Formulation and Characterization of Conjugate Vaccines to Reduce Opioid Use Disorders Suitable for Pharmaceutical Manufacturing and Clinical Evaluation.

This study focused on formulating conjugate vaccines targeting oxycodone and heroin for technology transfer, good manufacturing practice (GMP), and clinical evaluation. Lead vaccines used the highly immunogenic carrier protein keyhole limpet hemocyanin (KLH), which poses formulation problems because of its size. To address this barrier to translation, an oxycodone-based hapten conjugated to GMP-grade subunit KLH (OXY-sKLH) and adsorbed on alum adjuvant was studied with regard to carbodiimide coupling reaction time, buffer composition, purification methods for conjugates, conjugate size, state of aggregation, and protein/alum ratio. Vaccine formulations were screened for post-immunization antibody levels and efficacy in reducing oxycodone distribution to the brain in rats. While larger conjugates were more immunogenic, their size prevented characterization of the haptenation ratio by standard analytical methods and sterilization by filtration. To address this issue, conjugation chemistry and vaccine formulation were optimized for maximal efficacy, and conjugate size was measured by dynamic light scattering prior to adsorption to alum. An analogous heroin vaccine (M-sKLH) was also optimized for conjugation chemistry, formulated in alum, and characterized for potency against heroin in rats. Finally, this study found that the efficacy of OXY-sKLH was preserved when co-administered with M-sKLH, supporting the proof of concept for a bivalent vaccine formulation targeting both heroin and oxycodone. This study suggests methods for addressing the unique formulation and characterization challenges posed by conjugating small molecules to sKLH while preserving vaccine efficacy.

Isothiocyanate-Functionalized Bifunctional Chelates and fac-[M(I)(CO)3](+) (M = Re, (99m)Tc) Complexes for Targeting uPAR in Prostate Cancer.

Developing new strategies to rapidly incorporate the fac-[M(I)(CO)3](+) (M = Re, (99m)Tc) core into biological targeting vectors in radiopharmaceuticals continues to expand as molecules become more complex and as efforts to minimize nonspecific binding increase. This work examines a novel isothiocyanate-functionalized bifunctional chelate based on 2,2'-dipicolylamine (DPA) specifically designed for complexing the fac-[M(I)(CO)3](+) core. Two strategies (postlabeling and prelabeling) were explored using the isothiocyanate-functionalized DPA to determine the effectiveness of assembly on the overall yield and purity of the complex with amine containing biomolecules. A model amino acid (lysine) examined (1) amine conjugation of isothiocyanate-functionalized DPA followed by complexation with fac-[M(I)(CO)3](+) (postlabeling) and (2) complexation of fac-[M(I)(CO)3](+) with isothiocyanate-functionalized DPA followed by amine conjugation (prelabeling). Conducted with stable Re and radioactive (99m)Tc analogs, both strategies formed the product in good to excellent yields under macroscopic and radiotracer concentrations. A synthetic peptide (AE105) which targets an emerging biomarker in CaP prognosis, urokinase-type plasminogen activator receptor (uPAR), was also explored using the isothiocyanate-functionalized DPA strategy. In vitro PC-3 (uPAR+) cell uptake assays with the (99m)Tc-labeled peptide (8a) showed 4.2 ± 0.5% uptake at 4 h. In a murine model bearing PC-3 tumor xenografts, in vivo biodistribution of 8a led to favorable tumor uptake (3.7 ± 0.7% ID/g) at 4 h p.i. with relatively low accumulation (<2% ID/g) in normal organs not associated with normal peptide excretion. These results illustrate the promise of the isothiocyanate-functionalized approach for labeling amine containing biological targeting vectors with fac-[M(I)(CO)3](+).

Clickable, hydrophilic ligand for fac-[M(I)(CO)3](+) (M = Re/(99m)Tc) applied in an S-functionalized α-MSH peptide.

