Drosophila Spaghetti and Doubletime Link the Circadian Clock and Light to Caspases,Apoptosis and Tauopathy

While circadian dysfunction and neurodegeneration are correlated, the mechanism for this is not understood. It is not known if age-dependent circadian dysfunction leads to neurodegeneration or vice-versa, and the proteins that mediate the effect remain unidentified. Here, we show that the knock-down of a regulator (spag) of the circadian kinase Dbt in circadian cells lowers Dbt levels abnormally, lengthens circadian rhythms and causes expression of activated initiator caspase (Dronc) in the optic lobes during the middle of the day or after light pulses at night. Likewise, reduced Dbt activity lengthens circadian period and causes expression of activated Dronc, and a loss-of-function mutation in Clk also leads to expression of activated Dronc in a light-dependent manner. Genetic epistasis experiments place Dbt downstream of Spag in the pathway, and Spag-dependent reductions of Dbt are shown to require the proteasome. Importantly, activated Dronc expression due to reduced Spag or Dbt activity occurs in cells that do not express the spag RNAi or dominant negative Dbt and requires PDF neuropeptide signaling from the same neurons that support behavioral rhythms. Furthermore, reduction of Dbt or Spag activity leads to Dronc-dependent Drosophila Tau cleavage and enhanced neurodegeneration produced by human Tau in a fly eye model for tauopathy. Aging flies with lowered Dbt or Spag function show markers of cell death as well as behavioral deficits and shortened lifespans, and even old wild type flies exhibit Dbt modification and activated caspase at particular times of day. These results suggest that Dbt suppresses expression of activated Dronc to prevent Tau cleavage, and that the circadian clock defects confer sensitivity to expression of activated Dronc in response to prolonged light. They establish a link between the circadian clock factors, light, cell death pathways and Tau toxicity, potentially via dysregulation of circadian neuronal remodeling in the optic lobes.     

Biological and Chemical Induction of Resistance to the Globodera tabacum solanacearum in Oriental and Flue-Cured Tobacco (Nicotiana tabacum L.)

The effects of acibenzolar-S-methyl (ASM) and four combinations of plant growth-promoting rhizobacteria (PGPR) on the reproduction of a tobacco cyst nematode, Globodera tabacum solanacearum, and growth of Nicotiana tabacum (cv. K326 and Xanthi) were tested under greenhouse and field conditions. The PGPR included combinations of Bacillus subtilis A13 with B. pumilis INR7, B. pumilis SE34, B. licheniformis IN937b, or B. amyloliquefaciens IN937a, respectively. Among the four rhizobacterial combinations, IN937a + A13 exhibited the most consistent reduction in G. t. solanacearum cysts under greenhouse and field conditions. No undesirable effects of IN937a + A13 were observed on tobacco growth under greenhouse and field conditions. Use of INR7 + A13 reduced G. t. solanacearum reproduction on flue-cured tobacco cv. K326 but not on oriental tobacco cv. Xanthi. Application of ASM reduced final numbers of G. t. solanacearum cysts, but also resulted in phytotoxicity mainly under the greenhouse conditions. When oriental tobacco seedlings were pre-grown in a IN937a + A13-treated soil-less medium, a single application of ASM at 200 mg/L one week after transplanting significantly reduced G. t. solanacearum reproduction in the field.     

Effects of Ph Gene-Associated versus Induced Resistance to Tobacco Cyst Nematode in Flue-Cured Tobacco

Effects of the systemic acquired resistance (SAR)-inducing compound acibenzolar-S-methyl (ASM) and the plant-growth promoting rhizobacterial mixture Bacillus subtilis A13 and B. amyloliquefaciens IN937a (GB99+GB122) were assessed on the reproduction of a tobacco cyst nematode (TCN- Globodera tabacum solanacearum) under greenhouse conditions. Two sets of two independent experiments were conducted, each involving soil or root sampling. Soil sample experiments included flue-cured tobacco cultivars with (Ph(p)+: NC71 and NC102) and without (Ph(p)-: K326 and K346) a gene (Ph(p)) suppressing TCN parasitism. Root sample experiments examined TCN root parasitism of NC71 and K326. Cultivars possessing the Ph(p) gene (Ph(p)+) were compared with Ph(p)- cultivars to assess the effects of resistance mediated via Ph(p) gene vs. induced resistance to TCN. GB99+GB122 consistently reduced nematode reproductive ratio on both Ph(p)+ and Ph(p)- cultivars, but similar effects of ASM across Ph(p)- cultivars were less consistent. In addition, ASM application resulted in leaf yellowing and reduced root weight. GB99+GB122 consistently reduced nematode development in roots of both Ph(p)+ and Ph(p)- cultivars, while similar effects of ASM were frequently less consistent. The results of this study indicate that GB99+GB122 consistently reduced TCN reproduction in all flue-cured tobacco cultivars tested, while the effects of ASM were only consistent in Ph(p)+ cultivars. Under most circumstances, GB99+GB122 suppressed nematode reproduction more consistently than ASM compared to the untreated control.     

