The electron transport system of Alcaligenes eutrophus H16

In a previous work (Kömen et al. 1991) it has been concluded that membrane fragments isolated from autotrophically grown Alcaligenes eutrophus H16 contain several iron-sulphur centres along with haems of a-, b-, c-, and d-type. These redox components have been proposed to be part of a branched respiratory chain leading to multiple membrane bound oxidases. Here, some of the respiratory activities catalyzed by membrane fragments from wild type cells of A. eutrophus (H16) and, for comparison, Paracoccus denitrificans, have been investigated through the use of electron transport inhibitors. Cyanide (CN^-) titration curves indicated that in A. eutrophus H16 oxidation of succinate and H₂ preferentially proceeds via the cytochrome c oxidase(s) branch (I ₅₀=2 · 10^-5 M) whereas the NADH dependent respiration started being inhibited at higher CN^- concentrations (I ₅₀=5 · 10^-4 M). In membranes isolated from both, cells harvested at late growth-phase (OD 12) and from a mutant deficient in cytochrome c oxidase activity (A. eutrophus RK1), respiration was insensitive to low CN^- concentrations (< 10^-4 M), and it was sustained by the high catalytic activities of two quinol oxidases. These alternative oxidases of b- (formally o-) and d-type showed different sensitivities to KCN (I ₅₀=10^-3 M and 10^-2 M, respectively). Interestingly, the cytochrome c oxidase(s) dependent respiration of H16 membranes was insensitive to antimycin A but largely inhibited by myxothiazol (10^-6 M). This, and previous work (Kömen et al. 1991), suggest that although the respiratory chain of A. eutrophus is endowed with a putative bc ₁ complex, its biochemical nature and role in respiration of this organism are apparently different from those of P. denitrificans. The peculiarity of the respiratory chain of A. eutrophus is confirmed by the rotenone insensitivity of the NADH oxidation in both protoplasts and membrane fragments from wild type and soluble hydrogenase deficient cells (HF14 and HF160). A tentative model of the respiratory chain of autotrophically grown A. eutrophus is presented.     

DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays

The nick translation and terminal transferase assays have been compared to test their relative efficiency in detecting DNA breakage in ejaculated human spermatozoa. The results have been correlated with the percentage of chromomycin A3 positive sperm, a fluorochrome that is indicative of the protamination state of sperm. Examination of the ejaculated sperm of 30 subjects revealed that the percentage of positivity to the nick translation and terminal transferase assays did not differ, even when using different fixatives. It is concluded that the inability of the two assays to distinguish the type of DNA damage, as is possible in somatic nuclei, is most probably linked to the unique nature of sperm chromatin. It is proposed that the presence of the damaged DNA may be the remnants of an imperfect spermiogenesis, probably related to an inadequate protamine deposition. This is supported by the strong correlation between the presence of DNA damage and underprotamination as evidenced by chromomycin A3. © Chapman & Hall     

Candida pruni sp. nov. is a new yeast species with antagonistic potential against brown rot of peaches

Brown rot caused by Monilinia spp. is among the most important postharvest diseases of commercially grown stone fruits, and application of antagonistic yeasts to control brown rot is one promising strategy alternative to chemical fungicides. In this research, new yeast strains were isolated and tested for their activity against peach brown rot caused by Monilinia fructicola. Three yeast strains were originally isolated from the surface of plums (cv Chinese Angelino) collected in the north of China. In artificially wounded inoculation tests, the yeast reduced the brown rot incidence to 20 %. The population of the yeast within inoculated wounds on peaches significantly increased at 25 °C from an initial level of 5.0 × 10⁶ to 4.45 × 10⁷ CFU per wound after 1 day. The antagonistic strains were belonging to a new species of the genus Candida by sequence comparisons of 26 S rDNA D1/D2 domain and internal transcribed spacer region. The strains are most closely related to C. asparagi, C. musae and C. fructus on the basis of the phylogenetic trees based on the D1/D2 region of 26S rDNA. However, the strains are notably different from C. asparagi, C. musae and C. fructus, in morphological and physiological characteristics. Therefore, the name Candida pruni is proposed for the novel species, with sp-Quan (=CBS12814^T = KCTC 27526^T = GCMC 6582^T) as the type strain. Our study showed that Candida pruni is a novel yeast species with potential biocontrol against brown rot caused by M. fructicola on peaches.     

