Involvement of cytochromes P450 (CYP) and other haem associated enzymes in the bioreduction of AQ4N, an antitumour prodrug.

The anthraquinone di-N-oxide AQ4N is a prodrug designed to be excluded from cell nuclei until metabolised in hypoxic tumour regions to AQ4, a DNA binder and potent inhibitor of topoisomerase II. The antitumour effects of AQ4N in rodent neoplasms are well characterised but the identity of enzymes responsible for the metabolism are unknown. The aims of the present work were to identify Cytochrome P450 (CYP) enzymes responsible for AQ4N metabolism in rat and human tissue and to conduct a preliminary investigation into the in vivo metabolism of AQ4N in tumour bearing rodents. AQ4N was found to undergo a two electron reduction to the mono-Noxide AQM followed by a subsequent two electron reduction to cytotoxic AQ4. The process occurred in the microsomes of rat and human liver, was cofactor dependent and was inhibited by air. In rats, CYPs 2B and 2E were found to anaerobically metabolise both AQ4N and AQM. Kinetically, AQ4N metabolism conformed to a Michaelis-Menten model whereas the metabolism of AQM was better described by a sigmoidal relationship. In addition, both semi purified Cytochrome P450 reductase (CPR) and purified Nitric oxide synthase (NOS) were both able to anaerobically metabolise AQ4N. Both enzymes required NADPH and CPR mediated metabolism was dependent on the presence of exogenous haem. In humans, the anaerobic metabolism of both AQ4N and AQM correlated with CYP 3A activity and not with the activities of CYP 1 AI 2C and 2D. AQM metabolism correlated also with the activity of CYP 2A. The involvement of CYP 3A was confirmed by the use of CYP specific inhibitors and by the use of cDNA transfected cell microsomes. Human kidney and colonic tumours were found to anaerobically metabolise AQ4N and tumour metabolism was inhibited by the CYP inhibitor carbon monoxide (CO). Finally, the in vivo metabolism of AQ4N was studied in C3H tumour bearing mice. Metabolites of AQ4N were found in all tissues studied but the AQ4! AQ4N ratio was highest in the tumours. Collectively, these findings have identified the enzymes responsible for the metabolism of AQ4N and its mono-N-oxide. Differences exist between the CYP isoforms responsible for metabolism in rodents and in man, in humans, CYP 3A enzymes predominantly metabolise AQ4N and this subfamily of CYP are known to be well expressed in a broad spectrum of human cancers. With this in mind, AQ4N based therapy should be considered as a rational treatment regime for patients bearing solid tumour burdens

Effectiveness of highly active antiretroviral therapy in HIV-positive children: evaluation at 12 months in a routine program in Cambodia.

OBJECTIVE: Increasing access to highly active antiretroviral therapy to reach all those in need in developing countries (scale up) is slowly expanding to HIV-positive children, but documented experience remains limited. We aimed to describe the clinical, immunologic, and virologic outcomes of pediatric patients with >12 months of highly active antiretroviral therapy in 2 routine programs in Cambodia. METHODS: Between June 2003 and March 2005, 212 children who were younger than 13 years started highly active antiretroviral therapy. Most patients started a standard first-line regimen of lamivudine, stavudine, and nevirapine, using split adult fixed-dosage combinations. CD4 percentage and body weight were monitored routinely. A cross-sectional virologic analysis was conducted in January 2006; genotype resistance testing was performed for patients with a detectable viral load. RESULTS: Mean age of the subjects was 6 years. Median CD4 percentage at baseline was 6. Survival was 92% at 12 months and 91% at 24 months; 13 patients died, and 4 were lost to follow-up. A total of 81% of all patients had an undetectable viral load. Among the patients with a detectable viral load, most mutations were associated with resistance to lamivudine and non-nucleoside reverse-transcriptase inhibitor drugs. Five patients had developed extensive antiretroviral resistance. Being an orphan was found to be a predictor of virologic failure. CONCLUSIONS: This study provides additional evidence of the effectiveness of integrating HIV/AIDS care with highly active antiretroviral therapy for children in a routine setting, with good virologic suppression and immunologic recovery achieved by using split adult fixed-dosage combinations. Viral load monitoring and HIV genotyping are valuable tools for the clinical follow-up of the patients. Orphans should receive careful follow-up and extra support

Community Needs Assessment Collaboration Following the July 2022 Flooding in Eastern Kentucky

Rapidly rising waters due to flash floods and thunderstorms on the night of July 27, 2022, resulted in hundreds of water rescues throughout 14 rural Appalachian Kentucky counties. Lives were lost, thousands were injured, homes and property were damaged or destroyed, and many roadways were unpassable. Community partners serving these counties collaborated to design and conduct an assessment to gain a better understanding of the needs of individuals residing in certain remote sections within the communities. The assessment, conducted three months after the flood, collected information regarding flooding impact on housing, physical and behavioral health, transportation, work, and finances

CRISPRi-based radiation modifier screen identifies long non-coding RNA therapeutic targets in glioma.

