Characterization of a thermostable archaeal polynucleotide kinase homologous to human Clp1

Clp1 proteins are essential components of the eukaryal mRNA 3′ cleavage-polyadenylation machinery. Human Clp1 has an additional function as an RNA-specific 5′-OH polynucleotide kinase, which is implicated in RNA end healing. Yeast Clp1 has no kinase activity, although it binds ATP. Here we report that Clp1-like proteins are extant in archaea. Purification and characterization of Pyrococcus horikoshii Clp1 (PhoClp1) reveals it to be a thermostable 5′-OH polynucleotide kinase optimally active at 55°C to 85°C. PhoClp1 catalyzes transfer of the gamma phosphate from ATP (K _m 16 μM) to either 5′-OH RNA or DNA ends, although it prefers RNA in a competitive situation. Increasing the monovalent salt concentration to 250 mM suppresses the DNA kinase without affecting RNA phosphorylation, suggesting that RNA is a likely substrate for this enzyme in vivo. Indeed, we show that expression of PhoClp1 in budding yeast can complement a lethal mutation in the 5′-OH RNA kinase module of tRNA ligase. PhoClp1 is a member of the P-loop phosphotransferase superfamily. Alanine mutations at the P-loop lysine (Lys49) and a conserved aspartate (Asp73) inactivate the kinase. Our studies fortify emerging evidence for an enzymatic RNA repair capacity in archaea and provide a new reagent for polynucleotide phosphorylation at high temperatures.     

Attenuation corrected-optical coherence tomography for quantitative assessment of skin wound healing and scar morphology

Imaging the structural modifications of underlying tissues is vital to monitor wound healing. Optical coherence tomography (OCT) images high-resolution sub-surface information, but suffers a loss of intensity with depth, limiting quantification. Hence correcting the attenuation loss is important. We performed swept source-OCT of full-thickness excision wounds for 300 days in mice skin. We used single-scatter attenuation models to determine and correct the attenuation loss in the images. The phantom studies established the correspondence of corrected-OCT intensity (reflectivity) with matrix density and hydration. We histologically validated the corrected-OCT and measured the wound healing rate. We noted two distinct phases of healing—rapid and steady-state. We also detected two compartments in normal scars using corrected OCT that otherwise were not visible in the OCT scans. The OCT reflectivity in the scar compartments corresponded to distinct cell populations, mechanical properties and composition. OCT reflectivity has potential applications in evaluating the therapeutic efficacy of healing and characterizing scars.     

Antimicrobial and bone repair effects of boric acid in a rat model of dry socket (alveolar osteitis) following dental extraction

BackgroundAlveolitis occurs after dental extraction without blood clot formation, leading to an inflammatory process and bacterial contamination. Boric acid (BA) demonstrates anti-inflammatory, antimicrobial, and osteogenic properties. This study aims to evaluate the possible antimicrobial effects and bone repair of BA in a rat model of alveolitis (dry socket).Methods33 male Wistar rats were submitted to the extraction of the upper right incisor and dry socket induction. They were first divided into two groups: dry socket (n = 17) and dry socket + 0.75 % BA (n = 16). Samples for the microbiological analysis were collected immediately after dental extraction, at the detection of clinical alveolitis, 7, and 14 days after BA application. For microCT and histological analysis, samples from euthanized rats were used in 14 and 28 days after alveolitis detection.ResultsHigher bacterial counts were found in 4–5 days after alveolitis induction, compared to the baseline in both experimental groups, decreasing significantly after 7 and 14 days of treatment with BA (P < 0.05). The microCT evaluation displayed increased bone volume, bone volume fraction, trabecular thickness, and bone mineral density in a time-dependent manner, regardless of BA treatment. On the other hand, the number of trabeculae and total bone porosity decreased over the 28 days of the experiment in the dry-socket group and both groups, respectively (P < 0.05). Histological analysis did not differ on bone repair in both experimental groups.ConclusionThis was the first report investigating the effects of BA in a rat model of alveolitis regarding microbiological and bone repair aspects. The BA local application decreased the total aerobic and facultative bacteria counts and does not seem to benefit the bone repair after alveolitis development. This study paves the way for more studies involving alveolitis and different BA applications.     

Neurokinin-1-tachykinin receptor agonist promotes diabetic fracture healing in rats with type 1 diabetes via modulation of Wnt/β-catenin signalling axis

Diabetes mellitus is an ill-famed metabolic disorder with varied repercussions including delayed fracture healing. Wnt/β-catenin axis is known to play a tight pivotal role in the bone healing process. Substance P (SubP) is a neuropeptide with established positive modulatory functions in fracture healing and associated neuronal milieu. In this study, we performed local delivery of recombinant adenovirus of Dickkopf-1 (DKK1) into the fracture site to understand the antagonizing the role of DKK1 against substance P. Rats were segregated into 4 groups: (i) Fractured non-diabetic rats; (ii) Fractured T1D rats; T1D was provoked by using STZ 50 mg/kg for 5 consecutive days; (iii) Fractured T1D + SubP (50 mg/ml/Kg; i.p.; 30 min prior to fracture procedure); (iv) Fractured T1D + SubP + Ad-DKK1. Bone radiographs were taken using a Faxitron X-ray machine and the residual gap size was measured using an electric caliper. Western blotting was also performed to determine the protein expression levels of osteogenic markers (RUNX2, OSTX and OSTC) bone resorption markers (OPG, RANKL and RANK) and also Wnt-signalling markers (β-catenin, LRP5 and GSK-3β). We observed that SubP promoted osteogenesis (as indicated by RUNX2, OSTX and OSTC upregulation) and mitigated the bone resorption (as indicated by optimized OPG/RANKL/RANK axis) via activated Wnt signalling (manifested by upmodulated β-catenin and LRP5, with downmodulated GSK-3β levels. Activation of endogenous SubP or administration of exogenous mimics might counter-protect the fractured bone against the deforming effects of T1D.     

