Shmuel Shaltiel

The ability of cells to synthesize and secrete proteins is essential for numerous cellular functions. Therefore, when mutations in one component of the secretory pathway result in a tissue-specific defect, a unique opportunity arises to examine the molecular mechanisms at play. The recent finding that a defect in the protein sedlin, whose yeast counterpart is involved in the first step of the secretory pathway, leads to a cartilage-specific disorder in humans raises numerous questions and interesting possibilities for understanding both the pathobiology involved and the role of membrane traffic in normal cartilage development.     

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.     

Positive selectable marker genes for routine plant transformation

Summary Plant genetic transformation technologies rely upon the selection and recovery of transformed cells. Selectable marker genes used so far have been either antibiotic resistance genes or herbicide tolerance genes. There is a need to apply alternative principles of selection, as more transgenic traits have to be incorporated into a transgenic crop and because of concern that the use of conventional marker genes may pose a threat to humans and the environment. New classes of marker genes are now available, conferring metabolic advantage of the transgenic cells over the non-transformed cells. The new selection systems, as described in this review, are being used with success and superior performance over the traditional marker systems.     

Procurement and Supply Management System for MDR-TB in Nigeria: Are the Early Warning Targets for Drug Stock Outs and Over Stock of Drugs Being Achieved?

Background

The World Health Organisation (WHO) introduced the twelve early warning indicators for monitoring and evaluating drug Procurement and Supply management (PSM) systems, intended to prevent drug stock-outs and overstocking. Nigeria- one of the high Multi Drug Resistant Tuberculosis (MDR-TB) burden countries, scaled-up treatment in 2012 with the concurrent implementation of a PSM system.

Method

We evaluated how well this system functioned using the WHO indicators, including all seven MDR-TB treatment centres in the country that were functional throughout 2013.

Results

The quantity of MDR-TB drugs ordered for 2013 matched the annual forecast and all central orders placed during the year were delivered in full and on time. Drug consumption was 81%–106% of the quantity allocated for routine consumption. Timely submission of complete inventory reports ranged from 86–100%, late submissions being 5–15 days late. Forty to 71% of treatment centres placed a drug order when stock was below the minimum level of three months. The proportion of drug orders received at the treatment centres in full and on time ranged from 29–80%, late orders being 1–19 days late.

Conclusion

The PSM was found to be performing well in terms of forecasting and procurement of MDR-TB drugs, but there were shortcomings in drug distribution, reporting at treatment centre level and in drug order placements. Despite these gaps, there were no stock outs. These findings indicate that where it matters most, namely ensuring that no drug stock outs affect patient management, the PSM system is effective. Addressing the observed shortcomings will help to strengthen the existing PSM system in anticipation of a growing MDR-TB case burden in the country.     

Environmental and Genetic Determinants of Colony Morphology in Yeast

Nutrient stresses trigger a variety of developmental switches in the budding yeast Saccharomyces cerevisiae. One of the least understood of such responses is the development of complex colony morphology, characterized by intricate, organized, and strain-specific patterns of colony growth and architecture. The genetic bases of this phenotype and the key environmental signals involved in its induction have heretofore remained poorly understood. By surveying multiple strain backgrounds and a large number of growth conditions, we show that limitation for fermentable carbon sources coupled with a rich nitrogen source is the primary trigger for the colony morphology response in budding yeast. Using knockout mutants and transposon-mediated mutagenesis, we demonstrate that two key signaling networks regulating this response are the filamentous growth MAP kinase cascade and the Ras-cAMP-PKA pathway. We further show synergistic epistasis between Rim15, a kinase involved in integration of nutrient signals, and other genes in these pathways. Ploidy, mating-type, and genotype-by-environment interactions also appear to play a role in the controlling colony morphology. Our study highlights the high degree of network reuse in this model eukaryote; yeast use the same core signaling pathways in multiple contexts to integrate information about environmental and physiological states and generate diverse developmental outputs.     

2‐D DIGE reveals changes in wheat xylanase inhibitor protein families due to Fusarium graminearum ΔTri5 infection and grain development

Wheat contains three different classes of proteinaceous xylanase inhibitors (XIs), i.e. Triticum aestivum xylanase inhibitors (TAXIs) xylanase‐inhibiting proteins (XIPs), and thaumatin‐like xylanase inhibitors (TLXIs) which are believed to act as a defensive barrier against phytopathogenic attack. In the absence of relevant data in wheat kernels, we here examined the response of the different members of the XI protein population to infection with a ΔTri5 mutant of Fusarium graminearum, the wild type of which is one of the most important wheat ear pathogens, in early developing wheat grain. Wheat ears were inoculated at anthesis, analyzed using 2‐D DIGE and multivariate analysis at 5, 15, and 25 days post anthesis (DPA), and compared with control samples. Distinct abundance patterns could be distinguished for different XI forms in response to infection with F. graminearum ΔTri5. Some (iso)forms were up‐regulated, whereas others were down‐regulated. This pathogen‐specific regulation of proteins was mostly visible at five DPA and levelled off in the samples situated further from the inoculation point. Furthermore, it was shown that most identified TAXI‐ and XIP‐type XI (iso)forms significantly increased in abundance from the milky (15 DPA) to the soft dough stages (25 DPA) on a per kernel basis, although the extent of increase differed greatly. Non‐glycosylated XIP forms increased more strongly than their glycosylated counterparts.     

