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1.  BMI1-mediated histone ubiquitylation promotes DNA double-strand break repair 
The Journal of Cell Biology  2010;191(1):45-60.
The polycomb repressor complex ubiquitylates γ-H2AX and other components of the DNA damage response pathway to facilitate genomic repair.
Polycomb group (PcG) proteins are major determinants of cell identity, stem cell pluripotency, and epigenetic gene silencing during development. The polycomb repressive complex 1, which contains BMI1, RING1, and RING2, functions as an E3-ubuiquitin ligase. We found that BMI1 and RING2 are recruited to sites of DNA double-strand breaks (DSBs) where they contribute to the ubiquitylation of γ-H2AX. In the absence of BMI1, several proteins dependent on ubiquitin signaling, including 53BP1, BRCA1, and RAP80, are impaired in recruitment to DSBs. Loss of BMI1 sensitizes cells to ionizing radiation to the same extent as loss of RNF8. The simultaneous depletion of both proteins revealed an additive increase in radiation sensitivity. These data uncover an unexpected link between the polycomb and the DNA damage response pathways, and suggest a novel function for BMI1 in maintaining genomic stability.
PMCID: PMC2953429  PMID: 20921134
2.  Adenomatous Colorectal Polyps in Patients Referred for Colonoscopy in a Regional Hospital in Kuwait 
Adenomatous colorectal polyps (ACPs) are known to be the precursor lesions for colorectal cancer. The aim of the study was to determine the prevalence, endoscopic and pathological features of ACPs in patients referred for colonoscopy.
Patients and Methods:
The endoscopic and histological reports of adult patients who underwent complete colonoscopy in the gastroenterology unit of a regional Kuwaiti hospital between January 2008 and December 2008 were retrospectively studied. The specimens of polyps were reviewed by an experienced pathologist who was blinded to the clinical or endoscopic information. Non-neoplastic polyps were not included in the analysis.
Of 530 eligible patients (mean age, 45 years; male-female ratio, 2:1), 54 (10%) had 103 ACPs. Of the patients with ACPs (mean age, 57 years), 43 (80%) were males and 36 (67%) were Kuwaitis. Histopathological examination of the most significant polyp in each patient revealed that 40 (74%) polyps were tubular adenomas (TAs); 11 (20%), tubulovillous (TV) adenomas; and 3 (6%), villous adenomas. High-grade dysplasia was noticed in 4 (10%) adenomas. Fifteen (2.8%) of the 530 patients had advanced ACPs. Logistic regression analysis of some variables and their association with ACPs found that age (P<0.001; OR, 1.9; CI, 1.5-2.3), history of adenoma (P=0.001; OR, 6.4; CI, .2.1-19.4) and being Kuwaitis (P=0.029; OR, 2.1; CI, 1.1-4.1) to be independently associated with ACPs.
The most common histological type of ACPs was tubular adenoma. Advancing age, being Kuwaiti nationals and prior removal of ACPs were significantly associated with the occurrence of ACPs.
PMCID: PMC3003207  PMID: 20616414
Colonoscopy; colorectal polyps; Kuwait
3.  Effect of rosiglitazone and ramipril on macrovasculopathy in patients with type 2 diabetes: needs longer treatment and/or higher doses? 
The aim of the study is to investigate whether standard doses of rosiglitazone (4 mg/daily) and ramipril (5 mg/daily) can reverse pre-clinical macrovasculopathy in newly diagnosed never treated type 2 diabetes (T2DM) patients.
In this randomized, double-blind, placebo-controlled study, 33 T2DM patients were randomized to rosiglitazone (4 mg/daily) or ramipril (5 mg/daily) or placebo for 1 year. Hemodynamic variables were measured at 3 treatment phases and pulse wave velocity (PWV) and augmentation index (AI) were measured throughout the treatment period.
In diabetic patients, PWV (P = 0.037) and AI (P = 0.005) with ramipril and AI (P < 0.001) with rosiglitazone were significantly reduced during overall treatment period from the baseline; however, these differences were not significant in comparison to placebo.
Discussion and conclusion
The present study showed that treatment with standard doses of rosiglitazone and ramipril are not adequate to reverse pre-clinical vasculopathy in T2DM. The lack of benefit in newly diagnosed T2DM may be because of the relatively short-term intervention and/or the use of lower doses of rosiglitazone/ramipril. Further trials are needed for a longer period of time, possibly with higher doses, to show whether rosiglitazone/ramipril can reverse pre-clinical vasculopathy in T2DM ( number, NCT00489229).
