Plasma cell-derived mtDAMPs activate the macrophage STING pathway, promoting myeloma progression.

Mitochondrial damage-associated molecular patterns (mtDAMPs) include proteins, lipids, metabolites, and DNA and have various context-specific immunoregulatory functions. Cell-free mitochondrial DNA (mtDNA) is recognized via pattern recognition receptors and is a potent activator of the innate immune system. Cell-free mtDNA is elevated in the circulation of trauma patients and patients with cancer; however, the functional consequences of elevated mtDNA are largely undefined. Multiple myeloma (MM) relies upon cellular interactions within the bone marrow (BM) microenvironment for survival and progression. Here, using in vivo models, we describe the role of MM cell-derived mtDAMPs in the protumoral BM microenvironment and the mechanism and functional consequence of mtDAMPs in myeloma disease progression. Initially, we identified elevated levels of mtDNA in the peripheral blood serum of patients with MM compared with those of healthy controls. Using the MM1S cells engrafted into nonobese diabetic severe combined immunodeficient gamma mice, we established that elevated mtDNA was derived from MM cells. We further show that BM macrophages sense and respond to mtDAMPs through the stimulator of interferon genes (STING) pathway, and inhibition of this pathway reduces MM tumor burden in the KaLwRij-5TGM1 mouse model. Moreover, we found that MM-derived mtDAMPs induced upregulation of chemokine signatures in BM macrophages, and inhibition of this signature resulted in egress of MM cells from the BM. Here, we demonstrate that malignant plasma cells release mtDNA, a form of mtDAMPs, into the myeloma BM microenvironment, which in turn activates macrophages via STING signaling. We establish the functional role of these mtDAMP-activated macrophages in promoting disease progression and retaining MM cells in the protumoral BM microenvironment.

PGC-1α induced mitochondrial biogenesis in stromal cells underpins mitochondrial transfer to melanoma.

INTRODUCTION: Progress in the knowledge of metabolic interactions between cancer and its microenvironment is ongoing and may lead to novel therapeutic approaches. Until recently, melanoma was considered a glycolytic tumour due to mutations in mitochondrial-DNA, however, these malignant cells can regain OXPHOS capacity via the transfer of mitochondrial-DNA, a process that supports their proliferation in-vitro and in-vivo. Here we study how melanoma cells acquire mitochondria and how this process is facilitated from the tumour microenvironment. METHODS: Primary melanoma cells, and MSCs derived from patients were obtained. Genes' expression and DNA quantification was analysed using Real-time PCR. MSC migration, melanoma proliferation and tumour volume, in a xenograft subcutaneous mouse model, were monitored through bioluminescent live animal imaging. RESULTS: Human melanoma cells attract bone marrow-derived stromal cells (MSCs) to the primary tumour site where they stimulate mitochondrial biogenesis in the MSCs through upregulation of PGC1a. Mitochondria are transferred to the melanoma cells via direct contact with the MSCs. Moreover, inhibition of MSC-derived PGC1a was able to prevent mitochondrial transfer and improve NSG melanoma mouse tumour burden. CONCLUSION: MSC mitochondrial biogenesis stimulated by melanoma cells is prerequisite for mitochondrial transfer and subsequent tumour growth, where targeting this pathway may provide an effective novel therapeutic approach in melanoma.

ROS-mediated PI3K activation drives mitochondrial transfer from stromal cells to hematopoietic stem cells in response to infection.

Hematopoietic stem cells (HSCs) undergo rapid expansion in response to stress stimuli. Here we investigate the bioenergetic processes which facilitate the HSC expansion in response to infection. We find that infection by Gram-negative bacteria drives an increase in mitochondrial mass in mammalian HSCs, which results in a metabolic transition from glycolysis toward oxidative phosphorylation. The initial increase in mitochondrial mass occurs as a result of mitochondrial transfer from the bone marrow stromal cells (BMSCs) to HSCs through a reactive oxygen species (ROS)-dependent mechanism. Mechanistically, ROS-induced oxidative stress regulates the opening of connexin channels in a system mediated by phosphoinositide 3-kinase (PI3K) activation, which allows the mitochondria to transfer from BMSCs into HSCs. Moreover, mitochondria transfer from BMSCs into HSCs, in the response to bacterial infection, occurs before the HSCs activate their own transcriptional program for mitochondrial biogenesis. Our discovery demonstrates that mitochondrial transfer from the bone marrow microenvironment to HSCs is an early physiologic event in the mammalian response to acute bacterial infection and results in bioenergetic changes which underpin emergency granulopoiesis.

