Expressional Profiling of TEX11, ESRα and BOLL Genes in Yak under Different Feeding Conditions

Previous studies have demonstrated that nutrition plays a crucial part in improving the reproductive potential of farm animals; however, there is currently no research on the transcription and expression profiling of genes in yaks under different feeding conditions. Therefore, this research was planned to compare the transcription and expression profiles of TEX11, ESRα, and BOLL in yaks under natural grazing with concentrate supplementation (NG + CS) and NG without concentrate supplementation. The transcription and expressional levels of TEX11, ESRα, and BOLL mRNA were explored from the testes of yaks using qPCR, Western blotting, immunofluorescence, and immunochemistry. The results of the qPCR illustrated that the transcription levels of TEX11, ESRα, and BOLL were upregulated in the NG + CS group compared to those in the NG group. Moreover, the results of the immunochemistry and immunofluorescence showed that the expression of TEX11, ESRα, and BOLL proteins increased after concentrate supplementation. Meanwhile, ESRα protein levels were lower in the testes and epididymides of yaks in the NG group than in those in the NG + CS group. Similarly, BOLL protein expression was higher in the testes and epididymides of the NG + CS group, but its expression was lower in the epididymides of the NG group. Furthermore, Western blotting showed that the molecular weights of ESRα and BOLL proteins were 64 kDa and 31 kDa, respectively. Finally, in the conclusion we summarize how a proper level of dietary energy supplementation can improve the reproductive potential of yaks by upregulating genes related to reproduction.

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Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2

Viruses ◽

10.3390/v13020308 ◽

2021 ◽

Vol 13 (2) ◽

pp. 308

Author(s):

Ying-Ray Lee ◽

Chia-Ming Chang ◽

Yuan-Chieh Yeh ◽

Chi-Ying F. Huang ◽

Feng-Mao Lin ◽

...

Keyword(s):

Protein Expression ◽

Western Blotting ◽

Expression Profiles ◽

Plaque Assay ◽

Virus Titer ◽

Enterovirus 71 ◽

Luciferase Reporter ◽

Pathogen Invasion

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

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Comparison of the sensitivity of Western blotting between PVDF and NC membranes

Scientific Reports ◽

10.1038/s41598-021-91521-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yufang Xiang ◽

Yuanyuan Zheng ◽

Shaobo Liu ◽

Gang Liu ◽

Zhi Li ◽

...

Keyword(s):

Western Blotting ◽

Polyvinylidene Fluoride ◽

Staining Intensity ◽

Detection Sensitivity ◽

Key Factors ◽

Post Translational Modifications ◽

Factors Affecting ◽

Molecular Weights ◽

Direct Blue ◽

The Relationship

AbstractWestern blotting (WB) is one of the most widely used techniques to identify proteins as well as post translational modifications of proteins. The selection of electroblotted membrane is one of the key factors affecting the detection sensitivity of the protein which is transferred from gel to membrane in WB. The most common used membranes are polyvinylidene fluoride (PVDF) and nitrocellulose (NC) membranes. Which membrane of these two is more suitable for WB has not been reported so far. Here, by incubating proteins which were transferred to PVDF or NC membranes with a series of antibodies and different types of lectins, we investigated the relationship between the binding ability of these two membranes to proteins or glycoproteins and the molecular weight of the target protein. The antibody re-probed ability of the two membranes was also explored. Moreover, we verified the above results by directly incubating proteins having different molecular weights onto PVDF or NC membranes. Bound proteins were stained with direct blue-71, and the staining intensity was quantitated by scanning and densitometry.

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Silencing of Vangl2 attenuates the inflammation promoted by Wnt5a via MAPK and NF-κB pathway in chondrocytes

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-021-02268-x ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Ke Zhang ◽

Zhuoying Li ◽

Yunyang Lu ◽

Linyi Xiang ◽

Jiadong Sun ◽

...