PMCID: PMC3262381  PMID: 22291490
rosiglitazone; ramipril; diabetic vasculopathy
4.  Anticoagulant therapy for deep vein thrombosis (DVT) in pregnancy 
Thromboembolic complications are much higher in pregnancy due to procoagulant changes. Heparin does not cross the placenta and the use of unfractionated heparin (UFH) is the current established practice in prophylaxis and treatment for thromboembolism in pregnancy.
To compare the effectiveness of anticoagulant therapies for the treatment of deep vein thrombosis in pregnancy. The anticoagulant drugs included are UFH, low molecular weight heparin (LMWH) and warfarin.
Search methods
We searched the Cochrane Pregnancy and Childbirth Group’s Trials Register (March 2010) and reference lists of retrieved studies.
Selection criteria
Randomised controlled trials comparing any combination of warfarin, UFH, LMWH and placebo in pregnant women.
Data collection and analysis
We used methods described in the Cochrane Handbooks for Systemic Reviews of Interventions for assessing the eligibility of studies identified by the search strategy. A minimum of two review authors independently assessed each study.
Main results
We did not identify any eligible studies for inclusion in the review.
We identified three potential studies; after assessing eligibility, we excluded all three as they did not meet the prespecified inclusion criteria. One study compared LMWH and UFH in pregnant women with previous thromboembolic events and, for most of these women, anticoagulants were used as thromboprophylaxis. There were only three women who had a thromboembolic event during the current pregnancy and it was unclear whether the anticoagulant was used as therapy or prophylaxis. We excluded one study because it included only women undergoing caesarean birth. The third study was not a randomised trial.
Authors’ conclusions
There is no evidence from randomised controlled trials on the effectiveness of anticoagulation for deep vein thrombosis in pregnancy. Further studies are required.
PMCID: PMC4238056  PMID: 20556784
Anticoagulants [*therapeutic use]; Heparin [therapeutic use]; Heparin, Low-Molecular-Weight [therapeutic use]; Pregnancy Complications, Hematologic [*drug therapy]; Venous Thrombosis [*drug therapy]; Warfarin [therapeutic use]; Female; Humans; Pregnancy
5.  Effect of intensive treatment of hyperglycemia on microvascular complications of type 2 diabetes in ACCORD: a randomized trial 
Lancet  2010;376(9739):419-430.
ACCORD is a parallel group, randomized trial designed to investigate whether intensive glycemic therapy with a target HbA1c of <6.0% versus standard therapy with a target of 7.0 to 7.9% reduces cardiovascular disease (CVD) morbidity, mortality, and microvascular complications in participants with type 2 diabetes.
Volunteers with established type 2 diabetes, HbA1c levels ≥ 7.5% and CVD or two or more CVD risk factors were recruited at 77 clinical sites across the U.S. and Canada. Instructional materials, behavioral counseling, glucose-lowering medications and self-monitoring supplies were provided by the study. Therapeutic regimens were individualized on the basis of randomized assignment and response to therapy. This investigation examines the effect of treatment to glycemic goals on occurrence of microvascular diabetes complications. Prespecified composite outcomes were: 1) dialysis or renal transplantation, or serum creatinine >291.7 micromol/L, or retinal photocoagulation or vitrectomy, and 2) these plus peripheral neuropathy. Thirteen prespecified secondary measures of kidney, eye, and peripheral nerve function were also evaluated. Randomization was performed at clinical sites using a central randomization routine available on the study website. Both investigators and participants were unmasked to treatment arm assignment.
A total of 10,251 participants were randomized (5,128 intensive and 5,123 standard) between January, 2001 and October, 2005. This analysis includes 10,234 patients (5,107 intensive and 5,108 standard). Intensive therapy was stopped before study end due to increased mortality, and patients were transitioned to standard therapy. Outcomes are reported at transition and at study end. At transition, the first composite outcome occurred in 443/5107 and 444/5108 participants in the intensive and standard arms, respectively (p= 0.99), and the second outcome in 1591/5107 and 1659/5108 participants in intensive and standard arms (p=0.20). Results were similar at study end. Secondary measures at study end favoring intensive therapy (p<0.05) included development of macroalbuminuria, cataract extraction, visual acuity, a score of >2.0 on the Michigan Neuropathy Screening Instrument, loss of ankle jerk and light touch.
Intensive glycemic treatment did not reduce the risk of advanced measures of microvascular outcomes, but delayed the onset of macroalbuminuria and some measures of eye complications and neuropathy. These benefits must be weighed against the increase in total and CVD-related mortality, increased weight gain, and higher risk for severe hypoglycemia.