Mitochondrial oxidative phosphorylation in cutaneous melanoma.

The Warburg effect in tumour cells is associated with the upregulation of glycolysis to generate ATP, even under normoxic conditions and the presence of fully functioning mitochondria. However, scientific advances made over the past 15 years have reformed this perspective, demonstrating the importance of oxidative phosphorylation (OXPHOS) as well as glycolysis in malignant cells. The metabolic phenotypes in melanoma display heterogeneic dynamism (metabolic plasticity) between glycolysis and OXPHOS, conferring a survival advantage to adapt to harsh conditions and pathways of chemoresistance. Furthermore, the simultaneous upregulation of both OXPHOS and glycolysis (metabolic symbiosis) has been shown to be vital for melanoma progression. The tumour microenvironment (TME) has an essential supporting role in promoting progression, invasion and metastasis of melanoma. Mesenchymal stromal cells (MSCs) in the TME show a symbiotic relationship with melanoma, protecting tumour cells from apoptosis and conferring chemoresistance. With the significant role of OXPHOS in metabolic plasticity and symbiosis, our review outlines how mitochondrial transfer from MSCs to melanoma tumour cells plays a key role in melanoma progression and is the mechanism by which melanoma cells regain OXPHOS capacity even in the presence of mitochondrial mutations. The studies outlined in this review indicate that targeting mitochondrial trafficking is a potential novel therapeutic approach for this highly refractory disease.

Predictors of COVID-19 vaccine hesitancy in the UK household longitudinal study.

Vaccine hesitancy could undermine efforts to control COVID-19. We investigated the prevalence of COVID-19 vaccine hesitancy in the UK and identified vaccine hesitant subgroups. The 'Understanding Society' COVID-19 survey asked participants (n = 12,035) their likelihood of vaccine uptake and reason for hesitancy. Cross-sectional analysis assessed vaccine hesitancy prevalence and logistic regression calculated odds ratios. Overall vaccine hesitancy was low (18% unlikely/very unlikely). Vaccine hesitancy was higher in women (21.0% vs 14.7%), younger age groups (26.5% in 16-24 year olds vs 4.5% in 75 + ) and those with lower education levels (18.6% no qualifications vs 13.2% degree qualified). Vaccine hesitancy was high in Black (71.8%) and Pakistani/Bangladeshi (42.3%) ethnic groups. Odds ratios for vaccine hesitancy were 13.42 (95% CI:6.86, 26.24) in Black and 2.54 (95% CI:1.19, 5.44) in Pakistani/Bangladeshi groups (compared to White British/Irish) and 3.54 (95% CI:2.06, 6.09) for people with no qualifications versus degree. Urgent action to address hesitancy is needed for some but not all ethnic minority groups.

Mitochondria and the Tumour Microenvironment in Blood Cancer.

The bone marrow (BM) is a complex organ located within the cavities of bones. The main function of the BM is to produce all the blood cells required for a normal healthy blood system. As with any major organ, many diseases can arise from errors in bone marrow function, including non-malignant disorders such as anaemia and malignant disorders such as leukaemias. This article will explore the role of the bone marrow, in normal and diseased haematopoiesis, with an emphasis on the requirement for intercellular mitochondrial transfer in leukaemia.

Distinct Commensal Bacterial Signature in the Gut Is Associated With Acute and Long-Term Protection From Ischemic Stroke.

