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1.
Nat Commun ; 12(1): 5808, 2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34608152

RESUMO

The nucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress, they are considered to be alarmones triggering stress adaptive processes. However, their cellular roles remain elusive. To elucidate the proteome-wide interactome of Ap3A and Ap4A and thereby gain insights into their cellular roles, we herein report the development of photoaffinity-labeling probes and their employment in chemical proteomics. We demonstrate that the identified ApnA interactors are involved in many fundamental cellular processes including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and cellular response mechanisms and only around half of them are known nucleotide interactors. Our results highlight common functions of these ApnAs across the domains of life, but also identify those that are different for Ap3A or Ap4A. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterization of their regulatory mechanisms in cells.


Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Proteômica , Trifosfato de Adenosina/metabolismo , Fosfatos de Dinucleosídeos/química , Endorribonucleases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , L-Lactato Desidrogenase/metabolismo , Fosfoglicerato Quinase/metabolismo , Marcadores de Fotoafinidade/síntese química , Marcadores de Fotoafinidade/química , Marcadores de Fotoafinidade/metabolismo , Ligação Proteica , Enzimas Ativadoras de Ubiquitina/metabolismo
2.
BMC Cancer ; 21(1): 1022, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34525951

RESUMO

BACKGROUND: Eukaryotic translation initiation factors (eIFs) are the key factors to synthesize translation initiation complexes during the synthesis of eukaryotic proteins. Besides, eIFs are especially important in regulating the immune function of tumor cells. However, the effect mechanism of eIFs in prostate cancer remains to be studied, which is precisely the purpose of this study. METHODS: In this study, three groups of prostate cancer cells were investigated. One group had its eIF5B gene knocked down; another group had its Programmed death 1 (PD-L1) overexpressed; the final group had its Wild-type p53-induced gene 1 (Wig1) overexpressed. Genetic alterations of the cancer cells were performed by plasmid transfection. The expression of PD-L1 mRNA was detected by quantitative real-time PCR (qRT-PCR), and the expressions of PD-L1 and eIF5B proteins were observed by western blot assays. Cell Counting Kit-8 (CCK-8), flow cytometry, Transwell and Transwell martrigel were used to investigated cell proliferation, apoptosis, migration and invasion, respectively. The effect of peripheral blood mononuclear cells (PBMCs) on tumor cells was observed, and the interaction between eIF5B and Wig1 was revealed by co-immunoprecipitation (CoIP) assay. Finally, the effects of interference with eIF5B expression on the growth, morphology, and immunity of the tumor, as well as PD-L1 expression in the tumor, were verified by tumor xenograft assays in vivo. RESULTS: Compared with normal prostate epithelial cells, prostate cancer cells revealed higher expressions of eIF5B and PD-L1 interference with eIF-5B expression can inhibit the proliferation, migration, invasion and PD-L1 expression of prostate cancer cells. Meanwhile, the cancer cell group with interference with eIF5B expression also demonstrated greater, apoptosis and higher vulnerability to PBMCs. CoIP assays showed that Wig1 could bind to eIF5B in prostate cancer cells, and its overexpression can inhibit the proliferation, migration, invasion and PD-L1 expression of cancer cells while promoting apoptosis. Moreover, interference with eIF5B expression can inhibit tumor growth, destroy tumor morphology, and suppress the proliferation of tumor cells. CONCLUSION: eIF5B can promote the expression of PD-L1 by interacting with Wig1. Besides, interference with eIF5B expression can inhibit the proliferation, migration, invasion and immunosuppressive response of prostate cancer cells. This study proposes a new target, eIF5B, for immunotherapy of prostate cancer.


Assuntos
Antígeno B7-H1/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Apoptose/genética , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Fatores de Iniciação em Eucariotos/genética , Citometria de Fluxo , Inativação Gênica , Genes p53/fisiologia , Humanos , Imunoprecipitação , L-Lactato Desidrogenase/metabolismo , Leucócitos Mononucleares/imunologia , Linfócitos do Interstício Tumoral , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/genética , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transfecção/métodos , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Vet Ophthalmol ; 24(5): 509-519, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34553825

