Characterization of Metabolic Patterns in Mouse Oocytes during Meiotic Maturation.

Well-balanced and timed metabolism is essential for making a high-quality egg. However, the metabolic framework that supports oocyte development remains poorly understood. Here, we obtained the temporal metabolome profiles of mouse oocytes during in vivo maturation by isolating large number of cells at key stages. In parallel, quantitative proteomic analyses were conducted to bolster the metabolomic data, synergistically depicting the global metabolic patterns in oocytes. In particular, we discovered the metabolic features during meiotic maturation, such as the fall in polyunsaturated fatty acids (PUFAs) level and the active serine-glycine-one-carbon (SGOC) pathway. Using functional approaches, we further identified the key targets mediating the action of PUFA arachidonic acid (ARA) on meiotic maturation and demonstrated the control of epigenetic marks in maturing oocytes by SGOC network. Our data serve as a broad resource on the dynamics occurring in metabolome and proteome during oocyte maturation.

SPLHRNMTF: robust orthogonal non-negative matrix tri-factorization with self-paced learning and dual hypergraph regularization for predicting miRNA-disease associations.

MicroRNAs (miRNAs) have been demonstrated to be closely related to human diseases. Studying the potential associations between miRNAs and diseases contributes to our understanding of disease pathogenic mechanisms. As traditional biological experiments are costly and time-consuming, computational models can be considered as effective complementary tools. In this study, we propose a novel model of robust orthogonal non-negative matrix tri-factorization (NMTF) with self-paced learning and dual hypergraph regularization, named SPLHRNMTF, to predict miRNA-disease associations. More specifically, SPLHRNMTF first uses a non-linear fusion method to obtain miRNA and disease comprehensive similarity. Subsequently, the improved miRNA-disease association matrix is reformulated based on weighted k-nearest neighbor profiles to correct false-negative associations. In addition, we utilize L 2 , 1 norm to replace Frobenius norm to calculate residual error, alleviating the impact of noise and outliers on prediction performance. Then, we integrate self-paced learning into NMTF to alleviate the model from falling into bad local optimal solutions by gradually including samples from easy to complex. Finally, hypergraph regularization is introduced to capture high-order complex relations from hypergraphs related to miRNAs and diseases. In 5-fold cross-validation five times experiments, SPLHRNMTF obtains higher average AUC values than other baseline models. Moreover, the case studies on breast neoplasms and lung neoplasms further demonstrate the accuracy of SPLHRNMTF. Meanwhile, the potential associations discovered are of biological significance.

Quantum Light Generation Based on GaN Microring toward Fully On-Chip Source.

An integrated quantum light source is increasingly desirable in large-scale quantum information processing. Despite recent remarkable advances, a new material platform is constantly being explored for the fully on-chip integration of quantum light generation, active and passive manipulation, and detection. Here, for the first time, we demonstrate a gallium nitride (GaN) microring based quantum light generation in the telecom C-band, which has potential toward the monolithic integration of quantum light source. In our demonstration, the GaN microring has a free spectral range of 330 GHz and a near-zero anomalous dispersion region of over 100 nm. The generation of energy-time entangled photon pair is demonstrated with a typical raw two-photon interference visibility of 95.5±6.5%, which is further configured to generate a heralded single photon with a typical heralded second-order autocorrelation g_{H}^{(2)}(0) of 0.045±0.001. Our results pave the way for developing a chip-scale quantum photonic circuit.

Alzheimer-like behavior and synaptic dysfunction in 3 × Tg-AD mice are reversed with calcineurin inhibition.

Alzheimer's disease is a progressive neurodegenerative disorder characterized by impairments in synaptic plasticity and cognitive performance. Current treatments are unable to achieve satisfactory therapeutic effects or reverse the progression of the disease. Calcineurin has been implicated as part of a critical signaling pathway for learning and memory, and neuronal calcineurin may be hyperactivated in AD. To investigate the effects and underlying mechanisms of FK506, a calcineurin inhibitor, on Alzheimer-like behavior and synaptic dysfunction in the 3 × Tg-AD transgenic mouse model of Alzheimer's disease, we investigated the effect of FK506 on cognitive function and synaptic plasticity in the 3 × Tg-AD transgenic mouse model of Alzheimer's disease. The results showed that FK506 treatment ameliorated cognitive deficits, as indicated by the decreased latency in the water maze, and attenuated tau hyperphosphorylation in 3 × Tg-AD mice. Treatment with FK506 also reduced the levels of certain markers of postsynaptic deficits, including PSD-95 and NR2B, and reversed the long-term potentiation deficiency and dendritic spine impairments in 3 × Tg-AD mice. These findings suggest that treatment with calcineurin inhibitors such as FK506 can be an effective therapeutic strategy to rescue synaptic deficit and cognitive impairment in familial Alzheimer's disease and related tauopathies.

