Closed-loop fluid-fluid immiscibility in binary lipid-sterol membranes.

We present two binary lipid-sterol membrane systems that exhibit fluid-fluid coexistence. Partial phase diagrams of binary mixtures of dimyristoylphosphatidylcholine with 25-hydroxyxholesterol and 27-hydroxycholesterol, determined from small-angle X-ray scattering and fluorescence microscopy studies, show closed-loop fluid-fluid immiscibility gaps, with the appearance of a single fluid phase both at higher and lower temperatures. Computer simulations suggest that this unusual phase behavior results from the ability of these oxysterol molecules to take different orientations in the membrane depending on the temperature.

Genome-wide analysis of the host intracellular network that regulates survival of Mycobacterium tuberculosis.

We performed a genome-wide siRNA screen to identify host factors that regulated pathogen load in human macrophages infected with a virulent strain of Mycobacterium tuberculosis. Iterative rounds of confirmation, followed by validation, identified 275 such molecules that were all found to functionally associate with each other through a dense network of interactions. This network then yielded to a molecular description of the host cell functional modules that were both engaged and perturbed by the pathogen. Importantly, a subscreen against a panel of field isolates revealed that the molecular composition of the host interface varied with both genotype and the phenotypic properties of the pathogen. An analysis of these differences, however, permitted identification of those host factors that were invariantly involved, regardless of the diversification in adaptive mechanisms employed by the pathogen. Interestingly, these factors were found to predominantly function through the regulation of autophagy.

Lysozyme-Sucrose Interactions in the Solid State: Glass Transition, Denaturation, and the Effect of Residual Water.

The freeze-drying of proteins, along with excipients, offers a solution for increasing the shelf-life of protein pharmaceuticals. Using differential scanning calorimetry, thermogravimetric analysis, sorption calorimetry, and synchrotron small-angle X-ray scattering (SAXS), we have characterized the properties at low (re)hydration levels of the protein lysozyme, which was freeze-dried together with the excipient sucrose. We observe that the residual moisture content in these samples increases with the addition of lysozyme. This results from an increase in equilibrium water content with lysozyme concentration at constant water activity. Furthermore, we also observed an increase in the glass transition temperature (Tg) of the mixtures with increasing lysozyme concentration. Analysis of the heat capacity step of the mixtures indicates that lysozyme does not participate in the glass transition of the sucrose matrix; as a result, the observed increase in the Tg of the mixtures is the consequence of the confinement of the amorphous sucrose domains in the interstitial space between the lysozyme molecules. Sorption calorimetry experiments demonstrate that the hydration behavior of this formulation is similar to that of the pure amorphous sucrose, while the presence of lysozyme only shifts the sucrose transitions. SAXS analysis of amorphous lysozyme-sucrose mixtures and unfolding of lysozyme in this environment show that prior to unfolding, the size and shape of lysozyme in a solid sucrose matrix are consistent with its native state in an aqueous solution. The results obtained from our study will provide a better understanding of the low hydration behavior of protein-excipient mixtures and support the improved formulation of biologics.

In Silico Functional and Structural Analysis of Non-synonymous Single Nucleotide Polymorphisms (nsSNPs) in Human Paired Box 4 Gene.

In human genome, members of Paired box (PAX) transcription factor family are highly sequence-specific DNA-binding proteins. Among PAX gene family members, PAX4 gene has significant role in growth, proliferation, differentiation, and insulin secretion of pancreatic ß-cells. Single nucleotide polymorphisms (SNPs) in PAX4 gene progress in the pathogenesis of various human diseases. Hence, the molecular mechanism of how these SNPs in PAX4 gene significantly progress diseases pathogenesis needs to be elucidated. For the reason, a series of bioinformatic analyzes were done to identify the SNPs of PAX4 gene that contribute in diseases pathogenesis. From the analyzes, 4145 SNPs (rsIDs) in PAX4 gene were obtained, where, 362 missense (8.73%), 169 synonymous (4.08%), and 2323 intron variants (56.04%). The rest SNPs were unspecified. Among the 362 missense variants, 118 nsSNPs were found as deleterious in SIFT analysis. Among those, 25 nsSNPs were most probably damaging and 23 were deleterious as observed in PolyPhen-2 and PROVEAN analyzes, respectively. Following all analyzes, 14 nsSNPs (rs149708455, rs115887120, rs147279315, rs35155575, rs370095957, rs373939873, rs145468905, rs121917718, rs2233580, rs3824004, rs372751660, rs369459316, rs375472849, rs372497946) were common and observed as deleterious, probably damaging, affective and diseases associated. Following structural analyzes, 11 nsSNPs guided proteins were found as most unstable and highly conserved. Among these, R20W, R39Q, R45Q, R60H, G65D, and A223D mutated proteins were highly harmful. Hence, the results from above-mentioned integrated comprehensive bioinformatic analyzes guide how different nsSNPs in PAX4 gene alter structural and functional characteristics of the protein that might progress diseases pathogenesis in human including type 2 diabetes.

