Differentiation of Fanconi anemia and aplastic anemia using mitomycin C test in Tunisia

Fanconi anemia (FA) is a recessive chromosomal instability syndrome that is clinically characterized by multiple symptoms. Chromosome breakage hypersensitivity to alkylating agents is the gold standard test for FA diagnosis. In this study, we provide a detailed laboratory protocol for accurate assessment of FA diagnosis based on mitomycin C (MMC) test. Induced chromosomal breakage study was successful in 171 out of 205 aplastic anemia (AA) patients. According to the sensitivity of MMC at 50 ng/ml, 38 patients (22.22%) were diagnosed as affected and 132 patients (77.17%) as unaffected. Somatic mosaicism was suspected in an 11-year-old patient with a FA phenotype. Twenty-six siblings of FA patients were also evaluated and five of them (19.23%) were diagnosed as FA. From this study, a standard protocol for diagnosis of FA was developed. It is routinely used as a diagnostic test of FA in Tunisia.     

Surface polysaccharides and quorum sensing are involved in the attachment and survival of Xanthomonas albilineans on sugarcane leaves

Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is a bacterial plant pathogen that is mainly spread by infected cuttings and contaminated harvesting tools. However, some strains of this pathogen are known to be spread by aerial means and are able to colonize the phyllosphere of sugarcane before entering the host plant and causing disease. The objective of this study was to identify the molecular factors involved in the survival or growth of X. albilineans on sugarcane leaves. We developed a bioassay to test for the attachment of X. albilineans on sugarcane leaves using tissue‐cultured plantlets grown in vitro. Six mutants of strain XaFL07‐1 affected in surface polysaccharide production completely lost their capacity to survive on the sugarcane leaf surface. These mutants produced more biofilm in vitro and accumulated more cellular poly‐β‐hydroxybutyrate than the wild‐type strain. A mutant affected in the production of small molecules (including potential biosurfactants) synthesized by non‐ribosomal peptide synthetases (NRPSs) attached to the sugarcane leaves as well as the wild‐type strain. Surprisingly, the attachment of bacteria on sugarcane leaves varied among mutants of the rpf gene cluster involved in bacterial quorum sensing. Therefore, quorum sensing may affect polysaccharide production, or both polysaccharides and quorum sensing may be involved in the survival or growth of X. albilineans on sugarcane leaves.     

The activation of the atypical PKC zeta in light‐induced retinal degeneration and its involvement in L‐DNase II control

Light‐induced retinal degeneration is characterized by photoreceptor cell death. Many studies showed that photoreceptor demise is caspase‐independent. In our laboratory we showed that leucocyte elastase inhibitor/LEI‐derived DNase II (LEI/L‐DNase II), a caspase‐independent apoptotic pathway, is responsible for photoreceptor death. In this work, we investigated the activation of a pro‐survival kinase, the protein kinase C (PKC) zeta. We show that light exposure induced PKC zeta activation. PKC zeta interacts with LEI/L‐DNase II and controls its DNase activity by impairing its nuclear translocation. These results highlight the role of PKC zeta in retinal physiology and show that this kinase can control caspase‐independent pathways.     

