[Research progress of femoral bone tunnel positioning in anterior cruciate ligament reconstruction].

Objective: To review the concept and methods of femoral bone tunnel positioning in anterior cruciate ligament (ACL) reconstruction, in order to provide a reference for clinical treatment. Methods: The relevant literature on the concept and methods of femoral bone tunnel positioning in ACL reconstruction in domestic and international research was extensively reviewed. Results: The position of the femoral bone tunnel is a key factor in determining the prognosis of ACL reconstruction. The concept of femoral bone tunnel positioning in ACL reconstruction has experienced isometric reconstruction, anatomical reconstruction, Ribbon-like theory, I.D.E.A.L. theory, and nearly isometric reconstruction theory. The femoral bone tunnel positioning technique is also changing with the in-depth study of the anatomy and biomechanics of the ACL, and each bone tunnel positioning technique has its own advantages and disadvantages. Over-The-Top technique is now mainly used for ACL revision; the clock-face positioning method is basically no longer applicable due to the large error, poor stability, and low retrievability; the bone landmarks positioning method (the lateral condyle of the femur's Resident's ridge and bifurcation ridge, and the the apex of the deep cartilage), which is now mostly used clinically due to the more constant anatomical landmarks. The quadrant method under X-ray fluoroscopy is more cumbersome to implement intraoperatively, so it is mainly used for academic research; computer navigation-assisted positioning has gradually become popular in recent years, which is highly accurate, avoids the influence of human factors on the positioning of the bone tunnel, and has a very good prospect of application; three-dimensional printing-assisted positioning technology, which is accurate in positioning, with a high degree of reproducibility and a short learning curve. Conclusion: The concept of femoral bone tunnel positioning for ACL reconstruction has undergone several evolutions, reflecting the deepening of the understanding of ACL and the improvement of the clinical results of reconstruction. The precision, personalization, and intelligence of positioning techniques are the focus of current and future development.

High flexion femoral side remnant preservation positioning technique: a new method for positioning the femoral tunnel in anterior cruciate ligament reconstruction.

PURPOSE: The aim of this study is to find a new method for femoral side preservation positioning in anterior cruciate ligament (ACL) reconstruction and test the accuracy and precision of this method. METHOD: Fifty patients with isolated ACL rupture (42 males and 8 females) who underwent single-bundle ACL reconstruction in our hospital between July 2022 and July 2023 were included. The lowest point of the cartilage margin of the lateral wall of the intercontinental fossa and the tibial plateau plumb line at 120° of knee flexion were used as the anatomical landmarks for positioning of the femoral tunnel for ACL reconstruction surgery. Femoral side remnant preservation was performed in all cases. Three-dimensional CT was performed 3 days postoperatively to collect the data, which were analyzed using Mimics 21.0 software. We measured the posterior cortical distance of the femoral condyle at 90° of knee flexion and the vertical distance from the center of the bone tunnel to the cortical extension line behind the femur. All femoral tunnel positions were marked on a 4 × 4 grid and visualized using the quadrant method. RESULTS: Using the new positioning method in 50 knees, the average distance of x was 25.26 ± 2.76% of t and the average distance of y was 23.69 ± 6.19% of h. This is close to the results of previous studies, where x was 24.2 ± 4.0% of t and the average distance of y was 21.6 ± 5.2% of h. Most femoral tunnel positions were located in the same area. The D values were distributed as follows: 60% in the range of 0 to 2 mm, 24% in the range of 2 to 4 mm, and 16% more than 4 mm. The E values were distributed as follows: 80% in the range of 0 to 4 mm and 20% more than 4 mm. CONCLUSION: In arthroscopic ACL reconstruction, the knee was flexed at 120° and the lowest point of the cartilage edge of the lateral wall of the intercondylar fossa and the tibial plateau plumb line were used as anatomical landmarks for the positioning of the femoral bone tunnel, which resulted in more accurate femoral bone tunnel positioning, better reproducibility, and better preservation of the femoral stump compared to traditional positioning methods.

