Adeno-Associated Virus Type 9-Mediated Gene Therapy of Choline Acetyltransferase-Deficient Mice.

The enzyme choline acetyltransferase (ChAT) synthesizes acetylcholine from acetyl-CoA and choline at the neuromuscular junction and at the nerve terminals of cholinergic neurons. Mutations in the ChAT gene (CHAT) result in a presynaptic congenital myasthenic syndrome (CMS) that often associates with life-threatening episodes of apnea. Knockout mice for Chat (Chat-/-) die at birth. To circumvent the lethality of this model, we crossed mutant mice possessing loxP sites flanking Chat exons 4 and 5 with mice that expressed Cre-ERT2. Injection of tamoxifen (Tx) at postnatal (P) day 11 in these mice induced downregulation of Chat, autonomic failure, weakness, and death. However, a proportion of Chatflox/flox-Cre-ERT2 mice receiving at birth an intracerebroventricular injection of 2 × 1013 vg/kg adeno-associated virus type 9 (AAV9) carrying human CHAT (AAV9-CHAT) survived a subsequent Tx injection and lived to adulthood without showing signs of weakness. Likewise, injection of AA9-CHAT by intracisternal injection at P28 after the onset of weakness also resulted in survival to adulthood. The expression of Chat in spinal motor neurons of Chatflox/flox-Cre-ERT2 mice injected with Tx was markedly reduced, but AAV-injected mice showed a robust recovery of ChAT expression, which was mainly translated by the human CHAT RNA. The biodistribution of the viral genome was widespread but maximal in the spinal cord and brain of AAV-injected mice. No significant histopathological changes were observed in the brain, liver, and heart of AAV-injected mice after 1 year follow-up. Thus, AAV9-mediated gene therapy may provide an effective and safe treatment for patients severely affected with CHAT-CMS.

Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-α-amino acids in vitro and in vivo.

The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-L-α-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Here we report that pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants accept α-hydroxy, α-thio and N-formyl-L-α-amino acids, as well as α-carboxy acid monomers that are precursors to polyketide natural products. These monomers are accommodated and accepted by the translation apparatus in vitro; those with reactive nucleophiles are incorporated into proteins in vivo. High-resolution structural analysis of the complex formed between one PylRS enzyme and a m-substituted 2-benzylmalonic acid derivative revealed an active site that discriminates prochiral carboxylates and accommodates the large size and distinct electrostatics of an α-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges substantially from the α-amine of proteinogenic amino acids. These enzymes or derivatives thereof could synergize with natural or evolved ribosomes and/or translation factors to generate diverse sequence-defined non-protein heteropolymers.

Biocatalytic control of site-selectivity and chain length-selectivity in radical amino acid halogenases.

Biocatalytic C-H activation has the potential to merge enzymatic and synthetic strategies for bond formation. FeII/αKG-dependent halogenases are particularly distinguished for their ability both to control selective C-H activation as well as to direct group transfer of a bound anion along a reaction axis separate from oxygen rebound, enabling the development of new transformations. In this context, we elucidate the basis for the selectivity of enzymes that perform selective halogenation to yield 4-Cl-lysine (BesD), 5-Cl-lysine (HalB), and 4-Cl-ornithine (HalD), allowing us to probe how site-selectivity and chain length selectivity are achieved. We now report the crystal structure of the HalB and HalD, revealing the key role of the substrate-binding lid in positioning the substrate for C4 vs C5 chlorination and recognition of lysine vs ornithine. Targeted engineering of the substrate-binding lid further demonstrates that these selectivities can be altered or switched, showcasing the potential to develop halogenases for biocatalytic applications.

Methylsulfonylmethane and Sesame Seed Oil Improve Dyslipidemia and Modulate Polyunsaturated Fatty Acid Metabolism in Two Mouse Models of Diabetes.