RESUMO

PURPOSE: To establish a physiologically relevant ex vivo model of equine corneal epithelial wound healing. METHODS: Fourteen equine corneas were randomly assigned to one of two groups: wounded (n = 8) or unwounded (n = 6) controls. In the wounded group, the axial corneal epithelium was removed by applying a 6 mm filter paper disk soaked in 1N-NaOH for 60 s. Corneas were subsequently cultured using an air-liquid interface model. Evaluation of corneal healing was performed daily, and culture medium was collected. Corneas were randomly assigned to undergo processing via histopathology and RNAscope in situ hybridization for interleukin-6 (IL-6) and alpha-smooth muscle actin (αSMA) expression at T24, T48, and T72 h after wounding. Media of the cultured corneas were evaluated for the presence of lactate dehydrogenase (LDH) by a colorimetric assay. RESULTS: The ulcerated area of the wounded corneas decreased over time and all corneas healed within 72 h. Histologically, normal corneal architecture was observed including healthy epithelium (in areas other than the ulcerated ones), minimal stromal edema, intact endothelium, and Descemet's membrane. IL-6 expression was increased in wounded corneas compared with unwounded controls. LDH expression was elevated for both wounded and unwounded corneas at T24 but decreased substantially and was not detected at T48 in media from wounded and unwounded corneas, respectively. No αSMA expression was detected from either wounded or unwounded corneas. CONCLUSIONS: The equine air-liquid interface, ex vivo, corneal epithelial wound healing model is effective and physiologically relevant. This model can be used in future studies evaluating various corneal therapies.


Assuntos
Lesões da Córnea/veterinária , Cavalos/lesões , Interleucina-6/metabolismo , L-Lactato Desidrogenase/metabolismo , Cicatrização , Animais , Colorimetria/veterinária , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Modelos Animais de Doenças , Feminino , Masculino , Cultura Primária de Células/veterinária
4.
Exp Eye Res ; 211: 108750, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34481822

RESUMO

Pseudomonas aeruginosa (P. aeruginosa) keratitis, a worldwide leading cause of corneal perforation and blindness, which is associated with contact lens usage. Increasing evidence has indicated that pyroptosis, a novel proinflammatory programmed cell death, is linked with ocular diseases, little is known about the role of noncanonical pyroptosis in microbial keratitis. Here, we first indicated the involvement of noncanonical pyroptosis in P. aeruginosa keratitis and investigated whether wedelolactone (WDL), a major active component of Eclipta prostrate known to target caspase-11, could alleviate P. aeruginosa keratitis development. We found the expression of caspase-4/5/11 and cleaved GSDMD in corneas of P. aeruginosa keratitis patients, animal models and lipopolysaccharide (LPS)-induced primary cultured human corneal keratocytes (piHCKs) were increased. Combining ciprofloxacin with WDL significantly ameliorated the severity of P. aeruginosa keratitis, as manifested by decreased inflammatory responses and reduced corneal epithelial defects. Consistent with these findings, WDL also dose-dependently alleviated LPS-induced noncanonical pyroptosis by reversing the increased expression of caspase-4/5 and GSDMD in piHCKs. In summary, our results demonstrated that by targeting the activation of caspase-4/5/11, wedelolactone inhibited the development of P. aeruginosa keratitis and suppressed the release of proinflammatory cytokines. Wedelolactone may be a promising anti-inflammatory candidate to combat P. aeruginosa keratitis.


Assuntos
Caspases/metabolismo , Lesões da Córnea/prevenção & controle , Úlcera da Córnea/prevenção & controle , Cumarínicos/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Piroptose/efeitos dos fármacos , Animais , Western Blotting , Caspases Iniciadoras/metabolismo , Proliferação de Células , Lesões da Córnea/metabolismo , Lesões da Córnea/microbiologia , Úlcera da Córnea/metabolismo , Úlcera da Córnea/microbiologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Infecções Oculares Bacterianas/metabolismo , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Bacterianas/prevenção & controle , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , L-Lactato Desidrogenase/metabolismo , Masculino , Microscopia de Fluorescência , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/prevenção & controle , Pseudomonas aeruginosa , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real
5.
Sci Rep ; 11(1): 18571, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535719