Circ_0000419 acts as a tumor suppressor in gastric cancer development via regulating miR-300/RGMB axis.

OBJECTIVE: Dysregulated circular RNAs (circRNAs) have been verified to function in the development of gastric cancer (GC). The current study was designed to investigate the role of circ_0000419 in GC progression, and the potential mechanistic pathway. METHODS: Relative expression of circ_0000419, microRNA-300 (miR-300) and Repulsive Guidance Molecule B (RGMB) was analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay. Cell metastasis, including migration and invasion, was assessed by wound healing and Transwell assays. Glucose consumption and lactate production were examined using kits. The association between miR-300 and circ_0000419 or RGMB was validated by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assays. Role of circ_0000419 in vivo was determined by xenograft experiment. RESULTS: Circ_0000419 and RGMB were downregulated, while miR-300 was upregulated in GC tissues and cells. Gain of circ_0000419 inhibited migration, invasion and glycolysis in GC cells, which was attenuated by introduction of miR-300 or silencing of RGMB. Circ_0000419 sponged miR-300, and RGMB was direct target of miR-300. Circ_0000419 overexpression could block GC tumor growth in vivo. CONCLUSION: Circ_0000419 inhibited GC cell migration, invasion and glycolysis through regulation of miR-300/RGMB axis, at least in part, affording a molecular target for GC treatment.

Hyperoside ameliorates cerebral ischaemic-reperfusion injury by opening the TRPV4 channel in vivo through the IP3-PKC signalling pathway.

CONTEXT: Hyperoside (Hyp), one of the active flavones from Rhododendron (Ericaceae), has beneficial effects against cerebrovascular disease. However, the effect of Hyp on vasodilatation has not been elucidated. OBJECTIVE: To explore the effect of Hyp on vasodilatation in the cerebral basilar artery (CBA) of Sprague-Dawley (SD) rats suffering with ischaemic-reperfusion (IR) injury. MATERIALS AND METHODS: Sprague-Dawley rats were randomly divided into sham, model, Hyp, Hyp + channel blocker and channel blocker groups. Hyp (50 mg/kg, IC50 = 18.3 µg/mL) and channel blocker were administered via tail vein injection 30 min before ischaemic, followed by 20 min of ischaemic and 2 h of reperfusion. The vasodilation, hyperpolarization, ELISA assay, haematoxylin-eosin (HE), Nissl staining and channel-associated proteins and qPCR were analysed. Rat CBA smooth muscle cells were isolated to detect the Ca2+ concentration and endothelial cells were isolated to detect apoptosis rate. RESULTS: Hyp treatment significantly ameliorated the brain damage induced by IR and evoked endothelium-dependent vasodilation rate (47.93 ± 3.09% vs. 2.99 ± 1.53%) and hyperpolarization (-8.15 ± 1.87 mV vs. -0.55 ± 0.42 mV) by increasing the expression of IP3R, PKC, transient receptor potential vanilloid channel 4 (TRPV4), IKCa and SKCa in the CBA. Moreover, Hyp administration significantly reduced the concentration of Ca2+ (49.08 ± 7.74% vs. 83.52 ± 6.93%) and apoptosis rate (11.27 ± 1.89% vs. 23.44 ± 2.19%) in CBA. Furthermore, these beneficial effects of Hyp were blocked by channel blocker. DISCUSSION AND CONCLUSIONS: Although Hyp showed protective effect in ischaemic stroke, more clinical trial certification is needed due to the difference between animals and humans.

[Molecular mechanism of ligustilide attenuating OGD/R injury in PC12 cells by inhibiting ferroptosis].

