The crystal structure of P. knowlesi DBPalpha DBL domain and its implications for immune evasion

Plasmodium vivax invasion of human erythrocytes requires that the ligand domain of the Duffy-binding protein (DBP) recognize its cognate erythrocyte receptor, making DBP a potential target for therapy. The recently determined crystal structure of the orthologous DBP ligand domain of the closely related simian malaria parasite Plasmodium knowlesi provides insight into the molecular basis for receptor recognition and raises important questions about the mechanism of immune evasion employed by the malaria parasite.     

The rate of polymerase release upon filling the gap between Okazaki fragments is inadequate to support cycling during lagging strand synthesis

Upon completion of synthesis of an Okazaki fragment, the lagging strand replicase must recycle to the next primer at the replication fork in under 0.1 s to sustain the physiological rate of DNA synthesis. We tested the collision model that posits that cycling is triggered by the polymerase encountering the 5′-end of the preceding Okazaki fragment. Probing with surface plasmon resonance, DNA polymerase III holoenzyme initiation complexes were formed on an immobilized gapped template. Initiation complexes exhibit a half-life of dissociation of approximately 15 min. Reduction in gap size to 1 nt increased the rate of dissociation 2.5-fold, and complete filling of the gap increased the off-rate an additional 3-fold (t_1/2  ∼ 2 min). An exogenous primed template and ATP accelerated dissociation an additional 4-fold in a reaction that required complete filling of the gap. Neither a 5′-triphosphate nor a 5′-RNA terminated oligonucleotide downstream of the polymerase accelerated dissociation further. Thus, the rate of polymerase release upon gap completion and collision with a downstream Okazaki fragment is 1000-fold too slow to support an adequate rate of cycling and likely provides a backup mechanism to enable polymerase release when the other cycling signals are absent. Kinetic measurements indicate that addition of the last nucleotide to fill the gap is not the rate-limiting step for polymerase release and cycling. Modest (approximately 7 nt) strand displacement is observed after the gap between model Okazaki fragments is filled. To determine the identity of the protein that senses gap filling to modulate affinity of the replicase for the template, we performed photo-cross-linking experiments with highly reactive and non-chemoselective diazirines. Only the α subunit cross-linked, indicating that it serves as the sensor.     

Whole-genome sequence analysis reveals differences in population management and selection of European low-input pig breeds

Background

A major concern in conservation genetics is to maintain the genetic diversity of populations. Genetic variation in livestock species is threatened by the progressive marginalisation of local breeds in benefit of high-output pigs worldwide. We used high-density SNP and re-sequencing data to assess genetic diversity of local pig breeds from Europe. In addition, we re-sequenced pigs from commercial breeds to identify potential candidate mutations responsible for phenotypic divergence among these groups of breeds.

Results

Our results point out some local breeds with low genetic diversity, whose genome shows a high proportion of regions of homozygosis (>50%) and that harbour a large number of potentially damaging mutations. We also observed a high correlation between genetic diversity estimates using high-density SNP data and Next Generation Sequencing data (r = 0.96 at individual level). The study of non-synonymous SNPs that were fixed in commercial breeds and also in any local breed, but with different allele, revealed 99 non-synonymous SNPs affecting 65 genes. Candidate mutations that may underlie differences in the adaptation to the environment were exemplified by the genes AZGP1 and TAS2R40. We also observed that highly productive breeds may have lost advantageous genotypes within genes involve in immune response – e.g. IL12RB2 and STAB1–, probably as a result of strong artificial in the intensive production systems in pig.

Conclusions

The high correlation between genetic diversity computed with the 60K SNP and whole genome re-sequence data indicates that the Porcine 60K SNP Beadchip provides reliable estimates of genomic diversity in European pig populations despite the expected bias. Moreover, this analysis gave insights for strategies to the genetic characterization of local breeds. The comparison between re-sequenced local pigs and re-sequenced commercial pigs made it possible to report candidate mutations to be responsible for phenotypic divergence among those groups of breeds. This study highlights the importance of low input breeds as a valuable genetic reservoir for the pig production industry. However, the high levels of ROHs, inbreeding and potentially damaging mutations emphasize the importance of the genetic characterization of local breeds to preserve their genomic variability.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-601) contains supplementary material, which is available to authorized users.     

Frequent Seizures Are Associated with a Network of Gray Matter Atrophy in Temporal Lobe Epilepsy with or without Hippocampal Sclerosis

Objective

Patients with temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) have diffuse subtle gray matter (GM) atrophy detectable by MRI quantification analyses. However, it is not clear whether the etiology and seizure frequency are associated with this atrophy. We aimed to evaluate the occurrence of GM atrophy and the influence of seizure frequency in patients with TLE and either normal MRI (TLE-NL) or MRI signs of HS (TLE-HS).