Near-isogenic lines for resistance to brown planthopper with the genetic background of Indica Group elite rice (Oryza sativa L.) variety 'IR64'.

The brown planthopper (BPH), Nilaparvata lugens Stål, is an insect pest that severely damages rice (Oryza sativa L.) in Asia, causing huge yield loss. Use of resistant variety is a cost-effective and eco-friendly strategy for maintaining BPH populations below the economic injury level. However, current BPH populations have been changed to virulence against resistant varieties. In this study, to estimate effective combinations among eight BPH resistance genes (BPH32, BPH17-ptb, BPH20, BPH17, BPH3, BPH25, BPH26 and qBPH6), eight near-isogenic lines with the genetic background of an Indica Group rice variety 'IR64' (IR64-NIL) were developed using marker-assisted selection. The genome recoveries of these NILs ranged from 89.3% to 98.8% and agronomic traits of them were similar to those of 'IR64'. In modified seed box screening test, resistance level of IR64-NILs was higher than that of 'IR64'. In antibiosis test, high adult mortalities of BPH (from 56.0% to 97.0%) were observed among NILs, in comparison with that of 'IR64'. Among IR64-NILs, the line carrying BPH17 showed the highest resistance level at all tests. Thus, these IR64-NILs with multiple BPH resistance genes could be valuable breeding lines for enhancing resistance levels by gene pyramiding and multiline variety.

Development of pyramided lines carrying brown planthopper resistance genes in the genetic background of Indica Group rice (Oryza sativa L.) variety 'IR64'.

The development of resistant rice (Oryza sativa L.) varieties is a key strategy for the eco-friendly control of brown planthopper (BPH: Nilaparvata lugens Stål). However, BPH outbreaks occur frequently owing to the evolution of virulent strains in the field and the rapid breakdown of monogenic resistance to BPH. Therefore, to enhance BPH resistance and gauge the effectiveness of gene pyramiding against strongly virulent BPH, we developed pyramided lines (PYLs) in the genetic background of 'IR64' carrying BPH resistance genes. We developed six IR64-PYLs (BPH3 + BPH17, BPH32 + BPH17, BPH32 + BPH20, BPH3 + BPH17-ptb, BPH20 + BPH3, and BPH17-ptb + BPH32) through marker-assisted selection. To assess the resistance of the IR64-PYLs, we conducted antibiosis test, honeydew test, and modified seedbox screening test (MSST) using strongly virulent BPH populations. The level of BPH resistance increased in all six IR64-PYLs compared to both 'IR64' and the corresponding NILs in MSST. Among them, IR64-BPH3 + BPH17 and IR64-BPH32 + BPH17 exhibited the highest resistance to BPH. However, the resistance level of most IR64-PYLs was not significantly higher than that of the corresponding NILs in antibiosis test. Thus, these PYLs could serve as a valuable resource for breeding programs aimed at improving resistance to virulent strains of BPH and enhancing their durability.

A Cyber-Physical Framework for Optimal Coordination of Connected and Automated Vehicles on Multi-Lane Freeways.

Uncoordinated driving behavior is one of the main reasons for bottlenecks on freeways. This paper presents a novel cyber-physical framework for optimal coordination of connected and automated vehicles (CAVs) on multi-lane freeways. We consider that all vehicles are connected to a cloud-based computing framework, where a traffic coordination system optimizes the target trajectories of individual vehicles for smooth and safe lane changing or merging. In the proposed framework, the vehicles are coordinated into groups or platoons, and their trajectories are successively optimized in a receding horizon control (RHC) approach. Optimization of the traffic coordination system aims to provide sufficient gaps when a lane change is necessary while minimizing the speed deviation and acceleration of all vehicles. The coordination information is then provided to individual vehicles equipped with local controllers, and each vehicle decides its control acceleration to follow the target trajectories while ensuring a safe distance. Our proposed method guarantees fast optimization and can be used in real-time. The proposed coordination system was evaluated using microscopic traffic simulations and benchmarked with the traditional driving (human-based) system. The results show significant improvement in fuel economy, average velocity, and travel time for various traffic volumes.