The aim of this study is to explore the mechanism of ligustilide, the main active constituent of essential oils of traditional Chinese medicine Angelicae Sinensis Radix, on alleviating oxygen-glucose deprivation/reperfusion(OGD/R) injury in PC12 cells from the perspective of ferroptosis. OGD/R was induced in vitro, and 12 h after ligustilide addition during reperfusion, cell viability was detected by cell counting kit-8(CCK-8) assay. DCFH-DA staining was used to detect the level of intracellular reactive oxygen species(ROS). Western blot was employed to detect the expression of ferroptosis-related proteins, glutathione peroxidase 4(GPX4), transferrin receptor 1(TFR1), and solute carrier family 7 member 11(SLC7A11), and ferritinophagy-related proteins, nuclear receptor coactivator 4(NCOA4), ferritin heavy chain 1(FTH1), and microtubule-associated protein 1 light chain 3(LC3). The fluorescence intensity of LC3 protein was analyzed by immunofluorescence staining. The content of glutathione(GSH), malondialdehyde(MDA), and Fe was detected by chemiluminescent immunoassay. The effect of ligustilide on ferroptosis was observed by overexpression of NCOA4 gene. The results showed that ligustilide increased the viability of PC12 cells damaged by OGD/R, inhibited the release of ROS, reduced the content of Fe and MDA and the expression of TFR1, NCOA4, and LC3, and improved the content of GSH and the expression of GPX4, SLC7A11, and FTH1 compared with OGD/R group. After overexpression of the key protein NCOA4 in ferritinophagy, the inhibitory effect of ligustilide on ferroptosis was partially reversed, indicating that ligustilide may alleviate OGD/R injury of PC12 cells by blocking ferritinophagy and then inhibiting ferroptosis. The mechanism by which ligustilide reduced OGD/R injury in PC12 cells is that it suppressed the ferroptosis involved in ferritinophagy.

Has_circ_0071803 promotes colorectal cancer progression by regulating miR-330-5p/MAPK signaling pathway.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. A lack of effective targeted therapies against CRC makes the treatment challenging. Here, we report a circular RNA (circRNA), has_circ_0071803, functioning as an oncogene in CRC. Circ_0071803 was upregulated in CRC tissues and cell lines, and its expression levels were inversely correlated with the prognosis and survival rate of patients with CRC. Circ_0071803 knockdown suppressed cell proliferation, migration, and invasion in CRC. Moreover, we found that circ_0071803 sponged miR-330-5p, thereby upregulating mitogen-activated protein kinase 1 (MAPK1) in CRC cells. The suppression of cell activities by circ_0071803 knockdown were rescued by miR-330-5p inhibition or MAPK1 overexpression. Collectively, our findings elucidate that circ_0071803 promotes CRC progression by regulating the miR-330-5p/MAPK1 pathway, providing potential therapeutic targets for designing effective targeted treatments.

[Mechanism of total flavonoids of Rhododendra simsii in alleviating ischemic brain injury].

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 µg·mL~(-1)) group, and TFR(30 µg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.

Analysis of the Changes and Possible Reasons in Aberrations before and after Surgery in Patients with Concomitant Exotropia.