Interaction with MEK causes nuclear export and downregulation of peroxisome proliferator-activated receptor gamma

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade plays a central role in intracellular signaling by many extracellular stimuli. One target of the ERK cascade is peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor that promotes differentiation and apoptosis. It was previously demonstrated that PPARgamma activity is attenuated upon mitogenic stimulation due to phosphorylation of its Ser84 by ERKs. Here we show that stimulation by tetradecanoyl phorbol acetate (TPA) attenuates PPARgamma's activity in a MEK-dependent manner, even when Ser84 is mutated to Ala. To elucidate the mechanism of attenuation, we found that PPARgamma directly interacts with MEKs, which are the activators of ERKs, but not with ERKs themselves, both in vivo and in vitro. This interaction is facilitated by MEKs' phosphorylation and is mediated by the basic D domain of MEK1 and the AF2 domain of PPARgamma. Immunofluorescence microscopy and subcellular fractionation revealed that MEK1 exports PPARgamma from the nucleus, and this finding was supported by small interfering RNA knockdown of MEK1 and use of a cell-permeable interaction-blocking peptide, which prevented TPA-induced export of PPARgamma from the nucleus. Thus, we show here a novel mode of downregulation of PPARgamma by its MEK-dependent redistribution from the nucleus to the cytosol. This unanticipated role for the stimulation-induced nuclear shuttling of MEKs shows that MEKs can regulate additional signaling components besides the ERK cascade.     

Extraordinary host switching in siphonostomatoid copepods and the demise of the Monstrilloida: integrating molecular data, ontogeny and antennulary morphology

Copepods exhibit an astounding variety of lifestyles, host associations and morphology, to the extent that their crustacean affinities may be obscured. Relationships among the ten copepod orders based on morphological characters remain equivocal. Here we test the ordinal status of the enigmatic Monstrilloida using SSU rDNA gene sequences, comparative morphological data (antennulary sensory interface) and ontogenetic data (caudal ramus setation patterns). Bayesian analysis unexpectedly revealed the Monstrilloida are nested within a fish-parasitic clade of the Siphonostomatoida and share a common ancestor with the stem species of the caligiform families (sea-lice). This unforeseen relationship is congruent with both antennulary and caudal ramus morphology. The divergence of the monstrilloids from an ectoparasitic, vertebrate-associated ancestor involved radical changes in host utilization, body plan and life cycle strategy, a combination rarely observed and probably unique in metazoan parasites. Adult monstrilloids secondarily returned to a free-living, predator-exposed mode of life and we postulate the pressure on maintaining a functional approaching-predator detection system has progenetically delayed the suppression (as in post-copepodid caligiform instars) of the 5-point antennulary sensory array. The homoplastic evolution of the frontal filament in Siphonostomatoida is discussed.     

Patterns of genetic diversity in the critically endangered Central American river turtle: human influence since the Mayan age?

We conducted a phylogeographic analysis of the strictly aquatic and critically endangered Central American river turtle, Dermatemys mawii, as part of a conservation management program for the species. We sampled 238 individuals from 15 different localities throughout the species range. Using sequence fragments from the mtDNA Cyt b and ND4 genes, we identified 16 different haplotypes. Overall, our results reveal a signal of phylogeographic structure throughout the range, which appears to have been secondarily blurred by extensive gene flow. Notably, this also applies to genetic structuring across three major hydrological basins that pose biogeographic breaks in other aquatic taxa. Divergence times of mtDNA haplotypes in D. mawii suggest that the main lineages split in the Pliocene–Pleistocene (3.73–0.227 MA) and demographic tests indicate that the species has undergone drastic demographic size fluctuations since this time period. One ancient haplotype (1D) was found to exhibit sequence divergence of up to 2% from other haplogroups. Divergence of this magnitude is indicative of species level differentiation in other turtle genera. Haplotype 1D was found in only two localities, Sarstun and Salinas, but specimens with other haplotypes were also found in those localities. It is not known whether the individuals with the 1D haplotype interbreed with non-1D individuals. Our results suggest that human activity, such as harvesting and long distance transport of animals, may have influenced the current patterns of genetic diversity. For more than 2000 years, D. mawii has been consumed by people from Middle American cultures, and the archeological record contains strong evidence that the Mayans transported animals between villages and far away from their natural distribution range. Therefore, the large-scale pattern of haplotype sharing even across hydrological barriers, the observed low haplotype diversity in some populations and the contemporary absence of a pronounced phylogeographic pattern is likely due to a combination of population expansions, gene flow, extensive human-mediated-movements and recent bottlenecks resulting from over-harvesting.