Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells

Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.     

Induction of oxidative stress and histopathological changes by sub-chronic doses of triazophos

The effect of triazophos (O, O-diethyl O-1-phenyl-1 H-1, 2, 4-triazol-3-yl phosphorothioate), a widely used insecticide was studied on the induction of oxidative stress and histological alterations at sub-chronic doses in male albino rats. Oral administration of triazophos at concentrations of 1.64, 3.2 and 8.2 mg/kg body wt for 30 days produced dose as well as time-dependent increase in the lipid peroxidation (determined by malondialdehyde levels) and glutathione-S-transferase (GST) activity in serum with aconcomitant decrease in ferric reducing ability of plasma (FRAP) and blood glutathione (GSH) content. Histopathological examination of liver of triazophos-treated rats showed significant and progressive degenerative changes as compared to control, which could be due to induction of oxidative stress. However, no significant histopathological changes were observed in spleen, kidney and brain at either dose of triazophos with respect to control. These results indicated that oral administration of triazophos was associated with enhanced lipid peroxidation and compromised antioxidant defence in rats in dose and time-dependent manner. Thus the present study demonstrated for the first time the role of oxidative stress as the important mechanism involved in the stimulation of hepatic histoarchitectural alterations at sub-chronic doses of triazophos in rats.     

Functional gene analysis suggests different acetogen populations in the bovine rumen and tammar wallaby forestomach

Reductive acetogenesis via the acetyl coenzyme A (acetyl-CoA) pathway is an alternative hydrogen sink to methanogenesis in the rumen. Functional gene-based analysis is the ideal approach for investigating organisms capable of this metabolism (acetogens). However, existing tools targeting the formyltetrahydrofolate synthetase gene (fhs) are compromised by lack of specificity due to the involvement of formyltetrahydrofolate synthetase (FTHFS) in other pathways. Acetyl-CoA synthase (ACS) is unique to the acetyl-CoA pathway and, in the present study, acetyl-CoA synthase genes (acsB) were recovered from a range of acetogens to facilitate the design of acsB-specific PCR primers. fhs and acsB libraries were used to examine acetogen diversity in the bovine rumen and forestomach of the tammar wallaby (Macropus eugenii), a native Australian marsupial demonstrating foregut fermentation analogous to rumen fermentation but resulting in lower methane emissions. Novel, deduced amino acid sequences of acsB and fhs affiliated with the Lachnospiraceae in both ecosystems and the Ruminococcaeae/Blautia group in the rumen. FTHFS sequences that probably originated from nonacetogens were identified by low "homoacetogen similarity" scores based on analysis of FTHFS residues, and comprised a large proportion of FTHFS sequences from the tammar wallaby forestomach. A diversity of FTHFS and ACS sequences in both ecosystems clustered between the Lachnospiraceae and Clostridiaceae acetogens but without close sequences from cultured isolates. These sequences probably originated from novel acetogens. The community structures of the acsB and fhs libraries from the rumen and the tammar wallaby forestomach were different (LIBSHUFF, P < 0.001), and these differences may have significance for overall hydrogenotrophy in both ecosystems.     

Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis

Carbohydrate-induced laminitis in horses is characterized by marked changes in the composition of the hindgut microbiota, from a predominantly Gram-negative population to one dominated by Gram-positive bacteria. The objective of this study was to monitor changes in the relative abundance of selected hindgut bacteria that have previously been implicated in the pathophysiology of equine laminitis using fluorescence in situ hybridization (FISH). Caecal cannulae were surgically implanted in five Standardbred horses and laminitis induced by oral administration of a bolus dose of oligofructose. Caecal fluid and faecal specimens were collected over a 48 h period at 2 to 4 h intervals post-oligofructose administration and subjected to FISH using probes specific for nine bacterial groups to determine changes in their relative abundance compared with total bacteria hybridizing to the generic EUBMIX probe. Additionally, hoof biopsies were taken over the course of the experiment at 6 h intervals and evaluated for histopathological changes consistent with laminitis, allowing changes in hindgut microbiota to be correlated with the onset of lesions in the foot. Of the microorganisms specifically targeted, streptococci of the Streptococcus bovis/equinus complex were the only bacteria that consistently proliferated in both caecal fluid and faeces immediately before the onset of histological signs of laminitis. Furthermore, lactobacilli, Enterobacteriaceae, Allisonella histaminiformans, enterococci, Bacteroides fragilis, Mitsuokella jalaludinii and Clostridium difficile did not establish significant populations in the hindgut before the onset of equine laminitis.     

Anti-tumor and anti-leishmanial evaluations of 1,3,4-oxadiazine, pyran derivatives derived from cross-coupling reactions of β-bromo-6H-1,3,4-oxadiazine derivatives

Cyanoacetylhydrazine reacted with the ω-bromoacetophenones 2a,b to give hydrazide-hydrazone derivatives 3a,b. The latter products were cyclized to the 1,3,4-oxadiazine derivatives 4a,b. Bromination of the latter products gave the 6-bromo-6H-1,3,4-oxadiazine derivatives 5a,b which underwent a series of cross-coupling reactions. The antitumor evaluation of the newly synthesized products against the three cancer cells namely breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and CNS cancer (SF-268) showed that some of them have high inhibitory effect towards three cell lines which is higher than the standard. Moreover, the anti-leishmanial activity of the newly synthesized product was tested on Leishmania donovani amastigotes showed that some compounds have high activity.     