The human eIF4E:4E-BP2 complex structure for studying hyperphosphorylation.

The cap-dependent mRNA translation is dysregulated in many kinds of cancers. The interaction between eIF4E and eIF4G through a canonical eIF4E-binding motif (CEBM) determines the efficacy of the cap-dependent mRNA translation. eIF4E-binding proteins (4E-BPs) share the CEBM and compete with eIF4G for the same binding surface of eIF4E and then inhibit the mRNA translation. 4E-BPs function as tumor repressors in nature. Hyperphosphorylation of 4E-BPs regulates the structure folding and causes the dissociation of 4E-BPs from eIF4E. However, until now, there has been no structure of the full-length 4E-BPs in complex with eIF4E. The regulation mechanism of phosphorylation is still unclear. In this work, we first investigate the interactions of human eIF4E with the CEBM and an auxiliary eIF4E-binding motif (AEBM) in eIF4G and 4E-BPs. The results unravel that the structure and interactions of the CEBM are highly conserved between eIF4G and 4E-BPs. However, the extended CEBM (ECEBM) in 4E-BPs forms a longer helix than that in eIF4G. The residue R62 in the ECEBM of 4E-BP2 forms salt bridges with E32 and E70 of eIF4E. The residue R63 of 4E-BP2 forms two special hydrogen bonds with N77 of eIF4E. Both of these interactions are missing in eIF4G. The AEBM of 4E-BPs folds into a ß-sheet conformation, which protects V81 inside a hydrophobic core in 4E-BP2. In eIF4G, the AEBM exists in a random coil state. The hydrophilic residues S637 and D638 of eIF4G open the hydrophobic core for solvents. The results show that the ECEBM and AEBM may be responsible for the competing advantage of 4E-BP2. Finally, based on our previous work (J. Zeng, F. Jiang and Y. D. Wu, J. Chem. Theory Comput., 2017, 13, 320), the human eIF4E:4E-BP2 complex (eIF4E:BP2P18-I88) including all reported phosphorylation sites is predicted. The eIF4E:BP2P18-I88 complex is different from the existing experimental eIF4E:eIF4G complex and provides an important structure for further studying the regulation mechanism of phosphorylation in 4E-BPs.

A novel selective ERK1/2 inhibitor, Laxiflorin B, targets EGFR mutation subtypes in non-small-cell lung cancer.

Extracellular regulated protein kinases 1/2 (ERK1/2) are key members of multiple signaling pathways, including the ErbB axis. Ectopic ERK1/2 activation contributes to various types of cancer, especially drug resistance to inhibitors of RTK, RAF and MEK, and specific ERK1/2 inhibitors are scarce. In this study, we identified a potential novel covalent ERK inhibitor, Laxiflorin B, which is a herbal compound with anticancer activity. However, Laxiflorin B is present at low levels in herbs; therefore, we adopted a semi-synthetic process for the efficient production of Laxiflorin B to improve the yield. Laxiflorin B induced mitochondria-mediated apoptosis via BAD activation in non-small-cell lung cancer (NSCLC) cells, especially in EGFR mutant subtypes. Transcriptomic analysis suggested that Laxiflorin B inhibits amphiregulin (AREG) and epiregulin (EREG) expression through ERK inhibition, and suppressed the activation of their receptors, ErbBs, via a positive feedback loop. Moreover, mass spectrometry analysis combined with computer simulation revealed that Laxiflorin B binds covalently to Cys-183 in the ATP-binding pocket of ERK1 via the D-ring, and Cys-178 of ERK1 through non-inhibitory binding of the A-ring. In a NSCLC tumor xenograft model in nude mice, Laxiflorin B also exhibited strong tumor suppressive effects with low toxicity and AREG and EREG were identified as biomarkers of Laxiflorin B efficacy. Finally, Laxiflorin B-4, a C-6 analog of Laxiflorin B, exhibited higher binding affinity for ERK1/2 and stronger tumor suppression. These findings provide a new approach to tumor inhibition using natural anticancer compounds.

