Background Sperm cryopreservation is widely used in the cattle industry,as it allows for disassociating the localiza-tion of sires and the collection of semen from the timing of artificial insemination.While freeze-th...Background Sperm cryopreservation is widely used in the cattle industry,as it allows for disassociating the localiza-tion of sires and the collection of semen from the timing of artificial insemination.While freeze-thawing is known to impair sperm DNA integrity,whether the damage induced consists of single-(SSB)or double-strand breaks(DSB)has not been determined.In addition,no previous study has addressed if DNA breaks preferentially reside in specific genome regions such as those forming the toroid linker regions,or are rather spread throughout the regions linked to protamines.The main aim of the present work,therefore,was to elucidate the type and localization of the DNA damage generated by cryopreservation and to evaluate its impact on artificial insemination outcomes in cattle.Results The incidence of SSB and DSB was evaluated in 12 ejaculates before and after cryopreservation with the Comet assay,and the localization of the DNA breaks was assessed using pulsed-field gel electrophoresis(PFGE).Before cryopreservation,the incidence of SSB was 10.99%±4.62%and involved 20.56%±3.04%of sperm cells,whereas these figures significantly(P<0.0001)increased up to 34.11%±3.48%and 53.36%±11.00%in frozen-thawed sperm.In contrast,no significant differences in the incidence of DSB were observed(P>0.990)before and after cryopreservation(before:incidence of 13.91%±1.75%of sperm DNA affecting 56.04%±12.49%of sperm cells;after:incidence of 13.55%±1.55%of sperm DNA involving 53.36%±11.00%of sperm cells).Moreover,PFGE revealed that the percentage of sperm DNA fragments whose length was shorter than a toroid(<31.5 kb)was greater(P<0.0001)after(27.00%±4.26%)than before freeze-thawing(15.57%±4.53%).These differences indicated that the DNA breaks induced by cryopreservation affect the regions condensed in protamines,which are structured in toroids.On the other hand,in vivo fertility rates were associated to the incidence of SSB and DSB in frozen-thawed sperm(P=0.032 and P=0.005),but not with the size of the DNA fragments resulting from these breaks(P>0.05).Conclusion Cryopreservation of bovine sperm generates single-strand DNA breaks,which are mainly located in protamine-condensed toroidal regions.The incidence of DNA breaks in cryopreserved sperm has an impact on cat-tle fertility,regardless of the size of generated fragments.展开更多
The aim of this study was to compare the sperm nuclear and acrosomal morphometry of three species of domestic artiodactyls; cattle （Bos taurus）, sheep （Ovis aries）, and pigs （Sus scrofa）. Semen smears of twenty ...The aim of this study was to compare the sperm nuclear and acrosomal morphometry of three species of domestic artiodactyls; cattle （Bos taurus）, sheep （Ovis aries）, and pigs （Sus scrofa）. Semen smears of twenty ejaculates from each species were fixed and labeled with a propidium iodide-Pisum sativum agglutinin （PI/PSA） combination. Digital images of the sperm nucleus, acrosome, and whole sperm head were captured and analyzed. The use of the PI/PSA combination and CASA-Morph fluorescence-based method allowed the capture, morphometric analysis, and differentiation of most sperm nuclei, acrosomes and whole heads, and the assessment of acrosomal integrity with a high precision in the three species studied. For the size of the head and nuclear area, the relationship between the three species may be summarized as bull 〉 ram 〉 boar. However, for the other morphometric parameters （length, width, and perimeter）, there were differences in the relationships between species for sperm nuclei and whole sperm heads. Bull sperm acrosomes were clearly smaller than those in the other species studied and covered a smaller proportion of the sperm head. The acrosomal morphology, small in the bull, large and broad in the sheep, and large, long, and with a pronounced equatorial segment curve in the boar, was species-characteristic. It was concluded that there are clear variations in the size and shape of the sperm head components between the three species studied, the acrosome being the structure showing the most variability, allowing a clear distinction of the spermatozoa of each species.展开更多
Neurovascular interactions are crucial for the normal development of the central nervous system. To study such interactions in primary cultures, we developed a procedure to simultaneously isolate neural progenitor and...Neurovascular interactions are crucial for the normal development of the central nervous system. To study such interactions in primary cultures, we developed a procedure to simultaneously isolate neural progenitor and endothelial cell fractions from embryonic mouse brains. Depending on the culture conditions endothelial cells were found to favor maintenance of the neuroprogenitor phenotype through the production of soluble factors, or to promote neuronal differentiation of neural progenitors through direct contact. These apparently opposing effects could reflect differential cellular interactions needed for the proper development of the brain.展开更多
The aberrant use of alcohol is a major factor in cancer progression and metastasis.Contributing mechanisms include the systemic effects of alcohol and the exchange of bioactive molecules between cancerous and non-canc...The aberrant use of alcohol is a major factor in cancer progression and metastasis.Contributing mechanisms include the systemic effects of alcohol and the exchange of bioactive molecules between cancerous and non-cancerous cells along the brain-gut-liver axis.Such interplay leads to changes in molecular,cellular,and biological functions resulting in cancer progression.Recent investigations have examined the role of extracellular vesicles(EVs)in cancer mechanisms in addition to their contribution as diagnostic biomarkers.Also,EVs are emerging as novel cell-free mediators in pathophysiological scenarios including alcohol-mediated gut microbiome dysbiosis and the release of nanosized EVs into the circulatory system.Interestingly,EVs in cancer patients are enriched with oncogenes,miRNA,lipids,and glycoproteins whose delivery into the hepatic microenvironment may be enhanced by the detrimental effects of alcohol.Proof-of-concept studies indicate that alcohol-associated liver disease is impacted by the effects of exosomes,including altered immune responses,reprogramming of stromal cells,and remodeling of the extracellular matrix.Moreover,the culmination of alcoholrelated changes in the liver likely contributes to enhanced hepatic metastases and poor outcomes for cancer patients.This review summarizes the numerous aspects of exosome communications between organs with emphasis on the relationship of EVs in alcohol-associated diseases and cancer metastasis.The potential impact of EV cargo and release along a multi-organ axis is highly relevant to the promotion of tumorigenic mechanisms and metastatic disease.It is hypothesized that EVs target recipient tissues to initiate the formation of prometastatic niches and cancer progression.The study of alcohol-associated mechanisms in metastatic cancers is expected to reveal a better understanding of factors involved in the growth of secondary malignancies as well as novel approaches for therapeutic interventions.展开更多