Objective: The objective is to observe the changes in aberrations before and after surgery in patients with common horizontal strabismus and to analyze the possible reasons for the changes. Methods: Forty eyes of 40 cases with concomitant exotropia who underwent strabismus correction at the Ophthalmology Department of Nantong University Hospital from October 2020 to July 2021 were included in this study, all of whom underwent unilateral lateral rectus recession combined with a medial rectus resection in the same eye. Aberration parameters were measured 1 day before surgery and 1 week, 1 month, 3 months, and 6 months after surgery. Differences in the indicators at each time period were compared by analysis of variance (ANOVA) of repeated measures data for a single factor, and data were analyzed using SPSS 25.0 statistical application software. Results: 5 mm pupil diameter: the preoperative and postoperative RMS of total aberration showed statistically significant difference (P < 0.01). Postoperation test (Bonferroni method) and preoperative comparison at each period after surgery showed statistically significant differences between 6 months after surgery (P=0.002) and preoperative comparison. The preoperative and postoperative comparison of RMS in LOAs was statistically significant (P < 0.01); postoperative test (Bonferroni method) and preoperative comparison showed that there were statistically significant differences between 1 week (P=0.033) and 6 months (P=0.002) after operation. The difference of RMS of defocus before and after operation was statistically significant (P < 0.01); postoperation test (Bonferroni method) and preoperative comparison showed that there was statistically significant difference between 6 months after operation (P=0.007) and preoperative comparison. There was statistically significant difference in preoperative and postoperative RMS of HOAs (P=0.013). Postoperative test (Bonferroni method) and preoperative comparison showed that there was statistically significant difference 6 months after surgery (P=0.03). The RMS of secondary astigmatism showed a statistically significant difference before and after operation (P=0.001), and the postoperation test (Bonferroni method) showed a statistically significant difference 6 months after operation (P=0.002). In 5 mm pupil diameter, the preoperative and postoperative RMS of total aberration showed statistically significant difference (P < 0.01), postoperative test (Bonferroni method) was used to compare each period after surgery with that before surgery, and there were statistically significant differences between 1 week after surgery (P=0.034), 3 months after surgery (P=0.033), and 6 months after surgery (P=0.003). The preoperative and postoperative comparison of RMS in LOAs was statistically significant (P < 0.01), postoperative test (Bonferroni method) was used to compare each period after surgery with that before surgery, and there were statistically significant differences between 1 week after surgery (P=0.04), 3 months after surgery (P=0.034), and 6 months after surgery (P=0.004). The difference of RMS of defocus before and after surgery was statistically significant (P=0.002), and the comparison between postoperation test (Bonferroni method) and preoperation showed that the difference was statistically significant 6 months after surgery (P=0.027). The RMS of astigmatism showed statistically significant difference before and after operation (P=0.002), and the postoperation test (Bonferroni method) showed statistically significant difference between 6 months after operation (P=0.009) and before operation. Conclusion: We found that horizontal rectus surgery had a transient effect on LOAs and almost no effect on HOAs. Long-term follow-up is recommended after strabismus surgery to observe eye position and binocular visual function. Because of the high prevalence of strabismus in adolescents, long-term observation of the eye axis and aberration is recommended.

Frustration and the Kinetic Repartitioning Mechanism of Substrate Inhibition in Enzyme Catalysis.

Substrate inhibition, whereby enzymatic activity decreases with excess substrate after reaching a maximum turnover rate, is among the most elusive phenomena in enzymatic catalysis. Here, based on a dynamic energy landscape model, we investigate the underlying mechanism by performing molecular simulations and frustration analysis for a model enzyme adenylate kinase (AdK), which catalyzes the phosphoryl transfer reaction ATP + AMP ⇋ ADP + ADP. Intriguingly, these reveal a kinetic repartitioning mechanism of substrate inhibition, whereby excess substrate AMP suppresses the population of an energetically frustrated, but kinetically activated, catalytic pathway going through a substrate (ATP)-product (ADP) cobound complex with steric incompatibility. Such a frustrated pathway plays a crucial role in facilitating the bottleneck product ADP release, and its suppression by excess substrate AMP leads to a slow down of product release and overall turnover. The simulation results directly demonstrate that substrate inhibition arises from the rate-limiting product-release step, instead of the steps for populating the catalytically competent complex as often suggested in previous works. Furthermore, there is a tight interplay between the enzyme conformational equilibrium and the extent of substrate inhibition. Mutations biasing to more closed conformations tend to enhance substrate inhibition. We also characterized the key features of single-molecule enzyme kinetics with substrate inhibition effect. We propose that the above molecular mechanism of substrate inhibition may be relevant to other multisubstrate enzymes in which product release is the bottleneck step.

[Protective effect of total flavonoids of Rhododendron simsii on focal cerebral ischemia-reperfusion injury through SOCE pathway in rats].

