Kinesin motor KIF16A regulates microtubule stability and actin-dependent spindle migration in mouse oocyte meiosis.

Kif16A, a member of the kinesin-3 family of motor proteins, has been shown to play crucial roles in inducing mitotic arrest, apoptosis, and mitotic cell death. However, its roles during oocyte meiotic maturation have not been fully defined. In this study, we report that Kif16A exhibits unique accumulation on the spindle apparatus and colocalizes with microtubule fibers during mouse oocyte meiotic maturation. Targeted depletion of Kif16A using gene-targeting siRNA disrupts the progression of the meiotic cell cycle. Furthermore, Kif16A depletion leads to aberrant spindle assembly and chromosome misalignment in oocytes. Our findings also indicate that Kif16A depletion reduces tubulin acetylation levels and compromises microtubule resistance to depolymerizing drugs, suggesting its crucial role in microtubule stability maintenance. Notably, we find that the depletion of Kif16A results in a notably elevated incidence of defective kinetochore-microtubule attachments and the absence of BubR1 localization at kinetochores, suggesting a critical role for Kif16A in the activation of the spindle assembly checkpoint (SAC) activity. Additionally, we observe that Kif16A is indispensable for proper actin filament distribution, thereby impacting spindle migration. In summary, our findings demonstrate that Kif16A plays a pivotal role in regulating microtubule and actin dynamics crucial for ensuring both spindle assembly and migration during mouse oocyte meiotic maturation.

Characterization of sexual maturity-associated N6-methyladenosine in boar testes.

BACKGROUND: The health and size of the testes are crucial for boar fertility. Testicular development is tightly regulated by epigenetics. N6-methyladenosine (m6A) modification is a prevalent internal modification on mRNA and plays an important role in development. The mRNA m6A methylation in boar testicular development still needs to be investigated. RESULTS: Using the MeRIP-seq technique, we identify and profile m6A modification in boar testes between piglets and adults. The results showed 7783 distinct m6A peaks in piglets and 6590 distinct m6A peaks in adults, with 2,471 peaks shared between the two groups. Enrichment of GO and KEGG analysis reveal dynamic m6A methylation in various biological processes and signalling pathways. Meanwhile, we conjointly analyzed differentially methylated and expressed genes in boar testes before and after sexual maturity, and reproductive related genes (TLE4, TSSK3, TSSK6, C11ORF94, PATZ1, PHLPP1 and PAQR7) were identified. Functional enrichment analysis showed that differential genes are associated with important biological functions, including regulation of growth and development, regulation of metabolic processes and protein catabolic processes. CONCLUSION: The results demonstrate that m6A methylation, differential expression and the related signalling pathways are crucial for boar testicular development. These results suggest a role for m6A modification in boar testicular development and provided a resource for future studies on m6A function in boar testicular development.

Decoupled responses of above- and below-ground beta-diversity to nitrogen enrichment in a typical steppe.

Increased atmospheric nitrogen (N) deposition affects biodiversity in terrestrial ecosystems. However, we do not know whether the effects of N on above-ground plant ß-diversity are coupled with changes occurring in the soil seed bank. We conducted a long-term N-addition experiment in a typical steppe and found that above-ground ß-diversity increased and then decreased with increasing N addition, whereas below-ground ß-diversity decreased linearly. This suggests decoupled dynamics of plant communities and their soil seed bank under N enrichment. Species substitution determined above- and below-ground ß-diversity change via an increasing role of deterministic processes with N addition. These effects were mostly driven by differential responses of the above-ground vegetation and the soil seed bank ß-diversities to N-induced changes in environmental heterogeneity, increased soil inorganic N concentrations and soil acidification. Our findings highlight the importance of considering above- and below-ground processes simultaneously for effectively conserving grassland ecosystems under N enrichment.

Analysis of fecal microbiome and metabolome changes in goats with pregnant toxemia.