Analysis of quality evaluation and optimal harvest period of Aurantii Fructus from different sources using UHPLC-Q-TOF-MS/MS.

INTRODUCTION: The active constituents in Aurantii Fructus sourced from different regions within Hunan Province exhibit variations, with certain samples demonstrating substandard quality. OBJECTIVES: The aim of this study is to conduct a comparative analysis of the chemical composition and quality of Aurantii Fructus from various sources, establish a robust methodology for quality evaluation, and determine the optimal harvesting period. MATERIALS AND METHODS: The components of Aurantii Fructus were qualitatively analyzed using a non-targeted metabolomics approach. Multivariate statistical analyses were conducted to identify potential markers, enabling qualitative and quantitative evaluation of the quality and optimal harvest period of Aurantii Fructus. RESULTS: Overall, 155 compounds were identified in Aurantii Fructus, with Huangpi exhibiting the highest number of components. Eleven potential markers were selected to assess the quality of Aurantii Fructus. The average content of Huangpi was the highest, indicating a high level of similarity. The samples' overall scores were ordered as follows: Huangpi > Xiangcheng > Choucheng > Daidai. Anren and Changde's Huangpi exhibited high contents, being rich in chemical components, resulting in favorable scores. Similarly, Changde's Xiangcheng displayed significant medicinal value. As the harvest time was delayed, there was an increase in fruit size, accompanied by thinner peels and a continuous decrease in the contents of potential markers. The best harvest period of Aurantii Fructus was within 1 week before and after the Lesser Heat. CONCLUSION: The present study establishes a precise and efficient method for evaluating the quality of Aurantii Fructus, thereby providing more comprehensive insights into its composition. This research lays the foundation for subsequent development and utilization of Aurantii Fructus.

[Transcriptional regulation mechanism of differential accumulation of flavonoids in different varieties of Lonicera macranthoides based on metabonomics and transcriptomics].

This study aims to explore the molecular regulatory mechanism of the differential accumulation of flavonoids between 'Xianglei' and the wild type of Lonicera macranthoides. The flowers, stems, and leaves of the two varieties of L. macranthoides were collected. Ultra-performance liquid chromatography-mass spectrometry(UPLC-MS) and high-throughput sequencing(RNA-seq) were employed to screen out the differential flavonoids, key differentially expressed genes(DEGs) and transcription factors(TFs). Fourteen DEGs were randomly selected for verification by qRT-PCR. The results showed that a total of 17 differential flavonoids were obtained, including naringin chalcone, apigenin, and quercetin. The transcriptomic analysis predicted 19 DEGs associated with flavonoids, including 2 genes encoding chitin synthase(CHS) and 3 genes encoding chalcone isomerase(CHI). The regulatory network analysis and weighted gene co-expression network analysis(WGCNA) screen out the key enzyme genes CHS1, FLS1, and HCT regulating the accumulation of flavonoids. MYB12 and LBD4 may be involved in the biosynthesis of flavonoids by regulating the expression of key enzyme genes CHS1, FLS1, and HCT. The qRT-PCR and RNA-seq results were similar regarding the expression patterns of the 14 randomly selected DEGs. This study preliminarily analyzed the transcriptional regulatory mechanism for the differential accumulation of flavonoids in the two varieties of L. macranthoides and laid a foundation for further elucidating the regulatory effects of key enzyme genes and TFs on the accumulation of flavonoids.

Changing patterns of the East Asian monsoon drive shifts in migration and abundance of a globally important rice pest.