The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction was used to incorporate alkyne-functionalized dipicolylamine (DPA) ligands (1 and 3) for fac-[M(I)(CO)3](+) (M = Re/(99m)Tc) complexation into an α-melanocyte stimulating hormone (α-MSH) peptide analogue. A novel DPA ligand with carboxylate substitutions on the pyridyl rings (3) was designed to increase the hydrophilicity and to decrease in vivo hepatobiliary retention of fac-[(99m)Tc(I)(CO)3](+) complexes used in single photon emission computed tomography (SPECT) imaging studies with targeting biomolecules. The fac-[Re(I)(CO)3(3)] complex (4) was used for chemical characterization and X-ray crystal analysis prior to radiolabeling studies between 3 and fac-[(99m)Tc(I)(OH2)3(CO)3](+). The corresponding (99m)Tc complex (4a) was obtained in high radiochemical yields, was stable in vitro for 24 h during amino acid challenge and serum stability assays, and showed increased hydrophilicity by log P analysis compared to an analogous complex with nonfunctionalized pyridine rings (2a). An α-MSH peptide functionalized with an azide was labeled with fac-[M(I)(CO)3](+) using both click, then chelate (CuAAC reaction with 1 or 3 followed by metal complexation) and chelate, then click (metal complexation of 1 and 3 followed by CuAAC with the peptide) strategies to assess the effects of CuAAC conditions on fac-[M(I)(CO)3](+) complexation within a peptide framework. The peptides from the click, then chelate strategy had different HPLC tR's and in vitro stabilities compared to those from the chelate, then click strategy, suggesting nonspecific coordination of fac-[M(I)(CO)3](+) using this synthetic route. The fac-[M(I)(CO)3](+)-complexed peptides from the chelate, then click strategy showed >90% stability during in vitro challenge conditions for 6 h, demonstrated high affinity and specificity for the melanocortin 1 receptor (MC1R) in IC50 analyses, and led to moderately high uptake in B16F10 melanoma cells. Log P analysis of the (99m)Tc-labeled peptides confirmed the enhanced hydrophilicity of the peptide bearing the novel, carboxylate-functionalized DPA chelate (10a') compared to the peptide with the unmodified DPA chelate (9a'). In vivo biodistribution analysis of 9a' and 10a' showed moderate tumor uptake in a B16F10 melanoma xenograft mouse model with enhanced renal uptake and surprising intestinal uptake for 10a' compared to predominantly hepatic accumulation for 9a'. These results, coupled with the versatility of CuAAC, suggests this novel, hydrophilic chelate can be incorporated into numerous biomolecules containing azides for generating targeted fac-[M(I)(CO)3](+) complexes in future studies.

Survival, mortality and morbidity outcomes after oesophagogastric cancer surgery in New South Wales, 2001-2008.

OBJECTIVES: To examine the relationship between hospital volume and patient outcomes for New South Wales hospitals performing oesophagectomy and gastrectomy for oesophagogastric cancer. DESIGN, SETTING AND PATIENTS: A retrospective, population-based cohort study of NSW residents diagnosed with a new case of invasive oesophageal or gastric cancer who underwent oesophagectomy or gastrectomy between 2001 and 2008 in NSW hospitals using linked de-identified data from the NSW Central Cancer Registry, the National Death Index and the NSW Admitted Patient Data Collection. A higher-volume hospital was defined as one performing > 6 relevant procedures per year. MAIN OUTCOME MEASURES: Odds ratios for > 21-day length of stay, 28-day unplanned readmission, 30-day mortality and 90-day mortality, and hazard ratios (HRs) for 5-year absolute and conditional survival. RESULTS: Oesophagectomy (908 patients) and gastrectomy (1621 patients) were undertaken in 42 and 84 hospitals, respectively, between 2001 and 2008. Median annual hospital volume ranged from 2 to 4 for oesophagectomies and ranged from 2 to 3 for gastrectomies. Controlling for known confounders, no associations between hospital volume and > 21-day length of stay and 28-day unplanned readmission were found. Overall 30-day mortality was 4.1% and 4.4% for oesophagectomy and gastrectomy, respectively. Five-year absolute survival was significantly better for patients who underwent oesophagectomy in higher-volume hospitals (adjusted HR for lower-volume hospitals, 1.28 [95% CI, 1.10-1.49]; P = 0.002) and for those with localised gastric cancer who underwent gastrectomy in higher-volume hospitals (adjusted HR for lower-volume hospitals, 1.83 [95% CI, 1.28-2.61]; P = 0.001). CONCLUSIONS: These data support initial surgery for oesophagogastric cancer in higher-volume hospitals.