BACKGROUND: Pregnancy toxemia is a common disease, which occurs in older does that are pregnant with multiple lambs in the third trimester. Most of the sick goats die within a few days, which can seriously impact the economic benefits of goat breeding enterprises. The disease is believed to be caused by malnutrition, stress, and other factors, that lead to the disorder of lipid metabolism, resulting in increased ketone content, ketosis, ketonuria, and neurological symptoms. However, the changes in gut microbes and their metabolism in this disease are still unclear. The objective of this experiment was to evaluate the effect of toxemia of pregnancy on the fecal microbiome and metabolomics of does. RESULTS: Eight pregnant does suspected of having toxemia of pregnancy (PT group) and eight healthy does during the same pregnancy (NC group) were selected. Clinical symptoms and pathological changes at necropsy were observed, and liver tissue samples were collected for pathological sections. Jugular venous blood was collected before morning feeding to detect biochemical indexes. Autopsy revealed that the liver of the pregnancy toxemia goat was enlarged and earthy yellow, and the biochemical results showed that the serum levels of aspartate aminotransferase (AST) and ß-hydroxybutyric acid (B-HB) in the PT group were significantly increased, while calcium (Ca) levels were significantly reduced. Sections showed extensive vacuoles in liver tissue sections. The microbiome analysis found that the richness and diversity of the PT microbiota were significantly reduced. Metabolomic analysis showed that 125 differential metabolites were screened in positive ion mode and enriched in 12 metabolic pathways. In negative ion mode, 100 differential metabolites were screened and enriched in 7 metabolic pathways. CONCLUSIONS: Evidence has shown that the occurrence of pregnancy toxemia is related to gut microbiota, and further studies are needed to investigate its pathogenesis and provide research basis for future preventive measures of this disease.

Transient pulmonary and gastric bleeding after iopamidol administration in a patient with marginal zone lymphoma: a case report.

BACKGROUND: Iopamidol is a non-ionic, water-soluble iodine contrast agent that is considered safe for intravenous or intra-arterial administration and is widely used both in the general population and in patients undergoing oncological treatment. While adverse reactions to iopamidol have been documented, to date, no pulmonary and gastric hemorrhages induced by iopamidol have been reported in oncology patients. We report the first case of this complication. CASE PRESENTATION: We report the case of a 60-year-old woman with marginal zone lymphoma who was receiving antineoplastic therapy. As part of the investigation for the condition, she underwent chest enhancement CT with iopamidol. Shortly thereafter(within five minutes), she experienced hemoptysis and hematemesis. She was intubated and admitted to the intensive care unit. Pre- and post-contrast images demonstrated the course of the hemorrhage. Flexible bronchoscopy and gastroscopy on the following day showed no active bleeding, and the patient recovered completely after antiallergy treatment. We speculate that contrast-induced hypersensitivity was the most likely cause of the transient pulmonary and gastric bleeding. CONCLUSION: Although rare, the complications of iopamidol, which may cause allergic reactions in the lungs and stomach, should be considered.

False positive findings associated with adenoviral vector-based vaccine underscore the regulatory necessity to eliminate abnormal toxicity test.

Accumulating evidence has shown that the abnormal toxicity test (ATT) is not suitable as a quality control batch release test for biologics and vaccines. The purpose of the current study was to explore the optimal ATT experimental design for an adenoviral vector-based vaccine product to avoid false positive results following the standard test conditions stipulated in the Pharmacopoeias. ATT were conducted in both mice and guinea pigs based on methods in Pharmacopeias, with modifications to assess effects of dose volume and amount of virus particles (VPs). The results showed intraperitoneal (IP) dosing at human relevant dose and volume (i.e., VPs), as required by pharmacopeia study design, resulted in false positive findings not associated with extraneous contaminants of a product. Considering many gene therapy products use adeno associated virus as the platform for transgene delivery, data from this study are highly relevant in providing convincing evidence to show the ATT is inappropriate as batch release test for biologics, vaccine and gene therapy products. In conclusion, ATT, which requires unnecessary animal usage and competes for resources which otherwise can be spent on innovative medicine research, should be deleted permanently as batch release test by regulatory authorities around the world.

Integrative proteomic and phosphoproteomic analysis in the female goat hypothalamus to study the onset of puberty.