SLOW WALKER2, a NOC1/MAK21 Homologue,Is Essential for Coordinated Cell Cycle Progression during Female Gametophyte Development in Arabidopsis

Morphogenesis requires the coordination of cell growth, division, and cell differentiation. Female gametogenesis in flowering plants, where a single haploid spore undergoes continuous growth and nuclear division without cytokinesis to form an eight-nucleate coenocytic embryo sac before cellularization, provides a good system to study the genetic control of such processes in multicellular organisms. Here, we report the characterization of an Arabidopsis (Arabidopsis thaliana) female gametophyte mutant, slow walker2 (swa2), in which the progression of the mitotic cycles and the synchrony of female gametophyte development were impaired, causing an arrest of female gametophytes at the two-, four-, or eight-nucleate stage. Delayed pollination test showed that a portion of the mutant ovules were able to develop into functional embryo sacs and could be fertilized. SWA2 encodes a nucleolar protein homologous to yeast NUCLEOLAR COMPLEX ASSOCIATED PROTEIN1 (NOC1)/MAINTENANCE OF KILLER21 that, together with NOC2, is involved in preribosome export from the nucleus to the cytoplasm. Similarly, SWA2 can physically interact with a putative Arabidopsis NOC2 homologue. SWA2 is expressed ubiquitously throughout the plant, at high levels in actively dividing tissues and gametophytes. Therefore, we conclude that SWA2 most likely plays a role in ribosome biogenesis that is essential for the coordinated mitotic progression of the female gametophyte.Morphogenesis requires tightly coordinated coupling of cellular activities, such as cell growth, cell division, and differentiation. In past decades, significant progress on cell cycle control has been achieved mostly in single-celled organisms and cultured mammalian cells. The elucidation of the cyclin/cyclin-dependent kinase checkpoint control, for example, provides insight into molecular mechanisms on how and when cells divide. Mechanisms coupling cell growth to environmental and developmental signals have also been investigated. Ribosome biogenesis, a key for rapid cell growth, is coupled with nutrient availability and stress signals via the TOR signaling pathway (Warner et al., 2001; Wullischleger et al., 2006). However, questions such as how the cell senses intrinsic cellular homeostatic signals remain to be addressed. For example, how ribosome dynamics and translational activities are measured and coupled to cytokinesis and cell differentiation, especially in the context of development of multicellular organisms.Female gametogenesis in Arabidopsis (Arabidopsis thaliana) is a unique system to address such questions in multicellular organisms. During female gametogenesis, the haploid functional megaspore undergoes continuous cell growth and three cycles of consecutive nuclear division without cytokinesis, giving rise to a giant eight-nucleate, coenocytic cell: the embryo sac. The size of the embryo sac increases about 6-fold without cytokinesis until it reaches its maximum during gametogenesis in maize (Zea mays; Dow and Mascarenhas, 1991). The two polar nuclei migrate toward the micropylar half of the embryo sac and eventually fuse to give rise to a diploid nucleus of the central cell. As the polar nuclei migrate, cellularization takes place simultaneously to divide the coenocytic embryo sac into seven cells of four cell fates: three antipodal cells, two synergid cells, one egg cell, and one central cell (Drews et al., 1998; Grossniklaus and Schneitz, 1998; Yang and Sundaresan, 2000; Wilson and Yang, 2004). Obviously, its