PrimPol-deficient cells exhibit a pronounced G2 checkpoint response following UV damage

PrimPol is a recently identified member of the archaeo-eukaryote primase (AEP) family of primase-polymerases. It has been shown that this mitochondrial and nuclear localized enzyme plays roles in the maintenance of both unperturbed replication fork progression and in the bypass of lesions after DNA damage. Here, we utilized an avian (DT40) knockout cell line to further study the consequences of loss of PrimPol (PrimPol^?/?) on the downstream maintenance of cells after UV damage. We report that PrimPol^?/? cells are more sensitive to UV-C irradiation in colony survival assays than Pol η-deficient cells. Although this increased UV sensitivity is not evident in cell viability assays, we show that this discrepancy is due to an enhanced checkpoint arrest after UV-C damage in the absence of PrimPol. PrimPol^?/? arrested cells become stalled in G2, where they are protected from UV-induced cell death. Despite lacking an enzyme required for the bypass and maintenance of replication fork progression in the presence of UV damage, we show that PrimPol^?/? cells actually have an advantage in the presence of a Chk1 inhibitor due to their slow progression through S-phase.     

Microbiome Composition and Function Drives Wound-Healing Impairment in the Female Genital Tract

The mechanism(s) by which bacterial communities impact susceptibility to infectious diseases, such as HIV, and maintain female genital tract (FGT) health are poorly understood. Evaluation of FGT bacteria has predominantly been limited to studies of species abundance, but not bacterial function. We therefore sought to examine the relationship of bacterial community composition and function with mucosal epithelial barrier health in the context of bacterial vaginosis (BV) using metaproteomic, metagenomic, and in vitro approaches. We found highly diverse bacterial communities dominated by Gardnerella vaginalis associated with host epithelial barrier disruption and enhanced immune activation, and low diversity communities dominated by Lactobacillus species that associated with lower Nugent scores, reduced pH, and expression of host mucosal proteins important for maintaining epithelial integrity. Importantly, proteomic signatures of disrupted epithelial integrity associated with G. vaginalis-dominated communities in the absence of clinical BV diagnosis. Because traditional clinical assessments did not capture this, it likely represents a larger underrepresented phenomenon in populations with high prevalence of G. vaginalis. We finally demonstrated that soluble products derived from G. vaginalis inhibited wound healing, while those derived from L. iners did not, providing insight into functional mechanisms by which FGT bacterial communities affect epithelial barrier integrity.     

VIP and CRF reduce ADAMTS expression and function in osteoarthritis synovial fibroblasts

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family is known to play an important role in the pathogenesis of osteoarthritis (OA), working on aggrecan degradation or altering the integrity of extracellular matrix (ECM). Thus, the main purpose of our study was to define the role of vasoactive intestinal peptide (VIP) and corticotrophin‐releasing factor (CRF), as immunoregulatory neuropeptides, on ADAMTS production in synovial fibroblasts (SF) from OA patients and healthy donors (HD). OA‐ and HD‐SF were stimulated with pro‐inflammatory mediators and treated with VIP or CRF. Both neuropeptides decreased ADAMTS‐4, ‐5, ‐7 and ‐12 expressions, aggrecanase activity, glycosaminoglycans (GAG), and cartilage oligomeric matrix protein (COMP) degradation after stimulation with fibronectin fragments (Fn‐fs) in OA‐SF. After stimulation with interleukin‐1β, VIP reduced ADAMTS‐4 and ‐5, and both neuropeptides decreased ADAMTS‐7 production and COMP degradation. Moreover, VIP and CRF reduced Runx2 and β‐catenin activation in OA‐SF. Our data suggest that the role of VIP and CRF on ADAMTS expression and cartilage degradation could be related to the OA pathology since scarce effects were produced in HD‐SF. In addition, their effects might be greater when a degradation loop has been established, given that they were higher after stimulation with Fn‐fs. Our results point to novel OA therapies based on the use of neuropeptides, since VIP and CRF are able to stop the first critical step, the loss of cartilage aggrecan and the ECM destabilization during joint degradation.     

Fluram-Kemptide-Lys8 Non-radioactive Assay for Protein Kinase A

The cAMP-dependent protein kinase (PKA) is the best understood member of the superfamily of serine–threonine protein kinases and is involved in controlling a variety of cellular processes. Measurements of PKA activity traditionally relied on the use of [³²P]-labeled ATP as the phosphate donor and a protein or peptide substrate as the phosphoaceptor. Recently non-isotopic assays for the PKA have been developed and this paper presents an improvement of a fluorometric assay for measuring the activity of PKA. Three peptides were synthesized with the following sequences: LRRASLG (Kemptide), LRRASLGK (Kemptide-Lys8) and LRRASLGGGLRRASLG (Bis-Kemptide), these have in common the substrate sequence recognized by the PKA (RRXS/TΨ), where X is any amino acid and Ψ is a hydrophobic amino acid. Optimal conditions were established for the non-radioactive assay to detect the PKA activity by phosphorylation of these three peptides that are covalently linked to fluorescamine at their N-terminus. The phosphorylated and non-phosphorylated peptides were easily separated by electrophoresis, identified and quantified with optical densitometry and ultraviolet light. The fluorescamine-labeled Kemptide-Lys8 substrate (Fluram-Kemptide-Lys8) was used to calculate the Km and V_max of the catalytic subunit of PKA from pig heart and showed a detection limit of 260 pmol, a linear range between 700 and 1150 pmol with a linear regression R ² = 0.956.     