RESUMO

The current standard preclinical oncology models are not able to fully recapitulate therapeutic targets and clinically relevant disease biology, evidenced by the 90% attrition rate of new therapies in clinical trials. Three-dimensional (3D) culture systems have the potential to enhance the relevance of preclinical models. However, the limitations of currently available cellular assays to accurately evaluate therapeutic efficacy in these models are hindering their widespread adoption. We assessed the compatibility of the lactate dehydrogenase (LDH) assay in 3D spheroid cultures against other commercially available readout methods. We developed a standardized protocol to apply the LDH assay to ex vivo cultures, considering the impact of culture growth dynamics. We show that accounting for growth rates and background release levels of LDH are sufficient to make the LDH assay a suitable methodology for longitudinal monitoring and endpoint assessment of therapeutic efficacy in both cell line-derived xenografts (xenospheres) and patient-derived explant cultures. This method has the added value of being non-destructive and not dependent on reagent penetration or manipulation of the parent material. The establishment of reliable readout methods for complex 3D culture systems will further the utility of these tumor models in preclinical and co-clinical drug development studies.


Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , L-Lactato Desidrogenase/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Humanos , Camundongos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas
6.
FASEB J ; 35(9): e21825, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383978

RESUMO

Ubiquitination is an essential post-translational modification that regulates protein stability or function. Its substrate specificity is dictated by various E3 ligases. The human C-terminal to LisH (CTLH) complex is a newly discovered multi-subunit really interesting new gene (RING) E3 ligase with only a few known ubiquitination targets. Here, we used mass spectrometry-based proteomic techniques to gain insight into CTLH complex function and ubiquitination substrates in HeLa cells. First, global proteomics determined proteins that were significantly increased, and thus may be substrates targeted for degradation, in cells depleted of CTLH complex member RanBPM. RanBPM-dependent ubiquitination determined using diGLY-enriched proteomics and the endogenous RanBPM interactome further revealed candidate ubiquitination targets. Three glycolysis enzymes alpha-enolase, L-lactate dehydrogenase A chain (LDHA), and pyruvate kinase M1/2 (PKM) had decreased ubiquitin sites in shRanBPM cells and were found associated with RanBPM in the interactome. Reduced polyubiquitination was validated for PKM2 and LDHA in cells depleted of RanBPM and CTLH complex RING domain subunit RMND5A. PKM2 and LDHA protein levels were unchanged, yet their activity was increased in extracts of cells with downregulated RanBPM. Finally, RanBPM deficient cells displayed enhanced glycolysis and deregulated central carbon metabolism. Overall, this study identifies potential CTLH complex ubiquitination substrates and uncovers that the CTLH complex inhibits glycolysis via non-degradative ubiquitination of PKM2 and LDHA.


Assuntos
Glicólise/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Animais , Linhagem Celular Tumoral , Células HeLa , Humanos , L-Lactato Desidrogenase/metabolismo , Camundongos , Proteômica/métodos , Especificidade por Substrato , Ubiquitina/metabolismo
7.
Molecules ; 26(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34443533

RESUMO

Quercetin is a polyphenolic compound, the effects of which raise scientists' doubts. The results of many experiments show that it has anticancer, antiinflammatory, and antioxidant properties, while other studies indicate its pro-oxidative and cytotoxic action. This compound can react with reactive oxygen species, and due to its chemical properties, it can be found in the hydrophobic-hydrophilic area of cells. These features of quercetin indicate that its action in cells will be associated with the modification of membranes and its participation in maintaining the redox balance. Therefore, this study distinguishes these two mechanisms and determines whether they are important for cell function. We check: (1) Whether the selected concentrations of quercetin are cytotoxic and destructive for SK-N-SH cell membranes (MTT, LDH, MDA tests) in situations with and without the applied oxidative stress; (2) what is the level of changes in the structural/mechanical properties of the lipid part of the membranes of these cells due to the presence of polyphenol molecules; and (3) whether the antioxidative action of quercetin protects the membrane against its modification. Our results show that changes in the stiffness/elasticity of the lipid part of the membrane constitute the decisive mechanism of action of quercetin, potentially influencing cellular processes whose initial stages are associated with membranes (e.g., reception of signals from the environment, transport).


