Investigating molecular recognition and biological function at interfaces using piscidins, antimicrobial peptides from fish

We studied amidated and non-amidated piscidins 1 and 3, amphipathic cationic antimicrobial peptides from fish, to characterize functional and structural similarities and differences between these peptides and better understand the structural motifs involved in biological activity and functional diversity among amidated and non-amidated isoforms. Antimicrobial and hemolytic assays were carried out to assess their potency and toxicity, respectively. Site-specific high-resolution solid-state NMR orientational restraints were obtained from (15)N-labeled amidated and non-amidated piscidins 1 and 3 in the presence of hydrated oriented lipid bilayers. Solid-state NMR and circular dichroism results indicate that the peptides are alpha-helical and oriented parallel to the membrane surface. This orientation was expected since peptide-lipid interactions are enhanced at the water-bilayer interface for amphipathic cationic antimicrobial peptides. (15)N solid-state NMR performed on oriented samples demonstrate that piscidin experiences fast, large amplitude backbone motions around an axis parallel to the bilayer normal. Under the conditions tested here, piscidin 1 was confirmed to be more antimicrobially potent than piscidin 3 and antimicrobial activity was not affected by amidation. In light of functional and structural similarities between piscidins 1 and 3, we propose that their topology and fast dynamics are related to their mechanism of action.     

Chronic variable stress in fathers alters paternal and social behavior but not pup development in the biparental California mouse (Peromyscus californicus)

Stress and chronically elevated glucocorticoid levels have been shown to disrupt parental behavior in mothers; however, almost no studies have investigated corresponding effects in fathers. The present experiment tested the hypothesis that chronic variable stress inhibits paternal behavior and consequently alters pup development in the monogamous, biparental California mouse (Peromyscus californicus). First-time fathers were assigned to one of three experimental groups: chronic variable stress (CVS, n = 8), separation control (SC, n = 7), or unmanipulated control (UC, n = 8). The CVS paradigm (3 stressors per day for 7 days) successfully stressed mice, as evidenced by increased baseline plasma corticosterone concentrations, increased adrenal mass, decreased thymus mass, and a decrease in body mass over time. CVS altered paternal and social behavior of fathers, but major differences were observed only on day 6 of the 7-day paradigm. At that time point, CVS fathers spent less time with their pairmate and pups, and more time autogrooming, as compared to UC fathers; SC fathers spent more time behaving paternally and grooming the female mate than CVS and UC fathers. Thus, CVS blocked the separation-induced increase in social behaviors observed in the SC fathers. Nonetheless, chronic stress in fathers did not appear to alter survival or development of their offspring: pups from the three experimental conditions did not differ in body mass gain over time, in the day of eye opening, or in basal or post-stress corticosterone levels. These results demonstrate that chronic stress can transiently disrupt paternal and social behavior in P. californicus fathers, but does not alter pup development or survival under controlled, non-challenging laboratory conditions.     

Acute effects of corticosterone injection on paternal behavior in California mouse (Peromyscus californicus) fathers

Glucocorticoids are thought to mediate the disruption of parental behavior in response to acute and chronic stress. Previous research supports their role in chronic stress; however, no study has experimentally tested the effects of acute glucocorticoid elevation on paternal behavior. We tested the prediction that acute corticosterone (CORT) increases would decrease paternal behavior in California mouse fathers and would lead to longer-term effects on reproductive success, as even short-term increases in CORT have been shown to produce lasting effects on the hypothalamic-pituitary-adrenal axis. First-time fathers were injected with 30 mg/kg CORT, 60 mg/kg CORT or vehicle, or left unmanipulated. Interactions between the male and its pup(s) were recorded 1.5–2 h after injection and scored for paternal and non-paternal behavior. Treatment groups were combined into control (unmanipulated + vehicle, n = 15) and CORT (30 mg/kg + 60 mg/kg, n = 16) for analysis based on resulting plasma CORT concentrations. CORT treatment did not alter paternal or non-paternal behaviors or any long-term measures (male body mass or temperature, pup growth rate, pup survival, interbirth interval, number or mass of pups born in the second litter). Fathers showed a significant rise in body mass at day 30 postpartum, followed by a decrease in body mass after the birth of the second litter; however, this pattern did not differ between the CORT and control groups. In summary, acute elevation of plasma CORT did not alter direct paternal behavior, body mass, or reproductive outcomes, suggesting that acute CORT elevation alone does not overtly disrupt paternal care in this biparental mammal.     

