EMS-induced mutagenesis in Choy sum (Brassica chinensis var. parachinensis) and selection for low light tolerance using abiotic stress indices.

BACKGROUND: Choy Sum (Brassica rapa ssp. chinensis var. parachinensis), grown in a controlled environment, is vulnerable to changes in indoor light quality and displays distinct photo-morphogenesis responses. The scarcity of Choy Sum germplasm for indoor cultivation necessitates the development of new cultivars. Hence, this study attempted to develop mutants through chemical mutagenesis and select low-light-tolerant mutants by using abiotic stress tolerance indices. RESULTS: A mutant population of Choy Sum created using 1.5% ethyl methane sulfonate (EMS) at 4 h was manually pollinated to obtain the M2 generation. 154 mutants with reduced hypocotyl length were initially isolated from 3600 M2 seedlings screened under low light (R: FR = 0.5). Five mutants that showed reduced plant height at mature stages were selected and screened directly for shade tolerance in the M3 generation. Principal component analysis based on phenotypic data distinguished the M3 mutants from the wild type. Abiotic stress tolerance indices such as relative stress index (RSI), stress tolerance index (STI), geometric mean productivity (GMP), yield stability index (YSI), and stress resistance index (SRI) showed significant (P < 0.05), and positive associations with leaf yield under shade. M3-12-2 was selected as a shade-tolerant mutant based on high values of STI, YSI, and SRI with low values for tolerance (TOL) and stress susceptibility index (SSI). CONCLUSIONS: The results demonstrate that mutation breeding can be used to create dominant mutants in Choy Sum. Furthermore, we show that screening for low light and selection based on abiotic tolerance indices allowed the identification of mutants with high resilience under shade. This method should apply to developing new cultivars in other crop plants that can be suitable for controlled environments with stable yield performance.

A computational approach to investigate constitutive activation of NF-κB.

Nuclear factor kappa B (NF-κB) signaling is the master regulator of inflammatory pathways; therefore, its regulation has been the subject of investigation since last two decades. Multiple models have been published that describes the dynamics of NF-κB activity by stimulated activation and feedback loops. However, there is also paramount evidence of the critical role of posttranslational modifications (PTMs) in the regulation of NF-κB pathway. With the premise that PTMs present alternate routes for activation or repression of the NF-κB pathway, we have developed a model including all PTMs known so far describing the system behavior. We present a pathway network model consisting of 171 proteins forming 315 molecular species and consisting of 482 reactions that describe the NF-κB activity regulation in totality. The overexpression or knockdown of interacting molecular partners that regulate NF-κB transcriptional activity by PTMs is used to infer the dynamics of NF-κB activity and offers qualitative agreement between model predictions and the experimental results heuristically. Finally, we have demonstrated an instance of NF-κB constitutive activation through positive upregulation of cytokines (the stimuli) and IKK complex (NF-κB activator), the characteristic features in several cancer types and metabolic disorders, and its reversal by employing combinatorial activation of PPARG, PIAS3, and P50-homodimer. For the first time, we have presented a NF-κB model that includes transcriptional regulation by PTMs and presented a theoretical strategy for the reversal of NF-κB constitutive activation. The presented model would be important in understanding the NF-κB system, and the described method can be used for other pathways as well.

Fluorescent ureterography with indocyanine green in laparoscopic colorectal surgery: A safe method to prevent intraoperative ureteric injury.

Intraoperative injuries to the ureter can occur in complicated colorectal and gynaecologic procedures in minimal access surgery. The majority of these go unrecognised at the time of the operation, which can be disastrous to the patient. The routine use of ureteric stents is controversial, with some studies showing that stents only enable detection of ureteric injury but do not prevent it. Fluorescent image-guided surgery with indocyanine green (ICG) to visualise the ureter is a relatively new technique. We report our method of visualisation of the ureter in two patients undergoing laparoscopic anterior resection and Hartmann procedure, respectively. After induction of anaesthesia, retrograde catheterisation of both ureters was performed by the urologist. 2.5 mg ICG was injected into each catheter at the start of the procedure. Both ureters were visualised very well throughout the procedure with no post-operative complications. This technique using ICG adds visual cues to make up for the loss of tactile feedback, making it a safe strategy to prevent intraoperative ureteric injury.