Clinical and molecular profile of young adults with early-onset colorectal cancer: Experience from four Australian tertiary centers.

BACKGROUND: Patients with early-onset colorectal cancer (EO-CRC) have unique characteristics. Contemporary data on the pathological and molecular features, and survival of EO-CRC are limited in the Australian context. AIM: To determine the demographic, histopathological and molecular characteristics of adults with EO-CRC, and their survival. METHODS: We conducted a retrospective study of adults aged 18-49 years with EO-CRC who were referred to the Illawarra Shoalhaven Local Health District, South Eastern Sydney Local Health District and Royal North Shore Hospital in New South Wales, Australia, between 2014 and 2018. RESULTS: Of 257 patients included, 94 (37%) patients presented with de novo metastatic CRC, 80% patients had near-average risk family history and 89% had a symptomatic presentation. In 159 patients with nonmetastatic disease at diagnosis, stage III disease (OR 3.88 [95% CI: 1.13-13.3]; p = .03) and the presence of perineural invasion (PNI) (OR 6.63 [95% CI: 2.21-19.84]; p = .001) were risk factors associated with the development of metastatic disease. Among 94 patients with de novo metastatic disease, 43 (43%) and 12 (14%) patients harbored a KRAS or BRAF V600E mutation, respectively. The median overall survival was 29.6 months (95% CI: 20.4-38.7). BRAF mutation was associated with inferior survival (HR 3.00 [95% CI: 1.30-6.94]; p = .01). CONCLUSION: The prevalence of KRAS and BRAF mutations in our cohort is similar to the overseas experience. Stage III disease at diagnosis, presence of PNI and BRAF mutation are adverse prognostic indicators. A better understanding of the molecular landscape is needed for this patient cohort, so as to better tailor prevention strategies, screening and treatment pathways.

Pharmacological mechanisms underlying the efficacy of antibodies generated by a vaccine to treat oxycodone use disorder.

Therapeutic vaccines offer a viable strategy to treat opioid use disorders (OUD) complementary to current pharmacotherapies. The candidate Oxy(Gly)4-sKLH vaccine targeting oxycodone displayed pre-clinical proof of efficacy, selectivity and safety, and it is now undergoing clinical evaluation. To further support its implementation in the clinic, this study tested critical in vivo neuropsychopharmacological properties of the Oxy(Gly)4-sKLH vaccine in rats. While repeated immunizations with Oxy(Gly)4-sKLH were necessary to maintain the antibody response overtime, exposure to free oxycodone did not boost oxycodone-specific antibody levels in vaccinated rats, limiting concerns of immune-related side effects. Immunization with Oxy(Gly)4-sKLH achieved sustained antibody titers over a period of five months following initial vaccination, supporting its potential for providing long-lasting protection. In vivo studies of selectivity showed that vaccination prevented oxycodone-induced but not methadone-induced antinociception, while still preserving the opioid antagonist naloxone's pharmacological effects. Vaccination did not interfere with fentanyl-induced antinociception or fentanyl distribution to the brain. These in vivo data confirm the previously reported in vitro selectivity profile of Oxy(Gly)4-sKLH. Vaccination extended oxycodone's half-life up to 25 h compared to control. While vaccination reduced the reinforcing efficacy of oxycodone in an intravenous self-administration model, signs of toxicity were not observed. These rodent studies confirm that active immunization with Oxy(Gly)4-sKLH induces highly specific and long-lasting antibodies which are effective in decreasing the reinforcing effects of oxycodone while preserving the efficacy of medications used to treat OUD and overdose.

Pharmacokinetic and pharmacodynamic interactions of echinacea and policosanol with warfarin in healthy subjects.

