In vitro studies on CNGRC-CPG2 fusion proteins for ligand-directed enzyme prodrug therapy for targeted cancer therapy

The sequence asparagine-glycine arginine (NGR), flanked by Cysteine (Cys) residues so as to form a disulfide-bridge (CNGRC), has previously been found to target and bind specifically to aminopeptidase N (APN), which is highly expressed on the surface of tumor cells. The goal of this study was to develop and evaluate the potential of fusion proteins carrying the CNGRC sequence linked to the enzyme carboxypeptidase G2 (CPG2) for targeted cancer therapy. We refer to this strategy as ligand-directed enzyme prodrug therapy (LDEPT). We constructed two forms of the CNGRC-CPG2 fusions, containing one or two copies of the cyclic NGR motif and designated CNGRC-CPG2 (X-CPG2) and CNGRC-CPG2-CNGRC (X-CPG2-X), respectively. binding assays of the purified constructs showed that both X-CPG2 and X-CPG2-X bound with high affinity to cancer cells expressing high levels of APN, compared to their binding to cells expressing low levels of APN. Further studies of the constructs to assess the therapeutic potential of LDEPT were carried out using cells expressing high and low levels of APN. Using methotrexate, it was demonstrated that cancer cell survival was significantly higher in the presence of the fusion proteins, due to the hydrolysis of this cytotoxic drug by CPG2. Conversely, when the prodrug ZD2767P was used, cancer cell killing was higher in the presence of the fused CPG2 constructs than in their absence, which is consistent with CPG2-mediated release of the cytotoxic drug from the prodrug. Furthermore, the doubly-fused CPG2 construct (X-CPG2-X) was significantly more effective than the singly-fused construct (X-CPG2)

Functional modelling of a novel mutation in BBS5.

BACKGROUND: Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy disorder with 18 known causative genes (BBS1-18). The primary clinical features are renal abnormalities, rod-cone dystrophy, post-axial polydactyly, learning difficulties, obesity and male hypogonadism. RESULTS: We describe the clinical phenotype in three Saudi siblings in whom we have identified a novel mutation in exon 12 of BBS5 (c.966dupT; p.Ala323CysfsX57). This single nucleotide duplication creates a frame shift results in a predicted elongated peptide. Translation blocking Morpholino oligonucleotides were used to create zebrafish bbs5 morphants. Morphants displayed retinal layering defects, abnormal cardiac looping and dilated, cystic pronephric ducts with reduced cilia expression. Morphants also displayed significantly reduced dextran clearance via the pronephros compared to wildtype embryos, suggesting reduced renal function in morphants. The eye, kidney and heart defects reported in morphant zebrafish resemble the human phenotype of BBS5 mutations. The pathogenicity of the novel BBS5 mutation was determined. Mutant mRNA was unable to rescue pleiotropic phenotypes of bbs5 morphant zebrafish and in cell culture we demonstrate a mislocalisation of mutant BBS5 protein which fails to localise discretely with the basal body. CONCLUSIONS: We conclude that this novel BBS5 mutation has a deleterious function that accounts for the multisystem ciliopathy phenotype seen in affected human patients

Production of "biobetter" variants of glucarpidase with enhanced enzyme activity

Glucarpidase, also known as carboxypeptidase G(2), is a Food and Drug Administration-approved enzyme used in targeted cancer strategies such as antibody-directed enzyme prodrug therapy (ADEPT). It is also used in drug detoxification when cancer patients have excessive levels of the anti-cancer agent methotrexate. The application of glucarpidase is limited by its potential immunogenicity and limited catalytic efficiency. To overcome these pitfalls, mutagenesis was applied to the glucarpidase gene of Pseudomonas sp. strain RS-16 to isolate three novels "biobetter" variants with higher specific enzyme activity. DNA sequence analysis of the genes for the variants showed that each had a single point mutation, resulting in the amino acid substitutions: I100 T, G123S and T239 A. K-m, V-max and K-cat measurements confirmed that each variant had increased catalytic efficiency relative to wild type glucarpidase. Additionally, circular dichroism studies indicated that they had a higher alpha-helical content relative to the wild type enzyme. However, three different software packages predicted that they had reduced protein stability, which is consistent with having higher activities as a tradeoff. The novel glucarpidase variants presented in this work could pave the way for more efficient drug detoxification and might allow dose escalation during chemotherapy. They also have the potential to increase the efficiency of ADEPT and to reduce the number of treatment cycles, thereby reducing the risk that patients will develop antibodies to glucarpidase

Causes and risk factors for common mental illnesses : the beliefs of paediatric hospital staff in the United Arab Emirates