Evaluation of radioactivity in soil and rock samples from an undiscovered sea beach in the southeastern coastline of Bangladesh and associated health risk.

This study marks the first-ever assessment of radiological hazards linked to the sands and rocks of Patuartek Sea Beach, situated along one of the world's longest sea beaches in Cox' Bazar of Bangladesh. Through the utilization of an HPGe detector, a comprehensive analysis of the activity concentrations of 226Ra, 232Th, and 40 K was conducted, and their activity ranged from 7 to 23 Bq/kg, 9-58 Bq/kg, and 172-340 Bq/kg, respectively, in soils, and 19-24 Bq/kg, 27-39 Bq/kg, and 340-410 Bq/kg, respectively, in rocks. Some sand samples exhibited elevated levels of 232Th, while the rock samples displayed higher levels of 40 K compared to the global average. The radiological hazard parameters were assessed, and no values surpassed the recommended limits set by several international organizations. Hence, the sands and rocks of Patuartek sea beach pose no significant radiological risk to the residents or tourists. The findings of this study provide crucial insights for the development of a radiological baseline map in the country, which is important due to the commissioning of the country's first nuclear power plant Rooppur Nuclear Power Plant. The data may also stimulate interest in the rare-earth minerals present in the area, which is important for the electronics industry, thorium-based nuclear fuel cycles.

Genome-Wide Identification and Functional Analysis of Lysine Histidine Transporter (LHT) Gene Families in Maize.

Amino acid transporters (AATs) are essential membrane proteins that transfer amino acids across cells. They are necessary for plant growth and development. The lysine histidine transporter (LHT) gene family in maize (Zea mays) has not yet been characterized. According to sequence composition and phylogenetic placement, this study found 15 LHT genes in the maize genome. The ZmLHT genes are scattered across the plasma membrane. The study also analyzed the evolutionary relationships, gene structures, conserved motifs, 3D protein structure, a transmembrane domain, and gene expression of the 15 LHT genes in maize. Comprehensive analyses of ZmLHT gene expression profiles revealed distinct expression patterns in maize LHT genes in various tissues. This study's extensive data will serve as a foundation for future ZmLHT gene family research. This study might make easier to understand how LHT genes work in maize and other crops.

Plasma membrane proteomic changes of Arabidopsis DRP1E during cold acclimation in association with the enhancement of freezing tolerance.

The freezing tolerance of plants that live in cold regions increases after exposure to low temperature, a process termed cold acclimation (CA). During CA, restructuring of the plasma membrane (PM) is important to enhance freezing tolerance. We have previously shown that the function of DYNAMIN-RELATED PROTEIN 1 E (DRP1E), which regulates endocytosis by pinching vesicles from the PM, is associated with the enhancement of freezing tolerance during CA in Arabidopsis. DRP1E is predicted to play a role in reconstituting the PM composition during CA. In this study, to test the validity of this hypothesis, we studied the changes in PM proteome patterns induced by drp1e mutation. In a detailed physiological analysis, after 3 days of CA, only young leaves showed significantly less increase in freezing tolerance in the mutant than in the wild type (WT). Using nano-liquid chromatography-tandem mass spectrometry, 496 PM proteins were identified. Among these proteins, 81 or 71 proteins were specifically altered in the WT or the mutant, respectively, in response to CA. Principal component analysis showed that the proteomic pattern differed between the WT and the mutant upon cold acclimation (CA), suggesting that DRP1E contributes to reconstruction of the PM during CA. Cluster analysis revealed that proteins that were significantly increased in the mutant after CA were biased toward glycosylphosphatidylinositol-anchored proteins, such as fasciclin-like arabinogalactan proteins. Thus, a primary target of DRP1E-associated PM reconstruction during CA is considered to be glycosylphosphatidylinositol-anchored proteins, which may be removed from the PM by DRP1E in young leaves after 3 days of CA.

Individual- and Community-Level Factors Associated with Diarrhea in Children Younger Than Age 5 Years in Bangladesh: Evidence from the 2014 Bangladesh Demographic and Health Survey.