Numerous insects including pests and beneficial species undertake windborne migrations over hundreds of kilometers. In East Asia, climate-induced changes in large-scale atmospheric circulation systems are affecting wind-fields and precipitation zones and these, in turn, are changing migration patterns. We examined the consequences in a serious rice pest, the brown planthopper (BPH, Nilaparvata lugens) in East China. BPH cannot overwinter in temperate East Asia, and infestations there are initiated by several waves of windborne spring or summer migrants originating from tropical areas in Indochina. The East Asian summer monsoon, characterized by abundant rainfall and southerly winds, is of critical importance for these northward movements. We analyzed a 42-year dataset of meteorological parameters and catches of BPH from a standardized network of 341 light-traps in South and East China. We show that south of the Yangtze River during summer, southwesterly winds have weakened and rainfall increased, while the summer precipitation has decreased further north on the Jianghuai Plain. Together, these changes have resulted in shorter migratory journeys for BPH leaving South China. As a result, pest outbreaks of BPH in the key rice-growing area of the Lower Yangtze River Valley (LYRV) have declined since 2001. We show that these changes to the East Asian summer monsoon weather parameters are driven by shifts in the position and intensity of the Western Pacific subtropical high (WPSH) system that have occurred during the last 20 years. As a result, the relationship between WPSH intensity and BPH immigration that was previously used to predict the size of the immigration to the LYRV has now broken down. Our results demonstrate that migration patterns of a serious rice pest have shifted in response to the climate-induced changes in precipitation and wind pattern, with significant consequences for the population management of migratory pests.

Effect analysis of multi-department cooperation in improving etiological submission rates before antibiotic treatment.

Increased bacterial drug resistance has become a serious global public health problem. The application of antibiotics involves various clinical departments, and the rational application of antibiotics is the key to improving their efficacy. To provide a basis for further improving the etiological submission rate and standardizing the rational use of antibiotics, this article discusses the intervention effect of multi-department cooperation in improving the etiological submission rate before antibiotic treatment. A total of 87 607 patients were divided into a control group (n = 45 890) and an intervention group (n = 41 717) according to whether multi-department cooperation management was implemented. The intervention group involved the patients hospitalized from August to December 2021, while the control group involved the patients hospitalized from August to December 2020. The submission rates of the two groups; the rates before antibiotic treatment at the unrestricted use level, the restricted use level, and the special use level in departments; and the timing of submission were compared and analysed. The overall differences in the etiological submission rates before antibiotic treatment at the unrestricted use level (20.70% vs 55.98%), the restricted use level (38.23% vs 66.58%), and the special use level (84.92% vs 93.14%) were statistically significant before and after intervention (P < .05). At a more specific level, the etiological submission rates of different departments before antibiotic treatment at the unrestricted use level, the restricted use level, and the special use level were improved, but the special activities of multi-department cooperation management did not improve the submission timing significantly. Multi-department cooperation can effectively improve the etiological submission rates before antimicrobial treatment, but it is necessary to improve measures for specific departments to improve long-term management and incentive and restraint mechanisms.

[Mining and identification of members of MYB transcription factor family in Lonicera macranthoides].

As a large family of transcription factors, the MYB family plays a vital role in regulating flower development. We studied the MYB family members in Lonicera macranthoides for the first time and identified three sequences of 1R-MYB, 47 sequences of R2R3-MYB, two sequences of 3R-MYB, and one sequence of 4R-MYB from the transcriptome data. Further, their physicochemical properties, conserved domains, phylogenetic relationship, protein structure, functional information, and expression were analyzed. The results show that the 53 MYB transcription factors had different conserved motifs, physicochemical properties, structures, and functions in wild type and 'Xianglei' cultivar of L. macranthoides, indicating their conservation and diversity in evolution. The transcript level of LmMYB was significantly different between the wild type and 'Xianglei' cultivar as well as between flowers and leaves, and some genes were specifically expressed. Forty-three out of 53 LmMYB sequences were expressed in both flowers and leaves, and 9 of the LmMYB members showed significantly different transcript levels between the wild type and 'Xianglei' cultivar, which were up-regulated in the wild type. The results provide a theoretical basis for further studying the specific functional mechanism of the MYB family.

Conformations and binding pockets of HRas and its guanine nucleotide exchange factors complexes in the guanosine triphosphate exchange process.