ABA-dependent amine oxidases-derived H2O2 affects stomata conductance

Recently we showed that ABA is at least partly responsible for the induction of the polyamine exodus pathway in Vitis vinifera plants. Both sensitive and tolerant plants employ this pathway to orchestrate stress responses, differing between stress adaptation and programmed cell death. Herein we show that ABA is an upstream signal for the induction of the polyamine catabolic pathway in Vitis vinifera. Thus, amine oxidases are producing H₂O₂ which signals stomata closure. Moreover, the previously proposed model for the polyamine catabolic pathway is updated and discussed.Key words: plant growth, abscissic acid, polyamines, amine oxidases, signaling, oxidative stress, programmed cell deathWe have shown that tobacco salinity induces an exodus of the polyamine (PA) spermidine (Spd) into the apoplast where it is oxidized by polyamine oxidase (PAO) generating hydrogen peroxide (H₂O₂). Depending on the size of H₂O₂, it signals either tolerance-effector genes or the programmed cell death syndrome¹ (PCD). PAs are ubiquitous and biologically active molecules. In the recent years remarkable progress has been accomplished regarding the regulation of PAs biosynthesis and catalysis, not only under normal physiological but also under stress conditions.¹ The most studied PAs are the diamine Putrescine (Put) and its derivatives the triamine Spd and the tetramine spermine (Spm). They are present in the cells in soluble form (S), or conjugated either to low molecular weight compounds (soluble hydrolyzed form, SH) or to “macro” molecules or cell walls (pellet hydrolyzed form, PH). In higher plants, Put is synthesized either directly from ornithine via ornithine decarboxylase (ODC; EC 4.1.1.17) or indirectly from arginine via arginine decarboxylase (ADC; EC 4.1.1.19). Spd and Spm are synthesized via Spd synthase (EC 2.5.1.16, SPDS) and Spm synthase (EC 2.5.1.22, SPMS), respectively, by sequential addition of aminopropyl groups to Put, catalyzed by S-adenosyl-L-methionine decarboxylase (SAMDC; EC 4.1.1.50).^2,3 In plants, PAs are present in the cytoplasm, as well as in cellular organelles.⁴ Recently it was shown that during stress, they are secreted into the apoplast where they are oxidized by amine oxidases (AOs), such as diamine oxidase for Put (DAO, E.C. 1.4.3.6) and polyamine oxidase (PAO, E.C. 1.4.3.4) for Spd and Spm.^1,5,6 Oxidation of PAs generates, amongst other products, H₂O₂^1,7,8 which is involved in cell signaling processes coordinated by abscissic acid (ABA),⁹ but also acts as efficient oxidant and, at high concentration, orchestrates the PCD syndrome.^6,10 Two types of PA catabolism by PAO are known in plants: the terminal and the back-conversion pathways. The terminal one takes place in the apoplast, produces except H₂O₂, 1,3-diaminopropane and an aldehyde depending on the species. On the other hand, the back-conversion pathway is intracellular (cytoplasm and peroxisomes) resulting to the production of H₂O₂ and the sequential production of Put by Spm via Spd.^1,7 Now we have shown that PA exodus also occurs in Vitis vinifera and this phenomenon is at least partially induced by abscissic acid (ABA).¹¹ Thus, exogenous application of ABA results to PA exodus into the apoplast of grapevine. PA is oxidized by an AO resulting to production of H₂O₂. When the titer of H₂O₂ is below a threshold, expression of tolerance-effector genes is induced, while when it exceeds this threshold the programmed cell death (PCD) syndrome is induced.     

On the use of an appropriate TdT-mediated dUTP–biotin nick end labeling assay to identify apoptotic cells

Apoptosis is an essential cellular mechanism involved in many processes such as embryogenesis, metamorphosis, and tissue homeostasis. DNA fragmentation is one of the key markers of this form of cell death. DNA fragmentation is executed by endogenous endonucleases such as caspase-activated DNase (CAD) in caspase-dependent apoptosis. The TUNEL (TdT-mediated dUTP–biotin nick end labeling) technique is the most widely used method to identify apoptotic cells in a tissue or culture and to assess drug toxicity. It is based on the detection of 3′-OH termini that are labeled with dUTP by the terminal deoxynucleotidyl transferase. Although the test is very reliable and sensitive in caspase-dependent apoptosis, it is completely useless when cell death is mediated by pathways involving DNA degradation that generates 3′-P ends as in the LEI/L-DNase II pathway. Here, we propose a modification in the TUNEL protocol consisting of a dephosphorylation step prior to the TUNEL labeling. This allows the detection of both types of DNA breaks induced during apoptosis caspase-dependent and independent pathways, avoiding underestimating the cell death induced by the treatment of interest.     

Lysigenous aerenchyma formation in maize root is confined to cortical cells by regulation of genes related to generation and scavenging of reactive oxygen species

To adapt to waterlogging, maize (Zea mays) forms lysigenous aerenchyma in root cortex as a result of ethylene-promoted programmed cell death (PCD). Respiratory burst oxidase homolog (RBOH) gene encodes a homolog of gp91^phox in NADPH oxidase, and has a role in the generation of reactive oxygen species (ROS). Recently, we found that during aerenchyma formation, RBOH was upregulated in all maize root tissues examined, whereas an ROS scavengingrelated metallothionein (MT) gene was downregulated specifically in cortical cells. Together these changes should lead to high accumulations of ROS in root cortex, thereby inducing PCD for aerenchyma formation. As further evidence of the involvement of ROS in root aerenchyma formation, the PCD was inhibited by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Based on these results, we propose a model of cortical cell-specific PCD for root aerenchyma formation.Key words: aerenchyma, ethylene, laser microdissection, maize (Zea mays), metallothionein, programmed cell death, reactive oxygen species, respiratory burst oxidase homologIn both wetland and non-wetland plants, lysigenous aerenchyma is formed in roots by creating gas spaces as a result of death and subsequent lysis of some cortical cells, and allows internal transport of oxygen from shoots to roots under waterlogged soil conditions.^1–3 In rice (Oryza sativa) and some other wetland plant species, lysigenous aerenchyma is constitutively formed under aerobic conditions, and is further enhanced under waterlogged conditions.⁴ On the other hand, in non-wetland plants, including maize (Zea mays), lysigenous aerenchyma does not normally form under well-drained soil conditions, but is induced by waterlogging.⁵ Ethylene is involved in lysigenous aerenchyma formation,^1–3,6,7 but the molecular mechanisms are unclear.We recently identified two reactive oxygen species (ROS)-related genes that were specifically regulated in maize root cortex by waterlogged conditions, but not in the presence of an ethylene perception inhibitor 1-methylcyclopropene (1-MCP).⁵ One was respiratory burst oxidase homolog (RBOH), which has a role in ROS generation and the other was metallothionein (MT), which has a role in ROS scavenging. These results suggest that ROS has a role in ethylene signaling in the PCD that occurs during lysigenous aerenchyma formation.     

