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1.
Sci Adv ; 7(7)2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33579713

RESUMO

Chromosomal integration of recombinant genes is desirable compared with expression from plasmids due to increased stability, reduced cell-to-cell variability, and elimination of the need for antibiotics for plasmid maintenance. Here, we present a new approach for tuning pathway gene expression levels via random integration and high-throughput screening. We demonstrate multiplexed gene integration and expression-level optimization for isobutanol production in Escherichia coli The integrated strains could, with far lower expression levels than plasmid-based expression, produce high titers (10.0 ± 0.9 g/liter isobutanol in 48 hours) and yields (69% of the theoretical maximum). Close examination of pathway expression in the top-performing, as well as other isolates, reveals the complexity of cellular metabolism and regulation, underscoring the need for precise optimization while integrating pathway genes into the chromosome. We expect this method for pathway integration and optimization can be readily extended to a wide range of pathways and chassis to create robust and efficient production strains.


Assuntos
Cromossomos Bacterianos , Engenharia Metabólica , Cromossomos Bacterianos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Engenharia Metabólica/métodos , Plasmídeos/genética
2.
Blood ; 137(12): 1591-1602, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33275650

RESUMO

Digital protein assays have great potential to advance immunodiagnostics because of their single-molecule sensitivity, high precision, and robust measurements. However, translating digital protein assays to acute clinical care has been challenging because it requires deployment of these assays with a rapid turnaround. Herein, we present a technology platform for ultrafast digital protein biomarker detection by using single-molecule counting of immune-complex formation events at an early, pre-equilibrium state. This method, which we term "pre-equilibrium digital enzyme-linked immunosorbent assay" (PEdELISA), can quantify a multiplexed panel of protein biomarkers in 10 µL of serum within an unprecedented assay incubation time of 15 to 300 seconds over a 104 dynamic range. PEdELISA allowed us to perform rapid monitoring of protein biomarkers in patients manifesting post-chimeric antigen receptor T-cell therapy cytokine release syndrome, with ∼30-minute sample-to-answer time and a sub-picograms per mL limit of detection. The rapid, sensitive, and low-input volume biomarker quantification enabled by PEdELISA is broadly applicable to timely monitoring of acute disease, potentially enabling more personalized treatment.


Assuntos
Citocinas/sangue , Doenças do Sistema Imunitário/sangue , Testes Imediatos , Biomarcadores/sangue , Proteínas Sanguíneas/análise , Ensaio de Imunoadsorção Enzimática , Desenho de Equipamento , Humanos
3.
Lab Chip ; 21(2): 331-343, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33211045

RESUMO

Despite widespread concern regarding cytokine storms leading to severe morbidity in COVID-19, rapid cytokine assays are not routinely available for monitoring critically ill patients. We report the clinical application of a digital protein microarray platform for rapid multiplex quantification of cytokines from critically ill COVID-19 patients admitted to the intensive care unit (ICU) at the University of Michigan Hospital. The platform comprises two low-cost modules: (i) a semi-automated fluidic dispensing/mixing module that can be operated inside a biosafety cabinet to minimize the exposure of the technician to the virus infection and (ii) a 12-12-15 inch compact fluorescence optical scanner for the potential near-bedside readout. The platform enabled daily cytokine analysis in clinical practice with high sensitivity (<0.4 pg mL-1), inter-assay repeatability (∼10% CV), and rapid operation providing feedback on the progress of therapy within 4 hours. This test allowed us to perform serial monitoring of two critically ill patients with respiratory failure and to support immunomodulatory therapy using the selective cytopheretic device (SCD). We also observed clear interleukin-6 (IL-6) elevations after receiving tocilizumab (IL-6 inhibitor) while significant cytokine profile variability exists across all critically ill COVID-19 patients and to discover a weak correlation between IL-6 to clinical biomarkers, such as ferritin and C-reactive protein (CRP). Our data revealed large subject-to-subject variability in patients' response to COVID-19, reaffirming the need for a personalized strategy guided by rapid cytokine assays.