The objective of this study was to identify alterations in lipids and polyunsaturated fatty acid (PUFA) metabolism in both the streptozotocin (STZ)-induced type 1 diabetic (T1D) mouse and the mutant db/db type 2 diabetic (T2D) mouse to establish a biological signature for the evaluation of natural products with purported lipid-altering activity. Eight-week-old male C57BL/6J mice were randomized to nondiabetic group or STZ-induced diabetic groups (n = 10/group). STZ-induced diabetic mice and 6-week-old male db/db mice (n = 10/group) were randomized to the following groups: (1) diabetic control, no treatment, (2) methylsulfonylmethane (MSM) treatment, (3) sesame seed oil (SSO) treatment, and (4) MSM+SSO combination treatment. Clinical parameters measured included weights, blood glucose, serum lipid panels, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection of free fatty acids in serum, liver, brain, and eyes. Blood glucose significantly decreased after 4 weeks of MSM treatment in T1D mice. Serum PUFA levels were significantly reduced in T2D mice compared with control mice. In contrast, treatment with SSO reversed this effect in T2D mice, exhibiting serum PUFA levels comparable to control mice. Serum triglycerides were significantly increased in both diabetic models compared to nondiabetic control, mimicking diabetes in people. High-density lipoprotein (HDL) was significantly increased in T1D receiving MSM+SSO and all T2D treatment groups. A corresponding significant decrease in non-HDL cholesterol was seen in T2D mice in all treatment groups. MSM+SSO treatment's effects on HDL and non-HDL cholesterol and PUFA metabolism could lead to improved clinical outcomes in diabetics by improving the lipid profile.

Gel Polymer Electrolytes Based on Cross-Linked Poly(ethylene glycol) Diacrylate for Calcium-Ion Conduction.

Calcium batteries are promising alternatives to lithium batteries owing to their high energy density, comparable reduction potential, and mineral abundance. However, to meet practical demands in high-performance applications, suitable electrolytes must be developed. Here, we report the synthesis and characterization of polymer gel electrolytes for calcium-ion conduction prepared by the photo-cross-linking of poly(ethylene glycol) diacrylate (PEGDA) in the presence of solutions of calcium salts in a mixture of ethylene carbonate (EC) and propylene carbonate (PC) solvents. The results show room-temperature conductivity between 10-5 and 10-4 S/cm, electrochemical stability windows of ∼3.8 V, full dissociation of the salt, and minimal coordination with the PEGDA backbone. Cycling in symmetric Ca metal cells proceeds but with increasing overpotentials, which can be attributed to interfacial impedance between the electrolyte and calcium surface, which inhibits charge transfer. Calcium may still be plated and stripped yielding high-purity deposits and no indication of significant electrolyte breakdown, indicating that high overpotentials are associated with an electrically insulating, yet ion-permeable solid electrolyte interface (SEI). This work provides a contribution to the study and understanding of polymer gel materials toward their improvement and application as electrolytes for calcium batteries.

Validation of LexisNexis Accurint in the Georgia Cancer Registry's Cancer Recurrence and Information Surveillance Program.

BACKGROUND: LexisNexis Accurint is a database of ~84 billion public records that includes an individual's location of residence. Its ability to track residences longitudinally has not been validated. This study used the Georgia Cancer Registry's (GCR's) Cancer Recurrence and Information Surveillance Program (CRISP) to validate the U.S. state of residence and to examine characteristics of patients not included or who had an inaccurate entry in LexisNexis. METHODS: The GCR is routinely linked to the National Death Index (NDI), providing information regarding the state of residence in which the patient died. We compared the state of residence reported in LexisNexis with the NDI gold standard state of residence at death. Multivariate logistic regression analyses estimated associations between demographic information and: (1) having a mismatch between LexisNexis and NDI and (2) being missed in LexisNexis. RESULTS: Of the 69,494 patients in the CRISP cohort, 65,890 (95%) were found in LexisNexis and 9,597 (14%) had died. Among a subset of patients who were deceased, the sensitivity of LexisNexis for identifying persons who left Georgia was 42% and the specificity was 89%. Minority groups were more likely to be missed in the LexisNexis database as well as to have discordance between LexisNexis and NDI state of residence at death. CONCLUSIONS: LexisNexis Accurint failed to identify the emigration of more than half of deceased CRISP patients who had left Georgia but correctly identified most who had remained. The validity of the state of residence is important for studies using LexisNexis as a tool for follow-up.

Accounting for red blood cell accessibility reveals distinct invasion strategies in Plasmodium falciparum strains.