PMCID: PMC4123233  PMID: 20594588
6.  Young Egyptians’ perceptions, attitudes and knowledge of injuries 
The objective of this study was to qualitatively evaluate young Egyptians’ perceptions, attitudes, knowledge and behaviour towards injuries before implementation of an extensive questionnaire about injuries among Egyptian youth. In 2008, five focus groups of three to nine participants each were conducted in Cairo, Egypt in Arabic to evaluate young Egyptians’ attitudes towards injuries, injury prevention, and their understanding of ‘accidents’ and fatalism. Participants were 14–26 years of age and were from medium to high socioeconomic status. Focus group participants noted that the concept of hadthah (‘accident’) signified an event determined by destiny, whereas esabah (‘injury’) was the result of human actions. The results of these focus groups indicate that young, educated Egyptians are interested in injury prevention programmes despite low confidence in the preventability of injuries.
PMCID: PMC3570976  PMID: 20587813
7.  Risk Factors and Immune Response to Hepatitis E viral Infection among Acute Hepatitis Patients in Assiut, Egypt 
Hepatitis E virus (HEV) infection is a common cause of acute viral hepatitis (AVH) in Egypt. We aimed to identify risk factors of HEV among acute hepatitis cases, measure HEV specific immune response to differentiate between symptomatic and asymptomatic infections. The study included symptomatic acute hepatitis (AH) patients (n=235) and asymptomatic contacts (n=200) to HEV cases. They completed a lifestyle questionnaire, screened for common hepatotropic viruses. Blood and serum samples were collected from patients and contacts after onset of disease and follow-up samples collected until convalescence. PBMC were separated and tested for specific HEV T-cell response by INF-ELISPOT assay. Serum samples were tested for IgM and IgG anti-hepatitis E virus by ELISA. IgM antibodies to HAV were detected in 19 patients (8.1%), 37 (15.7%) with HBV, 10 (4.3%) with HCV. HEV infection was identified in 42 (16%) patients with AVH. Of the 200 contacts, 14 (7%) had serological evidence of recent HEV asymptomatic infection, showed stronger CMI responses than HEV infected subjects (2540 ± 28 and 182 ± 389 ISCs/106 cells, respectively; P <0.05). In conclusion, HEV is a major cause of AVH in Egypt. Asymptomatic HEV patients are likely to have stronger immune responses including CMI responses, than symptomatic cases.
PMCID: PMC3399527  PMID: 22053611
8.  Comparison of headless screws used in the treatment of proximal nonunion of scaphoid bone 
International Orthopaedics  2010;35(7):1031-1035.
Screws with different levels of compression force are available for scaphoid fixation and it is known that the Acutrak screw generates greater compression than the Herbert screw. We retrospectively compared two types of headless compression screw for their effectiveness in the repair of scaphoid nonunion. Twenty-nine cases of proximal scaphoid nonunion were surgically treated with non-vascularised bone graft: the Acutrak screw was used in 17 patients and the cannulated Herbert screw in 12 patients. Wrist range of motion, Mayo wrist score, grip strength and QuickDASH scores were indicators used for the functional evaluation. Radiographic findings were assessed for consolidation of nonunion and signs of arthrosis. The mean follow-up time was 49.2 months (range 12–96). Statistically, there was no significant difference between the Acutrak and Herbert screw types in terms of functional evaluation and time required for consolidation. Greater compression did not influence the functional outcome, consolidation rate or time to consolidation. The need for greater compression in the treatment of proximal scaphoid nonunions is thus questionable because it may increase the risk of proximal fragment communition.
PMCID: PMC3167405  PMID: 20890757
9.  Appendicitis epiploicae: a rare cause of acute abdomen 
BMJ Case Reports  2010;2010:bcr08.2009.2171.
Acute appendicitis is one of the most common causes of right lower quadrant acute abdominal pain in adults. Some other conditions, including appendicitis epiploicae, can simulate an acute abdomen. Appendicitis epiploicae or epiploic appendicitis usually originates in the sigmoid colon and rarely from other parts of colon. We report a case of a 20-year-old man with appendicitis epiploicae of the caecum, who underwent surgery for acute appendicitis. Analysis of this uncommon condition, together with a review of the pertinent literature, are presented.
PMCID: PMC3047281  PMID: 22736317
10.  Dicationic phenyl-2, 2′-bichalcophenes and analogues as antiprotozoal agents 
Bioorganic & medicinal chemistry  2010;19(2):978-984.
A series of phenyl-2, 2′-bichalcophene diamidines 1a-h were synthesized from the corresponding dinitriles either via a direct reaction with LiN(TMS)2, followed by deprotection with ethanolic HCl or through the bis-O-acetoxyamidoxime followed by hydrogenation in acetic acid and EtOH over Pd-C. These diamidines show a wide range of DNA affinities as judged from their ΔTm values which are remarkably sensitive to replacement of a furan unit with a thiophene one. These differences are explained in terms of the effect of subtle changes in geometry of the diamidines on binding efficacy. Five of the eight compounds were highly active (below 6 nM IC50) in vitro against Trypanosoma brucei rhodesiense (T. b. r.) and four gave IC50values less than 7 nM against Plasmodium falciparum (P. f.). Only one of the compounds was as effective as reference compounds in the T. b. r. mouse model for the acute phase of African trypanosomiasis.