Background: Diarrheal disease is among the leading causes of morbidity and mortality among children younger than age 5 years in Bangladesh. Objective: The objective of this study is to assess the prevalence of diarrhea among children younger than age 5 years and its associated risk factors. Methods: Data were sourced from the Bangladesh Demographic and Health Survey, a nationally representative study conducted in 2014. We used multilevel logistic regression models to identify factors associated with diarrheal disease. Results: Children aged 6 to11 months (odds ratio = 2.26; 95% CI, 1.50-3.42), and 12 to 23 months (odds ratio = 2.31; 95% CI, 1.62-3.31) were more likely to have diarrhea than older children. Other significant risk factors for diarrheal infection included households without access to drinking water (odds ratio = 1.39; 95% CI, 1.03-1.88) and mothers lacking mass media access (odds ratio = 1.32; 55% CI, 1.01-1.73). Conclusions: Childhood diarrhea in Bangladesh was associated with individual- and community-level factors. The finding of this study suggests that diarrhea prevention programs in the country can effectively be delivered by targeting young children through expanding community-based education and increasing access to health information through mass media.

Functional dairy products as a source of bioactive peptides and probiotics: current trends and future prospectives.

Milk is an incredibly healthy food world-wide. However, the 'lactase deficient' individuals cannot digest milk's carbohydrate lactose. A large part of the world population is depriving of highly beneficial milk proteins like casein, lactoalbumin, lactoglobulin, etc. due to lactose intolerance. Production of functional foods and bioactive peptides from milk with natural antioxidants and the addition of probiotics could be the best alternative to extend the use of milk functionalities. Among different probiotics, the lactic acid bacteria (LAB) like Lactobacillus delbrueckii sub sp. bulgaricus, Streptococcus thermophilus and some species of Bifidobacteria and their metabolites (paraprobiotics and postbiotics) have been given more preference to add in milk-derived functional foods. These species are generally considered as heat-tolerant, highly proteolytic, and peptidolytic towards milk proteins and they liberate smaller molecules of bioactive peptides during fermentation and other processes that stimulate the enzyme lactase to help people in digestion of milk carbohydrate lactose. Moreover, the incorporation of natural antioxidants in yoghurt and other dairy products prevents the rancidity of milk fat. The level of bioactive peptides produced in milk-derived functional foods can be determined by capillary zone electrophoresis, mass spectrometry, fractionation, and other modern assessment techniques. Commercial production of functional probiotic products with bioactive peptides could significantly contribute to reduce milk spoilage, enhance health benefits as well as the growth of the agro-processing industry.

Bi-Level Coordinated Merging of Connected and Automated Vehicles at Roundabouts.

Traditional uncoordinated traffic flows in a roundabout can lead to severe traffic congestion, travel delay, and the increased fuel consumption of vehicles. An interesting way to mitigate this would be through cooperative control of connected and automated vehicles (CAVs). In this paper, we propose a novel solution, which is a roundabout control system (RCS), for CAVs to attain smooth and safe traffic flows. The RCS is essentially a bi-level framework, consisting of higher and lower levels of control, where in the higher level, vehicles in the entry lane approaching the roundabout will be made to form clusters based on traffic flow volume, and in the lower level, the vehicles' optimal sequences and roundabout merging times are calculated by solving a combinatorial optimization problem using a receding horizon control (RHC) approach. The proposed RCS aims to minimize the total time taken for all approaching vehicles to enter the roundabout, whilst minimally affecting the movement of circulating vehicles. Our developed strategy ensures fast optimization, and can be implemented in real-time. Using microscopic simulations, we demonstrate the effectiveness of the RCS, and compare it to the current traditional roundabout system (TRS) for various traffic flow scenarios. From the results, we can conclude that the proposed RCS produces significant improvement in traffic flow performance, in particular for the average velocity, average fuel consumption, and average travel time in the roundabout.

A single seed treatment mediated through reactive oxygen species increases germination, growth performance, and abiotic stress tolerance in Arabidopsis and rice.

