Charcoal production in the Mopane woodlands of Mozambique: what are the trade-offs with other ecosystem services?

African woodlands form a major part of the tropical grassy biome and support the livelihoods of millions of rural and urban people. Charcoal production in particular is a major economic activity, but its impact on other ecosystem services is little studied. To address this, our study collected biophysical and social datasets, which were combined in ecological production functions, to assess ecosystem service provision and its change under different charcoal production scenarios in Gaza Province, southern Mozambique. We found that villages with longer histories of charcoal production had experienced declines in wood suitable for charcoal, firewood and construction, and tended to have lower perceived availabilities of these services. Scenarios of future charcoal impacts indicated that firewood and woody construction services were likely to trade-off with charcoal production. However, even under the most extreme charcoal scenario, these services were not completely lost. Other provisioning services, such as wild food, medicinal plants and grass, were largely unaffected by charcoal production. To reduce the future impacts of charcoal production, producers must avoid increased intensification of charcoal extraction by avoiding the expansion of species and sizes of trees used for charcoal production. This is a major challenge to land managers and policymakers in the area.This article is part of the themed issue ‘Tropical grassy biomes: linking ecology, human use and conservation’.     

How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?

RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%–40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more than fourfold. To achieve >85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools.     

Septins recognize micron-scale membrane curvature

How cells recognize membrane curvature is not fully understood. In this issue, Bridges et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201512029) discover that septins, a component of the cytoskeleton, recognize membrane curvature at the micron scale, a common morphological hallmark of eukaryotic cellular processes.Eukaryotic cells have dedicated proteins that sense membranes, depending on their curvature (Antonny, 2011). Sensors of membrane curvature are important because they organize a wide variety of cellular functions, including vesicle trafficking and organelle shaping (McMahon and Gallop, 2005). Curvature-sensing proteins, for example, the Bin-Amphiphysin-Rvs (BAR) domain–containing proteins, have been mostly described to work at the nanometer scale (Zimmerberg and Kozlov, 2006). In contrast, a clear mechanism of sensing membrane curvature at the micron scale in eukaryotic cells has not been described. In this issue, Bridges et al. discover that septins, a poorly understood component of the cytoskeleton, recognize plasma membrane curvature at the micron scale and serve as landmarks for eukaryotic cells to know their local shape.Septins are an evolutionarily conserved family of GTP-binding proteins that assemble into nonpolar filaments and rings (John et al., 2007; Sirajuddin et al., 2007; Bertin et al., 2008). Septins have been implicated in diverse membrane organization events where micron-scale curvature takes place (Saarikangas and Barral, 2011; Mostowy and Cossart, 2012), including the cytokinetic furrow, the annulus of spermatozoa, the base of cellular protrusions (e.g., cilium and dendritic spines), and the phagocytic cup surrounding invasive bacterial pathogens (Fig. 1). However, the precise role of septin–membrane interactions remains elusive. It was first suggested in 1999 that the interaction of human septins with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is important for septin localization (Zhang et al., 1999). More recently, work using recombinant septins from budding yeast Saccharomyces cerevisiae assembled on PI(4,5)P2 lipid monolayers showed that septins interact with membrane to facilitate filament assembly (Bridges et al., 2014). Membrane-facilitated septin assembly has also been observed using phospholipid liposomes, and in this case septins were also shown to induce membrane tubulation (Tanaka-Takiguchi et al., 2009). Given that (a) septins can interact with membrane, (b) septin assembly is membrane facilitated, and (c) septin assemblies are associated with a variety of membrane organization events from yeast to mammals, Bridges et al. (2016) hypothesized that septins serve as a mechanism to recognize membrane curvature.Open in a separate windowFigure 1.Morphological hallmarks of eukaryotic cells characterized by micron-scale membrane curvature and septin assembly. Septins have been implicated in membrane organization events where micron-scale curvature takes place. (A) A septin ring acts as a scaffold for cytokinesis proteins and forms a diffusion barrier at the cytokinetic furrow of a dividing cell. (B) A septin ring forms a diffusion barrier at the annulus of a mammalian spermatozoon, which separates the anterior and posterior tail. (C) A septin ring forms a diffusion barrier at the base of a cilium to separate the ciliary membrane from the plasma membrane. (D) In neurons, a septin-dependent diffusion barrier can localize at the base of dendritic spine necks. (E) During phagocytosis, a cup is formed at the plama membrane; septin rings assemble at the base of the phagocytic cup to regulate entry.In their new work, Bridges et al. (2016) provide several lines of evidence to support the hypothesis that septins recognize micron-scale curvature. First, using the filamentous fungus Ashbya gossypii, they performed in vivo localization studies and showed that the fungal septin Cdc11a concentrates in regions of positive micron-scale curvature and that the degree of concentration is proportional to the degree of curvature. Moreover, septins localize to curved membranes that also recruit septin-interacting proteins (e.g., the signaling protein Hsl7). These findings indicate that, by acting as curvature-sensing proteins, septins can localize signaling platforms in the cell. To test if septins can differentiate among micron-scale curvatures, Bridges et al. (2016) developed an elegant model system for septin assembly in vitro. They decorated silica beads with anionic phospholipid bilayers and measured the interaction affinity between purified fungal septin complexes and beads of different curvatures. Interestingly, septins were maximally recruited to “intermediate” sized beads (1.0–3.0 µm in diameter), with little to no recruitment to either very large (5.0–6.5 µm in diameter) or very small (0.3 µm in diameter) beads. These results indicate that septin filaments preferentially localize to a curvature (κ) of 0.7–2.0 µm⁻¹ in the absence of other cellular factors. To provide additional information on the mechanism of sensing, the authors purified mutant septin complexes that fail to polymerize into filaments and showed that the affinity of septins for micron-scale membrane curvature does not require filament formation per se. However, septins must polymerize into filaments for stable membrane association. Collectively, in vivo experiments using A. gossypii and in vitro experiments using silica beads highlight that septins have the intrinsic ability to recognize membrane curvature at the micron scale.Finally, to study the recognition of micron-scale membrane curvature beyond fungi, Bridges et al. (2016) turn their attention to human septins. Using tissue culture cells, they observe that the abundance of septins is associated with the degree of membrane curvature. To confirm these observations in vitro, they purified human septins and analyzed their binding affinity to silica beads with phospholipid bilayers. As seen with A. gossypii septins, human septins also showed a preference for beads ∼1.0 µm in diameter, strongly suggesting an evolutionarily conserved property of septins for sensing membrane curvature at the micron scale.Based on their findings, Bridges et al. (2016) propose that septins provide eukaryotic cells with a mechanism to recognize curvature at the micron scale. This feature differentiates septins from other sensor proteins that strictly detect curvature at the nanometer scale (e.g., BAR domain–containing proteins). However, it is likely that septins do more than recognize membrane, and the precise role of septins in membrane recognition remains unknown. The highly conserved structural and biochemical properties of septins suggest they organize, stabilize, and functionalize membrane domains (Caudron and Barral, 2009; Kusumi et al., 2012; Bridges and Gladfelter, 2015). Although we are far from knowing the full repertoire of septin function, this new work by Bridges et al. (2016) reminds us that understanding how membranes can specify septin assembly is essential to understand the role of septins in eukaryotic cells.     

