[Role of Orai 1-mediated store-operated calcium entry in the immune function of CD4+ T cells in septic mice].

Objective: To investigate the role of Orai1-mediated store-operated calcium entry in the immune damage of CD4+ T cells in septic mice. Methods: Sepsis mouse model was established by cecal ligation and puncture(CLP). Balb/c mice of clean grade were sacrificed 1, 3, and 5 days after operation. Spleen samples were harvested at given intervals. Splenic CD4+ T cells were selected by immunomagnetic beads and the expression of Orai1 protein was detected by western blotting, the storage operated calcium entry (SOCE) was detected by flow cytometry, the apoptosis of CD4+ T cells was detected by flow cytometry, the proliferation of CD4+ T cells was detected by CCK-8, and the IFN-γ and IL-4 were detected by enzyme-linked immunosorbent assay (ELISA). Then the expression of Orai1 protein was regulated to further detect the SOCE and immune function of splenic CD4+ T cells in mice. The experiment was divided into 4 groups, sham group, CLP3 group, Orai1 down group (Orai1-down group) and Orai1 up regulation group (Orai1-up group). Results: The relative expression of Orai1 protein in splenic CD4+ T cells in sham group was 1.03±0.16. Compared with sham group, Orai1 protein levels in CLP Group were all significantly lower (F=19.64, P=0.000 5). The increased value of splenic CD4+ T cells fluorescence intensity in sham group was 494±41. Compared with sham group, the levels of SOCE in CLP Group were all lower (F=30.01, P=0.001). The ratio of early and late apoptosis of CD4+ T cells in sham group was 8.7%±1.5%. Compared with sham group, the early and late apoptosis rates of CLP Group were significantly higher (F=32.29, P=0.000 1). The OD of sham group was 0.81±0.10 at 450 nm. Compared with sham group, the proliferation ability of splenic CD4+ T cells in CLP Group were significantly decreased (F=7.26, P=0.001 8). Compared with sham group, the secretion of IFN-γ and IL-4 by CD4+ T cells and the ratio of IFN-γ/IL-4 in CLP Group were all significantly decreased (F=19.690, 6.183, 11.230, all P<0.05). Compared with CLP3 group, the increased value of fluorescence intensity of CD4+ T cells was significantly decreased, the early and late apoptosis ratio of CD4+ T cells was significantly increased, the OD450 nm value of CD4+ T cells was decreased, the multiplication capacity of splenic CD4+ T cells were decreased, the level of IFN-γ and IL-4 secreted by T cells were decreased, and the value of IFN-γ/IL-4 in orai1-down group was decreased (t=4.819, 7.952, 2.988, 28.760, 3.140, 7.670, all P<0.05). However, Orail-up group showed the opposite trend. Conclusion: Orai1-mediated store-operated calcium entry can alleviate the immune dysfunction of CD4+ T cells in septic mice.

Effect of mannose on the lung function of rats with acute pancreatitis.

The present study aimed to investigate the mechanisms by which mannose protects the lung injury induced in rats with acute pancreatitis (AP). An AP combined with Acute Lung Injury (ALI) model was established. A total of 90 healthy adult male Sprague-Dawley rats (300±50g weight) were randomly divided into three groups: sham operation group (SO group), severe acute pancreatitis lung injury group (SAP group), and mannose intervention group (MT group). Subsequently, each group was divided into two subgroups based on the time passed from intervention, namely 6 and 12 h. Each subgroup comprised 15 rats. The ratio of wet/dry weight of the lung tissue exhibited no significant change at different time points in the SO group. This parameter was significantly increased in the SAP group compared with the SO group at each time point of the treatment (P less than 0.05) and it was significantly lower in the MT group than that in the AP group (P less than 0.05) and it was significantly increased in the AP group at each time (P less than 0.05) compared with the SO group. The levels of TNF-α in the lung tissue in the SO group exhibited no significant change at different time points, but they were significantly decreased in the MT group at each time point (P less than 0.05) compared with the SAP group. The mannose receptor (MR) mRNA and protein levels in the lung tissues exhibited no significant change at different time points. The mRNA and protein levels of MR in the SAP group were significantly decreased at each time point (P less than 0.05) compared with the SO group. The mRNA and protein levels of MR, in the lung tissue of the MT group were significantly increased at each time point compared with the SAP group (P less than 0.05). Mannose could reduce the injury caused to the lung tissue of rats with severe acute pancreatitis by up-regulation of the expression of MR mRNA and protein.