PMCID: PMC3064985  PMID: 21194955
diamidines; bichalcophenes; Antiprotozoal agents; Stille coupling; Heck coupling
11.  High-sensitivity O-glycomic analysis of mice deficient in core 2 β1,6-N-acetylglucosaminyltransferases 
Glycobiology  2010;21(1):82-98.
Core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT), which exists in three isoforms, C2GnT1, C2GnT2 and C2GnT3, is one of the key enzymes in the O-glycan biosynthetic pathway. These isoenzymes produce core 2 O-glycans and have been correlated with the biosynthesis of core 4 O-glycans and I-branches. Previously, we have reported mice with single and multiple deficiencies of C2GnT isoenzyme(s) and have evaluated the biological and structural consequences of the loss of core 2 function. We now present more comprehensive O-glycomic analyses of neutral and sialylated glycans expressed in the colon, small intestine, stomach, kidney, thyroid/trachea and thymus of wild-type, C2GnT2 and C2GnT3 single knockouts and the C2GnT1–3 triple knockout mice. Very high-quality data have emerged from our mass spectrometry techniques with the capability of detecting O-glycans up to at least 3500 Da. We were able to unambiguously elucidate the types of O-glycan core, branching location and residue linkages, which allowed us to exhaustively characterize structural changes in the knockout tissues. The C2GnT2 knockout mice suffered a major loss of core 2 O-glycans as well as glycans with I-branches on core 1 antennae especially in the stomach and the colon. In contrast, core 2 O-glycans still dominated the O-glycomic profile of most tissues in the C2GnT3 knockout mice. Analysis of the C2GnT triple knockout mice revealed a complete loss of both core 2 O-glycans and branched core 1 antennae, confirming that the three known isoenzymes are entirely responsible for producing these structures. Unexpectedly, O-linked mannosyl glycans are upregulated in the triple deficient stomach. In addition, our studies have revealed an interesting terminal structure detected on O-glycans of the colon tissues that is similar to the RM2 antigen from glycolipids.
PMCID: PMC2998984  PMID: 20855471
C2GnT; core 2 O-glycan; knockout mice; mass spectrometry; murine O-glycome
12.  Identification of Autoantibody-Negative Autoimmune Type 2 Diabetic Patients 
Diabetes Care  2010;34(1):168-173.
Islet autoimmunity has long been recognized in the pathogenesis of type 1 diabetes and is becoming increasingly acknowledged as a component in the pathogenesis of type 2 diabetes. Islet reactive T cells and autoantibodies have been demonstrated in type 1 diabetes, whereas islet autoimmunity in type 2 diabetes has been limited to islet autoantibodies. In this study, we investigated whether islet reactive T cells might also be present in type 2 diabetic patients and how islet reactive T cells correlate with β-cell function.
Adult phenotypic type 2 diabetic patients (n = 36) were screened for islet reactive T-cell responses using cellular immunoblotting and five islet autoantibodies (islet cell antibody, GADA, insulin autoantibody, insulinoma-associated protein-2 autoantibody, and zinc transporter autoantibody).
We identified four subgroups of adult phenotypic type 2 diabetic patients based on their immunological status (Ab−T−, Ab+T−, Ab−T+, and Ab+T+). The Ab−T+ type 2 diabetic patients demonstrated T-cell responses similar to those of the Ab+T+ type 2 diabetic patients. Data were adjusted for BMI, insulin resistance, and duration of diabetes. Significant differences (P < 0.02) were observed among groups for fasting and glucagon-stimulated C-peptide responses. T-cell responses to islet proteins were also demonstrated to fluctuate less than autoantibody responses.
We have identified a group of adult autoimmune phenotypic type 2 diabetic patients who are Ab−T+ and thus would not be detected using autoantibody testing alone. We conclude that islet autoimmunity may be more prevalent in adult phenotypic type 2 diabetic patients than previously estimated.
PMCID: PMC3004952  PMID: 20855551
13.  ‘An interventional urology list’ – a novel concept for UK urological services 
Almost all patients in the UK with obstructed and/or infected kidneys are referred to interventional radiology for percutaneous nephrostomy and/or placement of an anterograde JJ stent. Although this ‘tradition’ is going strong in the UK, urologists throughout the world have evolved their practice to encompass such interventional procedures in their remit. We have set up a local anaesthetic list ‘interventional urology list’ in our ESWL suite. We present our 4-year experience and discuss the benefits that this interventional list brings to our patients, our trainees, our interventional radiology colleagues and to the hospital trust.
