Molecular and physiological study of water-deficit stress on selected Eragrostis species

Bibliography: leaves 60-73.Eragrostis nindensis and Eragrostis tef are wild and domestic grasses respectively that belong to the subfamily Eragrostideae. E. nindensis is desiccation tolerant while E. tef is desiccation sensitive. The responses of these plants to water-deficit stress were studied using molecular and physiological approaches. A cDNA library of E. nindensis was screened to identify differentially expressed genes during dehydration. Physiological studies included monitoring changes in photosynthesis, respiration, ultrastructure and membrane integrity of plants during dehydration and rehydration. The differential screening of the cDNA library, using a radio-labelled cDNA from hydrated and dehydrated leaves respectively, revealed two genes, referred as Nin-19 and Nin-44, that were differentially expressed in dehydrated leaves of E. nindensis. These genes were sequenced and partially characterized. Nin-19 did not show considerable identity with any known genes and was not studied any further. Nin-44 was identified as a dehydrin-like gene with approximately 99 % identity to seven water-deficit stress responsive genes on a section of about 60 bp near the 3' end. As its sequence was found to represent a partial insert size, two forward and reverse primers were designed to find the full length through RT-PCR. Despite repeated attempts, no products that could be used in subsequent procedures were achieved using this technique. Hence, further characterization of this gene also could not be performed and different approaches were suggested. The physiological studies showed that E. nindensis is desiccation tolerant but E. tefis not, the latter dying below RWC of about 33 %. Difference among plants in physiological responses became evident after 6 days of dehydration treatment, which resulted in a decline of RWC to 65%, 39% and 33% in E. nindensis, E.tif(R) (red-seeded) and E. tef (W) (white-seeded) plants respectively. A significant decrease in photosynthesis, transpiration, stomatal conductance, and an increase in electrolyte leakage occurred in all species after 6 days of dehydration, but leaves of E. tef (W) did not recover from this level of dehydration when watered. Instead, new leaves were observed to re-grow from the stem nodes. The leaves of red-seeded variety of E. tef did recover fully from RWC of 39 %. After a further 3 days dehydration both varieties of E. tef died. On the other hand, E. nindensis was found to survive extreme water-deficit (-10 % RWC tested here) and recovered full physiological activity when watered. The electrolyte leakage study on these plants indicated major injury on E. tef(W), being intermediate in E. tef(R) and very low in E. nindensis, which coincided with the trend of declining in RWC and other metabolic activities measured. The ultrastructural study on E. tef varieties also showed evidence of the damage caused by dehydration, but the difference among these species was not significant enough to indicate the level of susceptibility of the plants to dehydration damage. The study demonstrated that E. tef varieties are not drought tolerant and showed a considerable difference in their responses to water-deficit stress with each other and with respect to E. nindensis. However, E. tef (R) seems to have a better control over transpiration and some form of repair mechanism operational at least until dehydration to 39 % RWC. This is proposed to be a better performing cereal in conditions of water stress. On the other hand E. nindnsis did not suffer major injury from the dehydration treatment and confirmed to be desiccation tolerant

Characterization of two, desiccation linked, Group 1 LEA proteins from the resurrection plant Xerophyta humilis

Studies on resurrection plants and other anhydrobiotic organisms show expression of Late Embryogenesis Abundant (LEA) proteins associated with desiccation tolerance. However, the precise role of these proteins has not been described. This study was undertaken to investigate expression, structure and function of XhLEA1-4S1 and XhLEA1-1S2, Group 1 LEA proteins from Xerophyta humilis, in order to shed light on their role in desiccation tolerance. Complementary DNA (cDNA) of these XhLEAs were cloned into bacterial expression vectors and the recombinant proteins expressed in E. coli. Antibodies were generated and used in determination of expression conditions and immunolocalization studies

Patient derived colonoids as drug testing platforms - Critical importance of oxygen concentration