AIMS: This study investigated the pharmacokinetic and pharmacodynamic interactions of echinacea and policosanol with warfarin in healthy subjects. METHODS: This was an open-label, randomized, three-treatment, cross-over, clinical trial in healthy male subjects (n= 12) of known CYP2C9 and VKORC1 genotype who received a single oral dose of warfarin alone or after 2 weeks of pre-treatment with each herbal medicine at recommended doses. Pharmacodynamic (INR, platelet activity) and pharmacokinetic (warfarin enantiomer concentrations) end points were evaluated. RESULTS: The apparent clearance of (S)-warfarin (90% CI of ratio; 1.01, 1.18) was significantly higher during concomitant treatment with echinacea but this did not lead to a clinically significant change in INR (90% CI of AUC of INR; 0.91, 1.31). Policosanol did not significantly affect warfarin enantiomer pharmacokinetics or warfarin response. Neither echinacea nor policosanol had a significant effect on platelet aggregation after 2 weeks of pre-treatment with the respective herbal medicines. CONCLUSION: Echinacea significantly reduced plasma concentrations of S-warfarin. However, neither echinacea nor policosanol significantly affected warfarin pharmacodynamics, platelet aggregation or baseline clotting status in healthy subjects.

Lessons Learned From Hurricane Maria in Puerto Rico: Practical Measures to Mitigate the Impact of a Catastrophic Natural Disaster on Radiation Oncology Patients.

PURPOSE: Although the wind, rain, and flooding of Hurricane Maria in Puerto Rico abated shortly after its landfall on September 20, 2017, the disruption of the electrical, communications, transportation, and medical infrastructure of the island was unprecedented in scope and caused lasting harm for many months afterward. A compilation of recommendations from radiation oncologists who were in Puerto Rico during the disaster, and from a panel of American Society for Radiation Oncology (ASTRO) cancer experts was created. METHODS AND MATERIALS: Radiation oncologists throughout Puerto Rico collaborated and improvised to continue treating patients in the immediate aftermath of the storm and as routine clinical operations were restored gradually. Empirical lessons from the experience of radiation therapy administration in this profoundly altered context of limited resources, impaired communication, and inadequate transportation were organized into a recommended template, applicable to any radiation oncology practice. ASTRO disease-site experts provided evidence-guidelines for mitigating the impact of a 2- to 3-week interruption in radiation therapy. RESULTS: Practical measures to mitigate the medical impact of a disaster are summarized within the framework of "Prepare, Communicate, Operate, Compensate." Specific measures include the development of an emergency operations plan tailored to specific circumstances, prospective coordination with other radiation oncology clinics before a disaster, ongoing communications with emergency management organizations, and routine practice of alternate methods to disseminate information among providers and patients. CONCLUSIONS: These recommendations serve as a starting point to assist any radiation oncology practice in becoming more resiliently prepared for a local or regional disruption from any cause. Disease-site experts provide evidence-based guidelines on how to mitigate the impact of a 2- to 3-week interruption in radiation therapy for lung, head and neck, uterine cervix, breast, and prostate cancers through altered fractionation or dose escalation.

Structural stability of neoangiogenic intramyocardial microvessels supports functional recovery in chronic ischemic myocardium.

We hypothesize that combining angiopoietin-1 (ANG-1) or ANG-2 with vascular endothelial growth factor (VEGF) improves myocardial perfusion and contractile function by modulating vascular adaptation of neoangiogenic microvessels in a chronic ischemic swine model. Four weeks after occlusion of the left circumflex coronary artery (LCx), animals were injected with AdVEGF(165) (n=6), AdVEGF(165)+AdANG-1 (n=6), AdVEGF(165)+AdANG-2 (n=6) or control vector (n=5) into the left ventricular posterolateral wall. Regional perfusion by fluorescent microspheres and segmental myocardial tissue velocity by tissue Doppler imaging (TDI) were assessed at baseline, 4 weeks post occlusion and 4 weeks post therapy. Despite similar vascular growth following VEGF+ANG-1 and VEGF+ANG-2 treatments, transmural myocardial contractility improved only when VEGF was paired with ANG-1. In contrast, regional systolic function deteriorated uniformly across subepicardial, mid-myocardial and subendocardial segments in VEGF and VEGF+ANG-2 treated groups. Contractile improvement was associated with enhanced vascular stability through augmented arteriole formation, tight structural integration between VE-cadherin and beta-catenin at endothelial junctions and improved cross-talk between endothelium and myocardium. Structural stability of developing intramyocardial microvessels contributes to systolic function during ischemic neovascularization. Coordinated regulation of angiogenic revascularization that supports vascular stability is a key aspect in improving therapeutic outcomes in ischemic myocardium.

