Exploring the spatial variation of mercury in the Gulf of St. Lawrence using northern gannets as fish samplers.

Mercury (Hg) is a ubiquitous and pervasive environmental contaminant with detrimental effects on wildlife, which originates from both natural and anthropogenic sources. Its distribution within ecosystems is influenced by various biogeochemical processes, making it crucial to elucidate the factors driving this variability. To explore these factors, we employed an innovative method to use northern gannets (Morus bassanus) as biological samplers of regurgitated fish in the Gulf of St. Lawrence. We assessed fish total Hg (THg) concentrations in relation to their geographical catch location as well as to pertinent biotic and anthropogenic factors. In small fish species, trophic position, calculated from compound-specific stable nitrogen isotopes in amino acids, emerged as the most influential predictor of THg concentrations. For large fish species, THg concentrations were best explained by δ13C, indicating higher concentrations in inshore habitats. No anthropogenic factors, such as pollution, shipping traffic, or coastal development, were significantly related to THg concentrations in fish. Moreover, previously published THg data in mussels sampled nearby were positively linked with THg concentrations in gannet prey, suggesting consistent mercury distribution across trophic levels in the Gulf of St. Lawrence. Our findings point to habitat-dependent variability in THg concentrations across multiple trophic levels. Our study could have many potential uses in the future, including the identification of vulnerability hotspots for fish populations and their predators, or assessing risk factors for seabirds themselves by using biologically relevant prey.

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients.

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or "empty" capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.

Development of Peptide Ligands for Targeted Capture of Host Cell Proteins from Cell Culture Production Harvests.

Capture of host cell proteins (HCPs) from cell culture production harvests is critical to ensure the maximum levels specified by international regulatory bodies of product purity for therapeutic monoclonal antibodies (mAbs). Peptide ligands that selectively target the whole spectrum of the HCPs, while letting the mAb product flow through unbound, are an ideal complement to the affinity-based capture step via Protein A chromatography. In this work, we describe the development of HCP-binding peptide ligands, especially focusing on the steps of (1) peptide selection via library screening and (2) quantification of HCP removal via proteomics by mass spectrometry.

Affibody-Binding Ligands.

While antibodies remain established therapeutic and diagnostic tools, other protein scaffolds are emerging as effective and safer alternatives. Affibodies in particular are a new class of small proteins marketed as bio-analytic reagents. They feature tailorable binding affinity, low immunogenicity, high tissue permeation, and high expression titer in bacterial hosts. This work presents the development of affibody-binding peptides to be utilized as ligands for their purification from bacterial lysates. Affibody-binding candidates were identified by screening a peptide library simultaneously against two model affibodies (anti-immunoglobulin G (IgG) and anti-albumin) with the aim of selecting peptides targeting the conserved domain of affibodies. An ensemble of homologous sequences identified from screening was synthesized on Toyopearl® resin and evaluated via binding studies to select sequences that afford high product binding and recovery. The affibody-peptide interaction was also evaluated by in silico docking, which corroborated the targeting of the conserved domain. Ligand IGKQRI was validated through purification of an anti-ErbB2 affibody from an Escherichia coli lysate. The values of binding capacity (~5 mg affibody per mL of resin), affinity (KD ~1 µM), recovery and purity (64-71% and 86-91%), and resin lifetime (100 cycles) demonstrate that IGKQRI can be employed as ligand in affibody purification processes.

Platelet-rich plasma lysate displays antibiofilm properties and restores antimicrobial activity against synovial fluid biofilms in vitro.

Infectious arthritis is difficult to treat in both human and veterinary clinical practice. Recent literature reports Staphylococcus aureus as well as other gram-positive and gram-negative isolates forming free-floating biofilms in both human and equine synovial fluid that are tolerant to traditional antimicrobial therapy. Using an in vitro equine model, we investigated the ability of platelet-rich plasma (PRP) formulations to combat synovial fluid biofilm aggregates. Synovial fluid was infected, and biofilm aggregates allowed to form over a 2-hour period. PRP was collected and processed into different formulations by platelet concentration, leukocyte presence, and activation or lysis. Infected synovial fluid was treated with different PRP formulations with or without aminoglycoside cotreatment. Bacterial load (colony-forming unit/mL) was determined by serial dilutions and plate counting at 8 hours posttreatment. All PRP formulations displayed antimicrobial properties; however, formulations containing higher concentrations of platelets without leukocytes had increased antimicrobial activity. Lysis of PRP and pooling of the PRP lysate (PRP-L) from multiple horses as compared to individual horses further increased antimicrobial activity. This activity was lost with the removal of the plasma component or inhibition of the proteolytic activity within the plasma. Fractionation of pooled PRP-L identified the bioactive components to be cationic and low-molecular weight (<10 kDa). Overall, PRP-L exhibited synergism with amikacin against aminoglycoside tolerant biofilm aggregates with greater activity against gram-positive bacteria. In conclusion, the use of PRP-L has the potential to augment current antimicrobial treatment regimens which could lead to a decrease in morbidity and mortality associated with infectious arthritis.