Molecular phylogeny and host-plant use (Lamiaceae) of the Thymogethes pollen beetles (Coleoptera)

The 24 members of the Euro-Asiatic genus Thymogethes are highly specialized pollen beetles associated as larvae with flowers of Lamiaceae Nepetoideae. All members of the genus were analysed in within the framework of an integrative taxonomy approach, which was aimed to reconstruct the phylogenetic relationships, as well as the possible pattern of evolution of their larval-host-plant association. Evidence from multiple molecular markers [COI; 16S; H3], combined with an estimation of divergence times using an average rate of 0.0177 substitutions/site/My among branches, placed the origin of the genus at a minimum of 9–10 Mya. This date of origin approximates the known evolution of the host plants in Euro-Mediterranean areas. Evidence from combined molecular and cladistic morphological analyses resulted in suitable agreement with the previously established morphology-based systematics of the genus, although members of the exilis species-group were split into three clades. The only disagreement between results of this new combined phylogeny and previous classification is in the exclusion of “Thymogethes” grenieri. This species is herein positioned outside the genus, based on molecular evidence. Our analysis depicts several Thymogethes species differentiating in the last few Mys, specifically those included in the T. lugubris species-group. Combined evidence from DNA, morphology and ancestral state parsimony reconstruction of larval-host-plant associations suggests that subtribe Menthinae likely represents the ancestral host plants, with a series of independent host shifts during the radiation of the clade, in association first with Menthinae and subsequently with Lavandulinae and Nepetinae. Steno-oligophagy is the most frequent (86%) condition, while strictly monophagous species are less numerous (14%).     

The Ediacaran emergence of bilaterians: congruence between the genetic and the geological fossil records

Unravelling the timing of the metazoan radiation is crucial for elucidating the macroevolutionary processes associated with the Cambrian explosion. Because estimates of metazoan divergence times derived from molecular clocks range from quite shallow (Ediacaran) to very deep (Mesoproterozoic), it has been difficult to ascertain whether there is concordance or quite dramatic discordance between the genetic and geological fossil records. Here, we show using a range of molecular clock methods that the major pulse of metazoan divergence times was during the Ediacaran, which is consistent with a synoptic reading of the Ediacaran macrobiota. These estimates are robust to changes in priors, and are returned with or without the inclusion of a palaeontologically derived maximal calibration point. Therefore, the two historical records of life both suggest that although the cradle of Metazoa lies in the Cryogenian, and despite the explosion of ecology that occurs in the Cambrian, it is the emergence of bilaterian taxa in the Ediacaran that sets the tempo and mode of macroevolution for the remainder of geological time.     

Molecular dynamic investigation of the interaction of supported affinity ligands with monoclonal antibodies

