Flow through Multiple Well Points System

Under natural geographical settings, there are regions all over the world where the native groundwater quality is brackish. However, recharging from the surface water bodies (i.e., rivers and canals) establishes freshwater lenses in the upper portions of these brackish aquifers. Skimming of these freshwater lenses is a viable technique for sustaining livelihood and agricultural practices in these regions. In the present chapter, various skimming methods have been discussed. In addition, one another type of problem has also been developed in certain pockets of these regions. The non-withdrawal of groundwater and the excess use of easily available surface water resources (i.e., canal water) have created severe water logging conditions. Subsequently, it leads to the creation of salt‐affected soils. One of such areas located in the southwestern Punjab (India) was taken under study. Keeping the region\u27s problems in mind and considering the merits and demerits of all the skimming methods, a much efficient and affordable technique named multiple well points system (MWPS) has been proposed. The continuous use of MWPS not only reclaimed the area but also improved the groundwater quality. The system was field tested and modified before recommending to the farmers. Later on, the MWPS\u27s feasibility as a groundwater recharging system was also demonstrated in the field and the results have shown that it is a viable technique for reviving freshwater lenses in the region

Cell biological mechanisms of activity-dependent synapse to nucleus translocation of CRTC1 in neurons.

Previous studies have revealed a critical role for CREB-regulated transcriptional coactivator (CRTC1) in regulating neuronal gene expression during learning and memory. CRTC1 localizes to synapses but undergoes activity-dependent nuclear translocation to regulate the transcription of CREB target genes. Here we investigate the long-distance retrograde transport of CRTC1 in hippocampal neurons. We show that local elevations in calcium, triggered by activation of glutamate receptors and L-type voltage-gated calcium channels, initiate active, dynein-mediated retrograde transport of CRTC1 along microtubules. We identify a nuclear localization signal within CRTC1, and characterize three conserved serine residues whose dephosphorylation is required for nuclear import. Domain analysis reveals that the amino-terminal third of CRTC1 contains all of the signals required for regulated nucleocytoplasmic trafficking. We fuse this region to Dendra2 to generate a reporter construct and perform live-cell imaging coupled with local uncaging of glutamate and photoconversion to characterize the dynamics of stimulus-induced retrograde transport and nuclear accumulation

Cold- and salinity stress-induced bipolar pea DNA helicase 47 is involved in protein synthesis and stimulated by phosphorylation with protein kinase C

Helicases are involved in the metabolism of nucleic acid; this is very sensitive to the abiotic stresses that reduce plant growth and productivity. However, the molecular targets responsible for this sensitivity have not been well studied. Here we report on the isolation and characterization of cold- and salinity stress-induced pea DNA helicase 47 (PDH47). The transcript of PDH47 was induced in both shoots and roots under cold (4°C) and salinity (300 mm NaCl) stress, but there was no change in response to drought stress. Tissue-specific differential regulation was observed under heat (37°C) stress. ABA treatment did not alter expression of PDH47 in shoots but induced its mRNA in roots, indicating a role for PDH47 in both the ABA-independent and ABA-dependent pathways in abiotic stress. The purified recombinant protein (47 kDa) contains ATP-dependent DNA and RNA helicase and DNA-dependent ATPase activities. With the help of photoaffinity labeling, PDH47 was labeled by [α-32P]-ATP. PDH47 is a unique bipolar helicase that contains both 3' to 5' and 5' to 3' directional helicase activities. Anti-PDH47 antibodies immunodeplete the activities of PDH47 and inhibit in vitro translation of protein. Furthermore, the PDH47 protein showed upregulation of protein synthesis. The activities of PDH47 are stimulated after phosphorylation by protein kinase C at Ser and Thr residues. Western blot analysis and in vivo immunostaining, followed by confocal microscopy, showed PDH47 to be localized in both the nucleus and cytosol. The discovery of cold- and salinity stress-induced DNA helicase should make an important contribution to a better understanding of DNA metabolism and stress signaling in plants. Its bipolar helicase activities may also be involved in distinct cellular processes in stressed conditions

Rapid degradation of mutant SLC25A46 by the ubiquitin-proteasome system results in MFN1/2-mediated hyperfusion of mitochondria.