Treatment of inflammatory bowel disease (IBD) is challenging, with a series of available drugs each helping only a fraction of patients. Patients may face time-consuming drug trials while the disease is active, thus there is an unmet need for biomarkers and assays to predict drug effect. It is well known that the intestinal epithelium is an important factor in disease pathogenesis, exhibiting physical, biochemical and immunologic driven barrier dysfunctions. One promising test system to study effects of existing or emerging IBD treatments targeting intestinal epithelial cells (IECs) is intestinal organoids (“mini-guts”). However, the fact that healthy intestinal epithelium is in a physiologically hypoxic state has largely been neglected, and studies with intestinal organoids are mainly performed at oxygen concentration of 20%. We hypothesized that lowering the incubator oxygen level from 20% to 2% would recapitulate better the in vivo physiological environment of colonic epithelial cells and enhance the translational value of intestinal organoids as a drug testing platform. In the present study we examine the effects of the key IBD cytokines and drug targets TNF/IL17 on human colonic organoids (colonoids) under atmospheric (20%) or reduced (2%) O2. We show that colonoids derived from both healthy controls and IBD-patients are viable and responsive to IBD-relevant cytokines at 2% oxygen. Because chemokine release is one of the important immunoregulatory traits of the epithelium that may be fine-tuned by IBD-drugs, we also examined chemokine expression and release at different oxygen concentrations. We show that chemokine responses to TNF/IL17 in organoids display similarities to inflamed epithelium in IBD-patients. However, inflammation-associated genes induced by TNF/IL17 were attenuated at low oxygen concentration. We detected substantial oxygen-dependent differences in gene expression in untreated as well as TNF/IL17 treated colonoids in all donors. Further, for some of the IBD-relevant cytokines differences between colonoids from healthy controls and IBD patients were more pronounced in 2% O2 than 20% O2. Our results strongly indicate that an oxygen concentration similar to the in vivo epithelial cell environment is of essence in experimental pharmacology

Genome-wide Phenotypic Profiling Identifies and Categorizes Genes Required for Mycobacterial Low Iron Fitness

Iron is vital for nearly all living organisms, but during infection, not readily available to pathogens. Infectious bacteria therefore depend on specialized mechanisms to survive when iron is limited. These mechanisms make attractive targets for new drugs. Here, by genome-wide phenotypic profiling, we identify and categorize mycobacterial genes required for low iron fitness. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), can scavenge host-sequestered iron by high-affinity iron chelators called siderophores. We take advantage of siderophore redundancy within the non-pathogenic mycobacterial model organism M. smegmatis (Msmeg), to identify genes required for siderophore dependent and independent fitness when iron is low. In addition to genes with a potential function in recognition, transport or utilization of mycobacterial siderophores, we identify novel putative low iron survival strategies that are separate from siderophore systems. We also identify the Msmeg in vitro essential gene set, and find that 96% of all growth-required Msmeg genes have a mutual ortholog in Mtb. Of these again, nearly 90% are defined as required for growth in Mtb as well. Finally, we show that a novel, putative ferric iron ABC transporter contributes to low iron fitness in Msmeg, in a siderophore independent manner

Patient derived colonoids as drug testing platforms-Critical importance of oxygen concentration

Treatment of inflammatory bowel disease (IBD) is challenging, with a series of available drugs each helping only a fraction of patients. Patients may face time-consuming drug trials while the disease is active, thus there is an unmet need for biomarkers and assays to predict drug effect. It is well known that the intestinal epithelium is an important factor in disease pathogenesis, exhibiting physical, biochemical and immunologic driven barrier dysfunctions. One promising test system to study effects of existing or emerging IBD treatments targeting intestinal epithelial cells (IECs) is intestinal organoids (“mini-guts”). However, the fact that healthy intestinal epithelium is in a physiologically hypoxic state has largely been neglected, and studies with intestinal organoids are mainly performed at oxygen concentration of 20%. We hypothesized that lowering the incubator oxygen level from 20% to 2% would recapitulate better the in vivo physiological environment of colonic epithelial cells and enhance the translational value of intestinal organoids as a drug testing platform. In the present study we examine the effects of the key IBD cytokines and drug targets TNF/IL17 on human colonic organoids (colonoids) under atmospheric (20%) or reduced (2%) O2. We show that colonoids derived from both healthy controls and IBD-patients are viable and responsive to IBD-relevant cytokines at 2% oxygen. Because chemokine release is one of the important immunoregulatory traits of the epithelium that may be fine-tuned by IBD-drugs, we also examined chemokine expression and release at different oxygen concentrations. We show that chemokine responses to TNF/IL17 in organoids display similarities to inflamed epithelium in IBD-patients. However, inflammation-associated genes induced by TNF/IL17 were attenuated at low oxygen concentration. We detected substantial oxygen-dependent differences in gene expression in untreated as well as TNF/IL17 treated colonoids in all donors. Further, for some of the IBD-relevant cytokines differences between colonoids from healthy controls and IBD patients were more pronounced in 2% O2 than 20% O2. Our results strongly indicate that an oxygen concentration similar to the in vivo epithelial cell environment is of essence in experimental pharmacology