A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

Background  

Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.     

COMMD1-mediated ubiquitination regulates CFTR trafficking

The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.     

Guano exposed: Impact of aerobic conditions on bat fecal microbiota

Bats and their associated guano microbiota provide important terrestrial and subterranean ecosystem services and serve as a reservoir for a wide range of epizootic and zoonotic diseases. Unfortunately, large‐scale studies of bats and their guano microbiotas are limited by the time and cost of sample collection, which requires specially trained individuals to work at night to capture bats when they are most active. Indirectly surveying bat gut microbiota through guano deposits could be a more cost‐effective alternative, but it must first be established whether the postdefecation exposure to an aerobic environment has a large impact on the guano microbial community. A number of recent studies on mammalian feces have shown that the impact of aerobic exposure is highly species specific; therefore, it is difficult to predict how exposure will affect the bat guano microbiota without empirical data. In our study, we collected fresh guano samples from 24 individuals of 10 bat species that are common throughout the arid environments of the American southwest and subjected the samples to 0, 1, and 12 hr of exposure. The biodiversity decreased rapidly after the shift from an anaerobic to an aerobic environment—much faster than previously reported in mammalian species. However, the relative composition of the core guano microbiota remained stable and, using highly sensitive targeted PCR methods, we found that pathogens present in the original, non‐exposed samples could still be recovered after 12 hr of exposure. These results suggest that with careful sample analysis protocols, a more efficient passive collection strategy is feasible; for example, guano could be collected on tarps placed near the roost entrance. Such passive collection methods would greatly reduce the cost of sample collection by allowing more sites or roosts to be surveyed with a fraction of trained personnel, time, and effort investments needed.     

Negative BOLD fMRI response in the visual cortex carries precise stimulus-specific information

Sustained positive BOLD (blood oxygen level-dependent) activity is employed extensively in functional magnetic resonance imaging (fMRI) studies as evidence for task or stimulus-specific neural responses. However, the presence of sustained negative BOLD activity (i.e., sustained responses that are lower than the fixation baseline) has remained more difficult to interpret. Some studies suggest that it results from local "blood stealing" wherein blood is diverted to neurally active regions without a concomitant change of neural activity in the negative BOLD regions. However, other evidence suggests that negative BOLD is a result of local neural suppression. In both cases, regions of negative BOLD response are usually interpreted as carrying relatively little, if any, stimulus-specific information (hence the predominant reliance on positive BOLD activity in fMRI). Here we show that the negative BOLD response resulting from visual stimulation can carry high information content that is stimulus-specific. Using a general linear model (GLM), we contrasted standard flickering stimuli to a fixation baseline and found regions of the visual cortex that displayed a sustained negative BOLD response, consistent with several previous studies. Within these negative BOLD regions, we compared patterns of fMRI activity generated by flickering Gabors that were systematically shifted in position. As the Gabors were shifted further from each other, the correlation in the spatial pattern of activity across a population of voxels (such as the population of V1 voxels that displayed a negative BOLD response) decreased significantly. Despite the fact that the BOLD signal was significantly negative (lower than fixation baseline), these regions were able to discriminate objects separated by less than 0.5 deg (at approximately 10 deg eccentricity). The results suggest that meaningful, stimulus-specific processing occurs even in regions that display a strong negative BOLD response.     

Deficiency of PTP1B in POMC neurons leads to alterations in energy balance and homeostatic response to cold exposure