This paper explored the protective effect of total flavonoids of Rhododendron simsii(TFR) on focal cerebral ischemia-reperfusion injury(CIRI) in rats and its relationship with the store-operated calcium entry(SOCE) pathway regulated by stromal intera-ction molecule(STIM) and calcium release-activated calcium modulator(Orai).Rats were randomly assigned into the sham group, model(middle cerebral artery occlusion, MCAO) group, TFR(60 mg·kg~(-1)) group, TFR(60 mg·kg~(-1))+SOCE pathway inhibitor 2-aminoethoxydiphenyl borate(2-APB, 2.5 mg·kg~(-1)) group, and 2-APB(2.5 mg·kg~(-1)) group.The rats in the sham group and MCAO group were administrated with normal saline, and those in the TFR group and TFR+2-APB group were administrated with TFR(60 mg·kg~(-1)) by gavage for 14 days until sampling.The rats in the 2-APB group and TFR+2-APB group were intraperitoneally injected with 2-APB(2.5 mg·kg~(-1)) after operation.The levels of interleukin-1(IL-1), interleukin-6(IL-6), and tumor necrosis factor-alpha(TNF-α) in serum were measured by ELISA.The cerebral infarction and the pathological status of ischemic brain tissue were detected via TTC staining and HE staining, respectively.The protein and mRNA levels of STIM1, STIM2, Orai1, cysteinyl aspartate specific proteinase 3(caspase-3), and protein kinase B(PKB) in brain tissue were respectively determined by Western blot and RT-qPCR.The growth of brain neurons in each group was observed via immunofluorescence method.The results showed that compared with the MCAO group, TFR lowered the levels of IL-1, IL-6 and TNF-α in serum and the score of neurological function, ameliorated the pathological injury of brain tissue, and decreased the infarct size.Moreover, TFR up-regulated the mRNA and protein levels of STIM1, STIM2, Orai1, and PKB, down-regulated those of caspase-3 in brain tissue, and increased the double-labeled positive cells under fluorescence microscope.However, the above effects were significantly weakened by the addition of 2-APB, a SOCE inhibitor.The results suggested that TFR may play a protective role against focal cerebral ischemia-reperfusion injury by up-regulating the expression of SOCE-related signal molecules, promoting neurogenesis around the ischemic area, improving the survival state of neurons, and redu-cing the activity of inflammatory mediators.

Extraction and Identification of Three New Urechis unicinctus Visceral Peptides and Their Antioxidant Activity.

The viscera of Urechis unicinctus with polypeptides, fatty acids, and amino acids are usually discarded during processing to food. In order to improve the utilization value of the viscera of Urechis unicinctus and avoid resource waste, antioxidant polypeptides were isolated from the viscera of Urechis unicinctus. First, a protein hydrolysate of Urechis unicinctus (UUPH) was prepared by ultrasonic-assisted enzymatic hydrolysis, and the degree of hydrolysis was as high as 79.32%. Subsequently, three new antioxidant peptides (P1, P2, and P3) were purified from UUPH using ultrafiltration and chromatography, and their amino acid sequences were identified as VTSALVGPR, IGLGDEGLRR, TKIRNEISDLNER, respectively. Then, the antioxidant activity of the polypeptide was predicted by the structure-activity relationship and finally verified by experiments on eukaryotic cells. The P1 peptide exhibited the strongest antioxidant activity among these three antioxidant peptides. Furthermore, P1, P2, and P3 have no toxic effect on RAW264.7 cells at the concentration of 0.01~2 mg/mL and can protect RAW264.7 cells from H2O2-induced oxidative damage in a concentration-dependent manner. These results suggested that these three new antioxidant peptides were isolated from the viscera of Urechis unicinctus, especially the P1 peptide, which might serve as potential antioxidants applied in health-derived food or beverages. This study further developed a new use of the by-product of Urechis unicinctus, which improved the comprehensive utilization of marine biological resources.

MicroRNA-4287 alleviates inflammatory response via targeting RIPK1 in osteoarthritis.

Studies have confirmed the regulatory effects of microRNAs (miRNAs) in osteoarthritis (OA) progression. MiR-4287 has been identified by a previous study as a downregulated miRNA in chondrocytes treated with IL-1ß and TNF-α. However, the function of the underlying mechanism of miR-4287 in OA is elusive. IL-1ß-treated chondrocytes were used as OA cell models. RNA expression was accessed using RT-qPCR. Cell Counting Kit-8 (CCK-8) assay was used to determine the chondrocytes' viability and proliferation. The protein levels of inflammation factors (IL-8, IL-6, and TNF-α), matrix metalloproteinases (MMP 1, MMP3, MMP13), and chondrogenic genes (COL2A1, SOX9, and Aggrecan) were detected using western blot analysis. Luciferase reporter assays were performed for interaction exploration. HE staining and Safranin O/Fast Green staining was used to access the pathological changes in OA mouse tissues and cartilage degeneration in OA mouse. MiR-4287 was downregulated in chondrocytes treated with IL-1ß and OA mouse models. MiR-4287 overexpression promoted the viability, and proliferation and attenuated the inflammation response and destruction of cartilage in IL-1ß-stimulated chondrocytes. Receptor-interacting protein kinase 1 (RIPK1) was a target gene of miR-4287 in chondrocytes. MiR-4287 negatively regulated RIPK1 expression. RIPK1 overexpression was revealed to reverse the miR-4287-mediated effects on proliferation and inflammatory response in IL-1ß-stimulated chondrocytes. Moreover, miR-4287 was demonstrated to inhibit the pathological changes, cartilage degeneration and inflammation response in OA mice models. In conclusion, miR-4287 is a critical molecule in OA development, which attenuates inflammatory response in vivo and in vitro by targeting RIPK1.

MiR-18a-3p improves cartilage matrix remodeling and inhibits inflammation in osteoarthritis by suppressing PDP1.

Osteoarthritis (OA) is a degenerative disease characterized by synovial inflammation. MiR-18a-3p was reported to be downregulated in knee anterior cruciate ligament of OA patients. In the present study, the specific functions and mechanism of miR-18a-3p in OA were explored. An in vitro model of OA was established using 10 ng/ml IL-1ß to treat ATDC5 cells, and medial meniscus instability surgery was performed on Wistar rats to establish in vivo rat model of OA. RT-qPCR revealed that miR-18a-3p was downregulated in IL-1ß-stimulated ATDC5 cells. MiR-18a-3p overexpression inhibited secretion of inflammatory cytokines and concentration of matrix metalloproteinases, as shown by ELISA and western blotting. The binding relation between miR-18a-3p and pyruvate dehydrogenase phosphatase catalytic subunit 1 (PDP1) was detected by luciferase reporter assays. MiR-18a-3p targeted PDP1 and negatively regulated PDP1 expression. Results of rescue assays revealed that PDP1 upregulation reserved the suppressive effect of miR-18a-3p overexpression on levels of inflammatory cytokines and matrix metalloproteinases in IL-1ß-stimulated ATDC5 cells. H&E staining was used to observe pathological changes of synovial tissues in the knee joint of Wistar rats. Safranin O-fast green/hematoxylin was used to stain cartilage samples of knee joints. MiR-18a-3p overexpression suppressed OA progression in vivo. Overall, miR-18a-3p improves cartilage matrix remodeling and suppresses inflammation in OA by targeting PDP1.

Blood-based Genomic Profiling of Circulating Tumor DNA from Patients with Advanced Pancreatic Cancer and its Value to Guide Clinical Treatment.

Objective: Pancreatic cancer (PC) is a malignant tumor with limited therapeutic choices and extremely poor prognosis. Personalized therapy based on gene alternations is a promising choice. Considering tumor heterogeneity, the practice of ctDNA analysis has drawn the attention. Here, we try to assess the applicability of ctDNA in PC. Methods and materials: Next generation sequencing (NGS) was performed from blood samples of 223 PC patients and tissue sample of 564 PC patients. Genomic data from the TCGA database were also utilized. In addition, two cases received personalized treatment based on ctDNA sequencing results were reported. Results: Based on ctDNA sequencing, the genomic features of PC was revealed. Totally, 68.2% of patients detected at least one reportable genomic alteration (GA) from ctDNA. The frequently altered genes were KRAS (53.5%), followed by TP53 (52.8%), and CDKN2A (15.1%). Cell cycle control (8%) and DNA damage response (8%) pathways enriched the most mutated genes. Compared with mutations from tissue samples and a tissue-genomic database, similar frequencies of GAs were detected from ctDNA. The first two highest frequent mutation of genes were the same, but some of mutated genes were inclined to be observed in ctDNA, like AR. And two cases who received personalized therapy achieved better clinical benefit. Conclusion: Blood-source ctDNA sequencing could be regarded as a meaningful complement to tissue testing, and might guide clinically therapeutic regimen.