Effective temperature shift strategy development and scale confirmation for simultaneous optimization of protein productivity and quality in Chinese hamster ovary cells

Temperature shifts to lower culture temperatures are frequently employed in the manufacturing of protein therapeutics in mammalian cells to improve productivity, viability, or quality attributes. The direction and extent to which a temperature shift affects productivity and quality may vary depending on the expression host and characteristics of the expressed protein. We demonstrated here that two Chinese hamster ovary (CHO) clones expressing different human monoclonal antibodies responded differently to a temperature shift despite sharing a common parental CHO cell line. Within a single CHO line, we observed a nonlinear response to temperature shift. A moderate shift to 35°C significantly decreased final titer relative to the unshifted control while a larger shift to 32°C significantly increased final titer by 25%. Therefore, we proposed a systematic empirical approach to assess the utility of a temperature shift for faster implementation during process development. By testing multiple shift parameters, we identified optimum shift conditions in shake flasks and successfully translated findings to benchtop bioreactors and 1,000-L bioreactor scale. Significant differences in final antibody titer and charge variants were observed with temperature shift increments as small as Δ1.5°C. Acidic charge variants decreased monotonically with decreasing shift temperature in both cell lines; however, final antibody titer required simultaneous optimization of shift day and temperature. Overall, we were able to show that a systematic approach to identify temperature shift parameters at small scales is useful to optimize protein production and quality for efficient and confident translation to large-scale production.     

Light-induced interaction between rhodopsin and GTP-binding protein leads to the hydrolysis of GTP in the rod outer segment

In the presence of exogenous GTP, vertebrate whole rod outer segments (ROS), with perforated plasma membranes in the "single particle" scattering range, elicit a light-induced light-scattering transient which we call the "G" signal. Here, we report on the characteristics of the "G" signal relative to the "binding" and "dissociation" signals reported by Kuhn and colleagues. Replacing GTP with guanylyl imidodiphosphate (GMP-PNP) does not give rise to the G signal. This indicates that hydrolysis of the terminal phosphate is required for the G signal and, in addition, GTP and GMP-PNP compete for the same binding site of the enzyme responsible for the G signal (i.e., GTP-binding protein). Also, neither GDP nor its nonhydrolyzable analogue, guanosine 5'-O-(2-thiodiphosphate), when present in ROS suspensions yield any light-scattering transient in the time period tested.     

Proteomics Analysis of Cancer Exosomes Using a Novel Modified Aptamer-based Array (SOMAscanTM) Platform