From May 2005 to May 2009, we have been running this list, twice-weekly, performing procedures such as nephrostomies, anterograde stents, nephrostograms and stent exchanges all under local anaesthetic.
A total of 580 procedures have been carried out on this list over this period. Our success rate for nephrostomy insertion is 96% with three failures, as a result of patient discomfort. No major complications and three minor complications were reported. We had four failed anterograde stenting procedures (out of 80). All other procedures including nephrostograms, stent exchanges/removals/insertions, as well as renal cyst aspiration and sclerotisation were successfully carried out.
Our results of percutaneous nephrostomy and antegrade stenting are favourable when compared with published data on nephrostomies. This novel set up has resulted in several improvements to the service we offer patients and also provided significant improvement in training for our residents. We encourage other departments to try and develop this type of ‘interventional urology list’.
PMCID: PMC3293267  PMID: 20977835
Interventional list; Urology; Percutaneous nephrostomy
14.  Homotypic gap junctional communication contributing to metastasis increases with PKA kinase activity and is unaffected by PI3K inhibition 
Cancer research  2010;70(23):10002-10011.
Loss of gap junctional intercellular communication (GJIC) between cancer cells is a common characteristic of malignant transformation. This communication is mediated by connexin proteins that make up the functional units of gap junctions. Connexins are highly regulated at the protein level and phosphorylation events play a key role in their trafficking and degradation. The metastasis suppressor BRMS1 up-regulates GJIC and decreases PI3K signaling. Based on these observations we set out to determine if there was a link between PI3K and GJIC in tumorigenic and metastatic cell lines. Treatment of cells with the well-known PI3K inhibitor LY294002, and its structural analog LY303511 which does not inhibit PI3K, increased homotypic GJIC; however, we found the effect to be independent of PI3K/AKT inhibition. Additionally, while levels of the connexin Cx43 remained unchanged, Cx43 relocalization from the cytosol to the plasma membrane was observed. Both LY294002 and LY303511 increased the activity of protein kinase A (PKA). Moreover, PKA blockade by the small molecule inhibitor H89 decreased the LY294002/LY303511 mediated increase in GJIC. Collectively, our findings demonstrate a connection between PKA activity and GJIC mediated by PI3K-independent mechanisms of LY294002 and LY303511. Manipulation of these signaling pathways could prove useful for anti-metastatic therapy.
PMCID: PMC3003438  PMID: 21098703
connexin 43; gap junction; protein kinase A; LY294002; LY303511
15.  Pathogenesis of Renal Injury in the Megabladder Mouse: A Genetic Model of Congenital Obstructive Nephropathy 
Pediatric research  2010;68(6):500-507.
Congenital obstructive nephropathy (CON) is the most common cause of chronic renal failure in children, often leading to end stage renal disease. The megabladder (mgb) mouse exhibits signs of urinary tract obstruction in utero resulting in the development of hydroureteronephrosis and progressive renal failure following birth. This study examined the development of progressive renal injury in homozygous mgb mice (mgb−/−). Renal ultrasound was utilized to stratify the disease state of mgb−/− mice, while surgical rescue was performed using vesicostomy. The progression of renal injury was characterized using a series of pathogenic markers including α-smooth muscle isoactin (α-SMA), TGF-β1, connective tissue growth factor (CTGF), E-cadherin, F4/80, Wilm’s Tumor 1 (WT-1), and paired box gene 2 (Pax2). This analysis indicated that mgb−/− mice are born with pathologic changes in kidney development that progressively worsen in direct correlation with the severity of hydronephrosis. The initiation and pattern of fibrotic development observed in mgb−/− kidneys appeared distinctive from prior animal models of obstruction. These observations suggest that the mgb mouse represents a unique small animal model for the study of CON.
PMCID: PMC3121911  PMID: 20736884
16.  Urban-rural differences in breast cancer incidence in Egypt (1999–2006) 
Breast (Edinburgh, Scotland)  2010;19(5):417-423.
To describe urban-rural differences in breast cancer incidence in Gharbiah, Egypt and to investigate if these differences could be explained by known risk factors of breast cancer.
We used data from the population-based cancer registry of Gharbiah, Egypt to assess breast cancer incidence from 1999 through 2006. The Egyptian census provided data on district-specific population, age, and urban-rural classification. Incidence patterns of breast cancer by district and age-specific urban-rural differences were analyzed.