Keyword(s):

Western Blotting ◽

Planar Cell Polarity ◽

Type Ii Collagen ◽

Mapk Signaling ◽

Dependent Manner ◽

Inflammatory Microenvironment ◽

Functional Roles ◽

Protein Levels ◽

Oa Chondrocytes

Abstract Background The Wnt planar cell polarity (PCP) pathway is implicated in osteoarthritis (OA) both in animals and in humans. Van Gogh-like 2 (Vangl2) is a key PCP protein that is required for the orientation and alignment of chondrocytes in the growth plate. However, its functional roles in OA still remain undefined. Here, we explored the effects of Vangl2 on OA chondrocyte in vitro and further elucidated the molecular mechanism of silencing Vangl2 in Wnt5a-overexpressing OA chondrocytes. Methods Chondrocytes were treated with IL-1β (10 ng/mL) to simulate the inflammatory microenvironment of OA. The expression levels of Vangl2, Wnt5a, MMPs, and related proinflammatory cytokines were measured by RT-qPCR. Small interfering RNA (siRNA) of Vangl2 and the plasmid targeting Wnt5a were constructed and transfected into ATDC5 cells. Then, the functional roles of silencing Vangl2 in the OA chondrocytes were investigated by Western blotting, RT-qPCR, and immunocytochemistry (ICC). Transfected OA chondrocytes were subjected to Western blotting to analyze the relationship between Vangl2 and related signaling pathways. Results IL-1β induced the production of Vangl2, Wnt5a, and MMPs in a time-dependent manner and the significantly increased expression of Vangl2. Vangl2 silencing effectively suppressed the expression of MMP3, MMP9, MMP13, and IL-6 at both gene and protein levels and upregulated the expression of type II collagen and aggrecan. Moreover, knockdown of Vangl2 inhibited the phosphorylation of MAPK signaling molecules (P38, ERK, and JNK) and P65 in Wnt5a-overexpressing OA chondrocytes. Conclusions For the first time, we demonstrate that Vangl2 is involved in the OA process. Vangl2 silencing can notably alleviate OA progression in vitro by inhibiting the expression of MMPs and increasing the formation of the cartilage matrix and can inhibit the proinflammatory effects of Wnt5a via MAPK and NF-κB pathway. This study provides new insight into the mechanism of cartilage inflammation.

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Effects of temperature on proliferation of myoblasts from donor piglets with different thermoregulatory maturities

BMC Molecular and Cell Biology ◽

10.1186/s12860-021-00376-4 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Katharina Metzger ◽

Dirk Dannenberger ◽

Armin Tuchscherer ◽

Siriluck Ponsuksili ◽

Claudia Kalbe

Keyword(s):

Muscle Development ◽

Muscle Growth ◽

Extreme Temperature ◽

Growth Inhibitor ◽

Muscle Cells ◽

The Body ◽

Farm Animals ◽

Neonatal Piglets ◽

Protein Levels

Abstract Background Climate change and the associated risk for the occurrence of extreme temperature events or permanent changes in ambient temperature are important in the husbandry of farm animals. The aim of our study was to investigate the effects of permanent cultivation temperatures below (35 °C) and above (39 °C, 41 °C) the standard cultivation temperature (37 °C) on porcine muscle development. Therefore, we used our porcine primary muscle cell culture derived from satellite cells as an in vitro model. Neonatal piglets have limited thermoregulatory stability, and several days after birth are required to maintain their body temperature. To consider this developmental step, we used myoblasts originating from thermolabile (five days of age) and thermostable piglets (twenty days of age). Results The efficiency of myoblast proliferation using real-time monitoring via electrical impedance was comparable at all temperatures with no difference in the cell index, slope or doubling time. Both temperatures of 37 °C and 39 °C led to similar biochemical growth properties and cell viability. Only differences in the mRNA expression of myogenesis-associated genes were found at 39 °C compared to 37 °C with less MYF5, MYOD and MSTN and more MYH3 mRNA. Myoblasts grown at 35 °C are smaller, exhibit higher DNA synthesis and express higher amounts of the satellite cell marker PAX7, muscle growth inhibitor MSTN and metabolic coactivator PPARGC1A. Only permanent cultivation at 41 °C resulted in higher HSP expression at the mRNA and protein levels. Interactions between the temperature and donor age showed that MYOD, MYOG, MYH3 and SMPX mRNAs were temperature-dependently expressed in myoblasts of thermolabile but not thermostable piglets. Conclusions We conclude that 37 °C to 39 °C is the best physiological temperature range for adequate porcine myoblast development. Corresponding to the body temperatures of piglets, it is therefore possible to culture primary muscle cells at 39 °C. Only the highest temperature of 41 °C acts as a thermal stressor for myoblasts with increased HSP expression, but it also accelerates myogenic development. Cultivation at 35 °C, however, leads to less differentiated myoblasts with distinct thermogenetic activity. The adaptive behavior of derived primary muscle cells to different cultivation temperatures seems to be determined by the thermoregulatory stability of the donor piglets.