Targeted capture of Chinese hamster ovary host cell proteins: Peptide ligand binding by proteomic analysis.

The clearance of host cell proteins (HCPs) is of crucial importance in biomanufacturing, given their diversity in composition, structure, abundance, and occasional structural homology with the product. The current approach to HCP clearance in the manufacturing of monoclonal antibodies (mAbs) relies on product capture with Protein A followed by removal of residual HCPs in flow-through mode using ion exchange or mixed-mode chromatography. Recent studies have highlighted the presence of "problematic HCP" species, which are either difficult to remove (Group I), can degrade the mAb product (Group II), or trigger immunogenic reactions (Group III). To improve the clearance of these species, we developed a family of synthetic peptides that target HCPs and exhibit low binding to IgG product. In this study, these peptides were conjugated onto chromatographic resins and evaluated in terms of HCP clearance and mAb yield, using an industrial mAb-producing CHO harvest as model supernatant. To gather detailed knowledge on the binding of individual HCPs, the unbound fractions were subjected to shotgun proteomic analysis by mass spectrometry. It was found that these peptide ligands exhibit superior HCP binding capability compared to those of the benchmark commercial resins commonly used in mAb purification. In addition, some peptide-based resins resulted in much lower losses of product yield compared to these commercial supports. The proteomic analysis showed effective capture of many "problematic HCPs" by the peptide ligands, especially some that are weakly bound by commercial media. Collectively, these results indicate that these peptides show great promise toward the development of next-generation adsorbents for safer and cost-effective manufacturing of biologics.

Multiplexed Competitive Screening of One-Bead-One-Component Combinatorial Libraries Using a ClonePix 2 Colony Sorter.

Screening solid-phase combinatorial libraries of bioactive compounds against fluorescently labeled target biomolecules is an established technology in ligand and drug discovery. Rarely, however, do screening methods include comprehensive strategies-beyond mere library blocking and competitive screening-to ensure binding selectivity of selected leads. This work presents a method for multiplexed solid-phase peptide library screening using a ClonePix 2 Colony Picker that integrates (i) orthogonal fluorescent labeling for positive selection against a target protein and negative selection against competitor species with (ii) semi-quantitative tracking of target vs. competitor binding for every library bead. The ClonePix 2 technology enables global at-a-glance evaluation and customization of the parameters for bead selection to ensure high affinity and selectivity of the isolated leads. A case study is presented by screening a peptide library against green-labeled human immunoglobulin G (IgG) and red-labeled host cell proteins (HCPs) using ClonePix 2 to select HCP-binding ligands for flow-through chromatography applications. Using this approach, 79 peptide ligand candidates (6.6% of the total number of ligands screened) were identified as potential HCP-selective ligands, enabling a potential rate of >3,000 library beads screened per hour.

Targeted Capture of Chinese Hamster Ovary Host Cell Proteins: Peptide Ligand Discovery.

The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of "problematic" HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography.

"The VIHsibilite Project": HIV-positive people in the Quebec press and community responses.

The VIHsibilite Project is a community-based action-research initiative that examines newspaper coverage of HIV/AIDS issues in Quebec from 1988 to 2004. Using standard qualitative research methods, and in consultation with an advisory committee of people directly impacted by HIV/AIDS news coverage, the project discerns trends in reporting on HIV/AIDS and undertakes discursive content analysis of these, aiming to better understand in what normative ways seropositive people are represented in print media, and, ultimately, to reduce the stigma attendant upon HIV infection. Preliminary findings include indications that seropositive women tend to be represented markedly differently from men in the news.