G protein-coupled receptor systems and their lipid environment in health disorders during aging

Cells, tissues and organs undergo phenotypic changes and deteriorate as they age. Cell growth arrest and hyporesponsiveness to extrinsic stimuli are all hallmarks of senescent cells. Most such external stimuli received by a cell are processed by two different cell membrane systems: receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). GPCRs form the largest gene family in the human genome and they are involved in most relevant physiological functions. Given the changes observed in the expression and activity of GPCRs during aging, it is possible that these receptors are directly involved in aging and certain age-related pathologies. On the other hand, both GPCRs and G proteins are associated with the plasma membrane and since lipid-protein interactions regulate their activity, they can both be considered to be sensitive to the lipid environment. Changes in membrane lipid composition and structure have been described in aged cells and furthermore, these membrane changes have been associated with alterations in GPCR mediated signaling in some of the main health disorders in elderly subjects. Although senescence could be considered a physiologic process, not all aging humans develop the same health disorders. Here, we review the involvement of GPCRs and their lipid environment in the development of the major human pathologies associated with aging such as cancer, neurodegenerative disorders and cardiovascular pathologies.     

Cleavage after residue Ala352 in the C-terminal extension is an early step in the maturation of the D1 subunit of Photosystem II in Synechocystis PCC 6803

We have investigated the pathway by which the 16 amino-acid C-terminal extension of the D1 subunit of photosystem two is removed in the cyanobacterium Synechocystis sp. PCC 6803 to leave Ala344 as the C-terminal residue. Previous work has suggested a two-step process involving formation of a processing intermediate of D1, termed iD1, of uncertain origin. Here we show by mass spectrometry that a synthetic peptide mimicking the C- terminus of the D1 precursor is cleaved by cellular extracts or purified CtpA processing protease after residue Ala352, making this a likely site for formation of iD1. Characteristics of D1 site-directed mutants with either the Leu353 residue replaced by Pro or with a truncation after Ala352 are in agreement with this assignment. Interestingly, analysis of various CtpA and CtpB null mutants further indicate that the CtpA protease plays a crucial role in forming iD1 but that, surprisingly, low levels of C-terminal processing occur in vivo in the absence of CtpA and CtpB, possibly catalysed by other related proteases. A possible role for two-step maturation of D1 in the assembly of PSII is discussed.     

