Poliserosite, artrite e doença respiratória em leitões: estudo de caso

O complexo da poliserosite em suínos é causado por múltiplos factores, em particular pelo agente Haemophilus parasuis (HPS), que tem vindo a causar vários problemas em suínos, quer ao nível respiratório, quer nas articulações. O agente HPS é o responsável pela Doença de Glässer (DG), caracterizada por uma inflamação generalizada da pleura, pericárdio, peritoneu, membranas sinoviais e meninges, acompanhada pela elevada presença de fibrina. HPS é normalmente um agente secundário causado por algum factor predisponente, como stress, pneumonias ou por algum vírus, em particular o Vírus do Síndrome Respiratório e Reprodutor Porcino (PRRSv).No caso das pneumonias verificou-se que o agente HPS é um invasor secundário oportunista e que causa doença em associação com outros agentes víricos ou bacterianos e está associado com maior prevalência de pneumonia por agentes respiratórios víricos, como o Síndrome Respiratório e Reprodutor Porcino (PRRS). Durante este estudo de caso acompanhou-se um lote de leitões desde o desmame até ao final da recria, em que foram feitas serologias e enviados leitões inteiros para laboratório de forma a isolar e identificar o agente. Foi feita a avaliação da morbilidade bem como o cálculo do índice de tosse e taxa de mortalidade nas primeiras quatro semanas de recria. Efectuaram-se necropsias dos leitões deste lote. Pretende-se, como objectivo deste estudo, definir o agente primário causador da patologia, neste caso o PRRSv, bem como a entrada da infecção bacteriana secundária causada pelo HPS. Procedeu-se à vacinação do efectivo frente ao PRRSv. Como profilaxia, devido à falha de protecção de imunidade maternal, fez-se a administração de tulatromicina a todos os leitões até todo o efectivo estar devidamente protegido frente ao PRRSv, conferindo assim uma boa imunidade maternal.The porcine polyserositis complex is caused by multiple factors, particularly by Haemophilus parasuis (HPS), which has been causing several problems in pigs, both at the respiratory and articular levels. HPS is the causative agent of Glässer's Disease (DG), characterized by a generalized inflammation of the pleura, pericardium, peritoneum, synovial membranes and meninges, toguether with an abundant presence of fibrin. HPS is usually a secondary agent enhanced by some predisposing factor, like stress, pneumonia or some viruses, such as Porcine Reproductive and Respiratory Syndrome virus (PRRSv). In pneumonia, it has been show that HPS is an opportunistic secondary invader causing disease in association with other viral or bacterial agents being PRRSv the most prevalent respiratory viral agent found together with HPS. During this study a batch of piglets was followed from weaning until the end of nursery season, when serologies were performed, samples were collected and whole piglets were sent to pathology in order to isolate and identify the agent. Health condition was evaluated as well as the calculation of cough index and mortality rate in the first four weeks of nursery. Necropsies of the piglets of this batch were also carried out.The objective of this study was to define the primary agent causing disease (in this case PRRSv), as well as the factors that predisposed to secondary bacterial infections caused by HPS. Following these findings all herd was vaccinated against PRRSv. Due to lack of protective maternal immunity, tulathromycin was administered in pigs, as prophylactic measure until all sows were adequately protected against PRRSv, thus conferring good maternal immunity to their offspring

Extracellular nanomatrix-induced self-organization of neural stem cells into miniature substantia nigra-like structures with therapeutic effects on Parkinsonian rats

Substantia nigra (SN) is a complex and critical region of the brain wherein Parkinson's disease (PD) arises from the degeneration of dopaminergic neurons. Miniature SN‐like structures (mini‐SNLSs) constructed from novel combination of nanomaterials and cell technologies exhibit promise as potentially curative cell therapies for PD. In this work, a rapid self‐organization of mini‐SNLS, with an organizational structure and neuronal identities similar to those of the SN in vivo, is achieved by differentiating neural stem cells in vitro on biocompatible silica nanozigzags (NZs) sculptured by glancing angle deposition, without traditional chemical growth factors. The differentiated neurons exhibit electrophysiological activity in vitro. Diverse physical cues and signaling pathways that are determined by the nanomatrices and lead to the self‐organization of the mini‐SNLSs are clarified and elucidated. In vivo, transplantation of the neurons from a mini‐SNLS results in an early and progressive amelioration of PD in rats. The sculptured medical device reported here enables the rapid and specific self‐organization of region‐specific and functional brain‐like structures without an undesirable prognosis. This development provides promising and significant insights into the screening of potentially curative drugs and cell therapies for PD