HIV and ethnicity in Canada: is the HIV risk-taking behaviour of young foreign-born MSM similar to Canadian born MSM?

There is a dearth of information on the HIV risk-taking behaviour of foreign-born men who have sex with men (MSM) in Canada. This study focused on identifying sexual risk behaviour among MSM who immigrated to Canada and compared them to MSM who were born in Canada. Baseline data from the Omega Cohort in Montreal and the Vanguard Project in Vancouver were combined to form four ethnicity/race analytical categories (n = 1,148): White born in Canada (WBIC), White born outside of Canada, non-White born in Canada (NBIC) and non-White born outside of Canada (NBOC). Psychological, demographic and sexual behaviour characteristics of the groups were similar except: NBOC were more likely to be unemployed, less likely to be tattooed, had fewer bisexual experiences and less likely worried of insufficient funds. WBOC were more likely to report unprotected sex with seropositives and more likely to have had unprotected sex while travelling. NBIC were more likely to have ever sold sex and to have had body piercing. WBOC are at high risk of acquiring as well as transmitting HIV. It is important to consider place of birth in addition to ethnicity when developing programmes to prevent the transmission of HIV.

Risk factors associated with HIV infection among young gay and bisexual men in Canada.

OBJECTIVE: To assess risk factors associated with HIV prevalence and incidence among gay and bisexual men in two prospective Canadian cohorts. METHODS: The Vanguard Project and the Omega Cohort are prospective cohort studies of gay and bisexual men ongoing in Vancouver and Montreal, respectively. For this analysis, baseline sociodemographic characteristics, sexual behavior, and substance use data from these two cohorts were combined. Assessment of risk factors for HIV seroprevalence and seroconversion were carried out using univariate and multivariate analysis. RESULTS: This analysis was based on 1373 gay and bisexual men aged 16 to 30 years. Men who were HIV-seropositive at baseline (n = 48) were more likely to report living in unstable housing, to have had less than a high school education, and to have been unemployed than those who were HIV-negative (n = 1325). HIV-positive men were also more likely to report having engaged in sexual risk behavior, including having had consensual sex at a younger age, having had at least 6 partners during the previous year, ever having been involved in the sex trade, and having engaged in unprotected receptive anal intercourse. With respect to substance use, HIV-positive men were more likely to report the use of crack, cocaine, heroin, and marijuana and to use injection drugs. Similarly, men who seroconverted during the course of the studies (n = 26) were more likely to report having less than a high school education and having lived in unstable housing at baseline. Compared with HIV-negative men, men who seroconverted were more likely to report ever having been involved in the sex trade and engaging in unprotected receptive anal intercourse. Reports of cocaine use and injection drug use were also significantly higher for men who seroconverted compared with HIV-negative men. CONCLUSIONS: Our data indicate that HIV-positive gay and bisexual men are more likely to be living in unstable conditions and to report more risky sexual and substance use behaviors than HIV-negative men.

Design and synthesis of a novel class of histone deacetylase inhibitors.

Histone deacetylase inhibitors (HDACs) have emerged as a novel class of antiproliferative agents. Utilizing structure-based design, the synthesis of a series of sulfonamide hydroxamic acids is described. Further optimization of this series by substitution of the terminal aromatic ring yielded HDAC inhibitors with good in vitro and in vivo activities.

Evaluation of the health risk associated with exposure to chloroform in indoor swimming pools.

The exposure of swimmers to chloroform (CHCl3) was investigated in indoor swimming pools of the Quebec City region along with the associated carcinogenic risk. Six training sessions involving 52 competition swimmers (11 to 20 yr old) were conducted in 3 different pools, while 12 adult leisure swimmers attended 5 sessions, each held in a different pool. For each session, water and ambient air CHCl3 concentrations were measured and CHCl3 levels in alveolar air samples (CHCl3 ALV) collected from swimmers prior to entering the swimming pool premises and after 15, 35, and 60 min of swimming. Mean water concentrations varied from 18 microg/L to 80 microg/L, while those in air ranged from 78 microg/m3 to 329 microg/m3. Multiple linear regression analyses revealed that CHCl3 ALV values in competition swimmers were strongly correlated to ambient air and water levels, and to a lesser degree to the intensity of training. Only ambient air concentration was positively correlated to CHCl3 ALV in the leisure group. Concentrations of CHCl3 metabolites bound to hepatic and renal macromolecules, estimated using a physiologically based pharmacokinetic (PBPK) model, were 1.6 and 1.9 times higher for the competition swimmers than for the leisure swimmers, respectively. The highest hepatic concentration predicted in competition swimmers, 0.22 microg CHCl3 equivalents/kg of tissue, was at least 10,000 times lower than the smallest no observed effect level for liver tumors in animals. Data indicate that the safety margin is therefore very large, for competitive swimmers as well as for leisure swimmers.