BackgroundLong non-coding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer treatments. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients.ResultsWe use CRISPR interference (CRISPRi) to screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modify cell growth in the presence of clinically relevant doses of fractionated radiation. Thirty-three of these lncRNA hits sensitize cells to radiation, and based on their expression in adult and pediatric gliomas, nine of these hits are prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1, a primate-conserved, nuclear-enriched lncRNA, inhibits the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a three-dimensional tissue substrate to model the invasive growth of glioma, we find that antisense oligonucleotides targeting lncGRS-1 selectively decrease tumor growth and sensitize glioma cells to radiation therapy.ConclusionsThese studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome to enhance radiation therapy

Evolution of Bacterial Phosphoglycerate Mutases: Non-Homologous Isofunctional Enzymes Undergoing Gene Losses, Gains and Lateral Transfers

The glycolytic phosphoglycerate mutases exist as non-homologous isofunctional enzymes (NISE) having independent evolutionary origins and no similarity in primary sequence, 3D structure, or catalytic mechanism. Cofactor-dependent PGM (dPGM) requires 2,3-bisphosphoglycerate for activity; cofactor-independent PGM (iPGM) does not. The PGM profile of any given bacterium is unpredictable and some organisms such as Escherichia coli encode both forms.To examine the distribution of PGM NISE throughout the Bacteria, and gain insight into the evolutionary processes that shape their phyletic profiles, we searched bacterial genome sequences for the presence of dPGM and iPGM. Both forms exhibited patchy distributions throughout the bacterial domain. Species within the same genus, or even strains of the same species, frequently differ in their PGM repertoire. The distribution is further complicated by the common occurrence of dPGM paralogs, while iPGM paralogs are rare. Larger genomes are more likely to accommodate PGM paralogs or both NISE forms. Lateral gene transfers have shaped the PGM profiles with intradomain and interdomain transfers apparent. Archaeal-type iPGM was identified in many bacteria, often as the sole PGM. To address the function of PGM NISE in an organism encoding both forms, we analyzed recombinant enzymes from E. coli. Both NISE were active mutases, but the specific activity of dPGM greatly exceeded that of iPGM, which showed highest activity in the presence of manganese. We created PGM null mutants in E. coli and discovered the ΔdPGM mutant grew slowly due to a delay in exiting stationary phase. Overexpression of dPGM or iPGM overcame this defect.Our biochemical and genetic analyses in E. coli firmly establish dPGM and iPGM as NISE. Metabolic redundancy is indicated since only larger genomes encode both forms. Non-orthologous gene displacement can fully account for the non-uniform PGM distribution we report across the bacterial domain

Safety and efficacy of an oxycodone vaccine: Addressing some of the unique considerations posed by opioid abuse

Among vaccines aimed at treating substance use disorders, those targeting opioids present several unique medication development challenges. 1) Opioid overdose is a common complication of abuse, so it is desirable for an opioid vaccine to block the toxic as well as the addictive effects of opioids. 2) It is important that an opioid vaccine not interfere with the action of opioid antagonists used to reverse opioid overdose or treat addiction. 3) Some opioids are immunosuppressive and chronic ongoing opioid use could interfere with vaccine immunogenicity. 4) Although antibody-bound oxycodone is unable to enter the brain because of its size, it might still be able to activate peripheral opioid receptors. To assess vaccine impact on opioid toxicity, rats vaccinated with oxycodone conjugated to keyhole limpet hemocyanin subunit dimer (OXY-dKLH) adsorbed to alum or controls vaccinated with dKLH were compared with regard to oxycodone-induced hotplate analgesia and oxycodone-induced respiratory depression and bradycardia. Vaccination shifted the dose-response curves to the right, representing protection, for each of these endpoints. Naloxone was equally effective in both OXY-dKLH and control groups, providing complete and rapid reversal of respiratory depression. The administration of a long-acting naltrexone formulation during vaccination did not impair vaccine immunogenicity in mice. Similarly, serum anti-oxycodone antibody titers were not altered by continuous morphine infusion during vaccination compared to opioid-naïve controls. Competitive ELISA assay showed negligible or low affinity of immune antiserum for endogenous opioids or opioid antagonists. In vitro receptor binding assays showed that antibody-bound oxycodone does not activate mu opioid receptors. These data support further study of OXY-dKLH as a potential treatment for oxycodone abuse and suggest that vaccination might also reduce the severity of oxycodone overdose