Plant-based release system of negative air ions and its application on particulate matter removal.

Particulate matter (PM, especially PM2.5 with diameter 2.5 microns) has been regarded as the major air pollutant. Negative air ions (NAIs) could electrically charge PM and remove it much more efficiently. In this study, a bio-generator of NAIs has been developed, which helps plants to generate NAIs at around 100 × 106 ions/cm3 under pulsed electrical field (PEF) treatment. By using the bio-generator, PM2.5 concentration in a growth chamber could be reduced from around 500 to near 0 µg/m3 within 5 minutes. It could also be used to remove continuously generated PM. Upon PEF treatment, genes encoding oxidoreductases and other enzymes were up-regulated, some of which might contribute to the generation of superoxide anions (one of NAIs). On the other hand, the emission of large numbers of electrons from the surface/edge of plant leaves has been detected upon PEF treatment and these electrons might be captured by surrounding air molecules to generate high concentration of NAIs.

Systems Metabolic Alteration in a Semi-Dwarf Rice Mutant Induced by OsCYP96B4 Gene Mutation.

Dwarfism and semi-dwarfism are among the most valuable agronomic traits in crop breeding, which were adopted by the "Green Revolution". Previously, we reported a novel semi-dwarf rice mutant (oscyp96b4) derived from the insertion of a single copy of Dissociator (Ds) transposon into the gene OsCYP96B4. However, the systems metabolic effect of the mutation is not well understood, which is important for understanding the gene function and developing new semi-dwarf mutants. Here, the metabolic phenotypes in the semi-dwarf mutant (M) and ectopic expression (ECE) rice line were compared to the wild-type (WT) rice, by using nuclear magnetic resonance (NMR) metabolomics and quantitative real-time polymerase chain reaction (qRT-PCR). Compared with WT, ECE of the OsCYP96B4 gene resulted in significant increase of γ-aminobutyrate (GABA), glutamine, and alanine, but significant decrease of glutamate, aromatic and branched-chain amino acids, and some other amino acids. The ECE caused significant increase of monosaccharides (glucose, fructose), but significant decrease of disaccharide (sucrose); induced significant changes of metabolites involved in choline metabolism (phosphocholine, ethanolamine) and nucleotide metabolism (adenosine, adenosine monophosphate, uridine). These metabolic profile alterations were accompanied with changes in the gene expression levels of some related enzymes, involved in GABA shunt, glutamate and glutamine metabolism, choline metabolism, sucrose metabolism, glycolysis/gluconeogenesis pathway, tricarboxylic acid (TCA) cycle, nucleotide metabolism, and shikimate-mediated secondary metabolism. The semi-dwarf mutant showed corresponding but less pronounced changes, especially in the gene expression levels. It indicates that OsCYP96B4 gene mutation in rice causes significant alteration in amino acid metabolism, carbohydrate metabolism, nucleotide metabolism, and shikimate-mediated secondary metabolism. The present study will provide essential information for the OsCYP96B4 gene function analysis and may serve as valuable reference data for the development of new semi-dwarf mutants.

Genetic Landscape of Common Epilepsies: Advancing towards Precision in Treatment.