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Using different levels of energy and protein and their effects on bodyweight and blood chemistry of ostriches

Veterinary Research Communications ◽

10.1007/s11259-021-09792-5 ◽

2021 ◽

Author(s):

Tahereh Nikravesh-Masouleh ◽

Alireza Seidavi ◽

Magdalena Solka ◽

Mohammad Dadashbeiki

Keyword(s):

Weight Gain ◽

Total Cholesterol ◽

Feed Intake ◽

Experimental Period ◽

Blood Chemistry ◽

Blood Parameters ◽

Bodyweight Gain ◽

Transferase Activity ◽

Dietary Energy ◽

Protein Levels

AbstractTo determine the effect of different dietary energy and protein levels on bodyweight and blood chemistry, 36 ostriches at 2 to 9 weeks of age for feeding conditions and 18 for blood chemistry parameters was used. The birds were divided into six treatment groups. Energy and protein levels of diet were 2400 and 2600 kcal/kg and 20%, 22%, and 24%, respectively. The feed intake and bodyweight gain were determined a weekly. Blood chemical parameters including glucose, HDL, LDL, total cholesterol, triglycerides, total protein, albumin, globulin, aspartate amino-transferase and alanine amino-transferase activity were determined. The highest weight gain during the whole experiment was observed in ostriches offered 2400 kcal · kg−1 dietary energy and 20% protein. The lowest level of total cholesterol and protein was observed in treatment V (2600 kcal · kg−1 dietary energy and 22% protein). The lowest level of glucose and triglycerides was noted after treatment I. The highest albumin and globulin concentrations were in treatment III (2400 kcal · kg−1 dietary energy and 24% protein) and treatment II (2400 kcal · kg−1 dietary energy and 22% protein), respectively. The energy level had no effect (P < 0.05) on feed intake and weight gain in all experimental period. The results of this study showed that with increasing energy and protein levels, most blood parameters increased in ostriches but total cholesterol did not.

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Ghrelin Gene Expression in Broiler Proventriculus Tissue are Changed by Feed Restriction, Different Dietary Energy and Protein Levels

American Journal of Animal and Veterinary Sciences ◽

10.3844/ajavsp.2010.175.179 ◽

2010 ◽

Vol 5 (3) ◽

pp. 175-179 ◽

Cited By ~ 1

Author(s):

Shokoufe Ghazanfari ◽

Mohammad Reza Nassiry ◽

Mojtaba Tahmooresp ◽

Abdolreza Salehi ◽

Karim Nobari

Keyword(s):

Gene Expression ◽

Feed Restriction ◽

Dietary Energy ◽

Protein Levels ◽

Ghrelin Gene

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Abstract 3653: Akt3 Offers Stronger Protection than Akt1 Against Brain Injury by Differentially Promoting Mtor Protein Levels In Both In Vitro and In Vivo Stroke Models

Stroke ◽

10.1161/str.43.suppl_1.a3653 ◽

2012 ◽

Vol 43 (suppl_1) ◽

Author(s):

Rong Xie ◽

Michelle Cheng ◽

Mei Li ◽

Robert Sapolsky ◽

Heng Zhao

Keyword(s):