Non-contiguous finished genome sequence and description of Halopiger goleamassiliensis sp. nov.

Halopiger goleamassiliensis strain IIH3^T sp. nov. is a novel, extremely halophilic archaeon within the genus Halopiger. This strain was isolated from an evaporitic sediment in El Golea Lake, Ghardaïa region (Algeria). The type strain is strain IIH3^T. H. goleamassiliensis is moderately thermophilic, neutrophilic, non-motile and coccus-shaped. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,906,923 bp long genome contains 3,854 protein-encoding genes and 49 RNA genes (1 gene is 16S rRNA, 1 gene is 23S rRNA, 3 genes are 5S rRNA, and 44 are tRNA genes).     

Induced change in <Emphasis Type="Italic">Arthrospira</Emphasis> sp. (<Emphasis Type="Italic">Spirulina</Emphasis>) intracellular and extracellular metabolites using multifactor stress combination approach

The interactive effects of light intensity, NaCl, nitrogen, and phosphorus on intracellular biomass content and extracellular polymeric substance production were assessed for Arthrospira sp. (Spirulina) in a two-phase culture process using principal component analysis and central composite face design. Under high light intensity (120 μmol photons m^?2?s^?1) and low NaCl (1 gL^?1), NaNO₃, and K₂HPO₄ (0.5 g L^?1), the carbohydrate content was maximized to 26.61%. Interaction of both K₂HPO₄ (1.6 gL^?1) and NaCl (1.19 gL^?1) with low NaNO₃ (0.5 gL^?1) achieved the maximum content of lipids (15.62%), while high NaCl (40 gL^?1), K₂HPO₄, and NaNO₃ (4.5 gL^?1) enhanced mainly total carotenoids (0.85%). Conversely, under low light intensity of 10 μmol photons m^?2?s^?1 combined with 11.76 gL^?1 of NaCl, 0.5 gL^?1 of NaNO₃, and 2.68 gL^?1 of K₂HPO₄, the phycobiliprotein content reached its highest level (16.09%). The maximum extracellular polymeric substance (EPS) production (0.902 gg^?1?DW) was triggered under moderate light of 57.25 μmol photons m^?2?s^?1 and interaction of high NaCl (40 gL^?1) and K₂HPO₄ (4.5 gL^?1) with low NaNO₃ (0.5 gL^?1). The maximization ratios of intracellular biomass content in terms of carbohydrate, lipid, total carotenoid, phycobiliprotein, and EPS production were 3.55-, 1.73-, 9.55-, 2.92-, and 1.46-fold, respectively, greater than those obtained at optimal growth conditions. This study demonstrated that the multiple stress factors applied to the adopted two-phase culture process could be a promising strategy to produce biomass enriched in various high-value compound.     

Polyamine anabolic/catabolic regulation along the woody grapevine plant axis

The distribution of the endogenous PA fractions throughout the entire perennial woody grapevine (Vitis vinifera L.) plant was studied, along with the expression profiles of the PA anabolic and catabolic genes and their substrates and secondary metabolites. Putrescine fractions increased with increasing leaf age, although the expression of its biosynthetic enzymes Arg and Orn decarboxylases decreased. Orn transport from young organs dramatically enhanced putrescine biosynthesis in older tissues, via the Orn decarboxylase pathway. S-adenosylmethionine decarboxylase and spermidine synthase genes were down-regulated during development in a tissue/organ-specific manner, as were spermidine and spermine levels. In contrast, amine oxidases, peroxidases and phenolics increased from the youngest to the fully developed vascular tissues; they also increased from the peripheral regions of leaves to the petioles. Hydrogen peroxide generated by amine oxidases accumulated for the covalent linkage of proteins via peroxidases during lignification. These results could be valuable for addressing further questions on the role of PAs in plant development.