Assuntos
Membrana Celular/efeitos dos fármacos , Neuroblastoma/patologia , Quercetina/farmacologia , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , L-Lactato Desidrogenase/metabolismo , Ozônio/farmacologia , Pressão , Temperatura
8.
Sci Rep ; 11(1): 17139, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429462

RESUMO

In human cells under stress conditions, misfolded polypeptides can form potentially cytotoxic insoluble aggregates. To eliminate aggregates, the HSP70 chaperone machinery extracts and resolubilizes polypeptides for triage to refolding or degradation. Yeast and bacterial chaperones of the small heat-shock protein (sHSP) family can bind substrates at early stages of misfolding, during the aggregation process. The co-aggregated sHSPs then facilitate downstream disaggregation by HSP70. Because it is unknown whether a human sHSP has this activity, we investigated the disaggregation role of human HSPB1. HSPB1 co-aggregated with unfolded protein substrates, firefly luciferase and mammalian lactate dehydrogenase. The co-aggregates formed with HSPB1 were smaller and more regularly shaped than those formed in its absence. Importantly, co-aggregation promoted the efficient disaggregation and refolding of the substrates, led by HSP70. HSPB1 itself was also extracted during disaggregation, and its homo-oligomerization ability was not required. Therefore, we propose that a human sHSP is an integral part of the chaperone network for protein disaggregation.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico/química , Humanos , L-Lactato Desidrogenase/metabolismo , Luciferases de Vaga-Lume/metabolismo , Chaperonas Moleculares/química , Multimerização Proteica , Desdobramento de Proteína
9.
Eur J Clin Invest ; 51(11): e13582, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34409593

RESUMO

BACKGROUND: A systematic analysis of concomitant arterial hypertension in COVID-19 patients and the impact of angiotensin-converting-enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARBs) have not been studied in a large multicentre cohort yet. We conducted a subanalysis from the international HOPE Registry (https://hopeprojectmd.com, NCT04334291) comparing COVID-19 in presence and absence of arterial hypertension. MATERIALS AND METHODS: Out of 5837 COVID-19 patients, 2850 (48.8%) patients had the diagnosis arterial hypertension. 1978/2813 (70.3%) patients were already treated with ACEI or ARBs. The clinical outcome of the present subanalysis included all-cause mortality over 40 days of follow-up. RESULTS: Patients with arterial hypertension suffered significantly more from different complications including respiratory insufficiency (60.8% vs 39.5%), heart failure (9.9% vs 3.1%), acute kidney injury (25.3% vs 7.3%), pneumonia (90.6% vs 86%), sepsis (14.7% vs 7.5%), and bleeding events (3.6% vs 1.6%). The mortality rate was 29.6% in patients with concomitant arterial hypertension and 11.3% without arterial hypertension (P < .001). Invasive and non-invasive respiratory supports were significantly more required in presence of arterial hypertension as compared without it. In the multivariate cox regression analysis, while age≥65, benzodiazepine, antidepressant at admission, elevated LDH or creatinine, respiratory insufficiency and sepsis might be a positive independent predictors of mortality, antiviral drugs, interferon treatment, ACEI or ARBs at discharge or oral anticoagulation at discharge might be an independent negative predictor of the mortality. CONCLUSIONS: The mortality rate and in-hospital complications might be increased in COVID-19 patients with a concomitant history of arterial hypertension. The history of ACEI or ARBs treatments does not seem to impact the outcome of these patients.


Assuntos
Injúria Renal Aguda/epidemiologia , COVID-19/epidemiologia , Insuficiência Cardíaca/epidemiologia , Hipertensão/epidemiologia , Pneumonia/epidemiologia , Insuficiência Respiratória/epidemiologia , Sepse/epidemiologia , Fatores Etários , Idoso , Antagonistas de Receptores de Angiotensina/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Antivirais/uso terapêutico , COVID-19/metabolismo , COVID-19/terapia , Creatinina/metabolismo , Feminino , Alemanha/epidemiologia , Mortalidade Hospitalar , Humanos , Hipertensão/tratamento farmacológico , Itália/epidemiologia , L-Lactato Desidrogenase/metabolismo , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Ventilação não Invasiva , Modelos de Riscos Proporcionais , Sistema de Registros , Respiração Artificial , SARS-CoV-2 , Índice de Gravidade de Doença , Espanha/epidemiologia
10.
Stroke ; 52(11): e706-e709, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34428931
11.
J Fish Biol ; 99(5): 1708-1718, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34392536