Diagnostics and therapeutic treatments based on monoclonal antibodies have been attaining an increasing importance in the past decades, but their large scale employment requires the optimization of purification processes. To obtain this goal, research is focusing on affinity chromatography techniques and the development of new synthetic ligands. In this work we present a computational investigation aimed at obtaining some guidelines for the rational design of affinity ligands, through the study of their interactions with both monoclonal antibodies (modeled as the FC domain of human IgG) and a model support material (agarose). The study was carried out performing molecular dynamics simulations of the support-spacer-ligand-IgG complex in explicit water. Binding energies between IgG and two supported ligands, a disubstituted derivative of trichlorotriazine and a tetrameric peptide, were determined with the linear interaction energy and MM-GBSA approaches. A detailed study of the possible binding sites of the considered ligands was performed exploiting docking protocols and MD simulations. It was found that both ligands bind IgG in the same site as protein A, which is the hinge region between the CH2 and CH3 domains of IgG. However this site is not easily accessible and requires a high mobility of the ligands. The energetic analysis revealed that van der Waals and electrostatic energies of interaction of the triazine ligand with the support are significant and comparable to those with the protein, so that they limit its capability to reach the protein binding site. A similar result was found also for the tetrameric peptide, which is however able to circumvent the problem; for steric reasons only two of its arms can interact at the same time with the agarose support, thus leaving the remaining two available to bind the protein. These results indicate that the interaction between ligand and support material is an important parameter, which should be considered in the computational and experimental design of ligands for affinity chromatography.     

Local Mechanical Stimuli Regulate Bone Formation and Resorption in Mice at the Tissue Level

Bone is able to react to changing mechanical demands by adapting its internal microstructure through bone forming and resorbing cells. This process is called bone modeling and remodeling. It is evident that changes in mechanical demands at the organ level must be interpreted at the tissue level where bone (re)modeling takes place. Although assumed for a long time, the relationship between the locations of bone formation and resorption and the local mechanical environment is still under debate. The lack of suitable imaging modalities for measuring bone formation and resorption in vivo has made it difficult to assess the mechanoregulation of bone three-dimensionally by experiment. Using in vivo micro-computed tomography and high resolution finite element analysis in living mice, we show that bone formation most likely occurs at sites of high local mechanical strain (p<0.0001) and resorption at sites of low local mechanical strain (p<0.0001). Furthermore, the probability of bone resorption decreases exponentially with increasing mechanical stimulus (R² = 0.99) whereas the probability of bone formation follows an exponential growth function to a maximum value (R² = 0.99). Moreover, resorption is more strictly controlled than formation in loaded animals, and ovariectomy increases the amount of non-targeted resorption. Our experimental assessment of mechanoregulation at the tissue level does not show any evidence of a lazy zone and suggests that around 80% of all (re)modeling can be linked to the mechanical micro-environment. These findings disclose how mechanical stimuli at the tissue level contribute to the regulation of bone adaptation at the organ level.     

Altered modulation of lamin A/C‐HDAC2 interaction and p21 expression during oxidative stress response in HGPS

Defects in stress response are main determinants of cellular senescence and organism aging. In fibroblasts from patients affected by Hutchinson–Gilford progeria, a severe LMNA‐linked syndrome associated with bone resorption, cardiovascular disorders, and premature aging, we found altered modulation of CDKN1A, encoding p21, upon oxidative stress induction, and accumulation of senescence markers during stress recovery. In this context, we unraveled a dynamic interaction of lamin A/C with HDAC2, an histone deacetylase that regulates CDKN1A expression. In control skin fibroblasts, lamin A/C is part of a protein complex including HDAC2 and its histone substrates; protein interaction is reduced at the onset of DNA damage response and recovered after completion of DNA repair. This interplay parallels modulation of p21 expression and global histone acetylation, and it is disrupted by LMNAmutations leading to progeroid phenotypes. In fact, HGPS cells show impaired lamin A/C‐HDAC2 interplay and accumulation of p21 upon stress recovery. Collectively, these results link altered physical interaction between lamin A/C and HDAC2 to cellular and organism aging. The lamin A/C‐HDAC2 complex may be a novel therapeutic target to slow down progression of progeria symptoms.     

Effects of Sirolimus treatment on patients with β-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β-Thalassemia). The objective of the present study was to analyse the activity of CD4⁺ and CD8⁺ T cells in β-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4⁺ and CD8⁺ T cells, respectively. The main results of the present study are that treatment of β-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4⁺ and CD8⁺ T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β-Thalassemia patients are considered prone to immune deficiency.