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.     

Decreased sensory responses in osteocalcin null mutant mice imply neuropeptide function

Osteocalcin, the most abundant member of the family of extracellular mineral binding gamma-carboxyglutamic acid proteins is synthesized primarily by osteoblasts. Its affinity for calcium ions is believed to limit bone mineralization. Several of the numerous hormones that regulate synthesis of osteocalcin, including glucocorticoids and parathyroid hormone, are also affected by stressful stimuli that require energy for an appropriate response. Based on our observations of OC responding to stressful sensory stimuli, the expression of OC in mouse and rat sensory ganglia was confirmed. It was thus hypothesized that the behavioral responses of the OC knockout mouse to stressful sensory stimuli would be abnormal. To test this hypothesis, behaviors related to sensory aspects of the stress response were quantified in nine groups of mice, aged 4-14 months, comparing knockout with their wild-type counterparts in six distinctly different behavioral tests. Resulting data indicated the following statistically significant differences: open field grooming frequency following saline injection, wild-type > knockout; paw stimulation with Von Frey fibers, knockout < wild-type; balance beam, knockout mobility < WT; thermal sensitivity to heat (tail flick), knockout < wild-type; and cold, knockout < wild-type. Insignificant differences in hanging wire test indicate that these responses are unrelated to reduced muscle strength. Each of these disparate environmental stimuli provided data indicating alterations of responses in knockout mice that suggest participation of osteocalcin in transmission of information about those sensory stimuli.     

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.     

Enhanced Expression of Rabies Virus Surface G-Protein in Escherichia coli using SUMO Fusion

Fusion systems are known to increase the expression of difficult to express recombinant proteins in soluble form to facilitate their purification. Rabies glycoprotein was also tough to express at sufficient level in soluble form in both E. coli and plant. The present work was aimed to over-express and purify this membrane protein from soluble extract of E. coli. Fusion of Small Ubiqutin like Modifier (SUMO) with rabies glycoprotein increased ~1.5 fold higher expression and ~3.0 fold solubility in comparison to non-fused in E. coli. The SUMO fusion also simplified the purification process. Previously engineered rabies glycoprotein gene in tobacco plants provides complete protection to mice, but the expression was very low for purification. Our finding demonstrated that the SUMO-fusion was useful for enhancing expression and solubility of the membrane protein and again proves to be a good alternative technology for applications in biomedical and pharmaceutical research.     

Epithelial topology

It is universally accepted that genetic control over basic aspects of cell and molecular biology is the primary organizing principle in development and homeostasis of living systems. However, instances do exist where important aspects of biological order arise without explicit genetic instruction, emerging instead from simple physical principles, stochastic processes, or the complex self-organizing interaction between random and seemingly unrelated parts. Being mostly resistant to direct genetic dissection, the analysis of such emergent processes falls into a grey area between mathematics, physics and molecular cell biology and therefore remains very poorly understood. We recently proposed a mathematical model predicting the emergence of a specific non-Gaussian distribution of polygonal cell shapes from the stochastic cell division process in epithelial cell sheets; this cell shape distribution appears to be conserved across a diverse set of animals and plants.1 The use of such topological models to study the process of cellular morphogenesis has a long history, starting almost a century ago, and many insights from those original works influence current experimental studies. Here we review current and past literature on this topic while exploring some new ideas on the origins and implications of topological order in proliferating epithelia.     

Two Tomato Expansin Genes Show Divergent Expression and Localization in Embryos during Seed Development and Germination

Expansins are plant proteins that can induce extension of isolated cell walls and are proposed to mediate cell expansion. Three expansin genes were expressed in germinating tomato (Lycopersicon esculentum Mill.) seeds, one of which (LeEXP4) was expressed specifically in the endosperm cap tissue enclosing the radicle tip. The other two genes (LeEXP8 and LeEXP10) were expressed in the embryo and are further characterized here. LeEXP8 mRNA was not detected in developing or mature seeds but accumulated specifically in the radicle cortex during and after germination. In contrast, LeEXP10 mRNA was abundant at an early stage of seed development corresponding to the period of rapid embryo expansion; it then decreased during seed maturation and increased again during germination. When gibberellin-deficient (gib-1) mutant seeds were imbibed in water, LeEXP8 mRNA was not detected, but a low level of LeEXP10 mRNA was present. Expression of both genes increased when gib-1 seeds were imbibed in gibberellin. Abscisic acid did not prevent the initial expression of LeEXP8 and LeEXP10, but mRNA abundance of both genes subsequently decreased during extended incubation. The initial increase in LeEXP8, but not LeEXP10, mRNA accumulation was blocked by low water potential, but LeEXP10 mRNA amounts fell after longer incubation. When seeds were transferred from abscisic acid or low water potential solutions to water, abundance of both LeEXP8 and LeEXP10 mRNAs increased in association with germination. The tissue localization and expression patterns of both LeEXP8 and LeEXP10 suggest developmentally specific roles during embryo and seedling growth.     