Epilepsy, a neurological disease characterized by recurrent seizures, is highly heterogeneous in nature. Based on the prevalence, epilepsy is classified into two types: common and rare epilepsies. Common epilepsies affecting nearly 95% people with epilepsy, comprise generalized epilepsy which encompass idiopathic generalized epilepsy like childhood absence epilepsy, juvenile myoclonic epilepsy, juvenile absence epilepsy and epilepsy with generalized tonic-clonic seizure on awakening and focal epilepsy like temporal lobe epilepsy and cryptogenic focal epilepsy. In 70% of the epilepsy cases, genetic factors are responsible either as single genetic variant in rare epilepsies or multiple genetic variants acting along with different environmental factors as in common epilepsies. Genetic testing and precision treatment have been developed for a few rare epilepsies and is lacking for common epilepsies due to their complex nature of inheritance. Precision medicine for common epilepsies require a panoramic approach that incorporates polygenic background and other non-genetic factors like microbiome, diet, age at disease onset, optimal time for treatment and other lifestyle factors which influence seizure threshold. This review aims to comprehensively present a state-of-art review of all the genes and their genetic variants that are associated with all common epilepsy subtypes. It also encompasses the basis of these genes in the epileptogenesis. Here, we discussed the current status of the common epilepsy genetics and address the clinical application so far on evidence-based markers in prognosis, diagnosis, and treatment management. In addition, we assessed the diagnostic predictability of a few genetic markers used for disease risk prediction in individuals. A combination of deeper endo-phenotyping including pharmaco-response data, electro-clinical imaging, and other clinical measurements along with genetics may be used to diagnose common epilepsies and this marks a step ahead in precision medicine in common epilepsies management.

Generating Cyan Fluorescence with De Novo Tripeptides: An In Vitro Mutation Study on the Role of Single Amino Acid Residues and Their Sequence.

Amino acids are natural choices as building blocks when developing biofunctional entities owing to their superior diversity and versatile physicochemical properties compared to nucleotide bases. A simple permutation of the amino acids creates a broad palette of proteins and these have been successfully engineered into useful biofunctional agents. For example, the intrinsic ultraviolet fluorescence of phenylalanine and tryptophan has been engineered to emit in the visible spectrum, which has broad applications for imaging/sensing probes, photothermal therapy agents, optogenetic switches, etc. Nature produces more colorful coats/furs, feathers/hairs, and eyes through various biochemical modifications of tyrosine-based pigmentation. However, it is challenging to modulate the fluorescence wavelength from the UV to the visible region through oligopeptides. Herein, we report an innovative approach to obtain cyan fluorescence by using de novo tripeptides containing glycine, tyrosine, and lysine, which form robust dimer structures under moderate oxidizing conditions. Through an in vitro mutation approach, we deduce that both the amino acids and their sequence play significant roles in modulating the fluorescence. We believe this work holds great promise for developing novel cell imaging and resonance energy-transfer-based fluorescent probes.

Lutein: A potential antibiofilm and antiquorum sensing molecule from green microalga Chlorella pyrenoidosa.

The increasing resistance of Pseudomonas aeruginosa towards antimicrobial agents has been a major cause for the escalation of untreatable diabetic foot ulcer cases around the globe. This demands research towards alternative natural products that inhibit biofilm formation by P. aeruginosa. The study focuses on enhancing as well as understanding the anti-biofilm property of lutein from Chlorella pyrenoidosa against MTCC strain of P. aeruginosa PAO1. C. pyrenoidosa was subjected to nutrient starvation (N-, S- and P-) and their growth, biomass, chlorophyll pigments and total carotenoids were estimated. Lutein extracted from nutrient starved C. pyrenoidosa were quantified using High Performance Liquid Chromatography (HPLC) and also used for quantification of biofilm formation, cell surface hydrophobicity (CSH), extracellular polymeric substances (EPS) and pyocyanin degradation. The results showed 20 µg/mL concentration of lutein showed maximum inhibition and degradation of biofilm formation, pyocyanin production, Cell Surface Hydrophobicity Extracellular Polymeric Substances, when compared to other concentrations. Azithromycin was used as a standard drug to compare the efficiency of lutein as a potential antibiofilm compound. Docking studies confirmed the interaction of lutein with the four proteins - LasI, LasR, RhlI and RhlR, involved in the quorum sensing mechanism during biofilm formation. Among them, RhlI protein was found to strongly interact and LasI exhibiting the least interaction with lutein. Gene expression analyses of las and rhl genes in P. aeruginosa PAO1 revealed a significant down regulation of both the genes in the cultures treated with different concentrations of lutein. Therefore, it can be understood that lutein is an effective antibiofilm agent and can be used in combination with generic drugs that are used for treating diseases such as diabetic foot ulcers, which are ineffective due to high biofilm forming capability of P. aeruginosa and other bacterial species.

Valine-glutamine (VQ) motif coding genes are ancient and non-plant-specific with comprehensive expression regulation by various biotic and abiotic stresses.