Endoplasmic reticulum stress markers are associated with obesity in nondiabetic subjects.

OBJECTIVE: Adipocyte and hepatocyte endoplasmic reticulum (ER) stress response is activated in dietary and genetic models of obesity in mice. We hypothesized that ER stress was also activated and associated with reduced insulin sensitivity (SI) in human obesity. RESEARCH DESIGN AND METHODS: We recruited 78 healthy, nondiabetic individuals over a spectrum of body mass index (BMI) who underwent oral and iv glucose tolerance tests, and fasting sc adipose and muscle biopsies. We tested expression of 18 genes and levels of total and phosphorylated eukaryotic initiation factor 2alpha, c-jun, and c-Jun N-terminal kinase 1 in adipose tissue. We compared gene expression in stromal vascular and adipocyte fractions in paired samples from 22 individuals, and tested clustering on gene and protein markers. RESULTS: Adipocyte expression of most markers of ER stress, including chaperones downstream of activating transcription factor 6, were significantly correlated with BMI and percent fat (r>0.5; P<0.00001). Phosphorylation of eukaryotic initiation factor 2alpha but not of c-Jun N-terminal kinase 1 or c-jun was increased with obesity. ER stress response (as elsewhere) was also increased with obesity in a second set of 86 individuals, and in the combined sample (n=161). The increase was only partially attributable to the stromal vascular fraction and macrophage infiltration. ER stress markers were only modestly correlated with S(I). Clustering algorithms supported ER stress activation with high BMI but not low SI. CONCLUSIONS: Multiple markers of ER stress are activated in human adipose with obesity, particularly for protective chaperones downstream of activating transcription factor 6alpha.

Activating transcription factor 6 (ATF6) sequence polymorphisms in type 2 diabetes and pre-diabetic traits.

Activating transcription factor 6 (ATF6) is located within the region of linkage to type 2 diabetes on chromosome 1q21-q23 and is a key activator of the endoplasmic reticulum stress response. We evaluated 78 single nucleotide polymorphisms (SNPs) spanning >213 kb in 95 people, from which we selected 64 SNPs for evaluation in 191 Caucasian case subjects from Utah and between 165 and 188 control subjects. Six SNPs showed nominal associations with type 2 diabetes (P = 0.001-0.04), including the nonsynonymous SNP rs1058405 (M67V) in exon 3 and rs11579627 in the 3' flanking region. Only rs1159627 remained significant on permutation testing. The associations were not replicated in 353 African-American case subjects and 182 control subjects, nor were ATF6 SNPs associated with altered insulin secretion or insulin sensitivity in nondiabetic Caucasian individuals. No association with type 2 diabetes was found in a subset of 44 SNPs in Caucasian (n = 2,099), Pima Indian (n = 293), and Chinese (n = 287) samples. Allelic expression imbalance was found in transformed lymphocyte cDNA for 3' untranslated region variants, thus suggesting cis-acting regulatory variants. ATF6 does not appear to play a major role in type 2 diabetes, but further work is required to identify the cause of the allelic expression imbalance.

Angiopoietin: a TIE(d) balance in tumor angiogenesis.

Angiopoietins (ANG-1 and ANG-2) and their TIE-2 receptor tyrosine kinase have wide-ranging effects on tumor malignancy that includes angiogenesis, inflammation, and vascular extravasation. These multifaceted pathways present a valuable opportunity in developing novel inhibition strategies for cancer treatment. However, the regulatory role of ANG-1 and ANG-2 in tumor angiogenesis remains controversial. There is a complex interplay between complementary yet conflicting roles of both the ANGs in shaping the outcome of angiogenesis. Embryonic vascular development suggests that ANG-1 is crucial in engaging interaction between endothelial and perivascular cells. However, recruitment of perivascular cells by ANG-1 has recently been implicated in its antiangiogenic effect on tumor growth. It is becoming clear that TIE-2 signaling may function in a paracrine and autocrine manner directly on tumor cells because the receptor has been increasingly found in tumor cells. In addition, alpha(5)beta(1) and alpha(v)beta(5) integrins were recently recognized as functional receptors for ANG-1 and ANG-2. Therefore, both the ligands may have wide-ranging functions in cellular activities that affect overall tumor development. Collectively, these TIE-2-dependent and TIE-2-independent activities may account for the conflicting findings of ANG-1 and ANG-2 in tumor angiogenesis. These uncertainties have impeded development of a clear strategy to target this important angiogenic pathway. A better understanding of the molecular basis of ANG-1 and ANG-2 activity in the pathophysiologic regulation of angiogenesis may set the stage for novel therapy targeting this pathway.

