Species Phylogeny versus Gene Trees: A Case Study of an Incongruent Data Matrix Based on Paphiopedilum Pfitz. (Orchidaceae)

The phylogeny of the genus Paphiopedilum based on the plastome is consistent with morphological analysis. However, to date, none of the analyzed nuclear markers has confirmed this. Topology incongruence among the trees of different nuclear markers concerns entire sections of the subgenus Paphiopedilum. The low-copy nuclear protein-coding gene PHYC was obtained for 22 species representing all sections and subgenera of Paphiopedilum. The nuclear-based phylogeny is supported by morphological characteristics and plastid data analysis. We assumed that an incongruence in nuclear gene trees is caused by ancestral homoploid hybridization. We present a model for inferring the phylogeny of the species despite the incongruence of the different tree topologies. Our analysis, based on six low-copy nuclear genes, is congruent with plastome phylogeny and has been confirmed by phylogenetic network analysis.     

Scleroderma-like Impairment in the Network of Telocytes/CD34+ Stromal Cells in the Experimental Mouse Model of Bleomycin-Induced Dermal Fibrosis

Considerable evidence accumulated over the past decade supports that telocytes (TCs)/CD34⁺ stromal cells represent an exclusive type of interstitial cells identifiable by transmission electron microscopy (TEM) or immunohistochemistry in various organs of the human body, including the skin. By means of their characteristic cellular extensions (telopodes), dermal TCs are arranged in networks intermingled with a multitude of neighboring cells and, hence, they are thought to contribute to skin homeostasis through both intercellular contacts and releasing extracellular vesicles. In this context, fibrotic skin lesions from patients with systemic sclerosis (SSc, scleroderma) appear to be characterized by a disruption of the dermal network of TCs, which has been ascribed to either cell degenerative processes or possible transformation into profibrotic myofibroblasts. In the present study, we utilized the well-established mouse model of bleomycin-induced scleroderma to gain further insights into the TC alterations found in cutaneous fibrosis. CD34 immunofluorescence revealed a severe impairment in the dermal network of TCs/CD34⁺ stromal cells in bleomycin-treated mice. CD31/CD34 double immunofluorescence confirmed that CD31⁻/CD34⁺ TC counts were greatly reduced in the skin of bleomycin-treated mice compared with control mice. Ultrastructural signs of TC injury were detected in the skin of bleomycin-treated mice by TEM. The analyses of skin samples from mice treated with bleomycin for different times by either TEM or double immunostaining and immunoblotting for the CD34/α-SMA antigens collectively suggested that, although a few TCs may transition to α-SMA⁺ myofibroblasts in the early disease stage, most of these cells rather undergo degeneration, and then are lost. Taken together, our data demonstrate that TC changes in the skin of bleomycin-treated mice mimic very closely those observed in human SSc skin, which makes this experimental model a suitable tool to (i) unravel the pathological mechanisms underlying TC damage and (ii) clarify the possible contribution of the TC loss to the development/progression of dermal fibrosis. In perspective, these findings may have important implications in the field of skin regenerative medicine.     

Synthesis of new polyesters containing phosphorus heterocycles and halogen

Two series of polyesters containing phenoxaphosphine rings and several halogens were synthesized by low-temperature solution polycondensation in the presence of triethylamine. One series, containing halogens only in the bisphenol moiety, was obtained from 2,8-dichloroformyl-10-phenylphenoxaphosphine-10-oxide and chlorinated bisphenol. In the second series, halogens belonged both to bisphenol and diacid dichlorides. For the last series, 2,8-dichloroformyl-10-(4-bromophenyl)phenoxaphosphine-10-oxide was prepared as a new monomer. All resulting polyesters were characterized by elemental analysis, reduced viscosity, IR and ¹H NMR spectroscopy, and thermogravimetric analysis. These polymers began to lose weight at about 400°C in air being seen as self-extinguishing ones.     

Production and purification of a fused recombinant protein gp-36 (HIV-2) from Escherichia coli

A fragment of the gp-36 gene of the Human Immunodeficiency Virus type 2 (HIV-2) was fused to a stabilizer sequence, which encodes for the first N-terminal 58 amino acids of the human interleukin-2. The fused protein was expressed under the control of the tryptophan promoter in Escherichia coli, and expressed as 20% of the total cellular protein. Transmission electron microscopy indicated that the fusion protein formed cytoplasmic insoluble inclusion bodies. Inclusion bodies were semipurified by a wash pellet cell procedure, rendering a material with a purity higher than 70% by SDS–polyacrylamide gel electrophoresis. After solubilization with urea, this preparation was further purified by gel-filtration chromatography up to 95% purity.     

Sample Preparation as a Critical Aspect of Blood Platelet Mitochondrial Respiration Measurements—The Impact of Platelet Activation on Mitochondrial Respiration

Blood platelets are considered as promising candidates as easily-accessible biomarkers of mitochondrial functioning. However, their high sensitivity to various stimulus types may potentially affect mitochondrial respiration and lead to artefactual outcomes. Therefore, it is crucial to identify the factors associated with platelet preparation that may lead to changes in mitochondrial respiration. A combination of flow cytometry and advanced respirometry was used to examine the effect of blood anticoagulants, the media used to suspend isolated platelets, respiration buffers, storage time and ADP stimulation on platelet activation and platelet mitochondria respiration. Our results clearly show that all the mentioned factors can affect platelet mitochondrial respiration. Briefly, (i) the use of EDTA as anticoagulant led to a significant increase in the dissipative component of respiration (LEAK), (ii) the use of plasma for the suspension of isolated platelets with MiR05 as a respiration buffer allows high electron transfer capacity and low platelet activation, and (iii) ADP stimulation increases physiological coupling respiration (ROUTINE). Significant associations were observed between platelet activation markers and mitochondrial respiration at different preparation steps; however, the fact that these relationships were not always apparent suggests that the method of platelet preparation may have a greater impact on mitochondrial respiration than the platelet activation itself.