The human Son of Sevenless (SOS) activates the signal-transduction protein Ras by forming the complex SOS·Ras and accelerating the guanosine triphosphate (GTP) exchange in Ras. Inhibition of SOS·Ras could regulate the function of Ras in cells and has emerged as an effective strategy for battling Ras related cancers. A key factor to the success of this approach is to understand the conformational change of Ras during the GTP exchange process. In this study, we perform an extensive molecular dynamics simulation to characterize the specific conformations of Ras without and with guanine nucleotide exchange factors (GEFs) of SOS, especially for the substates of State 1 of HRasGTP∙Mg2+ . The potent binding pockets on the surfaces of the RasGDP∙Mg2+ , the S1.1 and S1.2 substates in State 1 of RasGTP∙Mg2+ and the ternary complexes with SOS are predicted, including the binding sites of other domains of SOS. These findings help to obtain a more thorough understanding of Ras functions in the GTP cycling process and provide a structural foundation for future drug design.

Bt cotton area contraction drives regional pest resurgence, crop loss, and pesticide use.

Genetically-modified crops expressing Bacillus thuringiensis (Bt) proteins have been widely cultivated, permitting an effective non-chemical control of major agricultural pests. While their establishment can enable an area-wide suppression of polyphagous herbivores, no information is available on the impact of Bt crop abandonment in entire landscape matrices. Here, we detail a resurgence of the cosmopolitan bollworm Helicoverpa armigera following a contraction of Bt cotton area in dynamic agro-landscapes over 2007-2019 in North China Plain. An 80% reduction in Bt cotton was mirrored in a 1.9-fold increase of ambient H. armigera population levels, culminating in 1.5-2.1-fold higher yield loss and a 2.0-4.4-fold increase in pesticide use frequency in non-Bt crops (i.e. maize, peanut, soybean). Our work unveils the fate of herbivorous insect populations following a progressive dis-use of insecticidal crop cultivars, and hints at how tactically deployed Bt crops could be paired with agro-ecological measures to mitigate the environmental footprint of crop production.

Effect of empirical antifungal treatment on mortality in non-neutropenic critically ill patients: a propensity-matched retrospective cohort study.

To evaluate the effect of empirical antifungal treatment (EAFT) on mortality in critically ill patients without invasive fungal infections (IFIs). This was a single-center propensity score-matched retrospective cohort study involving non-transplanted, non-neutropenic critically ill patients with risk factors for invasive candidiasis (IC) in the absence of IFIs. We compared all-cause hospital mortality and infection-attributable hospital mortality in patients who was given EAFT for suspected IC as the cohort group and those without any systemic antifungal agents as the control group. Among 640 eligible patients, 177 patients given EAFT and 177 control patients were included in the analyses. As compared with controls, EAFT was not associated with the lower risks of all-cause hospital mortality [odds ratio (OR), 0.911; 95% CI, 0.541-1.531; P = 0.724] or infection-attributable hospital mortality (OR, 1.149; 95% CI, 0.632-2.092; P = 0.648). EAFT showed no benefit of improvement of infection at discharge, duration of mechanical ventilation, and antibiotic-free days. However, the later initiation of EAFT was associated with higher risks of all-cause hospital mortality (OR, 1.039; 95% CI, 1.003 to 1.076; P = 0.034) and infection-attributable hospital mortality (OR, 1.046; 95% CI, 1.009 to 1.085; P = 0.015) in patients with suspected IC. This effect was also found in infection-attributable hospital mortality (OR, 1.042; 95% CI, 1.005 to 1.081; P = 0.027) in septic patients with suspected IC. EAFT failed to decrease hospital mortality in non-neutropenic critically ill patients without IFIs. The timing may be critical for EAFT to improve mortality in these patients with suspected IC. ChiCTR2000038811, registered on Oct 3, 2020.

Identification of functional substates of KRas during GTP hydrolysis with enhanced sampling simulations.