Risk behaviours and HIV infection among men having sexual relations with men: baseline characteristics of participants in the Omega Cohort Study, Montreal, Quebec, Canada.

OBJECTIVE: To describe characteristics of men having sex with men (MSM) participating in the Omega Cohort, to describe HIV-positive participants at baseline interview, and to estimate HIV incidence. METHODS: The Omega Cohort is a study on the incidence and psychosocial determinants of HIV infection among MSM living in Montreal. MSM complete a questionnaire and are tested for HIV every six months. RESULTS: During the previous six months, 31% and 12% of 810 participants (mean age = 33 years) reported unprotected anal sex with regular and casual partners, respectively. Eight participants (0.98%) were HIV-infected at baseline. HIV incidence was 0.89 per 100 person-years (7/787 person-years) [95% confidence interval: 0.36-1.83]. CONCLUSION: A significant proportion of participants reported current risk behaviours. Despite this, HIV incidence is relatively low. It is important to target MSM who do not practice safe sex and to encourage those practicing safe sex to sustain these behaviours.

Association of hepatitis B virus infection with other sexually transmitted infections in homosexual men. Omega Study Group.

OBJECTIVES: This study determined the prevalence and factors associated with hepatitis B virus (HBV) infection among men who have sex with men. METHODS: At the baseline visit of an HIV study among men who have sex with men, we asked about HBV vaccination status and tested for HBV markers. RESULTS: Of 625 subjects, 48% had received at least 1 dose of HBV vaccine. Of 328 unvaccinated men, 41% had 1 or more HBV markers. HBV prevalence increased markedly with age and was associated with many sexual and drug-related behaviors. In a multivariate model, 7 variables were independently associated with HBV infection: ulcerative sexually transmitted diseases (odds ratio [OR] = 10.1; 95% confidence interval [CI] = 2.6, 54); injection drug use (OR = 5.2; 95% CI = 1.2, 26); gonorrhea or chlamydia (OR = 4.0; 95% CI = 1.9, 8.9); sexual partner with HIV/AIDS (OR = 3.6; 95% CI = 1.8, 7.1); 50 or more casual partners (OR = 3.4; 95% CI = 1.6, 7.1); received money for sex (OR = 3.0; 95% CI = 1.2, 7.8); and 20 or more regular partners (OR = 2.5; 95% CI = 1.1, 6.1). CONCLUSIONS: In Montreal, men who have sex with men are at risk for HBV infection, but a substantial proportion remain unvaccinated; new strategies are required to improve coverage. Men who have sex with men and who have a sexually transmitted infection, especially a genito-ulcerative infection, appear to be at particularly high risk for HBV infection.

Correlates of risky behaviors among young and older men having sexual relations with men in Montréal, Québec, Canada. Omega Study Group.

OBJECTIVE: To assess characteristics associated with 1) age and 2) recent unprotected anal sex with casual partners among men having sexual relations with men (MSM) participating in the Omega Cohort, Montréal, Québec, Canada. METHODS: The Omega Cohort is a study of the incidence and psychosocial determinants of HIV infection among MSM living in Montréal. MSM complete a questionnaire and are tested for HIV every 6 months. RESULTS: Thirteen percent of young MSM (< 30 years of age, n = 355) and 12% of older MSM (> or = 30 years of age, n = 455) reported recent unprotected anal sex with casual partners. The predictors of this latter behavior were: not living with a male sexual partner, unprotected anal sex with regular partner, > 5 casual partners, alcohol/drug use before anal sex, and having difficulties with procedures needed for safe sex. Among young MSM, additional predictors were: to have been living in Montreal for less than 1 year and to have exchanged money for sex. Among older MSM, additional predictors were: female sexual partners, unprotected anal sex with an HIV-infected partner, and feeling invulnerable to AIDS. CONCLUSION: Young Omega participants do not have more risky behaviors than older participants. Some predictors of recent risk behaviors with casual partners were different between the two groups. Prevention programs should be adapted consequently.

