Enhanced discrimination of malignant from benign pancreatic disease by measuring the CA 19-9 antigen on specific protein carriers

The CA 19-9 assay detects a carbohydrate antigen on multiple protein carriers, some of which may be preferential carriers of the antigen in cancer. We tested the hypothesis that the measurement of the CA 19-9 antigen on individual proteins could improve performance over the standard CA 19-9 assay. We used antibody arrays to measure the levels of the CA 19-9 antigen on multiple proteins in serum or plasma samples from patients with pancreatic adenocarcinoma or pancreatitis. Sample sets from three different institutions were examined, comprising 531 individual samples. The measurement of the CA 19-9 antigen on any individual protein did not improve upon the performance of the standard CA 19-9 assay (82% sensitivity at 75% specificity for early-stage cancer), owing to diversity among patients in their CA 19-9 protein carriers. However, a subset of cancer patients with no elevation in the standard CA 19-9 assay showed elevations of the CA 19-9 antigen specifically on the proteins MUC5AC or MUC16 in all sample sets. By combining measurements of the standard CA 19-9 assay with detection of CA 19-9 on MUC5AC and MUC16, the sensitivity of cancer detection was improved relative to CA 19-9 alone in each sample set, achieving 67-80% sensitivity at 98% specificity. This finding demonstrates the value of measuring glycans on specific proteins for improving biomarker performance. Diagnostic tests with improved sensitivity for detecting pancreatic cancer could have important applications for improving the treatment and management of patients suffering from this disease.     

Sperm competitive advantage of a rare mitochondrial haplogroup linked to differential expression of mitochondrial oxidative phosphorylation genes

Maternal inheritance of mitochondria creates a sex‐specific selective sieve through which mitochondrial mutations harmful to males but not females accumulate and contribute to sexual differences in longevity and disease susceptibility. Because eggs and sperm are under disruptive selection, sperm are predicted to be particularly vulnerable to the genetic load generated by maternal inheritance, yet evidence for mitochondrial involvement in male fertility is limited and controversial. Here, we exploit the coexistence of two divergent mitochondrial haplogroups (A and B2) in a Neotropical arachnid to investigate the role of mitochondria in sperm competition. DNA profiling demonstrated that B2‐carrying males sired more than three times as many offspring in sperm competition experiments than A males, and this B2 competitive advantage cannot be explained by female mitochondrial haplogroup or male nuclear genetic background. RNA‐Seq of testicular tissues implicates differential expression of mitochondrial oxidative phosphorylation (OXPHOS) genes in the B2 competitive advantage, including a 22‐fold upregulation of atp8 in B2 males. Previous comparative genomic analyses have revealed functionally significant amino acid substitutions in differentially expressed genes, indicating that the mitochondrial haplogroups differ not only in expression but also in DNA sequence and protein functioning. However, mitochondrial haplogroup had no effect on sperm number or sperm viability, and, when females were mated to a single male, neither male haplogroup, female haplogroup nor the interaction between male/female haplogroup significantly affected female reproductive success. Our findings therefore suggest that mitochondrial effects on male reproduction may often go undetected in noncompetitive contexts and may prove more important in nature than is currently appreciated.     

A multinomial model for estimating the size of a whale population from incomplete census data

This paper adapts the removal method of population size estimation to the problem of estimating the size of the western Arctic stock of bowhead whales. The whales are counted during their spring migration as they pass two census camps located near Point Barrow, Alaska. Whales seen at the first camp are "removed" from the population of concern to the second camp, where only whales missed by the first camp are counted. If both camps were in operation throughout the migration and if the probability of missing a whale were constant, the removal method would provide a population size estimate based on a trinomial model in which the size of the population would be the number of trials, whales counted by each camp would provide the observed cell totals, and whales missed by both camps would represent an unobserved cell total. Since the probability of missing a whale depends on visibility, we model the population size as the sum of the number of trials of several independent trinomial distributions, each of which represents a particular visibility condition occurring during the census. To account for the fact that watch cannot be maintained at both camps throughout the migration, we derive a confidence interval estimate of the number of trials under a more general model allowing for incomplete observation of totals within particular cells as well as for completely unobserved cells.     

