A Rapid Method to Characterize Mouse IgG Antibodies and Isolate Native Antigen Binding IgG B Cell Hybridomas

B cell hybridomas are an important source of monoclonal antibodies. In this paper, we developed a high-throughput method to characterize mouse IgG antibodies using surface plasmon resonance technology. This assay rapidly determines their sub-isotypes, whether they bind native antigen and their approximate affinities for the antigen using only 50 μl of hybridoma cell culture supernatant. Moreover, we found that mouse hybridomas secreting IgG antibodies also have membrane form IgG expression without Igα. Based on this surface IgG, we used flow cytometry to isolate rare γ2a isotype switched variants from a γ2b antibody secreting hybridoma cell line. Also, we used fluorescent antigen to single cell sort antigen binding hybridoma cells from bulk mixture of fused hybridoma cells instead of the traditional multi-microwell plate screening and limiting dilution sub-cloning thus saving time and labor. The IgG monoclonal antibodies specific for the native antigen identified with these methods are suitable for in vivo therapeutic uses, but also for sandwich ELISA assays, histology, flow cytometry, immune precipitation and x-ray crystallography.     

Patterns of embryonic cell secretion with special reference to double yolk function during early development of the starfish Pisaster ochraceus

The patterns of secretion utilized by embryonic cells during early development in the starfish Pisaster ochraceus were studied by transmission electron microscopy and morphometry. In addition to exocytosis of cortical granules, exocytosis and micro-apocrine secretion-like blebbing were performed by secretory vacuoles and secretory vesicles. In 2-cell and 4-cell embryos, as well the 22-hour blastula, secretory vacuole exocytosis (VAE) was the most frequent of the secretory types. In the early to middle gastrula, VAE declined and secretory vacuole blebbing (VAB) appeared. Both VAE and VAB almost disappeared in 5-day gastrulae, and secretory vesicle exocytosis (VEE) as well as the secretory vesicle blebbing (VEB) became dominant. VEB was the only mechanism of secretion in bipinnaria. With regard to yolk granules, Y1, Y2, and Y3 granules underwent lysosome-induced utilization (LIU). In addition, Y3 yolk underwent lysosome-induced sparseness (LIS), followed by Y3-Y5B, the pathway that assumes the formation of Y4 and Y5 intermediate yolk patterns and is completed by the blebbing of Y5 granules in the early to middle gastrulae and 5-day gastrulae. These findings demonstrated the high complexity of the embryonic secretion machinery in sea stars.     

An appraisal of the fossil record for the Cirolanidae (Malacostraca: Peracarida: Isopoda: Cymothoida), with a description of a new cirolanid isopod crustacean from the early Miocene of the Vienna Basin (Western Carpathians)

Abstract:  Isopod crustaceans are rarely preserved in the fossil record. Herein, an appraisal of the fossil record for the cirolanid isopods is presented. Five genera are briefly discussed, including Bathynomus, Brunnaega, Palaega, Pseudopalaega and Cirolana. A key for the cirolanid genera known to date from the fossil record is provided based mostly on pleotelson characters. From the early Miocene of the Slovak part of the Vienna Basin, Cirolana feldmanni sp. nov. is described being only the fifth fossil Cirolana species known to date and one of the few with preserved appendages. The material exhibits preservation suggesting biphasic moulting; the mode of preservation suggests a rather short time between shedding the posterior and anterior parts of the exoskeleton instead of hours or even days known in extant taxa. As no subsequent transport or physical disturbance was inferred, the specimens can be stated as in situ preservation. From the palaeoecological point of view, it is concluded that Cirolana feldmanni sp. nov. is the first unequivocal fossil deep‐water Cirolana as suggested by the accompanied fauna.     

Putting Brain Training to the Test in the Workplace: A Randomized,Blinded, Multisite,Active-Controlled Trial

Background

Cognitive training (CT) is effective at improving cognitive outcomes in children with and without clinical impairment as well as older individuals. Yet whether CT is of any preventative health benefit to working age adults is controversial. Our objective was therefore to investigate the real-world efficacy of CT in the workplace, involving employees from across the working-age spectrum and addressing many of the design issues that have limited trials to date.

Methods and Findings

135 white collar employees of a large Australian public sector organization were randomised to either 16 weeks (20 minutes three times per week) of online CT or an active control (AC) program of equal length and structure. Cognitive, wellbeing and productivity outcome measures were analysed across three timepoints: baseline, immediately after training and 6 months post-training. CT effects on cognitive outcomes were limited, even after planned subgroup analyses of cognitive capacity and age. Unexpectedly, we found that our AC condition, which comprised viewing short documentaries about the natural world, had more impact. Compared to the CT group, 6 months after the end of training, those in the AC group experienced a significant increase in their self-reported Quality of Life (Effect Size g = .34 vs −.15; TIME×GROUP p = .003), decrease in stress levels (g = .22 vs −.19; TIME x GROUP p = .03), and overall improvement in Psychological Wellbeing (g = .32 vs −.06; TIME×GROUP p = .02).