RESUMO

The ontogenesis of catabolic abilities and energy metabolism during endogenous nutritional periods of tongue sole was investigated. In this work, trypsin-like proteases (TRY) and triglyceride lipase (LIP) activities were measured to assess the capacities to catabolize proteins and lipids, respectively. Meanwhile, specific enzymes including pyruvate kinase (PK), glutamic oxalo acetic transaminase (GOT) and glutamate dehydrogenase (GDH), and hydroxyacyl CoA dehydrogenase (HOAD) as well as their ratios were assayed to evaluate the abilities to use energy substrates of carbohydrates, amino acids and fatty acids, respectively, for energy production. In addition, activities of citrate synthase (CS) and lactate dehydrogenase (LDH) and LDH/CS ratio were calculated to analyse the evolution of aerobic and anaerobic pathways. The study found that hatching occurred at 38.8 h after fertilization (HAF), mouth-opening day of eleuteroembryo appeared at 3 days after hatching (DAH), and the most rapid embryonic growth was observed in blastula stage before hatching. Enzymatic assay revealed that except for PK which appeared in cleavage stage onwards, all the other enzymes functioned after fertilization, preparing well for the coming embryogenesis of tongue sole. By comparing the average specific activity of enzyme in each period, it can be found that the highest value occurred at 3 DAH (for TRY, LIP, PK and LDH), 2 DAH (for GDH), fertilized egg (for GOT) and segmentation stage (for HOAD and CS), and the lowest value occurred at fertilized egg (for HOAD, CS and GDH), cleavage stage (for TRY, PK and LDH), gastrula stage (for GOT) and hatching day (for LIP). Based on the changeable patterns of metabolic enzymatic activities and ratios, it is concluded that metabolic capacities on three energy substrates displayed stage-specific traits, and the dominant energy substrate was fatty acids before segmentation stage, amino acids until hatching day and carbohydrate during eleuteroembryo period. As for energy production mode, aerobic pathway appeared to increase greater in fertilized egg and gastrula stage, whereas anaerobic pathway played a predominant role during cleavage stage, blastula stage, segmentation stage and eleuteroembryo stage. These results are valuable to elucidate the nutritional requirements of embryonic stages in tongue sole and to further understand their energy metabolic mechanisms.


Assuntos
Desenvolvimento Embrionário , Metabolismo Energético , Animais , Citrato (si)-Sintase/metabolismo , L-Lactato Desidrogenase/metabolismo , Língua/metabolismo
12.
Life Sci ; 282: 119847, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34293399

RESUMO

AIMS: Thymic carcinoma is a rare type of cancer without an established standard pharmaceutical treatment. This study investigated the antitumor effect of dimethyl itaconate (DI), a cell-permeable derivative of itaconate, on human thymic carcinoma cell line. MAIN METHODS: Human thymic carcinoma cell line Ty82 was used to evaluate the effect of DI on cell viability. Western blotting and immunohistochemistry were performed to determine the molecular mechanism of antitumor effects of DI on Ty82. KEY FINDINGS: DI suppressed cell growth and promoted apoptosis of Ty82. The suppressive effect of DI on Ty82 was mediated by the downregulation of lactate dehydrogenase A (LDHA), and the subsequent decrease in the activity of mechanistic target of rapamycin (mTOR). DI exhibited synergistic antitumor effects with a specific inhibitor of large neutral amino acid transporter 1 (LAT1), an amino acid transporter currently being investigated as a novel target for cancer therapy. SIGNIFICANCE: Our findings demonstrate that DI is a novel potential strategy for thymic carcinoma treatment.


Assuntos
Antineoplásicos/farmacologia , L-Lactato Desidrogenase/metabolismo , Proteínas de Neoplasias/metabolismo , Succinatos/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Timoma , Neoplasias do Timo , Linhagem Celular Tumoral , Humanos , Timoma/tratamento farmacológico , Timoma/enzimologia , Timoma/patologia , Neoplasias do Timo/tratamento farmacológico , Neoplasias do Timo/enzimologia , Neoplasias do Timo/patologia
13.
Int J Mol Sci ; 22(14)2021 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-34299084

RESUMO

During pregnancy, freely floating placental villi are adapted to fluid shear stress due to placental perfusion with maternal plasma and blood. In vitro culture of placental villous explants is widely performed under static conditions, hoping the conditions may represent the in utero environment. However, static placental villous explant culture dramatically differs from the in vivo situation. Thus, we established a flow culture system for placental villous explants and compared commonly used static cultured tissue to flow cultured tissue using transmission and scanning electron microscopy, immunohistochemistry, and lactate dehydrogenase (LDH) and human chorionic gonadotropin (hCG) measurements. The data revealed a better structural and biochemical integrity of flow cultured tissue compared to static cultured tissue. Thus, this new flow system can be used to simulate the blood flow from the mother to the placenta and back in the most native-like in vitro system so far and thus can enable novel study designs.