ABUNDANCE AND POPULATION TREND (1978-2001) OF WESTERN ARCTIC BOWHEAD WHALES SURVEYED NEAR BARROW, ALASKA

The 2001 survey of western Arctic (Bering, Chukchi, and Beaufort seas) bowhead whales was conducted from 5 April to 7 June near Barrow, Alaska. Visual observers recorded a total of 3,295 “new” (not seen before) and 532 “conditional” (possibly seen before) whales in 1,130 h of watch effort, including 121 new calves (3.7% of the new whales). Concurrent with the visual survey, passive acoustic surveillance was conducted almost continuously from 16 April to 31 May, resulting in 27,023 locations of vocalizing bowhead whales. The estimated number of whales within 4 km of the perch (N₄) was 7,025 (SE = 1,068). The estimated proportion of the whales within 4 km of the perch (P₄) was 0.862 (SE = 0.044, computed by a moving blocks bootstrap). Combining these, the abundance estimate (N₄/P₄) for 2001 is 10,470 (SE = 1, 351) with a 95% confidence interval of 8, 100–13, 500. The estimated annual rate of increase (ROI) of the population from 1978 to 2001 is 3.4% (95% CI 1.7%‐5%). Reports from hunters and results of an aerial survey in June 2001 indicate whales continued to pass Barrow after the survey had ended. In 2001 51% (572 h) of the watch was scored as occurring during “fair‐excellent” visibility conditions, somewhat lower than the average for all surveys since 1978. Sea ice in the leads and fog were the principle environmental factors affecting visibility for all years. The estimated rate of increase and the fact that the number of calves counted in 2001 is the highest ever recorded suggest a steady recovery of this population. Other populations of large balaenids, notably the North Atlantic right whale, have failed to recover despite 70 yr of protection. The recovery of the howhead whale is likely attributable to low anthropogenic mortality, a relatively pristine habitat, and a well‐managed subsistence hunt. Nonetheless, offshore oil development, increasing shipping traffic, changes in the Bering Sea ecosystem, sea ice retreat, and possibly killer whale predation within its range could impact this bowhead population and should be carefully monitored.     

Natural and newly synthesized hydroxy-1-aryl-isochromans: a class of potential antioxidants and radical scavengers

We investigated the antioxidant and radical scavenging activity of polyphenolic isochromans. To assess the relation between structure and scavenging properties the natural occurring 1-(3'-methoxy-4'-hydroxy)phenyl-6,7-dihydroxy-isochroman (ISO-3, three OH groups) was compared with three newly synthesized derivatives that differ in their degree of hydroxylation by substitution with methoxy-groups (ISO-4: four OH groups; ISO-2: two OH groups and ISO-0: fully methoxylated). We found that ISO-4 is a 2-fold better scavenger for the artificial radical 1,1-diphenyl-2-picrylhydrazyl (DPPH, 100 microM) with an EC50=10.3 microM compared to the natural ISO-3 (EC50=22.4 microM) and to ISO-2 (EC50=25.1 microM), while ISO-0 did not react with DPPH. The scavenging capacity for superoxide enzymatically generated in a hypoxanthin-xanthinoxidase reaction was the highest for ISO-4 (EC50=34.3 microM) compared to those of ISO-3 (EC50=84.0 microM) and ISO-2 (EC50=91.8 microM), while ISO-0 was inactive. In analogy, ISO-4 scavenged peroxynitrite (ONOO-, EC25=23.0 microM) more effective than ISO-3, ISO-2 and ISO-0.When C6 rat glioma cells loaded with the reactive oxygen/nitrogen (ROS/RNS)-sensitive fluorochrome 2,7-dichlorodihydrofluorescein, were exposed to hydrogen peroxide, the lowest stress level as indicated by the fluorescence signal was detected when the cells were pretreated with ISO-4 or ISO-2 but to a much lesser extent with ISO-3, while ISO-0 did not show any effect. All tested hydroxyisochromans superceded the scavenging effect of trolox.The excellent radical and ROS/RNS scavenging features of the hydroxy-1-aryl isochromans and their simple synthesis let these compounds appear to be interesting candidates for pharmaceutical interventions that protect against the deleterious action of ROS/RNS.     