Factors associated with hepatitis B vaccination among men having sexual relations with men in Montreal, Quebec, Canada. Omega Study Group.

OBJECTIVE: To determine factors associated with hepatitis B virus (HBV) vaccination status among HIV-uninfected men who have affective and sexual relations with men (MASM) in Montreal, Quebec, Canada. METHODS: The Omega Cohort is a study of the incidence and psychosocial determinants of HIV infection among MASM in Montreal. Participants complete a questionnaire and are HIV-tested every 6 months. At baseline, we also performed testing for HBV markers and collected data on HBV vaccination history. RESULTS: Forty-six percent of 653 participants had received at least one dose of HBV vaccination, whereas 28% were completely vaccinated. Lack of vaccination was associated with injection drug use, having > or =20 regular lifetime partners, living outside Montreal, not having sex in bathhouses, and not having consulted a physician aware of the participant's sexual orientation. Among vaccinated MASM, incomplete vaccination was associated with having <20 lifetime casual partners, trading sex for drugs, having given goods for sex, having had unprotected anal sex with regular partners, and having no history of a previous sexually transmitted disease. CONCLUSION: A significant proportion of Montreal's MASM, some of whom are at risk of contracting HBV through sexual and parenteral transmission, have not been vaccinated for HBV. Men who have affective and sexual relations with men should be educated about the risk of HBV transmission and the seriousness of the disease.

Bioactive isocoumarins and related metabolites from conifer endophytes.

Six new [1-4, 7, 9] and two known [6 and 8] metabolites have been isolated from culture filtrates of conifer endophyte strains of Canoplea elegantula. Their structures have been determined by spectroscopic means. Compound 1 was toxic to spruce budworm cells, and both 1 and 3 were toxic to spruce budworm larvae.

Differential diagnostic dilemmas in malignant fine-needle aspirates of liver: a practical approach to final diagnosis.

We retrospectively reviewed two hundred malignant fine-needle aspirates of liver. Of these 49.5% were metastatic neoplasms, 32% were hepatocellular carcinomas, and 18.5% presented with diagnostic dilemmas. In less than half of these diagnostic challenges, differential diagnosis was between hepatocellular carcinoma and metastatic adenocarcinoma. The remainder of cases involved a variety of metastatic neoplasms. Cytomorphology in association with immunocytochemistry resolved the diagnostic problems in about half of the problematic cases. Three cases were undifferentiated and remained unclassifiable. We conclude that approximately 80% of malignant lesions of liver can be correctly diagnosed by thorough cytomorphologic analysis and good clinical correlation, and 20% pose differential diagnostic dilemmas.

Evaluation of dermal and respiratory chloroform exposure in humans.

Chloroform is a known contaminant of chlorinated drinking water and of swimming pool water disinfected with chlorine or one of its derivatives. Few data exist regarding the importance of dermal and inhalation exposure routes to the chloroform body burden resulting from domestic and recreational use of chlorinated water. In our experimental study involving 11 male swimmers, we quantified the body burden resulting from exposure to various concentrations of chloroform in water and air of an indoor swimming pool, during a daily 55-min exercise period. From the first to the sixth exercise period, CHCl3 mean concentration in water was increased from 159 micrograms/l to 553 micrograms/l. Corresponding mean air CHCl3 level ranged from 597 ppb to 1630 ppb. To dissociate the dermal exposure route from that of inhalation, swimmers used scuba tanks during an additional exercise period. Chloroform concentrations were measured in alveolar air before and after each exercise period, as well as after 35 min of physical activity. Chloroform levels in water and air were measured every 10 min. We examined the relationship between alveolar air concentration (a measure of body burden) at 35 and 55 min and environmental chloroform concentrations by using multiple regression models. The natural logarithm of alveolar air concentration was strongly correlated with aqueous chloroform concentration both at 35 (p2 < 0.001, r2 = 0.75) and 55 min (p < 0.001, r2 = 0.86). The relationship with air concentrations was also statistically significant (35 min: p < 0.001, r2 = 0.58, 55 min: p < 0.001, r2 = 0.63).(ABSTRACT TRUNCATED AT 250 WORDS)