High glucose causes apoptosis of rabbit corneal epithelial cells involving activation of PERK-eIF2α-CHOP-caspase-12 signaling pathway.

AIM: To investigate the effect of high concentration of glucose (HCG) on double stranded RNA-activated protein kinase-like ER kinase (PERK)-eukaryotic initiation factor-2α (eIF2α)-transcription factor C/EBP homologous protein (CHOP)-cysteine aspartate specific proteinase (caspase-12) signaling pathway activation and apoptosis in rabbit corneal epithelial cells (RCECs). METHODS: RCECs were treated by different concentrations of glucose for 0-48h. The expressions of PERK, p-PERK, eIF2α, p-eIF2α, 78 kDa glucose-regulated protein 78 (GRP78), CHOP, B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-2-associated X protein (Bax) and caspase-12 were determined by Western blot. Apoptosis was detected by TUNEL assay. Meanwhile, the function of PERK-eIF2α-CHOP-caspase-12 signaling pathway activation in high glucose-induced apoptosis was evaluated using PERK inhibitor, GSK2606414. RESULTS: HCG significantly promoted the expression of p-PERK, p-eIF2α, GRP78, CHOP, Bax and cleaved caspase-12 in RCECs (P<0.05), while remarkably decreased the expression of Bcl-2 and caspase-12 (P<0.05), and the alterations caused by glucose were in concentration- and time-dependent manners. Meanwhile, PERK and eIF2α expressions were not affected in all groups (P>0.05). TUNEL assay showed that the apoptosis rate of RCECs in the HCG group increased significantly in contrast with that in the normal concentration of glucose or osmotic pressure control group (P<0.05), and the apoptosis rate increased with the increase of glucose concentration within limits (P<0.05). GSK2606414 down-regulated the expression of p-PERK and p-eIF2α in the HCG group (P<0.05), while still did not affect the expression of PERK and eIF2α among groups (P>0.05). Correspondingly, GSK2606414 also significantly reduced the apoptosis rate induced by high glucose (P<0.05). CONCLUSION: HCG activates PERK-eIF2α-CHOP-caspase-12 signaling pathway and promotes apoptosis of RCECs.

Role of substrate-product frustration on enzyme functional dynamics.

Natural enzymes often have enormous catalytic power developed by evolution. Revealing the underlying physical strategy used by enzymes to achieve high catalysis efficiency is one of the central focuses in the field of biological physics. Our recent work demonstrated that multisubstrate enzymes can utilize steric frustration encountered in the substrate-product cobound complex to overcome the bottleneck of the enzymatic cycle [W. Li et al., Phys. Rev. Lett. 122, 238102 (2019)10.1103/PhysRevLett.122.238102]. However, the key atomic-level interactions by which the steric frustration contributes to the enzymatic cycle remain elusive. In this work we study the microscopic mechanism for the role of the substrate-product frustration on the key physical steps in the enzymatic cycle of adenylate kinase (AdK), a multisubstrate enzyme catalyzing the reversible phosphoryl transfer reaction ATP+AMP⇋ADP+ADP. By using atomistic molecular dynamics simulations with enhanced sampling, we showed that the competitive interactions from the phosphate groups of the substrate ATP and product ADP in the ATP-ADP cobound complex of the AdK lead to local frustration in the binding pockets. Such local frustration disrupts the hydrogen bond network around the binding pockets, which causes lowered barrier height for the opening of the enzyme conformations and expedited release of the bottleneck product ADP. Our results directly demonstrated from the atomistic level that the local frustration in the active sites of the enzyme can be utilized to facilitate the key physical steps of the enzymatic cycle, providing numerical evidence to the predictions of the previous theoretical work.