Methods

We evaluated a group of 172 consecutive patients with unilateral TLE-HS or TLE-NL as defined by hippocampal volumetry and signal quantification (122 TLE-HS and 50 TLE-NL) plus a group of 82 healthy individuals. Voxel-based morphometry was performed with VBM8/SPM8 in 3T MRIs. Patients with up to three complex partial seizures and no generalized tonic-clonic seizures in the previous year were considered to have infrequent seizures. Those who did not fulfill these criteria were considered to have frequent seizures.

Results

Patients with TLE-HS had more pronounced GM atrophy, including the ipsilateral mesial temporal structures, temporal lobe, bilateral thalami and pre/post-central gyri. Patients with TLE-NL had more subtle GM atrophy, including the ipsilateral orbitofrontal cortex, bilateral thalami and pre/post-central gyri. Both TLE-HS and TLE-NL showed increased GM volume in the contralateral pons. TLE-HS patients with frequent seizures had more pronounced GM atrophy in extra-temporal regions than TLE-HS with infrequent seizures. Patients with TLE-NL and infrequent seizures had no detectable GM atrophy. In both TLE-HS and TLE-NL, the duration of epilepsy correlated with GM atrophy in extra-hippocampal regions.

Conclusion

Although a diffuse network GM atrophy occurs in both TLE-HS and TLE-NL, this is strikingly more evident in TLE-HS and in patients with frequent seizures. These findings suggest that neocortical atrophy in TLE is related to the ongoing seizures and epilepsy duration, while thalamic atrophy is more probably related to the original epileptogenic process.     

Tyrosine-specific MAPK phosphatases and the control of ERK signaling in PC12 cells

Background

Spatio-temporal control of extracellular signal-regulated kinase (ERK) activity, a critical determinant of the cell's response to growth factors, requires timely dephosphorylation of its regulatory tyrosine and/or threonine residue by MAPK phosphatases. We studied the physiological role of kinase interaction motif (KIM)-containing protein tyrosine phosphatases (PTPs) in the control of EGF- and NGF-induced ERK activity in neuroendocrine PC12 cells.

Results

We found a single KIM-containing PTP to be endogenously expressed in rat PC12 cells: the transmembrane PTPRR isoform termed PCPTP1. Protein knock-down of PCPTP1, or fourfold overexpression of its mouse orthologue, PTPBR7, left EGF- and NGF-induced ERK1/2 activity in PC12 cells unaltered. Ectopic expression of cytosolic PTPRR isoforms, however, resulted in reduced EGF-induced ERK1/2 activity, an effect that was dependent on the phosphatase activity and the KIM-domain of these PTPs.

Conclusion

The finding that robust changes in tyrosine-specific MAPK phosphatase expression levels have minor effects on temporal ERK1/2 activity control in PC12 cells suggests that dual-specificity MAPK phosphatases may act as major regulators of growth factor-induced ERK1/2 signaling in these cells.     

Ubiquitin crosstalk connecting cellular processes

Cullin 4 (Cul4), a member of the evolutionally conserved cullin protein family, serves as a scaffold to assemble multisubunit ubiquitin E3 ligase complexes. Cul4 interacts with the Ring finger-containing protein ROC1 through its C-terminal cullin domain and with substrate recruiting subunit(s) through its N-terminus. Previous studies have demonstrated that Cul4 E3 ligase ubiquitylates key regulators in cell cycle control and mediates their degradation through the proteasomal pathway, thus contributing to genome stability. Recent studies from several groups have revealed that Cul4 E3 ligase can target histones for ubiquitylation, and importantly, ubiquitylation of histones may facilitate the cellular response to DNA damage. Therefore, histone ubiquitylation by Cul4 E3 ligase constitutes a novel mechanism through which Cul4 regulates chromatin function and maintains genomic integrity. We outline these studies and suggest that histone ubiquitylation might play important roles in Cul4-regualted chromatin function including the cellular response to DNA damage and heterochromatin gene silencing.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Comparison of linkage disequilibrium and haplotype diversity on macro- and microchromosomes in chicken

Background

Toll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens.

Results

The study presents association between TLR gene mutations and increased susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 – Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further in silico analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs.

Conclusion

The most critical positions that may alter the pathogen recognition ability of TLR were: the 9^th amino acid position in LRR motif (TLR1–LRR10) and 4^th residue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.