Assuntos
Gonadotropina Coriônica/metabolismo , Vilosidades Coriônicas/crescimento & desenvolvimento , L-Lactato Desidrogenase/metabolismo , Trofoblastos/citologia , Técnicas de Cultura de Células , Vilosidades Coriônicas/metabolismo , Feminino , Citometria de Fluxo , Humanos , Gravidez , Trofoblastos/metabolismo
14.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202757

RESUMO

The aim of this study was to investigate the effect of the application of homocysteine as well as its effect under the condition of aerobic physical activity on the activities of matrix metalloproteinases (MMP), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) in cardiac tissue and on hepato-renal biochemical parameters in sera of rats. Male Wistar albino rats were divided into four groups (n = 10, per group): C: 0.9% NaCl 0.2 mL/day subcutaneous injection (s.c.); H: homocysteine 0.45 µmol/g b.w./day s.c.; CPA saline (0.9% NaCl 0.2 mL/day s.c.) and a program of physical activity on a treadmill; and HPA homocysteine (0.45 µmol/g b.w./day s.c.) and a program of physical activity on a treadmill. Subcutaneous injection of substances was applied 2 times a day at intervals of 8 h during the first two weeks of experimental protocol. Hcy level in serum was significantly higher in the HPA group compared to the CPA group (p < 0.05). Levels of glucose, proteins, albumin, and hepatorenal biomarkers were higher in active groups compared with the sedentary group. It was demonstrated that the increased activities of LDH (mainly caused by higher activity of isoform LDH2) and mMDH were found under the condition of homocysteine-treated rats plus aerobic physical activity. Independent application of homocysteine did not lead to these changes. Physical activity leads to activation of MMP-2 isoform and to increased activity of MMP-9 isoform in both homocysteine-treated and control rats.


Assuntos
Hiper-Homocisteinemia/metabolismo , Rim/metabolismo , L-Lactato Desidrogenase/metabolismo , Fígado/metabolismo , Malato Desidrogenase/metabolismo , Metaloproteinases da Matriz/metabolismo , Miocárdio/metabolismo , Condicionamento Físico Animal , Animais , Biomarcadores , Pesos e Medidas Corporais , Ativação Enzimática , Hiper-Homocisteinemia/etiologia , Miocárdio/enzimologia , Especificidade de Órgãos , Ratos , Fatores de Tempo
15.
Anticancer Res ; 41(7): 3535-3542, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230149

RESUMO

BACKGROUND/AIM: No biomarkers that predict the benefit from anti-vascular endothelial growth factor (VEGF) antibodies have been identified. It is necessary to discover biomarkers that can identify patients who are more likely to benefit from bevacizumab-containing treatment, especially those who are more likely to benefit from treatment with bevacizumab beyond progression (BBP). Levels of serum lactate dehydrogenase (LDH), reported to be an indirect marker of hypoxia and angiogenesis, may be a useful marker for monitoring the efficacy of suppression of angiogenesis. PATIENTS AND METHODS: The clinical data of 91 patients with unresectable metastatic colorectal cancer who were treated with bevacizumab-containing chemotherapy as first-line treatment were collected and studied. RESULTS: In the second-line treatment, the bevacizumab plus chemotherapy group showed significantly better progression-free survival (PFS) in comparison to the chemotherapy-alone group in patients with low post-first-line-treatment serum LDH levels. On the other hand, no significant differences in the PFS rate were observed between the two groups in patients with high post-first-line-treatment serum LDH levels. CONCLUSION: The post-first-line-treatment serum LDH levels may, therefore, be useful marker for predicting the efficacy of treatment with BBP.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bevacizumab/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , L-Lactato Desidrogenase/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Inibidores da Angiogênese/uso terapêutico , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto Jovem
16.
Molecules ; 26(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200394

RESUMO

Zinc is an effective anti-inflammatory and antioxidant trace element. The aim of this study was to analyse the protective effect of zinc and zinc-prolactin systems as additives of preservation solutions in the prevention of nephron damage caused during ischemia. The study used a model for storing isolated porcine kidneys in Biolasol®. The solution was modified with the addition of Zn at a dose of 1 µg/L and Zn: 1 µg/L with prolactin (PRL): 0.1 µg/L. After 2 h and 48 h of storage, the levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, sodium, potassium, creatinine and total protein were determined. Zinc added to the Biolasol® composition at a dose of 1 µg/L showed minor effectiveness in the protection of nephrons. In turn, Zn2+ added to Biolasol + PRL (PRL: 0.1 µg/L) acted as a prolactin inhibitor. We do not recommend the addition of Zn(II) (1 µg/L) and Zn(II) (1 µg/L) + PRL (0.1 µg/L) to the Biolasol solution.


Assuntos
Isquemia/metabolismo , Rim/metabolismo , Prolactina/metabolismo , Zinco/metabolismo , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Creatinina/metabolismo , Criopreservação/métodos , L-Lactato Desidrogenase/metabolismo , Preservação de Órgãos/métodos , Perfusão/métodos , Potássio/metabolismo , Sódio/metabolismo , Suínos
17.
PLoS One ; 16(7): e0255154, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324560

RESUMO

BACKGROUND: COVID-19 has been reported in over 40million people globally with variable clinical outcomes. In this systematic review and meta-analysis, we assessed demographic, laboratory and clinical indicators as predictors for severe courses of COVID-19. METHODS: This systematic review was registered at PROSPERO under CRD42020177154. We systematically searched multiple databases (PubMed, Web of Science Core Collection, MedRvix and bioRvix) for publications from December 2019 to May 31st 2020. Random-effects meta-analyses were used to calculate pooled odds ratios and differences of medians between (1) patients admitted to ICU versus non-ICU patients and (2) patients who died versus those who survived. We adapted an existing Cochrane risk-of-bias assessment tool for outcome studies. RESULTS: Of 6,702 unique citations, we included 88 articles with 69,762 patients. There was concern for bias across all articles included. Age was strongly associated with mortality with a difference of medians (DoM) of 13.15 years (95% confidence interval (CI) 11.37 to 14.94) between those who died and those who survived. We found a clinically relevant difference between non-survivors and survivors for C-reactive protein (CRP; DoM 69.10 mg/L, CI 50.43 to 87.77), lactate dehydrogenase (LDH; DoM 189.49 U/L, CI 155.00 to 223.98), cardiac troponin I (cTnI; DoM 21.88 pg/mL, CI 9.78 to 33.99) and D-Dimer (DoM 1.29mg/L, CI 0.9 to 1.69). Furthermore, cerebrovascular disease was the co-morbidity most strongly associated with mortality (Odds Ratio 3.45, CI 2.42 to 4.91) and ICU admission (Odds Ratio 5.88, CI 2.35 to 14.73). DISCUSSION: This comprehensive meta-analysis found age, cerebrovascular disease, CRP, LDH and cTnI to be the most important risk-factors that predict severe COVID-19 outcomes and will inform clinical scores to support early decision-making.


Assuntos
COVID-19/patologia , Proteína C-Reativa/metabolismo , COVID-19/metabolismo , Transtornos Cerebrovasculares/metabolismo , Transtornos Cerebrovasculares/virologia , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Troponina I/metabolismo
18.
Molecules ; 26(11)2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198937

RESUMO

Diabetic nephropathy is reported to occur as a result of the interactions between several pathophysiological disturbances, as well as renal oxidative stress and inflammation. We examined the effect of Malaysian propolis (MP), which has anti-hyperglycemic, antioxidant and anti-inflammatory properties, on diabetes-induced nephropathy. Diabetic rats were either treated with distilled water (diabetic control (DC) group), MP (300 mg/kg b.w./day), metformin (300 mg/kg b.w./day) or MP + metformin for four weeks. We found significant increases in serum creatinine, urea and uric acid levels, decreases in serum sodium and chloride levels, and increase in kidney lactate dehydrogenase activity in DC group. Furthermore, malondialdehyde level increased significantly, while kidney antioxidant enzymes activities, glutathione level and total antioxidant capacity decreased significantly in DC group. Similarly, kidney immunoexpression of nuclear factor kappa B, tumor necrosis factor-α, interleukin (IL)-1ß and caspase-3 increased significantly, while IL-10 immunoexpression decreased significantly in DC group relative to normal control group. Histopathological observations for DC group corroborated the biochemical data. Intervention with MP, metformin or both significantly mitigated these effects and improved renal function, with the best outcome following the combined therapy. MP attenuates diabetic nephropathy and exhibits combined beneficial effect with metformin.


