypercholesterolemia. Elevated serum levels of aspartate transaminase, alanine aminotransferase, and alkaline phosphatase were restored to normal or greatly reduced in treated rats, indicating normalization of liver function. Non-viral insulin minicircle DNA-based TA1m mediated glucose-dependent insulin production in liver may represent a safe and promising approach to treat T1DM.

An algorithm for glaucoma screening in clinical settings and its preliminary performance profile.

PURPOSE: To devise and evaluate a screening algorithm for glaucoma in clinical settings. METHODS: Screening included examination of the optic disc for vertical cupping (≥0.4) and asymmetry (≥0.15), Goldmann applanation tonometry (≥21 mmHg, adjusted or unadjusted for central corneal thickness), and automated perimetry. In the diagnostic step, retinal nerve fiber layer imaging was performed using scanning laser polarimetry. Performance of the screening protocol was assessed in an eye hospital-based program in which 124 non-physician personnel aged 40 years or above were examined. A single ophthalmologist carried out the examinations and in equivocal cases, a glaucoma subspecialist's opinion was sought. RESULTS: Glaucoma was diagnosed in six cases (prevalence 4.8%; 95% confidence interval, 0.01-0.09) of whom five were new. The likelihood of making a definite diagnosis of glaucoma for those who were screened positively was 8.5 times higher than the estimated baseline risk for the reference population; the positive predictive value of the screening protocol was 30%. Screening excluded 80% of the initial population. CONCLUSION: Application of a formal screening protocol (such as our algorithm or its equivalent) in clinical settings can be helpful in detecting new cases of glaucoma. Preliminary performance assessment of the algorithm showed its applicability and effectiveness in detecting glaucoma among subjects without any visual complaint.

Update on the management of ulcerative colitis.

The present treatment goals for inflammatory bowel diseases (IBD) especially ulcerative colitis (UC) include rapid induction of clinical remission, steroid-free maintenance of clinical remission, mucosal healing and improvement of quality of life in UC patients. Immunomodulators have been reserved for steroid- dependent or steroid- refractory UC patients. Among these agents, azathioprine/6-mercaptopurine should be used for maintenance of remission in quiescent UC. Calcineurin inhibitors can be prescribed as a short-term rescue therapy in steroid- refractory UC patients, but the long term efficacy of these agents remains unclear. According to retrospective studies, methotraxate is not recommended for inducing and maintaining remission in UC. Novel biological therapies targeting different specific immunological pathways continue to be developed and introduced for a variety of clinical scenarios in IBD. Infliximab is currently used for induction and maintenance therapy in patients who have moderately to severely active UC with an inadequate response to conventional agents such as aminosalicylates, corticosteroids, or immunomodulators. Other anti-TNF agents and biologic therapies are undergoing evaluation in clinical trials for their efficacy in IBD. Most patients who start biologics should continue treatment for the foreseeable future and potential consequences of discontinuation should be discussed with individual patients. Currently, data do not exist to administer biologics as first-line therapy in UC. Emerging data suggest that biologics may have the potential to prevent complications and limit disease progression. If such benefits are proven, biologics may be used in the future to modulate subclinical inflammation and to prevent the development of clinical disease.

Association between irritable bowel syndrome and restless legs syndrome: a comparative study with control group.

BACKGROUND/AIMS: As a common gastrointestinal (GI) disorder, irritable bowel syndrome (IBS) has been reported to be associated with some psychological and neurological factors. This study aimed to evaluate the prevalence rate of restless legs syndrome (RLS) in a sample of IBS patients and to compare this prevalence with that of matched healthy controls. METHODS: This prospective comparative study was conducted in Tehran, Iran during 2010-2011. Based on the Rome III criteria, a total number of 225 definite IBS patients and 262 age- and sex-matched healthy controls were recruited in the final assessment to compare the prevalence rate of RLS between the 2 groups. RESULTS: RLS was significantly more frequent in IBS group (25.3% vs 6.5%, P < 0.001) which led to an odds ratio (OR) of 4.89 (95% CI, 2.75-8.70). IBS patients with co-morbid RLS significantly suffered more from stomach pain (96.5% vs 86.3%, OR = 4.36 [95% CI, 1.00-19.12]), nausea (40.4% vs 21.4%, OR = 2.48 [95% CI, 1.30-4.73]) and vomiting (10.5% vs 2.4%, OR = 4.82 [95% CI, 1.31-17.76]). CONCLUSIONS: By enrolling a considerable number of IBS patients and healthy controls, our study showed a significantly higher prevalence of RLS in IBS patients. Surprisingly, a higher prevalence rate of RLS was also accompanied with a more severe discomfort and stomach pain in IBS patients. It seems that screening patients with IBS for RLS may lead to greater identification of RLS and improved treatment for both conditions.