Conclusions

CT does not appear to positively impact cognition or wellbeing amongst white collar office workers; however, short time-out respite activities may have value in the promotion of psychological wellbeing. Given looming challenges to workplace productivity, further work-based interventional research targeting employee mental health is recommended.

Trial Registration

This trial was registered with the Australian New Zealand Clinical Trials Registry: ACTRN12610000604000 (http://www.anzctr.org.au/TrialSearch.aspx).     

The Association between Pulse Wave Velocity and Cognitive Function: The Sydney Memory and Ageing Study

Objectives

Pulse wave velocity (PWV) is a measure of arterial stiffness and its increase with ageing has been associated with damage to cerebral microvessels and cognitive impairment. This study examined the relationship between carotid-femoral PWV and specific domains of cognitive function in a non-demented elderly sample.

Method

Data were drawn from the Sydney Memory and Ageing Study, a cohort study of non-demented community-dwelling individuals aged 70–90 years, assessed in successive waves two years apart. In Wave 2, PWV and cognitive function were measured in 319 participants. Linear regression was used to analyse the cross-sectional relationship between arterial stiffness and cognitive function in the whole sample, and separately for men and women. Analysis of covariance was used to assess potential differences in cognition between subjects with PWV measurements in the top and bottom tertiles of the cohort. Covariates were age, education, body mass index, pulse rate, systolic blood pressure, cholesterol, depression, alcohol, smoking, hormone replacement therapy, apolipoprotein E ε4 genotype, use of anti-hypertensive medications, history of stroke, transient ischemic attack, myocardial infarction, angina, diabetes, and also sex for the whole sample analyses.

Results

There was no association between PWV and cognition after Bonferroni correction for multiple testing. When examining this association for males and females separately, an association was found in males, with higher PWV being associated with lower global cognition and memory, however, a significant difference between PWV and cognition between males and females was not found.

Conclusion

A higher level of PWV was not associated with lower cognitive function in the whole sample.     

Recruitment, genetic counseling, and BRCA testing for underserved women at a public hospital

Genetic counseling and testing for heritable susceptibility to breast cancer caused by mutations in BRCA genes are largely unavailable to underserved women in the United States. Starting in 2002 the UCSF Cancer Risk Program offered this service free of charge to poor and medically indigent women at San Francisco General Hospital (SFGH). One recruitment strategy was a single-page questionnaire in four languages administered to women waiting for mammograms at SFGH. This report analyzes our first 3 years of experience with the recruitment questionnaire and compares the patient demographics and BRCA test results at SFGH with a more typical population undergoing genetic counseling and testing at UCSF's Mt. Zion Hospital (MZH). To our knowledge this is the first comprehensive clinical service for hereditary breast cancer in a U.S. public hospital. The ethnic mix of all 350 patients counseled was Caucasian 49% (approximately 20% of Caucasians reported Ashkenazi Jewish ancestry), Latina, 26%; African American, 13%; and Asian/other, 12%. Compared to the MZH population, SFGH patients were more ethnically diverse, less educated and more likely to be unemployed. Of 72 patients tested for BRCA mutations, 51 (71%) were negative, 5 were BRCA1 positive, and 12 were BRCA2 positive. Four (1 Caucasian, 1 Latina, 2 African American) had a total of 13 BRCA variants of unknown significance (VUS). The ratio of BRCA1/BRCA2 positive SFGH patients (5/12) was reversed compared to MZH (119/91). We evaluated 4573 recruitment questionnaires and 280 (6%) were judged to represent a high risk of heritable cancer. After additional screening and referral negotiation, 74 were scheduled for counseling. We judged the recruitment questionnaire to be a feasible, efficient, and reasonably cost-effective way to identify women at high risk of hereditary cancer in a traditionally underserved population. Underserved populations present special challenges for genetic counselors because of large, geographically dispersed families, cultural taboos about cancer diagnoses, and social marginalization. Despite these complexities, the clinical service at SFGH has been well accepted by patients and staff. Our successful venture can serve as a model for other public hospitals contemplating this clinical service.     

Towards an evolutionary ecology of life in soil

The soil-microbe system is one of the most diverse components of the terrestrial ecosystem. The origin of this diversity, and its relation to the life-sustaining processes that are mediated by the resident microbial community, is still poorly understood. The inherent complexities necessitate a theoretical framework that integrates ecological and evolutionary approaches and which embraces the physical heterogeneity of the soil environment. Such a framework is currently lacking, although recent advances in theory and experimentation are beginning to identify the essential ingredients. Here, we review and evaluate the relevance of current modelling approaches, and propose a new synthesis of an evolutionary ecology of life in soil. Key elements include an account of dispersal, horizontal gene transfer, and the consideration of the physical and biological components of soil as an integrated complex adaptive system.     