Assuntos
Anti-Inflamatórios/administração & dosagem , Antioxidantes/administração & dosagem , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Metformina/administração & dosagem , Própole/administração & dosagem , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Creatinina/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Sinergismo Farmacológico , L-Lactato Desidrogenase/metabolismo , Masculino , Metformina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Própole/farmacologia , Ratos , Estreptozocina , Regulação para Cima , Ureia/sangue , Ácido Úrico/sangue
19.
Mol Med Rep ; 24(3)2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34296301

RESUMO

Ischemia/reperfusion (I/R)­induced liver injury remains a primary concern in liver transplantation and hepatectomy. Previous studies have indicated that microRNAs (miRs) are involved in multiple pathophysiological processes, including liver I/R. miR­140­5p reportedly inhibits inflammatory responses and apoptosis in several diseases; however, the role of miR­140­5p in liver I/R remains unknown. The present study aimed to investigate the potential role and mechanism of miR­140­5p on liver I/R injury. Mouse liver I/R and mouse AML12 cell hypoxia/reoxygenation (H/R) models were established. miR­140­5p mimics, inhibitor or agonists were used to overexpress or inhibit miR­140­5p in vitro and in vivo. Reverse transcription­quantitative polymerase chain reaction was used to detect miR­140­5p expression. Liver and cell injury were evaluated using several biochemical assays. The association between miR­140­5p and calpain­1 (CAPN1) was confirmed using a dual­luciferase reporter assay. The results revealed that miR­140­5p expression was decreased in the mouse model of liver I/R injury and AML12 cells subjected to H/R, while overexpressed miR­140­5p reduced liver injury in vivo and cell injury in vitro. In addition, CAPN1 was determined to be a target of miR­140­5p; overexpressed CAPN1 abrogated the effect of miR­140­5p on H/R­induced cell injury. The present study indicated that miR­140­5p protected against liver I/R by targeting CAPN1, which may provide a novel therapeutic target for liver I/R injury.


Assuntos
Calpaína/metabolismo , Fígado/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Alanina Transaminase/sangue , Animais , Apoptose/genética , Calpaína/genética , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/genética , Citocinas/metabolismo , Modelos Animais de Doenças , L-Lactato Desidrogenase/metabolismo , Fígado/lesões , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Traumatismo por Reperfusão/patologia , Proteína X Associada a bcl-2/metabolismo
20.
Nat Commun ; 12(1): 4546, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315884

RESUMO

The NLRP3 inflammasome mediates the production of proinflammatory cytokines and initiates inflammatory cell death. Although NLRP3 is essential for innate immunity, aberrant NLRP3 inflammasome activation contributes to a wide variety of inflammatory diseases. Understanding the pathways that control NLRP3 activation will help develop strategies to treat these diseases. Here we identify WNK1 as a negative regulator of the NLRP3 inflammasome. Macrophages deficient in WNK1 protein or kinase activity have increased NLRP3 activation and pyroptosis compared with control macrophages. Mice with conditional knockout of WNK1 in macrophages have increased IL-1ß production in response to NLRP3 stimulation compared with control mice. Mechanistically, WNK1 tempers NLRP3 activation by balancing intracellular Cl- and K+ concentrations during NLRP3 activation. Collectively, this work shows that the WNK1 pathway has a critical function in suppressing NLRP3 activation and suggests that pharmacological inhibition of this pathway to treat hypertension might have negative clinical implications.


Assuntos
Cloretos/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Animais , Caspase 1/metabolismo , Feminino , Imidazóis/farmacologia , Imunidade Inata/efeitos dos fármacos , Interleucina-1beta/metabolismo , L-Lactato Desidrogenase/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Potássio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Piroptose/efeitos dos fármacos , Pirrolidinas/farmacologia , Tamoxifeno/farmacologia , Proteína Quinase 1 Deficiente de Lisina WNK/antagonistas & inibidores
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