Physical interactions between specifically regulated subpopulations of the MCM and RNR complexes prevent genetic instability.

The helicase MCM and the ribonucleotide reductase RNR are the complexes that provide the substrates (ssDNA templates and dNTPs, respectively) for DNA replication. Here, we demonstrate that MCM interacts physically with RNR and some of its regulators, including the kinase Dun1. These physical interactions encompass small subpopulations of MCM and RNR, are independent of the major subcellular locations of these two complexes, augment in response to DNA damage and, in the case of the Rnr2 and Rnr4 subunits of RNR, depend on Dun1. Partial disruption of the MCM/RNR interactions impairs the release of Rad52 -but not RPA-from the DNA repair centers despite the lesions are repaired, a phenotype that is associated with hypermutagenesis but not with alterations in the levels of dNTPs. These results suggest that a specifically regulated pool of MCM and RNR complexes plays non-canonical roles in genetic stability preventing persistent Rad52 centers and hypermutagenesis.

Impairment of the non-catalytic subunit Dpb2 of DNA Pol ɛ results in increased involvement of Pol δ on the leading strand.

The generally accepted model assumes that leading strand synthesis is performed by Pol ε, while lagging-strand synthesis is catalyzed by Pol δ. Pol ε has been shown to target the leading strand by interacting with the CMG helicase [Cdc45 Mcm2-7 GINS(Psf1-3, Sld5)]. Proper functioning of the CMG-Pol ɛ, the helicase-polymerase complex is essential for its progression and the fidelity of DNA replication. Dpb2p, the essential non-catalytic subunit of Pol ε plays a key role in maintaining the correct architecture of the replisome by acting as a link between Pol ε and the CMG complex. Using a temperature-sensitive dpb2-100 mutant previously isolated in our laboratory, and a genetic system which takes advantage of a distinct mutational signature of the Pol δ-L612M variant which allows detection of the involvement of Pol δ in the replication of particular DNA strands we show that in yeast cells with an impaired Dpb2 subunit, the contribution of Pol δ to the replication of the leading strand is significantly increased.

Quantitative Analysis of Nucleoside Triphosphate Pools in Mouse Muscle Using Hydrophilic Interaction Liquid Chromatography Coupled with Tandem Mass Spectrometry Detection.

Defects in deoxyribonucleoside triphosphate (dNTP) metabolism are associated with a number of mitochondrial DNA (mtDNA) depletion syndromes (MDS). These disorders affect the muscles, liver, and brain, and the concentrations of dNTPs in these tissues are already normally low and are, therefore, difficult to measure. Thus, information about the concentrations of dNTPs in tissues of healthy animals and animals with MDS are important for mechanistic studies of mtDNA replication, analysis of disease progression, and the development of therapeutic interventions. Here, we present a sensitive method for the simultaneous analysis of all four dNTPs as well as all four ribonucleoside triphosphates (NTPs) in mouse muscles using hydrophilic interaction liquid chromatography coupled with triple quadrupole mass spectrometry. The simultaneous detection of NTPs allows them to be used as internal standards for the normalization of dNTP concentrations. The method can be applied for measuring dNTP and NTP pools in other tissues and organisms.

Optimization of 'on farm' hydropriming conditions in wheat: Soaking time and water volume have interactive effects on seed performance.