Expression of Ndi1p, an alternative NADH:ubiquinone oxidoreductase, increases mitochondrial membrane potential in a C. elegans model of mitochondrial disease

The NADH:ubiquinone oxidoreductase or complex I of the mitochondrial respiratory chain is an intricate enzyme with a vital role in energy metabolism. Mutations affecting complex I can affect at least three processes; they can impair the oxidation of NADH, reduce the enzyme's ability to pump protons for the generation of a mitochondrial membrane potential and increase the production of damaging reactive oxygen species. We have previously developed a nematode model of complex I-associated mitochondrial dysfunction that features hallmark characteristics of mitochondrial disease, such as lactic acidosis and decreased respiration. We have expressed the Saccharomyces cerevisiae NDI1 gene, which encodes a single subunit NADH dehydrogenase, in a strain of Caenorhabditis elegans with an impaired complex I. Expression of Ndi1p produces marked improvements in animal fitness and reproduction, increases respiration rates and restores mitochondrial membrane potential to wild type levels. Ndi1p functionally integrates into the nematode respiratory chain and mitigates the deleterious effects of a complex I deficit. However, we have also shown that Ndi1p cannot substitute for the absence of complex I. Nevertheless, the yeast Ndi1p should be considered as a candidate for gene therapy in human diseases involving complex I.     

An exodeoxyribonuclease from Streptomyces coelicolor: expression, purification and biochemical characterization

Streptomyces coelicolor A3(2) produces several intra and extracellular enzymes with deoxyribonuclease activities. The examined N-terminal amino acid sequence of one of extracellular DNAases (TVTSVNVNGLL) and database search on S. coelicolor genome showed a significant homology to the putative secreted exodeoxyribonuclease. The corresponding gene (exoSc) was amplified, cloned, expressed in Escherichia coli, purified to homogeneity and characterized. Exonuclease recExoSc degraded chromosomal, linear dsDNA with 3'-overhang ends, linear ssDNA and did not digest linear dsDNA with blunt ends, supercoiled plasmid ds nor ssDNA. The substrate specificity of recExoSc was in the order of dsDNA>ssDNA>3'-dAMP. The purified recExoSc was not a metalloprotein and exhibited neither phosphodiesterase nor RNase activity. It acted as 3'-phosphomonoesterase only at 3'-dAMP as a substrate. The optimal temperature for its activity was 57 degrees C in Tris-HCl buffer at optimal pH=7.5 for either ssDNA or dsDNA substrates. It required a divalent cation (Mg(2+), Co(2+), Ca(2+)) and its activity was strongly inhibited in the presence of Zn(2+), Hg(2+), chelating agents or iodoacetate.     

Apoptosis and inhibition of gap-junctional intercellular communication induced by LA-12, a novel hydrophobic platinum(IV) complex

A new hydrophobic platinum(IV) complex, LA-12, a very efficient anticancer drug lacking cross-resistance with cisplatin (CDDP), is now being tested in clinical trials. Here we investigated the apoptogenic activity of LA-12 and its effect on gap-junctional intercellular communication (GJIC) in the rat liver epithelial cell line WB-F344. LA-12 induced apoptosis much more efficiently than did CDDP due to a combination of rapid penetration into the cell and attack on DNA, leading to fast activation of p53 and caspase-3. Exposure of WB-F344 cells to LA-12 led to rapid induction of the time- and dose-dependent decrease in GJIC. On the molecular level, loss of GJIC induced by LA-12 was mediated by activation of extracellular signal-regulated kinase (ERK)-1 and ERK-2, as demonstrated by the use of inhibitors of ERK activation. Inhibition of GJIC was linked to rapid hyperphosphorylation of connexin-43 and disappearance of connexon clusters from membranes, which was not observed in the case of CDDP.     

Lactobacillus salivarius CTC2197 prevents Salmonella enteritidis colonization in chickens.

A rifampin-resistant Lactobacillus salivarius strain, CTC2197, was assessed as a probiotic in poultry, by studying its ability to prevent Salmonella enteritidis C-114 colonization in chickens. When the probiotic strain was dosed by oral gavage together with S. enteritidis C-114 directly into the proventriculus in 1-day-old Leghorn chickens, the pathogen was completely removed from the birds after 21 days. The same results were obtained when the probiotic strain was also administered through the feed and the drinking water apart from direct inoculation into the proventriculus. The inclusion of L. salivarius CTC2197 in the first day chicken feed revealed that a concentration of 10(5) CFU g(-1) was enough to ensure the colonization of the gastrointestinal tract of the birds after 1 week. However, between 21 and 28 days, L. salivarius CTC2197 was undetectable in the gastrointestinal tract of some birds, showing that more than one dose would be necessary to ensure its presence till the end of the rearing time. Freeze-drying and freezing with glycerol or skim milk as cryoprotective agents, appeared to be suitable methods to preserve the probiotic strain. The inclusion of the L. salivarius CTC2197 in a commercial feed mixture seemed to be a good way to supply it on the farm, although the strain showed sensitivity to the temperatures used during the feed mixture storage and in the chicken incubator rooms. Moreover, survival had been improved after several reinoculations in chicken feed mixture.