BACKGROUND: Valine-glutamine (VQ) motif containing proteins play important roles in abiotic and biotic stress responses in plants. However, little is known about the origin and evolution as well as comprehensive expression regulation of the VQ gene family. RESULTS: In this study, we systematically surveyed this gene family in 50 plant genomes from algae, moss, gymnosperm and angiosperm and explored their presence in other species from animals, bacteria, fungi and viruses. No VQs were detected in all tested algae genomes and all genomes from moss, gymnosperm and angiosperm encode varying numbers of VQs. Interestingly, some of fungi, lower animals and bacteria also encode single to a few VQs. Thus, they are not plant-specific and should be regarded as an ancient family. Their family expansion was mainly due to segmental duplication followed by tandem duplication and mobile elements. Limited contribution of gene conversion was detected to the family evolution. Generally, VQs were very much conserved in their motif coding region and were under purifying selection. However, positive selection was also observed during species divergence. Many VQs were up- or down-regulated by various abiotic / biotic stresses and phytohormones in rice and Arabidopsis. They were also co-expressed with some of other stress-related genes. All of the expression data suggest a comprehensive expression regulation of the VQ gene family. CONCLUSIONS: We provide new insights into gene expansion, divergence, evolution and their expression regulation of this VQ family. VQs were detectable not only in plants but also in some of fungi, lower animals and bacteria, suggesting the evolutionary conservation and the ancient origin. Overall, VQs are non-plant-specific and play roles in abiotic / biotic responses or other biological processes through comprehensive expression regulation.

Molecular mechanisms underlying the impact of mutations in SOD1 on its conformational properties associated with amyotrophic lateral sclerosis as revealed with molecular modelling.

BACKGROUND: So far, little is known about the molecular mechanisms of amyotrophic lateral sclerosis onset and progression caused by SOD1 mutations. One of the hypotheses is based on SOD1 misfolding resulting from mutations and subsequent deposition of its cytotoxic aggregates. This hypothesis is complicated by the fact that known SOD1 mutations of similar clinical effect could be distributed over the whole protein structure. RESULTS: In this work, a measure of hydrogen bond stability in conformational states was studied with elastic network analysis of 35 SOD1 mutants. Twenty-eight hydrogen bonds were detected in nine of 35 mutants with their stability being significantly different from that with the wild-type. These hydrogen bonds were formed by the amino acid residues known from the literature to be located in contact between SOD1 aggregates. Additionally, residues disposed between copper binding sites of both protein subunits were found from the models to form a stiff core, which can be involved in mechanical impulse transduction between these active centres. CONCLUSIONS: The modelling highlights that both stability of the copper binding site and stability of the dimer can play an important role in ALS progression.

Correction to: selected articles from Belyaev Conference 2017: structural biology.

After publication of the article [1], it has been brought to our attention that there is a discrepancy between the publication date on the pdf and online formats. The date on the pdf is 6th February 2018 and online is 5th February 2018. The correct publication date is the one on the pdf, 6th February 2018.

The OsPS1-F gene regulates growth and development in rice by modulating photosynthetic electron transport rate.

KEY MESSAGE: Ds insertion in rice OsPS1-F gene results in semi-dwarf plants with reduced tiller number and grain yield, while genetic complementation with OsPS1-F rescued the mutant phenotype. Photosynthetic electron transport is regulated in the chloroplast thylakoid membrane by multi-protein complexes. Studies about photosynthetic machinery and its subunits in crop plants are necessary, because they could be crucial for yield enhancement in the long term. Here, we report the characterization of OsPS1-F (encoding Oryza sativa PHOTOSYSTEM 1-F subunit) using a single copy Ds insertion rice mutant line. The homozygous mutant (osps1-f) showed striking difference in growth and development compared to the wild type (WT), including, reduction in plant height, tiller number, grain yield as well as pale yellow leaf coloration. Chlorophyll concentration and electron transport rate were significantly reduced in the mutant compared to the WT. OsPS1-F gene was highly expressed in rice leaves compared to other tissues at different developmental stages tested. Upon complementation of the mutant with proUBI::OsPS1-F, the observed mutant phenotypes were rescued. Our results illustrate that OsPS1-F plays an important role in regulating proper growth and development of rice plants.