Assuntos
COVID-19/imunologia , Síndrome da Liberação de Citocina/sangue , Citocinas/sangue , Tecnologia Digital/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Monitorização Fisiológica/métodos , Análise Serial de Proteínas/métodos , Algoritmos , Biomarcadores/sangue , Proteína C-Reativa/análise , COVID-19/sangue , Estado Terminal , Síndrome da Liberação de Citocina/imunologia , Desenho de Equipamento , Ferritinas/análise , Interleucina-10/sangue , Interleucina-1beta/sangue , Interleucina-6/sangue , Limite de Detecção , Monitorização Fisiológica/instrumentação , SARS-CoV-2 , Fator de Necrose Tumoral alfa/sangue
4.
medRxiv ; 2020 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-32587979

RESUMO

Despite widespread concern for cytokine storms leading to severe morbidity in COVID-19, rapid cytokine assays are not routinely available for monitoring critically ill patients. We report the clinical application of a machine learning-based digital protein microarray platform for rapid multiplex quantification of cytokines from critically ill COVID-19 patients admitted to the intensive care unit (ICU) at the University of Michigan Hospital. The platform comprises two low-cost modules: (i) a semi-automated fluidic dispensing/mixing module that can be operated inside a biosafety cabinet to minimize the exposure of technician to the virus infection and (ii) a 12-12-15 inch compact fluorescence optical scanner for the potential near-bedside readout. The platform enabled daily cytokine analysis in clinical practice with high sensitivity (<0.4pg/mL), inter-assay repeatability (~10% CV), and near-real-time operation with a 10 min assay incubation. A cytokine profiling test with the platform allowed us to observe clear interleukin-6 (IL-6) elevations after receiving tocilizumab (IL-6 inhibitor) while significant cytokine profile variability exists across all critically ill COVID-19 patients and to discover a weak correlation between IL-6 to clinical biomarkers, such as Ferritin and CRP. Our data revealed large subject-to-subject variability in a patient's response to anti-inflammatory treatment for COVID-19, reaffirming the need for a personalized strategy guided by rapid cytokine assays.

5.
Mol Cell ; 75(3): 644-660.e5, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31398325

RESUMO

Cell-cell communication via ligand-receptor signaling is a fundamental feature of complex organs. Despite this, the global landscape of intercellular signaling in mammalian liver has not been elucidated. Here we perform single-cell RNA sequencing on non-parenchymal cells isolated from healthy and NASH mouse livers. Secretome gene analysis revealed a highly connected network of intrahepatic signaling and disruption of vascular signaling in NASH. We uncovered the emergence of NASH-associated macrophages (NAMs), which are marked by high expression of triggering receptors expressed on myeloid cells 2 (Trem2), as a feature of mouse and human NASH that is linked to disease severity and highly responsive to pharmacological and dietary interventions. Finally, hepatic stellate cells (HSCs) serve as a hub of intrahepatic signaling via HSC-derived stellakines and their responsiveness to vasoactive hormones. These results provide unprecedented insights into the landscape of intercellular crosstalk and reprogramming of liver cells in health and disease.


Assuntos
Comunicação Celular/genética , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Análise de Sequência de RNA , Animais , Reprogramação Celular/genética , Modelos Animais de Doenças , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Ligantes , Fígado/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Transdução de Sinais/genética , Análise de Célula Única
6.
Lab Chip ; 19(18): 3065-3076, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31389447

RESUMO

Widespread commercial and clinical adaptation of biomedical microfluidic technology has been limited in large part due to the lack of mass producibility of polydimethylsiloxane (PDMS) and glass-based devices commonly as reported in the literature. Here, we present a batch-fabricated, robust, and mass-producible immunophenotyping microfluidic device using silicon micromachining processes. Our Si and glass-based microfluidic device, named the silicon microfluidic immunophenotyping assay (SiMIPA), consists of a highly porous (∼40%) silicon membrane that can selectively separate microparticles below a certain size threshold. The device is capable of isolating and stimulating specific leukocyte populations, and allows for measuring their secretion of cell signaling proteins by means of a no-wash homogeneous chemiluminescence-based immunoassay. The high manufacturing throughput (∼170 devices per wafer) makes a large quantity of SiMIPA chips readily available for clinically relevant applications, which normally require large dataset acquisitions for statistical accuracy. With 30 SiMIPA chips, we performed in vitro immunomodulatory drug screening on isolated leukocyte subsets, yielding 5 data points at 6 drug concentrations. Furthermore, the excellent structural integrity of the device allowed for samples and reagents to be loaded using a micropipette, greatly simplifying the experimental protocol.


Assuntos
Fatores Imunológicos/farmacologia , Imunofenotipagem , Leucócitos/efeitos dos fármacos , Silício/química , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Humanos , Células Jurkat , Leucócitos/imunologia , Técnicas Analíticas Microfluídicas/instrumentação , Tamanho da Partícula , Porosidade , Propriedades de Superfície
7.
Lab Chip ; 19(14): 2425-2434, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31187105

RESUMO

Recent advances in transcriptomic analysis at single-cell resolution reveal cell-to-cell heterogeneity in a biological sample with unprecedented resolution. Partitioning single cells in individual micro-droplets and harvesting each cell's mRNA molecules for next-generation sequencing has proven to be an effective method for profiling transcriptomes from a large number of cells at high throughput. However, the assays to recover the full transcriptomes are time-consuming in sample preparation and require expensive reagents and sequencing cost. Many biomedical applications, such as pathogen detection, prefer highly sensitive, reliable and low-cost detection of selected genes. Here, we present a droplet-based microfluidic platform that permits seamless on-chip droplet sorting and merging, which enables completing multi-step reaction assays within a short time. By sequentially adding lysis buffers and reactant mixtures to micro-droplet reactors, we developed a novel workflow of single-cell reverse transcription loop-mediated-isothermal amplification (scRT-LAMP) to quantify specific mRNA expression levels in different cell types within one hour. Including single cell encapsulation, sorting, lysing, reactant addition, and quantitative mRNA detection, the fully on-chip workflow provides a rapid, robust, and high-throughput experimental approach for a wide variety of biomedical studies.


Assuntos
Dispositivos Lab-On-A-Chip , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Análise de Célula Única/instrumentação , Humanos , Células Jurkat , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
Metab Eng ; 54: 232-243, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31034921

RESUMO

Microbes can be engineered to synthesize a wide array of bioproducts, yet production phenotype evaluation remains a frequent bottleneck in the design-build-test cycle where strain development requires iterative rounds of library construction and testing. Here, we present Syntrophic Co-culture Amplification of Production phenotype (SnoCAP). Through a metabolic cross-feeding circuit, the production level of a target molecule is translated into highly distinguishable co-culture growth characteristics, which amplifies differences in production into highly distinguishable growth phenotypes. We demonstrate SnoCAP with the screening of Escherichia coli strains for production of two target molecules: 2-ketoisovalerate, a precursor of the drop-in biofuel isobutanol, and L-tryptophan. The dynamic range of the screening can be tuned by employing an inhibitory analog of the target molecule. Screening based on this framework requires compartmentalization of individual producers with the sensor strain. We explore three formats of implementation with increasing throughput capability: confinement in microtiter plates (102-104 assays/experiment), spatial separation on agar plates (104-105 assays/experiment), and encapsulation in microdroplets (105-107 assays/experiment). Using SnoCAP, we identified an efficient isobutanol production strain from a random mutagenesis library, reaching a final titer that is 5-fold higher than that of the parent strain. The framework can also be extended to screening for secondary metabolite production using a push-pull strategy. We expect that SnoCAP can be readily adapted to the screening of various microbial species, to improve production of a wide range of target molecules.


Assuntos
Engenharia Metabólica , Mutagênese , Fenótipo , Técnicas de Cocultura , Escherichia coli/genética , Escherichia coli/metabolismo , Testes Genéticos
9.
Nat Commun ; 9(1): 1030, 2018 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-29531246