Focal adhesion kinase: predictor of tumour response and risk factor for recurrence after neoadjuvant chemoradiation in rectal cancer

Rectal cancer represents about 30% of colorectal cancers, being around 50% locally advanced at presentation. Chemoradiation (CRT) followed by total mesorectal excision is the standard of care for these locally advanced stages. However, it is not free of adverse effects and toxicity and the complete pathologic response rate is between 10% and 30%. This makes it extremely important to define factors that can predict response to this therapy. Focal adhesion kinase (FAK) expression has been correlated with worse prognosis in several tumours and its possible involvement in cancer radio‐ and chemosensitivity has been suggested; however, its role in rectal cancer has not been analysed yet. To analyse the association of FAK expression with tumour response to CRT in locally advanced rectal cancer. This study includes 73 patients with locally advanced rectal cancer receiving standard neoadjuvant CRT followed by total mesorectal excision. Focal adhesion kinase protein levels were immunohistochemically analysed in the pre‐treatment biopsies of these patients and correlated with tumour response to CRT and patients survival. Low FAK expression was significantly correlated with local and distant recurrence (P = 0.013). Low FAK expression was found to be a predictive marker of tumour response to neoadjuvant therapy (P = 0.007) and patients whose tumours did not express FAK showed a strong association with lower disease‐free survival (P = 0.01). Focal adhesion kinase expression predicts neoadjuvant CRT response in rectal cancer patients and it is a clinically relevant risk factor for local and distant recurrence.     

Role of the clam Neilonella sulculata as substrate for the hydroid Bougainvillia macloviana on muddy bottoms of San Jorge Gulf (Argentina,Southwest Atlantic)

The clam Neilonella sulculata is the dominant bivalve on muddy bottoms along the Argentine continental shelf (Southwest Atlantic). The medusa stage of the hydroid Bougainvillia macloviana is one of the most abundant species in the gelatinous zooplankton of the Magellan region. Two benthic surveys carried out in 2007 and 2009 in San Jorge Gulf gave us the opportunity to analyse (1) the interaction between N. sulculata and its epibiont, B. macloviana, and (2) the role of the clam as hard substrate in a depositional area characterized by silt/clay sediments. Bougainvillia macloviana has a high prevalence on the posterior region of live clams. The presence of gonophores shows that these small colonies are able to reproduce there. Hydroid colonies are very rare on dead valves of N. sulculata and also on almost all other live bivalve species in the study area. Specimens of N. sulculata with hydroids were slightly but significantly larger than those without them, both in 2007 and 2009. The high prevalence of B. macloviana on live individuals of N. sulculata and its almost total absence on other clam species suggest that this association may be critical at least for the epibiont and not the result of chance alone.     

Circadian rhythm of autophagy proteins in hippocampus is blunted by sleep fragmentation

Autophagy is essential for normal cellular survival and activity. Circadian rhythms of autophagy have been studied in several peripheral organs but not yet reported in the brain. Here, we measured the circadian rhythm of autophagy-related proteins in mouse hippocampus and tested the effect of sleep fragmentation (SF). Expressions of the autophagy-related proteins microtubule‐associated protein 1 light chain 3 (LC3) and beclin were determined by western blotting and immunohistochemistry. Both the hippocampal LC3 signal and the ratio of its lipid-conjugated form LC3-II to its cytosolic form LC3-I showed a 24 h rhythm. The peak was seen at ZT6 (1 pm) and the nadir at ZT16 (1 am). The LC3 immunoreactivity in hippocampal CA1 pyramidal neurons also distributed differently, with more diffuse cytoplasmic appearance at ZT16. Chronic SF had a mild effect to disrupt the 24 h rhythm of LC3 and beclin expression. Interestingly, a greater effect of SF was seen after 24 h of recovery sleep when LC3-II expression was attenuated at both the peak and trough of circadian activities. Overall, the results show for the first time that the hippocampus has a distinct rhythm of autophagy that can be altered by SF.     

To Smell you Better: Prior Food Deprivation Increases Herbivore Insect Responsiveness to Host Plant Odor Cues

Hunger plays a crucial role in insect feeding behavior, however food deprivation is rarely considered when insect responses to plant host and related chemical stimuli are investigated. Here we assessed, by means of experiments with Y-tube olfactometer, the effect of food deprivation time on the response of a specialist (Xanthogaleruca luteola) and a generalist (Diabrotica speciosa) herbivore beetle species (Coleoptera: Chrysomelidae) to odor cues of their respective host plants. Increasing food deprivation periods enhanced responses to host plant odor in both species, with insects remaining for longer in the olfactometer arm carrying plant odor than in the control, moving less frequently between olfactometer arms, and being more efficient in moving towards the plant odor as their first choice. These trends were less significant in the generalist species, which also required a longer fasting threshold (48 h) in comparison with the specialist (8 h). Our results, showing that prior food deprivation time can influence insect herbivore responsiveness to plant stimuli and that those effects may vary between species, highlight the risk of neglecting this factor in studies involving insect responses to host or chemical stimuli.