haploid nature and coupling of cell growth, division, and cell fates make the female gametophyte a nice system to investigate how these cellular activities are coordinated in development.The temporal and spatial control of cell growth, the mitotic division cycles, and cell fate specification during female gametogenesis have been the focus of sexual plant reproduction research. Recently, genetic studies have identified gametophytic mutations that start to shed light on the genetic and molecular control of these processes. Mutations in genes involved in diverse cellular functions, including ANDARTA (Howden et al., 1998), GAMETOPHYTIC FACTOR1 (GFA1; Christensen et al., 1997), HADAD (Moore et al., 1997), LETHAL OVULE2 (Sheridan and Huang, 1997), LYSOPHOSPHATIDYL ACYLTRANSFERASE (Kim et al., 2005), NOMEGA (Kwee and Sundaresan, 2003), PROLIFERA (Springer et al., 1995), SLOW WALKER1 (SWA1; Shi et al., 2005), SUCCINATE DEHYDROGENASE (Leon et al., 2007), and TISTRYA (Howden et al., 1998), all result in defective gametophytic cell divisions, implying that progression of the mitotic cycle is critical for the formation of a functional female gametophyte. Loss-of-function mutations in the Arabidopsis RETINOBLASTOMA-RELATED PROTEIN1, a key negative regulator controlling the G1/S transition of the cell cycle, result in uncontrolled nuclear proliferation and cell fates, giving rise to embryo sacs with supernumerary nuclei that are irregular in size and partially enclosed by cell wall-like structures (Ebel et al., 2004). Loss of functions in CYTOKININ INDEPENDENT1 (Hejatko et al., 2003), DIANA/AGAMOUS-LIKE61 (Bemer et al., 2008), AGAMOUS-LIKE80 (Portereiko et al., 2006a), and NUCLEAR FUSION DEFECTIVE1 (Portereiko et al., 2006b) affect polar nuclear fusion and central cell development.Accumulating data suggest a key role of the nucleolus in cell survival and proliferation (Cockell and Gasser, 1999; Shaw and Doonan, 2005). A number of nucleolar proteins have been discovered to be involved in linking cell proliferation control and ribosome biogenesis in yeast (Srivastava and Pollard, 1999; Du and Stillman, 2002; Jorgensen et al., 2002; Zhang et al., 2002; Bernstein et al., 2007). Mutations in genes involved in RNA processing, including SWA1 (Shi et al., 2005), GFA1/CLO1, and ATROPOS (ATO; Moll et al., 2008; Liu et al., 2009; Yagi et al., 2009), lead to slow progression of the division cycle during female gametogenesis. Intriguingly, mutation in LACHESIS (LIS), coding for a putative splicing factor, promotes egg cell fate in the synergid and the central cell at the expense of the synergid and central cell fate (Groß-Hardt et al., 2007), suggesting that LIS plays a pivotal role in suppressing the egg cell fate in the synergid and the central cell as well as the central cell fate in antipodal cells. Similarly, cell fate changes have also been observed in gfa1/clo1 and ato mutants (Moll et al., 2008). These data imply that RNA processing and ribosome biogenesis play a key role in coordinating cell cycle progression and cell fate. Here, we report the genetic and molecular characterization of a swa2 mutation that impairs cell growth and cell division in Arabidopsis. SWA2 encodes a nucleolar protein homologous to yeast NUCLEOLAR COMPLEX ASSOCIATED PROTEIN1 (NOC1)/MAINTENANCE OF KILLER21 (MAK21) that is essential for ribosome biogenesis in yeast. We also show that SWA2 interacts physically with NOC2 homologues in yeast cells. Together, these data indicate that SWA2 is most likely involved in ribosome biogenesis and essential for cell cycle progression in female gametophyte development in Arabidopsis.     

