Chemical-based fecal source tracking methods: current status and guidelines for evaluation

Fecal source tracking is a rapidly evolving field for which there have been a number of method evaluation studies, workshops, review articles and a book that synthesize information about method efficacy. Chemicals that are specific to human wastewater offer several potential advantages over biologically based methods, but have received less scrutiny. More than 35 chemical analytes have been found to consistently occur in human waste streams and here we review these potential human-origin indicators in context of seven evaluation criteria. Some chemical methods offer advantages over microbial methods: they are generally faster to prepare and analyze, more source-specific because they are not confounded by regrowth in the environment, and some may be more geographically and temporally stable. However, they often require specialized equipment and are usually more expensive regarding sample preparation and analysis. Additionally, most chemicals that are specific to human waste-streams occur at concentrations low enough to be diluted below detection limits once the waste-stream enters the ambient environment. These two factors will likely result in chemical measures being used more often as cross-validation supplements or initial screening approaches, rather than replacements for microbial measures. Cross-validation supplements include several chemicals that are highly specific to human sources and can be important contributors when certainties about human sources are critical, such as in drinking water applications. At least one set of chemicals, fecal sterols and stanols, may have potential for identification of other sources in addition to humans. Of all the chemicals examined to date, optical brighteners (OBs) in detergents have shown considerable promise, especially for screening purposes. Optical brighteners are not as sensitive as most microbial assessments, but can be measured with a hand-held fluorometer, providing near real-time and relatively inexpensive tracking of signals in the field, if the human fecal source contains an OB concentration large enough to produce a measurable signal.     

Functional,Non-Clonal IgMa-Restricted B Cell Receptor Interactions with the HIV-1 Envelope gp41 Membrane Proximal External Region

The membrane proximal external region (MPER) of HIV-1 gp41 has several features that make it an attractive antibody-based vaccine target, but eliciting an effective gp41 MPER-specific protective antibody response remains elusive. One fundamental issue is whether the failure to make gp41 MPER-specific broadly neutralizing antibodies like 2F5 and 4E10 is due to structural constraints with the gp41 MPER, or alternatively, if gp41 MPER epitope-specific B cells are lost to immunological tolerance. An equally important question is how B cells interact with, and respond to, the gp41 MPER epitope, including whether they engage this epitope in a non-canonical manner i.e., by non-paratopic recognition via B cell receptors (BCR). To begin understanding how B cells engage the gp41 MPER, we characterized B cell-gp41 MPER interactions in BALB/c and C57BL/6 mice. Surprisingly, we found that a significant (∼7%) fraction of splenic B cells from BALB/c, but not C57BL/6 mice, bound the gp41 MPER via their BCRs. This strain-specific binding was concentrated in IgM^hi subsets, including marginal zone and peritoneal B1 B cells, and correlated with enriched fractions (∼15%) of gp41 MPER-specific IgM secreted by in vitro-activated splenic B cells. Analysis of Igh^a (BALB/c) and Igh^b (C57BL/6) congenic mice demonstrated that gp41 MPER binding was controlled by determinants of the Igh^a locus. Mapping of MPER gp41 interactions with IgM^a identified MPER residues distinct from those to which mAb 2F5 binds and demonstrated the requirement of Fc C_H regions. Importantly, gp41 MPER ligation produced detectable BCR-proximal signaling events, suggesting that interactions between gp41 MPER and IgM^a determinants may elicit partial B cell activation. These data suggest that low avidity, non-paratopic interactions between the gp41 MPER and membrane Ig on naïve B cells may interfere with or divert bnAb responses.     

Enigma Prevents Cbl-c-Mediated Ubiquitination and Degradation of RETMEN2A

The Cbl proteins (Cbl, Cbl-b, and Cbl-c) are a highly conserved family of RING finger ubiquitin ligases (E3s) that function as negative regulators of tyrosine kinases in a wide variety of signal transduction pathways. In this study, we identify a new Cbl-c interacting protein, Enigma (PDLIM7). This interaction is specific to Cbl-c as Enigma fails to bind either of its closely related homologues, Cbl and Cbl-b. The binding between Enigma and Cbl-c is mediated through the LIM domains of Enigma as removal of all three LIM domains abrogates this interaction, while only LIM1 is sufficient for binding. Here we show that Cbl-c binds wild-type and MEN2A isoforms of the receptor tyrosine kinase, RET, and that Cbl-c enhances ubiquitination and degradation of activated RET. Enigma blocks Cbl-c-mediated RETMEN2A ubiquitination and degradation. Cbl-c decreased downstream ERK activation by RETMEN2A and co-expression of Enigma blocked the Cbl-c-mediated decrease in ERK activation. Enigma showed no detectable effect on Cbl-c-mediated ubiquitination of activated EGFR suggesting that this effect is specific to RET. Through mapping studies, we show that Cbl-c and Enigma bind RETMEN2A at different residues. However, binding of Enigma to RETMENA prevents Cbl-c recruitment to RETMEN2A. Consistent with these biochemical data, exploratory analyses of breast cancer patients with high expression of RET suggest that high expression of Cbl-c correlates with a good outcome, and high expression of Enigma correlates with a poor outcome. Together, these data demonstrate that Cbl-c can ubiquitinate and downregulate RETMEN2A and implicate Enigma as a positive regulator of RETMEN2A through blocking of Cbl-mediated ubiquitination and degradation.     

