The above-ground coarse wood productivity of 104 Neotropical forest plots

The net primary production of tropical forests and its partitioning between long‐lived carbon pools (wood) and shorter‐lived pools (leaves, fine roots) are of considerable importance in the global carbon cycle. However, these terms have only been studied at a handful of field sites, and with no consistent calculation methodology. Here we calculate above‐ground coarse wood carbon productivity for 104 forest plots in lowland New World humid tropical forests, using a consistent calculation methodology that incorporates corrections for spatial variations in tree‐size distributions and wood density, and for census interval length. Mean wood density is found to be lower in more productive forests. We estimate that above‐ground coarse wood productivity varies by more than a factor of three (between 1.5 and 5.5 Mg C ha^?1 a^?1) across the Neotropical plots, with a mean value of 3.1 Mg C ha^?1 a^?1. There appear to be no obvious relationships between wood productivity and rainfall, dry season length or sunshine, but there is some hint of increased productivity at lower temperatures. There is, however, also strong evidence for a positive relationship between wood productivity and soil fertility. Fertile soils tend to become more common towards the Andes and at slightly higher than average elevations, so the apparent temperature/productivity relationship is probably not a direct one. Coarse wood productivity accounts for only a fraction of overall tropical forest net primary productivity, but the available data indicate that it is approximately proportional to total above‐ground productivity. We speculate that the large variation in wood productivity is unlikely to directly imply an equivalent variation in gross primary production. Instead a shifting balance in carbon allocation between respiration, wood carbon and fine root production seems the more likely explanation.     

Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension

Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood. Here we show that inhibiting the expression of the small GTPase Cdc42 or preventing its activation with a dominant negative Cdc42 construct in human neuroendocrine cells impaired the release process by compromising fusion pore enlargement. Consequently the mode of vesicle exocytosis was shifted from full-collapse fusion to kiss-and-run. Remarkably, Cdc42-knockdown cells showed reduced membrane tension, and the artificial increase of membrane tension restored fusion pore enlargement. Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation. We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force.     

Construction of a Highly Active Xylanase Displaying Oleaginous Yeast: Comparison of Anchoring Systems

Three Yarrowia lipolytica cell wall proteins (YlPir, YlCWP1 and YlCBM) were evaluated for their ability to display the xylanase TxXYN from Thermobacillus xylanilyticus on the cell surface of Y. lipolytica. The fusion proteins were produced in Y. lipolytica JMY1212, a strain engineered for mono-copy chromosomal insertion, and enabling accurate comparison of anchoring systems. The construction using YlPir enabled cell bound xylanase activity to be maximised (71.6 U/g). Although 48% of the activity was released in the supernatant, probably due to proteolysis at the fusion zone, this system is three times more efficient for the anchoring of TxXYN than the YlCWP1 system formerly developed for Y. lipolytica. As far as we know it represents the best displayed xylanase activity ever published. It could be an attractive alternative anchoring system to display enzymes in Y. lipolytica.     

State of art and limitations in genetic engineering to induce stable chondrogenic phenotype

Current protocols for chondrocyte expansion and chondrogenic differentiation of stem cells fail to reduce phenotypic loss and to mitigate hypertrophic tendency. To this end, cell genetic manipulation is gaining pace as a means of generating cells with stable chondrocyte phenotype. Herein, we provide an overview of candidate genes that either induce cartilage regeneration or inhibit cartilage degeneration. We further discuss in vitro, ex vivo and in vivo viral transduction and non-viral transfection strategies for targeted cells (chondrocytes, mesenchymal stem cells, induced pluripotent stem cells and synovial cells), along with the most representative results obtained in pre-clinical models and in clinical trials. We highlight current challenges and associated risks that slowdown clinical acceptance and commercialisation of gene transfer technologies.     

SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction

The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.     

Nippostrongylus-Induced Intestinal Hypercontractility Requires IL-4 Receptor Alpha-Responsiveness by T Cells in Mice

Gut-dwelling helminthes induce potent IL-4 and IL-13 dominated type 2 T helper cell (T_H2) immune responses, with IL-13 production being essential for Nippostrongylus brasiliensis expulsion. This T_H2 response results in intestinal inflammation associated with local infiltration by T cells and macrophages. The resulting increased IL-4/IL-13 intestinal milieu drives goblet cell hyperplasia, alternative macrophage activation and smooth muscle cell hypercontraction. In this study we investigated how IL-4-promoted T cells contributed to the parasite induced effects in the intestine. This was achieved using pan T cell-specific IL-4 receptor alpha-deficient mice (iLck^creIL-4Rα^−/lox) and IL-4Rα-responsive control mice. Global IL-4Rα^−/− mice showed, as expected, impaired type 2 immunity to N. brasiliensis. Infected T cell-specific IL-4Rα-deficient mice showed comparable worm expulsion, goblet cell hyperplasia and IgE responses to control mice. However, impaired IL-4-promoted T_H2 cells in T cell-specific IL-4Rα deficient mice led to strikingly reduced IL-4 production by mesenteric lymph node CD4⁺ T cells and reduced intestinal IL-4 and IL-13 levels, compared to control mice. This reduced IL-4/IL-13 response was associated with an impaired IL-4/IL-13-mediated smooth muscle cell hypercontractility, similar to that seen in global IL-4Rα^−/− mice. These results demonstrate that IL-4-promoted T cell responses are not required for the resolution of a primary N. brasiliensis infection. However, they do contribute significantly to an important physiological manifestation of helminth infection; namely intestinal smooth muscle cell-driven hypercontractility.     

The dileucine motif at the COOH terminus of human multidrug resistance P-glycoprotein is important for folding but not activity

P-glycoprotein (P-gp, ABCB1) actively transports a broad range of cytotoxic compounds out of the cell. The COOH terminus of P-gp contains a dileucine motif (Leu(1260)-Leu(1261)) and a conserved phenylalanine (Phe(1268)). Similar residues in SUR1 (ABCC8) were reported to be important plasma membrane-targeting signals (Sharma, N., Crane, A., Clement, J. P. t., Gonzalez, G., Babenko, A. P., Bryan, J., and Aguilar-Bryan, L. (1999) J. Biol. Chem. 274, 20628-20632). Here, we used alanine-scanning mutagenesis to test whether these residues were essential for trafficking of P-gp to the cell surface. Mutant L1260A expressed a 150-kDa immature protein that did not reach the cell surface and was sensitive to digestion by Endo H(f). By contrast, mutants L1261A, F1268A, and wild-type P-gps expressed the 170-kDa mature proteins at the cell surface. Mutation of Leu(1260) to Gly, Ile, Trp, Lys, or Glu also resulted in the expression of the 150-kDa immature protein. All of the mutants, however, expressed the 170-kDa protein in the presence of the drug substrate/specific chemical chaperone cyclosporin A. Mutant L1260A P-gp exhibited drug-stimulated ATPase activities similar to that of wild-type enzyme after rescue with cyclosporin A. Deletion of the last 22 amino acids (Q(1259)-Q(1280)) also caused misprocessing. The mutant, however, was rescued by expression in the presence of cyclosporin A and conferred resistance to colchicine in transfected cells. These results show that the dileucine motif is not a plasma membrane targeting signal. The COOH terminus is required for proper folding of P-gp but not for activity.     

Characterization of Superoxide dismutase genes from Gram-positive bacteria by polymerase chain reaction using degenerate primers

Abstract An internal fragment representing approximately 85% of sod genes from seven Gram-positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae , and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes , and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens.