A quinolinyl resveratrol derivative alleviates acute ischemic stroke injury by promoting mitophagy for neuroprotection via targeting CK2α'.

Ischemic stroke (IS) is a serious threat to human health. The naturally derived small molecule (E)-5-(2-(quinolin-4-yl) ethenyl) benzene-1,3-diol (RV01) is a quinolinyl analog of resveratrol with great potential in the treatment of IS. The aim of this study was to investigate the potential mechanisms and targets for the protective effect of the RV01 on IS. The mouse middle cerebral artery occlusion and reperfusion (MCAO/R) and oxygen-glucose deprivation and reperfusion (OGD/R) models were employed to evaluate the effects of RV01 on ischemic injury and neuroprotection. RV01 was found to significantly increase the survival of SH-SY5Y cells and prevent OGD/R-induced apoptosis in SH-SY5Y cells. Furthermore, RV01 reduced oxidative stress and mitochondrial damage by promoting mitophagy in OGD/R-exposed SH-SY5Y cells. Knockdown of CK2α' abolished the RV01-mediated promotion on mitophagy and alleviation on mitochondrial damage as well as neuronal injury after OGD/R. These results were further confirmed by molecular docking, drug affinity responsive target stability and cellular thermal shift assay analysis. Importantly, in vivo study showed that treatment with the CK2α' inhibitor CX-4945 abolished the RV01-mediated alleviation of cerebral infarct volume, brain edema, cerebral blood flow and neurological deficit in MCAO/R mice. These data suggest that RV01 effectively reduces damage caused by acute ischemic stroke by promoting mitophagy through its interaction with CK2α'. These findings offer valuable insights into the underlying mechanisms through which RV01 exerts its therapeutic effects on IS.

Kellerin alleviates cerebral ischemic injury by inhibiting ferroptosis via targeting Akt-mediated transcriptional activation of Nrf2.

BACKGROUND: Ischemic stroke (IS) is characterized as a detrimental cerebrovascular disease with high mortality and disability. Ferroptosis is a novel mechanism involved in neuronal death. There is a close connection between IS and ferroptosis, and inhibiting ferroptosis may provide an effective strategy for treating IS. Our previous investigations have discovered that kellerin, the active compound of Ferula sinkiangensis K. M. Shen, possesses the capability to shield against cerebral ischemia injury. PURPOSE: Our objective is to clarify the relationship between the neuroprotective properties of kellerin against IS and its ability to modulate ferroptosis, and investigate the underlying regulatory pathway. STUDY DESIGN: We investigated the impact and mechanism of kellerin in C57BL/6 mice underwent middle cerebral artery occlusion/reperfusion (MCAO/R) as well as SH-SY5Y cells exposed to oxygen-glucose deprivation/ re-oxygenation (OGD/R). METHODS: The roles of kellerin on neurological severity, cerebral infarction and edema were investigated in vivo. The regulatory impacts of kellerin on ferroptosis, mitochondrial damage and Akt/Nrf2 pathway were explored. Molecular docking combined with drug affinity responsive target stability assay (DARTS) and cellular thermal shift assay (CETSA) were performed to analyze the potential target proteins for kellerin. RESULTS: Kellerin protected against IS and inhibited ferroptosis in vivo. Meanwhile, kellerin improved the neuronal damage caused by OGD/R and suppressed ferroptosis by inhibiting the production of mitochondrial ROS in vitro. Further we found that kellerin directly interacted with Akt and enhanced its phosphorylation, leading to the increase of Nrf2 nuclear translocation and its downstream antioxidant genes expression. Moreover, kellerin's inhibitory effect on ferroptosis and mitochondrial ROS release was eliminated by inhibiting Akt/Nrf2 pathway. CONCLUSIONS: Our study firstly demonstrates that the neuroprotective properties of kellerin against IS are related to suppressing ferroptosis through inhibiting the production of mitochondrial ROS, in which its modulation on Akt-mediated transcriptional activation of Nrf2 plays an important role. This finding shed light on the potential mechanism that kellerin exerts therapeutic effects in IS.

Mechanism of Circ_HECW2 regulating osteoblast apoptosis in osteoporosis by attenuating the maturation of miR-1224-5p.