Background and Purpose- Commensal gut bacteria have a profound impact on stroke pathophysiology. Here, we investigated whether modification of the microbiota influences acute and long-term outcome in mice subjected to stroke. Methods- C57BL/6 male mice received a cocktail of antibiotics or single antibiotic. After 4 weeks, fecal bacterial density of the 16S rRNA gene was quantitated by qPCR, and phylogenetic classification was obtained by 16S rRNA gene sequencing. Infarct volume and hemispheric volume loss were measured 3 days and 5 weeks after middle cerebral artery occlusion, respectively. Neurological deficits were tested by the Tape Test and the open field test. Results- Mice treated with a cocktail of antibiotics displayed a significant reduction of the infarct volume in the acute phase of stroke. The neuroprotective effect was abolished in mice recolonized with a wild-type microbiota. Single antibiotic treatment with either ampicillin or vancomycin, but not neomycin, was sufficient to reduce the infarct volume and improved motorsensory function 3 days after stroke. This neuroprotective effect was correlated with a specific microbial population rather than the total bacterial density. In particular, random forest analysis trained for the severity of the brain damage revealed that Bacteroidetes S24.7 and the enzymatic pathway for aromatic metabolism discriminate between large versus small infarct size. Additionally, the microbiota signature in the ampicillin-treated mice was associated with a reduced gut inflammation, long-term favorable outcome shown by an amelioration of the stereotypic behavior, and a reduction of brain tissue loss in comparison to control and was predictive of a regulation of short-chain fatty acids and tryptophan pathways. Conclusions- The findings highlight the importance of the intestinal microbiota in short- and long-term outcomes of ischemic stroke and raises the possibility that targeted modification of the microbiome associated with specific microbial enzymatic pathways may provide a preventive strategy in patients at high risk for stroke. Visual Overview- An online visual overview is available for this article.

Endogenous Protection from Ischemic Brain Injury by Preconditioned Monocytes.

Exposure to low-dose lipopolysaccharide (LPS) before cerebral ischemia is neuroprotective in stroke models, a phenomenon termed preconditioning (PC). Although it is well established that LPS-PC induces central and peripheral immune responses, the cellular mechanisms modulating ischemic injury remain unclear. Here, we investigated the role of immune cells in the brain protection afforded by PC and tested whether monocytes may be reprogrammed by ex vivo LPS exposure, thus modulating inflammatory injury after cerebral ischemia in male mice. We found that systemic injection of low-dose LPS induces a Ly6Chi monocyte response that protects the brain after transient middle cerebral artery occlusion (MCAO) in mice. Remarkably, adoptive transfer of monocytes isolated from preconditioned mice into naive mice 7 h after transient MCAO reduced brain injury. Gene expression and functional studies showed that IL-10, inducible nitric oxide synthase, and CCR2 in monocytes are essential for neuroprotection. This protective activity was elicited even if mouse or human monocytes were exposed ex vivo to LPS and then injected into male mice after stroke. Cell-tracking studies showed that protective monocytes are mobilized from the spleen and reach the brain and meninges, where they suppress postischemic inflammation and neutrophil influx into the brain parenchyma. Our findings unveil a previously unrecognized subpopulation of splenic monocytes capable of protecting the brain with an extended therapeutic window and provide the rationale for cell therapies based on the delivery of autologous or allogeneic protective monocytes in patients after ischemic stroke.SIGNIFICANCE STATEMENT Inflammation is a key component of the pathophysiology of the brain in stroke, a leading cause of death and disability with limited therapeutic options. Here, we investigate endogenous mechanisms of protection against cerebral ischemia. Using lipopolysaccharide (LPS) preconditioning (PC) as an approach to induce ischemic tolerance in mice, we found generation of neuroprotective monocytes within the spleen, from which they traffic to the brain and meninges, suppressing postischemic inflammation. Importantly, systemic LPS-PC can be mimicked by adoptive transfer of in vitro-preconditioned mouse or human monocytes at translational relevant time points after stroke. This model of neuroprotection may facilitate clinical efforts to increase the efficacy of BM mononuclear cell treatments in acute neurological diseases such as cerebral ischemia.

