Roles of Hsp104 and trehalose in solubilisation of mutant huntingtin in heat shocked Saccharomyces cerevisiae cells

Inhibition of huntingtin aggregation, either in the nucleus and/or in the cytosol, has been identified as a major strategy to ameliorate the symptoms of Huntington's disease. Chaperones and other protein stabilisers would thus be key players in ensuring the correct folding of the amyloidogenic protein and its expression in the soluble form. By transient activation of the global heat stress response in Saccharomyces cerevisiaeBY4742, we show that heterologous expression of mutant huntingtin (103Q-htt) could be modulated so that the protein was partitioned off in the soluble fraction of the cytosol. This led to lower levels of reactive oxygen species and improved cell viability. Previous reports had speculated on the relationship between trehalose and the heat shock response in ensuring enhanced cell survival but no direct evidence of such an interaction was available. Using mutants of an isogenic strain which do not express the major trehalose synthetic or metabolising enzymes or the chaperone, heat shock protein 104 (Hsp104), we were able to identify the functions of Hsp104 and the osmoprotectant trehalose in solubilising mutant huntingtin. We propose that the beneficial effect of the protein refolding machinery in solubilising the aggregation-prone protein is exerted by maintaining a tight balance between the trehalose synthetic enzyme, trehalose-6-phosphate synthase 1 and Hsp104. This ensures that the level of the osmoprotectant, trehalose, does not exceed the limit beyond which it is reported to inhibit protein refolding.     

An Antitubulin Agent BCFMT Inhibits Proliferation of Cancer Cells and Induces Cell Death by Inhibiting Microtubule Dynamics

Using cell based screening assay, we identified a novel anti-tubulin agent (Z)-5-((5-(4-bromo-3-chlorophenyl)furan-2-yl)methylene)-2-thioxothiazolidin-4-one (BCFMT) that inhibited proliferation of human cervical carcinoma (HeLa) (IC(50), 7.2±1.8 μM), human breast adenocarcinoma (MCF-7) (IC(50), 10.0±0.5 μM), highly metastatic breast adenocarcinoma (MDA-MB-231) (IC(50), 6.0±1 μM), cisplatin-resistant human ovarian carcinoma (A2780-cis) (IC(50), 5.8±0.3 μM) and multi-drug resistant mouse mammary tumor (EMT6/AR1) (IC(50), 6.5±1μM) cells. Using several complimentary strategies, BCFMT was found to inhibit cancer cell proliferation at G2/M phase of the cell cycle apparently by targeting microtubules. In addition, BCFMT strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. At its half maximal proliferation inhibitory concentration (10 μM), BCFMT reduced the rates of growing and shortening phases of microtubules in MCF-7 cells by 37 and 40%, respectively. Further, it increased the time microtubules spent in the pause (neither growing nor shortening detectably) state by 135% and reduced the dynamicity (dimer exchange per unit time) of microtubules by 70%. In vitro, BCFMT bound to tubulin with a dissociation constant of 8.3±1.8 μM, inhibited tubulin assembly and suppressed GTPase activity of microtubules. BCFMT competitively inhibited the binding of BODIPY FL-vinblastine to tubulin with an inhibitory concentration (K(i)) of 5.2±1.5 μM suggesting that it binds to tubulin at the vinblastine site. In cultured cells, BCFMT-treatment depolymerized interphase microtubules, perturbed the spindle organization and accumulated checkpoint proteins (BubR1 and Mad2) at the kinetochores. BCFMT-treated MCF-7 cells showed enhanced nuclear accumulation of p53 and its downstream p21, which consequently activated apoptosis in these cells. The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug resistance cells by suppressing microtubule dynamics and indicated that the compound may have chemotherapeutic potential.     

Potential use of the essential oil ofTrachyspermum ammi against seed-borne fungi of Guar (Cyamopsis tetragonoloba L. (Taub.))

Essential oils isolated from leaves and seeds of seven umbelliferous plants were tested against the growth ofAspergillus flavus. Those from seeds ofTrachyspermum ammi, Cuminum cyminum, Carum carvi, Daucus carota and from leaves ofAnethum graveolens exhibited antifungal activity against the test fungus. Amongst these, oil from seeds ofTrachyspermum ammi was most toxic. Its minimum inhibitory concentration was 300 ppm, at which it exhibited fungistatic but not phytotoxic properties, when tested at 200, 300 and 400 ppm. The fungitoxic potency ofTrachyspermum seed oil remained unchanged after a long storage period and at high inoculum density of the test fungus. The oil was thermostable and was more efficaceous than the fungicides Agrosan G.N., Benlate, Ceresan, Dithane M-45 and Thiovit commonly used for the control of plant diseases.     

Functional analysis of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> subpopulations associated with artemisinin resistance in Cambodia

Background

Plasmodium falciparum malaria is one of the most widespread parasitic infections in humans and remains a leading global health concern. Malaria elimination efforts are threatened by the emergence and spread of resistance to artemisinin-based combination therapy, the first-line treatment of malaria. Promising molecular markers and pathways associated with artemisinin drug resistance have been identified, but the underlying molecular mechanisms of resistance remains unknown. The genomic data from early period of emergence of artemisinin resistance (2008–2011) was evaluated, with aim to define k13 associated genetic background in Cambodia, the country identified as epicentre of anti-malarial drug resistance, through characterization of 167 parasite isolates using a panel of 21,257 SNPs.

Results

Eight subpopulations were identified suggesting a process of acquisition of artemisinin resistance consistent with an emergence-selection-diffusion model, supported by the shifting balance theory. Identification of population specific mutations facilitated the characterization of a core set of 57 background genes associated with artemisinin resistance and associated pathways. The analysis indicates that the background of artemisinin resistance was not acquired after drug pressure, rather is the result of fixation followed by selection on the daughter subpopulations derived from the ancestral population.