The growth of the malaria parasite Plasmodium falciparum in human blood causes all the symptoms of malaria. To proliferate, non-motile parasites must have access to susceptible red blood cells, which they invade using pairs of parasite ligands and host receptors that define invasion pathways. Parasites can switch invasion pathways, and while this flexibility is thought to facilitate immune evasion, it may also reflect the heterogeneity of red blood cell surfaces within and between hosts. Host genetic background affects red blood cell structure, for example, and red blood cells also undergo dramatic changes in morphology and receptor density as they age. The in vivo consequences of both the accessibility of susceptible cells, and their heterogeneous susceptibility, remain unclear. Here, we measured invasion of laboratory strains of P. falciparum relying on distinct invasion pathways into red blood cells of different ages. We estimated invasion efficiency while accounting for red blood cell accessibility to parasites. This approach revealed different tradeoffs made by parasite strains between the fraction of cells they can invade and their invasion rate into them, and we distinguish "specialist" strains from "generalist" strains in this context. We developed a mathematical model to show that generalist strains would lead to higher peak parasitemias in vivo compared to specialist strains with similar overall proliferation rates. Thus, the ecology of red blood cells may play a key role in determining the rate of P. falciparum parasite proliferation and malaria virulence.

"Cure" Versus "Clinical Remission": The Impact of a Medication Description on the Willingness of People Living with HIV to Take a Medication.

Many people living with HIV (PLWHIV) state that they would be willing to take significant risks to be "cured" of the virus. However, how they interpret the word "cure" in this context is not clear. We used a randomized survey to examine whether PLWHIV had a different willingness to take a hypothetical HIV medication if it causes flu-like symptoms, but provides: (a) cure, (b) remission that was labeled "cure", or (c) remission. PLWHIV (n = 454) were more willing to take a medication that provided a "cure" versus a "remission" if the side effects lasted less than 1 year. PLWHIV were more willing to take a medication that provided a remission that was labeled "cure" versus a "remission" (p = 0.01) if the side effects lasted 2 weeks. Clinicians and researchers should be aware of the impact of the word "cure" and ensure that PLWHIV fully understand the possible outcomes of their treatment options.

Nanotechnology in regenerative ophthalmology.

In recent years, regenerative medicine is gaining momentum and is giving hopes for restoring function of diseased, damaged, and aged tissues and organs and nanotechnology is serving as a catalyst. In the ophthalmology field, various types of allogenic and autologous stem cells have been investigated to treat some ocular diseases due to age-related macular degeneration, glaucoma, retinitis pigmentosa, diabetic retinopathy, and corneal and lens traumas. Nanomaterials have been utilized directly as nanoscaffolds for these stem cells to promote their adhesion, proliferation and differentiation or indirectly as vectors for various genes, tissue growth factors, cytokines and immunosuppressants to facilitate cell reprogramming or ocular tissue regeneration. In this review, we reviewed various nanomaterials used for retina, cornea, and lens regenerations, and discussed the current status and future perspectives of nanotechnology in tracking cells in the eye and personalized regenerative ophthalmology. The purpose of this review is to provide comprehensive and timely insights on the emerging field of nanotechnology for ocular tissue engineering and regeneration.

YpeB dimerization may be required to stabilize SleB for effective germination of Bacillus anthracis spores.

BACKGROUND: Bacillus cells faced with unfavorable environmental conditions undergo an asymmetric division process ultimately leading to the formation of the bacterial spore. In some instances the spore serves as an infectious agent; such is the case with the spore of Bacillus anthracis and the disease anthrax. Spores are resistant to a variety of environment conditions including traditional decontamination techniques due to the formation of specialized cellular structures. One such structure, the spore cortex, is a thick layer of modified peptidoglycan that contributes to spore dormancy through maintenance of the dehydrated state of the spore core. During spore germination, degradation of the cortex is required to facilitate complete hydration of the core and a return to vegetative growth. Degradation of the cortex is accomplished through the action of germination-specific lytic enzymes. One of these enzymes, SleB, has been previously shown to require the presence of the YpeB protein for its stable incorporation and subsequent function in spores of B. anthracis. The focus of the present study is to identify protein interactions of YpeB through in vivo chemical cross-linking and two-hybrid analysis. RESULTS: Conserved residues within YpeB PepSY domains were altered to facilitate implementation of a site-specific chemical cross-linker, 4-Azidophenacyl bromide. Analyses of crosslinked-spore extracts suggests that YpeB exists as a dimer or larger multimer within the spore, potentially mediated through interactions of the C-terminal domains. Spores expressing stable truncated forms of YpeB were crosslinked and corresponding truncated dimers were detected. Further characterization of individual YpeB domains using bacterial two-hybrid analysis indicated a possible role for both N-and C-terminal domains in YpeB oligomerization. CONCLUSIONS: The YpeB protein likely exists as dimer or higher-order multimer in the dormant spore. Both the N- and C-terminal YpeB domains contribute to multimerization. SleB likely also exists as an oligomer, and SleB and YpeB may be found together within a protein complex. Disassembly of this complex during spore germination likely allows SleB to become active in spore cortex degradation. Further study of this protein complex may contribute to the development of methods to inhibit or stimulate germination, allowing more effective spore decontamination.