We have used a novel affinity-based proteomics technology to examine the protein signature of small secreted extracellular vesicles called exosomes. The technology uses a new class of protein binding reagents called SOMAmers® (slow off-rate modified aptamers) and allows the simultaneous precise measurement of over 1000 proteins. Exosomes were highly purified from the Du145 prostate cancer cell line, by pooling selected fractions from a continuous sucrose gradient (within the density range of 1.1 to 1.2 g/ml), and examined under standard conditions or with additional detergent treatment by the SOMAscan^TM array (version 3.0). Lysates of Du145 cells were also prepared, and the profiles were compared. Housekeeping proteins such as cyclophilin-A, LDH, and Hsp70 were present in exosomes, and we identified almost 100 proteins that were enriched in exosomes relative to cells. These included proteins of known association with cancer exosomes such as MFG-E8, integrins, and MET, and also those less widely reported as exosomally associated, such as ROR1 and ITIH4. Several proteins with no previously known exosomal association were confirmed as exosomally expressed in experiments using individual SOMAmer® reagents or antibodies in micro-plate assays. Western blotting confirmed the SOMAscan^TM-identified enrichment of exosomal NOTCH-3, L1CAM, RAC1, and ADAM9. In conclusion, we describe here over 300 proteins of hitherto unknown association with prostate cancer exosomes and suggest that the SOMAmer®-based assay technology is an effective proteomics platform for exosome-associated biomarker discovery in diverse clinical settings.Prostate carcinoma is the most frequent male cancer, with an estimated 240,000 newly diagnosed individuals and 28,000 deaths in the United States during 2012 (National Cancer Institute (NIH)). Methods for detecting this cancer are based on a combination of physical examination through digital rectal examination, clinical imaging, quantification of circulating levels of prostate specific antigen (PSA),¹ and transrectal ultrasound-guided biopsy. As a non-invasive test, PSA measurement is still widely used, but it remains insensitive, as around 15% of men with normal levels of PSA will have prostate cancer according to biopsy results (1), and 60% of men with elevated PSA levels may have other, noncancerous conditions but be subjected to further, unnecessary investigations and interventions (2). PSA may be of better utility in monitoring disease progression (2). An ability to diagnose the disease more specifically at an early stage is likely to save lives and alleviate the healthcare burden and morbidities arising from misdiagnosis. In addition, methods for monitoring the course of the disease in a non-invasive and perhaps predictive manner would offer increased patient benefit, enabling early detection of imminent relapse under hormone therapy, for example. Therefore there is a clinical need for improved molecular approaches for disease diagnosis and monitoring in these settings.Small vesicles termed exosomes are present in body fluids, including serum, plasma, urine, and seminal plasma (3–7), and their isolation and examiniation may prove useful as a minimally invasive means of obtaining a complex set of disease markers. Exosomes are secreted by most, if not all, cell types and are generally accepted as derived principally from multivesicular bodies of the late endocytic tract (8), although examples of plasma membrane budding nanovesicles of similar phenotype have also been described (9). Exosomes are particularly enriched in membrane proteins and in factors related to such endosomal compartments. They also contain proteins found in the cytosol, but they poorly represent components of organelles such as the mitochondria, nucleus, and endoplasmic reticulum (10). Exosomes also comprise an assortment of coding and noncoding RNA. There has been considerable global effort toward defining disease-related alterations in exosomal RNA. However, it is well established that aberrant alterations in cancer cells in response to metabolic, hypoxic, or other forms of stress are reflected in protein changes in the exosomes produced (11–13). Thus exosomes from diseased origins can be distinguished from those of a normal phenotype based on their protein profiles alone.Proteomics studies using mass spectrometry (MS) have previously been conducted on prostate cancer exosomes/microvesicles obtained from cell lines (14, 15), xenotransplantation models (16), or ex vivo biofluids (17). Hundreds of proteins with putative associations with exosomes/microvesicles have been identified. These studies highlight several interesting candidate markers of potential biomarker utility that are currently being explored. However, global proteomic approaches of this nature can have two major limitations. Although the most abundant proteins are more likely to be identified by MS, it is difficult to infer information about relative abundances of proteins in complex samples when using these methods. Secondly, given the often exacting, difficult-to-reproduce, and time-consuming workflows involved, such technologies are poorly suited for the analysis of a large number of samples. Multiplex protein array methodologies have the potential to overcome such issues and offer quantification and options for more rapid sample throughput. However, most platforms are based on antibodies, and these arrays are typically limited to <100 proteins, principally because the cross-reactivity of secondary antibodies can negatively affect assay specificity (18).A recently developed proteomics platform, termed SOMAscan^TM, provides a new generation of protein detection technologies. The platform is capable of the simultaneous quantitative analysis of 1129 proteins per sample in its current form. It is also an approach well suited to handling large numbers of specimens required for well-powered clinical studies (19). The key to this technology, which is described in detail by Gold et al. (20, 21), is the use of slow off-rate modified aptamers (SOMAmers) containing chemically modified nucleotides. This confers greater stability, expanded target range, and improved affinity for the target proteins. This multiplex platform has been applied successfully to small volumes (∼15 μl) of plasma specimens from chronic renal disease patients (20), serum specimens from mesothelioma (22) or lung cancer patients (19), tissue lysates (23), and cerebrospinal fluid (24). However, to date, the compatibility of this array technology with exosomes as the specimen has not been investigated.The purpose of the current study was to examine the utility of this evolving technology in profiling the protein repertoire of exosomes. Research was conducted using highly pure exosomes isolated from a prostate cancer cell line, and we compared this sample to the protein profile of the parent cells. By so doing, we obtained evidence of the compatibility of the platform with this difficult, membranous sample and identified several proteins of previously unknown association with exosomes. In summary, SOMAscan^TM is a versatile tool for probing the composition of exosomes and is a suitable platform to provide a high-throughput approach for exosome-based biomarker discovery in prostate cancer and other clinical settings.     