BACKGROUND: Puberty marks the end of childhood and achieve sexual maturation and fertility. The role of hypothalamic proteins in regulating puberty onset is unclear. We performed a comprehensive differential proteomics and phosphoproteomics analysis in prepubertal and pubertal goats to determine the roles of hypothalamic proteins and phosphoproteins during the onset of puberty. RESULTS: We used peptide and posttranslational modifications peptide quantification and statistical analyses, and identified 69 differentially expressed proteins from 5,057 proteins and 576 differentially expressed phosphopeptides from 1574 phosphorylated proteins. Combined proteomic and phosphoproteomics, 759 correlated proteins were identified, of which 5 were differentially expressed only at the protein level, and 201 were only differentially expressed at the phosphoprotein level. Pathway enrichment analyses revealed that the majority of correlated proteins were associated with glycolysis/gluconeogenesis, Fc gamma R-mediated phagocytosis, focal adhesion, GABAergic synapse, and Rap1 signaling pathway. These pathways are related to cell proliferation, neurocyte migration, and promoting the release of gonadotropin-releasing hormone in the hypothalamus. CTNNB1 occupied important locations in the protein-protein interaction network and is involved in focal adhesion. CONCLUSION: The results demonstrate that the proteins differentially expression only at the protein level or only differentially expressed at the phosphoprotein level and their related signalling pathways are crucial in regulating puberty in goats. These differentially expressed proteins and phosphorylated proteins may constitute the proteomic backgrounds between the two different stages.

CircKDM5B sponges miR-128 to regulate porcine blastocyst development by modulating trophectoderm barrier function.

Circular RNAs (circRNAs), which exert critical functions in the regulation of transcriptional and post-transcriptional gene expression, are found in mammalian cells but their functions in mammalian preimplantation embryo development remain poorly understood. Here, we showed that circKDM5B mediated miRNA-128 (miR-128) to regulate porcine early embryo development. We screened circRNAs potentially expressed in porcine embryos through an integrated analysis of sequencing data from mouse and human embryos, as well as porcine oocytes. An authentic circRNA originating from histone demethylase KDM5B (referred to as circKDM5B) was abundantly expressed in porcine embryos. Functional studies revealed that circKDM5B knockdown not only significantly reduced blastocyst formation but also decreased the number of total cells and trophectoderm (TE) cells. Moreover, the knockdown of circKDM5B resulted in the disturbance of tight junction assembly and impaired paracellular sealing within the TE epithelium. Mechanistically, miR-128 inhibitor injection could rescue the early development of circKDM5B knockdown embryos. Taken together, the findings revealed that circKDM5B functions as a miR-128 sponge, thereby facilitating early embryonic development in pigs through the modulation of gene expression linked to tight junction assembly.

Double helix point spread function with variable spacing for precise 3D particle localization.

To extend the axial depth of nanoscale 3D-localization microscopy, we propose here a splicing-type vortex singularities (SVS) phase mask, which has been meticulously optimized with a Fresnel approximation imaging inverse operation. The optimized SVS DH-PSF has proven to have high transfer function efficiency with adjustable performance in its axial range. The axial position of the particle was computed by using both the main lobes' spacing and the rotation angle, an improvement of the localization precision of the particle. Concretely, the proposed optimized SVS DH-PSF, with a smaller spatial extent, can effectively reduce the overlap of nanoparticle images and realize the 3D localization of multiple nanoparticles with small spacing, with respect to PSFs for large axial 3D localization. Finally, we successfully conducted extensive experiments on 3D localization for tracking dense nanoparticles at 8µm depth with a numerical aperture of 1.4, demonstrating its great potential.

Butylparaben weakens female fertility via causing oocyte meiotic arrest and fertilization failure in mice.

Butylparaben is an ubiquitous environmental endocrine disruptor, that is commonly used in cosmetics and personal care product due to its anti-microbial properties. Butylparaben has been shown to cause developmental toxicity, endocrine and metabolic disorders and immune diseases. However, little is known about the impact on female fertility, especially oocyte quality. In the present study, we reported that butylparaben influenced female fertility by showing the disturbed oocyte meiotic capacity and fertilization potential. Specifically, butylparaben results in the oocyte maturation arrest by impairing spindle/chromosome structure and microtubule stability. Besides, butylparaben results in fertilization failure by impairing the dynamics of Juno and ovastacin and the sperm binding ability. Last, single-cell transcriptome analysis showed that butylparaben-induced oocyte deterioration was caused by mitochondrial dysfunction, which led to the accumulation of ROS and occurrence of apoptosis. Collectively, our study indicates that mitochondrial dysfunction and redox perturbation is the major cause of the weakened female fertility expoesd to butylparaben.