Negative Air Ions and Their Effects on Human Health and Air Quality Improvement.

Negative air ions (NAIs) have been discovered for more than 100 years and are widely used for air cleaning. Here, we have carried out a comprehensive reviewing on the effects of NAIs on humans/animals, and microorganisms, and plant development. The presence of NAIs is credited for increasing psychological health, productivity, and overall well-being but without consistent or reliable evidence in therapeutic effects and with controversy in anti-microorganisms. Reports also showed that NAIs could help people in relieving symptoms of allergies to dust, mold spores, and other allergens. Particulate matter (PM) is a major air pollutant that affects human health. Experimental data showed that NAIs could be used to high-efficiently remove PM. Finally, we have reviewed the plant-based NAI release system under the pulsed electric field (PEF) stimulation. This is a new NAI generation system which releases a huge amount of NAIs under the PEF treatment. The system may be used to freshen indoor air and reduce PM concentration in addition to enriching oxygen content and indoor decoration at home, school, hospital, airport, and other indoor areas.

Oligomerization of the microtubule-associated protein tau is mediated by its N-terminal sequences: implications for normal and pathological tau action.

Despite extensive structure-function analyses, the molecular mechanisms of normal and pathological tau action remain poorly understood. How does the C-terminal microtubule-binding region regulate microtubule dynamics and bundling? In what biophysical form does tau transfer trans-synaptically from one neuron to another, promoting neurodegeneration and dementia? Previous biochemical/biophysical work led to the hypothesis that tau can dimerize via electrostatic interactions between two N-terminal 'projection domains' aligned in an anti-parallel fashion, generating a multivalent complex capable of interacting with multiple tubulin subunits. We sought to test this dimerization model directly. Native gel analyses of full-length tau and deletion constructs demonstrate that the N-terminal region leads to multiple bands, consistent with oligomerization. Ferguson analyses of native gels indicate that an N-terminal fragment (tau(45-230) ) assembles into heptamers/octamers. Ferguson analyses of denaturing gels demonstrates that tau(45-230) can dimerize even in sodium dodecyl sulfate. Atomic force microscopy reveals multiple levels of oligomerization by both full-length tau and tau(45-230) . Finally, ion mobility-mass spectrometric analyses of tau(106-144) , a small peptide containing the core of the hypothesized dimerization region, also demonstrate oligomerization. Thus, multiple independent strategies demonstrate that the N-terminal region of tau can mediate higher order oligomerization, which may have important implications for both normal and pathological tau action. The microtubule-associated protein tau is essential for neuronal development and maintenance, but is also central to Alzheimer's and related dementias. Unfortunately, the molecular mechanisms underlying normal and pathological tau action remain poorly understood. Here, we demonstrate that tau can homo-oligomerize, providing novel mechanistic models for normal tau action (promoting microtubule growth and bundling, suppressing microtubule shortening) and pathological tau action (poisoning of oligomeric complexes).

Downregulation of miRNAs during Delayed Wound Healing in Diabetes: Role of Dicer.

Delayed wound healing is a major complication associated with diabetes and is a result of a complex interplay among diverse deregulated cellular parameters. Although several genes and pathways have been identified to be mediating impaired wound closure, the role of microRNAs (miRNAs) in these events is not very well understood. Here, we identify an altered miRNA signature in the prolonged inflammatory phase in a wound during diabetes, with increased infiltration of inflammatory cells in the basal layer of the epidermis. Nineteen miRNAs were downregulated in diabetic rat wounds (as compared with normal rat wound, d 7 postwounding) together with inhibited levels of the central miRNA biosynthesis enzyme, Dicer, suggesting that in wounds of diabetic rats, the decreased levels of Dicer are presumably responsible for miRNA downregulation. Compared with unwounded skin, Dicer levels were significantly upregulated 12 d postwounding in normal rats, and this result was notably absent in diabetic rats that showed impaired wound closure. In a wound-healing specific quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) array, 10 genes were significantly altered in the diabetic rat wound and included growth factors and collagens. Network analyses demonstrated significant interactions and correlations between the miRNA predicted targets (regulators) and the 10 wound-healing specific genes, suggesting altered miRNAs might fine-tune the levels of these genes that determine wound closure. Dicer inhibition prevented HaCaT cell migration and affected wound closure. Altered levels of Dicer and miRNAs are critical during delayed wound closure and offer promising targets to address the issue of impaired wound healing.