Amyotrophic lateral sclerosis is a motoneuron degenerative disease that is challenging to diagnose and presents with considerable variability in survival.Early identification and enhanced understanding of symptomatic ...Amyotrophic lateral sclerosis is a motoneuron degenerative disease that is challenging to diagnose and presents with considerable variability in survival.Early identification and enhanced understanding of symptomatic patterns could aid in diagnosis and provide an avenue for monitoring disease progression.Use of the m SOD1 G93 A mouse model provides control of the confounding environmental factors and genetic heterogeneity seen in amyotrophic lateral sclerosis patients,while investigating underlying disease-induced changes.In the present study,we performed a longitudinal behavioral assessment paradigm and identified an early hindlimb symptom,resembling the common gait abnormality foot drop,along with an accompanying forelimb compensatory mechanism in the m SOD1 G93 A mouse.Following these initial changes,m SOD1 mice displayed a temporary hindlimb compensatory mechanism resembling an exaggerated steppage gait.As the disease progressed,these compensatory mechanisms were not sufficient to sustain fundamental locomotor parameters and more severe deficits appeared.We next applied these initial findings to investigate the inherent variability in B6 SJL m SOD1 G93 A survival.We identified four behavioral variables that,when combined in a cluster analysis,identified two subpopulations with different disease progression rates：a fast progression group and a slow progression group.This behavioral assessment paradigm,with its analytical approaches,provides a method for monitoring disease progression and detecting m SOD1 subgroups with different disease severities.This affords researchers an opportunity to search for genetic modifiers or other factors that likely enhance or slow disease progression.Such factors are possible therapeutic targets with the potential to slow disease progression and provide insight into the underlying pathology and disease mechanisms.展开更多
Background:The analysis of chromatin integrity has become an important determinant of sperm quality.In frozenthawed bovine sperm,neither the sequence of post-thaw injury events nor the dynamics of different types of s...Background:The analysis of chromatin integrity has become an important determinant of sperm quality.In frozenthawed bovine sperm,neither the sequence of post-thaw injury events nor the dynamics of different types of sperm DNA breaks are well understood.The aim of the present work was to describe such sperm degradation aftermath focusing on DNA damage dynamics,and to assess if this parameter can predict pregnancy rates in cattle.Results:A total of 75 cryopreserved ejaculates from 25 Holstein bulls were evaluated at two post-thawing periods(0-2 h and 2-4 h),analyzing global and double-stranded DNA damage through alkaline and neutral Comet assays,chromatin deprotamination and decondensation,sperm motility,viability,acrosomal status,and intracellular levels of total ROS,superoxides and calcium.Insemination of 59,605 females was conducted using sperm from the same bulls,thus obtaining the non-return to estrus rates after 90 d(NRR).Results showed an increased rate of double-stranded breaks in the first period(0-2 h:1.29±1.01%/h vs.2-4 h:0.13±1.37%/h;P<0.01),whereas the rate of sperm with moderate+high single-stranded breaks was higher in the second period(0-2 h:3.52±7.77%/h vs.2-4h:21.06±11.69%/h;P<0.0001).Regarding sperm physiology,viability decrease rate was different between the two periods(0-2 h:-4.49±1.79%/h vs.2-4 h:-2.50±3.39%/h;P=0.032),but the progressive motility decrease rate was constant throughout post-thawing incubation(0-2 h:-4.70±3.42%/h vs.2-4 h:-1.89±2.97%/h;P>0.05).Finally,whereas no correlations between bull fertility and any dynamic parameter were found,there were correlations between the NRR and the basal percentage of highly-damaged sperm assessed with the alkaline Comet(Rs=-0.563,P=0.003),between NRR and basal progressive motility(Rs=0.511,P=0.009),and between NRR and sperm with high ROS at 4 h post-thaw(Rs=0.564,P=0.003).Conclusion:The statistically significant correlations found between intracellular ROS,sperm viability,sperm motility,DNA damage and chromatin deprotamination suggested a sequence of events all driven by oxidative stress,where viability and motility would be affected first and sperm chromatin would be altered at a later stage,thus suggesting that bovine sperm should be used for fertilization within 2 h post-thaw.Fertility correlations supported that the assessment of global DNA damage through the Comet assay may help predict bull fertility.展开更多
The distribution, diversity, and abundance of sea cucumbers in intertidal zones of Aurora were studied from February to June 2013 to obtain baseline data. A one-hour timed-search survey covering an approximate one kil...The distribution, diversity, and abundance of sea cucumbers in intertidal zones of Aurora were studied from February to June 2013 to obtain baseline data. A one-hour timed-search survey covering an approximate one kilometer over a ten-meter wide area was conducted in six coastal municipalities to obtain a comprehensive list of sea cucumber species found in the zones. Only four sampling stations (Baler, Dipaculao, Dilasag, and Dingalan) were selected for belt transect survey, 50 × 50 m, based on the availability of coralline, sandy, muddy sand, and rocky habitats which were laid perpendicular and parallel to each intertidal zone. Timed-search survey revealed a total of 15 species of sea cucumbers distributed among two Orders (Order Aspidochirotida and Order Apodida) and four Families (Family Holothuriidae, Family Stichopodidae, Family Synaptidae, and Family Chirotidae) were recorded. Synapta maculata is most common and has widespread distribution. Belt-tranect surveys showed Holothuria leucospilota is among the most frequently occurring species both during day time (58%) and night time (75%). The Dilasag sampling station had the most diverse species (0.71) while the Dipaculao sampling station had the densest species of Holothuria leucospilota (10,014 in<span style="white-space:nowrap;">∙ha<sup><span style="white-space:nowrap;">−1</sup>) and abundant (95.91%) species.展开更多
The diversity and abundance of bivalves and gastropods were studied from April to July 2015. Two sampling stations, Tinib and Esteves, in Casiguran, Aurora were selected based on the presence of mangrove forest, bival...The diversity and abundance of bivalves and gastropods were studied from April to July 2015. Two sampling stations, Tinib and Esteves, in Casiguran, Aurora were selected based on the presence of mangrove forest, bivalves, gastropods, muddy substrate, and gleaners for the establishment of a transect line. Each station was laid with 100 meters transect line overlaid continuously with 2 m × 2 m quadrat along each transect. A total of 50 sampling units covering 0.02 ha area per sampling station were surveyed. The transect line recorded six species of bivalves distributed among five families and 12 species of gastropods belonging to eight families. Three species of bivalves and five species of gastropods were common to both sampling stations. However, Tinib station had most diverse species of bivalves (0.55) while Esteves station had most diverse species of gastropods (0.43). Both stations revealed Dendrostrea folium (43%) (bivalve) and Cerithidea cingulata (93%) (gastropods) as most frequently occurring species. Dendostrea folium also emerged as the densest (10,640 ind<span style="white-space:nowrap;">∙ha<sup><span style="white-space:nowrap;">−1</sup> and 6850 ind<span style="white-space:nowrap;">∙ha<sup><span style="white-space:nowrap;">−1</sup>) and most abundant (58.52% and 77.84%) bivalve species. For gastropods, Terebralia sulcata (249,250 ind<span style="white-space:nowrap;">∙ha<sup><span style="white-space:nowrap;">−1</sup>) was the densest and most abundant (96.97%) in Tinib while Terebralia palustris was the densest (747,050 ind<span style="white-space:nowrap;">∙ha<sup><span style="white-space:nowrap;">−1</sup>) and most abundant (70.44%) in Esteves. Pooled results for bivalve species maintained Dendrostrea folium as the densest (8750 ind<span style="white-space:nowrap;">∙ha<sup><span style="white-space:nowrap;">−1</sup>) and most abundant (64.81%). However, for gastropods, Terebralia palustris emerged as the densest (373,525 ind<span style="white-space:nowrap;">∙ha<sup><span style="white-space:nowrap;">−1</sup>) and most abundant (56.70%) species. The mangrove forests of Casiguran, Aurora thus contain a variety of bivalves and gastropods with Tinib station dominated by bivalves while Esteves station dominated by gastropods.展开更多