RESUMO

Directed evolution has long been a key strategy to generate enzymes with desired properties like high selectivity, but experimental barriers and analytical costs of screening enormous mutant libraries have limited such efforts. Here, we describe an ultrahigh-throughput dual-channel microfluidic droplet screening system that can be used to screen up to ~107 enzyme variants per day. As an example case, we use the system to engineer the enantioselectivity of an esterase to preferentially produce desired enantiomers of profens, an important class of anti-inflammatory drugs. Using two types of screening working modes over the course of five rounds of directed evolution, we identify (from among 5 million mutants) a variant with 700-fold improved enantioselectivity for the desired (S)-profens. We thus demonstrate that this screening platform can be used to rapidly generate enzymes with desired enzymatic properties like enantiospecificity, chemospecificity, and regiospecificity.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/genética , Archaeoglobus fulgidus/enzimologia , Evolução Molecular Direcionada/métodos , Esterases/química , Esterases/genética , Microfluídica/métodos , Proteínas Arqueais/metabolismo , Archaeoglobus fulgidus/química , Archaeoglobus fulgidus/genética , Esterases/metabolismo , Evolução Molecular , Ibuprofeno/química , Ibuprofeno/metabolismo , Cinética , Modelos Moleculares , Estereoisomerismo , Especificidade por Substrato
10.
Lab Chip ; 17(21): 3664-3671, 2017 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-28967663

RESUMO

Co-encapsulation of two distinct particles within microfluidic droplets provides the means to achieve various high-throughput single-cell assays, such as biochemical reactions and cell-cell interactions in small isolated volumes. However, limited by the Poisson statistics, the co-encapsulation rate of the conventional co-flow approach is low even under optimal conditions. Only up to 13.5% of droplets precisely contain a pair of two distinct particles, while the rest, either being empty or encapsulating unpaired particles become wastes. Thus, the low co-encapsulation efficiency makes droplet-based assays impractical in biological applications involving low abundant bioparticles. In this paper, we present a highly promising droplet merging strategy to increase the co-encapsulation efficiency. Our method first enriches droplets exactly encapsulating a single particle via fluorescence or scattering-light activated sorting. Then, two droplets, each with a distinct particle, are precisely one-to-one paired and merged in a novel microwell device. This deterministic approach overcomes the Poisson statistics limitation facing conventional stochastic methods, yielding an up to 90% post-sorting particle capture rate and an overall 88.1% co-encapsulation rate. With its superior single-particle pairing performance, our system provides a promising technological platform to enable highly efficient microdroplet assays.

11.
Nano Lett ; 17(4): 2374-2380, 2017 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-28296413

RESUMO

Label-free, nanoparticle-based plasmonic optical biosensing, combined with device miniaturization and microarray integration, has emerged as a promising approach for rapid, multiplexed biomolecular analysis. However, limited sensitivity prevents the wide use of such integrated label-free nanoplasmonic biosensors in clinical and life science applications where low-abundance biomolecule detection is needed. Here, we present a nanoplasmofluidic device integrated with microelectrodes for rapid, label-free analysis of a low-abundance cell signaling protein, detected by AC electroosmosis-enhanced localized surface plasmon resonance (ACE-LSPR) biofunctional nanoparticle imaging. The ACE-LSPR device is constructed using both bottom-up and top-down sensor fabrication methods, allowing the seamless integration of antibody-conjugated gold nanorod (AuNR) biosensor arrays with microelectrodes on the same microfluidic platform. Applying an AC voltage to microelectrodes while scanning the scattering light intensity variation of the AuNR biosensors results in significantly enhanced biosensing performance. The AC electroosmosis (ACEO) based enhancement of the biosensor performance enables rapid (5-15 min) quantification of IL-1ß, a pro-inflammatory cytokine biomarker, with a sensitivity down to 158.5 fg/mL (9.1 fM) for spiked samples in PBS and 1 pg/mL (58 fM) for diluted human serum. Together with the optimized detection sensitivity and speed, our study presents the first critical step toward the application of nanoplasmonic biosensing technology to immune status monitoring guided by low-abundance cytokine measurement.


Assuntos
Técnicas Biossensoriais/métodos , Citocinas/sangue , Eletro-Osmose/instrumentação , Dispositivos Lab-On-A-Chip , Biomarcadores/sangue , Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ouro/química , Humanos , Limite de Detecção , Nanotecnologia , Nanotubos/química , Imagem Óptica/métodos , Tamanho da Partícula , Ressonância de Plasmônio de Superfície
12.
Adv Drug Deliv Rev ; 95: 90-103, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26408791

RESUMO

Systemic inflammatory disorders resulting from infection, trauma, surgery, and severe disease conditions pose serious threats to human health leading to organ dysfunction, organ failure, and mortality. The highly complex and dynamic nature of the immune system experiencing acute inflammation makes immunomodulatory therapy blocking pro-inflammatory cytokines very challenging. Successful therapy requires the ability to determine appropriate anti-cytokine drugs to be delivered at a right dose in a timely manner. Label-free micro- and nano-biosensors hold the potential to overcome the current challenges, enabling cytokine-targeted treatments to be tailored according to the immune status of an individual host with their unique cytokine biomarker detection capabilities. This review studies the recent progress in label-free cytokine biosensors, summarizes their performances and potential merits, and discusses future directions for their advancements to meet challenges towards personalized anti-cytokine drug delivery.