Conclusions

Functional analysis of artemisinin resistance subpopulations illustrates the strong interplay between ubiquitination and cell division or differentiation in artemisinin resistant parasites. The relationship of these pathways with the P. falciparum resistant subpopulation and presence of drug resistance markers in addition to k13, highlights the major role of admixed parasite population in the diffusion of artemisinin resistant background. The diffusion of resistant genes in the Cambodian admixed population after selection resulted from mating of gametocytes of sensitive and resistant parasite populations.

     

Control of annual gonadal cycles in Indian songbirds

Abstract

Reproduction is a part of life cycle with great environmental dependence. In contrast to temperate avian species, which mostly breed during summer, the Indian songbirds have more flexible breeding programs and exhibit a spectrum of reproductive strategies with the breeding season scattered all over the year. Control of breeding cycles in the Indian songbirds, therefore, are broadly viewed in light of two strategies (i) birds showing strong photoperiodic component in regulation of reproductive and post-reproductive events (ii) birds that do not exhibit typical photoperiodic regulation indicating the involvement of an inherent rhythm of reproduction. Both circadian and circannual rhythms have been demonstrated to regulate annual gonadal cycles of Indian songbirds. While photoperiod continues to be a predominant proximate factor for timing of breeding in majority of Indian songbirds investigated so far, some studies reveal the role of non photoperiodic cues such as the food availability, temperature, rainfall, etc. in timing/modulating the timing of breeding. The conversion or non-conversion of thyroxine to triiodothyronine may act as a long or short photoperiod signal and may up or downregulate the synthesis and release of GnRH-I in hypothalamus, FSH and LH in anterior pituitary and gonadal steroids in gonads causing gonadal growth or regression, respectively.     

Towards molecular breeding of reproductive traits in cereal crops

The transition from vegetative to reproductive phase, flowering per se , floral organ development, panicle structure and morphology, meiosis, pollination and fertilization, cytoplasmic male sterility (CMS) and fertility restoration, and grain development are the main reproductive traits. Unlocking their genetic insights will enable plant breeders to manipulate these traits in cereal germplasm enhancement. Multiple genes or quantitative trait loci (QTLs) affecting flowering (phase transition, photoperiod and vernalization, flowering per se ), panicle morphology and grain development have been cloned, and gene expression research has provided new information about the nature of complex genetic networks involved in the expression of these traits. Molecular biology is also facilitating the identification of diverse CMS sources in hybrid breeding. Few Rf (fertility restorer) genes have been cloned in maize, rice and sorghum. DNA markers are now used to assess the genetic purity of hybrids and their parental lines, and to pyramid Rf or tms (thermosensitive male sterility) genes in rice. Transgene(s) can be used to create de novo CMS trait in cereals. The understanding of reproductive biology facilitated by functional genomics will allow a better manipulation of genes by crop breeders and their potential use across species through genetic transformation.     

Organelle positioning in muscles requires cooperation between two KASH proteins and microtubules

Striated muscle fibers are characterized by their tightly organized cytoplasm. Here, we show that the Drosophila melanogaster KASH proteins Klarsicht (Klar) and MSP-300 cooperate in promoting even myonuclear spacing by mediating a tight link between a newly discovered MSP-300 nuclear ring and a polarized network of astral microtubules (aMTs). In either klar or msp-300(ΔKASH), or in klar and msp-300 double heterozygous mutants, the MSP-300 nuclear ring and the aMTs retracted from the nuclear envelope, abrogating this even nuclear spacing. Anchoring of the myonuclei to the core acto-myosin fibrillar compartment was mediated exclusively by MSP-300. This protein was also essential for promoting even distribution of the mitochondria and ER within the muscle fiber. Larval locomotion is impaired in both msp-300 and klar mutants, and the klar mutants were rescued by muscle-specific expression of Klar. Thus, our results describe a novel mechanism of nuclear spacing in striated muscles controlled by the cooperative activity of MSP-300, Klar, and astral MTs, and demonstrate its physiological significance.     

NmeCas9 is an intrinsically high-fidelity genome-editing platform

Background

The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery.

Results

Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5′-N₄GATT-3′), for NmeCas9 genome editing in human cells.

Conclusions

Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

     

Repeated Administration of Dexamethasone Increases Phosphoinositide-Specific Phospholipase C Activity and mRNA and Protein Expression of the Phospholipase C β1 Isozyme in Rat Brain

Altered hypothalamic-pituitary-adrenal (HPA) function has been shown to be associated with changes in mood and behavior. The enzyme phosphoinositide-specific phospholipase C (PI-PLC), an important component of the PI signal transduction system, plays a major role in mediating various physiological functions. In the present study, we investigated the effects of a single dose and of repeated administration (0.5 or 1.0 mg/kg for 10 days) of dexamethasone (DEX), a synthetic glucocorticoid, on PI-PLC activity and on expression of PLC isozymes (beta1, delta1, and gamma1) in rat brain. Repeated administration of DEX (1.0 mg/kg) caused a significant increase in PI-PLC activity and in protein expression of the PLC beta1 isozyme in both membrane and cytosol fractions of cortex and hippocampus; however, the repeated administration of a smaller dose of DEX (0.5 mg/kg) caused these changes only in hippocampus but not in cortex. The increase in PLC beta1 protein was associated with an increase in its mRNA level, as measured by competitive RT-PCR. A single administration of DEX (0.5 or 1.0 mg/kg) to rats had no significant effects on PI-PLC activity or on the protein expression of PLC isozymes. These results suggest that DEX up-regulates PI-PLC in rat brain, which presumably is due to a selective increase in expression of the PLC beta1 isozyme, and that these changes in PI-PLC may be related to HPA axis-mediated changes in mood and behavior.