Redesigning the procaspase‐8 dimer interface for improved dimerization

Caspase‐8 is a cysteine directed aspartate‐specific protease that is activated at the cytosolic face of the cell membrane upon receptor ligation. A key step in the activation of caspase‐8 depends on adaptor‐induced dimerization of procaspase‐8 monomers. Dimerization is followed by limited autoproteolysis within the intersubunit linker (IL), which separates the large and small subunits of the catalytic domain. Although cleavage of the IL stabilizes the dimer, the uncleaved procaspase‐8 dimer is sufficiently active to initiate apoptosis, so dimerization of the zymogen is an important mechanism to control apoptosis. In contrast, the effector caspase‐3 is a stable dimer under physiological conditions but exhibits little enzymatic activity. The catalytic domains of caspases are structurally similar, but it is not known why procaspase‐8 is a monomer while procaspase‐3 is a dimer. To define the role of the dimer interface in assembly and activation of procaspase‐8, we generated mutants that mimic the dimer interface of effector caspases. We show that procaspase‐8 with a mutated dimer interface more readily forms dimers. Time course studies of refolding also show that the mutations accelerate dimerization. Transfection of HEK293A cells with the procaspase‐8 variants, however, did not result in a significant increase in apoptosis, indicating that other factors are required in vivo. Overall, we show that redesigning the interface of procaspase‐8 to remove negative design elements results in increased dimerization and activity in vitro, but increased dimerization, by itself, is not sufficient for robust activation of apoptosis.     

Trap Array Configuration Influences Estimates and Precision of Black Bear Density and Abundance

Spatial capture-recapture (SCR) models have advanced our ability to estimate population density for wide ranging animals by explicitly incorporating individual movement. Though these models are more robust to various spatial sampling designs, few studies have empirically tested different large-scale trap configurations using SCR models. We investigated how extent of trap coverage and trap spacing affects precision and accuracy of SCR parameters, implementing models using the R package secr. We tested two trapping scenarios, one spatially extensive and one intensive, using black bear (Ursus americanus) DNA data from hair snare arrays in south-central Missouri, USA. We also examined the influence that adding a second, lower barbed-wire strand to snares had on quantity and spatial distribution of detections. We simulated trapping data to test bias in density estimates of each configuration under a range of density and detection parameter values. Field data showed that using multiple arrays with intensive snare coverage produced more detections of more individuals than extensive coverage. Consequently, density and detection parameters were more precise for the intensive design. Density was estimated as 1.7 bears per 100 km² and was 5.5 times greater than that under extensive sampling. Abundance was 279 (95% CI = 193–406) bears in the 16,812 km² study area. Excluding detections from the lower strand resulted in the loss of 35 detections, 14 unique bears, and the largest recorded movement between snares. All simulations showed low bias for density under both configurations. Results demonstrated that in low density populations with non-uniform distribution of population density, optimizing the tradeoff among snare spacing, coverage, and sample size is of critical importance to estimating parameters with high precision and accuracy. With limited resources, allocating available traps to multiple arrays with intensive trap spacing increased the amount of information needed to inform parameters with high precision.     

Environmental variables associated with the distribution and occupancy of habitat specialist tadpoles in naturally acidic,oligotrophic waterbodies

Environmental factors play an integral role, either directly or indirectly, in structuring faunal assemblages. Water chemistry, predation, hydroperiod and competition influence tadpole assemblages within waterbodies. We surveyed aquatic predators, habitat refugia, water height and water chemistry variables (pH, salinity and turbidity) at 37 waterbodies over an intensive 22‐day field survey to determine which environmental factors influence the relative abundance and occupancy of two habitat specialist anuran tadpole species in naturally acidic, oligotrophic waterbodies within eastern Australian wallum communities. The majority of tadpoles found were of Litoria olongburensis (wallum sedge frog) and Crinia tinnula (wallum froglet) species, both habitat specialists that are associated with wallum waterbodies and listed as Vulnerable under the IUCN Red List. Tadpoles of two other species (Litoria fallax (eastern sedge frog), and Litoria cooloolensis (cooloola sedge frog)) were recorded from two waterbodies. Tadpoles of Litoria gracilenta (graceful treefrog) were recorded from one waterbody. Relative abundance and occupancy of L. olongburensis tadpoles were associated with pH and water depth. Additionally, L. olongburensis tadpole relative abundance was negatively associated with turbidity. Waterbody occupancy by C. tinnula tadpoles was negatively associated with predatory fish and water depth and positively associated with turbidity. Variables associated with relative abundance of C. tinnula tadpoles were inconclusive and further survey work is required to identify these environmental factors. Our results show that the ecology of specialist and non‐specialist tadpole species associated with ‘unique’ (e.g. wallum) waterbodies is complex and species specific, with specialist species likely dominating unique habitats.     

Quantitative Imaging with a Mobile Phone Microscope

Use of optical imaging for medical and scientific applications requires accurate quantification of features such as object size, color, and brightness. High pixel density cameras available on modern mobile phones have made photography simple and convenient for consumer applications; however, the camera hardware and software that enables this simplicity can present a barrier to accurate quantification of image data. This issue is exacerbated by automated settings, proprietary image processing algorithms, rapid phone evolution, and the diversity of manufacturers. If mobile phone cameras are to live up to their potential to increase access to healthcare in low-resource settings, limitations of mobile phone–based imaging must be fully understood and addressed with procedures that minimize their effects on image quantification. Here we focus on microscopic optical imaging using a custom mobile phone microscope that is compatible with phones from multiple manufacturers. We demonstrate that quantitative microscopy with micron-scale spatial resolution can be carried out with multiple phones and that image linearity, distortion, and color can be corrected as needed. Using all versions of the iPhone and a selection of Android phones released between 2007 and 2012, we show that phones with greater than 5 MP are capable of nearly diffraction-limited resolution over a broad range of magnifications, including those relevant for single cell imaging. We find that automatic focus, exposure, and color gain standard on mobile phones can degrade image resolution and reduce accuracy of color capture if uncorrected, and we devise procedures to avoid these barriers to quantitative imaging. By accommodating the differences between mobile phone cameras and the scientific cameras, mobile phone microscopes can be reliably used to increase access to quantitative imaging for a variety of medical and scientific applications.     