Identification of L-Valine-initiated-germination-active genes in Bacillus subtilis using Tn-seq.

Bacterial endospores can survive harsh environmental conditions and long-term dormancy in the absence of nutrients, but can rapidly germinate under favorable conditions. In the present study, we employed transposon sequencing (Tn-seq) to identify genes with previously uncharacterized roles in spore germination. Identified genes that encoded spore inner membrane proteins were chosen for study of defined mutants, which exhibited delayed germination in several assays in response to varying germinants. Significantly slowed release of DPA indicated that mutants were affected in Stage I of germination. Several mutants exhibited phenotypic traits consistent with failure of a GerA germinant receptor-mediated response, while others appeared to have a more general loss of response to varied germinants. Use of a gerA-lacZ transcriptional fusion and quantitative western blotting of GerAC allowed mutants to be classified based upon normal or decreased gerA transcription and normal or reduced GerA accumulation. Fourteen genes were identified to have newly described roles within Bacillus spore germination. A more complete understanding of this process can contribute to the development of better spore decontamination procedures.

Efficient transposon mutagenesis mediated by an IPTG-controlled conditional suicide plasmid.

BACKGROUND: Transposon mutagenesis is highly valuable for bacterial genetic and genomic studies. The transposons are usually delivered into host cells through conjugation or electroporation of a suicide plasmid. However, many bacterial species cannot be efficiently conjugated or transformed for transposon saturation mutagenesis. For this reason, temperature-sensitive (ts) plasmids have also been developed for transposon mutagenesis, but prolonged incubation at high temperatures to induce ts plasmid loss can be harmful to the hosts and lead to enrichment of mutants with adaptive genetic changes. In addition, the ts phenotype of a plasmid is often strain- or species-specific, as it may become non-ts or suicidal in different bacterial species. RESULTS: We have engineered several conditional suicide plasmids that have a broad host range and whose loss is IPTG-controlled. One construct, which has the highest stability in the absence of IPTG induction, was then used as a curable vector to deliver hyperactive miniTn5 transposons for insertional mutagenesis. Our analyses show that these new tools can be used for efficient and regulatable transposon mutagenesis in Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. In P. aeruginosa PAO1, we have used this method to generate a Tn5 insertion library with an estimated diversity of ~ 108, which is ~ 2 logs larger than the best transposon insertional library of PAO1 and related Pseudomonas strains previously reported. CONCLUSION: We have developed a number of IPTG-controlled conditional suicide plasmids. By exploiting one of them for transposon delivery, a highly efficient and broadly useful mutagenesis system has been developed. As the assay condition is mild, we believe that our methodology will have broad applications in microbiology research.

Clostridium difficile Lipoprotein GerS Is Required for Cortex Modification and Thus Spore Germination.