Effectiveness of bifenthrin (Onyx) and carbaryl (Sevin SL) for protecting individual, high-value conifers from bark beetle attack (Coleoptera: Curculionidae: Scolytinae) in the Western United States

High-value trees, such as those located in residential, recreational, or administrative sites, are particularly susceptible to bark beetle (Coleoptera: Curculionidae: Scolytinae) attack as a result of increased amounts of stress associated with drought, soil compaction, mechanical injury, or vandalism. Tree losses in these unique environments generally have a substantial impact. The value of these individual trees, cost of removal, and loss of esthetics may justify protection until the main thrust of a bark beetle infestation subsides. This situation emphasizes the need for ensuring that effective insecticides are available for individual tree protection. In this study, we assess the efficacy of bifenthrin (Onyx) and carbaryl (Sevin SL) for protecting: ponderosa pine, Pinus ponderosa Dougl. ex. Laws., from western pine beetle, Dendroctonus brevicomis LeConte, in California; mountain pine beetle, Dendroctonus ponderosae Hopkins in South Dakota; and Ips spp. in Arizona; lodgepole pine, Pinus contorta Dougl. ex Loud., from D. ponderosae in Montana; pinyon, Pinus edulis Engelm. in Colorado and Pinus monophylla Torr. and Frem. in Nevada from pinyon ips, Ips confusus (LeConte); and Engelmann spruce, Picea engelmannii Parry ex. Engelm. from spruce beetle, Dendroctonus rufipennis (Kirby) in Utah. Few trees were attacked by Ips spp. in Arizona and that study was discontinued. Sevin SL (2.0%) was effective for protecting P. ponderosa, P. contorta, and P. monophylla for two field seasons. Estimates of efficacy could not be made during the second field season in P. edulis and P. engelmannii due to insufficient mortality in untreated, baited control trees. Two field seasons of efficacy was demonstrated in P. ponderosa/D. brevicomis and P. monophylla for 0.06% Onyx. We conclude that Onyx is an effective individual tree protection tool, but repeated annual applications may be required in some systems if multiyear control is desired.     

Analysis of the overdispersed clock in the short-term evolution of hepatitis C virus: Using the E1/E2 gene sequences to infer infection dates in a single source outbreak

The assumption of a molecular clock for dating events from sequence information is often frustrated by the presence of heterogeneity among evolutionary rates due, among other factors, to positively selected sites. In this work, our goal is to explore methods to estimate infection dates from sequence analysis. One such method, based on site stripping for clock detection, was proposed to unravel the clocklike molecular evolution in sequences showing high variability of evolutionary rates and in the presence of positive selection. Other alternatives imply accommodating heterogeneity in evolutionary rates at various levels, without eliminating any information from the data. Here we present the analysis of a data set of hepatitis C virus (HCV) sequences from 24 patients infected by a single individual with known dates of infection. We first used a simple criterion of relative substitution rate for site removal prior to a regression analysis. Time was regressed on maximum likelihood pairwise evolutionary distances between the sequences sampled from the source individual and infected patients. We show that it is indeed the fastest evolving sites that disturb the molecular clock and that these sites correspond to positively selected codons. The high computational efficiency of the regression analysis allowed us to compare the site-stripping scheme with random removal of sites. We demonstrate that removing the fast-evolving sites significantly increases the accuracy of estimation of infection times based on a single substitution rate. However, the time-of-infection estimations improved substantially when a more sophisticated and computationally demanding Bayesian method was used. This method was used with the same data set but keeping all the sequence positions in the analysis. Consequently, despite the distortion introduced by positive selection on evolutionary rates, it is possible to obtain quite accurate estimates of infection dates, a result of especial relevance for molecular epidemiology studies.