A biophysically-defined hyaluronic acid-based compound accelerates migration and stimulates the production of keratinocyte-derived neuromodulators

Hyaluronic acid (HA) preparations are widely used in clinical practice and recent data suggest that commercially available HA-based compounds promote ulcer re-epithelialization and induce pain relief. However, the pathophysiological basis of these effects remains poorly understood. In the present study, we investigated the biophysical, biomolecular and functional properties of a HA preparation combined with a pool of collagen precursor synthetic aminoacids, namely l-proline, l-leucine, l-lysine and glycine (Aminogam®). Hydrodynamic characterization of Aminogam® by size exclusion chromatography-triple detector array (SEC-TDA) revealed an average molecular weight in the range of 700–1700 kDa. Rheological measurements of the 1700kDa M_w lot showed a pseoudoplastic behaviour with a zero-shear viscosity (η₀) equal to 90 ± 9 Pa?s at 25°C and 55 ± 6 Pa?s at 37°C. Automated time-lapse videomicroscopy studies in a fibroblast-free system demonstrated that 1% (v/v) Aminogam® significantly reduced the healing time of wounded keratinocyte monolayers. In AKGOS assays, Aminogam® stimulated cellular locomotion (chemokinesis) and directional migration (chemotaxis) of keratinocytes. Analysis of microarray data suggested that keratinocytes had a functional neuroendocrine machinery, and this was confirmed by testing the secretion of six neuroactive molecules by ELISA, namely α-MSH, β-endorphins, melatonin, substance P, cortisol, and neurotensin. Interestingly, Aminogam® regulated the production of several neuropeptides, including β-endorphins. In conclusion, our data shed light on the epithelial-dependent mechanisms that underlie the efficacy of Aminogam®, particularly in reference to wound healing and nociception.     

Mitochondrial dysfunction and oxidative stress in diabetic wound

Diabetic wounds nowadays have become a major health challenge with the changes of the disease spectrum. Mitochondria are closely associated with stubborn nonhealing diabetic wounds for their vital role in energy metabolism, redox homeostasis, and signal transduction. There is significant mitochondrial dysfunction and oxidative stress in diabetic wounds. However, the contribution of mitochondrial dysfunction in oxidative stress induced nonhealing diabetic wound is still not fully understood. In this review, we will briefly summarize the current knowledge of the reported signaling pathways and therapeutic strategies involved in mitochondrial dysfunction in diabetic wounds. The findings provide further understanding of strategies that focus on mitochondria in diabetic wound treatment.     

Wound‐healing evaluation of entrapped active agents into protein microspheres over cellulosic gauzes

The use of active ingredients in wound management have evolved alongside the pharmaceutical agents and dressings used to deliver them. However, the development of gauzes, dressings with specific properties, still remains a challenge for several medical applications. A new methodology for the controlled release of active components for the healing of burn wounds is proposed herein. Cotton and non‐woven bandages have been cationised to promote the attachment of protein microspheres. The active agents, piroxicam and vegetable oil, were entrapped into the microspheres using ultrasound energy. Active agents were released from the microspheres by a change in pH. Wound healing was assessed through the use of standardised burn wounds induced by a cautery in human full‐thickness skin equivalents (EpidermFT). The best re‐epithelialisation and fastest wound closure was observed in wounds treated with proteinaceous microspheres attached to gauzes, after six days of healing, in comparison with commercial collagen dressing and other controls. Furthermore, the ability of these materials to reduce the inflammation process, together with healing improvement, makes these biomaterials suitable for wound‐dressing applications.     

Host genetic factors in American cutaneous leishmaniasis: a critical appraisal of studies conducted in an endemic area of Brazil

American cutaneous leishmaniasis (ACL) is a vector-transmitted infectious disease with an estimated 1.5 million new cases per year. In Brazil, ACL represents a significant public health problem, with approximately 30,000 new reported cases annually, representing an incidence of 18.5 cases per 100,000 inhabitants. Corte de Pedra is in a region endemic for ACL in the state of Bahia (BA), northeastern Brazil, with 500-1,300 patients treated annually. Over the last decade, population and family-based candidate gene studies were conducted in Corte de Pedra, founded on previous knowledge from studies on mice and humans. Notwithstanding limitations related to sample size and power, these studies contribute important genetic biomarkers that identify novel pathways of disease pathogenesis and possible new therapeutic targets. The present paper is a narrative review about ACL immunogenetics in BA, highlighting in particular the interacting roles of the wound healing gene FLI1 with interleukin-6 and genes SMAD2 and SMAD3 of the transforming growth factor beta signalling pathway. This research highlights the need for well-powered genetic and functional studies on Leishmania braziliensis infection as essential to define and validate the role of host genes in determining resistance/susceptibility regarding this disease.