Honesty mediates the relationship between serotonin and reaction to unfairness

How does one deal with unfair behaviors? This subject has long been investigated by various disciplines including philosophy, psychology, economics, and biology. However, our reactions to unfairness differ from one individual to another. Experimental economics studies using the ultimatum game (UG), in which players must decide whether to accept or reject fair or unfair offers, have also shown that there are substantial individual differences in reaction to unfairness. However, little is known about psychological as well as neurobiological mechanisms of this observation. We combined a molecular imaging technique, an economics game, and a personality inventory to elucidate the neurobiological mechanism of heterogeneous reactions to unfairness. Contrary to the common belief that aggressive personalities (impulsivity or hostility) are related to the high rejection rate of unfair offers in UG, we found that individuals with apparently peaceful personalities (straightforwardness and trust) rejected more often and were engaged in personally costly forms of retaliation. Furthermore, individuals with a low level of serotonin transporters in the dorsal raphe nucleus (DRN) are honest and trustful, and thus cannot tolerate unfairness, being candid in expressing their frustrations. In other words, higher central serotonin transmission might allow us to behave adroitly and opportunistically, being good at playing games while pursuing self-interest. We provide unique neurobiological evidence to account for individual differences of reaction to unfairness

Concussion-associated gene variants and history of concussion in elite male rugby athletes

Occurrence of and outcomes following a concussion are probably affected by the interaction of multiple genes in a polygenic manner [1,2]. This study investigated whether suspected concussion-associated polygenic profiles of elite rugby athletes with a history of previous concussion (RAC) differed from rugby athletes with no history of previous concussion (RANC). We hypothesised that concussion-associated risk genotypes would be underrepresented in RANC compared to RAC. Participants were from the RugbyGene project, comprising elite male rugby athletes (RA) (185 white males; mean (standard deviation) height 1.86 (0.07) m, mass 102 (12.6) kg, age 26.4 (5.1) yr) competing at an elite level in rugby union (n = 165) and league (n = 20) in the UK, Ireland, Italy and South Africa. Concussion history was collected using a self-reported concussion history questionnaire. PCR of genomic DNA was used to determine genotypes using TaqMan probes, and total genotype scores (TGS) were calculated, then groups were compared using χ2 and odds ratio (OR) statistics. In addition, multifactor dimensionality reduction (MDR) was used to identify genetic interactions. Seventy-eight percent of RA reported a history of sustaining at least one concussion and 54% of RA reported sustaining multiple (≥2) concussions from rugby. For BDNF-AS rs6265, the GG genotype was more common in RAC compared to RANC (69.7% vs 61.0%, P = 0.006, OR = 9.90, 95% CI = 01.81-54.06) (Fig. 1). The GG genotype of BDNF-AS rs6265 was more common in RAC compared to RANC (70.7% vs. 61.0%, P = 0.041, OR 4.44, 95% CI = 1.04-120.97) (Fig. 1). However, TGS did not differ between RANC and RAC (Fig. 2A) recovery duration and family history of neurological conditions (P > 0.05). Receiver operating characteristic curve (ROC) and area under the curve (AUC) analysis confirmed the TGS algorithm could not identify concussion history (AUC = 0.436; 95% CI = 0.338-0.534; P = 0.218; Fig. 2B). MDR could not identify a model to predict concussion history, recovery duration and family history of neurological conditions with a sufficiently powerful cross-validation statistic (P ≤ 0.05). These findings support the growing evidence that incidence and recovery from concussion could be influenced by an athlete’s genetic predisposition. Such knowledge could be used in the future and when additional relevant variants have been identified, to inform individualised management strategies for athletes in possession of risk genotypes.Peer reviewe