Depletion of O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine in three-dimensional collagen cultures of normal human breast epithelial cells.

O6-Alkylguanine--DNA alkyltransferase (AGT) is a protein which removes the promutagenic O6-alkylguanine lesion induced in DNA by alkylating agents. Our results demonstrate that freshly isolated organoids from reduction mammoplasty specimens contain significant levels of AGT activity. AGT activity in breast epithelial cells shows interindividual variation. Constitutive levels of AGT activity remain unchanged during short-term serum-free culture of breast epithelial cells inside three-dimensional rat-tail collagen gel matrix. In the present study, we optimized conditions for depleting AGT activity in human breast epithelial cells cultured in three-dimensional collagen gel matrix using O6-methylguanine and O6-benzylguanine which are substrates for AGT. AGT activity was efficiently inactivated by exposure of cells to O6-methylguanine or O6-benzylguanine. Inactivation with O6-benzylguanine was more rapid, of greater magnitude and consistency and occurred at lower concentrations than with O6-methylguanine. Near-complete inactivation (> 99.5%) of AGT activity was reproducibly achieved with 50 microM O6-benzylguanine. In contrast, 500 microM O6-methylguanine was needed to obtain a maximal effect and this reduced AGT activity by only 53-93% of control. Within 30 min of adding the free base, 50 microM O6-benzylguanine depleted 95% of the levels of AGT compared to 30% inhibition with 500 microM O6-methylguanine. The profile for restoration of AGT activity was different following a 24 h incubation and subsequent removal of each of the guanine derivatives. AGT activity levels remained undetectable for at least 2 days after removal of 50 microM O6-benzylguanine from the medium and recovered to only 53% of control values after an additional 3 days. AGT activity levels remained undetectable for at least 2 days after removal of 50 microM O6-benzylguanine from the medium and recovered to only 53% of control values after an additional 3 days. In contrast, following removal of 500 microM O6-methylguanine, the activity was restored from its nadir of 16% of control values reaching pretreatment levels after 5 days. These results suggest that treatment with O6-benzylguanine may be used to modulate the incidence of transforming mutations in cultured human breast epithelial cells treated with chemical carcinogens which give rise to O6-alkylguanine adducts.     

Locked intramedullary nailing for complex non-union of the tibia

Ten patients with complex non-union of the tibia were treated by locked intramedullary nailing. These patients had scarred skin as a result of initial severe open fractures, multiple debridement or fasciotomies with external fixators and skin grafts applied. Seven of the patients also had previous osteomyelitis or pin track infections. Fully pain-free walking was achieved in all patients and radiological union in nine patients without the need for a bone graft. Four patients developed infection after nailing, of which three resolved with treatment.     

The hypoglycaemic and insulinotropic activity of Tinospora crispa: studies with human and rat islets and HIT-T15 B cells

Summary In Malaysia, Tinospora crispa extract is taken orally by Type 2 (non-insulin-dependent) diabetic patients to treat hyperglycaemia. We have evaluated the claimed hypoglycaemic property by adding aqueous extract to the drinking water of normal and alloxan-diabetic rats. After one week, fasting blood glucose levels were significantly (p<0.01) lower and serum insulin levels were significantly (p<0.01) higher in treated diabetic animals (10.4±1.0 mmol/l and 12.8±1.1 U/ml respectively) compared to untreated diabetic controls (17.4±1.7 mmol/l and 8.0±0.7 U/ml respectively). The insulinotropic action of T. crispa was further investigated in vitro using isolated human or rat islets of Langerhans and HIT-T15 cells. In static incubations with rat islets and HIT-T15 B cells, the extract induced a dosage dependent stimulation and potentiation of basal and glucose-stimulated insulin secretion respectively. This insulinotropic effect was also evident in perifused human and rat islets and HIT-T5 B-cells. The observations that (i) in all three models insulin secretory rates rapidly returned to basal levels on removal of the extract and (ii) in rat islets, a second challenge with T. crispa induced an additional, stimulated response, are all consistent with physiological release of insulin by B cells. Moreover, the rate of HIT-T15 glucose utilisation was not affected by incubation with T. crispa, suggesting that the cells were viable throughout. These are the first studies to provide biochemical evidence which substantiates the traditional claims for an oral hypoglycaemic effect of Tinospora crispa, and which also show that the hypoglycaemic effect is associated with increased insulin secretion.     

