Microsatellite markers based genetic diversity and bottleneck studies in Zanskari pony

Genetic diversity in Zanskari pony breed was evaluated at 48 microsatellite loci using fifty adult, healthy and unrelated animals. Allele frequency data was used to detect genetic diversity and bottleneck. The estimated average number of alleles (±s.e.) was 8.5208±2.5010 with a total of 409 alleles. A high level of genetic diversity within this breed was observed in terms of number of alleles, observed heterozygosity (0.6763±0.1704), expected Leven's heterozygosity (0.7724±0.795), expected Nei's heterozygosity (0.7644±0.0787) and polymorphism information content (>0.5). In-breeding coefficient (F(is)) was 0.115±0.0209, suggesting moderately high in-breeding in Zanskari breed. Although analysis of bottleneck revealed no bottleneck in recent past but population of Zanskari ponies has decreased drastically and only a few thousand pure-bred animals are left. The information is useful for proposing effective population management strategies for future.     

PHYRN: a robust method for phylogenetic analysis of highly divergent sequences

Both multiple sequence alignment and phylogenetic analysis are problematic in the "twilight zone" of sequence similarity (≤ 25% amino acid identity). Herein we explore the accuracy of phylogenetic inference at extreme sequence divergence using a variety of simulated data sets. We evaluate four leading multiple sequence alignment (MSA) methods (MAFFT, T-COFFEE, CLUSTAL, and MUSCLE) and six commonly used programs of tree estimation (Distance-based: Neighbor-Joining; Character-based: PhyML, RAxML, GARLI, Maximum Parsimony, and Bayesian) against a novel MSA-independent method (PHYRN) described here. Strikingly, at "midnight zone" genetic distances (~7% pairwise identity and 4.0 gaps per position), PHYRN returns high-resolution phylogenies that outperform traditional approaches. We reason this is due to PHRYN's capability to amplify informative positions, even at the most extreme levels of sequence divergence. We also assess the applicability of the PHYRN algorithm for inferring deep evolutionary relationships in the divergent DANGER protein superfamily, for which PHYRN infers a more robust tree compared to MSA-based approaches. Taken together, these results demonstrate that PHYRN represents a powerful mechanism for mapping uncharted frontiers in highly divergent protein sequence data sets.     

CXCL12/CXCR4 Protein Signaling Axis Induces Sonic Hedgehog Expression in Pancreatic Cancer Cells via Extracellular Regulated Kinase- and Akt Kinase-mediated Activation of Nuclear Factor κB: IMPLICATIONS FOR BIDIRECTIONAL TUMOR-STROMAL INTERACTIONS*

Recent evidence suggests a major role of tumor-stromal interactions in pancreatic cancer pathobiology. The chemokine CXCL12 (stromal cell-derived factor 1 (SDF-1)), abundantly produced by stromal cells, promotes progression, metastasis, and chemoresistance of pancreatic cancer cells. On the other hand, pancreatic tumor cell-derived sonic hedgehog (SHH) acts predominantly on stromal cells to induce desmoplasia and, thus, has a paracrine effect on tumorigenesis and therapeutic outcome. In this study, we examined the association between these two proteins of pathological significance in pancreatic cancer. Our data demonstrate that CXCL12 leads to a dose- and time-dependent up-regulation of SHH in pancreatic cancer cells. CXCL12-induced SHH up-regulation is specifically mediated through the receptor CXCR4 and is dependent on the activation of downstream Akt and ERK signaling pathways. Both Akt and ERK cooperatively promote nuclear accumulation of NF-κB by inducing the phosphorylation and destabilization of its inhibitory protein, IκB-α. Using dominant negative IκB-α, a SHH promoter (deletion mutant) reporter, and chromatin immunoprecipitation assays, we demonstrate that CXCL12 exposure enhances direct binding of NF-κB to the SHH promoter and that suppression of NF-κB activation abrogates CXCL12-induced SHH expression. Finally, our data demonstrate a strong correlative expression of CXCR4 and SHH in human pancreatic cancer tissues, whereas their expression is not observed in the normal pancreas. Altogether, our data reveal a novel mechanism underlying aberrant SHH expression in pancreatic cancer and identify a molecular link facilitating bidirectional tumor-stromal interactions.     