As the hub of major signaling pathways, Ras proteins are implicated in 19% of tumor-caused cancers due to perturbations in their conformational and/or catalytic properties. Despite numerous studies, the functions of the conformational substates for the most important isoform, KRas, remain elusive. In this work, we perform an extensive simulation analysis on the conformational landscape of KRas in its various chemical states during the GTP hydrolysis cycle: the reactant state KRasGTP·Mg2+, the intermediate state KRasGDP·Pi·Mg2+ and the product state KRasGDP·Mg2+. The results from enhanced sampling simulations reveal that State 1 of KRasGTP·Mg2+ has multiple stable substates in solution, one of which might account for interacting with GEFs. State 2 of KRasGTP·Mg2+ features two substates "Tyr32in" and "Tyr32out", which are poised to interact with effectors and GAPs, respectively. For the intermediate state KRasGDP·Pi·Mg2+, Gln61 and Pi are found to assume a broad set of conformations, which might account for the weak oncogenic effect of Gln61 mutations in KRas in contrast to the situation in HRas and NRas. Finally, the product state KRasGDP·Mg2+ has more than two stable substates in solution, pointing to a conformation-selection mechanism for complexation with GEFs. Based on these results, some specific inhibition strategies for targeting the binding sites of the high-energy substates of KRas during GTP hydrolysis are discussed.

[Cloning and function analysis of chalcone isomerase gene and chalcone synthase gene in Lonicera macranthoides].

In order to explore the functions of genes of key rate-limiting enzymes chalcone isomerase(CHI) and chalcone synthase(CHS) in the biosynthesis of flavonoids in Lonicera macranthoides, this study screened and cloned the cDNA sequences of CHI and CHS genes from the transcriptome data of conventional variety and 'Xianglei' of L. macranthoides. Online bioinformatics analysis software was used to analyze the characteristics of the encoded proteins, and quantitative reverse-transcription polymerase chain reaction(qRT-PCR) to detect the expression of CHI and CHS in different parts of the varieties at different flowering stages. The content of luteo-loside was determined by high performance liquid chromatography(HPLC) and the correlation with the expression of the two genes was analyzed. The results showed that the CHI and CHS of the two varieties contained a 627 bp and 1170 bp open reading frame(ORF), respectively, and the CHI protein and CHS protein were stable, hydrophilic, and non-secretory. qRT-PCR results demonstrated that CHI and CHS of the two varieties were differentially expressed in stems and leaves at different flowering stages, particularly the key stages. Based on HPLC data, luteoloside content was in negative correlation with the relative expression of the genes. Thus, CHI and CHS might regulate the accumulation of flavonoids in L. macranthoides, and the specific functions should be further studied. This study cloned CHI and CHS in L. macranthoides and analyzed their expression for the first time, which laid a basis for investigating the molecular mechanism of the differences in flavonoids such as luteoloside in L. macranthoides and variety breeding.

Genetic alterations and functional networks of m6A RNA methylation regulators in pancreatic cancer based on data mining.

BACKGROUND: Pancreatic cancer is a fatal malignancy of the digestive system and the 5-year survival rate remains low. Therefore, new molecular therapeutic targets are required to improve treatments, prognosis, and the survival of patients. N6-methyladenosine (m6A) is the most prevalent reversible methylation in mammalian messenger RNA (mRNA) and has critical roles in the tumorigenesis and metastasis of various malignancies. However, the role of m6A in pancreatic cancer is still unclear. Exploring genetic alterations and functional networks of m6A regulators in pancreatic cancer may provide new strategies for its treatment. METHODS: In this study, we used data from the Cancer Genome Atlas (TCGA) database and other public databases through cBioPortal, LinkedOmics, UALCAN, GEPIA, STRING, and the database for annotation, visualization, and integrated discovery (DAVID) to systematically analyze the molecular alterations and functions of 20 main m6A regulators in pancreatic cancer. RESULTS: We found that m6A regulators had widespread genetic alterations, and that their expression levels were significantly correlated with pancreatic cancer malignancy. Moreover, m6A regulators were associated with the prognosis of pancreatic cancer patients. CONCLUSIONS: m6A regulators play a crucial part in the occurrence and development of pancreatic cancer. Our study will guide further studies of m6A RNA modification in pancreatic cancer and could potentially provide new strategies for pancreatic cancer treatment.

Pak2 reduction induces a failure of early embryonic development in mice.