Clostridium difficile, also known as Clostridioides difficile, is a Gram-positive, spore-forming bacterium that is a leading cause of antibiotic-associated diarrhea. C. difficile infections begin when its metabolically dormant spores germinate to form toxin-producing vegetative cells. Successful spore germination depends on the degradation of the cortex, a thick layer of modified peptidoglycan that maintains dormancy. Cortex degradation is mediated by the SleC cortex lytic enzyme, which is thought to recognize the cortex-specific modification muramic-δ-lactam. C. difficile cortex degradation also depends on the Peptostreptococcaceae-specific lipoprotein GerS for unknown reasons. In this study, we tested whether GerS regulates production of muramic-δ-lactam and thus controls the ability of SleC to recognize its cortex substrate. By comparing the muropeptide profiles of ΔgerS spores to those of spores lacking either CwlD or PdaA, both of which mediate cortex modification in Bacillus subtilis, we determined that C. difficile GerS, CwlD, and PdaA are all required to generate muramic-δ-lactam. Both GerS and CwlD were needed to cleave the peptide side chains from N-acetylmuramic acid, suggesting that these two factors act in concert. Consistent with this hypothesis, biochemical analyses revealed that GerS and CwlD directly interact and that CwlD modulates GerS incorporation into mature spores. Since ΔgerS, ΔcwlD, and ΔpdaA spores exhibited equivalent germination defects, our results indicate that C. difficile spore germination depends on cortex-specific modifications, reveal GerS as a novel regulator of these processes, and highlight additional differences in the regulation of spore germination in C. difficile relative to B. subtilis and other spore-forming organisms.IMPORTANCE The Gram-positive, spore-forming bacterium Clostridium difficile is a leading cause of antibiotic-associated diarrhea. Because C. difficile is an obligate anaerobe, its aerotolerant spores are essential for transmitting disease, and their germination into toxin-producing cells is necessary for causing disease. Spore germination requires the removal of the cortex, a thick layer of modified peptidoglycan that maintains spore dormancy. Cortex degradation is mediated by the SleC hydrolase, which is thought to recognize cortex-specific modifications. Cortex degradation also requires the GerS lipoprotein for unknown reasons. In our study, we tested whether GerS is required to generate cortex-specific modifications by comparing the cortex composition of ΔgerS spores to the cortex composition of spores lacking two putative cortex-modifying enzymes, CwlD and PdaA. These analyses revealed that GerS, CwlD, and PdaA are all required to generate cortex-specific modifications. Since loss of these modifications in ΔgerS, ΔcwlD, and ΔpdaA mutants resulted in spore germination and heat resistance defects, the SleC cortex lytic enzyme depends on cortex-specific modifications to efficiently degrade this protective layer. Our results further indicate that GerS and CwlD are mutually required for removing peptide chains from spore peptidoglycan and revealed a novel interaction between these proteins. Thus, our findings provide new mechanistic insight into C. difficile spore germination.

Directed evolution of the Escherichia coli cAMP receptor protein at the cAMP pocket.

The Escherichia coli cAMP receptor protein (CRP) requires cAMP binding to undergo a conformational change for DNA binding and transcriptional regulation. Two CRP residues, Thr(127) and Ser(128), are known to play important roles in cAMP binding through hydrogen bonding and in the cAMP-induced conformational change, but the connection between the two is not completely clear. Here, we simultaneously randomized the codons for these two residues and selected CRP mutants displaying high CRP activity in a cAMP-producing E. coli. Many different CRP mutants satisfied the screening condition for high CRP activity, including those that cannot form any hydrogen bonds with the incoming cAMP at the two positions. In vitro DNA-binding analysis confirmed that these selected CRP mutants indeed display high CRP activity in response to cAMP. These results indicate that the hydrogen bonding ability of the Thr(127) and Ser(128) residues is not critical for the cAMP-induced CRP activation. However, the hydrogen bonding ability of Thr(127) and Ser(128) was found to be important in attaining high cAMP affinity. Computational analysis revealed that most natural cAMP-sensing CRP homologs have Thr/Ser, Thr/Thr, or Thr/Asn at positions 127 and 128. All of these pairs are excellent hydrogen bonding partners and they do not elevate CRP activity in the absence of cAMP. Taken together, our analyses suggest that CRP evolved to have hydrogen bonding residues at the cAMP pocket residues 127 and 128 for performing dual functions: preserving high cAMP affinity and keeping CRP inactive in the absence of cAMP.

Genetic markers of repolarization and arrhythmic events after acute coronary syndromes.