Inhibitory Effects of 3-Methylcholanthrene Exposure on Porcine Oocyte Maturation.

3-methylcholanthrene (3-MC) is a highly toxic environmental pollutant that impairs animal health. 3-MC exposure can cause abnormal spermatogenesis and ovarian dysfunction. However, the effects of 3-MC exposure on oocyte maturation and embryo development remain unclear. This study revealed the toxic effects of 3-MC exposure on oocyte maturation and embryo development. 3-MC with different concentrations of 0, 25, 50, and 100 µM was applied for in vitro maturation of porcine oocytes. The results showed that 100 µM 3-MC significantly inhibited cumulus expansion and the first polar body extrusion. The rates of cleavage and blastocyst of embryos derived from 3-MC-exposed oocytes were significantly lower than those in the control group. Additionally, the rates of spindle abnormalities and chromosomal misalignments were higher than those in the control group. Furthermore, 3-MC exposure not only decreased the levels of mitochondria, cortical granules (CGs), and acetylated α-Tubulin, but also increased the levels of reactive oxygen species (ROS), DNA damage, and apoptosis. The expression of cumulus expansion and apoptosis-related genes was abnormal in 3-MC-exposed oocytes. In conclusion, 3-MC exposure disrupted the nuclear and cytoplasmic maturation of porcine oocytes through oxidative stress.

Phase optimization algorithm for 3D particle localization with large axial depth.

We propose an optimization algorithm based on Fresnel approximation (FA) imaging to optimize an extended-axial-depth point spread function (PSF) for 3D particle localization. The transfer function efficiency of the PSF is improved by repeatedly imposing constraints in the object plane, the spatial domain, and the Fourier domain. During the iterative calculation, the effective photon number or Cramer-Rao lower bound is used as the termination condition of the iteration. The algorithm allows flexible adjustment of the peak intensity ratio of the two main lobes. Moreover, the transfer function efficiency can be balanced by increasing the weight of the modulation function of the expected PSF at each axial position. The twin-Airy (TA) PSF optimized by the FA optimization algorithm does not require complex post-processing, whereas post-processing is an essential step for the unoptimized TA-PSF. The optimization algorithm is significant for extended-axial-depth PSFs used for 3D particle localization, as it improves localization precision and temporal resolution.

Maternal DDB1 regulates apoptosis and lineage differentiation in porcine preimplantation embryos.

CONTEXT: Maternal-effect genes (MEGs) play a critical role in modulating both cellular and molecular biology events in preimplantation embryonic development. Damage-specific DNA binding protein 1 (DDB1) is a gene that participates in meiotic resumption, ovulation, and embryonic stem cell maintenance. Its function in preimplantation development is not well-studied. AIMS: We aimed to explore the expression pattern, genomic heritage, and potential molecular mechanisms of DDB1 in preimplantation embryos in porcine. METHODS: In this study, RNA interference, microinjection, RT-qPCR, immunofluorescence staining and single-cell RNA sequencing were used to explore the molecular function of DDB1 in porcine preimplantation embryos. KEY RESULTS: DDB1 was found to be expressed in germinal vesicle (GV) and Meiosis II (MII) oocytes and in preimplantation embryos. We confirmed it is a MEG. DDB1 -deficient blastocysts had a significantly reduced number of trophectoderm cells, an increased apoptotic cell number and increased apoptosis index. According to a next-generation sequencing (NGS) analysis, 236 genes (131 upregulated and 105 downregulated) significantly changed in the DDB1 -deficient morula. The myeloid leukaemia factor 1 (MLF1 ) and yes-associated protein 1 (YAP1 ) expressions were significantly upregulated and downregulated respectively, in the DDB1 -deficient morula. In combination with the decreased expression of TEAD4 , CDX2 , GATA3 , OCT4 , and NANOG and the increased expression of SOX2 in the blastocyst, DDB1 may play a role in determining lineage differentiation and pluripotency maintenance. CONCLUSIONS: DDB1 is a MEG and it plays a crucial role in porcine preimplantation embryonic development. IMPLICATIONS: This study provides a theoretical basis for further understanding the molecular mechanisms of preimplantation embryo development.