Seed priming is a simple and cost effective method to obtain a better plant stand under diverse environmental conditions. The current study was designed to determine the optimal priming duration and water volume for wheat seed. For this experiment, three wheat genotypes with distinct genetic and adaptive backgrounds were chosen. Seeds of each genotype were hydroprimed for 7 durations, i.e. 1, 2, 4, 8, 12, 16, and 20 hours, in three different water volumes, i.e. half, equal, and double volume with respect to seed weight and then surface dried for 1 hour. The control was unprimed (dry) seed. The germination characteristics and seedling vigour potential of hydroprimed seeds were evaluated in the lab by recording several parameters such as germination percentage and speed, seedling growth, and vigour indices at two different temperature levels. The results showed that optimal duration for hydropriming of wheat seed is 12 hours with an equal volume with respect to original seed weight, closely followed by 8 hours with double volume. Reduction in seed performance was observed at 16 and 20 hours priming particularly at double volume treatment. Effect of temperature on seed germination showed improvement in seedling vigour at 25°C when compared to 20°C, although effect on germination percentage was non-significant. Volume of water and priming duration showed significant interactive effects demonstrating that a higher volume can give equivalent results at a shorter duration and vice versa. Another experiment was also conducted to compare the on-farm priming (surface dried seed) with conventional priming (seed re-dried to original moisture) taking 3 potential durations i.e. 8, 12 and 16 hours. Results revealed that both priming methods were statistically at par in terms of germination percentage, while, surface drying resulted in better seedling vigour and speed of germination.

Factors and perceptions associated with energy drink consumption among undergraduate students attending a rural university: a descriptive study.

Objectives: This study examined the prevalence, patterns, and knowledge of Energy Drink (ED) consumption among undergraduate students. Participants: Participants included students (n = 373) attending a medium-sized rural university in Texas. Methods: Students were surveyed anonymously using convenience sampling and a cross-sectional design with 15 items structured questionnaire. Results: Nearly 90% of the study participants (N = 373) were between 18-24 years. Among the consumers (n = 165), the majority were females (80%) and Caucasian (73%). About 60% of them lived on campus, 22% were engaged in some type of sports activity, and primarily represented the freshmen (42%) and sophomore (25%) population. Having apriori knowledge of the negative health effects of EDs was associated with their consumption [OR: 0.40, CI: (0.22, 0.72)]. Conclusions: Our findings highlight the need to establish programs and policies on campus to address ED consumption issues and create educational campaigns to inform the undergraduate population attending a rural university.

Nanotechnology for sustainable agro-food systems: The need and role of nanoparticles in protecting plants and improving crop productivity.

The rapid population growth and environmental challenges in agriculture need innovative and sustainable solutions to meet the growing need for food worldwide. Recent nanotechnological advances found its broad applicability in agriculture's protection and post-harvesting. Engineered nanomaterials play a vital role in plant regulation, seed germination, and genetic manipulation. Their size, surface morphology, properties, and composition were designed for controlled release and enhanced properties in agriculture and the food industry. Nanoparticles can potentially be applied for the targeted and controlled delivery of fertilizers, pesticides, herbicides, plant growth regulators, etc. This help to eliminate the use of chemical-based pesticides and their water solubility, protect agrochemicals from breakdown and degradation, improve soil health, and naturally control crop pathogens, weeds, and insects, ultimately leading to enhanced crop growth and production capacity in the food industry. They can be effectively utilized for nano-encapsulation, seed germination, genetic manipulation, etc., for protecting plants and improving crop productivity, safe and improved food quality, and monitoring climate conditions. Nanoparticles played a crucial role in the uptake and translocation processes, genetically modifying the crops, high seed germination, and productivity. In this article, we have reviewed some important applications of nanoparticles for sustainable agro-food systems. The need and role of nanotechnology concerning challenges and problems faced by agriculture and the food industry are critically discussed, along with the limitations and future prospects of nanoparticles.

dNTP concentrations do not increase in mammalian cells in response to DNA damage.

A recent publication reported a uniform ∼5- to 6-fold increase in dNTP pools 30 min after exposure to ionizing radiation. Das et al. were not able to reproduce these results. Their data instead agree with earlier publications reporting no increase in dNTP pools in mammalian cells in response to DNA damage.

Role of Salicylic Acid in Combating Heat Stress in Plants: Insights into Modulation of Vital Processes.