Overall, incidence rate of breast cancer was three to four times higher in urban areas than in rural areas (60.9/105/year for urban areas versus 17.8/105/year for rural areas; IRR = 3.73, 95% CI = 3.30, 4.22). Urban areas had consistently higher incidence of breast cancer across all age-groups for all years. Higher incidence of breast cancer was also seen in the more developed districts of Tanta and El-Mehalla.
Higher incidence of breast cancer in urban and more developed populations might be related to higher xenoestrogens, as well as other endocrine disruptors and genotoxic substances.
PMCID: PMC3209663  PMID: 20452771
Breast cancer; incidence; urban-rural; xenoestrogens; Egypt
Transplantation  2010;90(8):836-842.
Assays for assessing human islet cell quality which provide results prior to transplantation would be very beneficial to improving outcomes for islet transplantation therapy. Parameters such as percent beta cell apoptosis and cell composition are found to vary markedly between different islet preparations, and may serve as markers of islet quality. We have developed fluorescence-based assays using laser scanning cytometry (LSC) for assessing beta cell apoptosis and islet cell composition on serial sections of intact isolated islets.
Isolated human islets were fixed in formalin and embedded in paraffin. Serial sections were immunostained for the pancreatic hormones, acinar and ductal cell markers. DNA fragmentation was used to label apoptotic cells. Stained cells were quantified using an iCys laser scanning cytometer.
Islet preparations from 102 human pancreatic islet isolations were analyzed. For the whole set of islet preparations we found a mean islet cell composition of 54.5±1.2% insulin positive; 33.9±1.2% glucagon; 12.1±0.7% somatostatin and 1.5±0.2% pancreatic polypeptide positive cells. The apoptotic beta cells were 2.85±0.4% with a range of 0.27% to 18.3%. The percentage of apoptotic beta cells correlated well (p<0.0001, n=59) with results obtained in vivo by transplantation of the corresponding islets in diabetic NODscid mice.
The analysis of whole, non-dissociated islets for cell composition and beta cell apoptosis using LSC is giving reliable and reproducible results and could be done both before islet transplantation, as well as on preserved cell blocks at any future time. Thus, they can be a powerful tool for islet quality assessment.
PMCID: PMC2959131  PMID: 20697327
Laser scanning cytometry; human pancreatic islets; islet cell composition; β cell apoptosis
18.  Discovery of Novel Benzo[a]phenoxazine SSJ-183 as a Drug Candidate for Malaria 
ACS Medicinal Chemistry Letters  2010;1(7):360-364.
Malaria is a serious infectious disease caused by protozoan parasites in tropical and subtropical regions. Even inhabitants of temperate zones are exposed to the danger of malaria infection because of travel and global warming. Novel, effective, safe, and inexpensive drugs are required to treat malaria and contribute to the global goal of eradication. A search for new antimalarial agents has been performed by the synthesis of new benzo[a]phenoxazines, followed by biological evaluations. The derivative SSJ-183 (5), having a 4-aminopyridine group, showed an IC50 value against Plasmodium falciparum of 7.6 nM and a selectivity index of >7300. Cure was achieved by three oral doses of 5 at 100 mg/kg to mice infected with the Plasmodium berghei ANKA strain. The safety of 5 was supported by acute toxicity testing in mice with single doses up to 2000 mg/kg po, chromosome aberration test, in vitro as well as in vivo micronucleus tests, and phototoxicity studies in mice. Thus, 5 is a promising candidate as a new antimalarial agent.
PMCID: PMC4007839  PMID: 24900219
Antimalarial activity; benzo[a]phenoxazine; Plasmodium falciparum; Plasmodium berghei; oral administration
19.  DNA Minor Groove Induced Dimerization of Heterocyclic Cations: Compound Structure, Binding Affinity and Specificity for a TTAA Site 
Journal of molecular biology  2010;402(5):847-864.
With the increasing number and variations of genome sequences available control of gene expression with synthetic, cell permeable molecules is within reach. The variety of sequence-specific-binding agents is, however, still quite limited. Many minor groove binding agents selectivity recognize AT over GC sequences but have less ability to distinguish among different AT sequences. The goal with this paper is to develop compounds that can bind selectively to different AT sequences. A number of studies indicate that AATT and TTAA sequences have significantly different physical and interaction properties and different requirements for minor groove recognition. Although it has been difficult to get minor groove binding at TTAA, DB293, a phenyl-furan-benzimidazole-diamidine, was found to bind as a strong, cooperative dimer at TTAA but with no selectivity over AATT. In order to improve selectivity, modifications were made to each unit of DB293. Binding affinities and stoichiometries obtained from biosensor-surface plasmon resonance experiments show that DB1003, a furan-furan-benzimidazole diamidine binds strongly to TTAA as a dimer and has selectivity (KTTAA/KAATT = 6). CD and DNAse I footprinting studies confirmed the preference of this compound for TTAA. In summary (i) a favorable stacking surface provided by the pi system, (ii) H-bond donors to interact with TA base pairs at the floor of the groove provided by a benzimidazole (or indole) –NH and amidines, and (iii) appropriate curvature of the dimer complex to match the curvature of the minor groove play important roles in differentiating the TTAA and AATT minor grooves.