Gender Role in Irritable Bowel Syndrome: A Comparison of Irritable Bowel Syndrome Module (ROME III) Between Male and Female Patients.

BACKGROUND/AIMS: Irritable bowel syndrome (IBS) is a widespread chronic health condition which is significantly more prevalent in women. We conducted a gender difference analysis by comparing findings of men and women to determine whether any significant differences exist or not. METHODS: This single-center study was conducted in Tehran, Iran during 2009-2010. IBS was diagnosed on the basis of Rome III criteria. A simple "10 point" objective questionnaire was used. RESULTS: A total number of 144 IBS patients including 44 (30.6%) males and 100 (69.4%) females with the mean age of 37.50 ± 11.50 years, were assessed. The only differently observed symptom was nausea which was significantly more prevalent in females (49% vs 18.2%, P < 0.001). The commonest subtype of IBS in male patients was diarrhea predominant IBS (38.6%); while, constipation predominant IBS was the most frequent type among females (38%). Moreover, the frequency of loose, mushy or watery stools within the last 3 months was significantly higher among males (2.11 ± 1.67 vs 1.37 ± 1.50, P = 0.009). CONCLUSIONS: We report that gender is important in IBS. Although qualitative comparison of different subtypes of IBS between male and female failed to meet the statistically significant level, the answers to the corresponding questions of ROME III IBS module suggest the higher prevalence of bowel movements and looser stool in males. Moreover, nausea was reported more often by females.

Pain and Its Determinants in Photorefractive Keratectomy.

PURPOSE: To assess the determinants of early postoperative pain in photorefractive keratectomy. DESIGN: A prospective cross-sectional study. METHODS: One hundred and four myopic-astigmatic patients undergoing bilateral standard photorefractive keratectomy were evaluated for early postoperative pain severity. On day 1 postoperatively, the level of pain experienced was reported by the patient on a visual analog scale of 0 to 10. At the preoperative interview, data were collected on clinical, demographic, and social characteristics to find potential pain determinants. RESULTS: The median reported pain level was 3. About 20% of subjects reported a pain score of 6 or higher, and 2.9% (6 eyes of 4 patients) reported the highest pain score. The presence of external eye inflammatory signs was associated with higher levels of pain (P < 0.001). Patients with a higher body mass index reported more severe pain (P = 0.006). An inverse association was found between pain and harmful lifestyle choices (P = 0.008). Demographic characteristics, history of contact lens wear, history of major operation, past experience of severe pain, knowledge about the operation's adverse effects, preoperative insomnia, preoperative anxiety, operative factors, and refractive indices were not related to the severity of pain experienced (all P > 0.05). CONCLUSIONS: The association of pain with ocular surface inflammation suggests that inflammatory processes have a role in early postoperative pain, supporting the use of anti-inflammatory agents for pain management. Prescription of weight-adjusted dosages of analgesics is recommended on the basis of the association between severity of postoperative pain and body mass index.

Non-alcoholic fatty liver disease and correlation of serum alanin aminotransferase level with histopathologic findings.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) has been recognized as the most common cause of chronic liver disease worldwide. It occurs in patients who do not consume alcohol in large amounts. Alanine aminotranferase (ALT) and aspartate aminotransferase (AST) are indicators of hepatocellular injury. OBJECTIVES: To determine correlation between histopathologic specifications of NAFLD in patients with little or no history of alcohol consumption and the serum level of ALT. PATIENTS AND METHODS: In a cross-sectional study carried out in two gastroenterology and hepatology clinics in Tehran, Iran, the medical records of those who had undergone liver biopsies between years 2005 and 2009 were reviewed. Clinical and laboratory information of biopsy-proven cases of NAFLD were obtained from 147 eligible medical records. The histopathologic, demographic, and laboratory data of the participants were also collected. Two groups of patients according to their serum ALT level (cut-point of 35 U/L) were defined. The quantitative pathologic grade of the biopsy specimens was determined based on Brunt scoring system. RESULTS: We studied 147 NAFLD patients including 127 men (86.4%) and 20 women (13.6%) with a mean ± SD age of 41.4 ± 11.2 years. Considering serum ALT, the mean ± SD quantitative grade of hepatosteatosis was 1.50 ± 0.67 and 1.74 ± 0.73 (p=0.136); advanced fibrosis (consisted of grade III and cirrhosis) was found in 4.5% (1/22) and 5.6% (7/125) of patients (p=0.327). CONCLUSIONS: We found that using the cut-off value of 35 U/L for serum ALT level, it has little contribution to predict NAFLD severity.