Aryl hydrocarbon receptor nuclear translocator (ARNT) gene as a positional and functional candidate for type 2 diabetes and prediabetic intermediate traits: Mutation detection, case-control studies, and gene expression analysis.

BACKGROUND: ARNT, a member of the basic helix-loop-helix family of transcription factors, is located on human chromosome 1q21-q24, a region which showed well replicated linkage to type 2 diabetes. We hypothesized that common polymorphisms in the ARNT gene might increase the susceptibility to type 2 diabetes through impaired glucose-stimulated insulin secretion. METHODS: We selected 9 single nucleotide polymorphisms to tag common variation across the ARNT gene. Additionally we searched for novel variants in functional coding domains in European American and African American samples. Case-control studies were performed in 191 European American individuals with type 2 diabetes and 187 nondiabetic European American control individuals, and in 372 African American individuals with type 2 diabetes and 194 African American control individuals. Metabolic effects of ARNT variants were examined in 122 members of 26 European American families from Utah and in 225 unrelated individuals from Arkansas. Gene expression was tested in 8 sibling pairs discordant for type 2 diabetes. RESULTS: No nonsynonymous variants or novel polymorphisms were identified. No SNP was associated with type 2 diabetes in either African Americans or European Americans, but among nondiabetic European American individuals, ARNT SNPs rs188970 and rs11204735 were associated with acute insulin response (AIRg; p = or < 0.005). SNP rs2134688 interacted with body mass index to alter beta-cell compensation to insulin resistance (disposition index; p = 0.004). No significant difference in ARNT mRNA levels was observed in transformed lymphocytes from sibling pairs discordant for type 2 diabetes. CONCLUSION: Common ARNT variants are unlikely to explain the linkage signal on chromosome 1q, but may alter insulin secretion in nondiabetic subjects. Our studies cannot exclude a role for rare variants or variants of small (< 1.6) effect size.

The non-linear and lagged short-term relationship between rainfall and leptospirosis and the intermediate role of floods in the Philippines.

BACKGROUND: Leptospirosis is a worldwide bacterial zoonosis. Outbreaks of leptospirosis after heavy rainfall and flooding have been reported. However, few studies have formally quantified the effect of weather factors on leptospirosis incidence. We estimated the association between rainfall and leptospirosis cases in an urban setting in Manila, the Philippines, and examined the potential intermediate role of floods in this association. METHODS/PRINCIPAL FINDINGS: Relationships between rainfall and the weekly number of hospital admissions due to leptospirosis from 2001 to 2012 were analyzed using a distributed lag non-linear model in a quasi-Poisson regression framework, controlling for seasonally varying factors other than rainfall. The role of floods on the rainfall-leptospirosis relationship was examined using an indicator. We reported relative risks (RRs) by rainfall category based on the flood warning system in the country. The risk of post-rainfall leptospirosis peaked at a lag of 2 weeks (using 0 cm/week rainfall as the reference) with RRs of 1.30 (95% confidence interval: 0.99-1.70), 1.53 (1.12-2.09), 2.45 (1.80-3.33), 4.61 (3.30-6.43), and 13.77 (9.10-20.82) for light, moderate, heavy, intense and torrential rainfall (at 2, 5, 16, 32 and 63 cm/week), respectively. After adjusting for floods, RRs (at a lag of 2 weeks) decreased at higher rainfall levels suggesting that flood is on the causal pathway between rainfall and leptospirosis. CONCLUSIONS: Rainfall was strongly associated with increased hospital admission for leptospirosis at a lag of 2 weeks, and this association was explained in part by floods.

Frequent Community Use of Antibiotics among a Low-Economic Status Population in Manila, the Philippines: A Prospective Assessment Using a Urine Antibiotic Bioassay.