Gene Transfer ◽

Western Blotting ◽

Ischemic Injury ◽

Akt Pathway ◽

Over Expression ◽

Protein Levels ◽

Akt Isoforms ◽

The Brain

Background and Objective: Akt is a serine-threonine kinase that plays critical role in promoting cell survival. Akt consists of three isoforms (Akt1, 2, 3), with Akt3 predominantly expressed in the brain. Although Akt pathway has been shown to mediate neuronal survival in cerebral ischemic injury, it is unclear how these Akt isoforms contribute to neuronal protection, and whether exogenous Akt can protect the brain against ischemic injury or not. In this study, we over-expressed Akt isoforms and its downstream signaling proteins such as FKHR and PRAS40 to investigate the role of the Akt pathway along with its potential relationship with the mTOR pathway in stroke. Methods: Sprauge Dawley rats (250∼280g) were used for all studies. A lentiviral vector consists of a CMV promoter driving IRES-eGFP was used to clone an active Akt 1 and 3 (cAKt 1 and 3), dominant-negative Akt (AktDN), active FKHR (AAA FKHR), and PRAS40. Lentivirus expressing these genes were added to primary mixed cortical cultures for two days prior to oxygen glucose deprivation (OGD) (MOI=1:5). Neuronal survival was measured by LDH release. Lentivirus were stereotaxically injected into the cortex, and rats were subjected to focal ischemia induced by distal MCA occlusion combined with bilateral CCA occlusion. Western blotting and immunofluorescent confocal microscopy were used to detect the expression of Akt isoforms and other proteins in both the Akt and mTOR pathways. Results: Western blotting analysis showed that both endogenous Akt1 and 3 proteins degraded as early as 1 h after stroke, while Akt2 protein remained unchanged until 24 h after stroke. In vitro studies showed that over-expression of both constitutively active cAkt1 and cAkt3 decreased LDH release after OGD, while AktDN worsened neuronal death ( P <0.05). In vivo over-expression of cAkt1, cAkt3 and PRAS40 reduced infarct size after stroke ( P <0.01). Gene transfer of cAkt1 and 3 also promoted protein levels of pAkt (phosphorylated Akt), pPRAS40, pFKHR, pPTEN, pmTOR, but not pGSK3β. Both in vitro and in vivo studies showed that over-expression of cAkt3 resulted in a stronger protection than cAkt1 ( P <0.05). Interestingly, cAkt3 gene transfer preserved both endogenous protein levels of Akt1 and 3, whereas cAkt1 gene transfer only preserved endogenous Akt1. Furthermore, cAkt3 promoted higher pmTOR levels than cAkt1. Treatment of rapamycin, an mTOR inhibitor, blocked the protective effects of both cAkt1 and cAkt3 both in vitro and in vivo. Conclusion: Lentiviral-mediated overexpression of cAkt3 confers stronger protection than that of cAkt1, by maintaining both endogenous Akt1 and Akt3, as well as promoting higher mTOR activities after stroke.

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Abstract 3126: Ischemic Postconditioning Facilitates Brain Recovery After Stroke by Promoting mTOR Activity

Stroke ◽

10.1161/str.43.suppl_1.a3126 ◽

2012 ◽

Vol 43 (suppl_1) ◽

Author(s):

Peng Wang ◽

Rong Xie ◽

Xunming Ji ◽

Heng Zhao

Keyword(s):