In the present era of climate change and global warming, high temperatures have increased considerably, posing a threat to plant life. Heat stress affects the biochemistry, physiology and molecular makeup of the plant by altering the key processes, i.e., photosynthesis, respiration and reproduction which reduces its growth and development. There is a dire need to manage this problem sustainably for plant conservation as well as the food security of the human population. Use of phytohormones to induce thermotolerance in plants can be a sustainable way to fight the adversities of heat stress. Phytohormone-induced thermotolerance proves to be a compelling approach to sustainably relieve the damaging effects of heat stress on plants. Salicylic acid (SA) is an essential molecule in biotic and abiotic defense response signal transduction pathways. When supplied externally, it imparts heat stress tolerance to the plants by different means, viz., increased Heat Shock Proteins (HSP) production, Reactive oxygen species (ROS) scavenging, protection of the reproductive system and enhancing photosynthetic efficiency. The effect of SA on plants is highly dependent on the concentration applied, plant species, plant age, type of tissues treated, and duration of the treatment. The present review paper summarizes the mechanism of thermotolerance induced by salicylic acid in plants under heat stress conditions. It includes the regulatory effects of SA on heat shock proteins, antioxidant metabolism, and maintenance of Ca2+ homeostasis under heat stress. This review combines the studies conducted to elucidate the role of SA in the modulation of different mechanisms which lead to heat stress tolerance in plants. It discusses the mechanism of SA in protecting the photosynthetic machinery and reproductive system during high-temperature stress.

Synthesis and characterization of Ni0.5Al0.5Fe2O4 nanoparticles for potent antifungal activity against dry rot of ginger (Fusarium oxysporum).

Current study signifies the use of nanoparticles as alternative in plant disease management to avoid harmful effect of pesticide and fungicide residue. Synthesis of nanoparticles (Ni0.5Al0.5Fe2O4) by hydrothermal method and studied their X-ray diffraction analysis (XRD), Raman spectra, and UV spectra and further successfully evaluated for antifungal activity against a soil and seed borne pathogenic fungus (Fusarium oxysporum).Among various pests, fungal pathogens are the main cause of crop destruction and we developed nanoparticles (Ni0.5Al0.5Fe2O4) which is successfully evaluated for antimycotic activity against dry rot (F. oxysporum) of ginger which causes 50-70% losses in the ginger plant. In vitro and in vivo analysis designated that the nanoparticles (Ni0.5Al0.5Fe2O4) has shown an excellent antifungal activity against F. oxysporum at 0.5 mg/ml concentration. Similarly, no disease incidence was recorded when Ni0.5Al0.5Fe2O4 nanoparticles used at 0.5 mg/ml concentration under in vivo conditions. In plants various environmental stresses (biotic and abiotic) leads to excessive production of reactive oxygen species (ROS) causing progressive oxidative damage and ultimately leads to cell death. The role of ROS in nanoparticles (Ni0.5Al0.5Fe2O4) represents by reduction in the growth inhibition of F. oxysporum. We speculated in light of these results that the cytotoxic effect of Ni0.5Al0.5Fe2O4 nanoparticles on F. oxysporum may be mediated through ROS. We can suggest the role of nanoparticles (Ni0.5Al0.5Fe2O4) gives a promising result as a fungicidal activity and could be a novel family of future new generation fungicide.

The development of compliance behavioral imperatives in public for management of covid-19.

The Covid-19 pandemic, which was declared a public health emergency on 30 January 2020, has made it crucial for humans to learn how to behave to control the pandemic's spread. Policymakers must assess human behaviour and their responses to pandemic breakouts to develop a strategy for limiting pandemics and their harm to society at large. The present study applying exploratory factor analysis assessed five aspects of human behaviour regarding Covid-19, namely compliance behaviour, avoidance behaviour, protective behaviour, informed behaviour, and risk perception. The study applying hierarchical regression discovered that by combining informed, protective, and avoidance behaviour, people can be convinced to embrace the compliance behaviour required by public authorities. Furthermore, higher risk perception also positively moderates the relationship between information and compliance behaviour.

Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex.

DNA replication is performed by replisome proteins, which are highly conserved from yeast to humans. The CMG [Cdc45-Mcm2-7-GINS(Psf1-3, Sld5)] helicase unwinds the double helix to separate the leading and lagging DNA strands, which are replicated by the specialized DNA polymerases epsilon (Pol ε) and delta (Pol δ), respectively. This division of labor was confirmed by both genetic analyses and in vitro studies. Exceptions from this rule were described mainly in cells with impaired catalytic polymerase ε subunit. The central role in the recruitment and establishment of Pol ε on the leading strand is played by the CMG complex assembled on DNA during replication initiation. In this work we analyzed the consequences of impaired functioning of the CMG complex for the division labor between DNA polymerases on the two replicating strands. We showed in vitro that the GINSPsf1-1 complex poorly bound the Psf3 subunit. In vivo, we observed increased rates of L612M Pol δ-specific mutations during replication of the leading DNA strand in psf1-1 cells. These findings indicated that defective functioning of GINS impaired leading strand replication by Pol ε and necessitated involvement of Pol δ in the synthesis on this strand with a possible impact on the distribution of mutations and genomic stability. These are the first results to imply that the division of labor between the two main replicases can be severely influenced by a defective nonpolymerase subunit of the replisome.

Chl1 helicase controls replication fork progression by regulating dNTP pools.

Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that MRC1 negatively interacts with CHL1, which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased RNR1 gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions.

Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples.

Information about the cellular concentrations of deoxyribonucleoside triphosphates (dNTPs) is instrumental for mechanistic studies of DNA replication and for understanding diseases caused by defects in dNTP metabolism. The dNTPs are measured by methods based on either HPLC or DNA polymerization. An advantage with the HPLC-based techniques is that the parallel analysis of ribonucleoside triphosphates (rNTPs) can serve as an internal quality control of nucleotide integrity and extraction efficiency. We have developed a Freon-free trichloroacetic acid-based method to extract cellular nucleotides and an isocratic reverse phase HPLC-based technique that is able to separate dNTPs, rNTPs and ADP in a single run. The ability to measure the ADP levels improves the control of nucleotide integrity, and the use of an isocratic elution overcomes the shifting baseline problems in previously developed gradient-based reversed phase protocols for simultaneously measuring dNTPs and rNTPs. An optional DNA-polymerase-dependent step is used for confirmation that the dNTP peaks do not overlap with other components of the extracts, further increasing the reliability of the analysis. The method is compatible with a wide range of biological samples and has a sensitivity better than other UV-based HPLC protocols, closely matching that of mass spectrometry-based detection.

Indian classical music with incremental variation in tempo and octave promotes better anxiety reduction and controlled mind wandering-A randomised controlled EEG study.

Studies have reported the benefits of music-listening in stress-reduction using musical pieces of specific scale or 'Raaga'. But the influence of lower-level musical properties (like tempo, octave, timbre, etc.) lack research backing. Carnatic music concerts use incremental modulations in tempo and octave (e.g.: 'Ragam-Tanam-Pallavi') to elevate the mood of audiences. Therefore, the current study aimed to examine the anxiolytic effect of this musical property. A randomised controlled cross-over study with 21 male undergraduate medical students was followed. 11 participants listened to 'Varying music' (VM: instrumental music with incremental variations in tempo and octave) and 10 listened to 'Stable music' (SM: instrumental music without such variations), thrice daily for 6 days, both clips recorded in Raaga-Kaapi and silence being the control intervention. Electroencephalography (EEG) and Electrocardiography (for heart rate variability or HRV) were done on all 6 days. Beck's Anxiety inventory and State-trait anxiety scale were administered on Day-1 and Day-6. A significant anxiety score reduction was seen only in VM. VM showed marked decrease in lower frequency EEG power in bilateral temporo-parieto-occipital regions compared to silence, whereas SM showed increase in higher frequencies. Relatively, VM showed more midline power reduction (i.e., lower default mode network or DMN activity) and SM showed greater left-dominant alpha/beta asymmetry (i.e., greater right brain activation). During both music interventions HRV remained stable, unlike silence intervention. We speculate that, gradual transition between lower-slower and higher-faster music portions of VM induces a 'controlled-mind wandering' state involving balanced switching between heightened mind wandering ('attention to self') and reduced mind wandering ('attention to music') states, respectively. Therefore, music-selection has remarkable influence on stress-management and warrants further research.