The widespread unregulated use of antibiotics without medical consultation contributes to the burden of antibiotic resistance in Southeast Asian countries. This study investigated antibiotic use before hospital consultation. In a prospective observational study from February 2, 2015, to July 2, 2015, we enrolled febrile patients attending the emergency room in San Lazaro Hospital, Manila, the Philippines. A urine sample was collected and a bioassay was used to detect antibiotic activity in urine using Bacillus stearothermophilus (ATCC7953), Escherichia coli (ATCC25922), and Streptococcus pyogenes (ATCC19615). Patients or caregivers reported their medication history, clinical information, and socioeconomic status. During the study period, 410 patients were enrolled. The median (interquartile range) age was 14 (7-23) years and 158 (39%) reported prior antibiotic use, predominantly a beta-lactam antibiotic. A total of 164 (40%, 95% confidence interval [CI]: 35-45) patients were urine bioassay positive with any of three organisms. The Bacillus assay was the most sensitive, detecting 162 (99%, 95% CI: 96-100) cases. Among bioassay positive patients, dengue (N = 91, 55%, 95% CI: 48-63) was the most frequent diagnosis, followed by other viral infections, including measles, rubella, and mumps (N = 17, 10%, 95% CI: 6-16). Patients with a positive bioassay were significantly more likely to be from the lowest-income group (adjusted odds ratio [AOR]: 1.7; 95% CI: 1.1-2.6) and required hospital admission (AOR: 2.1; 95% CI: 1.3-3.5). Unnecessary antibiotic use for febrile illnesses before hospital consultation is common in a low-income, highly populated urban community in Manila. Education targeting this group should be implemented to reduce unnecessary antibiotic use.

Polymorphisms in the glucokinase-associated, dual-specificity phosphatase 12 (DUSP12) gene under chromosome 1q21 linkage peak are associated with type 2 diabetes.

Linkage of type 2 diabetes to chromosome 1q21-q23 is well replicated across populations. In an initial 50-kb marker map (580 markers) across the linked region, one of the two strongest associations observed in Utah Caucasians was at marker rs1503814 (P < 0.00001 in pools, P < 0.004 in individuals). Based on this association, we typed additional markers and screened for sequence variation in the nearby DUSP12 gene. The strongest associations mapped to a highly conserved nongenic sequence just telomeric to rs1503814 and extended 10 kb telomeric through the DUSP12 gene and into the 5' end of the adjacent ATF6 gene. No coding variant could explain the association in the DUSP12 gene. An extended haplotype encompassing markers from -8,379 to +10,309 bp relative to the ATG start was more common in Caucasian case (0.381) than control subjects (0.285, P = 0.005) and was uniquely tagged by a 194-bp allele at either of two simple tandem repeat variants or by the T allele at marker +7,580. Markers -8,379 and +7,580 were nominally associated with type 2 diabetes in African-American subjects (P < 0.05), but with different alleles. Marker rs1503814 was strongly associated with postchallenge insulin levels among family members (P = 0.000002), but sequence variation in this region was not associated with type 2 diabetes in three other populations of European ancestry. Our data suggest that sequences in or upstream of DUSP12 may contribute to type 2 diabetes susceptibility, but the lack of replication suggests a small effect size.

Effects of four decades of fire manipulation on woody vegetation structure in Savanna.

The amount of carbon stored in savannas represents a significant uncertainty in global carbon budgets, primarily because fire causes actual biomass to differ from potential biomass. We analyzed the structural response of woody plants to long-term experimental burning in savannas. The experiment uses a randomized block design to examine fire exclusion and the season and frequency of burn in 192 7-ha experimental plots located in four different savanna ecosystems. Although previous studies would lead us to expect tree density to respond to the fire regime, our results, obtained from four different savanna ecosystems, suggest that the density of woody individuals was unresponsive to fire. The relative dominance of small trees was, however, highly responsive to fire regime. The observed shift in the structure of tree populations has potentially large impacts on the carbon balance. However, the response of tree biomass to fire of the different savannas studied were different, making it difficult to generalize about the extent to which fire can be used to manipulate carbon sequestration in savannas. This study provides evidence that savannas are demographically resilient to fire, but structurally responsive.

Contribution of the Microenvironmental Niche to Glioblastoma Heterogeneity.