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Role of p14ARF in TWIST-mediated senescence in prostate epithelial cells

Recently, TWIST, a basic helix-loop-helix transcription factor, is suggested to be an oncogene because of its over-expression in many types of human cancer and its positive role in promoting cell survival. The aim of this study was to investigate the role of TWIST on the growth of human epithelial cells. Using two immortalized human prostate epithelial cell lines, we demonstrated that inactivation of TWIST by small RNA technology led to the promotion of cellular senescence and growth arrest, suggesting that TWIST plays a key role in the continuous proliferation of immortalized cells. Over-expression of TWIST, in contrast, resulted in suppression of cellular senescence in response to genotoxic damage and promotion of cell proliferation with DNA damage accumulation, indicating that TWIST promotes genomic instability. In addition, we also found that the TWIST-mediated cellular senescence was regulated through its negative effect on p14ARF and subsequent suppression of MDM2/p53 and Chk1/2 DNA damage response pathways. Our results suggest that over-expression of TWIST results in down-regulation of p14ARF, which leads to the impairment of DNA damage checkpoint in response to genotoxic stress. This negative effect of TWIST on DNA damage response facilitates uncontrolled cell proliferation with genomic instability and tumorigenesis in non-malignant cells. © The Author 2007. Published by Oxford University Press. All rights reserved.link_to_OA_fulltex

Role of MEK/ERK pathway in the MAD2-mediated cisplatin sensitivity in testicular germ cell tumour cells

Testicular germ cell tumour (TGCT) is the most common malignancy in young males. Although most TGCTs are sensitive to cisplatin-based chemotherapy, significant numbers of TGCT patients still relapse and die each year because of the development of resistance to cisplatin. Previously, we first reported that a key regulator of the mitotic checkpoint, mitotic arrest deficient-2 (MAD2), was a mediator of cisplatin sensitivity in human cancer cells. In this study, we investigated whether MAD2 played a role in cellular sensitivity to cisplatin in TGCT cells and the underlying molecular mechanisms responsible. Using 10 TGCT cell lines, we found that increased MAD2 expression was correlated with cellular sensitivity to cisplatin, which was associated with activation of the MEK pathway. Treatment of cells expressing high levels of MAD2 with an MEK inhibitor, U0126, led to cellular protection against cisplatin-induced apoptosis. Inactivation of MAD2 by transfecting a dominant-negative construct in TGCT cells with high levels of MAD2 resulted in the suppression of MEK pathway and resistance to cisplatin-induced cell death. These results support previous suggestion on the involvement of mitotic checkpoint in DNA damage response in human cancer cells and demonstrate a possible molecular mechanism responsible for the MAD2-mediated sensitivity to cisplatin in TGCT cells. Our results also suggest that downregulation of MAD2 may be an indicator for identification of TGCT cancer cells that are potentially resistant to cisplatin-based therapy. © 2006 Cancer Research.link_to_OA_fulltex

Effect on Germline Mutation Rate in a High-Risk Chinese Breast Cancer Cohort after Compliance with The National Comprehensive Cancer Network (NCCN) 2023 v.1 Testing Criteria

Background: The National Comprehensive Cancer Network (NCCN) testing criteria for the high-penetrance breast cancer susceptibility genes, specifically BRCA1, BRCA2, CDH1, PALB2, PTEN, and TP53, have been recently modified in 2023 to 2023 v.1. The following criteria have been changed: (1) from a person diagnosed with breast cancer at ≤45 to ≤50; (2) from aged 45–50 of personal breast diagnosis to any age of diagnosis with multiple breast cancers; and (3) from aged ≥51 of personal breast diagnosis to any age of diagnosis with family history listed in NCCN 2022 v.2. Methods: High-risk breast cancer patients (n = 3797) were recruited from the Hong Kong Hereditary Breast Cancer Family Registry between 2007 and 2022. Patients were grouped according to NCCN testing criteria 2023 v.1 and 2022 v.2. A 30-gene panel for hereditary breast cancer was performed. The mutation rates on high-penetrance breast cancer susceptibility genes were compared. Results: About 91.2% of the patients met the 2022 v.2 criteria, while 97.5% of the patients met the 2023 v.1 criteria. An extra 6.4% of the patients were included after the revision of the criteria, and 2.5% of the patients did not meet both testing criteria. The germline BRCA1/2 mutation rates for patients meeting the 2022 v.2 and 2023 v.1 criteria were 10.1% and 9.6%, respectively. The germline mutation rates of all 6 high-penetrance genes in these two groups were 12.2% and 11.6%, respectively. Among the additional 242 patients who were included using the new selection criteria, the mutation rates were 2.1% and 2.5% for BRCA1/2 and all 6 high-penetrance genes, respectively. Patients who did not meet both testing criteria were those with multiple personal cancers, a strong family history of cancers not listed in the NCCN, unclear pathology information, or the patient’s voluntary intention to be tested. The mutation rates of BRCA1/2 and the 6 high-penetrance genes in these patients were 5.3% and 6.4%, respectively. Conclusion: This study provided a real-world application of the revision of NCCN guidelines and its effect on the germline mutation rate in the Chinese population. Applying the updated criteria for further genetic investigation would increase the positive detection rate, and potentially more patients would benefit. The balance between the resource and outcome requires careful consideration