PMCID: PMC2988403  PMID: 20713062
TTAA; Sequence-recognition; DNA; Biosensor-SPR; Cooperative dimer
20.  Comparing BRIN-BD11 culture producing insulin using different type of microcarriers 
Cytotechnology  2010;62(5):423-430.
This research was conducted to examine the growth profile, growth kinetics, and insulin-secretory responsiveness of BRIN-BD11 cells grown in optimized medium on different types of microcarriers (MCs). Comparisons were made on modified polystyrene (Hillex® II) and crosslinked polystyrene Plastic Plus (PP) from Solohill Engineering. The cell line producing insulin was cultured in a 25 cm2 T-flask as control while MCs based culture was implemented in a stirred tank bioreactor with 1 L working volume. For each culture type, the viable cell number, glucose, lactate, glutamate, and insulin concentrations were measured and compared. Maximum viable cell number was obtained at 1.47 × 105 cell/mL for PP microcarrier (PPMCs) culture, 1.35 × 105 cell/mL Hillex® II (HIIMCs) culture and 0.95 × 105 cell/mL for T-flask culture, respectively. The highest insulin concentration has been produced in PPMCs culture (5.31 mg/L) compared to HIIMCs culture (2.01 mg/L) and T-flask culture (1.99 mg/L). Therefore overall observation suggested that PPMCs was likely preferred to be used for BRIN-BD11 cell culture as compared with Hillex® II MCs.
PMCID: PMC2993864  PMID: 20953703
Insulin; BRIN-BD11; Solohill; Microcarrier; Bioreactor
Oncogene  2010;30(13):1586-1596.
Our previous studies demonstrated that protein kinase D (PKD), a serine/threonine kinase implicated in various cell processes, is up-regulated in basal cell carcinoma (BCC), supporting a possible tumorigenic role for PKD in skin. Since the greatest risk factor for BCC is sun exposure, the ability of ultraviolet B (UVB) irradiation to activate PKD in primary mouse keratinocytes was investigated. Using western analysis with two autophosphorylation-specific antibodies, we show for the first time that UVB activated PKD in a time- and dose-dependent manner. UVB-induced PKD activation was verified using an in vitro kinase assay. Furthermore, activation was reduced by antioxidant pretreatment, suggesting a link with oxidative stress. UVB-induced PKD activation was mediated primarily by Src family tyrosine kinases rather than protein kinase C (PKC), and in fact, UVB did not alter PKC-mediated transphosphorylation. UVB induced apoptosis dose-dependently, and this death could be prevented by overexpression of wild-type PKD, but not mutant PKD or the empty adenovirus. Indeed, a mutant that cannot be phosphorylated by Src kinases exacerbated UVB-elicited apoptosis. Thus, our data indicate that UVB irradiation of keratinocytes induces Src-mediated activation of PKD, which protects cells from UVB-stimulated apoptosis, providing a possible explanation for the observed up-regulation of PKD in BCC.
PMCID: PMC3069139  PMID: 21132013
Apoptosis; Epidermis; Protein kinase C (PKC); Protein kinase D (PKD); Skin; Src
22.  Tiam1/Rac1 signaling pathway mediates palmitate-induced, ceramide-sensitive generation of superoxides and lipid peroxides and the loss of mitochondrial membrane potential in pancreatic β-cells 
Biochemical pharmacology  2010;80(6):874-883.
The phagocytic NADPH-oxidase [NOX] has been implicated in the generation of superoxides in the pancreatic β-cell. Herein, using normal rat islets and clonal INS 832/13 cells, we tested the hypothesis that activation of the small G-protein Rac1, which is a member of the NOX holoenzyme, is necessary for palmitate [PA]-induced generation of superoxides in pancreatic β-cells. Incubation of isolated β-cells with PA potently increased the NOX activity culminating in a significant increase in the generation of superoxides and lipid peroxides in these cells; such effects of PA were attenuated by diphenyleneiodonium [DPI], a known inhibitor of NOX. In addition, PA caused a transient, but significant activation [i.e., GTP-bound form] of Rac1 in these cells. NSC23766, a selective inhibitor of Rac1, but not Cdc42 or Rho activation, inhibited Rac1 activation and the generation of superoxides and lipid peroxides induced by PA. Fumonisin B-1 [FB-1], which inhibits de novo synthesis of ceramide [CER] from PA, also attenuated PA-induced superoxide and lipid peroxide generation and NOX activity implicating intracellularly generated CER in the metabolic effects of PA; such effects were also demonstrable in the presence of the cell-permeable C2-CER. Further, NSC23766 prevented C2-CER-induced Rac1 activation and production of superoxides and lipid peroxides. Lastly, C2-CER, but not its inactive analogue, significantly reduced the mitochondrial membrane potential, which was prevented to a large degree by NSC23766. Together, our findings suggest that Tiam1/Rac1 signaling pathway regulates PA-induced, CER-dependent superoxide generation and mitochondrial dysfunction in pancreatic β-cells.