High Concentration of Glucose Increases Reactive Oxygen Species Generation and Apoptosis Induced by Endoplasmic Reticulum Stress Pathway in Rabbit Corneal Epithelial Cells.

OBJECTIVE: To investigate the effect of high concentration of glucose on reactive oxygen species (ROS) production in rabbit corneal epithelial cells (RECEs) and explore whether the increased ROS initiates the apoptosis process of RECEs through oxidative stress and endoplasmic reticulum (ER) stress pathway. METHODS: RECEs were treated by different concentrations of glucose for a while, and then the production of ROS was detected by flow cytometry. The expressions of PERK, p-PERK, Akt, p-Akt, and CHOP were determined by western blot, and the cell viability was measured by Cell Counting Kit-8 (CCK-8). Flow cytometry was used to detect the early apoptosis rate. Meanwhile, the effects of N-acetyl-L-cysteine (NAC), an active oxygen inhibitor, on the experimental results were observed. RESULTS: Compared with the normal glucose concentration group, the fluorescence intensity of ROS in the high concentration (1 mM glucose) of glucose group was significantly increased (P < 0.05). NAC-inhibited ROS production was induced by high concentration of glucose (P < 0.05).Western blot demonstrated that the expressions of the p-PERK and CHOP increased significantly (P < 0.05), the p-Akt expression decreased (P < 0.05), and the PERK and Akt expressions did not change significantly in the high concentration of glucose group compared to the normal concentration group. CCK-8 results revealed that compared with the normal concentration of glucose group, the cell activity of the high concentration of glucose group decreased. For the cells in the high concentration of glucose group, the cell survival rate of NAC-treated cells was higher than that of untreated (P < 0.05). The flow cytometry results indicated that the early apoptosis rate of the cells in the high concentration of glucose group increased in contrast with that in the normal concentration of glucose group (P < 0.05). Treating the cells in the high concentration of glucose group with NAC could reduce the cell apoptosis resulted from high glucose (P < 0.05). CONCLUSIONS: High concentration of glucose may induce the formation of ROS which leads to oxidative stress and ER stress in RECEs and even leads to cell apoptosis. The reactive oxygen inhibitor, NAC, can play a protective character in the high concentration of glucose environment. These results might provide theoretical basis for the study of the diabetes-related dry eye.

The anti-tumor effect of pachymic acid on osteosarcoma cells by inducing PTEN and Caspase 3/7-dependent apoptosis.

Pachymic acid (PA) is a lanostane type triterpenoid isolated from Poria cocos, which possesses an anti-tumor effect in breast cancer, prostate cancer, lung cancer, and bladder cancer cells. In this study, we investigated the effect of PA on the growth and apoptosis of human immortalized cell line (HOS) and primary osteosarcoma cells by a Cell Counting Kit-8 (CCK-8) and Annexin V and propidium iodide (PI) staining, respectively. Western blot was used to measure the expression of cleaved Caspase 3, PTEN, and AKT, as well as the AKT phosphorylation. The Caspase 3 activity was determined using the Caspase-3 Colorimetric Assay Kit. From the results, PA significantly reduced cell proliferation in a concentration- and time-dependent manner. PA also induced cell apoptosis in a dose-dependent fashion. PA treatment led to increased Caspase 3 activation and PTEN expression, as well as reduced AKT phosphorylation. Moreover, Ac-DEVD-CHO (a Caspase 3/7 inhibitor) pre-treatment or PTEN knockdown partially blocked the effects of PA on cell proliferation and apoptosis. Caspase 3/7 inhibitor had an additive effect with PTEN knockdown. Collectively, our results suggested that induction of apoptosis by PA was mediated in part by PTEN/AKT signaling and Caspase 3/7 activity. This study provides evidence that PA might be useful in the treatment of human osteosarcoma.