The adipose tissue-derived hormone leptin regulates energy balance through catabolic effects on central circuits, including proopiomelanocortin (POMC) neurons. Leptin activation of POMC neurons increases thermogenesis and locomotor activity. Protein tyrosine phosphatase 1B (PTP1B) is an important negative regulator of leptin signaling. POMC neuron-specific deletion of PTP1B in mice results in reduced high-fat diet-induced body weight and adiposity gain due to increased energy expenditure and greater leptin sensitivity. Mice lacking the leptin gene (ob/ob mice) are hypothermic and cold intolerant, whereas leptin delivery to ob/ob mice induces thermogenesis via increased sympathetic activity to brown adipose tissue (BAT). Here, we examined whether POMC PTP1B mediates the thermoregulatory response of CNS leptin signaling by evaluating food intake, body weight, core temperature (T(C)), and spontaneous physical activity (SPA) in response to either exogenous leptin or 4-day cold exposure (4°C) in male POMC-Ptp1b-deficient mice compared with wild-type controls. POMC-Ptp1b(-/-) mice were hypersensitive to leptin-induced food intake and body weight suppression compared with wild types, yet they displayed similar leptin-induced increases in T(C). Interestingly, POMC-Ptp1b(-/-) mice had increased BAT weight and elevated plasma triiodothyronine (T(3)) levels in response to a 4-day cold challenge, as well as reduced SPA 24 h after cold exposure, relative to controls. These data show that PTP1B in POMC neurons plays a role in short-term cold-induced reduction of SPA and may influence cold-induced thermogenesis via enhanced activation of the thyroid axis.     

Two msbB genes encoding maximal acylation of lipid A are required for invasive Shigella flexneri to mediate inflammatory rupture and destruction of the intestinal epithelium

Shigella flexneri is a Gram-negative pathogen that invades and causes inflammatory destruction of the human colonic epithelium, thus leading to bloody diarrhea and dysentery. A type III secretion system that delivers effector proteins into target eukaryotic cells is largely responsible for cell and tissue invasion. However, the respective role of this invasive phenotype and of lipid A, the endotoxin of the Shigella LPS, in eliciting the inflammatory cascade that leads to rupture and destruction of the epithelial barrier, was unknown. We investigated whether genetic detoxification of lipid A would cause significant alteration in pathogenicity. We showed that S. flexneri has two functional msbB genes, one carried by the chromosome (msbB1) and the other by the virulence plasmid (msbB2), the products of which act in complement to produce full acyl-oxy-acylation of the myristate at the 3' position of the lipid A glucosamine disaccharide. A mutant in which both the msbB1 and msbB2 genes have been inactivated was impaired in its capacity to cause TNF-alpha production by human monocytes and to cause rupture and inflammatory destruction of the epithelial barrier in the rabbit ligated intestinal loop model of shigellosis, indicating that lipid A plays a significant role in aggravating inflammation that eventually destroys the intestinal barrier. In addition, neutralization of TNF-alpha during invasion by the wild-type strain strongly impaired its ability to cause rupture and inflammatory destruction of the epithelial lining, thus indicating that TNF-alpha is a major effector of epithelial destruction by Shigella.     

Familial Sinistrals Avoid Exact Numbers

We report data from an internet questionnaire of sixty number trivia. Participants were asked for the number of cups in their house, the number of cities they know and 58 other quantities. We compare the answers of familial sinistrals – individuals who are left-handed themselves or have a left-handed close blood-relative – with those of pure familial dextrals – right-handed individuals who reported only having right-handed close blood-relatives. We show that familial sinistrals use rounder numbers than pure familial dextrals in the survey responses. Round numbers in the decimal system are those that are multiples of powers of 10 or of half or a quarter of a power of 10. Roundness is a gradient concept, e.g. 100 is rounder than 50 or 200. We show that very round number like 100 and 1000 are used with 25% greater likelihood by familial sinistrals than by pure familial dextrals, while pure familial dextrals are more likely to use less round numbers such as 25, 60, and 200. We then use Sigurd’s (1988, Language in Society) index of the roundness of a number and report that familial sinistrals’ responses are significantly rounder on average than those of pure familial dextrals. To explain the difference, we propose that the cognitive effort of using exact numbers is greater for the familial sinistral group because their language and number systems tend to be more distributed over both hemispheres of the brain. Our data support the view that exact and approximate quantities are processed by two separate cognitive systems. Specifically, our behavioral data corroborates the view that the evolutionarily older, approximate number system is present in both hemispheres of the brain, while the exact number system tends to be localized in only one hemisphere.