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.

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.

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.

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.

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

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

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.

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](+).

Diagnostic Accuracy of Recombinant Immunoglobulin-like Protein A-Based IgM ELISA for the Early Diagnosis of Leptospirosis in the Philippines.

BACKGROUND: Leptospirosis is an important but largely under-recognized public health problem in the tropics. Establishment of highly sensitive and specific laboratory diagnosis is essential to reveal the magnitude of problem and to improve treatment. This study aimed to evaluate the diagnostic accuracy of a recombinant LigA protein based IgM ELISA during outbreaks in the clinical-setting of a highly endemic country. METHODOLOGY/PRINCIPAL FINDINGS: A prospective study was conducted from October 2011 to September 2013 at a national referral hospital for infectious diseases in Manila, Philippines. Patients who were hospitalized with clinically suspected leptospirosis were enrolled. Plasma and urine were collected on admission and/or at discharge and tested using the LigA-IgM ELISA and a whole cell-based IgM ELISA. Sensitivity and specificity of these tests were evaluated with cases diagnosed by microscopic agglutination test (MAT), culture and LAMP as the composite reference standard and blood bank donors as healthy controls: the mean+3 standard deviation optical density value of healthy controls was used as the cut-off limit (0.062 for the LigA-IgM ELISA and 0.691 for the whole cell-based IgM ELISA). Of 304 patients enrolled in the study, 270 (89.1%) were male and the median age was 30.5 years; 167 (54.9%) were laboratory confirmed. The sensitivity and ROC curve AUC for the LigA-IgM ELISA was significantly greater than the whole cell-based IgM ELISA (69.5% vs. 54.3%, p<0.01; 0.90 vs. 0.82, p<0.01) on admission, but not at discharge. The specificity of LigA-IgM ELISA and whole cell-based IgM ELISA were not significantly different (98% vs. 97%). Among 158 MAT negative patients, 53 and 28 were positive by LigA- and whole cell-based IgM ELISA, respectively; if the laboratory confirmation was re-defined by LigA-IgM ELISA and LAMP, the clinical findings were more characteristic of leptospirosis than the diagnosis based on MAT/culture/LAMP. CONCLUSIONS/SIGNIFICANCE: The newly developed LigA-IgM ELISA is more sensitive than the whole cell-based IgM based ELISA. Although the final diagnosis must be validated by more specific tests, LigA-IgM ELISA could be a useful diagnostic test in a real clinical-setting, where diagnosis is needed in the early phase of infection.

A cross-sectional study of autoantibody profiles in the Waikato systemic sclerosis cohort, New Zealand.

The autoantibody profiles in New Zealand systemic sclerosis patients have not previously been reported. The aim of this study was to evaluate the autoantibody profiles of patients in the Waikato Hospital Systemic Sclerosis Clinic cohort. The EUROLINE (IgG) Systemic Sclerosis panel test kit (which tests for Scl-70, CENP-A, CENP-B, RP11, RP155, Fib, NOR90, Th/To, PM100, PM75, Ku, PDGFR and Ro-52) was selected for the purpose of this study. All patients attending the Waikato Hospital Systemic Sclerosis clinic were invited to participate. These patients were categorised by systemic sclerosis subtypes [1]. Results were compared with previously published data, including the EUSTAR database. Sixty patients (56 female) were recruited, with a median age of 61 years (range 29-81 years). Forty-one had limited cutaneous systemic sclerosis (lcSSc). Of these lcSSc patients, 31 (75.6%) were positive for CENP-A and CENP-B (anti-centromere) antibodies, 12 (29.3%) for Ro-52 antibodies, 5 (12.2%) for RP11 and RP155, 4 (9.8%) for Scl-70 and 1 (2.4%) each for anti-Fib and Th/To antibodies. Fifteen patients had diffuse cutaneous systemic sclerosis (dcSSc), of which 7 patients (47.6%) were positive for RP11 and RP155, 4 (26.7%) for Scl-70. Three dcSSc patients did not have either of these two major antibodies, but of these 15 dcSSc patients, 4 patients (26.7%) were positive also for Ro-52, 2 (13.3%) for anti-Ku, and 1 (6.7%) each for anti-Fib and NOR90. Four patients had overlap syndrome (OLS), 1 had CENP-A and CENP-B antibodies, 1 had Ro-52 autoantibodies 1 had anti-Ku antibodies. Three patients had no autoantibodies. This is the first study to look at the autoantibody profile of SSc patients in New Zealand. A higher prevalence of antibodies against centromere and RNA polymerase III was demonstrated in our group compared with the EUSTAR database suggesting that antibody prevalence may vary geographically.

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.

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.

Sleep active cortical neurons expressing neuronal nitric oxide synthase are active after both acute sleep deprivation and chronic sleep restriction.

Non-rapid eye movement (NREM) sleep electroencephalographic (EEG) delta power (~0.5-4 Hz), also known as slow wave activity (SWA), is typically enhanced after acute sleep deprivation (SD) but not after chronic sleep restriction (CSR). Recently, sleep-active cortical neurons expressing neuronal nitric oxide synthase (nNOS) were identified and associated with enhanced SWA after short acute bouts of SD (i.e., 6h). However, the relationship between cortical nNOS neuronal activity and SWA during CSR is unknown. We compared the activity of cortical neurons expressing nNOS (via c-Fos and nNOS immuno-reactivity, respectively) and sleep in rats in three conditions: (1) after 18-h of acute SD; (2) after five consecutive days of sleep restriction (SR) (18-h SD per day with 6h ad libitum sleep opportunity per day); (3) and time-of-day matched ad libitum sleep controls. Cortical nNOS neuronal activity was enhanced during sleep after both 18-h SD and 5 days of SR treatments compared to control treatments. SWA and NREM sleep delta energy (the product of NREM sleep duration and SWA) were positively correlated with enhanced cortical nNOS neuronal activity after 18-h SD but not 5days of SR. That neurons expressing nNOS were active after longer amounts of acute SD (18h vs. 6h reported in the literature) and were correlated with SWA further suggest that these cells might regulate SWA. However, since these neurons were active after CSR when SWA was not enhanced, these findings suggest that mechanisms downstream of their activation are altered during CSR.