This study was conducted to determine the compatibility of ectomycorrhizal (ECM) fungi associated with eucalypts on dipterocarps. Two nodal cuttings of Shorea contorta (Vid.) were rooted in a non-mist system for two m...This study was conducted to determine the compatibility of ectomycorrhizal (ECM) fungi associated with eucalypts on dipterocarps. Two nodal cuttings of Shorea contorta (Vid.) were rooted in a non-mist system for two months, and later inoculated with vegetative mycelia of three strains of Pisolithus collected under eucalypts and a strain of Scleroderma from dipterocarps. Inoculated rooted cuttings were planted in irradiated potting mix and raised in the nursery for five months. Root colonization prior to outplanting ranged from 31% to 38% and ECM fungi did not colonize the uninoculated ones. The cuttings were outplanted in a logged-over dipterocarp forest in Bislig, Surigao Sur following a Randomized Complete Block Design with four blocks;each block was planted with 50 seedlings. Two years after outplanting, Pisolithus strain from New Caledonia (H6394) promoted the highest height (46 cm) and diameter (0.48 cm) increment. Height was increased by 17% and diameter by 7% relative to the uninoculated control. By contrast, Pisolithus strain from the Philippines (H615) gave the shortest (26 cm) height increment and smallest (0.42 cm) diameter. Diameter growth of cuttings inoculated with Scleroderma D01 (from the Philippines) and a Pisolithus strain from Australia (H445) was bigger (0.47 cm) than the uninoculated treated cuttings (0.45 cm). In terms of survival, the uninoculated cuttings gave the highest (90%) percent survival while the lowest (60%) was those inoculated with Pisolithus strain from New Caledonia. It is possible that the indigenous ECM fungi infected easily the roots of the uninoculated cuttings thus contributing to the high survival rate (90%). During outplanting, fruit bodies of S. columnare were present in the field site. The results, however, show that ECM fungi were able to colonize the roots and that some strains promoted the growth and survival of S. contorta planted in a logged-over dipterocarp forest in Bislig, Surigao, Philippines.展开更多
OBJECTIVE: To investigate the neural differentiation capacity of water extraction of velvet antler.METHODS: Velvet antler(Cervus Nippon Temminck) polypeptide(VAP) was used to differentiate neural stem cells(NSCs) towa...OBJECTIVE: To investigate the neural differentiation capacity of water extraction of velvet antler.METHODS: Velvet antler(Cervus Nippon Temminck) polypeptide(VAP) was used to differentiate neural stem cells(NSCs) towards neurons in the study. Firstly, we obtain the polypeptides of VAP by water extraction. Secondly, we observed the morphology, assayed the factors in the media by enzyme-linked immunosorbent assay, and detected the special neural molecules by immuno fl uorescence staining. NSCs were cultured on the cell climbing film. After neuronal differentiation, differentiated NSCs were mounted for immunocytochemistry with immunofluorescence technique.RESULTS: The differentiating cells look like neuron,some special factors, such as Glial cell line-derived neurotrophic factor, nerve growth factor, in the media can be detected while differentiated neuron-like cells can express the special neural molecules.CONCLUSION: Differentiation of NSCs towards neurons can be induced by velvet antler polypeptide.展开更多
OBJECTIVE:To investigate the mechanism of action of the Lingbao Huxin Dan(灵宝护心丹)in treating bradycardia arrhythmia with coronary heart disease(BACHD)by network pharmacology.METHODS:The active ingredients of the L...OBJECTIVE:To investigate the mechanism of action of the Lingbao Huxin Dan(灵宝护心丹)in treating bradycardia arrhythmia with coronary heart disease(BACHD)by network pharmacology.METHODS:The active ingredients of the Lingbao Huxin Dan were screened on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and Bioinformatics tools designed for the analysis of molecular mechanisms of Chinese medicine platform;target prediction was conducted with the SwissTargetPrediction database,and Cytoscape 3.8 was used to construct a drug ingredient-target network.The Genecards,Online Mendelian Inheritance in Man,and DrugB ank databases were searched for disease targets.Venn plots were used to display the common targets of BA-CHD and active ingredients.The STRING platform was used to construct a protein-protein interaction network.The Metascape data platform was used for Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis to construct a signaling pathway network of the active ingredients of the Lingbao Huxin Dan.RESULTS:There were 121 active ingredients,899 related targets,39 targets important in BA-CHD and 14 targets which intersected between the active ingredients and BA-CHD.There were 27 core therapeutic ingredients,153 biological processes,18 cell ingredients and 20 molecular functions obtained by GO enrichment analysis.The KEGG pathway analysis yielded 19 signaling pathways.CONCLUSION:RBA-CHD may treat BA-CHD by regulating adrenergic receptor beta-1,alpha 1-αadrenergic receptor,calcium voltage-gated channel subunit alpha1 C,alpha-1β-adrenergic receptor,nitric oxide synthase 2,beta-2 adrenergic receptor,voltagedependent calcium channel subunit alpha-2/delta-1,angiotensin-converting enzyme,Raf-1 proto-oncogene serine/threonine-protein kinase,and other targets,potentially by affecting adrenergic receptor binding and calcium channel opening,to regulate the activity of cardiomyocytes.展开更多
Background:Genetic selection in cattle has been directed to increase milk production.This,coupled to the fact that the vast majority of bovine artificial inseminations(AI)are performed using cryopreserved sperm,have l...Background:Genetic selection in cattle has been directed to increase milk production.This,coupled to the fact that the vast majority of bovine artificial inseminations(AI)are performed using cryopreserved sperm,have led to a reduction of fertility rates over the years.Thus,seeking sensitive and specific sperm biomarkers able to predict fertility rates is of vital importance to improve cattle reproductive efficiency.In humans,sperm chromatin condensation evaluated through chromomycin A3(CMA3)has recently been purported to be a powerful biomarker for sperm functional status and male infertility.The objectives of the present study were:a)to set up a flow cytometry method for simultaneously evaluating chromatin condensation and sperm viability,and b)to test whether this parameter could be used as a predictor of in vivo fertility in bulls.The study included pools of three independent cryopreserved ejaculates per bull from 25 Holstein males.Reproductive outcomes of each sire were determined by non-return rates,which were used to classify bulls into two groups(highly fertile and subfertile).Results:Chromatin condensation status of bovine sperm was evaluated through the combination of CMA3 and Yo-Pro-1 staining and flow cytometry.Sperm quality parameters(morphology,viability,total and progressive motility)were also assessed.Pearson correlation coefficients and ROC curves were calculated to assess their capacity to predict in vivo fertility.Sperm morphology,viability and total motility presented an area under the ROC curve(AUC)of 0.54,0.64 and 0.68,respectively(P>0.05),and thus were not able to discriminate between fertile and subfertile individuals.Alternatively,while the percentage of progressively motile sperm showed a significant predictive value,with an AUC of 0.73(P=0.05),CMA3/Yo-Pro-1 staining even depicted superior results for the prediction of in vivo fertility in bulls.Specifically,the percentage of viable sperm with poor chromatin condensation showed better accuracy and precision to predict in vivo fertility,with an AUC of 0.78(P=0.02).Conclusions:Chromatin condensation evaluated through CMA3/Yo-Pro-1 and flow cytometry is defined here as a more powerful tool than conventional sperm parameters to predict bull in vivo fertility,with a potential ability to maximising the efficiency of dairy breeding industry.展开更多