Background Children and adolescents with chronic physical health conditions are vulnerable to poor mental health outcomes. The measurement of mental health literacy of health professionals working with such populations is important because of their role in promoting early and appropriate help-seeking. This study sought to determine the beliefs regarding the causes of and risks factors for three types of mental illnesses amongst health professionals in United Arab Emirates. Method A culturally validated mental health literacy survey presenting three vignettes of fictional characters meeting diagnostic criteria for posttraumatic stress disorder, depression with suicidal thoughts and psychosis was distributed. The survey measured health care professionals’ beliefs regarding the causes of and risk factors for these disorders. Results A total of 317 health care professional (> 90% nurses) were surveyed from across the UAE. Although 43.8% correctly endorsed exposure to a ‘traumatic event’ as the most likely cause for developing posttraumatic stress disorder, there was a more limited understanding of the contribution of biopsychosocial factors to the development of the mental illness, particularly for psychosis. Participant socio-demographic variables were associated with attributions of religious or spiritual beliefs and personal weakness as causal and/or vulnerability factors in the development of depression with suicidal thoughts and psychosis. Conclusions Efforts to improve mental health systems and health care providers in UAE and other similar Middle Eastern countries requires targeted mental health literacy programs that seek to integrate biopsychosocial models of mental illness and their treatment with the positive aspects of religious and cultural beliefs that are dominant in this region

The Therapeutic Potential of Anthocyanins : Current Approaches Based on Their Molecular Mechanism of Action

Anthocyanins are natural phenolic pigments with biological activity. They are well-known to have potent antioxidant and antiinflammatory activity, which explains the various biological effects reported for these substances suggesting their antidiabetic and anticancer activities, and their role in cardiovascular and neuroprotective prevention. This review aims to comprehensively analyze different studies performed on this class of compounds, their bioavailability and their therapeutic potential. An in-depth look in preclinical, in vitro and in vivo, and clinical studies indicates the preventive effects of anthocyanins on cardioprotection, neuroprotection, antiobesity as well as their antidiabetes and anticancer effects

Molecular identification of adenoviruses associated with respiratory infection in Egypt from 2003 to 2010.

BACKGROUND: Human adenoviruses of species B, C, and E (HAdV-B, -C, -E) are frequent causative agents of acute respiratory infections worldwide. As part of a surveillance program aimed at identifying the etiology of influenza-like illness (ILI) in Egypt, we characterized 105 adenovirus isolates from clinical samples collected between 2003 and 2010. METHODS: Identification of the isolates as HAdV was accomplished by an immunofluorescence assay (IFA) and confirmed by a set of species and type specific polymerase chain reactions (PCR). RESULTS: Of the 105 isolates, 42% were identified as belonging to HAdV-B, 60% as HAdV-C, and 1% as HAdV-E. We identified a total of six co-infections by PCR, of which five were HAdV-B/HAdV-C co-infections, and one was a co-infection of two HAdV-C types: HAdV-5/HAdV-6. Molecular typing by PCR enabled the identification of eight genotypes of human adenoviruses; HAdV-3 (n = 22), HAdV-7 (n = 14), HAdV-11 (n = 8), HAdV-1 (n = 22), HAdV-2 (20), HAdV-5 (n = 15), HAdV-6 (n = 3) and HAdV-4 (n = 1). The most abundant species in the characterized collection of isolates was HAdV-C, which is concordant with existing data for worldwide epidemiology of HAdV respiratory infections. CONCLUSIONS: We identified three species, HAdV-B, -C and -E, among patients with ILI over the course of 7 years in Egypt, with at least eight diverse types circulating

Endoscopic ultrasound-guided ethanol and radiofrequency ablation of pancreatic insulinomas: a systematic literature review.

Background: Insulinoma is the most common neuroendocrine neoplasm of the pancreas, characterised by hypoglycaemic symptoms. Endoscopic ultrasound-guided radiofrequency ablation (EUS-RFA) and ethanol ablation (EUS-EA) are novel methods for treating insulinoma.We aimed to perform a systematic review to assess the efficacy and safety of EUS-guided ablation techniques for pancreatic insulinomas. Methods: We systematically searched for articles detailing EUS-guided ablations of insulinomas. We performed a qualitative analysis and summarised data on the efficacy and safety of EUS-RFA and EUS-EA techniques. Results: In total, we identified 35 case reports and case series describing 75 patients with insulinomas treatment with EUS-guided ablation. Twenty-seven patients were treated with EUS-RFA, 47 patients with EUS-EA, and 1 patient received EUS-EA and EUS-RFA in the same session. In total, 84 insulinomas were ablated (EUS-RFA: 31, EUS-EA: 53). Most insulinomas were in the head of the pancreas (40%). The clinical success rate for EUS-guided ablation techniques was 98.5%. The median glucose level was 1.95 (Q1-Q3: 1.69-2.13) mmol/L before ablation compared to 6.20 (Q1-Q3: 5.30-7.05) mmol/L after treatment. The median insulin and C-peptide levels before and after RFA/EA were 230 (Q1-Q2: 120-257) pmol/L and 41 (Q1-Q2 35-42) pmol/L; 2077 (Q1-Q2 1644-2459) pmol/L and 819 (Q1-Q2 696-1072) pmol/L, respectively. There were eleven adverse events: seven abdominal pain, two mild acute pancreatitis, one necrotising acute pancreatitis and one local hematoma. All patients recovered, and there were no periprocedural deaths. Conclusions: EUS-guided ablation of insulinoma seems to be a safe and effective treatment and is an alternative to surgical resection in selected cases