A Digital Intervention Addressing Alcohol Use Problems (the "Daybreak" Program): Quasi-Experimental Randomized Controlled Trial.

BACKGROUND: Alcohol use is prevalent in many societies and has major adverse impacts on health, but the availability of effective interventions limits treatment options for those who want assistance in changing their patterns of alcohol use. OBJECTIVE: This study evaluated the new Daybreak program, which is accessible via mobile app and desktop and was developed by Hello Sunday Morning to support high-risk drinking individuals looking to change their relationship with alcohol. In particular, we compared the effect of adding online coaching via real-time chat messages (intervention group) to an otherwise self-guided program (control group). METHODS: We designed the intervention as a randomized control trial, but as some people (n=48; 11.9%) in the control group were able to use the online coaching, the main analysis comprised all participants. We collected online surveys at one-month and three-months follow-up. The primary outcome was change in alcohol risk (measured with the alcohol use disorders identification test-consumption [AUDIT-C] score), but other outcomes included the number of standard drinks per week, alcohol-related days out of role, psychological distress (Kessler-10), and quality of life (EUROHIS-QOL). Markers of engagement with the program included posts to the site and comments on the posts of others. The primary analysis used Weighted Generalized Estimating Equations. RESULTS: We recruited 398 people to the intervention group (50.2%) and 395 people to the control group (49.8%). Most were female (71%) and the mean age was 40.1 years. Most participants were classified as probably dependent (550, 69%) on the AUDIT-10, with 243 (31%) classified with hazardous or harmful consumption. We followed up with 334 (42.1%) participants at one month and 293 (36.9%) at three months. By three months there were significant improvements in AUDIT-C scores (down from mean 9.1 [SD 1.9] to 5.8 [SD 3.1]), alcohol consumed per week (down from mean 37.1 [SD 28.3] to mean 17.5 [SD 18.9]), days out of role (down from mean 1.6 [SD 3.6] to 0.5 [SD 1.6]), quality of life (up from 3.2 [SD 0.7] to 3.6 [SD 0.7]) and reduced distress (down from 24.8 [SD 7.0] to 19.0 [SD 6.6]). Accessing online coaching was not associated with improved outcomes, but engagement with the program (eg, posts and comments on the posts of others) were significantly associated with improvements (eg, in AUDIT-C, alcohol use and EUROHIS-QOL). Reduced alcohol use was found for both probably dependent (estimated marginal mean of 40.8 to 20.1 drinks) and hazardous or harmful alcohol users (estimated marginal mean of 22.9 to 11.9 drinks). CONCLUSIONS: Clinically significant reductions in alcohol use were found, as well as reduced alcohol risk (AUDIT-C) and days out of role. Importantly, improved alcohol-related outcomes were found for both hazardous or harmful and probably dependent drinkers. Since October 2016, Daybreak has reached more than 50,000 participants. Therefore, there is the potential for the program to have an impact on alcohol-related problems at a population health level, importantly including an effect on probably dependent drinkers. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry: ACTRN12618000010291; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=373110. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/9982.

STING-Licensed Macrophages Prime Type I IFN Production by Plasmacytoid Dendritic Cells in the Bone Marrow during Severe Plasmodium yoelii Malaria.

Malaria remains a global health burden causing significant morbidity, yet the mechanisms underlying disease outcomes and protection are poorly understood. Herein, we analyzed the peripheral blood of a unique cohort of Malawian children with severe malaria, and performed a comprehensive overview of blood leukocytes and inflammatory mediators present in these patients. We reveal robust immune cell activation, notably of CD14+ inflammatory monocytes, NK cells and plasmacytoid dendritic cells (pDCs) that is associated with very high inflammation. Using the Plasmodium yoelii 17X YM surrogate mouse model of lethal malaria, we report a comparable pattern of immune cell activation and inflammation and found that type I IFN represents a key checkpoint for disease outcomes. Compared to wild type mice, mice lacking the type I interferon (IFN) receptor exhibited a significant decrease in immune cell activation and inflammatory response, ultimately surviving the infection. We demonstrate that pDCs were the major producers of systemic type I IFN in the bone marrow and the blood of infected mice, via TLR7/MyD88-mediated recognition of Plasmodium parasites. This robust type I IFN production required priming of pDCs by CD169+ macrophages undergoing activation upon STING-mediated sensing of parasites in the bone marrow. pDCs and macrophages displayed prolonged interactions in this compartment in infected mice as visualized by intravital microscopy. Altogether our findings describe a novel mechanism of pDC activation in vivo and precise stepwise cell/cell interactions taking place during severe malaria that contribute to immune cell activation and inflammation, and subsequent disease outcomes.