Background MicroRNAs(miRNAs)are small,single-stranded,non-coding RNA molecules of 22–24 nucleotides that regulate gene expression.In the last decade,miRNAs have been described in sperm of several mammals,including ca...Background MicroRNAs(miRNAs)are small,single-stranded,non-coding RNA molecules of 22–24 nucleotides that regulate gene expression.In the last decade,miRNAs have been described in sperm of several mammals,including cattle.It is known that miRNAs can act as key gene regulators of early embryogenesis in mice and humans;however,little is known about the content,expression,and function of sperm-borne miRNAs in early bovine embryo.In this study,total sperm RNA was isolated from 29 cryopreserved sperm samples(each coming from a separate bull)using a RNeasy kit and treatment with DNase I.RNA concentration and purity were determined through an Epoch spectrophotometer and an Agilent Bioanalyzer.The expression of 10 candidate miRNAs in bovine sperm(bta-miR-10a,bta-miR-10b,bta-miR-138,bta-miR-146b,bta-miR-19b,bta-miR-26a,bta-miR-34a,bta-miR-449a,bta-miR-495 and btamiR-7),previously identified in testis and/or epididymis,was evaluated with RT-qPCR.The cel-miR-39-3p was used as a spike-in exogenous control.Nonparametric Mann–Whitney tests were run to evaluate which miRNAs were differentially expressed between bulls with high fertility[HF;non-return rates(NRR)ranging from 39.5 to 43.5]and those with subfertility(SF;NRR ranging from 33.3 to 39.3).Several sperm functionality parameters(e.g.,viability,membrane stability or oxygen consumption,among others)were measured by multiplexing flow cytometry and oxygen sensing technologies.Results RNA concentration and purity(260/280 nm ratio)(mean±SD)from the 29 samples were 99.3±84.6 ng/μL and 1.97±0.72,respectively.Bioanalyzer results confirmed the lack of RNA from somatic cells.In terms of the presence or absence of miRNAs,and after applying the Livak method,8 out of 10 miRNAs(bta-miR-10b,-138,-146b,-19b,-26a,-449a,-495,-7)were consistently detected in bovine sperm,whereas the other two(bta-miR-10a,and-34a)were absent.Interestingly,the relative expression of one miRNA(bta-miR-138)in sperm was significantly lower in the SF than in the HF group(P=0.038).In addition to being associated to fertility potential,the presence of this miRNA was found to be negatively correlated with sperm oxygen consumption.The expression of three other miRNAs(bta-miR-19b,bta-miR-26a and bta-miR-7)was also correlated with sperm function variables.Conclusions In conclusion,although functional validation studies are required to confirm these results,this study suggests that sperm bta-miR-138 is involved in fertilization events and beyond,and supports its use as a fertility biomarker in cattle.展开更多
Abnormal immune response/inflammation is present in patients of amyotrophic lateral sclerosis (ALS). Autoimmune-related inflammation has been thought to be involved in the pathogenesis of ALS. However, how the abnorma...Abnormal immune response/inflammation is present in patients of amyotrophic lateral sclerosis (ALS). Autoimmune-related inflammation has been thought to be involved in the pathogenesis of ALS. However, how the abnormal immune responses are initiated, what specific immune cells and how these immune cells are involved in this disease have not been well understood. This is partly owing to two facts of ALS: late diagnosis and chronic nature. The late diagnosis makes it difficult to conclude whether the abnormal immune responses/inflammation is the cause or result of the disease. The chronic nature makes it difficult to determine the best timing for the detection of such autoimmune responses. To resolve these two challenges for research, the authors introduced motor nerve injury (facial nerve axotomy, FNA) into a pre-symptomatic mouse ALS model (8-week-old SOD1G93A mice), which induces a readily detectable immune response in a predictable time period (3-14 days). The authors found that pre-symptomatic SOD1G93A mice showed a higher basal level of T cell activation and Th17 cells than WT mice, which can be further increased by FNA. However, why these pro-inflammatory Th lymphocyte subsets are preferentially elicited in ALS has been elusive. Recently, several studies support that the programmed necrosis (necroptosis), a new type of cell death, is present in ALS. Because necroptosis results in the release of pro-inflammatory stimuli, we speculate that initial motoneuron (MN) necroptosis may be the cause of abnormal immune responses in the development of ALS, and subsequently, inflammation/immune response serve as an amplifier to cause more MN death. Here, the authors reviewed recent studies concerning the type of MN death, the inflammation/immune responses and the research strategies for ALS. With the available evidences from the literature, the authors present a hypothesized working model to indicate the possible connections among necroptosis, immune responses and MN death in the development of ALS and suggest the future studies for searching the potential therapy for ALS.展开更多
Alzheimer’s disease(AD)is the most common form of dementia.At the present time,however,AD still lacks effective treatments.Our recent studies showed that chronic treatment with anesthetic propofol attenuated brain ca...Alzheimer’s disease(AD)is the most common form of dementia.At the present time,however,AD still lacks effective treatments.Our recent studies showed that chronic treatment with anesthetic propofol attenuated brain caspase-3 activation and improved cognitive function in aged mice.Accumulation ofβ-amyloid protein(Aβ)is a major component of the neuropathogenesis of AD dementia and cognitive impairment.We therefore set out to determine the effects of chronic treatment with propofol on Aβlevels in brain tissues of aged mice.Propofol(50 mg/kg)was administrated to aged(18 month-old)wild-type mice once a week for 8 weeks.The brain tissues of mice were harvested one day after the final propofol treatment.The harvested brain tissues were then subjected to enzyme-linked immunosorbent assay(ELISA)and Western blot analysis.Here we report that the propofol treatment reduced Aβ(Aβ40 and Aβ42)levels in the brain tissues of the aged mice.Moreover,the propofol treatment decreased the levels ofβ-site amyloid precursor protein cleaving enzyme(the enzyme for Aβgeneration),and increased the levels of neprilysin(the enzyme for Aβdegradation)in the brain tissues of the aged mice.These results suggested that the chronic treatment with propofol might reduce brain Aβlevels potentially via decreasing brain levels ofβ-site amyloid precursor protein cleaving enzyme,thus decreasing Aβgeneration;and via increasing brain neprilysin levels,thus increasing Aβdegradation.These preliminary findings from our pilot studies have established a system and postulated a new hypothesis for future research.展开更多
基金Ministry of Science,Innovation and Universities,Spain(NextGeneration EU fundsMaría Zambrano Program 124/MTAI/22+2 种基金and PID2020-113320RB-I00)Agency for Management of University and Research Grants,Regional Government of Catalonia,Spain(2021-SGR-00900)Catalan Institution for Research and Advanced Studies(ICREA).