BACKGROUND: There is a genetic contribution to the risk of ventricular arrhythmias in survivors of acute coronary syndromes (ACS). We wished to explore the role of 33 candidate single nucleotide polymorphisms (SNPs) in prolonged repolarization and sudden death in patients surviving ACS. METHODS: A total of 2,139 patients (1680 white ethnicity) surviving an admission for ACS were enrolled in the prospective Coronary Disease Cohort Study. Extensive clinical, echocardiographic, and neurohormonal data were collected for 12 months, and clinical events were recorded for a median of 5 years. Each SNP was assessed for association with sudden cardiac death (SCD)/cardiac arrest (CA) and prolonged repolarization at 3 time-points: index admission, 1 month, and 12 months postdischarge. RESULTS: One hundred six SCD/CA events occurred during follow-up (6.3%). Three SNPs from 3 genes (rs17779747 [KCNJ2], rs876188 [C14orf64], rs3864180 [GPC5]) were significantly associated with SCD/CA in multivariable models (after correction for multiple testing); the minor allele of rs17779747 with a decreased risk (hazard ratio [HR] 0.68 per copy of the minor allele, 95% CI 0.50-0.92, P = .012), and rs876188 and rs386418 with an increased risk (HR 1.52 [95% CI 1.10-2.09, P = .011] and HR 1.34 [95% CI 1.04-1.82, P = .023], respectively). At 12 months postdischarge, rs10494366 and rs12143842 (NOS1AP) were significant predictors of prolonged repolarization (HR 1.32 [95% CI 1.04-1.67, P = .022] and HR 1.30 [95% CI 1.01-1.66, P = .038], respectively), but not at earlier time-points. CONCLUSION: Three SNPs were associated with SCD/CA. Repolarization time was associated with variation in the NOS1AP gene. This study demonstrates a possible role for SNPs in risk stratification for arrhythmic events after ACS.

Canterbury Health, Ageing and Life Course (CHALICE) study: rationale, design and methodology.

AIMS: New Zealand's ageing population threatens the financial sustainability of our current model of health service delivery. The Canterbury Health, Ageing and Life Course (CHALICE) study aims to develop a comprehensive and flexible database of important determinants of health to inform new models. This paper describes the design, methodology, and first 300 participants of CHALICE. METHODS: Commencing August 2010, CHALICE is a multidisciplinary prospective random cohort study and biobank of 1,000 Canterbury adults aged 49-51 years at inception, stratified by self-identified Maori (n=200) and non-Maori (n=800) ethnicity. Assessment covers sociodemographic, physical, cognition, mental health, clinical history, family and social, cardiovascular, and lifestyle domains. Detailed follow-up assessment occurs every 5 years, with a brief postal follow-up assessment undertaken annually. RESULTS: For the first 300 participants (44 Maori, 256 non-Maori), the participation rate is 63.7%. Overall, 53.3% of participants are female, 75.3% are living in married or de facto relationships, and 19.0% have university degrees. These sociodemographic profiles are comparable with the 2006 Census, Canterbury region, 50-54 years age group percentages (50.7%, 77.2%, and 14.3%, respectively). CONCLUSIONS: CHALICE has been designed to provide quality data that will inform policy development and programme implementation across a broad spectrum of health indicators.

Hyperuricaemia and gout in New Zealand rural and urban Maori and non-Maori communities.

BACKGROUND: There are few current data on the prevalence of hyperuricaemia and gout in New Zealand, particularly among the indigenous Maori population. AIMS: To determine the prevalence of gout and hyperuricaemia in rural and urban Maori and non-Maori community samples and describe the treatment and comorbidities of participants with gout. METHODS: Participants aged 20-64 years were recruited by random selection from the electoral roll. Maori samples were selected from among those identified as being of Maori descent on the roll and who self-identified as being of Maori ethnicity at interview. Personal medical history, blood pressure, anthropometrics, fasting lipids, glucose, HbA1c and urate were recorded. RESULTS: There were 751 participants. Mean serum urate (SU) was 0.30 mmol/L (0.06-0.69 mmol/L). Maori had a significantly higher prevalence of hyperuricaemia (SU > 0.40 mmol/L) compared with non-Maori (17.0% vs 7.5%, P = 0.0003). A total of 57 participants had a history of gout, with a higher prevalence in Maori compared with non-Maori (10.3% vs 2.3%, P < 0.0001). Of the participants, 18/57 (31.6%) with gout were receiving urate-lowering therapy, but in 38.9%, SU was >0.36 mmol/L. Participants with gout were more likely to have metabolic syndrome, diabetes, cardiac disease or hypertension. CONCLUSIONS: Gout and hyperuricaemia were more prevalent in Maori, and participants with gout were more likely to have comorbidities. There was not a higher overall adjusted cardiovascular disease risk in Maori participants with gout. Despite the high prevalence of gout, management remains suboptimal.

Impact of age, phenotype and cardio-renal function on plasma C-type and B-type natriuretic peptide forms in an adult population.