Rearing the Mexican beetle Zygogramma bicolorata (Chrysomelidae: Coleoptera) on a semi-synthetic diet

A semi-synthetic diet utilising parthenium leaf powder was evaluated for rearing the Mexican beetle Zygogramma bicolorata Pallister under in vitro conditions at 26±1°C, 65% RH. Significant differences were observed in the biological parameters of the beetle viz., the grub survival (54.3%), pupation (47.2%), fecundity (54.6/day) and total developmental period (37.7 days), when reared on the semi-synthetic diet compared to those when reared on natural parthenium leaves (91.5, 85.5, 74.8 and 29.8, respectively). The feasibility of utilising the semi-synthetic diet for mass production of the beetle is discussed.     

Anthranilic sulfonamide CCK1/CCK2 dual receptor antagonists II: Tuning of receptor selectivity and in vivo efficacy

In the previous article we demonstrated how certain CCK2R-selective anthranilic amides could be structurally modified to afford high-affinity, selective CCK1R activity. We now describe our efforts at modulating and optimizing the CCK1R and CCK2R affinities aimed at producing compounds with good pharmacokinetics properties and in vivo efficacy in rat models of gastric acid and pancreatic amylase secretion.     

Edgetic perturbation models of human inherited disorders

Cellular functions are mediated through complex systems of macromolecules and metabolites linked through biochemical and physical interactions, represented in interactome models as ‘nodes’ and ‘edges’, respectively. Better understanding of genotype‐to‐phenotype relationships in human disease will require modeling of how disease‐causing mutations affect systems or interactome properties. Here we investigate how perturbations of interactome networks may differ between complete loss of gene products (‘node removal’) and interaction‐specific or edge‐specific (‘edgetic’) alterations. Global computational analyses of ～50 000 known causative mutations in human Mendelian disorders revealed clear separations of mutations probably corresponding to those of node removal versus edgetic perturbations. Experimental characterization of mutant alleles in various disorders identified diverse edgetic interaction profiles of mutant proteins, which correlated with distinct structural properties of disease proteins and disease mechanisms. Edgetic perturbations seem to confer distinct functional consequences from node removal because a large fraction of cases in which a single gene is linked to multiple disorders can be modeled by distinguishing edgetic network perturbations. Edgetic network perturbation models might improve both the understanding of dissemination of disease alleles in human populations and the development of molecular therapeutic strategies.     

Role of Interferon Gamma Release Assay in Active TB Diagnosis among HIV Infected Individuals

Background

A rapid and specific test is urgently needed for tuberculosis (TB) diagnosis especially among human immunodeficiency virus (HIV) infected individuals. In this study, we assessed the sensitivity of Interferon gamma release assay (IGRA) in active tuberculosis patients who were positive for HIV infection and compared it with that of tuberculin skin test (TST).

Methodology/Principal Findings

A total of 105 HIV-TB patients who were naïve for anti tuberculosis and anti retroviral therapy were included for this study out of which 53 (50%) were culture positive. Of 105 tested, QuantiFERON-TB Gold in-tube (QFT-G) was positive in 65% (95% CI: 56% to 74%), negative in 18% (95% CI: 11% to 25%) and indeterminate in 17% (95% CI: 10% to 24%) of patients. The sensitivity of QFT-G remained similar in pulmonary TB and extra-pulmonary TB patients. The QFT-G positivity was not affected by low CD4 count, but it often gave indeterminate results especially in individuals with CD4 count <200 cells/µl. All of the QFT-G indeterminate patients whose sputum culture were positive, showed ≤0.25 IU/ml of IFN-γ response to phytohemagglutinin (PHA). TST was performed in all the 105 patients and yielded the sensitivity of 31% (95% CI: 40% to 22%). All the TST positives were QFT-G positives. The sensitivity of TST was decreased, when CD4 cell counts declined.

Conclusions/Significance

Our study shows neither QFT-G alone or in combination with TST can be used to exclude the suspicion of active TB disease. However, unlike TST, QFT-G yielded fewer false negative results even in individuals with low CD4 count. The low PHA cut-off point for indeterminate results suggested in this study (≤0.25 IU/ml) may improve the proportion of valid QFT-G results.     

Design,synthesis and anticancer activity of piperazine hydroxamates and their histone deacetylase (HDAC) inhibitory activity

Six compounds were synthesized with piperazine in linker region and hydroxamate as Zinc Binding Group (ZBG). They were screened against three cancer cell-lines (NCIH460; HCT116; U251). Compounds 5c and 5f with GI₅₀ value of 9.33 ± 1.3 μM and 12.03 ± 4 μM, respectively, were tested for their inhibitory potential on hHDAC8. Compound 5c had IC₅₀ of 33.67 μM. Compounds were also screened for their anticancer activity against HL60 human promyelocytic leukemia cell line due to the presence of pharmacophoric features of RR inhibitors in them. Compound 5c had IC₅₀ of 0.6 μM at 48 h.     

Active Site Mutations in Mammalian DNA Polymerase δ Alter Accuracy and Replication Fork Progression

DNA polymerase δ (pol δ) is one of the two main replicative polymerases in eukaryotes; it synthesizes the lagging DNA strand and also functions in DNA repair. In previous work, we demonstrated that heterozygous expression of the pol δ L604G variant in mice results in normal life span and no apparent phenotype, whereas a different substitution at the same position, L604K, is associated with shortened life span and accelerated carcinogenesis. Here, we report in vitro analysis of the homologous mutations at position Leu-606 in human pol δ. Four-subunit human pol δ variants that harbor or lack 3′ → 5′-exonucleolytic proofreading activity were purified from Escherichia coli. The pol δ L606G and L606K holoenzymes retain catalytic activity and processivity similar to that of wild type pol δ. pol δ L606G is highly error prone, incorporating single noncomplementary nucleotides at a high frequency during DNA synthesis, whereas pol δ L606K is extremely accurate, with a higher fidelity of single nucleotide incorporation by the active site than that of wild type pol δ. However, pol δ L606K is impaired in the bypass of DNA adducts, and the homologous variant in mouse embryonic fibroblasts results in a decreased rate of replication fork progression in vivo. These results indicate that different substitutions at a single active site residue in a eukaryotic polymerase can either increase or decrease the accuracy of synthesis relative to wild type and suggest that enhanced fidelity of base selection by a polymerase active site can result in impaired lesion bypass and delayed replication fork progression.