文摘Background Sperm cryopreservation is widely used in the cattle industry,as it allows for disassociating the localiza-tion of sires and the collection of semen from the timing of artificial insemination.While freeze-thawing is known to impair sperm DNA integrity,whether the damage induced consists of single-(SSB)or double-strand breaks(DSB)has not been determined.In addition,no previous study has addressed if DNA breaks preferentially reside in specific genome regions such as those forming the toroid linker regions,or are rather spread throughout the regions linked to protamines.The main aim of the present work,therefore,was to elucidate the type and localization of the DNA damage generated by cryopreservation and to evaluate its impact on artificial insemination outcomes in cattle.Results The incidence of SSB and DSB was evaluated in 12 ejaculates before and after cryopreservation with the Comet assay,and the localization of the DNA breaks was assessed using pulsed-field gel electrophoresis(PFGE).Before cryopreservation,the incidence of SSB was 10.99%±4.62%and involved 20.56%±3.04%of sperm cells,whereas these figures significantly(P<0.0001)increased up to 34.11%±3.48%and 53.36%±11.00%in frozen-thawed sperm.In contrast,no significant differences in the incidence of DSB were observed(P>0.990)before and after cryopreservation(before:incidence of 13.91%±1.75%of sperm DNA affecting 56.04%±12.49%of sperm cells;after:incidence of 13.55%±1.55%of sperm DNA involving 53.36%±11.00%of sperm cells).Moreover,PFGE revealed that the percentage of sperm DNA fragments whose length was shorter than a toroid(<31.5 kb)was greater(P<0.0001)after(27.00%±4.26%)than before freeze-thawing(15.57%±4.53%).These differences indicated that the DNA breaks induced by cryopreservation affect the regions condensed in protamines,which are structured in toroids.On the other hand,in vivo fertility rates were associated to the incidence of SSB and DSB in frozen-thawed sperm(P=0.032 and P=0.005),but not with the size of the DNA fragments resulting from these breaks(P>0.05).Conclusion Cryopreservation of bovine sperm generates single-strand DNA breaks,which are mainly located in protamine-condensed toroidal regions.The incidence of DNA breaks in cryopreserved sperm has an impact on cat-tle fertility,regardless of the size of generated fragments.
文摘The aim of this study was to compare the sperm nuclear and acrosomal morphometry of three species of domestic artiodactyls; cattle （Bos taurus）, sheep （Ovis aries）, and pigs （Sus scrofa）. Semen smears of twenty ejaculates from each species were fixed and labeled with a propidium iodide-Pisum sativum agglutinin （PI/PSA） combination. Digital images of the sperm nucleus, acrosome, and whole sperm head were captured and analyzed. The use of the PI/PSA combination and CASA-Morph fluorescence-based method allowed the capture, morphometric analysis, and differentiation of most sperm nuclei, acrosomes and whole heads, and the assessment of acrosomal integrity with a high precision in the three species studied. For the size of the head and nuclear area, the relationship between the three species may be summarized as bull 〉 ram 〉 boar. However, for the other morphometric parameters （length, width, and perimeter）, there were differences in the relationships between species for sperm nuclei and whole sperm heads. Bull sperm acrosomes were clearly smaller than those in the other species studied and covered a smaller proportion of the sperm head. The acrosomal morphology, small in the bull, large and broad in the sheep, and large, long, and with a pronounced equatorial segment curve in the boar, was species-characteristic. It was concluded that there are clear variations in the size and shape of the sperm head components between the three species studied, the acrosome being the structure showing the most variability, allowing a clear distinction of the spermatozoa of each species.
文摘Neurovascular interactions are crucial for the normal development of the central nervous system. To study such interactions in primary cultures, we developed a procedure to simultaneously isolate neural progenitor and endothelial cell fractions from embryonic mouse brains. Depending on the culture conditions endothelial cells were found to favor maintenance of the neuroprogenitor phenotype through the production of soluble factors, or to promote neuronal differentiation of neural progenitors through direct contact. These apparently opposing effects could reflect differential cellular interactions needed for the proper development of the brain.
基金by Biomedical Laboratory Research and Development,VA Office of Research and Development,No.BX004127(to McVicker BL and Tobi M).
文摘The aberrant use of alcohol is a major factor in cancer progression and metastasis.Contributing mechanisms include the systemic effects of alcohol and the exchange of bioactive molecules between cancerous and non-cancerous cells along the brain-gut-liver axis.Such interplay leads to changes in molecular,cellular,and biological functions resulting in cancer progression.Recent investigations have examined the role of extracellular vesicles(EVs)in cancer mechanisms in addition to their contribution as diagnostic biomarkers.Also,EVs are emerging as novel cell-free mediators in pathophysiological scenarios including alcohol-mediated gut microbiome dysbiosis and the release of nanosized EVs into the circulatory system.Interestingly,EVs in cancer patients are enriched with oncogenes,miRNA,lipids,and glycoproteins whose delivery into the hepatic microenvironment may be enhanced by the detrimental effects of alcohol.Proof-of-concept studies indicate that alcohol-associated liver disease is impacted by the effects of exosomes,including altered immune responses,reprogramming of stromal cells,and remodeling of the extracellular matrix.Moreover,the culmination of alcoholrelated changes in the liver likely contributes to enhanced hepatic metastases and poor outcomes for cancer patients.This review summarizes the numerous aspects of exosome communications between organs with emphasis on the relationship of EVs in alcohol-associated diseases and cancer metastasis.The potential impact of EV cargo and release along a multi-organ axis is highly relevant to the promotion of tumorigenic mechanisms and metastatic disease.It is hypothesized that EVs target recipient tissues to initiate the formation of prometastatic niches and cancer progression.The study of alcohol-associated mechanisms in metastatic cancers is expected to reveal a better understanding of factors involved in the growth of secondary malignancies as well as novel approaches for therapeutic interventions.
基金supported by a grant from National Institute of Health(NIH)Grant No.NS040433
文摘Amyotrophic lateral sclerosis is a motoneuron degenerative disease that is challenging to diagnose and presents with considerable variability in survival.Early identification and enhanced understanding of symptomatic patterns could aid in diagnosis and provide an avenue for monitoring disease progression.Use of the m SOD1 G93 A mouse model provides control of the confounding environmental factors and genetic heterogeneity seen in amyotrophic lateral sclerosis patients,while investigating underlying disease-induced changes.In the present study,we performed a longitudinal behavioral assessment paradigm and identified an early hindlimb symptom,resembling the common gait abnormality foot drop,along with an accompanying forelimb compensatory mechanism in the m SOD1 G93 A mouse.Following these initial changes,m SOD1 mice displayed a temporary hindlimb compensatory mechanism resembling an exaggerated steppage gait.As the disease progressed,these compensatory mechanisms were not sufficient to sustain fundamental locomotor parameters and more severe deficits appeared.We next applied these initial findings to investigate the inherent variability in B6 SJL m SOD1 G93 A survival.We identified four behavioral variables that,when combined in a cluster analysis,identified two subpopulations with different disease progression rates：a fast progression group and a slow progression group.This behavioral assessment paradigm,with its analytical approaches,provides a method for monitoring disease progression and detecting m SOD1 subgroups with different disease severities.This affords researchers an opportunity to search for genetic modifiers or other factors that likely enhance or slow disease progression.Such factors are possible therapeutic targets with the potential to slow disease progression and provide insight into the underlying pathology and disease mechanisms.