CONTEXT: In contrast to the cardiac hormones, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), variations in plasma concentrations of C-type natriuretic peptide (CNP) in healthy adults are ill-defined, limiting their clinical application. OBJECTIVE: Our objective was to define the effect of age, phenotype (gender, height, BMI), and cardiac and renal function on plasma CNPs in an adults population without renal or cardiovascular disease. DESIGN AND SETTING: This was a prospective cross-sectional observational study of adult volunteers, aged 21-80 years, randomly selected from the electoral roll. SUBJECTS AND METHODS: Plasma CNP and its associated aminoterminal propeptide (NTproCNP) were measured in 258 subjects and related to age, gender, height and plasma creatinine. Subgroup analyses seeking associations with cardiac function (plasma BNP and NTproBNP) and bone turnover bone-specific alkaline phosphatase (bALP) were also determined. RESULTS: Plasma concentrations of CNPs in men continued to decline from adolescent values to reach a nadir in the 5th decade after which values increased. Similar but less marked changes occurred in women. In both sexes, NTproCNP was inversely and independently correlated with height. In contrast to B-type natriuretic peptides (BNPs), NTproCNP was higher in men, significantly related to creatinine and positively related to bALP. CONCLUSIONS: Gender- and age-specific changes affect CNPs in adults. Inverse associations of NTproCNP with adult height, positive correlation with creatinine - and in contrast to CNP - no association with BNP are further unique findings distinguishing NTproCNP, which need to be considered in future studies.

Genetic variation in the renin-angiotensin-aldosterone system is associated with cardiovascular risk factors and early mortality in established coronary heart disease.

This study examined renin-angiotensin-aldosterone (RAAS) system gene variants for associations with cardiovascular risk factors and outcomes in coronary heart disease. Coronary disease patients (n=1186) were genotyped for 21 single-nucleotide polymorphisms (SNPs) within angiotensinogen (AGT), angiotensin-converting enzyme (ACE), angiotensin-II type-1 receptor (AGTR1) and aldosterone synthase (CYP11B2). Associations with all-cause mortality and cardiovascular readmissions were assessed over a median of 3.0 years. The AGT M235T 'T' allele was associated with a younger age of clinical coronary disease onset (P=0.006), and the AGT rs2478545 minor allele was associated with lower circulating natriuretic peptides (P=0.0001-P=0.001) and E/E(1) (P=0.018). Minor alleles of AGT SNPs rs1926723 and rs11122576 were associated with more frequent history of renal disease (P0.04) and type-2 diabetes (P0.02), higher body mass index (P0.02) and greater mortality (P0.007). AGT rs11568054 minor allele carriers had more frequent history of renal disease (P=0.04) and higher plasma creatinine (P=0.033). AGT rs6687360 minor allele carriers exhibited worse survival (P=0.02). ACE rs4267385 was associated with older clinical coronary disease onset (P=0.008) and hypertension (P=0.013) onset, increased plasma creatinine (P=0.01), yet greater mortality (P=0.044). Less history of hypertension was observed with the AGTR1 rs12685977 minor allele (P=0.039). Genetic variation within the RAAS was associated with cardiovascular risk factors and accordingly poorer survival.

Characterization of a unique conformational epitope on free immunoglobulin kappa light chains that is recognized by an antibody with therapeutic potential.

The murine mAb, K-1-21, recognizes a conformational epitope expressed on free Ig kappa light chains (FκLCs) and also on cell membrane-associated FκLCs found on kappa myeloma cells. This has led to the development of a chimeric version of K-1-21, MDX-1097, which is being assessed in a Phase II clinical trial for the treatment of multiple myeloma. The epitope recognized by K-1-21 is of particular interest, especially in the context that it is not expressed on heavy chain-associated light chains such as in an intact Ig molecule. Using epitope excision techniques we have localized the K-1-21 epitope to a region spanning residues 104-110 of FκLC. This short strand of residues links the variable and constant domains, and is a flexible region that adopts different conformations in FκLC and heavy chain-associated light chain. We tested this region using site-directed mutations and found that the reactivity of K-1-21 for FκLC was markedly reduced. Finally, we applied in silico molecular docking to generate a model that satisfied the experimental data. Given the clinical potential of the Ag, this study may aid the development of next generation compounds that target the membrane form of FκLC expressed on the surface of myeloma plasma cells.