Enhancement of E-Peroxone process with waste-tire carbon composite cathode for tinidazole degradation.

The cathode is the key component in the electro-peroxone process (E-Peroxone), which is popularly constructed with carbon materials. This study developed an innovative method to fabricate a cathode with waste-tire carbon (WTC) whose performance was evaluated for the degradation of tinidazole (TNZ), an antibiotic frequently detected in water. It was found that the addition of WTC in the cathode can significantly promote the yield of H2O2 and the current efficiency: around 2.7 times that of commercial carbon black at the same loading. The critical influencing factors were studied, including the current density, ozone concentration, initial pH value, chlorine ions and initial TNZ concentration. The scavenger tests demonstrated the possible involvement of •OH and O2•-. Some transformation products of TNZ were identified with UPLC-MS and the degradation pathway was proposed accordingly. These results demonstrated the potential of WTC for developing E-Peroxone cathodes.

METTL3-mediated m6A methylation negatively modulates autophagy to support porcine blastocyst development‡.

N6-methyladenosine (m6A) catalyzed by METTL3 regulates the maternal-to-zygotic transition in zebrafish and mice. However, the role and mechanism of METTL3-mediated m6A methylation in blastocyst development remains unclear. Here, we show that METTL3-mediated m6A methylation sustains porcine blastocyst development via negatively modulating autophagy. We found that reduced m6A levels triggered by METTL3 knockdown caused embryonic arrest during morula-blastocyst transition and developmental defects in trophectoderm cells. Intriguingly, overexpression of METTL3 in early embryos resulted in increased m6A levels and these embryos phenocopied METTL3 knockdown embryos. Mechanistically, METTL3 knockdown or overexpression resulted in a significant increase or decrease in expression of ATG5 (a key regulator of autophagy) and LC3 (an autophagy marker) in blastocysts, respectively. m6A modification of ATG5 mRNA mainly occurs at 3'UTR, and METTL3 knockdown enhanced ATG5 mRNA stability, suggesting that METTL3 negatively regulated autophagy in an m6A dependent manner. Furthermore, single-cell qPCR revealed that METTL3 knockdown only increased expression of LC3 and ATG5 in trophectoderm cells, indicating preferential inhibitory effects of METTL3 on autophagy activity in the trophectoderm lineage. Importantly, autophagy restoration by 3MA (an autophagy inhibitor) treatment partially rescued developmental defects of METTL3 knockdown blastocysts. Taken together, these results demonstrate that METTL3-mediated m6A methylation negatively modulates autophagy to support blastocyst development.

Splicing exponential point spread function design for localization of nanoparticles.

We propose a point spread function for three-dimensional localization of nanoparticles. The axial detection range of the point spread function can be simply changed by adjusting the design parameters. In addition, the spatial extent of the point spread function can also be changed by adjusting the design parameters, which is an advantage other point spread functions do not have. We used our point spread functions and the existing point spread functions for dense multi-particle imaging, which proved the advantage that the point spread function with a smaller spatial extent we designed can effectively reduce the overlap between the point spread functions. The three-dimensional process of the fluorescent microsphere penetrating HT-22 cell membrane was successfully recorded, which verified the effectiveness of this method.

Carbon limitation overrides acidification in mediating soil microbial activity to nitrogen enrichment in a temperate grassland.