基金the European Union’s Horizon 2020 Research and Innovation scheme under the Marie Sklodowska-Curie grant agreement No.801342(Tecniospring INDUSTRY,TECSPR-19-1-0003)the Ministry of Science and Innovation,Spain(AGL2017-88329-R and PID2020-113320RBI00)+2 种基金the Catalan Agency for Management of University and Research Grants,Regional Government of Catalonia,Spain(2017-SGR-1229)the Catalan Institution for Research and Advanced Studies(ICREA)La Maratóde TV3 Foundation(214/857-202039)。
文摘Background:The analysis of chromatin integrity has become an important determinant of sperm quality.In frozenthawed bovine sperm,neither the sequence of post-thaw injury events nor the dynamics of different types of sperm DNA breaks are well understood.The aim of the present work was to describe such sperm degradation aftermath focusing on DNA damage dynamics,and to assess if this parameter can predict pregnancy rates in cattle.Results:A total of 75 cryopreserved ejaculates from 25 Holstein bulls were evaluated at two post-thawing periods(0-2 h and 2-4 h),analyzing global and double-stranded DNA damage through alkaline and neutral Comet assays,chromatin deprotamination and decondensation,sperm motility,viability,acrosomal status,and intracellular levels of total ROS,superoxides and calcium.Insemination of 59,605 females was conducted using sperm from the same bulls,thus obtaining the non-return to estrus rates after 90 d(NRR).Results showed an increased rate of double-stranded breaks in the first period(0-2 h:1.29±1.01%/h vs.2-4 h:0.13±1.37%/h;P<0.01),whereas the rate of sperm with moderate+high single-stranded breaks was higher in the second period(0-2 h:3.52±7.77%/h vs.2-4h:21.06±11.69%/h;P<0.0001).Regarding sperm physiology,viability decrease rate was different between the two periods(0-2 h:-4.49±1.79%/h vs.2-4 h:-2.50±3.39%/h;P=0.032),but the progressive motility decrease rate was constant throughout post-thawing incubation(0-2 h:-4.70±3.42%/h vs.2-4 h:-1.89±2.97%/h;P>0.05).Finally,whereas no correlations between bull fertility and any dynamic parameter were found,there were correlations between the NRR and the basal percentage of highly-damaged sperm assessed with the alkaline Comet(Rs=-0.563,P=0.003),between NRR and basal progressive motility(Rs=0.511,P=0.009),and between NRR and sperm with high ROS at 4 h post-thaw(Rs=0.564,P=0.003).Conclusion:The statistically significant correlations found between intracellular ROS,sperm viability,sperm motility,DNA damage and chromatin deprotamination suggested a sequence of events all driven by oxidative stress,where viability and motility would be affected first and sperm chromatin would be altered at a later stage,thus suggesting that bovine sperm should be used for fertilization within 2 h post-thaw.Fertility correlations supported that the assessment of global DNA damage through the Comet assay may help predict bull fertility.
文摘The distribution, diversity, and abundance of sea cucumbers in intertidal zones of Aurora were studied from February to June 2013 to obtain baseline data. A one-hour timed-search survey covering an approximate one kilometer over a ten-meter wide area was conducted in six coastal municipalities to obtain a comprehensive list of sea cucumber species found in the zones. Only four sampling stations (Baler, Dipaculao, Dilasag, and Dingalan) were selected for belt transect survey, 50 × 50 m, based on the availability of coralline, sandy, muddy sand, and rocky habitats which were laid perpendicular and parallel to each intertidal zone. Timed-search survey revealed a total of 15 species of sea cucumbers distributed among two Orders (Order Aspidochirotida and Order Apodida) and four Families (Family Holothuriidae, Family Stichopodidae, Family Synaptidae, and Family Chirotidae) were recorded. Synapta maculata is most common and has widespread distribution. Belt-tranect surveys showed Holothuria leucospilota is among the most frequently occurring species both during day time (58%) and night time (75%). The Dilasag sampling station had the most diverse species (0.71) while the Dipaculao sampling station had the densest species of Holothuria leucospilota (10,014 in<span style="white-space:nowrap;">∙ha<sup><span style="white-space:nowrap;">−1</sup>) and abundant (95.91%) species.
文摘The diversity and abundance of bivalves and gastropods were studied from April to July 2015. Two sampling stations, Tinib and Esteves, in Casiguran, Aurora were selected based on the presence of mangrove forest, bivalves, gastropods, muddy substrate, and gleaners for the establishment of a transect line. Each station was laid with 100 meters transect line overlaid continuously with 2 m × 2 m quadrat along each transect. A total of 50 sampling units covering 0.02 ha area per sampling station were surveyed. The transect line recorded six species of bivalves distributed among five families and 12 species of gastropods belonging to eight families. Three species of bivalves and five species of gastropods were common to both sampling stations. However, Tinib station had most diverse species of bivalves (0.55) while Esteves station had most diverse species of gastropods (0.43). Both stations revealed Dendrostrea folium (43%) (bivalve) and Cerithidea cingulata (93%) (gastropods) as most frequently occurring species. Dendostrea folium also emerged as the densest (10,640 ind<span style="white-space:nowrap;">∙ha<sup><span style="white-space:nowrap;">−1</sup> and 6850 ind<span style="white-space:nowrap;">∙ha<sup><span style="white-space:nowrap;">−1</sup>) and most abundant (58.52% and 77.84%) bivalve species. For gastropods, Terebralia sulcata (249,250 ind<span style="white-space:nowrap;">∙ha<sup><span style="white-space:nowrap;">−1</sup>) was the densest and most abundant (96.97%) in Tinib while Terebralia palustris was the densest (747,050 ind<span style="white-space:nowrap;">∙ha<sup><span style="white-space:nowrap;">−1</sup>) and most abundant (70.44%) in Esteves. Pooled results for bivalve species maintained Dendrostrea folium as the densest (8750 ind<span style="white-space:nowrap;">∙ha<sup><span style="white-space:nowrap;">−1</sup>) and most abundant (64.81%). However, for gastropods, Terebralia palustris emerged as the densest (373,525 ind<span style="white-space:nowrap;">∙ha<sup><span style="white-space:nowrap;">−1</sup>) and most abundant (56.70%) species. The mangrove forests of Casiguran, Aurora thus contain a variety of bivalves and gastropods with Tinib station dominated by bivalves while Esteves station dominated by gastropods.