BACKGROUND: Osteoporosis (OP) poses a significant clinical challenge with escalating morbidity. This study explores Circ_HECW2 expression in OP patients and its regulatory role in lipopolysaccharide (LPS)-induced osteoblast apoptosis. METHODS: Circ_HECW2 expression in OP patient serum and healthy controls was quantified using RT-qPCR. Diagnostic value of Circ_HECW2 for OP was assessed via ROC curve. Pearson's correlation model examined associations between indicators. Human osteoblasts HFOB1.19, treated with LPS, were analyzed for Circ_HECW2, pre-miR-1224, miR-1224-5p, and PDK2 mRNA levels. TUNEL assay determined cell apoptosis and Western blot assessed cleaved-caspase-3 protein levels. RNase R resistance assay and actinomycin D assay confirmed Circ_HECW2's cyclic structure. RNA pull-down and dual-luciferase reporter assay verified binding relationships between Circ_HECW2 and miR-1224 and between miR-1224-5p and PDK2. RESULTS: Circ_HECW2 exhibited elevated expression in OP patients with diagnostic significance and a negative correlation with lumbar T-score. LPS co-culture increased Circ_HECW2 expression in HFOB1.19 cells, significantly elevating apoptosis index and cleaved-caspase-3. Circ_HECW2 downregulation inhibited HFOB1.19 apoptosis, reduced pre-miR-1224 expression, and elevated mature miR-1224-5p. Circ_HECW2 bound to pre-miR-1224, and inhibiting miR-1224-5p reversed the effect of Circ_HECW2 downregulation on osteoblast apoptosis. miR-1224-5p targeted PDK2 transcription. CONCLUSION: Circ_HECW2, highly expressed in OP, holds diagnostic significance and reflects disease severity. Circ_HECW2 reduces mature miR-1224-5p by binding to pre-miR-1224, upregulating PDK2, and facilitating LPS-induced osteoblast apoptosis.

Emodin treatment of papillary thyroid cancer cell lines in vitro inhibits proliferation and enhances apoptosis via downregulation of NF­κB and its upstream TLR4 signaling.

Thyroid cancer is one of the most common types of endocrine malignancy. In addition to surgical treatment, it is very important to find new treatment methods. The aim of the present study was to evaluate the effect of 1,3,8-trihydroxy-6-methylanthraquinone (emodin) on cellular NF-κB components and the upstream regulatory pathway of toll-like receptor 4 (TLR4) signaling, as well as the invasion and migration of papillary thyroid carcinoma (PTC) cells. The protein expression of NF-κB components p65 and p50 and their phosphorylated (p-) forms in the sections of PTC tissues was measured by individual immunohistochemical assays. PTC cell lines TPC-1 and IHH4 were exposed to 20 and 40 µM emodin for 24 h. The levels of the NF-κB components p65, p50, c-Rel, p-p65 and p-p50, elements in TLR4 signaling, including TLR4, MYD88 innate immune signal transduction adaptor (MyD88), interferon regulatory factor 3, AKT and MEK, and proliferative and apoptotic biomarkers, including c-Myc, cyclin D1, proliferating cell nuclear antigen, Bcl-2 and Bax, were evaluated by western blotting and immunofluorescent assays. The invasion and migration of PTC cell lines exposed to emodin were tested by plate colony and wound healing assay. Compared with hyperplasia tissue, the expression levels of NF-κB components p65 and p50, and p-p65 and p-p50 in PTC tissue were significantly increased. Treatment of PTC cell lines with emodin lead to significantly reduced levels of the aforementioned NF-κB components, accompanied by markedly downregulated TLR4 signaling. MYD 88-dependent and -independent pathways, are also significantly down-regulated. Downregulation of proliferative factors and activation of apoptotic factors were observed in the cell lines following treatment with emodin. Consequently, inhibition of the invasion and migration activities were observed in the emodin-treated PTC cells. Emodin could inhibit proliferation and promote apoptosis of PTC cells, which is dependent on the downregulation of cellular NF-κB and the TLR4 signaling pathway.

Clinical characteristics of radiation-induced optic neuropathy: A single-center retrospective study.