Model for assessment of proficiency of human immunodeficiency virus type 1 sequencing-based genotypic antiretroviral assays

Use of sequencing-based genotyping as a diagnostic assay for human immunodeficiency virus (HIV) antiretroviral resistance is increasing. Periodic evaluation of the proficiency of laboratories performing this assay should be established. It is important to identify components of the assay that influence the generation of reliable sequencing data and that should and can be monitored. A model was developed to determine what parameters were reasonable and feasible for assessing the performance of genotyping assays. Ten laboratories using the genotyping platform, HIV-1 Genotyping System (HGS) v. 1 and software versions 1.1 or 2.0, participated in two rounds of testing. For each round, each group was sent a panel consisting of three clinical samples to sequence in real time. Six months later, seven laboratories using the TRUGENE HIV-1 Genotyping Kit participated in a separate round, working with both panels at the same time. Analysis of the data showed that one main indicator of genotyping proficiency was achievement of > or =98% sequence homology of a sample tested to a group consensus sequence for that sample. A second was concordant identification of codons at sites identified with resistance mutations in the sample, although scoring of these criteria is still undetermined from this study. These criteria are applicable to all sequence-based genotyping platforms and have been used as a baseline for assessing the performance of genotyping for the determination of antiretroviral resistance in our ongoing proficiency program.     

CGS 22745: A selective orally active inhibitor of 5-lipoxygenase

CGS 22745, and aralkyl hydroxamic acid, inhibited 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B₄ (LTB₄) synthesis in guinea pig leukocytes (IC₅₀=0.6M). The compound did not appreciably affect cyclooxygenase (ram seminal vesicles), 12-lipoxygenase and thromboxane synthase (human platelets) or 15-lipoxygenase (human neutrophils). CGS 22745 inhibited A23187-induced formation of LTB₄ in blood (IC₅₀'s of 4.3, 0.56 and 3.2 M for human, dog and rat, respectively). At 1 mg/kg i.v. in dogs, it caused 96% inhibition of A23187-stimulated LTB₄ formationex vivo after 5 min. Its effective biological half-life was >160 min. In dogs at 3 and 10 mg/kg p.o., CGS 22745 inhibitedex vivo A23187-stimulated LTB₄ formation at 3 hr by 48% and 97%, respectively. The inhibition persisted up to 6 hr (26% at 3 mg/kg; 49% at 10 mg/kg). CGS 22745 (3, 10 and 30 mg/kg p.o.) inhibited exudate formation, mononuclear cells and PMN accumulation in a dose-dependent manner during the late phase (48 and 72 hr) of carrageenan-induced pleurisy in the rat.     

Anticancer Mechanism of Curcumin on Human Glioblastoma

Glioblastoma (GBM) is the most malignant brain tumor and accounts for most adult brain tumors. Current available treatment options for GBM are multimodal, which include surgical resection, radiation, and chemotherapy. Despite the significant advances in diagnostic and therapeutic approaches, GBM remains largely resistant to treatment, with a poor median survival rate between 12 and 18 months. With increasing drug resistance, the introduction of phytochemicals into current GBM treatment has become a potential strategy to combat GBM. Phytochemicals possess multifarious bioactivities with multitarget sites and comparatively marginal toxicity. Among them, curcumin is the most studied compound described as a potential anticancer agent due to its multi-targeted signaling/molecular pathways properties. Curcumin possesses the ability to modulate the core pathways involved in GBM cell proliferation, apoptosis, cell cycle arrest, autophagy, paraptosis, oxidative stress, and tumor cell motility. This review discusses curcumin’s anticancer mechanism through modulation of Rb, p53, MAPK, P13K/Akt, JAK/STAT, Shh, and NF-κB pathways, which are commonly involved and dysregulated in preclinical and clinical GBM models. In addition, limitation issues such as bioavailability, pharmacokinetics perspectives strategies, and clinical trials were discussed.     

