Rectangular method: a modified technique for sampling the ischemic border zone in a rat model of transient middle cerebral artery occlusion.

To date, there have been three common methods for sampling the cerebral ischemic border zone in a rat model of transient middle cerebral artery occlusion (tMCAO): the "two o'clock method", the "diagonal method", and the "parallel line method". However, these methods have their own advantages and limitations. Here, we propose a modified technique (the "rectangular method") for sampling the ischemic border zone. A rat tMCAO model was prepared under the support of a compact small animal anesthesia machine. Cerebral blood flow was monitored by high-resolution laser Doppler to control the quality of modeling, and 2,3,5-triphenyl tetrazolium chloride (TTC) staining was used for cerebral infarction location assessment. Superoxide dismutase 2 (SOD2), cysteinyl aspartate specific proteinase (caspase)-3, caspase-9, and heat shock protein 70 (HSP70) were used to verify the reliability and reproducibility of the rectangular method. The expression of biomarkers (SOD2, caspase-3, caspase-9, and HSP70) in the traditional (two o'clock method after TTC staining) and modified (rectangular method) groups were increased. There were no significant differences between the groups. The rectangular method proposed herein is based on a modification of the diagonal method and parallel line method, which could provide a directly observable infarct borderline and a sufficient sampling area for subsequent experimental operations regardless of the cerebral infarct location. The assessed biomarkers (SOD2, caspase-3, caspase-9, and HSP70) demonstrated the reliability and reproducibility of the rectangular method, which may facilitate inter-laboratory comparisons.

CDK4/6 inhibitor palbociclib promotes SARS-CoV-2 cell entry by down-regulating SKP2 dependent ACE2 degradation.

Coronavirus disease 2019 (COVID-19) outbreak has become a global pandemic. CDK4/6 inhibitor palbociclib was reported to be one of the top-scored repurposed drugs to treat COVID-19. As the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry, expression level of angiotensin-converting enzyme 2 (ACE2) is closely related to SARS-CoV-2 infection. In this study, we demonstrated that palbociclib and other methods could arrest cells in G0/G1 phase and up-regulate ACE2 mRNA and protein levels without altering its subcellular localization. Palbociclib inhibited ubiquitin-proteasome and lysosomal degradation of ACE2 through down-regulating S-phase kinase-associated protein 2 (SKP2). In addition, increased ACE2 expression induced by palbociclib and other cell cycle arresting compounds facilitated pseudotyped SARS-CoV-2 infection. This study suggested that ACE2 expression was down-regulated in proliferating cells. Cell cycle arresting compounds could increase ACE2 expression and facilitate SARS-CoV-2 cell entry, which may not be suitable therapeutic agents for the treatment of SARS-CoV-2 infection.

Rectangular method: a modified technique for sampling the ischemic border zone in a rat model of transient middle cerebral artery occlusion

To date, there have been three common methods for sampling the cerebral ischemic border zone in a rat model of transient middle cerebral artery occlusion (tMCAO): the "two o'clock method", the "diagonal method", and the "parallel line method". However, these methods have their own advantages and limitations. Here, we propose a modified technique (the "rectangular method") for sampling the ischemic border zone. A rat tMCAO model was prepared under the support of a compact small animal anesthesia machine. Cerebral blood flow was monitored by high-resolution laser Doppler to control the quality of modeling, and 2,3,5-triphenyl tetrazolium chloride (TTC) staining was used for cerebral infarction location assessment. Superoxide dismutase 2 (SOD2), cysteinyl aspartate specific proteinase (caspase)-3, caspase-9, and heat shock protein 70 (HSP70) were used to verify the reliability and reproducibility of the rectangular method. The expression of biomarkers (SOD2, caspase-3, caspase-9, and HSP70) in the traditional (two o'clock method after TTC staining) and modified (rectangular method) groups were increased. There were no significant differences between the groups. The rectangular method proposed herein is based on a modification of the diagonal method and parallel line method, which could provide a directly observable infarct borderline and a sufficient sampling area for subsequent experimental operations regardless of the cerebral infarct location. The assessed biomarkers (SOD2, caspase-3, caspase-9, and HSP70) demonstrated the reliability and reproducibility of the rectangular method, which may facilitate inter-laboratory comparisons.

Reduced expression of Raf kinase inhibitor protein correlates with poor prognosis in pancreatic cancer.

AIM: Raf kinase inhibitory protein (RKIP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. But its function in pancreatic cancer was not yet clarified completely. The aim of this study was to investigate the involvement of RKIP in pancreatic cancer. METHODS: RKIP expression was investigated retrospectively by immunohistochemistry in paraffin-embedded primary tumor tissue samples from a series (n = 99) of consecutive patients with pancreatic cancer. Survival was calculated using Kaplan-Meier curves. Parameters found to be of prognostic significance in univariate analysis were verified in a multivariate Cox regression model. RESULTS: RKIP expression was high in normal pancreatic epithelium and retained to varying degrees in pancreatic cancer tissues. However, in tumor tissues with lymph node metastasis (P = 0.008) and high UICC stage (P = 0.006), RKIP expression was highly significantly reduced or lost. Furthermore, the reduced expression of RKIP significantly correlated with both poor overall and disease-free survival (P = 0.008 and 0.01, respectively). Multivariate analyses revealed RKIP to be an independent prognosticator. CONCLUSION: These findings suggest that RKIP could be a promising marker for predicting a better prognosis in pancreatic cancer.