Purpose: To observe the clinical and imaging characteristics of radiation-induced optic neuropathy (RION). Methods: We retrospectively reviewed the clinical data of 43 patients (69 eyes) who were diagnosed with RION at the Chinese PLA General Hospital from 2010 to 2021. Results: The latency from radiotherapy to onset of visual loss ranged from 1 to 132 (36.33 â€‹± â€‹30.48) months. Optic disc pallor and optic disc edema were found in 27.0% (10/37) and 8.1% (3/37) of the eyes, respectively, within 2 months. After treatment, the best corrected visual acuity (BCVA) was restored in 24.6% (17/69) of the eyes and the final BCVA improved in 13.0% (9/69) of the eyes. An 82.5% (33/40) of the eyes with magnetic resonance imaging (MRI) showed enhancement of the affected optic nerve, mostly (69.7%) in the intracranial segment, and 36.4% (12/33) of the eyes with expansion and T2-high signals also showed enhancement of the affected optic nerve. The superior retinal nerve fiber layer (RNFL) and the outer circle superior quadrant (OS) of the inner limiting membrane to retinal pigment epithelium (ILM-RPE) layer thinned significantly during the first month. The center of the ILM-RPE layer thickened significantly during the first two months and the inner circle temporal quadrant (IT) of the ILM-RPE layer thickened significantly from the third to sixth month. The RNFL thinned significantly after 6 months except for the temporal quadrant, and the average inner circle superior quadrant (IS) and outer circle of the ILM-RPE layer thinned significantly after 6 months. There was no significant difference between hyperbaric oxygen therapy (HBOT) and high-dose intravenous methylprednisolone (IVMP) therapy in improving BCVA recovery or final BCVA (P â€‹> â€‹0.05). Conclusions: The structural damage of the RNFL and ILM-RPE layer occurred during the first month, the RNFL showed progressive thinning during the follow-up period, while the ILM-RPE layer showed thinning during the first month, thickening from the third to sixth month, and thinning after 6 months. There was a discrete region of enhancement of the optic nerve, often with expansion and high-T2 signals on MRI. HBOT and high-dose IVMP therapy were hardly effective for treating RION in the non-acute stage.

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor.

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant diseases. CAR-T cells are genetically modified T cells that express a chimeric protein that recognizes and binds to a cell surface target, resulting in the killing of the target cell. Traditional CAR-T cell manufacturing methods are labor-intensive, expensive, and may carry the risk of contamination. The CliniMACS Prodigy, an automated cell processor, allows for manufacturing cell therapy products at a clinical scale in a closed system, minimizing the risk of contamination. Processing occurs semi-automatically under the control of a computer and thus minimizes human involvement in the process, which saves time and reduces variability and errors. This manuscript and video describes the T cell transduction (TCT) process for manufacturing CAR-T cells using this processor. The TCT process involves CD4+/CD8+ T cell enrichment, activation, transduction with a viral vector, expansion, and harvest. Using the Activity Matrix, a functionality that allows ordering and timing of these steps, the TCT process can be customized extensively. We provide a walk-through of CAR-T cell manufacturing in compliance with current Good Manufacturing Practice (cGMP) and discuss required release testing and preclinical experiments that will support an Investigational New Drug (IND) application. We demonstrate the feasibility and discuss the advantages and disadvantages of using a semi-automatic process for clinical CAR-T cell manufacturing. Finally, we describe an ongoing investigator-initiated clinical trial that targets pediatric B-cell malignancies [NCT05480449] as an example of how this manufacturing process can be applied in a clinical setting.

In vivo hematopoietic stem cell modification by mRNA delivery.

Hematopoietic stem cells (HSCs) are the source of all blood cells over an individual's lifetime. Diseased HSCs can be replaced with gene-engineered or healthy HSCs through HSC transplantation (HSCT). However, current protocols carry major side effects and have limited access. We developed CD117/LNP-messenger RNA (mRNA), a lipid nanoparticle (LNP) that encapsulates mRNA and is targeted to the stem cell factor receptor (CD117) on HSCs. Delivery of the anti-human CD117/LNP-based editing system yielded near-complete correction of hematopoietic sickle cells. Furthermore, in vivo delivery of pro-apoptotic PUMA (p53 up-regulated modulator of apoptosis) mRNA with CD117/LNP affected HSC function and permitted nongenotoxic conditioning for HSCT. The ability to target HSCs in vivo offers a nongenotoxic conditioning regimen for HSCT, and this platform could be the basis of in vivo genome editing to cure genetic disorders, which would abrogate the need for HSCT.