Western Blotting ◽

Brain Plasticity ◽

Focal Ischemia ◽

Mtor Pathway ◽

Synaptic Function ◽

Ischemic Postconditioning ◽

Protein Levels ◽

Left Lateral Ventricle ◽

Brain Recovery ◽

Mtor Activity

Introduction: Ischemic postconditioning represents a series of brief occlusions of blood flow before complete restoration of reperfusion after brain stroke. We have shown that ischemic postconditioning reduces infarct size in focal ischemia in rats and improves neurological deficits, but the underlying protective mechanisms are not fully understood. The mammalian target of rapamycin (mTOR) pathway plays a key role in cell growth, differentiation and survival. We studied the hypothesis that the mTOR pathway is involved in the protective effect of ischemic postconditioning and brain recovery after stroke in rats. Methods: Focal ischemia was induced by 30 min of bilateral CCA occlusion and permanent distal MCA occlusion in rats. Ischemic postconditioning was induced by 3 cycles of 30 sec reperfusion and 10 sec occlusion at the end of stroke. Rapamycin, an mTOR inhibitor, was injected into the left lateral ventricle 1 hour before stroke onset. For the behavior test, home cage and vibrissa-elicited limb use tests were used and performed until 21d after stroke. Peri-infarct tissues were collected 1 and 3 weeks after stroke for Western blotting and immunostaining. Molecular markers related with synaptic transmission and neuronal growth and plasticity, including Gap-43, synaptophysin, MAP-2 and PSD-95 proteins were detected. Protein levels of phosphorylated mTOR (p-MTOR) and 4EPB-1 in the mTOR pathway, and the upstream molecule, Akt, were also measured. Results: Ischemic postconditioning improved neurological function when measured 2 weeks after stroke (n=6, p<0.05), and reduced brain injury size by 34.2% (P<0.05). These protective effects were abolished by rapamycin treatment. The results of Western blotting showed that postconditioning substantially promoted the protein level of Gap-43, MAP-2 and PSD-95, but not synaptophysin. Rapamycin significantly inhibited Gap-43 levels at 1 and 3 weeks after stroke, and inhibited Map-2 level at 1 week (P<0.05). Postconditioning significantly increased the protein levels of p-Akt, p-mTOR, p-4EBP-1 compared with control ischemia (p<0.05) at 1 week after stroke injury. Rapamycin attenuated p-mTOR levels 1 and 3 weeks after stroke, and inhibited p-4EBP-1 level at 1 week (p<0.05), but had no effects on the expression level of p-Akt and Akt. Conclusion: Ischemic postconditioning improved brain function, which is consistent with the improved expression of proteins related with synaptic function and brain plasticity. This protection appears to be achieved by the enhanced mTOR activity.

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RNA Sequencing in Comparison to Immunohistochemistry for Measuring Cancer Biomarkers in Breast Cancer and Lung Cancer Specimens

Biomedicines ◽

10.3390/biomedicines8050114 ◽

2020 ◽

Vol 8 (5) ◽

pp. 114

Author(s):

Maxim Sorokin ◽

Kirill Ignatev ◽

Elena Poddubskaya ◽

Uliana Vladimirova ◽

Nurshat Gaifullin ◽

...

Keyword(s):

Breast Cancer ◽

Lung Cancer ◽

Rna Sequencing ◽

Expression Profiles ◽

Correlation Study ◽

Cancer Tissue ◽

Transcriptional Level ◽

Tissue Samples ◽

Protein Levels ◽

Formalin Fixed Paraffin Embedded

RNA sequencing is considered the gold standard for high-throughput profiling of gene expression at the transcriptional level. Its increasing importance in cancer research and molecular diagnostics is reflected in the growing number of its mentions in scientific literature and clinical trial reports. However, the use of different reagents and protocols for RNA sequencing often produces incompatible results. Recently, we published the Oncobox Atlas of RNA sequencing profiles for normal human tissues obtained from healthy donors killed in road accidents. This is a database of molecular profiles obtained using uniform protocol and reagents settings that can be broadly used in biomedicine for data normalization in pathology, including cancer. Here, we publish new original 39 breast cancer (BC) and 19 lung cancer (LC) RNA sequencing profiles obtained for formalin-fixed paraffin-embedded (FFPE) tissue samples, fully compatible with the Oncobox Atlas. We performed the first correlation study of RNA sequencing and immunohistochemistry-measured expression profiles for the clinically actionable biomarker genes in FFPE cancer tissue samples. We demonstrated high (Spearman’s rho 0.65–0.798) and statistically significant (p < 0.00004) correlations between the RNA sequencing (Oncobox protocol) and immunohistochemical measurements for HER2/ERBB2, ER/ESR1 and PGR genes in BC, and for PDL1 gene in LC; AUC: 0.963 for HER2, 0.921 for ESR1, 0.912 for PGR, and 0.922 for PDL1. To our knowledge, this is the first validation that total RNA sequencing of archived FFPE materials provides a reliable estimation of marker protein levels. These results show that in the future, RNA sequencing can complement immunohistochemistry for reliable measurements of the expression biomarkers in FFPE cancer samples.

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