SCL25A46 is a mitochondrial carrier protein that surprisingly localizes to the outer membrane and is distantly related to Ugo1. Here we show that a subset of SLC25A46 interacts with mitochondrial dynamics components and the MICOS complex. Decreased expression of SLC25A46 results in increased stability and oligomerization of MFN1 and MFN2 on mitochondria, promoting mitochondrial hyperfusion. A mutation at L341P causes rapid degradation of SLC25A46, which manifests as a rare disease, pontocerebellar hypoplasia. The E3 ubiquitin ligases MULAN and MARCH5 coordinate ubiquitylation of SLC25A46 L341P, leading to degradation by organized activities of P97 and the proteasome. Whereas outer mitochondrial membrane-associated degradation is typically associated with apoptosis or a specialized type of autophagy termed mitophagy, SLC25A46 degradation operates independently of activation of outer membrane stress pathways. Thus SLC25A46 is a new component in mitochondrial dynamics that serves as a regulator for MFN1/2 oligomerization. Moreover, SLC25A46 is selectively degraded from the outer membrane independently of mitophagy and apoptosis, providing a framework for mechanistic studies in the proteolysis of outer membrane proteins

Protein palmitoylation plays an important role in Trichomonas vaginalis adherence

The flagellated protozoan parasite Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted infection worldwide. As an obligate extracellular pathogen, adherence to epithelial cells is critical for parasite survival within the human host and a better understanding of this process is a prerequisite for the development of therapies to combat infection. In this sense, recent work has shown S-acylation as a key modification that regulates pathogenesis in different protozoan parasites. However, there are no reports indicating whether this post-translational modification is a mechanism operating in T. vaginalis. In order to study the extent and function of S-acylation in T. vaginalis biology, we undertook a proteomic study to profile the full scope of S-acylated proteins in this parasite and reported the identification of 363 proteins involved in a variety of biological processes such as protein transport, pathogenesis related and signaling, among others. Importantly, treatment of parasites with the palmitoylation inhibitor 2-bromopalmitate causes a significant decrease in parasite: Parasite aggregation as well as adherence to host cells suggesting that palmitoylation could be modifying proteins that are key regulators of Trichomonas vaginalis pathogenesis.Fil: Nievas, Yésica Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Vashisht, Ajay A.. University of California; Estados UnidosFil: Corvi, Maria Martha. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Metz, Sebastián Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Johnson, Patricia J. University of California; Estados UnidosFil: Wohlschlegel, James A.. University of California; Estados UnidosFil: de Miguel, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin

In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis.

UnlabelledToxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis.ImportanceMost intracellular pathogens reside inside a membrane-bound vacuole within their host cell that is extensively modified by the pathogen to optimize intracellular growth and avoid host defenses. In Toxoplasma, this vacuole is modified by a host of secretory GRA proteins, many of which remain unidentified. Here we demonstrate that in vivo biotinylation of proximal and interacting proteins using the promiscuous biotin ligase BirA* is a powerful approach to rapidly identify vacuolar GRA proteins. We further demonstrate that one factor identified by this approach, GRA39, plays an important role in the ability of the parasite to replicate within its host cell and cause disease

Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal

MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans microRNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We extensively characterized proteins associated with wild-type and mutant ALG-1 and found that the mutant ALG-1(anti) protein fails to interact with numerous miRISC cofactors, including proteins known to be necessary for target repression. In addition, alg-1(anti) mutants dramatically overaccumulated microRNA* (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA* strands, with some microRNA* strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA* strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA* strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA* strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA* ejection steps of miRISC maturation

Gcn5 and Sirtuins Regulate Acetylation of the Ribosomal Protein Transcription Factor Ifh1

SummaryBackgroundIn eukaryotes, ribosome biosynthesis involves the coordination of ribosomal RNA and ribosomal protein (RP) production. In S. cerevisiae, the regulation of ribosome biosynthesis occurs largely at the level of transcription. The transcription factor Ifh1 binds at RP genes and promotes their transcription when growth conditions are favorable. Although Ifh1 recruitment to RP genes has been characterized, little is known about the regulation of promoter-bound Ifh1.ResultsWe used a novel whole-cell-extract screening approach to identify Spt7, a member of the SAGA transcription complex, and the RP transactivator Ifh1 as highly acetylated nonhistone species. We report that Ifh1 is modified by acetylation specifically in an N-terminal domain. These acetylations require the Gcn5 histone acetyltransferase and are reversed by the sirtuin deacetylases Hst1 and Sir2. Ifh1 acetylation is regulated by rapamycin treatment and stress and limits the ability of Ifh1 to act as a transactivator at RP genes.ConclusionsOur data suggest a novel mechanism of regulation whereby Gcn5 functions to titrate the activity of Ifh1 following its recruitment to RP promoters to provide more than an all-or-nothing mode of transcriptional regulation. We provide insights into how the action of histone acetylation machineries converges with nutrient-sensing pathways to regulate important aspects of cell growth