Metadata Correction: Alcohol Consumption Reduction Among a Web-Based Supportive Community Using the Hello Sunday Morning Blog Platform: Observational Study.

[This corrects the article DOI: 10.2196/jmir.9605.].

Alcohol Consumption Reduction Among a Web-Based Supportive Community Using the Hello Sunday Morning Blog Platform: Observational Study.

BACKGROUND: Alcohol misuse is a major social and public health issue in Australia, with an estimated cost to the community of Aus $30 billion per annum. Until recently, a major barrier in addressing this significant public health issue is the fact that the majority of individuals with alcohol use disorders and alcohol misuse are not receiving treatment. OBJECTIVE: This study aimed to assess whether alcohol consumption changes are associated with participation in Hello Sunday Morning's blog platform, an online forum discussing experiences in abstaining from alcohol. METHODS: The study reports on Hello Sunday Morning participants who signed up for a 3-month period of abstinence from November 2009 to November 2016. The sample comprised 1917 participants (female: 1227/1917, 64.01%; male: 690/1917, 35.99%). Main outcome measures were Alcohol Use Disorders Identification Test (AUDIT) scores, mood, program engagement metrics, and slip-ups. RESULTS: Individuals who reported hazardous (preprogram AUDIT mean 11.92, SD 2.25) and harmful consumption levels (preprogram AUDIT mean 17.52, SD 1.08) and who engaged in the Hello Sunday Morning program reported a significant decrease in alcohol consumption, moving to lower risk consumption levels (hazardous, mean 7.59, SD 5.70 and harmful, mean 10.38, SD 7.43), 4 months following program commencement (P<.001). Those who reported high-risk or dependent consumption levels experienced the biggest reduction (preprogram mean 25.38, SD 4.20), moving to risky consumption (mean 15.83, SD 11.11), 4 months following program commencement (P<.001). These reductions in risk were maintained by participants in each group, 7 months following program commencement. Furthermore, those who engaged in the program more (as defined by more sign-ins, blogs posted, check-ins completed, and engagement with the community through likes and following) had lower alcohol consumption. Finally, those who experienced more slip-ups had lower alcohol consumption. CONCLUSIONS: Participation in an online forum can support long-term behavior change in individuals wishing to change their drinking behavior. Importantly, reductions in AUDIT scores appeared larger for those drinking at high-risk and hazardous levels before program commencement. This has promising implications for future models of alcohol reduction treatment, as online forums are an anonymous, accessible, and cost-effective alternative or adjunct to treatment-as-usual. Further research is needed into the specific mechanisms of change within a Web-based supportive community, as well as the role of specific mood states in predicting risky drinking behavior.

Spatio-temporal profile, phenotypic diversity, and fate of recruited monocytes into the post-ischemic brain.