Temporal Analysis of Image-Rivalry Suppression

During binocular rivalry, perception alternates between two different images presented one to each eye. At any moment, one image is visible, dominant, while the other is invisible, suppressed. Alternations in perception during rivalry could involve competition between eyes, eye-rivalry, or between images, image-rivalry, or both. We measured response criteria, sensitivities, and thresholds to brief contrast increments to one of the rival stimuli in conventional rivalry displays and in a display in which the rival stimuli swapped between the eyes every 333 ms–swap rivalry–that necessarily involves image rivalry. We compared the sensitivity and threshold measures in dominance and suppression to assess the strength of suppression. We found that response criteria are essentially the same during dominance and suppression for the two sorts of rivalry. Critically, we found that swap-rivalry suppression is weak after a swap and strengthens throughout the swap interval. We propose that image rivalry is responsible for weak initial suppression immediately after a swap and that eye rivalry is responsible for the stronger suppression that comes later.     

The coronavirus endoribonuclease Nsp15 interacts with retinoblastoma tumor suppressor protein

Coronaviruses encode an endoribonuclease, Nsp15, which has a poorly defined role in infection. Sequence analysis revealed a retinoblastoma protein-binding motif (LXCXE/D) in the majority of the Nsp15 of the severe acute respiratory syndrome coronavirus (SARS-CoV) and its orthologs in the alpha and beta coronaviruses. The endoribonuclease activity of the SARS-CoV Nsp15 (sNsp15) was stimulated by retinoblastoma protein (pRb) in vitro, and the two proteins can be coimmunoprecipitated from cellular extracts. Mutations in the pRb-binding motif rendered sNsp15 to be differentially modified by ubiquitin in cells, and cytotoxicity was observed upon its expression. Expression of the sNsp15 in cells resulted in an increased abundance of pRb in the cytoplasm, decreased overall levels of pRb, an increased proportion of cells in the S phase of the cell cycle, and an enhanced expression from a promoter normally repressed by pRb. The endoribonuclease activity of the mouse hepatitis virus (MHV) A59 Nsp15 was also increased by pRb in vitro, and an MHV with mutations in the LXCXE/D-motif, named vLC, exhibited a smaller plaque diameter and reduced the virus titer by ～1 log. Overexpression of pRb delayed the viral protein production by wild-type MHV but not by vLC. This study reveals that pRb and its interaction with Nsp15 can affect coronavirus infection and adds coronaviruses to a small but growing family of RNA viruses that encode a protein to interact with pRb.     

Structural Plasticity of the Protein Plug That Traps Newly Packaged Genomes in Podoviridae Virions

Bacterial viruses of the P22-like family encode a specialized tail needle essential for genome stabilization after DNA packaging and implicated in Gram-negative cell envelope penetration. The atomic structure of P22 tail needle (gp26) crystallized at acidic pH reveals a slender fiber containing an N-terminal “trimer of hairpins” tip. Although the length and composition of tail needles vary significantly in Podoviridae, unexpectedly, the amino acid sequence of the N-terminal tip is exceptionally conserved in more than 200 genomes of P22-like phages and prophages. In this paper, we used x-ray crystallography and EM to investigate the neutral pH structure of three tail needles from bacteriophage P22, HK620, and Sf6. In all cases, we found that the N-terminal tip is poorly structured, in stark contrast to the compact trimer of hairpins seen in gp26 crystallized at acidic pH. Hydrogen-deuterium exchange mass spectrometry, limited proteolysis, circular dichroism spectroscopy, and gel filtration chromatography revealed that the N-terminal tip is highly dynamic in solution and unlikely to adopt a stable trimeric conformation at physiological pH. This is supported by the cryo-EM reconstruction of P22 mature virion tail, where the density of gp26 N-terminal tip is incompatible with a trimer of hairpins. We propose the tail needle N-terminal tip exists in two conformations: a pre-ejection extended conformation, which seals the portal vertex after genome packaging, and a postejection trimer of hairpins, which forms upon its release from the virion. The conformational plasticity of the tail needle N-terminal tip is built in the amino acid sequence, explaining its extraordinary conservation in nature.