基金funded by the INCO-DC,International Cooperation with Developing Coun-tries Contract#ERBIC18CT-98319 entitled“Harnessing mycorrhizal symbiosis in mixed Acacia mangium and dipterocarp plantations in Malaysia and in the Philip-pines”,National Institute of Molecular Biology and Bio-technology,University of the Philippines Los Banos and the Department of Environment and Natural Resources,Caraga Region,Butuan City,Mindanao island,Philip-pines.
文摘This study was conducted to determine the compatibility of ectomycorrhizal (ECM) fungi associated with eucalypts on dipterocarps. Two nodal cuttings of Shorea contorta (Vid.) were rooted in a non-mist system for two months, and later inoculated with vegetative mycelia of three strains of Pisolithus collected under eucalypts and a strain of Scleroderma from dipterocarps. Inoculated rooted cuttings were planted in irradiated potting mix and raised in the nursery for five months. Root colonization prior to outplanting ranged from 31% to 38% and ECM fungi did not colonize the uninoculated ones. The cuttings were outplanted in a logged-over dipterocarp forest in Bislig, Surigao Sur following a Randomized Complete Block Design with four blocks;each block was planted with 50 seedlings. Two years after outplanting, Pisolithus strain from New Caledonia (H6394) promoted the highest height (46 cm) and diameter (0.48 cm) increment. Height was increased by 17% and diameter by 7% relative to the uninoculated control. By contrast, Pisolithus strain from the Philippines (H615) gave the shortest (26 cm) height increment and smallest (0.42 cm) diameter. Diameter growth of cuttings inoculated with Scleroderma D01 (from the Philippines) and a Pisolithus strain from Australia (H445) was bigger (0.47 cm) than the uninoculated treated cuttings (0.45 cm). In terms of survival, the uninoculated cuttings gave the highest (90%) percent survival while the lowest (60%) was those inoculated with Pisolithus strain from New Caledonia. It is possible that the indigenous ECM fungi infected easily the roots of the uninoculated cuttings thus contributing to the high survival rate (90%). During outplanting, fruit bodies of S. columnare were present in the field site. The results, however, show that ECM fungi were able to colonize the roots and that some strains promoted the growth and survival of S. contorta planted in a logged-over dipterocarp forest in Bislig, Surigao, Philippines.
基金Supported by the National Natural Sciences Foundation of China(No.81470171)Basic Science Research Fund from Ministry of Finance of China(No.ZZ2012008 and ZZ2015014)
文摘OBJECTIVE: To investigate the neural differentiation capacity of water extraction of velvet antler.METHODS: Velvet antler(Cervus Nippon Temminck) polypeptide(VAP) was used to differentiate neural stem cells(NSCs) towards neurons in the study. Firstly, we obtain the polypeptides of VAP by water extraction. Secondly, we observed the morphology, assayed the factors in the media by enzyme-linked immunosorbent assay, and detected the special neural molecules by immuno fl uorescence staining. NSCs were cultured on the cell climbing film. After neuronal differentiation, differentiated NSCs were mounted for immunocytochemistry with immunofluorescence technique.RESULTS: The differentiating cells look like neuron,some special factors, such as Glial cell line-derived neurotrophic factor, nerve growth factor, in the media can be detected while differentiated neuron-like cells can express the special neural molecules.CONCLUSION: Differentiation of NSCs towards neurons can be induced by velvet antler polypeptide.
基金Training Project of National Administration of Traditional Chinese Medicine“Natural Training Project of Backbone of Traditional Chinese Medicine”(SATCM,No.2019-128)the Fundamental Research Funds for the Central Public Welfare Research Institutes:the Application of Panoramic Tissue Scanning and Quantitative Analysis Systems in the Research of Traditional Chinese Medicine for Disease Prevention and Treatment”(No.JBGS2021005)。
文摘OBJECTIVE:To investigate the mechanism of action of the Lingbao Huxin Dan(灵宝护心丹)in treating bradycardia arrhythmia with coronary heart disease(BACHD)by network pharmacology.METHODS:The active ingredients of the Lingbao Huxin Dan were screened on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and Bioinformatics tools designed for the analysis of molecular mechanisms of Chinese medicine platform;target prediction was conducted with the SwissTargetPrediction database,and Cytoscape 3.8 was used to construct a drug ingredient-target network.The Genecards,Online Mendelian Inheritance in Man,and DrugB ank databases were searched for disease targets.Venn plots were used to display the common targets of BA-CHD and active ingredients.The STRING platform was used to construct a protein-protein interaction network.The Metascape data platform was used for Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis to construct a signaling pathway network of the active ingredients of the Lingbao Huxin Dan.RESULTS:There were 121 active ingredients,899 related targets,39 targets important in BA-CHD and 14 targets which intersected between the active ingredients and BA-CHD.There were 27 core therapeutic ingredients,153 biological processes,18 cell ingredients and 20 molecular functions obtained by GO enrichment analysis.The KEGG pathway analysis yielded 19 signaling pathways.CONCLUSION:RBA-CHD may treat BA-CHD by regulating adrenergic receptor beta-1,alpha 1-αadrenergic receptor,calcium voltage-gated channel subunit alpha1 C,alpha-1β-adrenergic receptor,nitric oxide synthase 2,beta-2 adrenergic receptor,voltagedependent calcium channel subunit alpha-2/delta-1,angiotensin-converting enzyme,Raf-1 proto-oncogene serine/threonine-protein kinase,and other targets,potentially by affecting adrenergic receptor binding and calcium channel opening,to regulate the activity of cardiomyocytes.
基金the support from the Ministry of ScienceInnovation and Universities,Spain (AGL2017–88329-R and FPU18/00666)+1 种基金Regional Government of Catalonia,Spain (2017-SGR-1229)University of Girona (Postdoc-Ud G2020)。
文摘Background:Genetic selection in cattle has been directed to increase milk production.This,coupled to the fact that the vast majority of bovine artificial inseminations(AI)are performed using cryopreserved sperm,have led to a reduction of fertility rates over the years.Thus,seeking sensitive and specific sperm biomarkers able to predict fertility rates is of vital importance to improve cattle reproductive efficiency.In humans,sperm chromatin condensation evaluated through chromomycin A3(CMA3)has recently been purported to be a powerful biomarker for sperm functional status and male infertility.The objectives of the present study were:a)to set up a flow cytometry method for simultaneously evaluating chromatin condensation and sperm viability,and b)to test whether this parameter could be used as a predictor of in vivo fertility in bulls.The study included pools of three independent cryopreserved ejaculates per bull from 25 Holstein males.Reproductive outcomes of each sire were determined by non-return rates,which were used to classify bulls into two groups(highly fertile and subfertile).Results:Chromatin condensation status of bovine sperm was evaluated through the combination of CMA3 and Yo-Pro-1 staining and flow cytometry.Sperm quality parameters(morphology,viability,total and progressive motility)were also assessed.Pearson correlation coefficients and ROC curves were calculated to assess their capacity to predict in vivo fertility.Sperm morphology,viability and total motility presented an area under the ROC curve(AUC)of 0.54,0.64 and 0.68,respectively(P>0.05),and thus were not able to discriminate between fertile and subfertile individuals.Alternatively,while the percentage of progressively motile sperm showed a significant predictive value,with an AUC of 0.73(P=0.05),CMA3/Yo-Pro-1 staining even depicted superior results for the prediction of in vivo fertility in bulls.Specifically,the percentage of viable sperm with poor chromatin condensation showed better accuracy and precision to predict in vivo fertility,with an AUC of 0.78(P=0.02).Conclusions:Chromatin condensation evaluated through CMA3/Yo-Pro-1 and flow cytometry is defined here as a more powerful tool than conventional sperm parameters to predict bull in vivo fertility,with a potential ability to maximising the efficiency of dairy breeding industry.