BACKGROUND: A key feature of the inflammatory response after cerebral ischemia is the brain infiltration of blood monocytes. There are two main monocyte subsets in the mouse blood: CCR2+Ly6Chi "inflammatory" monocytes involved in acute inflammation, and CX3CR1+Ly6Clo "patrolling" monocytes, which may play a role in repair processes. We hypothesized that CCR2+Ly6Chi inflammatory monocytes are recruited in the early phase after ischemia and transdifferentiate into CX3CR1+Ly6Clo "repair" macrophages in the brain. METHODS: CX3CR1GFP/+CCR2RFP/+ bone marrow (BM) chimeric mice underwent transient middle cerebral artery occlusion (MCAo). Mice were sacrificed from 1 to 28 days later to phenotype and map subsets of infiltrating monocytes/macrophages (Mo/MΦ) in the brain over time. Flow cytometry analysis 3 and 14 days after MCAo in CCR2-/- mice, which exhibit deficient monocyte recruitment after inflammation, and NR4A1-/- BM chimeric mice, which lack circulating CX3CR1+Ly6Clo monocytes, was also performed. RESULTS: Brain mapping of CX3CR1GFP/+ and CCR2RFP/+ cells 3 days after MCAo showed absence of CX3CR1GFP/+ Mo/MΦ but accumulation of CCR2RFP/+ Mo/MΦ throughout the ischemic territory. On the other hand, CX3CR1+ cells accumulated 14 days after MCAo at the border of the infarct core where CCR2RFP/+ accrued. Whereas the amoeboid morphology of CCR2RFP/+ Mo/MΦ remained unchanged over time, CX3CR1GFP/+ cells exhibited three distinct phenotypes: amoeboid cells with retracted processes, ramified cells, and perivascular elongated cells. CX3CR1GFP/+ cells were positive for the Mo/MΦ marker Iba1 and phenotypically distinct from endothelial cells, smooth muscle cells, pericytes, neurons, astrocytes, or oligodendrocytes. Because accumulation of CX3CR1+Ly6Clo Mo/MΦ was absent in the brains of CCR2 deficient mice, which exhibit deficiency in CCR2+Ly6Chi Mo/MΦ recruitment, but not in NR4A1-/- chimeric mice, which lack of circulating CX3CR1+Ly6Clo monocytes, our data suggest a local transition of CCR2+Ly6Chi Mo/MΦ into CX3CR1+Ly6Clo Mo/MΦ phenotype. CONCLUSIONS: CX3CR1+Ly6Clo arise in the brain parenchyma from CCR2+Ly6Chi Mo/MΦ rather than being de novo recruited from the blood. These findings provide new insights into the trafficking and phenotypic diversity of monocyte subtypes in the post-ischemic brain.

Inducible nitric oxide synthase in neutrophils and endothelium contributes to ischemic brain injury in mice.

NO produced by inducible NO synthase (iNOS) contributes to ischemic brain injury, but the cell types expressing iNOS and mediating tissue damage have not been elucidated. To examine the relative contribution of iNOS in resident brain cells and peripheral leukocytes infiltrating the ischemic brain, we used bone marrow (BM) chimeric mice in which the middle cerebral artery was occluded and infarct volume was determined 3 d later. iNOS(-/-) mice engrafted with iNOS(+/+) BM exhibited larger infarcts (44 ± 2 mm(3); n = 13; mean ± SE) compared with autologous transplanted iNOS(-/-) mice (24 ± 3 mm(3); n = 10; p < 0.01), implicating blood-borne leukocytes in the damage. Furthermore, iNOS(+/+) mice transplanted with iNOS(-/-) BM had large infarcts (39 ± 6 mm(3); n = 13), similar to those of autologous transplanted iNOS(+/+) mice (39 ± 4 mm(3); n = 14), indicating the resident brain cells also play a role. Flow cytometry and cell sorting revealed that iNOS is highly expressed in neutrophils and endothelium but not microglia. Surprisingly, postischemic iNOS expression was enhanced in the endothelium of iNOS(+/+) mice transplanted with iNOS(-/-) BM and in leukocytes of iNOS(-/-) mice with iNOS(+/+) BM, suggesting that endothelial iNOS suppresses iNOS expression in leukocytes and vice versa. To provide independent evidence that neutrophils mediate brain injury, neutrophils were isolated and transferred to mice 24 h after stroke. Consistent with the result in chimeric mice, transfer of iNOS(+/+), but not iNOS(-/-), neutrophils into iNOS(-/-) mice increased infarct volume. The findings establish that iNOS in both neutrophils and endothelium mediates tissue damage and identify these cell types as putative therapeutic targets for stroke injury.