Effect of Physical Exercise on Bone Density and Remodeling in Egyptian Type 1 Diabetic Osteopenic Adolescents

<p>Abstract</p> <p>Background</p> <p>The study was planned to assess effect of physical exercise on bone remodeling in type I diabetics with osteopenia.</p> <p>Methods</p> <p>Twenty-four type I diabetes mellitus (DM1) with osteopenia (10 females and 14 males) were compared to thirty-eight age- and sex-matched healthy control individuals (20 females and 18 males) for biochemical and radiologic parameters of bone mass. Laboratory investigations included serum and urinary calcium, inorganic phosphorus, alkaline phosphatase, and serum "procollagen type 1 N-terminal propeptide (P1NP). Bone densitometry was assessed at neck femur using Dual Energy X-ray Absorptiometry (DEXA). Serum P1NP and DEXA were reevaluated after a planned exercise program.</p> <p>Results</p> <p>Patients and controls were comparable with respect to serum as well as urinary biochemical parameters of bone mass namely; calcium, phosphorus and total serum alkaline phosphatase. Osteopenic DM1 patients displayed lower mean serum P1NP than control group (20.11 ± 6.72 ugdL versus 64.96 ± 34.89 ugdL; p < 0.05). A significant correlation was observed between BMD and degree of glycemic control reflected by serum glycated hemoglobin (r = -0.44, p, 0.030). Bone densitometry correlated with serum P1NP (r = -0.508, p, 0.011). After a planned regular exercise for 3 months, serum P1NP and BMD levels increased with percentage change of 40.88 ± 31.73 and 3.36 ± 2.94, respectively. Five patients resumed normal densitometry and they were all males.</p> <p>Conclusion</p> <p>Diabetic osteopenic patients displayed lower serum levels of procollagen type 1 N-terminal propeptide which reflects poor bone formation. A 3-months planned exercise program was associated with improvement of bone densitometry and significant increment of serum P1NP.</p

Mutations in the Beta Propeller WDR72 Cause Autosomal-Recessive Hypomaturation Amelogenesis Imperfecta

Healthy dental enamel is the hardest and most highly mineralized human tissue. Though acellular, nonvital, and without capacity for turnover or repair, it can nevertheless last a lifetime. Amelogenesis imperfecta (AI) is a collective term for failure of normal enamel development, covering diverse clinical phenotypes that typically show Mendelian inheritance patterns. One subset, known as hypomaturation AI, is characterised by near-normal volumes of organic enamel matrix but with weak, creamy-brown opaque enamel that fails prematurely after tooth eruption. Mutations in genes critical to enamel matrix formation have been documented, but current understanding of other key events in enamel biomineralization is limited. We investigated autosomal-recessive hypomaturation AI in a consanguineous Pakistani family. A whole-genome SNP autozygosity screen identified a locus on chromosome 15q21.3. Sequencing candidate genes revealed a point mutation in the poorly characterized WDR72 gene. Screening of WDR72 in a panel of nine additional hypomaturation AI families revealed the same mutation in a second, apparently unrelated, Pakistani family and two further nonsense mutations in Omani families. Immunohistochemistry confirmed intracellular localization in maturation-stage ameloblasts. WDR72 function is unknown, but as a putative β propeller is expected to be a scaffold for protein-protein interactions. The nearest homolog, WDR7, is involved in vesicle mobilization and Ca2+-dependent exocytosis at synapses. Vesicle trafficking is important in maturation-stage ameloblasts with respect to secretion into immature enamel and removal of cleaved enamel matrix proteins via endocytosis. This raises the intriguing possibility that WDR72 is critical to ameloblast vesicle turnover during enamel maturation

Discovery of mating in the major African livestock pathogen Trypanosoma congolense

The protozoan parasite, Trypanosoma congolense, is one of the most economically important pathogens of livestock in Africa and, through its impact on cattle health and productivity, has a significant effect on human health and well being. Despite the importance of this parasite our knowledge of some of the fundamental biological processes is limited. For example, it is unknown whether mating takes place. In this paper we have taken a population genetics based approach to address this question. The availability of genome sequence of the parasite allowed us to identify polymorphic microsatellite markers, which were used to genotype T. congolense isolates from livestock in a discrete geographical area of The Gambia. The data showed a high level of diversity with a large number of distinct genotypes, but a deficit in heterozygotes. Further analysis identified cryptic genetic subdivision into four sub-populations. In one of these, parasite genotypic diversity could only be explained by the occurrence of frequent mating in T. congolense. These data are completely inconsistent with previous suggestions that the parasite expands asexually in the absence of mating. The discovery of mating in this species of trypanosome has significant consequences for the spread of critical traits, such as drug resistance, as well as for fundamental aspects of the biology and epidemiology of this neglected but economically important pathogen