PMCID: PMC2919057  PMID: 20493824
NADPH Oxidase; Rac1; Tiam1; palmitate; ceramide; oxidative stress; pancreatic β-cells
23.  PhoB regulates both environmental and virulence gene expression in Vibrio cholerae 
Molecular microbiology  2010;77(6):1595-1605.
Vibrio cholerae is a facultative pathogen that thrives in two nutritionally disparate environments, aquatic and human small intestine. Phosphate (Pi) is an essential nutrient that is limited in aquatic ecosystems and of unknown availability in the small intestine. Here we show that the Pi (Pho) regulon, which is controlled by the Pi-specific transporter (Pst) and two-component system PhoBR, is required for V. cholerae survival in both environments, though for differing reasons. While induction of Pi acquisition systems including Pst is critical for survival in the aquatic environment, regulation of virulence genes by PhoB and not Pi transport per se is required for colonization of the small intestine. We show that PhoB regulates virulence genes by directly controlling expression of a key upstream transcriptional regulator, tcpPH. Thus, the Pho regulon includes virulence genes and represents a diverse gene set essential to pathogenic V. cholerae throughout its life cycle.
PMCID: PMC2981138  PMID: 20659293
24.  Genetic diversity for grain nutrients in wild emmer wheat: potential for wheat improvement 
Annals of Botany  2010;105(7):1211-1220.
Background and Aims
Micronutrient malnutrition, particularly zinc and iron deficiency, afflicts over three billion people worldwide due to low dietary intake. In the current study, wild emmer wheat (Triticum turgidum ssp. dicoccoides), the progenitor of domesticated wheat, was tested for (1) genetic diversity in grain nutrient concentrations, (2) associations among grain nutrients and their relationships with plant productivity, and (3) the association of grain nutrients with the eco-geographical origin of wild emmer accessions.
A total of 154 genotypes, including wild emmer accessions from across the Near Eastern Fertile Crescent and diverse wheat cultivars, were characterized in this 2-year field study for grain protein, micronutrient (zinc, iron, copper and manganese) and macronutrient (calcium, magnesium, potassium, phosphorus and sulphur) concentrations.
Key Results
Wide genetic diversity was found among the wild emmer accessions for all grain nutrients. The concentrations of grain zinc, iron and protein in wild accessions were about two-fold greater than in the domesticated genotypes. Concentrations of these compounds were positively correlated with one another, with no clear association with plant productivity, suggesting that all three nutrients can be improved concurrently with no yield penalty. A subset of 12 populations revealed significant genetic variation between and within populations for all minerals. Association between soil characteristics at the site of collection and grain nutrient concentrations showed negative associations between soil clay content and grain protein and between soil-extractable zinc and grain zinc, the latter suggesting that the greatest potential for grain nutrient minerals lies in populations from micronutrient-deficient soils.
Wild emmer wheat germplasm offers unique opportunities to exploit favourable alleles for grain nutrient properties that were excluded from the domesticated wheat gene pool.
PMCID: PMC2887062  PMID: 20202969
grain quality; iron; macronutrient; micronutrient; protein; Triticum turgidum ssp. dicoccoides; wheat improvement; zinc
25.  Bilateral symptomatic petrous apex effusion 
Petrous apex effusions can present with aural fullness, hearing loss and dizziness. Although they can be followed-up when asymptomatic, clinical management of symptomatic patients is controversial. In this study, we present clinical and radiological findings of a 24-year-old patient with bilateral petrous apex effusion. She had been complaining of bilateral aural fullness and dizziness for 2 years. Radiological examinations revealed bilateral petrous apex effusion. After medical treatment, her symptoms gradually disappeared. In all previous published studies, unilateral petrous apex effusions were reported. To our best knowledge, this is the first patient with trapped fluid in bilateral petrous apex.
PMCID: PMC3450305  PMID: 23120711
Petrous apex; Effusion; Computed tomography; Magnetic resonance imaging

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