Novel pan-neuronal Cre-transgenic line for conditional ablation of genes in the nervous system

Tissue-specific gene ablation is accomplished by combining conventional gene targeting approaches with site-specific recombinases such as the Cre/loxP system. Despite the use of a cardiac-specific rat myosin light chain II promoter, our transgenic line (CRE3) had little or no Cre expression in the heart; however, strong Cre activity was detected in the brain as early as gestation day E11.5. This was determined by several methods including crossing our mouse line with a lacZ indicator line (ROSA26). Transgenic Cre, in this mouse line, mediated DNA recombination of loxP-flanked genes selectively in neurons throughout the gray matter of the brain, cerebellum, spinal cord, as well as retina, dorsal, and sympathetic ganglia. Cre protein was also detected by immunohistochemistry exclusively in neurons, but not in other types of cells or tissues. Thus, our transgenic CRE3 mice provide pan-neuronal expression of CRE for carrying out conditional deletion of genes in neurons and their progenitors.     

Maternal inheritance, sexual conflict and the maladapted male

Females differ from males in transmitting not only nuclear genes but also cytoplasmic genetic elements (CGEs), including DNA in mitochondria, chloroplasts and microorganisms that are present in the cell. Until recently, evolutionary research has adopted a nucleocentric approach in which organelles have been viewed as subservient energy suppliers. In this article, we propose that a more equitable view of nuclear genes and organelle genomes will lead to a better understanding of the dynamics of sexual selection and the constraints on male adaptation. Maternal inheritance of CGEs intensifies sexually-antagonistic coevolution and provides a parsimonious explanation for the relatively high frequency in males of such apparently maladaptive traits as infertility, homosexuality and baldness.     

Current understanding of the regulation of methionine biosynthesis in plants

Plants can provide most of the nutrients for the human diet. However, the major crops are often deficient in some of the nutrients. Thus, malnutrition, with respect to micronutrients such as vitamin A, iron, and zinc, but also macronutrients such as the essential amino acids lysine and methionine, affects more than 40% of the world's population. Recent advances in molecular biology, but also the grasp of biochemical pathways, metabolic fluxes, and networks can now be exploited to produce crops enhanced in key nutrients to increase the nutritional value of plant-derived foods and feeds. Some of the predictions appear to be accurate, while others not, reflecting the fact that plant metabolism is more complex than presently understood. A good example for a complex regulation is the methionine biosynthetic pathway in plants. The nutritional importance of Met and cysteine has motivated extensive studies of their roles in plant molecular physiology, especially regarding to their transport, synthesis, and accumulation in plants. Recent studies have demonstrated that Met metabolism is regulated differently in various plant species.     

Unraveling what makes a monoclonal antibody difficult-to-express: From intracellular accumulation to incomplete folding and degradation via ERAD

Although most therapeutic monoclonal antibodies (mAbs) can routinely be produced in the multigram per litre range, some mAb candidates turn out to be difficult-to-express (DTE). In addition, the class of more complex biological formats is permanently increasing and mammalian expression systems like Chinese hamster ovary (CHO) cell lines can show low performance. Hence, there is an urgent need to identify any rate limiting processing step during cellular synthesis. Therefore, we assessed the intracellular location of the DTE antibody mAb2 by fluorescence and electron microscopy (EM) and revealed an accumulation of the antibody, which led to an aberrant morphology of the endoplasmic reticulum (ER). Analysis of underlying cellular mechanisms revealed that neither aggregation nor antibody assembly, but folding represented the reason for hampered secretion. We identified that the disulfide bridge formation within the antibody light chain (LC) was impaired due to less recognition by protein disulfide isomerase (PDI). As a consequence, the DTE molecule was degraded intracellularly by the ubiquitin proteasome system via ER-associated degradation (ERAD). This study revealed that with the continuous emergence of DTE therapeutic protein candidates, special attention needs to be drawn to optimization processes to ensure manufacturability.