MAD2 interacts with DNA repair proteins and negatively regulates DNA damage repair

MAD2 (mitotic arrest deficient 2) is a key regulator of mitosis. Recently, it had been suggested that MAD2-induced mitotic arrest mediates DNA damage response and that upregulation of MAD2 confers sensitivity to DNA-damaging anticancer drug-induced apoptosis. In this study, we report a potential novel role of MAD2 in mediating DNA nucleotide excision repair through physical interactions with two DNA repair proteins, XPD (xeroderma pigmentosum complementation group D) and ERCC1. First, overexpression of MAD2 resulted in decreased nuclear accumulation of XPD, a crucial step in the initiation of DNA repair. Second, immunoprecipitation experiments showed that MAD2 was able to bind to XPD, which led to competitive suppression of binding activity between XPD and XPA, resulting in the prevention of physical interactions between DNA repair proteins. Third, unlike its role in mitosis, the N-terminus domain seemed to be more important in the binding activity between MAD2 and XPD. Fourth, phosphorylation of H2AX, a process that is important for recruitment of DNA repair factors to DNA double-strand breaks, was suppressed in MAD2-overexpressing cells in response to DNA damage. These results suggest a negative role of MAD2 in DNA damage response, which may be accounted for its previously reported role in promoting sensitivity to DNA-damaging agents in cancer cells. However, the interaction between MAD2 and ERCC1 did not show any effect on the binding activity between ERCC1 and XPA in the presence or absence of DNA damage. Our results suggest a novel function of MAD2 by interfering with DNA repair proteins. © 2008 Elsevier Ltd. All rights reserved.link_to_subscribed_fulltex

NHE7 upregulation potentiates the uptake of small extracellular vesicles by enhancing maturation of macropinosomes in hepatocellular carcinoma

Abstract Background Small extracellular vesicles (sEVs) mediate intercellular communication that contributes to hepatocellular carcinoma (HCC) progression via multifaceted pathways. The success of cell entry determines the effect of sEV on recipient cells. Here, we aimed to delineate the mechanisms underlying the uptake of sEV in HCC. Methods Macropinocytosis was examined by the ability of cells to internalize dextran and sEV. Macropinocytosis was analyzed in Na(+)/H(+) exchanger 7 (NHE7)‐knockdown and ‐overexpressing cells. The properties of cells were studied using functional assays. pH biosensor was used to evaluate the intracellular and endosomal pH. The expression of NHE7 in patients’ liver tissues was examined by immunofluorescent staining. Inducible silencing of NHE7 in established tumors was performed to reveal the therapeutic potential of targeting NHE7. Results The data revealed that macropinocytosis controlled the internalization of sEVs and their oncogenic effect on recipient cells. It was found that metastatic HCC cells exhibited the highest efficiency of sEV uptake relative to normal liver cells and non‐metastatic HCC cells. Attenuation of macropinocytic activity by 5‐(N‐ethyl‐N‐isopropyl)‐amiloride (EIPA) limited the entry of sEVs and compromised cell aggressiveness. Mechanistically, we delineated that high level of NHE7, a sodium‐hydrogen exchanger, alkalized intracellular pH and acidized endosomal pH, leading to the maturation of macropinosomes. Inducible inhibition of NHE7 in established tumors developed in mice delayed tumor development and suppressed lung metastasis. Clinically, NHE7 expression was upregulated and linked to dismal prognosis of HCC. Conclusions This study advances the understanding that NHE7 enhances sEV uptake by macropinocytosis to promote the malignant properties of HCC cells. Inhibition of sEV uptake via macropinocytosis can be exploited as a treatment alone or in combination with conventional therapeutic approaches for HCC