Impact of Tibial Tubercle Osteotomy on Final Outcome in Revision Total Knee Arthroplasty: Our Experience and Technique in Pakistan

BackgroudDue to extensive fibrosis during revision surgery, adequate exposure is essential and it can be achieved with several extensile approach options, such as tibial tubercle osteotomy. Information regarding surgical exposure during revision arthroplasty is limited in developing countries, such as Pakistan, due to the lack of adequate data collection and follow-up. Therefore, the purpose of this study was to evaluate the impact of tibial tubercle osteotomy on final outcome of revision total knee arthroplasty (TKA).MethodsA total of 231 revision TKAs were performed between January 2008 and December 2017. Twenty-nine patients underwent tibial tubercle osteotomy for adequate exposure during revision surgery. Of these, 27 patients with complete follow-up were included in our study. Factors examined include age at the time of revision surgery, gender, comorbidities, arthroplasty site (right or left), body mass index (BMI), and primary indications for the tibial tubercle osteotomy during revision TKA. Functional outcome was measured by using Knee Society score (KSS) at 3 months and the final follow-up. All statistical analysis was done using SPSS version 20.0 with a p-value < 0.05 considered significant.ResultsOut of 27 patients, 6 patients (22.2%) were men and 21 patients (77.7%) were women. Right knee revision arthroplasty was performed in 15 patients (55.5%), left knee revision arthroplasty was performed in 12 patients (44.4%), and bilateral revision surgery was performed in only 1 patient (3.7%). The mean BMI was 29.2 kg/m². We used a constrained condylar knee in 20 patients (74%), a rotating hinge knee in 5 patients (18.5%), and mobile bearing tray plus metaphyseal sleeves in 2 patients (7.4%). The KSS was 52.21 ± 4.05 preoperatively, and 79.42 ± 2.2 and 80.12 ± 1.33 at 3 months and 12 months, respectively. Radiological union was achieved in all patients at 3 months. Of 27 patients, only 1 patient (3.7%) had proximal migration of the osteotomy site at 6 months: the patient was asymptomatic and union was also achieved and, therefore, no surgical intervention was performed.ConclusionsTibial tubercle osteotomy during revision TKA can be a safe and reliable technique with superior outcomes and minimal complication rates.     

Prevalence of HIV and sexually transmitted and blood-borne infections,and related preventive and risk behaviours,among gay,bisexual and other men who have sex with men in Montreal,Toronto and Vancouver: results from the Engage Study

ObjectivesThe last Canadian biobehavioural surveillance study of HIV and other sexually transmitted and blood-borne infections (STBBI) among gay, bisexual and other men who have sex with men (GBM) was conducted in 2010. We designed a study to measure STBBI prevalence among GBM in metropolitan Montreal, Toronto and Vancouver and to document related preventive and risk behaviours.MethodsThe Engage Cohort Study used respondent-driven sampling (RDS) to recruit GBM who reported sex with another man in the past 6 months. At baseline, we examined recruitment characteristics of the samples, and the RDS-II-adjusted distributions of socio-demographics, laboratory-confirmed HIV and other STBBI prevalence, and related behaviours, with a focus on univariate differences among cities.ResultsA total of 2449 GBM were recruited from February 2017 to August 2019. HIV prevalence was lower in Montreal (14.2%) than in Toronto (22.2%) or Vancouver (20.4%). History of syphilis infection was similar across cities (14–16%). Vancouver had more HIV-negative/unknown participants who reported never being HIV tested (18.6%) than Toronto (12.9%) or Montreal (11.5%). Both Montreal (74.9%) and Vancouver (78.8%) had higher proportions of men who tested for another STBBI in the past 6 months than Toronto (67.4%). Vancouver had a higher proportion of men who used pre-exposure prophylaxis (PrEP) in the past 6 months (18.9%) than Toronto (11.1%) or Montreal (9.6%).ConclusionThe three largest cities of Canada differed in HIV prevalence, STBBI testing and PrEP use among GBM. Our findings also suggest the need for scale-up of both PrEP and STI testing among GBM in Canada.