Small molecule NPT-440-1 inhibits ionic flux through Aß1-42 pores: Implications for Alzheimer's disease therapeutics.

Increased levels of soluble amyloid-beta (Aß) oligomers are suspected to underlie Alzheimer's disease (AD) pathophysiology. These oligomers have been shown to form multi-subunit Aß pores in bilayers and induce uncontrolled, neurotoxic, ion flux, particularly calcium ions, across cellular membranes that might underlie cognitive impairment in AD. Small molecule interventions that modulate pore activity could effectively prevent or ameliorate their toxic activity. Here we examined the efficacy of a small molecule, NPT-440-1, on modulating amyloid pore permeability. Co-incubation of B103 rat neuronal cells with NPT-440-1 and Aß1-42 prevented calcium influx. In purified lipid bilayers, we show that a 10-15min preincubation, prior to membrane introduction, was required to prevent conductance. Thioflavin-T and circular dichroism both suggested a reduction in Aß1-42 ß-sheet content during this incubation period. Combined with previous studies on site-specific amino acid substitutions, these results suggest that pharmacological modulation of Aß1-42 could prevent amyloid pore-mediated AD pathogenesis.

Ion Channel Formation by Tau Protein: Implications for Alzheimer's Disease and Tauopathies.

Tau is a microtubule associated protein implicated in the pathogenesis of several neurodegenerative diseases. Because of the channel forming properties of other amyloid peptides, we employed planar lipid bilayers and atomic force microscopy to test tau for its ability to form ion permeable channels. Our results demonstrate that tau can form such channels, but only under acidic conditions. The channels formed are remarkably similar to amyloid peptide channels in their appearance, physical and electrical size, permanence, lack of ion selectivity, and multiple channel conductances. These channels differ from amyloid channels in their voltage dependence and resistance to blockade by zinc ion. These channels could explain tau's pathologic role in disease by lowering membrane potential, dysregulating calcium, depolarizing mitochondria, or depleting energy stores. Tau might also combine with amyloid beta peptides to form toxic channels.

Disordered amyloidogenic peptides may insert into the membrane and assemble into common cyclic structural motifs.

Aggregation of disordered amyloidogenic peptides into oligomers is the causative agent of amyloid-related diseases. In solution, disordered protein states are characterized by heterogeneous ensembles. Among these, ß-rich conformers self-assemble via a conformational selection mechanism to form energetically-favored cross-ß structures, regardless of their precise sequences. These disordered peptides can also penetrate the membrane, and electrophysiological data indicate that they form ion-conducting channels. Based on these and additional data, including imaging and molecular dynamics simulations of a range of amyloid peptides, Alzheimer's amyloid-ß (Aß) peptide, its disease-related variants with point mutations and N-terminal truncated species, other amyloidogenic peptides, as well as a cytolytic peptide and a synthetic gel-forming peptide, we suggest that disordered amyloidogenic peptides can also present a common motif in the membrane. The motif consists of curved, moon-like ß-rich oligomers associated into annular organizations. The motif is favored in the lipid bilayer since it permits hydrophobic side chains to face and interact with the membrane and the charged/polar residues to face the solvated channel pores. Such channels are toxic since their pores allow uncontrolled leakage of ions into/out of the cell, destabilizing cellular ionic homeostasis. Here we detail Aß, whose aggregation is associated with Alzheimer's disease (AD) and for which there are the most abundant data. AD is a protein misfolding disease characterized by a build-up of Aß peptide as senile plaques, neurodegeneration, and memory loss. Excessively produced Aß peptides may directly induce cellular toxicity, even without the involvement of membrane receptors through Aß peptide-plasma membrane interactions.