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.     

The Ctp Type IVb Pilus Locus of Agrobacterium tumefaciens Directs Formation of the Common Pili and Contributes to Reversible Surface Attachment

Agrobacterium tumefaciens can adhere to plant tissues and abiotic surfaces and forms biofilms. Cell surface appendages called pili play an important role in adhesion and biofilm formation in diverse bacterial systems. The A. tumefaciens C58 genome sequence revealed the presence of the ctpABCDEFGHI genes (cluster of type IV pili; Atu0216 to Atu0224), homologous to tad-type pilus systems from several bacteria, including Aggregatibacter actinomycetemcomitans and Caulobacter crescentus. These systems fall into the type IVb pilus group, which can function in bacterial adhesion. Transmission electron microscopy of A. tumefaciens revealed the presence of filaments, significantly thinner than flagella and often bundled, associated with cell surfaces and shed into the external milieu. In-frame deletion mutations of all of the ctp genes, with the exception of ctpF, resulted in nonpiliated derivatives. Mutations in ctpA (a pilin homologue), ctpB, and ctpG decreased early attachment and biofilm formation. The adherence of the ctpA mutant could be restored by ectopic expression of the paralogous pilA gene. The ΔctpA ΔpilA double pilin mutant displayed a diminished biovolume and lower biofilm height than the wild type under flowing conditions. Surprisingly, however, the ctpCD, ctpE, ctpF, ctpH, and ctpI mutants formed normal biofilms and showed enhanced reversible attachment. In-frame deletion of the ctpA pilin gene in the ctpCD, ctpE, ctpF, ctpH, and ctpI mutants caused the same attachment-deficient phenotype as the ctpA single mutant. Collectively, these findings indicate that the ctp locus is involved in pilus assembly and that nonpiliated mutants, which retain the CtpA pilin, are proficient in attachment and adherence.     

Agrobacterium tumefaciens ExoR Controls Acid Response Genes and Impacts Exopolysaccharide Synthesis,Horizontal Gene Transfer,and Virulence Gene Expression

Agrobacterium tumefaciens is a facultative plant pathogen and the causative agent of crown gall disease. The initial stage of infection involves attachment to plant tissues, and subsequently, biofilms may form at these sites. This study focuses on the periplasmic ExoR regulator, which was identified based on the severe biofilm deficiency of A. tumefaciens exoR mutants. Genome-wide expression analysis was performed to elucidate the complete ExoR regulon. Overproduction of the exopolysaccharide succinoglycan is a dramatic phenotype of exoR mutants. Comparative expression analyses revealed that the core ExoR regulon is unaffected by succinoglycan synthesis. Several findings are consistent with previous observations: genes involved in succinoglycan biosynthesis, motility, and type VI secretion are differentially expressed in the ΔexoR mutant. In addition, these studies revealed new functional categories regulated by ExoR, including genes related to virulence, conjugation of the pAtC58 megaplasmid, ABC transporters, and cell envelope architecture. To address how ExoR exerts a broad impact on gene expression from its periplasmic location, a genetic screen was performed to isolate suppressor mutants that mitigate the exoR motility phenotype and identify downstream components of the ExoR regulatory pathway. This suppression analysis identified the acid-sensing two-component system ChvG-ChvI, and the suppressor mutant phenotypes suggest that all or most of the characteristic exoR properties are mediated through ChvG-ChvI. Subsequent analysis indicates that exoR mutants are simulating a response to acidic conditions, even in neutral media. This work expands the model for ExoR regulation in A. tumefaciens and underscores the global role that this regulator plays on gene expression.     

Parallel multiplicity and error discovery rate (EDR) in microarray experiments

Background  

In microarray gene expression profiling experiments, differentially expressed genes (DEGs) are detected from among tens of thousands of genes on an array using statistical tests. It is important to control the number of false positives or errors that are present in the resultant DEG list. To date, more than 20 different multiple test methods have been reported that compute overall Type I error rates in microarray experiments. However, these methods share the following dilemma: they have low power in cases where only a small number of DEGs exist among a large number of total genes on the array.     

Exploration of inhibitors for diaminopimelate aminotransferase

Bacteria and higher plants make l-lysine from diaminopimelic acid (DAP). In mammals l-lysine is an essential amino acid that must be acquired from the diet as the biosynthetic pathway is absent for this key constituent of proteins. Recently, ll-diaminopimelate aminotransferase (ll-DAP-AT), a pyridoxal-5′-phosphate (PLP)-dependent enzyme, was reported to catalyze a key step in the route to l-lysine in plants and Chlamydia. Specific inhibitors of this enzyme could thus potentially serve as herbicides or antibiotics that are non-toxic to mammals. In this work, 29,201 inhibitors were screened against ll-DAP-AT and the IC₅₀ values were determined for the top 46 compounds. An aryl hydrazide and rhodanine derivatives were further modified to generate 20 analogues that were also tested against ll-DAP-AT. These analogues provide additional structure–activity relationships (SAR) that are useful in guiding further design of inhibitors.