Comparative insights using the molecular homology model of BDNF (Brain derived neurotrophic factor) of Varanus komodoensis and the known NGF (Nerve growth factor) structure of Naja atra

BDNF (Brain derived neurotrophic factor) is a secretion protein and a member of the neurotrophin family of growth factors. Structural and functional characterization of BDNF Varanus komodoensis is of interest while its structure remains unknown. Thus, a homology molecular model of BDNF was constructed for gleaning possible structural insights. The model was compared with the structure of the homologous NGF (Nerve growth factor, another member of neuro-trophin family) from Naja atra. Comparative structural analysis of the models showed structural similarities with their predicted cavities for the interpretation of potential functional analogy.     

Design,synthesis and pharmacological evaluation of some novel indanone derivatives as acetylcholinesterase inhibitors for the management of cognitive dysfunction

The present study reports the effect of indanone derivatives on scopolamine induced deficit cholinergic neurotransmission serving as promising leads for the therapeutics of cognitive dysfunction. Eleven compounds 54–64 have been designed, synthesised and evaluated against behavioural alterations using step down passive avoidance protocol at a dose of 0.5?mg/kg with Donepezil (1) as the reference standard. All the synthesised compounds were evaluated for their in vitro acetylcholinesterase (AChE) inhibition at five different concentrations using mice brain homogenate as the source of the enzyme. Compounds 54, 56, 59 and 64 displayed appreciable activity with an IC₅₀ value of 14.06?µM, 12.30?µM, 14.06?µM and 12.01?µM, respectively towards acetylcholinesterase inhibition. The molecular docking study performed to predict the binding mode of the compounds suggested that these compounds could bind appreciably to the amino acids present at the active site of recombinant human acetylcholinesterase (rhAChE). The behavioural, biochemical and in silico pharmacokinetic studies were in concordance with each other.     

Acute pharmacogenetic activation of medial prefrontal cortex excitatory neurons regulates anxiety-like behaviour

The medial prefrontal cortex (mPFC) is implicated in anxiety-like behaviour. In rodent models, perturbations of mPFC neuronal activity through pharmacological manipulations, optogenetic activation of mPFC neurons or cell-type specific pharmacogenetic inhibition of somatostatin interneurons indicate conflicting effects on anxiety-like behaviour. In the present study we examined the effects of pharmacogenetic activation of Ca²⁺/calmodulin-dependent protein kinase α (CamKIIα)-positive excitatory neurons on anxiety-like behaviour. We used clozapine-N-oxide (CNO) to pharmacogenetically activate virally delivered CamKIIα-hM3Dq-DREADD in mPFC excitatory neurons. The effects of acute CNO or vehicle treatment on anxiety-like behaviour in the open field and elevated plus maze tests were examined in rats virally infected with either CamKIIα-hM3Dq-DREADD or CamKIIα-GFP. In addition, the effects of acute CNO treatment on the expression of the neuronal activity marker c-Fos were examined in the mPFC as well as downstream target neuronal circuits using immunohistochemistry. Acute pharmacogenetic activation of mPFC excitatory neurons evoked a significant decrease in anxiety-like behaviour selectively on the elevated plus maze task, but not the open field test. Acute CNO treatment resulted in enhanced c-Fos-immunopositive cell number in the infralimbic, prelimbic and cingulate subdivisions of the mPFC. This was also accompanied by enhanced c-Fos-immunopositive cell number in multiple downstream circuits of the mPFC in CNO-treated hM3Dq animals. Acute pharmacogenetic activation of mPFC excitatory neurons reduces anxiety-like behaviour in a task-specific fashion accompanied by enhanced c-Fos expression in the mPFC and multiple target circuits implicated in the regulation of anxiety-like behaviour.     

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.