Development of new indole-derived neuroprotective agents

There is a great deal of interest in neurotrophin therapy to prevent neuronal degeneration. The present study aimed at synthesizing new functionalized indole derivatives with structures justifying neuroprotective activity using L-tryptophan (TRP) as starting material. The potential neuroprotective effect of these newly synthesized agents against acrylamide (ACR) induced neurotoxicity was investigated in adult female rats. The novel indole derivatives, indolylmethyl pyridine derivatives 9a,b, pyrimidinylindolyl propanone derivatives 12a-c, pyrazolylindolyl propanone derivatives 14a,b, and indolyl tetrazolopropanoic acid derivative 17 were synthesized and their chemical structures were confirmed by studying their analytical and spectral data. The administration of ACR [ip, 50mgkg(-1) body weight (b. wt.)] alone resulted in significant increase in brain malondialdehyde level (MDA) and lactate dehydrogenase (LDH) activity whereas it caused significant decrease in brain monoamines levels and antioxidant enzymes activity. Treatment with the indole derivatives 9b, 12c, 14a, and 17 (ip, 50mgkg(-1) b. wt.) prior to ACR produced neuroprotective activity with various intensities depending on the structure of each compound. Compound 17 in which the tetrazole ring was attached to the TRP moiety ranked as the strongest neuroprotective agent. All the tested compounds have been shown to possess antioxidant properties offering promising efficacy against oxidative stress induced by ACR administration.     

Cytotoxicity and gene expression profiles of novel synthesized steroid derivatives as chemotherapeutic anti-breast cancer agents

Anti-cancer agents which combine two biologically active compounds in one such as steroidal heterocyclic derivatives attain both hormone and cytotoxic effects on cancer cells. The aim of the present study is to synthesize and evaluate new potential chemotherapeutic anti-breast cancer agents. Several pyridazino-, pyrimido-, quinazolo-, oxirano- and thiazolo-steroid derivatives were synthesized. The structure of the novel steroid derivatives was confirmed using the analytical and spectral data. The most structurally promising of the novel synthesized steroid derivatives, compounds 8, 12, 17, 20, 22c, 24c, 30a and 30b, were investigated individually as anti-breast cancer agents against human breast cancer cells (MCF-7) using sulforhodamine B (SRB) assay. The tested compounds 17, 20, 22c and 8 showed potent broad spectrum cytotoxic activity in vitro after 48 h incubation. Compound 17 (IC(50)=2.5 μM) exhibited more inhibitory influence on MCF-7 growth than the reference drug doxorubicin (Dox) (IC(50)=4.5 μM) after 48 h incubation. Also, the present study showed that all the tested steroid derivatives exhibited significant depletion with various intensities in gene expression of breast cancer related genes (VEGF, CYP19 and hAP-2γ). Noteworthy, compounds 17, 20 and 22c showed the most pronounced effect in this respect.     

Anesthesiologists' and surgeons' perceptions about routine pre-operative testing in low-risk patients: application of the Theoretical Domains Framework (TDF) to identify factors that influence physicians' decisions to order pre-operative tests

ABSTRACT: BACKGROUND: Routine pre-operative tests for anesthesia management are often ordered by bothanesthesiologists and surgeons for healthy patients undergoing low-risk surgery. TheTheoretical Domains Framework (TDF) was developed to investigate determinants ofbehaviour and identify potential behaviour change interventions. In this study, the TDF is used to explore anaesthesiologists' and surgeons' perceptions of ordering routine tests forhealthy patients undergoing low-risk surgery. METHODS: Sixteen clinicians (eleven anesthesiologists and five surgeons) throughout Ontario wererecruited. An interview guide based on the TDF was developed to identify beliefs about preoperativetesting practices. Content analysis of physicians' statements into the relevanttheoretical domains was performed. Specific beliefs were identified by grouping similarutterances of the interview participants. Relevant domains were identified by noting thefrequencies of the beliefs reported, presence of conflicting beliefs, and perceived influence onthe performance of the behaviour under investigation. RESULTS: Seven of the twelve domains were identified as likely relevant to changing clinicians'behaviour about pre-operative test ordering for anesthesia management. Key beliefs wereidentified within these domains including: conflicting comments about who was responsiblefor the test-ordering (Social/professional role and identity); inability to cancel tests orderedby fellow physicians (Beliefs about capabilities and social influences); and the problem withtests being completed before the anesthesiologists see the patient (Beliefs about capabilitiesand Environmental context and resources). Often, tests were ordered by an anesthesiologistbased on who may be the attending anesthesiologist on the day of surgery while surgeonsordered tests they thought anesthesiologists may need (Social influences). There were alsoconflicting comments about the potential consequences associated with reducing testing,from negative (delay or cancel patients' surgeries), to indifference (little or no change inpatient outcomes), to positive (save money, avoid unnecessary investigations) (Beliefs aboutconsequences). Further, while most agreed that they are motivated to reduce orderingunnecessary tests (Motivation and goals), there was still a report of a gap between theirmotivation and practice (Behavioural regulation). CONCLUSION: We identified key factors that anesthesiologists and surgeons believe influence whether theyorder pre-operative tests routinely for anesthesia management for a healthy adults undergoinglow-risk surgery. These beliefs identify potential individual, team, and organisation targetsfor behaviour change interventions to reduce unnecessary routine test ordering.