Assuntos
Técnicas Biossensoriais , Citocinas/imunologia , Inflamação/terapia , Humanos , Imunoensaio , Inflamação/imunologia , Microfluídica , Nanomedicina , Medicina de Precisão
13.
ACS Nano ; 9(4): 4173-81, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25790830

RESUMO

Precise monitoring of the rapidly changing immune status during the course of a disease requires multiplex analysis of cytokines from frequently sampled human blood. However, the current lack of rapid, multiplex, and low volume assays makes immune monitoring for clinical decision-making (e.g., critically ill patients) impractical. Without such assays, immune monitoring is even virtually impossible for infants and neonates with infectious diseases and/or immune mediated disorders as access to their blood in large quantities is prohibited. Localized surface plasmon resonance (LSPR)-based microfluidic optical biosensing is a promising approach to fill this technical gap as it could potentially permit real-time refractometric detection of biomolecular binding on a metallic nanoparticle surface and sensor miniaturization, both leading to rapid and sample-sparing analyte analysis. Despite this promise, practical implementation of such a microfluidic assay for cytokine biomarker detection in serum samples has not been established primarily due to the limited sensitivity of LSPR biosensing. Here, we developed a high-throughput, label-free, multiarrayed LSPR optical biosensor device with 480 nanoplasmonic sensing spots in microfluidic channel arrays and demonstrated parallel multiplex immunoassays of six cytokines in a complex serum matrix on a single device chip while overcoming technical limitations. The device was fabricated using easy-to-implement, one-step microfluidic patterning and antibody conjugation of gold nanorods (AuNRs). When scanning the scattering light intensity across the microarrays of AuNR ensembles with dark-field imaging optics, our LSPR biosensing technique allowed for high-sensitivity quantitative cytokine measurements at concentrations down to 5-20 pg/mL from a 1 µL serum sample. Using the nanoplasmonic biosensor microarray device, we demonstrated the ability to monitor the inflammatory responses of infants following cardiopulmonary bypass (CPB) surgery through tracking the time-course variations of their serum cytokines. The whole parallel on-chip assays, which involved the loading, incubation, and washing of samples and reagents, and 10-fold replicated multianalyte detection for each sample using the entire biosensor arrays, were completed within 40 min.


Assuntos
Citocinas/sangue , Imunoensaio/métodos , Nanotecnologia/métodos , Ressonância de Plasmônio de Superfície/métodos , Ponte Cardiopulmonar , Humanos , Imunidade , Lactente , Monitorização Fisiológica
14.
Lab Chip ; 14(7): 1230-45, 2014 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-24525555

RESUMO

Cellular analysis plays important roles in various biological applications, such as cell biology, drug development, and disease diagnosis. Conventional cellular analysis usually measures the average response from a whole cell group. However, bulk measurements may cause misleading interpretations due to cell heterogeneity. Another problem is that current cellular analysis may not be able to differentiate various subsets of cell populations, each exhibiting a different behavior than the others. Single-cell analysis techniques are developed to analyze cellular properties, conditions, or functional responses in a large cell population at the individual cell level. Integrating optics with microfluidic platforms provides a well-controlled microenvironment to precisely control single cell conditions and perform non-invasive high-throughput analysis. This paper reviews recent developments in optofluidic technologies for various optics-based single-cell analyses, which involve single cell manipulation, treatment, and property detection. Finally, we provide our views on the future development of integrated optics with microfluidics for single-cell analysis and discuss potential challenges and opportunities of this emerging research field in biological applications.


Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/tendências , Retratos como Assunto
15.
Anim Reprod Sci ; 123(3-4): 221-33, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21183296

RESUMO

The development of the testes includes changes in cell morphology and endocrine levels that are essential for the maturation of males. A large number of novel proteins are expressed throughout testis development and play important roles in spermatogenesis. Differences in protein expressions during the development of porcine testes have not been systematically studied. The purpose of this study was to investigate differential protein expression in porcine testes during postnatal development. Testes from four pigs each at 1wk, 3mo, and 1yr of age were used for a proteomic analysis. Expression levels of 264 protein spots were quantified using the Melanie 3 software. In total, 108 protein spots showed more than 2-fold differences (P<0.05) among developmental stages, and 90 of them were successfully identified by mass spectrometry. The proteins were sorted based on whether the expression levels increased with age (36.1%), decreased with age (38.0%), or fluctuated among different developmental stages (25.9%). In total, 69 unique gene products were further classified according to their gene ontology annotations. A majority of the proteins are organelle proteins (41%) with the nucleus and mitochondria being the main organelles. About 45% of the proteins have a protein binding domain and are likely involved in protein-protein interactions. Finally, a large proportion of these differentially expressed proteins are involved in cellular (25%) and metabolic (22%) processes. Identifying these differentially expressed proteins should be valuable for exploring developmental biology and the pathology of male reproduction.


Assuntos
Proteínas/análise , Sus scrofa/crescimento & desenvolvimento , Sus scrofa/metabolismo , Testículo/química , Testículo/metabolismo , Animais , Animais Recém-Nascidos , Análise por Conglomerados , Eletroforese em Gel Bidimensional , Imuno-Histoquímica , Masculino , Espectrometria de Massas , Metaboloma , Proteínas/metabolismo , Proteoma/análise , Proteoma/metabolismo , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
16.
Anim Reprod Sci ; 112(1-2): 136-49, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-18538515

RESUMO

A sublethal environmental stress, high-hydrostatic pressure (HHP) was reported to significantly improve the motility, viability and fertility parameters of frozen bull and boar semen. However, the mechanism of how HHP treatment improves survival rates at sperm cryopreservation remains unclear. The purpose of this study was to evaluate the effect of HHP treatment of fresh boar semen on the protein profile of boar sperm before and after freezing. Fresh, extended semen of eight boars was split, one part was treated with 200, 300 or 400bar for 90min using a custom made pressuring device before the start of the semen freezing procedure, and the other part was prepared without HHP treatment. After thawing, samples were checked for motility. The effect of HHP treatment on the post-thaw motility of frozen semen was significant (P=0.02). Post-thaw motility of each treatment groups increased compared to control (46% vs. 52%, 56% and 56%; control vs. 200bar, 300bar and 400bar treatments). Samples for protein analysis were collected from the 300bar treatment group before HHP treatment at room temperature (25+/-3 degrees C), at 5 degrees C of the cooling process and after thawing with or without HHP treatment. The sperm were lysed using a urea-pyranoside-dithiothreitol buffer to extract their proteins for protein analysis. Approximately 800microg total proteins were assayed by two-dimensional gel electrophoresis and stained with colloidal Coomassie blue. The levels of 125 protein spots were quantified. The results revealed that the levels of 7 protein spots differed significantly among treatments. The identities of various protein constituents were identified by mass spectrometry and database searching. Ubiquinol-cytochrome c reductase complex core protein 1, perilipin, and carbohydrate-binding protein AWN precursor were identified as HHP response proteins being significantly higher in HHP-treated samples. Testis-specific glyceraldehyde 3-phosphate dehydrogenase, outer dense fiber of sperm tails 2 isoform 10, cytosolic 5'-nucleotidase 1B, and quinone oxidoreductase represented the cooling and freezing related proteins. The differing levels of these identified proteins could be valuable for further exploring the protective mechanism of the HHP treatment in frozen-thawed porcine sperm.


Assuntos
Criopreservação/veterinária , Pressão Hidrostática , Proteínas/análise , Preservação do Sêmen/veterinária , Espermatozoides/química , Suínos , 5'-Nucleotidase/análise , Animais , Eletroforese em Gel Bidimensional , Gliceraldeído-3-Fosfato Desidrogenases/análise , Temperatura Alta , Masculino , NAD(P)H Desidrogenase (Quinona)/análise , Preservação do Sêmen/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Motilidade dos Espermatozoides , Espermatozoides/fisiologia , Espectrometria de Massas em Tandem
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