Hydroxyl radical (•OH) is considered to be the most damaging among reactive oxygen species. Although afew studies have reported on its effects on growth and stress adaptation of plants, no detailed studies have been performed using •OH in germination and early seedling growth under abiotic stresses. Here we report a single seed treatment with •OH on germination and seedling growth of Arabidopsis and rice under non-stressed (ambient) and various abiotic-stressed conditions (chilling, high temperature, heat, and salinity). The treatment resulted in faster seed germination and early seedling growth under non-stressed conditions, and, interestingly, these effects were more prominent under abiotic stresses. In addition, Arabidopsis seedlings from treated seeds showed faster root growth and developed more lateral roots. These results show apositive and potential practical use for •OH in model and crop plants for direct seeding in the field, as well as improvement of tolerance against emerging stresses. Abbreviations: AUC: area under curve; MGT: mean germination time; t50: time to reach 50% germination; U7525: time for uniform germination from 25% to 75%; ROS: reactive oxygen species; GSI: germination speed index; SI: stress index; DI: dormancy index.

Device-to-Device Aided Cooperative Relaying Scheme Exploiting Spatial Modulation: An Interference Free Strategy.

In this paper, a novel interference free dual-hop device-to-device (D2D) aided cooperative relaying strategy (CRS) based on spatial modulation (SM) (termed D2D-CRS-SM) is proposed. In D2D-CRS-SM, two cellular users (e.g., a near user (NU) and a relay-aided far user (FU)) and a pair of D2D transmitter (D1)-receivers (D2) are served in two time-slots. Two different scenarios are investigated considering information reception criteria at the NU. Irrespective of the scenarios, the base station (BS) exploits SM to map information bits into two sets: modulation bits and antenna index, in phase-1. In the first scenario, the BS maps FU information as the modulation bits and NU information as antenna index, whereas modulation bits correspond to NU information and the antenna index carries FU's information in scenario-2. The iterative-maximum ratio combining (i-MRC) technique is then used by NU and D1 to de-map their desired information bits. During phase-2, D1 also exploits SM to forward FU's information received from BS and its own information bits to the D2D receiver D2. Then, FU and D2 retrieve their desired information by using i-MRC. Due to exploiting SM in both phases, interference free information reception is possible at each receiving node without allocating any fixed transmit power. The performance of D2D-CRS-SM is studied in terms of bit-error rate and spectral efficiency considering M-ary phase shift keying and quadrature amplitude modulation. Finally, the efficiency of D2D-CRS-SM is demonstrated via the Monte Carlo simulation.

Large-Scale Phosphoproteomic Study of Arabidopsis Membrane Proteins Reveals Early Signaling Events in Response to Cold.

Cold stress is one of the major factors limiting global crop production. For survival at low temperatures, plants need to sense temperature changes in the surrounding environment. How plants sense and respond to the earliest drop in temperature is still not clearly understood. The plasma membrane and its adjacent extracellular and cytoplasmic sites are the first checkpoints for sensing temperature changes and the subsequent events, such as signal generation and solute transport. To understand how plants respond to early cold exposure, we used a mass spectrometry-based phosphoproteomic method to study the temporal changes in protein phosphorylation events in Arabidopsis membranes during 5 to 60 min of cold exposure. The results revealed that brief cold exposures led to rapid phosphorylation changes in the proteins involved in cellular ion homeostasis, solute and protein transport, cytoskeleton organization, vesical trafficking, protein modification, and signal transduction processes. The phosphorylation motif and kinase-substrate network analysis also revealed that multiple protein kinases, including RLKs, MAPKs, CDPKs, and their substrates, could be involved in early cold signaling. Taken together, our results provide a first look at the cold-responsive phosphoproteome changes of Arabidopsis membrane proteins that can be a significant resource to understand how plants respond to an early temperature drop.

Effects of processing techniques on drying characteristics, physicochemical properties and functional compounds of green and red chilli (Capsicum annum L.) powder.

This study was aimed to investigate the effect of processing techniques on the characteristics of green and red chilli powder. Four samples, such as pretreated green chilli paste (PTGP), pretreated green chilli longitudinal slit (PTGL), pretreated whole red chilli (PTWR) and untreated green chilli paste (UTGP), were prepared and dried at 60 °C in a cabinet dryer. The pretreatment was blanching in acetic acid solution and soaking immediately in a combined solution of Na2S2O5 and CaCl2. Pretreated samples took a shorter drying time than the untreated sample in reducing moisture content from 86.31 to 8%. Pretreatment before drying resulted in retaining total chlorophyll (~ 86%), phenolic compounds (~ 32%), green color, and pungency of chilli. Analysis result indicated that more than 60% retention of ß-carotene was found while retention of ascorbic acid was comparable. Conclusively, this research reveals a good nutritional profile in cabinet dried green chilli powder, which may open the scope for commercial production.