Differentiation of subspecies and sexes of Beringian Dunlins using morphometric measures

Five subspecies of Dunlins (Calidris alpina) that breed in Beringia are potentially sympatric during the non‐breeding season. Studying their ecology during this period requires techniques to distinguish individuals by subspecies. Our objectives were to determine (1) if five morphometric measures (body mass, culmen, head, tarsus, and wing chord) differed between sexes and among subspecies (C. a. actites, arcticola, kistchinski, pacifica, and sakhalina), and (2) if these differences were sufficient to allow for correct classification of individuals using equations derived from discriminant function analyses. We conducted analyses using morphometric data from 10 Dunlin populations breeding in northern Russia and Alaska, USA. Univariate tests revealed significant differences between sexes in most morphometric traits of all subspecies, and discriminant function equations predicted the sex of individuals with an accuracy of 83–100% for each subspecies. We provide equations to determine sex and subspecies of individuals in mixed subspecies groups, including the (1) Western Alaska group of arcticola and pacifica (known to stage together in western Alaska) and (2) East Asia group of arcticola, actites, kistchinski, and sakhalina (known to winter together in East Asia). Equations that predict the sex of individuals in mixed groups had classification accuracies between 75% and 87%, yielding reliable classification equations. We also provide equations that predict the subspecies of individuals with an accuracy of 22–96% for different mixed subspecies groups. When the sex of individuals can be predetermined, the accuracy of these equations is increased substantially. Investigators are cautioned to consider limitations due to age and feather wear when using these equations during the non‐breeding season. These equations will allow determination of sexual and subspecies segregation in non‐breeding areas, allowing implementation of taxonomic‐specific conservation actions.     

Regulation of mitosis by the NIMA kinase involves TINA and its newly discovered partner,An-WDR8, at spindle pole bodies

The NIMA kinase is required for mitotic nuclear pore complex disassembly and potentially controls other mitotic-specific events. To investigate this possibility, we imaged NIMA–green fluorescent protein (GFP) using four-dimensional spinning disk confocal microscopy. At mitosis NIMA-GFP locates to spindle pole bodies (SPBs), which contain Cdk1/cyclin B, followed by Aurora, TINA, and the BimC kinesin. NIMA promotes NPC disassembly in a spatially regulated manner starting near SPBs. NIMA is also required for TINA, a NIMA-interacting protein, to locate to SPBs during initiation of mitosis, and TINA is then necessary for locating NIMA back to SPBs during mitotic progression. To help expand the NIMA-TINA pathway, we affinity purified TINA and found it to uniquely copurify with An-WDR8, a WD40-domain protein conserved from humans to plants. Like TINA, An-WDR8 accumulates within nuclei during G2 but disperses from nuclei before locating to mitotic SPBs. Without An-WDR8, TINA levels are greatly reduced, whereas TINA is necessary for mitotic targeting of An-WDR8. Finally, we show that TINA is required to anchor mitotic microtubules to SPBs and, in combination with An-WDR8, for successful mitosis. The findings provide new insights into SPB targeting and indicate that the mitotic microtubule-anchoring system at SPBs involves WDR8 in complex with TINA.     

Calculation of effective diffusivities for biofilms and tissues.

In this study we describe a scheme for numerically calculating the effective diffusivity of cellular systems such as biofilms and tissues. This work extends previous studies in which we developed the macroscale representations of the transport equations for cellular systems based on the subcellular-scale transport and reaction processes. A finite-difference model is used to predict the effective diffusivity of a cellular system on the basis of the subcellular-scale geometry and transport parameters. The effective diffusivity is predicted for a complex three-dimensional structure that is based on laboratory observations of a biofilm, and these numerical predictions are compared with predictions from a simple analytical solution and with experimental data. Our results indicate that, under many practical circumstances, the simple analytical solution can be used to provide reasonable estimates of the effective diffusivity.     

Migration timing and distance from shore of southbound eastern Pacific gray whales (Eschrichtius robustus) off Ensenada,Baja California,Mexico

Eastern Pacific gray whales were monitored off Ensenada, Mexico, during the southbound migration. The objectives were to determine southbound migration timing and width of the migration corridor during three seasons (2003–2006). Migration timing was determined by fitting a generalized additive model to the shore counts for each season and estimating the 10, 50, and 90 percentiles of the fitted curves. To estimate abundance from shore‐based counts, a probability density function for the shore based distances was estimated by a product of a gamma distribution fit to the boat survey distance data for 2006/2007 and a half‐normal detection function using combined data of the three seasons. The parameters of the gamma distribution were corrected to account for less boat survey effort carried out 20–40 km than 0–20 km from shore. The onset of the migration off Ensenada was in late December/early January and ended around 13 February. The median date was 23–26 January for the first and third season and a week early for the second season. Boat surveys indicated a wide (20 km) migration corridor but most gray whales traveled within 9.9 km from shore. The estimated total number of whales during watch hours was 2,298 (95% CI = 1,536–4,447).