Elimination of rNMPs from mitochondrial DNA has no effect on its stability.

Ribonucleotides (rNMPs) incorporated in the nuclear genome are a well-established threat to genome stability and can result in DNA strand breaks when not removed in a timely manner. However, the presence of a certain level of rNMPs is tolerated in mitochondrial DNA (mtDNA) although aberrant mtDNA rNMP content has been identified in disease models. We investigated the effect of incorporated rNMPs on mtDNA stability over the mouse life span and found that the mtDNA rNMP content increased during early life. The rNMP content of mtDNA varied greatly across different tissues and was defined by the rNTP/dNTP ratio of the tissue. Accordingly, mtDNA rNMPs were nearly absent in SAMHD1-/- mice that have increased dNTP pools. The near absence of rNMPs did not, however, appreciably affect mtDNA copy number or the levels of mtDNA molecules with deletions or strand breaks in aged animals near the end of their life span. The physiological rNMP load therefore does not contribute to the progressive loss of mtDNA quality that occurs as mice age.

SAMHD1 Limits the Efficacy of Forodesine in Leukemia by Protecting Cells against the Cytotoxicity of dGTP.

The anti-leukemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphate (dNTP), protects cells against the effects of dNTP imbalances. SAMHD1-deficient cells induce intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine act synergistically to kill cells lacking SAMHD1. Using mass cytometry, we find that these compounds kill SAMHD1-deficient malignant cells in patients with chronic lymphocytic leukemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation are unaffected. We therefore propose to use forodesine as a precision medicine for leukemia, stratifying patients by SAMHD1 genotype or expression.

High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase.

Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion.

Mec1 Is Activated at the Onset of Normal S Phase by Low-dNTP Pools Impeding DNA Replication.

The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G1 phase may not be sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use suboptimal dNTP pools to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.

Every dog has its prey: Range-wide assessment of links between diet patterns, livestock depredation and human interactions for an endangered carnivore.

Livestock depredation is the most ubiquitous type of negative interaction between humans and carnivores. We conducted a range-wide assessment linking diet patterns of the endangered dhole Cuon alpinus, with livestock consumption and human-dhole interactions. We first performed a reanalysis of dhole diet data from all published studies (1973-2013) incorporating a recently-developed non-linear correction factor for quantifying prey biomass consumed. We then determined the relative livestock numbers consumed by dholes over time across its range, compared these with earlier estimates, and investigated the relative importance of wild vs. non-wild prey in dhole diet. Using information from >70 studies, we explored links between livestock consumption by dholes, availability of wild versus non-wild prey, sympatric depredation-prone carnivores, and people's perception of dholes as livestock predators. We found that (a) dhole diet profiles varied regionally, (b) dholes consumed fewer livestock compared to estimates generated using other, widely used methods, (c) livestock consumption by dholes was associated with wild and non-wild prey densities, and number of co-predator species, and (d) people's negative perception of dholes was associated with pack sizes, levels of livestock depredation and number of sympatric carnivore species. Global efforts for dhole conservation should involve different strategies based on region-specific realities that account for ecological context as well as human perceptions, which would require well-designed studies of dhole social and population dynamics, and human-dhole interactions. We also call for more such range-wide assessments of livestock depredation by wild canids, complemented with direct investigations of human-canid interactions.