Physicochemical and microbiological characteristics of honey obtained through sugar feeding of bees.

This study was planned to characterize the physicochemical and antioxidant properties, and microbiological quality of honey obtained from the sandbar pumpkin field. In this study, four sugar supplemented and one control (without sugar fed) honey sample was used. Results revealed that all samples exhibited appropriate maturity considering their low moisture content (~ 19%) and high total solids (~ 80%) and TSS (~ 79%). Total acidity (< 40 meq/kg) and pH (~ 4.5) directed the absenteeism of detrimental fermentation. Ash (~ 0.29%) and electrical conductivity (~ 700 µS/cm) were reasonable and distinctive of dark yellowish-brown honey, which is buttressed by color attributes. Reducing sugars, glucose, fructose, and sucrose values ranged from 68.98 to 75.82%, 26.01 to 33.84%, 34.93 to 38.70%, and 1.74 to 5.96%, respectively. Proline (~ 400 mg/kg), HMF (< 40 mg/kg) and diastase action (~ 14° Gothe) were found within accepted limits, and also possesses good antioxidants in terms of total phenol (~ 160 mg GAE/100 g), total flavonoid (4.67-6.25 mg CE/100 g), and DPPH-RSA (30.65-35.97%). The microbial study revealed that the total viable count ranged between 33.33 and 27.66 CFU/g, while yeasts and mold count varied between 14.33 and 12 CFU/g. Principle component analysis (PCA) results revealed that all the studied parameters could be used effectively to discriminate the honey sample. The overall results signpost a new information regarding the quality i.e. processing, maturity, freshness and composition of honey obtained from the sandbar pumpkin field.

Binding of bis-ANS to Bacillus subtilis lipase: a combined computational and experimental investigation.

8-Anilino-1-naphthalene sulfonate (ANS) and its covalent dimer bis-ANS are widely used for titrating hydrophobic surfaces of proteins. Interest to understand the nature of interaction of these dyes with proteins was seriously pursued. However as the techniques used in these studies varied, they often provided varied information regarding stoichiometry, binding affinity, actual binding sites etc. In the present study, we used combination of computation methods (docking and MD simulation) and experimental methods (mutations, steady-state and time-resolved fluorescence) to investigate bis-ANS interaction with Bacillus subtilis lipase. We identified seven binding sites for bis-ANS on lipase using computational docking and MD simulation and verified these data using a set of single amino acid substituted mutants. Docking and MD simulation studies indicated that the binding sites were various indentations and grooves on protein surface with hydrophobic characteristics. Both hydrophobic and ionic interactions were involved in each of these binding events. We further examine the fluorescence properties of bis-ANS bound to mutant lipases that either gained or lost a binding site. Our results indicated that neither gain nor loss of single binding site caused any change in fluorescence lifetimes (and their relative amplitudes) of mutant lipase-bound bis-ANS in comparison to that bound to wild type; hence, it suggested that nature of bis-ANS binding to each of the sites in lipase was very similar.

Altered chromatin condensation of heat-stressed spermatozoa perturbs the dynamics of DNA methylation reprogramming in the paternal genome after in vitro fertilisation in cattle.

Shortly after penetration of the oocyte, sperm DNA is actively demethylated, which is required for totipotent zygotic development. Aberrant DNA methylation is thought to be associated with altered chromatin condensation of spermatozoa. The objectives of this study were to investigate the dynamics of DNA methylation reprogramming in the paternal pronucleus and subsequent fertilisation potential of heat-stressed bull spermatozoa having altered chromatin condensation. Hence, bovine zygotes (n=1239) were collected at three different time points (12, 18 and 24h post insemination, hpi), and stained with an antibody against 5-methylcytosine. Fluorescence intensities of paternal and maternal pronuclei were measured by ImageJ. DNA methylation patterns in paternal pronuclei derived from heat-stressed spermatozoa did not differ between time points (P>0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24hpi, respectively. Moreover, heat-stressed spermatozoa showed a highly reduced (P<0.01) fertilisation rate compared with non-heat-stressed or normal control spermatozoa (53.7% vs 70.2% or 81.5%, respectively). Our data show that the normal pattern of active DNA demethylation followed by de novo methylation in the paternal pronucleus is perturbed when oocytes are fertilised with heat-stressed spermatozoa, which may be responsible for decreased fertilisation potential.