Mechanistic Insights into Radical-Mediated Oxidation of Tryptophan from ab Initio Quantum Chemistry Calculations and QM/MM Molecular Dynamics Simulations.

An assessment of the mechanisms of (•)OH and (•)OOH radical-mediated oxidation of tryptophan was performed using density functional theory calculations and ab initio plane-wave Quantum Mechanics/Molecular Mechanics (QM/MM) molecular dynamics simulations. For the (•)OH reactions, addition to the pyrrole ring at position 2 is the most favored site with a barrierless reaction in the gas phase. The subsequent degradation of this adduct through a H atom transfer to water was intermittently observed in aqueous-phase molecular dynamics simulations. For the (•)OOH reactions, addition to the pyrrole ring at position 2 is the most favored pathway, in contrast to the situation in the model system ethylene, where concerted addition to the double bond is preferred. From the (•)OOH position 2 adduct QM/MM simulations show that formation of oxy-3-indolanaline occurs readily in an aqueous environment. The observed transformation starts from an initial rupture of the O-O bond followed by a H atom transfer with the accompanying loss of an (•)OH radical to solution. Finally, classical molecular dynamics simulations were performed to equate observed differential oxidation rates of various tryptophan residues in monoclonal antibody fragments. It was found that simple parameters derived from simulation correlate well with the experimental data.

Specific catalysis of asparaginyl deamidation by carboxylic acids: kinetic, thermodynamic, and quantitative structure-property relationship analyses.

Asparaginyl (Asn) deamidation could lead to altered potency, safety, and/or pharmacokinetics of therapeutic protein drugs. In this study, we investigated the effects of several different carboxylic acids on Asn deamidation rates using an IgG1 monoclonal antibody (mAb1*) and a model hexapeptide (peptide1) with the sequence YGKNGG. Thermodynamic analyses of the kinetics data revealed that higher deamidation rates are associated with predominantly more negative ΔS and, to a lesser extent, more positive ΔH. The observed differences in deamidation rates were attributed to the unique ability of each type of carboxylic acid to stabilize the energetically unfavorable transition-state conformations required for imide formation. Quantitative structure property relationship (QSPR) analysis using kinetic data demonstrated that molecular descriptors encoding for the geometric spatial distribution of atomic properties on various carboxylic acids are effective determinants for the deamidation reaction. Specifically, the number of O-O and O-H atom pairs on carboxyl and hydroxyl groups with interatomic distances of 4-5 Å on a carboxylic acid buffer appears to determine the rate of deamidation. Collectively, the results from structural and thermodynamic analyses indicate that carboxylic acids presumably form multiple hydrogen bonds and charge-charge interactions with the relevant deamidation site and provide alignment between the reactive atoms on the side chain and backbone. We propose that carboxylic acids catalyze deamidation by stabilizing a specific, energetically unfavorable transition-state conformation of l-asparaginyl intermediate II that readily facilitates bond formation between the γ-carbonyl carbon and the deprotonated backbone nitrogen for cyclic imide formation.

Reactions of benzene and 3-methylpyrrole with the •OH and •OOH radicals: an assessment of contemporary density functional theory methods.