[Correlation between Movement Distance of Small Intestinal Contents and Survival Time in Female Rats after Feeding].

OBJECTIVES: To study the correlation between the movement distance of small intestinal contents and survival time in female SD rat models after one-time satiation, and to evaluate its application value for postmortem interval estimation. METHODS: Adult female SD rats were randomly divided into postprandial groups （1, 2, 3, 4 and 5 h after feeding） and control group. The postprandial groups were fed for 1 h, meanwhile control group was kept fasting. All rats were sacrificed at the given time. The contents in stomach and small intestine were observed, described, compared and photographed, and the movement distance of small intestinal contents was measured. The data of postprandial groups were analysed by one-way analysis of variance. RESULTS: The stomach and duodenum of control group were empty with a little thin and yellow small intestinal liquid. The gastral cavities of 1 h postprandial group were full of undigested food. The evolutionary changes of character, colour and content were observed in the gastric and small intestinal contents of other postprandial groups. The movement distance of intestinal contents increased while the empty part decreased gradually. The differences among the postprandial groups were statistically significant （P<0.05）. CONCLUSIONS: After a 24 h fasting with free drinking and the following 1 h feeding, an ideal animal model can be established successfully on female SD rats, which can provide an experimental basis for postmortem interval estimation based on the changes of small intestinal contents in forensic practice.

[Age Estimation of Adult Living Donor by Pubic MSCT Three-dimensional Reconstruction].

OBJECTIVES: To establish a method for the age estimation of adult living donor based on pubic MSCT three-dimensional reconstruction and verify its accuracy and reliability. METHODS: The volume rendering （VR） image data of pubic symphysis surface were collected from 300 volunteers aged over 17 years old. According to different age groups, the age estimation of these volunteers was performed by the method and formula of pubic symphysis surface. RESULTS: In the 300 volunteers, the difference between biological age and actual age was <1 year in 117 cases, >1-2 years in 178 cases, >2 years in 5 cases. CONCLUSIONS: MSCT three-dimensional reconstruction technology of pubic symphysis surface can be used to estimate the age of adult living donor, which can provide a high accurate and reliable result.

Expression and clinical significance of rhubarb on serum amylase and TNF-alpha of rat model of acute pancreatitis.

The aim of this study was to evaluate the therapeutic effect of rhubarb extract on acute pancreatitis. Ninety-six healthy Sprague Dawley rats, weighing 301±5.12 g were randomly divided into 4 groups: sham surgery (group A), acute pancreatitis model (group B), acute pancreatitis with normal saline (group C), and acute pancreatitis model with rhubarb (group D). The levels of serum amylase (AMY) and TNF-α were measured at 1st, 6th, 12th and 24th hour after modeling, and the pancreatic tissue were used to observe the pathologic changes. Compared to the sham group, the serum AMY and serum tumor necrosis factor (TNF-α) levels were significantly increased in the other groups (p <0.05). Compared to the model group and the saline group, the serum AMY, serum TNF-α level and pathological changes of rats in the rhubarb group were significantly lower (p <0.05). The serum AMY and TNF-α levels increased in acute pancreatitis. The rhubarb reduced the serum AMY and TNF-α level in rats with acute pancreatitis and reduced the pathological changes of pancreas and other tissues.

Long non-coding RNA ANCR promotes progression of NSCLC by inhibiting E-Ca expression.

The article "Long non-coding RNA ANCR promotes progression of NSCLC by inhibiting E-Ca expression, by T. Zhou, J.-J. Fang, Y.-X. Zhou, Z.-P. Li, L. Jiang, W.-W. Han, Z.-H. Zhu, published in Eur Rev Med Pharmacol Sci 2020; 24 (3): 1250-1257-DOI: 10.26355/eurrev_202002_20178-PMID: 32096155" has been withdrawn from the authors. They stated that "during the last few months, new experimental data have been obtained and analyzed, so we want to rewrite the paper to further test the cell experiment and give more evidence to support our paper. It is difficult to repeat the part of the cell experiment, and the results need to be further improved. In addition, the internal reference pictures in the paper are wrong and need to be re experimented and modified. This needs more detailed studies and maybe a long time, so we want to withdraw our paper and resubmit it when it is ready". The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/20178.