Higher ecosystem nitrogen (N) inputs resulting from human activities often suppress soil microbial biomass and respiration, thereby altering biogeochemical cycling. Soil acidification and carbon (C) limitation may drive these microbial responses, yet their relative importance remains elusive, which limits our understanding of the longer term effects of increasing N inputs. In a field experiment with continuous N addition at seven different rates from 0 to 50 g N m-2  year-1 over 6 years in a temperate grassland of Inner Mongolia, China, we examined the responses of soil microbial biomass and respiration to changes in soil acidity and C availability by adding lime and/or glucose to soil samples. Soil microbial biomass and respiration did only weakly respond to increasing soil pH, but increased strongly in response to higher C availability with increasing N addition rates. Soil net N immobilization increased in response to glucose addition, and soil microbial biomass increased at higher rates than microbial respiration along the gradient of previous N addition rates, both suggesting increasingly reinforced microbial C limitation with increasing N addition. Our results provide clear evidence for strong N-induced microbial C limitation, but only little support for soil acidity effects within the initial pH range of 4.73-7.86 covered by our study. Field data support this conclusion by showing reduced plant C allocation belowground in response to N addition, resulting in soil microbial C starvation over the long term. In conclusion, soil microbial biomass and respiration under N addition were strongly dependent on C availability, most likely originating from plant belowground C inputs, and was much less affected by changes in soil pH. Our data help clarify a long-standing debate about how increasing N input rates affect soil microbial biomass and respiration, and improve the mechanistic understanding of the linkages between ecosystem N enrichment and C cycling.

Particles 3D tracking with large axial depth by using the 2π-DH-PSF.

We propose a 2π-double-helix point spread function (2π-DH-PSF) using the Fresnel zone approach that can rotate 2π rad. When 16 Fresnel zones are used, the particles can be tracked in the axial range of 10 µm in a 100× microscopy imaging system (NA=1.4, λ=514nm). We measured the diffusion coefficient of nanospheres in different concentrations of glycerol with the 2π-DH-PSF, and the error between the measured results and theoretical value was within 10%, indicating the superior performance of 2π-DH-PSF in 3D localization imaging of nanoparticles. When combined with the defocus phase, the rotation angle can reach 4π rad, four times that of the conventional DH-PSF.

Three-dimensional diffusion coefficient measurement by a large depth-of-field rotating point spread function.

A prominent challenge in single-molecule localization microscopy is the real-time, fast, and accurate localization of nano-objects moving in three-dimensional (3D) samples. A well-established method for 3D single-molecule localization is the double-helix pointspread-function (DH-PSF) engineering, which uses additional optical elements to make the PSF exhibit different rotation angles with different nanoparticle depths. However, the compact main lobe size, effective detection depth, and precise conversion between rotation angle and depth are necessary, posing challenges to the DH-PSF generation method. Here we generate a more compact DH-PSF using Fresnel-zone-based spiral phases, and the pure phase mask achieves high transmission efficiency. The final generated DH-PSFs have a linear rotation rate at each axial position, showing a more accurate rotation angle and depth conversion. The Cramer-Rao lower limit calculation results show that the axial depth of DH-PSF extends to ∼11µm with an axial localization precision of ∼45nm at 3000 photons and average background noise of 15. We measured the diffusion coefficient of nanospheres in different concentrations of glycerol using the generated DH-PSF. The measured results are within 6% error from the theoretical values, indicating the superior performance of the DH-PSF for nanoparticle diffusion coefficient measurements.

Social support status and associated factors among people living with HIV/AIDS in Kunming city, China.

BACKGROUND: People living with HIV/AIDS not only require effective treatment for the alleviation of physical discomfort but also require social support to help them address difficulties in life and relieve their psychological anxiety and uneasiness. The social support network is of tremendous importance in helping people living with HIV/AIDS maintain good physical and mental health. This study aims to analyse the social support status among people living with HIV/AIDS in Kunming and explore associated factors. METHOD: The Social Support Rating Scale (SSRS) was used, and a questionnaire survey was conducted using convenience sampling to select people living with HIV/AIDS from 14 counties of Kunming. It collected information on general demographic information and social support status. Univariate and multivariate linear regression models were used to explore the associated factors. RESULTS: A total of 990 valid questionnaires were completed. Data from all participants were analysed. Univariate analysis suggested that the factors associated with social support may include marital status, monthly income, and antiretroviral therapy. On the other hand, factors including monthly income and antiretroviral therapy accounted for the social support total score in the multivariate analysis. CONCLUSION: Social support among people living with HIV/AIDS in Kunming was generally low. This study identified a number of factors associated with social support among people living with HIV/AIDS. Based on our findings, appropriate interventions should be introduced to provide social support for those living with HIV/AIDS.