基金the Ministry of Science and Innovation,Spain(IJC2019-039615-I and PID2020-113320RB-I00)the European Union’s Horizon 2020 research and innovation scheme under the Marie Sklodowska-Curie grant agreement No.801342(Techniospring INDUSTRY+2 种基金TECSPR-19-1-0003)the Regional Government of Catalonia(2017-SGR-1229 and 2021-SGR-00900)the Catalan Institution for Research and Advanced Studies(ICREA).
文摘Background MicroRNAs(miRNAs)are small,single-stranded,non-coding RNA molecules of 22–24 nucleotides that regulate gene expression.In the last decade,miRNAs have been described in sperm of several mammals,including cattle.It is known that miRNAs can act as key gene regulators of early embryogenesis in mice and humans;however,little is known about the content,expression,and function of sperm-borne miRNAs in early bovine embryo.In this study,total sperm RNA was isolated from 29 cryopreserved sperm samples(each coming from a separate bull)using a RNeasy kit and treatment with DNase I.RNA concentration and purity were determined through an Epoch spectrophotometer and an Agilent Bioanalyzer.The expression of 10 candidate miRNAs in bovine sperm(bta-miR-10a,bta-miR-10b,bta-miR-138,bta-miR-146b,bta-miR-19b,bta-miR-26a,bta-miR-34a,bta-miR-449a,bta-miR-495 and btamiR-7),previously identified in testis and/or epididymis,was evaluated with RT-qPCR.The cel-miR-39-3p was used as a spike-in exogenous control.Nonparametric Mann–Whitney tests were run to evaluate which miRNAs were differentially expressed between bulls with high fertility[HF;non-return rates(NRR)ranging from 39.5 to 43.5]and those with subfertility(SF;NRR ranging from 33.3 to 39.3).Several sperm functionality parameters(e.g.,viability,membrane stability or oxygen consumption,among others)were measured by multiplexing flow cytometry and oxygen sensing technologies.Results RNA concentration and purity(260/280 nm ratio)(mean±SD)from the 29 samples were 99.3±84.6 ng/μL and 1.97±0.72,respectively.Bioanalyzer results confirmed the lack of RNA from somatic cells.In terms of the presence or absence of miRNAs,and after applying the Livak method,8 out of 10 miRNAs(bta-miR-10b,-138,-146b,-19b,-26a,-449a,-495,-7)were consistently detected in bovine sperm,whereas the other two(bta-miR-10a,and-34a)were absent.Interestingly,the relative expression of one miRNA(bta-miR-138)in sperm was significantly lower in the SF than in the HF group(P=0.038).In addition to being associated to fertility potential,the presence of this miRNA was found to be negatively correlated with sperm oxygen consumption.The expression of three other miRNAs(bta-miR-19b,bta-miR-26a and bta-miR-7)was also correlated with sperm function variables.Conclusions In conclusion,although functional validation studies are required to confirm these results,this study suggests that sperm bta-miR-138 is involved in fertilization events and beyond,and supports its use as a fertility biomarker in cattle.
文摘Abnormal immune response/inflammation is present in patients of amyotrophic lateral sclerosis (ALS). Autoimmune-related inflammation has been thought to be involved in the pathogenesis of ALS. However, how the abnormal immune responses are initiated, what specific immune cells and how these immune cells are involved in this disease have not been well understood. This is partly owing to two facts of ALS: late diagnosis and chronic nature. The late diagnosis makes it difficult to conclude whether the abnormal immune responses/inflammation is the cause or result of the disease. The chronic nature makes it difficult to determine the best timing for the detection of such autoimmune responses. To resolve these two challenges for research, the authors introduced motor nerve injury (facial nerve axotomy, FNA) into a pre-symptomatic mouse ALS model (8-week-old SOD1G93A mice), which induces a readily detectable immune response in a predictable time period (3-14 days). The authors found that pre-symptomatic SOD1G93A mice showed a higher basal level of T cell activation and Th17 cells than WT mice, which can be further increased by FNA. However, why these pro-inflammatory Th lymphocyte subsets are preferentially elicited in ALS has been elusive. Recently, several studies support that the programmed necrosis (necroptosis), a new type of cell death, is present in ALS. Because necroptosis results in the release of pro-inflammatory stimuli, we speculate that initial motoneuron (MN) necroptosis may be the cause of abnormal immune responses in the development of ALS, and subsequently, inflammation/immune response serve as an amplifier to cause more MN death. Here, the authors reviewed recent studies concerning the type of MN death, the inflammation/immune responses and the research strategies for ALS. With the available evidences from the literature, the authors present a hypothesized working model to indicate the possible connections among necroptosis, immune responses and MN death in the development of ALS and suggest the future studies for searching the potential therapy for ALS.
基金This research was supported by R21AG038994,RO1 GM088801 and RO1AG041274 from National lnstitutes of Health,Bethesda,Maryland,Investigator-initiated Research grant from Alzheimer's Association,Chicago,llinois,and Cure Alzheimer's Fund,Wellesley,Massachusetts to Zhongcong Xie.
文摘Alzheimer’s disease(AD)is the most common form of dementia.At the present time,however,AD still lacks effective treatments.Our recent studies showed that chronic treatment with anesthetic propofol attenuated brain caspase-3 activation and improved cognitive function in aged mice.Accumulation ofβ-amyloid protein(Aβ)is a major component of the neuropathogenesis of AD dementia and cognitive impairment.We therefore set out to determine the effects of chronic treatment with propofol on Aβlevels in brain tissues of aged mice.Propofol(50 mg/kg)was administrated to aged(18 month-old)wild-type mice once a week for 8 weeks.The brain tissues of mice were harvested one day after the final propofol treatment.The harvested brain tissues were then subjected to enzyme-linked immunosorbent assay(ELISA)and Western blot analysis.Here we report that the propofol treatment reduced Aβ(Aβ40 and Aβ42)levels in the brain tissues of the aged mice.Moreover,the propofol treatment decreased the levels ofβ-site amyloid precursor protein cleaving enzyme(the enzyme for Aβgeneration),and increased the levels of neprilysin(the enzyme for Aβdegradation)in the brain tissues of the aged mice.These results suggested that the chronic treatment with propofol might reduce brain Aβlevels potentially via decreasing brain levels ofβ-site amyloid precursor protein cleaving enzyme,thus decreasing Aβgeneration;and via increasing brain neprilysin levels,thus increasing Aβdegradation.These preliminary findings from our pilot studies have established a system and postulated a new hypothesis for future research.
