Local targets of T-stellate cells in the ventral cochlear nucleus.

T-stellate cells in the ventral cochlear nucleus (VCN) are known to have local axon collaterals that terminate in the vicinity of their dendrites and cell bodies within the same isofrequency lamina in parallel with the auditory nerve fibers that innervate them. Excitatory synaptic connections between stellate cells within an isofrequency lamina are hypothesized to be involved in the nitric oxide-mediated upregulation of T-stellate responses to their auditory input. This could serve as a mechanism of variable gain control in the enhancement of responses to vowel spectral peaks. Previous studies have provided indirect evidence for these possible synaptic interconnections between T-stellate cells, but unequivocal identification has yet to be established. Here, we used retrograde neuronal tracing with adeno-associated viral vector or biotinylated dextran amine injected into the inferior colliculus (IC) to detect the postsynaptic target of T-stellate cells within the VCN. We show that backfilled T-stellate cell axons make monosynapatic connections on the labeled cell bodies and dendrites of other labeled T-stellate cells within an isofrequency lamina. Electron microscopy revealed that T-stellate terminals can also make synapses on structures not retrogradely labeled from the IC. Glycine antibodies combined with the viral labeling indicated that these nonbackfilled structures that the labeled T-stellate terminals were synapsing on are most likely the cell bodies and dendrites of two size categories of glycinergic VCN cells, whose sizes and relative numbers indicated they are the D- and L-stellate cells. These cells are known to provide inhibitory inputs back onto T-stellate cells. Our data indicate that, in addition to their auditory nerve input, T-stellate cells provide a second modulatable excitatory input to both inhibitory and excitatory cells in a VCN isofrequency lamina and may play a significant role in acoustic information processing.

Effects of an Experimental vs. Traditional Military Training Program on 2-Mile Run Performance During the Army Physical Fitness Test.

Stone, BL, Heishman, AD, and Campbell, JA. The effects of an experimental vs. traditional military training program on 2-mile run performance during the army physical fitness test. J Strength Cond Res 34(12): 3431-3438, 2020-The purpose of this study was to compare the effects of an experimental vs. traditional military run training on 2-mile run ability in the Army Reserve Officers' Training Corps cadets. Fifty college-aged cadets were randomly placed into 2 groups and trained for 4 weeks with either an experimental running program (EXP, n = 22) comprised rating of perceived exertion (RPE) intensity-specific, energy system-based intervals or with traditional military running program (TRA, n = 28) using a crossover study design. A 2-mile run assessment was performed just before the start, at the end of the first 4 weeks, and again after the second 4 weeks of training after crossover. The EXP program significantly decreased 2-mile run times (961.3 ± 155.8 seconds to 943.4 ± 140.2 seconds, p = 0.012, baseline to post 1), whereas the TRA group experienced a significant increase in run times (901.0 ± 79.2 vs. 913.9 ± 82.9 seconds) over the same training period. There was a moderate effect size (d = 0.61, p = 0.07) for the experimental run program to "reverse" the adverse effects of the traditional program within the 4-week training period (post 1 to post 2) after treatment crossover. Thus, for short-term training of military personnel, RPE intensity-specific running program comprising aerobic and anaerobic system development can enhance 2-mile run performance superior to a traditional program while reducing training volume (60 minutes per session vs. 43.2 minutes per session, respectively). Future research should extend the training period to determine efficacy of this training approach for long-term improvement of aerobic capacity and possible reduction of musculoskeletal injury.

Simulation of attenuated total reflection infrared absorbance spectra: applications to automotive clear coat forensic analysis.

Attenuated total reflection (ATR) is a widely used sampling technique in infrared (IR) spectroscopy because minimal sample preparation is required. Since the penetration depth of the ATR analysis beam is quite shallow, the outer layers of a laminate or multilayered paint sample can be preferentially analyzed with the entire sample intact. For this reason, forensic laboratories are taking advantage of ATR to collect IR spectra of automotive paint systems that may consist of three or more layers. However, the IR spectrum of a paint sample obtained by ATR will exhibit distortions, e.g., band broadening and lower relative intensities at higher wavenumbers, compared with its transmission counterpart. This hinders library searching because most library spectra are measured in transmission mode. Furthermore, the angle of incidence for the internal reflection element, the refractive index of the clear coat, and surface contamination due to inorganic contaminants can profoundly influence the quality of the ATR spectrum obtained for automotive paints. A correction algorithm to allow ATR spectra to be searched using IR transmission spectra of the paint data query (PDQ) automotive database is presented. The proposed correction algorithm to convert transmission spectra from the PDQ library to ATR spectra is able to address distortion issues such as the relative intensities and broadening of the bands, and the introduction of wavelength shifts at lower frequencies, which prevent library searching of ATR spectra using archived IR transmission data.

Regulation of endothelial cell barrier function by antibody-driven affinity modulation of platelet endothelial cell adhesion molecule-1 (PECAM-1).