Glioblastoma is the most aggressive cancer of the brain. The dismal prognosis is largely attributed to the heterogeneous nature of the tumor, which in addition to intrinsic molecular and genetic changes is also influenced by the microenvironmental niche in which the glioma cells reside. The cancer stem cells (CSCs) hypothesis suggests that all cancers arise from CSCs that possess the ability to self-renew and initiate tumor formation. CSCs reside in specialized niches where interaction with the microenvironment regulates their stem cell behavior. The reciprocal interaction between glioma stem cells (GSCs) and cells from the microenvironment, such as endothelial cells, immune cells, and other parenchymal cells, may also promote angiogenesis, invasion, proliferation, and stemness of the GSCs and be likely to have an underappreciated role in their responsiveness to therapy. This crosstalk may also promote molecular transition of GSCs. Hence the inherent plasticity of GSCs can be seen as an adaptive response, changing according to the signaling cue from the niche. Given the association of GSCs with tumor recurrence and treatment sensitivity, understanding this bidirectional crosstalk between GSCs and its niche may provide a framework to identify more effective therapeutic targets and improve treatment outcome.

Clinical utility of hypo- and hyperpigmentation of skin in diffuse cutaneous systemic sclerosis.

AIM: Cutaneous involvement is an early manifestation of systemic sclerosis (SSc). Localized areas of 'salt and pepper skin' (S&P) may develop. We hypothesize that S&P skin occurs frequently in diffuse cutaneous (dc) SSc which can be used in its early diagnosis and may correlate with joint contractures. METHODS: Sixty-five patients were recruited for this study. The demographic profiles of SSc were ascertained from hospital records. These patients fulfilled the 2013 American College of Rheumatology/European League Against Rheumatism classification criteria. Patients were examined for skin pigmentary changes, modified Rodnan skin score (mRSS), telengiectasias, calcinosis, arthritis and joint contractures and pruritus. RESULTS: Sixty-five patients (59 female) were recruited with median age of 62.87 years. Forty-four had limited cutaneous SSc, 16 dcSSc, five had scleroderma overlap syndrome. Multivariate stepwise logistic regression indicated that mRSS severity and the presence of contractures were independently (P < 0.05) associated with dcSSc. The strong positive association between S&P and mRSS severity may explain the non-significance of S&P in this analysis. If mRSS severity is not included in the logistic regression analysis, the presence of contractures and S&P (odds ratio = 15.1) show significant (P < 0.01) independent associations with the dcSSc subtype. S&P skin and pruritus were similar in patients with Scl-70 and anti-RNA polymerase antibodies. Anti-centromere antibodies were negatively associated with S&P (χ2 = 7.89, P = 0.005). CONCLUSION: Our study demonstrates strong association of S&P skin with dcSSc (69%), increased risk of pruritus and contractures. Its presence can be used as another clinical tool to diagnose dcSSc in early stages. Observing for S&P skin changes does not require much training.

Influence of bidentate ligand donor types on the formation and stability in 2 + 1 fac-[MI(CO)3]+ (M = Re, 99mTc) complexes.

In the last two decades, a number of chelate strategies have been proposed for the fac-[MI(CO)3]+ (M = Re, 99mTc) core in radiopharmaceutical applications. However, the development of new ligands/complexes with improved function and in vivo performance has been limited in recent years. Expanding on our previous studies using the 2 + 1 labeling strategy, a series of bidentate ligands (neutral vs. anionic) containing an aromatic amine in combination with monodentate pyridine analogs or imidazole were explored to determine the influence of the bidentate and monodentate ligands on the formation and stability of the respective complexes. The 2 + 1 complexes with Re and 99mTc were synthesized in two steps and characterized by standard radio/chemical methods. X-ray characterization and density functional theory analysis of the Re 2 + 1 complexes with the complete bidentate series with 4-dimethylaminopyridine were conducted, indicating enhanced ligand binding energies of the neutral over anionic ligands. In the 99mTc studies, anionic bidentate ligands had significantly higher formation yields of the 2 + 1 product, but neutral ligands appear to have increased stability in an amino acid challenge assay. Both bidentate series exhibited improved stability by increasing the basicity of the pyridine ligands.