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HiBiT is an engineered luciferase’s 11-amino-acid component that can be introduced as a tag at either terminus of a protein of interest. When the LgBiT component and a substrate are present,HiBiT and LgBiT dimerize forming a functional luciferase. The HiBiT technology has been extensively used for high-throughput protein turnover studies in cells. Here,we have adapted the use of the HiBiT technology to quantify messenger RNA (mRNA) translation temporally in vitro in the rabbit reticulocyte system and in cellulo in HEK293 cells constitutively expressing LgBiT. The assay system can uniquely detect differences in cap,5?UTR,modified nucleotide composition,coding sequence optimization and poly(A) length,and their effects on mRNA translation over time. Importantly,using these assays we established the optimal mRNA composition varied depending on the encoded protein of interest,highlighting the importance of screening methods tailored to the protein of interest,and not reliant on reporter proteins. Our findings demonstrated that HiBiT can be easily and readily adapted to monitor real-time mRNA translation in live cells and offers a novel and highly favourable method for the development of mRNA-based therapeutics. View Publication

Genetic variations in the Trpm8 gene that encodes the cold receptor TRPM8 have been linked to protection against polygenic migraine,a disabling condition primarily affecting women. Noteworthy,TRPM8 has been recently found in brain areas related to emotional processing,suggesting an unrecognized role in migraine comorbidities. Here,we use mouse behavioural models to investigate the role of Trpm8 in migraine-related phenotypes. Subsequently,we test the efficacy of rapamycin,a clinically relevant TRPM8 agonist,in these behavioural traits and in human induced pluripotent stem cell (iPSC)-derived sensory neurons. We report that Trpm8 null mice exhibited impulsive and depressive-like behaviours,while also showing frequent pain-like facial expressions detected by an artificial intelligence algorithm. In a nitroglycerin-induced migraine model,Trpm8 knockout mice of both sexes developed anxiety and mechanical hypersensitivity,whereas wild-type females also displayed depressive-like phenotype and hypernociception. Notably,rapamycin alleviated pain-related behaviour through both TRPM8-dependent and independent mechanisms but lacked antidepressant activity,consistent with a peripheral action. The macrolide ionotropically activated TRPM8 signalling in human sensory neurons,emerging as a new candidate for intervention. Together,our findings underscore the potential of TRPM8 for migraine relief and its involvement in affective comorbidities,emphasizing the importance of addressing emotional symptoms to improve clinical outcomes for migraine sufferers,especially in females. The online version contains supplementary material available at 10.1186/s10194-025-02082-4. View Publication

The enteric pathogen Lawsonia intracellularis is one of the main causes of diarrhea and compromised weight gain in pigs worldwide. Traditional cell-line cultures have been used to study L. intracellularis pathogenesis. However,these systems fail to reproduce the epithelial changes observed in the intestines of L. intracellularis-infected pigs,specifically,the changes in intestinal cell constitution and gene expression. A more physiologically accurate and state-of-the-art model is provided by swine enteroids derived from stem cell-containing crypts from healthy pigs. The objective of this study was to verify the feasibility of two-dimensional swine enteroids as in vitro models for L. intracellularis infection. We established both three- and two-dimensional swine enteroid cultures derived from intestinal crypts. The two-dimensional swine enteroids were infected by L. intracellularis in four independent experiments. Enteroid-infected samples were collected 3 and 7 d postinfection for analysis using real-time quantitative PCR and L. intracellularis immunohistochemistry. In this study,we show that L. intracellularis is capable of infecting and replicating intracellularly in two-dimensional swine enteroids derived from ileum. View Publication