Long non-coding RNA ANCR promotes progression of NSCLC by inhibiting E-Ca expression.

OBJECTIVE: This study aimed to investigate whether long-chain non-coding ANCR is involved in the progression of non-small cell LCa (NSCLC) and its possible molecular mechanisms. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was applied to examine ANCR expression in 48 cases of NSCLC and adjacent normal tissues. In addition, ANCR level in patients of different tumor staging was analyzed. The Kaplan-Meier method was applied to analyze the interplay between ANCR expression and the prognosis of patients with NSCLC. Subsequently, qRT-PCR was performed to detect ANCR level in LCa cell lines. After knocking down ANCR in A549 cells, ANCR and E-Ca mRNA expression were examined by qRT-PCR, while the expression levels of epithelial-mesenchymal transition (EMT)-related proteins were detected by Western blot. At the same time, cell viability and migration ability were analyzed through cell counting kit-8 (CCK-8) and cell wound healing assay, respectively. RNA immunoprecipitation (RIP) test was performed to verify the binding of ANCR to EZH2. After knocking down EZH2 in A549 cells, E-Ca messenger ribonucleic acid (mRNA) expression was detected. Additionally, Chromatin immunoprecipitation (ChIP) assay was performed to detect the binding of EZH2 to the E-Ca promoter region. When E-Ca and ANCR were simultaneously knocked down in A549 cells, Western blot investigation was performed to examine the expression of EMT-related proteins, while CCK-8 and wound healing assays were applied to figure out the changes in cell viability and cell migration capacity. RESULTS: ANCR level was conspicuously higher in NSCLC tissues than that in normal tissues, and that in T3 and T4 tumors was also higher than that in T1 and T2. Meanwhile, ANCR expression in the tissues of patients with lymph node metastasis was conspicuously higher than those without metastasis. Survival analysis revealed that the overall survival of patients with NSCLC with high expression of ANCR was conspicuously lower than patients with low expression of ANCR. The qRT-PCR study verified that ANCR was highly expressed in the LCa cell line A549. After knocking down ANCR in A549 cells, ANCR and E-Ca mRNA levels were found conspicuously decreased, and so were the expression levels of EMT-related proteins, as well as the cell viability and migration ability. The RIP assay result indicated that ANCR can indeed bind to EZH2. E-Ca mRNA expression was elevated after the knockdown of EZH2 in A549 cells. In addition, the result of CHIP test demonstrated that EZH2 could combine with E-Ca. Simultaneous down-regulation of ANCR and E-Ca in A549 cells could reverse the influence of knocking down ANCR alone on cell viability and migration ability. CONCLUSIONS: Long-chain non-coding RNA ANCR was highly expressed in NSCLC tissues and could enhance the viability and malignancy of NSCLC cells by inhibiting the expression of E-Ca, thereby promoting the progression of NSCLC.

A comparative study of transfection methods for RNA interference in bone marrow-derived murine dendritic cells.

Selective gene silencing using RNA interference (RNAi) has been shown to be an efficient method for manipulation of cellular functions. In this study, we compare three previously established methods for transfection of murine bone marrow-derived DC (BM-DC). We tested the efficacy of electroporation with the Mouse Nucleofector kit((R)) from Amaxa Biosystems and lipid-based transfection methods using transfection reagents from Santa Cruz Biotechnology or Genlantis. To analyse the transfection efficacy we used FITC-conjugated siRNA as a positive control together with CD80 and CD86 specific siRNA. We show that electroporation using the Mouse Nucleofector kit((R)) from Amaxa Biosystems was not an efficient method to transfect BM-DC with siRNA in our hands. Transfection with Santa Cruz Biotechnology reagents resulted in up to 59% FITC-siRNA positive cells, but did not result in effective silencing of CD80 surface expression. In contrast, the most effective method was the lipid-based method using the siRNA transfection reagent GeneSilencer((R)) from Genlantis. This protocol resulted in up to 92% FITC-siRNA positive cells after 4 h which declined to 62% and 59% 24 and 48 h post-transfection, respectively. The transfected BM-DC remained CD11c positive, expressed high MHC class II and intermediate CD40 and were functional as APC. In conclusion, this protocol was effective for manipulation of murine BM-DC function through the use of specific siRNA and such methods can be important for the future study of DC-T cell interactions.