PECAM-1 is a 130-kDa member of the immunoglobulin (Ig) superfamily that is expressed on the surface of platelets and leukocytes, and at the intracellular junctions of confluent endothelial cell monolayers. Previous studies have shown that PECAM-1/PECAM-1 homophilic interactions play a key role in leukocyte transendothelial migration, in allowing PECAM-1 to serve as a mechanosensory complex in endothelial cells, in its ability to confer cytoprotection to proapoptotic stimuli, and in maintaining endothelial cell junctional integrity. To examine the adhesive properties of full-length PECAM-1 in a native lipid environment, we purified it from platelets and assembled it into phospholipid nanodiscs. PECAM-1-containing nanodiscs retained not only their ability to bind homophilically to PECAM-1-expressing cells, but exhibited regulatable adhesive interactions that could be modulated by ligands that bind membrane- proximal Ig Domain 6. This property was exploited to enhance the rate of barrier restoration in endothelial cell monolayers subjected to inflammatory challenge. The finding that the adhesive properties of PECAM-1 are regulatable suggests novel approaches for controlling endothelial cell migration and barrier function in a variety of vascular permeability disorders.

Did mineral surface chemistry and toxicity contribute to evolution of microbial extracellular polymeric substances?

Modern ecological niches are teeming with an astonishing diversity of microbial life in biofilms closely associated with mineral surfaces, which highlights the remarkable success of microorganisms in conquering the challenges and capitalizing on the benefits presented by the mineral-water interface. Biofilm formation capability likely evolved on early Earth because biofilms provide crucial cell survival functions. The potential toxicity of mineral surfaces toward cells and the complexities of the mineral-water-cell interface in determining the toxicity mechanisms, however, have not been fully appreciated. Here, we report a previously unrecognized role for extracellular polymeric substances (EPS), which form biofilms in shielding cells against the toxicity of mineral surfaces. Using colony plating and LIVE/DEAD staining methods in oxide suspensions versus oxide-free controls, we found greater viability of wild-type, EPS-producing strains of Pseudomonas aeruginosa PAO1 compared to their isogenic knockout mutant with defective biofilm-producing capacity. Oxide toxicity was specific to its surface charge and particle size. High resolution transmission electron microscopy (HRTEM) images and assays for highly reactive oxygen species (hROS) on mineral surfaces suggested that EPS shield via both physical and chemical mechanisms. Intriguingly, qualitative as well as quantitative measures of EPS production showed that toxic minerals induced EPS production in bacteria. By determining the specific toxicity mechanisms, we provide insight into the potential impact of mineral surfaces in promoting increased complexity of cell surfaces, including EPS and biofilm formation, on early Earth.

Cortical granule exocytosis in C. elegans is regulated by cell cycle components including separase.

In many organisms, cortical granules undergo exocytosis following fertilization, releasing cargo proteins that modify the extracellular covering of the zygote. We identified cortical granules in Caenorhabditis elegans and have found that degranulation occurs in a wave that initiates in the vicinity of the meiotic spindle during anaphase I. Previous studies identified genes that confer an embryonic osmotic sensitivity phenotype, thought to result from abnormal eggshell formation. Many of these genes are components of the cell cycle machinery. When we suppressed expression of several of these genes by RNAi, we observed that cortical granule trafficking was disrupted and the eggshell did not form properly. We conclude that osmotic sensitivity phenotypes occur because of defects in trafficking of cortical granules and the subsequent formation of an impermeable eggshell. We identified separase as a key cell cycle component that is required for degranulation. Separase localized to cortically located filamentous structures in prometaphase I upon oocyte maturation. After fertilization, separase disappeared from these structures and appeared on cortical granules by anaphase I. RNAi of sep-1 inhibited degranulation in addition to causing extensive chromosomal segregation failures. Although the temperature-sensitive sep-1(e2406) allele exhibited similar inhibition of degranulation, it had minimal effects on chromosome segregation. These observations lead us to speculate that SEP-1 has two separable yet coordinated functions: to regulate cortical granule exocytosis and to mediate chromosome separation.

Collagen reorganization at the tumor-stromal interface facilitates local invasion.