BACKGROUND: The quality of the early embryo is vital to embryonic development and implantation. As a highly conserved serine/threonine kinase, p21-activated kinase 2 (Pak2) participates in diverse biologic processes, especially in cytoskeleton remodeling and cell apoptosis. In mice, Pak2 knock out and endothelial depletion of Pak2 showed embryonic lethality. However, the role of Pak2 in preimplantation embryos remains unelucidated. METHODS: In the present work, Pak2 was reduced using a specific small interfering RNA in early mouse embryos, validating the unique roles of Pak2 in spindle assembly and DNA repair during mice early embryonic development. We also employed immunoblotting, immunostaining, in vitro fertilization (IVF) and image quantification analyses to test the Pak2 knockdown on the embryonic development progression, spindle assembly, chromosome alignment, oxidative stress, DNA lesions and blastocyst cell apoptosis. Areas in chromatin with γH2AX were detected by immunofluorescence microscopy and serve as a biomarker of DNA damages. RESULTS: We found that Pak2 knockdown significantly reduced blastocyst formation of early embryos. In addition, Pak2 reduction led to dramatically increased abnormal spindle assembly and chromosomal aberrations in the embryos. We noted the overproduction of reactive oxygen species (ROS) with Pak2 knockdown in embryos. In response to DNA double strand breaks (DSBs), the histone protein H2AX is specifically phosphorylated at serine139 to generate γH2AX, which is used to quantitative DSBs. In this research, Pak2 knockdown also resulted in the accumulation of phosphorylated γH2AX, indicative of increased embryonic DNA damage. Commensurate with this, a significantly augmented rate of blastocyst cell apoptosis was detected in Pak2-KD embryos compared to their controls. CONCLUSIONS: Collectively, our data suggest that Pak2 may serve as an important regulator of spindle assembly and DNA repair, and thus participate in the development of early mouse embryos.

The effect of cell isolation methods on the human transcriptome profiling and microbial transcripts of peripheral blood.

The expression of human and microbial genes serves as biomarkers for disease and health. Blood RNA is an important biological resource for precision medicine and translational medicine. However, few studies have assessed the human transcriptome profiles and microbial communities composition and diversity of peripheral blood from different cell isolation methods, which could affect the reproducibility of researches. We collected peripheral blood from three healthy donors and processed it immediately. We used RNA sequencing to investigate the effect of three leukocyte isolation methods including buffy coat (BC) extraction, red blood cell (RBC) lysis and peripheral blood mononuclear cell (PBMC) isolation with the comparison with whole blood (WB), through analyzing the sensitivity of gene detection, the whole transcriptome profiling and microbial composition and diversity. Our data showed that BC extraction with high globin mRNA mapping rate had similar transcriptome profiles with WB, while RBC lysis and PBMC isolation depleted RBCs effectively. With the efficient depletion of RBC and distinct compositions of leukocyte subsets, RNA-seq of RBC lysis and PBMC isolation uniquely detected genes from specific cell types, like granulocytes and NK cells. In addition, we observed that the microbial composition and diversity were more affected by individuals than isolation methods. Our results showed that blood cell isolations could largely influence the sensitivity of detection of human genes and transcriptome profile.

Impact of storage conditions on peripheral leukocytes transcriptome.

Leukocytes reflect the physiological and pathological states of each individual, and transcriptomic data of leukocytes have been used to reflect health conditions. Since the overall impact of ex vivo conditions on the leukocyte transcriptome before RNA stabilization remains unclear, we evaluated the influence of temporary storage conditions on the leukocyte transcriptome through RNA sequencing. We collected peripheral blood with EDTA tubes, which were processed immediately or stored either at 4 °C or room temperature (RT, 18-22 °C) for 2 h, 6 h and 24 h. Total cellular RNA was extracted from 42 leukocyte samples after red blood cells lysis for subsequent RNA sequencing. We applied weighted gene co-expression network analysis to construct co-expression networks of mRNA and lncRNA among the samples, and then performed gene ontology (GO) term enrichment to explore possible biological processes affected by storage conditions. Storage conditions change the gene expression of peripheral leukocytes. Comparing with fresh leukocytes, storage for 24 h at 4 °C and RT affected 1515 (1.51%) and 10,823 (10.82%) genes, respectively. Pathway enrichment analysis identified nucleosome assembly enriched in up-regulated genes at both conditions. When blood was stored at RT for 24 h, genes involved in apoptotic signaling pathway, negative regulation of cell cycle and lymphocyte activation were upregulated, while the relative proportion of neutrophils was significantly decreased. Temporary storage conditions profoundly affect the gene expression profiles of leukocytes and might further change cell viability and state. Storage of blood samples at 4 °C within 6 h largely maintains their original transcriptome.