Endothelial microparticles: missing link in endothelial dysfunction?

Vascular homeostasis is an integral reflection of quiescent, but competent endothelium. Endothelial microparticles (EMPs) are circulating submicron-sized membranous vesicles released by endothelium that are increasingly recognized and play a multitude of biological roles reflecting competency of endothelial function and vascular biology. EMPs have been shown to act as primary and secondary messengers of vascular inflammation, thrombosis, vasomotor response, angiogenesis, and endothelial survival. These often-neglected vesicles are emerging as potentially useful indicators of dysfunctioning endothelium, whereby differential presence of EMPs is linked to disease manifestation. In this review, we outline the evolving understanding of EMPs from mere passive vesiculation to active messenger in pathophysiological responses. Current evidence implicating EMPs in cardiopulmonary, renal, cerebral, and metabolic disorders warrants vigorous clinical validation. While studies of EMPs face significant technical challenges that await standardization and consolidation, unravelling its biological significance in endothelial dysfunction could herald the advent of novel non-invasive surveillance platforms for vascular health, risk stratification, and disease prognostication.

Impact of prandial status on the comparison of capillary glucose meter and venous plasma glucose measurements in healthy volunteers.

BACKGROUND: There is a negative glucose gradient between the capillary and venous systems, produced by glucose uptake into peripheral tissues. This gradient is augmented by oral glucose ingestion in healthy volunteers; thus prandial status may impact on capillary glucose meter performance. Our primary aim was to investigate whether the (capillary-venous plasma) glucose difference changed in relation to prandial status, in healthy volunteers. METHODS: Glucose was measured fasting and also one hour after an ad libitum breakfast, in 103 healthy volunteers. Duplicate capillary (finger stick) measurements were undertaken at both time points, using both the FreeStyle Lite and AccuChek Performa meters. Simultaneous venous (antecubital fossa) samples were centrifuged immediately after collection and plasma glucose was measured using the laboratory hexokinase method. Results were compared by Bland-Altman difference analysis. RESULTS: The mean (95% CI) pre- and postprandial (capillary-plasma) glucose differences (mmol/L) were calculated for each meter. For the Freestyle Lite, the preprandial difference was -0.51 (-0.58 to -0.45) and postprandial difference was 0.81 (0.69-0.94). Corresponding differences for the Performa were -0.13 (-0.20 to -0.06) and 1.19 (1.07-1.31), respectively. T-test comparison of participants' paired pre- and postprandial (capillary-plasma) glucose differences confirmed a significant meal-related change in glucose estimation for both meters (P < 0.0001). Also, both meters read highest at lower glucose concentrations. CONCLUSIONS: In healthy volunteers, both glucose meters showed a systematic positive bias one hour after breakfast. The significance of this finding in diabetes remains to be determined.

Different expression of large-conductance calcium-activated K+ channels in human internal mammary and radial arteries.

AIMS: In this study, we investigated and compared the electrophysiological and molecular properties of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels between human internal mammary arteries (IMA) and radial arteries (RA). METHODS AND RESULTS: IMA and RA sections were obtained from 79 patients (including 9 females) undergoing coronary artery bypass graft surgery. We examined the effects of K(+) channel blockers tetraethylammonium (TEA), iberiotoxin (IBTX), and 4-aminopyridine (4-AP) on isolated smooth muscle cells (SMCs) using patch clamping. Both TEA (1 mM) and IBTX (0.1 µM) significantly decreased K(+) currents in IMA SMCs and RA SMCs, while 4-AP (1 mM) only had a weak effect. IBTX had a greater K(+)-blocking effect on IMA SMCs than on RA SMCs. Consistently, TEA and IBTX evoked significant constriction of both intact vascular rings. IBTX had a greater constrictor effect on IMA rings (18.5 ± 6.7%, n= 8) than on RA rings (10.6 ± 3.1%, n= 8), P< 0.05. RT-PCR and western blot analysis demonstrated that gene and protein expression of the α-subunit of BK(Ca) channels from IMA was greater than that from RA. CONCLUSION: The density of BK(Ca) channels is greater in human IMA than in RA resulting in greater BK(Ca) currents in SMCs from IMA. This may partly explain the finding of less spasm in IMA grafts than in RA grafts. Our results may be of value in determining the best anti-spasm agent to use peri-operatively.

Laparoscopic pancreaticoduodenectomy procedure: Pushing the limits of laparoscopy

Whipple operation, or open pancreaticoduodenectomy, is perhaps one of the most technically challenging operations in the abdomen. The overall perioperative mortality rate is about 2% to 5% and with operative morbidity of about 25% to 45%. In the past, minimally invasive techniques were only used for laparoscopy, staging of pancreatic cancer, and palliative procedures for unresectable pancreatic cancer. Recent advances in operative techniques and instrumentation have empowered laparoscopic surgeons to perform virtually all procedures in the pancreas, including the Whipple procedure. Presented is the first case of laparoscopic pancreaticoduodenectomy. Key words: laparoscopy, laparoscopic pancreaticoduodenectomy