Survey of zona pellucida antigens for immunocontraception of cats

The purpose of this study was to screen a panel of native zona pellucida (ZP) antigens isolated from five mammalian species for immunocontraceptive activity in the cat (Felis catus). Native soluble-isolated ZP (SIZP) was prepared from the ovaries of cows (bZP), cats (fZP), ferrets (feZP), dogs (cZP), and mink (mZP). Vaccines were constructed using SIZP from each of the above species encapsulated in liposomes suspended in saline and emulsified with Freund's complete adjuvant (SpayVac). Female cats were immunized once (n = 3 cats per group). Serum was collected for determination of antibody titers against SIZP and for binding of antibodies to feline ovaries. All cats responded to immunization by producing anti-SIZP antibodies. The most immunogenic SIZP in cats was from mink, followed by feZP, cZP, and fZP in descending order. Antibodies had low reactivity for fZP, and no reactivity against feline ovaries was detected by immunohistochemistry. A breeding trial was commenced 20 weeks after immunization. All cats became pregnant, averaging 4.1 +/- 0.7 viable kittens per litter. We have previously shown that porcine SIZP is not an effective antigen for immunocontraception of cats. In this study, SIZP from five other mammalian species were immunogenic in the cat, but ZP antibodies failed to bind to fZP in situ, and fertility was not impeded.     

Characterization of Bison bison major histocompatibility complex class IIa haplotypes

American bison (Bison bison) and domestic cattle (Bos taurus and Bos indicus) evolved from a common ancestor 1–1.4 million years ago. Nevertheless, they show dramatic differences in their susceptibility to infectious diseases, including malignant catarrhal fever (MCF). Although bison are highly susceptible to ovine herpesvirus-2 (OvHV-2) associated MCF, about 20% of healthy domesticated and wild bison are positive for OvHV-2 antibody. We are interested in testing the hypothesis that, within the bison population, the polymorphism of major histocompatibility complex (MHC) class II genes influences resistance to MCF. However, since little was known about the MHC class II genes of bison, it was necessary to first characterize class II haplotypes present in Bi. bison (Bibi). Thus, the MHC class II haplotypes carried by 14 bison were characterized by the PCR-based cloning and sequencing of their DRB3, DQA, and DQB alleles. Twelve MHC class II haplotypes were identified in the 14 bison. These haplotypes comprised six previously reported and six new Bibi-DRB3 alleles, along with 11 Bibi-DQA and 10 Bibi-DQB alleles. For each bison class II allele, it was possible to identify closely related cattle sequences. The closest bison and bovine DQA, DQB, and DRB3 alleles, on average, differed by only 1.3, 3.5, and 5.8 amino acids, respectively. Furthermore, bison MHC haplotypes with both nonduplicated and duplicated DQ genes were identified; these haplotypes appear to have originated from the same ancestral haplotypes as orthologous cattle haplotypes. This study was supported by USDA-Agricultural Research Service grant CWU-5348-32000-018-00D. While working on this project, Dr. Bharat Bhushan was supported by a fellowship from the World-Bank-sponsored National Agricultural Technology Project of the Indian Council of Agricultural Research, Indian Ministry of Agriculture, New Delhi, India     

Ontogeny and regulation of matrix metalloproteinase activity in the zebrafish embryo by in vitro and in vivo zymography

Remodeling of the extracellular matrix (ECM) during development, angiogenesis, wound healing, tumor metastasis, and other morphogenetic processes depends on the exquisitely regulated activities of matrix metalloproteinases (MMPs). Yet very little is known about the activity patterns of these proteases in vivo. We have employed fluorescent MMP-substrates, both in vitro and in vivo, to characterize patterns of MMP activity in the zebrafish embryo. Qualitatively similar patterns of degradation are detected using native Type I or Type IV collagen substrates, suggesting that multiple MMPs are being regulated concomitantly. MMP activity is observed primarily in ECM-rich structures predicted to be undergoing active remodeling, such as the perichordal sheath and somite boundaries. Patterns of Type I and Type IV collagen hydrolysis are similar, but not identical in embryos of any given stage. Conventional gelatin zymography shows MMPs present in embryos as early as 3-somites (11 h) and our in vivo assays detect Type IV collagen degradation at somite boundaries as early as 4-somites (11.5 h). However, we are unable to detect significant in vitro activity using homogenates made from embryos prior to Prim-16 (31 h). Mixed lysate assays demonstrate that this is the result of endogenous inhibitors present in early embryos, suggesting a model of matrix remodeling regulated by spatially heterogeneous MMP inhibition.