Evaluation of mandibular contour in patients with significant facial asymmetry.

Most previous studies on facial asymmetry have not specifically differentiated mandible deviation from structural asymmetry of the mandible. The purpose of this study was to assess the symmetry of the mandible by examining its contour in a cohort of patients with significant facial asymmetry. Eleven cases of facial asymmetry with chin deviation ≥10mm were enrolled. A voxel-paired median plane (optimal symmetry plane, OSP) and two landmark-based median planes were generated. The OSP was created by computing the best pairing of the bony voxels on the two sides. One side of the mandibular contour was mirrored onto the other side using the test plane. The contour differences were measured by distance and by area ratio. They were examined both in frontal and frontal downward inclined view. The contour symmetry of the mandible was that revealed by the plane that presented the best symmetry. The results showed that the OSP worked best in bisecting the contour into two symmetrical halves. Contour analysis showed relatively small discrepancies between the two sides. In conclusion, the mandibles retained an acceptable contour symmetry despite the presence of significant mandibular deviations. It is suggested that proper mandibular alignment be the primary objective in the correction of facial asymmetry.

Portable system for quantifying motor abnormalities in Parkinson's disease.

In order to quantify motor disabilities in Parkinson's disease (PD), we designed a compact, portable, neurophysiological system based upon a personal computer to measure tremor, bradykinesia, and muscle tone. Tremor was detected by solid state accelerometers and translated into a digital signal. The system displayed the root mean square displacements and frequency distribution of the tremor in the horizontal and vertical planes, along with a reconstructed graphic image of the displacement. Bradykinesia was measured using a panel that detects release and depression of switches in response to auditory and visual signals; the system calculated subjects' reaction times and movement times in milliseconds. Tone at the elbow was measured by strapping the upper extremity to a lightweight low-friction cradle and then passively moving the cradle with an instrumented handle. Signals representing torque and arm angle were processed by the computer and displayed in real time on the screen with stiffness as a mean slope in Nm/degree. In clinical tests, quantitative measures of tremor, movement time and rigidity were significantly abnormal in PD patients compared to control subjects. We conclude that this system is a convenient and accurate method to quantitate important aspects of the parkinsonian syndrome, and may be applied to quantitate other movement disorders.

The IFN regulatory factor family participates in regulation of Fas ligand gene expression in T cells.

TCR engagement leads to the transcriptional activation of cytokine genes and activation-induced cell death. Activated T cells undergo apoptosis upon expression and ligation of Fas ligand (FasL) to Fas/APO-1 (CD95) receptor. FasL expression is under the transcriptional regulation of multiple factors. The present study demonstrates that TCR-inducible FasL expression is also under the direct influence of the IFN regulatory factor (IRF) transcription factor family. Deletion and mutagenesis of a putative IRF-1 binding site in the FasL promoter results in deficient expression of FasL. EMSAs demonstrate specific FasL promoter binding by IRF-1 and IRF-2. Forced expression of either IRF-1 or IRF-2 leads to FasL promoter activation in T cells and FasL expression in heterologous cells. Finally, suppression of IRF-1 expression in T cells results in deficient TCR-induced FasL expression. These results confirm that the IRF family participates in the regulation of FasL gene expression.

Magnetic stimulation of the human motor cortex: ipsilateral and contralateral facilitation effects.

Voluntary contraction of a muscle greatly increases the amplitude and decreases the latency of the motor potentials evoked by electromagnetic coil brain stimulation (facilitation). Facilitation has also been observed with contraction of a nearby ipsilateral and a contralateral homologous muscle. We studied these facilitatory relationships in 5 normal subjects in small hand, forearm and upper arm muscles using surface-recorded compound motor action potentials, single motor unit recordings, and post-stimulus time histograms. There was no evidence for spread of facilitation between any pair of muscles if the muscle from which motor evoked potentials (MEPs) were recorded was completely at rest during brain stimulation, although this sometimes required training to accomplish or could not be achieved. Thus, although spread of facilitation has been observed by others under these conditions, we did not find this effect. There may be significant interindividual variations in the degree of facilitatory spread.