Chronic compressive cervical spinal cord injury (cCSCI),a debilitating condition,lacks effective treatment options. Addressing this gap,our study introduces a novel rat model of cCSCI developed through spinal cord compression via synthetic polyether sheet implantation,closely mimicking human pathology. We evaluated the model’s fidelity utilizing a comprehensive series of behavioral,electrophysiological,and histological assessments. Our research also explored the therapeutic potential of oligodendrogenic neural progenitor cells (oNPCs) derived from induced pluripotent stem cells. Transplanted oNPCs successfully integrated into the host spinal cord,differentiated into neurons,astrocytes,and oligodendrocytes,and demonstrated a remarkable capacity for enhancing neuroplasticity. Electrophysiological analyses revealed significant improvements in motor evoked potentials and a rectification of the excitability imbalance posttransplantation,indicating substantial recovery of motor circuits. Histological findings complemented these results,showing enhanced remyelination and a reduction in excitatory transmitter expression in the residual gray matter. Functionally,the transplantation of oNPCs led to marked improvements in grip strength,locomotor abilities,and sensory functions,surpassing those seen with standard treatments. This study not only provides a novel and reliable rat model of cCSCI for further research but also highlights the potential of oNPCs as a transformative approach for spinal cord injury therapy,suggesting their significant role in neural regeneration and repair. View Publication

The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)-kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors,namely insulin receptor substrate-2 (IRS2),Src homology 2 domain-containing inositol 5'-phosphatase (SHIP),Grb2-associated binder-1 (Gab1),and the Epo receptor (EpoR). Using different in vitro systems,we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors,Akt,FKHRL1,and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR),through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR,or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors,but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors,the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally,our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCF(SKP2),which,in turn,down-regulates p27(Kip1) cyclin-dependent kinase (CDK) inhibitor via proteasome degradation. View Publication

Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here,we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging. View Publication

Efficient delivery of therapeutics across the neuroprotective blood-brain barrier (BBB) remains a formidable challenge for central nervous system drug development. High-fidelity in vitro models of the BBB could facilitate effective early screening of drug candidates targeting the brain. In this study,we developed a microfluidic BBB model that is capable of mimicking in vivo BBB characteristics for a prolonged period and allows for reliable in vitro drug permeability studies under recirculating perfusion. We derived brain microvascular endothelial cells (BMECs) from human induced pluripotent stem cells (hiPSCs) and cocultured them with rat primary astrocytes on the two sides of a porous membrane on a pumpless microfluidic platform for up to 10 days. The microfluidic system was designed based on the blood residence time in human brain tissues,allowing for medium recirculation at physiologically relevant perfusion rates with no pumps or external tubing,meanwhile minimizing wall shear stress to test whether shear stress is required for in vivo-like barrier properties in a microfluidic BBB model. This BBB-on-a-chip model achieved significant barrier integrity as evident by continuous tight junction formation and in vivo-like values of trans-endothelial electrical resistance (TEER). The TEER levels peaked above 4000 $$ textperiodcentered cm(2) on day 3 on chip and were sustained above 2000 $$ textperiodcentered cm(2) up to 10 days,which are the highest sustained TEER values reported in a microfluidic model. We evaluated the capacity of our microfluidic BBB model to be used for drug permeability studies using large molecules (FITC-dextrans) and model drugs (caffeine,cimetidine,and doxorubicin). Our analyses demonstrated that the permeability coefficients measured using our model were comparable to in vivo values. Our BBB-on-a-chip model closely mimics physiological BBB barrier functions and will be a valuable tool for screening of drug candidates. The residence time-based design of a microfluidic platform will enable integration with other organ modules to simulate multi-organ interactions on drug response. Biotechnol. Bioeng. 2016;9999: 1-11. textcopyright 2016 Wiley Periodicals,Inc. View Publication

In several clinical contexts,the measurement of ATP concentration in T lymphocytes has been proposed as a biomarker of immune status,predictive of secondary infections. However,the use of such biomarker in lymphopenic patients requires some adaptations in the ATP dosage protocol. We used blood from healthy volunteers to determine the optimal experimental settings. We investigated technical aspects such as the type of anticoagulant for blood sampling,the effect of freeze and thaw cycles,the reagent and sample mixing sequence,and the optimal dilution buffer. We also shortened the incubation time to 8h,and even showed that a 30min incubation may be sufficient. To evaluate the ATP rise upon lymphocyte activation,the optimal dose of stimulant was defined to be 4μg/mL of phytohaemagglutinin. Lastly,we determined that the number of T cells needed for this measurement was as low as 50,000,which is compatible with the existing lymphopenia in clinical settings. This optimized protocol appears ready to be assessed in lymphopenic patients to further investigate the interconnection between T lymphocyte metabolism and impaired phenotype and functions. View Publication