A high-level quantum chemistry investigation has been carried out for the addition and abstraction reactions by the radicals (•)OH and (•)OOH to and from the model alkenes 3-methylpyrrole and benzene. These models were chosen to reflect the functionalities contained in the side chain of the amino acid tryptophan. The W1BD procedure was used to calculate benchmark barriers and reaction energies for the smaller model system of (•)OOH addition to ethylene. It was found that the CBS-QB3 methodology compares best with the W1BD benchmark, demonstrating a mean absolute deviation (MAD) from W1BD of 3.9 kJ mol(-1). For the reactions involving the (•)OH radical and benzene or 3-methylpyrrole, addition is favored over abstraction in all cases. In particular the CBS-QB3 calculations suggest a barrierless addition reaction of the (•)OH radical to position two of 3-methylpyrrole. For the analogous addition and abstraction reactions involving the (•)OOH radical, the same order of reactivity was found, albeit with higher barriers. A number of other processes involving the addition of the (•)OOH radical were also investigated. The main findings of these studies determined that the initial (•)OOH barrier of stepwise addition to 3-methylpyrrole (+18.8 kJ mol(-1)) is significantly smaller than the concerted addition barrier (+71.5 kJ mol(-1)). This conclusion contrasts starkly with the situation for ethylene in which it is well established that the concerted process has the smaller barrier. A considerable variety of contemporary density functional theory procedures have been tested to examine their accuracy in predicting the CBS-QB3 results. It was found that the best overall performing method was UBMK with an MAD of 7.3 kJ mol(-1). A number of other functionals additionally performed well. They included UM06, RM06, UXYG3 and RXYG3, all of which have MADs of less than 8 kJ mol(-1).

COX-1-derived PGE2 and PGE2 type 1 receptors are vital for angiotensin II-induced formation of reactive oxygen species and Ca(2+) influx in the subfornical organ.

Regulation of blood pressure by angiotensin II (ANG II) is a process that involves the reactive oxygen species (ROS) and calcium. We have shown that ANG-II type 1 receptor (AT1R) and prostaglandin E2 (PGE2) type 1 receptors (EP1R) are required in the subfornical organ (SFO) for ROS-mediated hypertension induced by slow-pressor ANG-II infusion. However, the signaling pathway associated with this process remains unclear. We sought to determine mechanisms underlying the ANG II-induced ROS and calcium influx in mouse SFO cells. Ultrastructural studies showed that cyclooxygenase 1 (COX-1) codistributes with AT1R in the SFO, indicating spatial proximity. Functional studies using SFO cells revealed that ANG II potentiated PGE2 release, an effect dependent on AT1R, phospholipase A2 (PLA2) and COX-1. Furthermore, both ANG II and PGE2 increased ROS formation. While the increase in ROS initiated by ANG II, but not PGE2, required the activation of the AT1R/PLA2/COX-1 pathway, both ANG II and PGE2 were dependent on EP1R and Nox2 as downstream effectors. Finally, ANG II potentiated voltage-gated L-type Ca(2+) currents in SFO neurons via the same signaling pathway required for PGE2 production. Blockade of EP1R and Nox2-derived ROS inhibited ANG II and PGE2-mediated Ca(2+) currents. We propose a mechanism whereby ANG II increases COX-1-derived PGE2 through the AT1R/PLA2 pathway, which promotes ROS production by EP1R/Nox2 signaling in the SFO. ANG II-induced ROS are coupled with Ca(2+) influx in SFO neurons, which may influence SFO-mediated sympathoexcitation. Our findings provide the first evidence of a spatial and functional framework that underlies ANG-II signaling in the SFO and reveal novel targets for antihypertensive therapies.

A second generation human haplotype map of over 3.1 million SNPs.

We describe the Phase II HapMap, which characterizes over 3.1 million human single nucleotide polymorphisms (SNPs) genotyped in 270 individuals from four geographically diverse populations and includes 25-35% of common SNP variation in the populations surveyed. The map is estimated to capture untyped common variation with an average maximum r2 of between 0.9 and 0.96 depending on population. We demonstrate that the current generation of commercial genome-wide genotyping products captures common Phase II SNPs with an average maximum r2 of up to 0.8 in African and up to 0.95 in non-African populations, and that potential gains in power in association studies can be obtained through imputation. These data also reveal novel aspects of the structure of linkage disequilibrium. We show that 10-30% of pairs of individuals within a population share at least one region of extended genetic identity arising from recent ancestry and that up to 1% of all common variants are untaggable, primarily because they lie within recombination hotspots. We show that recombination rates vary systematically around genes and between genes of different function. Finally, we demonstrate increased differentiation at non-synonymous, compared to synonymous, SNPs, resulting from systematic differences in the strength or efficacy of natural selection between populations.