BACKGROUND: Stromal-epithelial interactions are of particular significance in breast tissue as misregulation of these interactions can promote tumorigenesis and invasion. Moreover, collagen-dense breast tissue increases the risk of breast carcinoma, although the relationship between collagen density and tumorigenesis is not well understood. As little is known about epithelial-stromal interactions in vivo, it is necessary to visualize the stroma surrounding normal epithelium and mammary tumors in intact tissues to better understand how matrix organization, density, and composition affect tumor formation and progression. METHODS: Epithelial-stromal interactions in normal mammary glands, mammary tumors, and tumor explants in three-dimensional culture were studied with histology, electron microscopy, and nonlinear optical imaging methodologies. Imaging of the tumor-stromal interface in live tumor tissue ex vivo was performed with multiphoton laser-scanning microscopy (MPLSM) to generate multiphoton excitation (MPE) of endogenous fluorophores and second harmonic generation (SHG) to image stromal collagen. RESULTS: We used both laser-scanning multiphoton and second harmonic generation microscopy to determine the organization of specific collagen structures around ducts and tumors in intact, unfixed and unsectioned mammary glands. Local alterations in collagen density were clearly seen, allowing us to obtain three-dimensional information regarding the organization of the mammary stroma, such as radiating collagen fibers that could not have been obtained using classical histological techniques. Moreover, we observed and defined three tumor-associated collagen signatures (TACS) that provide novel markers to locate and characterize tumors. In particular, local cell invasion was found predominantly to be oriented along certain aligned collagen fibers, suggesting that radial alignment of collagen fibers relative to tumors facilitates invasion. Consistent with this observation, primary tumor explants cultured in a randomly organized collagen matrix realigned the collagen fibers, allowing individual tumor cells to migrate out along radially aligned fibers. CONCLUSION: The presentation of these tumor-associated collagen signatures allowed us to identify pre-palpable tumors and see cells at the tumor-stromal boundary invading into the stroma along radially aligned collagen fibers. As such, TACS should provide indications that a tumor is, or could become, invasive, and may serve as part of a strategy to help identify and characterize breast tumors in animal and human tissues.

Involvement of the actin cytoskeleton and homotypic membrane fusion in ER dynamics in Caenorhabditis elegans.

The endoplasmic reticulum (ER) is the major intracellular membrane system. The ER is essential for protein and lipid biosynthesis, transport of proteins along the secretory pathway, and calcium storage. Here, we describe our investigations into the dynamics and regulation of the ER in the early Caenorhabditis elegans embryo. Using a GFP fusion to the ER-resident signal peptidase SP12, we observed the morphological transitions of the ER through fertilization and the early cell-cycles in living embryos. These transitions were tightly coordinated with the division cycle: upon onset of mitosis, the ER formed structured sheets that redispersed at the initiation of cleavage. Although microtubules were not required for the transition of the ER between these different states, the actin cytoskeleton facilitated the dispersal of the ER at the end of mitosis. The ER had an asymmetric distribution in the early embryo, which was dependent on the establishment of polarity by the PAR proteins. The small GTPase ARF-1 played an essential role in the ER dynamics, although this function appeared to be unrelated to the role of ARF-1 in vesicular traffic. In addition, the ER-resident heat shock protein BiP and a homologue of the AAA ATPase Cdc48/p97 were found to be crucial for the ER transitions. Both proteins have been implicated in homotypic ER membrane fusion. We provide evidence that homotypic membrane fusion is required to form the sheet structure in the early embryo.

The C. elegans hook protein, ZYG-12, mediates the essential attachment between the centrosome and nucleus.

The centrosome and nucleus are intimately associated in most animal cells, yet the significance of this interaction is unknown. Mutations in the zyg-12 gene of Caenorhabditis elegans perturb the attachment of the centrosome to the nucleus, giving rise to aberrant spindles and ultimately, DNA segregation defects and lethality. These phenotypes indicate that the attachment is essential. ZYG-12 is a member of the Hook family of cytoskeletal linker proteins and localizes to both the nuclear envelope (via SUN-1) and centrosomes. ZYG-12 is able to bind the dynein subunit DLI-1 in a two-hybrid assay and is required for dynein localization to the nuclear envelope. Loss of dynein function causes a low percentage of defective centrosome/nuclei interactions in both Drosophila and Caenorhabditis elegans. We propose that dynein and ZYG-12 move the centrosomes toward the nucleus, followed by a ZYG-12/SUN-1-dependent anchorage.

An integrated haplotype map of the human major histocompatibility complex.

Numerous studies have clearly indicated a role for the major histocompatibility complex (MHC) in susceptibility to autoimmune diseases. Such studies have focused on the genetic variation of a small number of classical human-leukocyte-antigen (HLA) genes in the region. Although these genes represent good candidates, given their immunological roles, linkage disequilibrium (LD) surrounding these genes has made it difficult to rule out neighboring genes, many with immune function, as influencing disease susceptibility. It is likely that a comprehensive analysis of the patterns of LD and variation, by using a high-density map of single-nucleotide polymorphisms (SNPs), would enable a greater understanding of the nature of the observed associations, as well as lead to the identification of causal variation. We present herein an initial analysis of this region, using 201 SNPs, nine classical HLA loci, two TAP genes, and 18 microsatellites. This analysis suggests that LD and variation in the MHC, aside from the classical HLA loci, are essentially no different from those in the rest of the genome. Furthermore, these data show that multi-SNP haplotypes will likely be a valuable means for refining association signals in this region.