A machine-learning model that incorporates CD45 surface expression predicts hematopoietic progenitor cell recovery after freeze-thaw.

BACKGROUND AIMS: Sufficient doses of viable CD34+ (vCD34) hematopoietic progenitor cells (HPCs) are crucial for engraftment. Additional-day apheresis collections can compensate for potential loss during cryopreservation but incur high cost and additional risk. To aid predicting such losses for clinical decision support, we developed a machine-learning model using variables obtainable on the day of collection. METHODS: In total, 370 consecutive autologous HPCs, apheresis-collected since 2014 at the Children's Hospital of Philadelphia, were retrospectively reviewed. Flow cytometry was used to assess vCD34% on fresh products and thawed quality control vials. The ratio of vCD34% thawed to fresh, which we call "post-thaw index," was used as an outcome measure, with a "poor" post-thaw index defined as <70%. HPC CD45 normalized mean fluorescence intensity (MFI) was calculated by dividing CD45 MFI of HPCs to the CD45 MFI of lymphocytes in the same sample. We trained XGBoost, k-nearest neighbor and random forest models for the prediction and calibrated the best model to minimize falsely-reassuring predictions. RESULTS: In total, 63 of 370 (17%) products had a poor post-thaw index. The best model was XGBoost, with an area under the receiver operator curve of 0.83 evaluated on an independent test data set. The most important predictor for a poor post-thaw index was the HPC CD45 normalized MFI. Transplants after 2015, based on the lower of the two vCD34% values, showed faster engraftment than older transplants, which were based on fresh vCD34% only (average 10.6 vs 11.7 days, P = 0.0006). CONCLUSIONS: Transplants taking into account post-thaw vCD34% improved engraftment time in our patients; however, it came at the cost of unnecessary multi-day collections. The results from applying our predictive algorithm retrospectively to our data suggest that more than one-third of additional-day collections could have been avoided. Our investigation also identified CD45 nMFI as a novel marker for assessing HPC health post-thaw.

Rambutan-liked Pickering emulsion stabilized by cellulose nanocrystals for enhancing anti-bacterial activity and anti-inflammatory effect of Chimonanthus nitens Oliv. essential oil.

Owing to volatility and poor water solubility, the medical application of Chimonanthus nitens Oliv. essential oil (CEO) in the fields of medicine was strictly limited. To tackle this problem, a novel CEO loaded rambutan-liked Pickering emulsion (CEO-RPE) with a spiky surface was effectively designed by coating with carboxymethyl cellulose sodium modified cellulose nanocrystals (CCN) as stabilizer. The effect of CCN concentration on the formation and stabilization of CEO-RPE was investigated. The results showed that CEO-RPE stabilized by 1 % CCN had a smaller droplet size and exhibited a rambutan-liked surface, and was stabilized against concentrated salt and high pH condition due to the steric barrier of CCN that covered in the droplet surface. Subsequently, the antibacterial performance of CEO-RPE was investigated against E. coli, S. aureus, P. aeruginosa, and S. pneumoniae by determining the minimum inhibitory concentration (MIC). The results showed that the CEO-RPE exhibited higher antibacterial activity compared to CEO, which could be attributed to its effective adhesion to the cell membrane of bacteria. In addition, the results of anti-inflammatory experiments showed that CEO-RPE also exhibited strong anti-inflammatory effect on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats. Therefore, the CCN stabilized rambutan-liked Pickering emulsion seemed to be a promising strategy to increase the antibacterial and anti-inflammatory activity of CEO.

Loureirin C inhibits ferroptosis after cerebral ischemia reperfusion through regulation of the Nrf2 pathway in mice.

BACKGROUND: Ischemic stroke (IS) is considered as a serious cerebral vascular disease. Ferroptosis is a novel type of regulated cell death (RCD), that closely related to the occurrence and progress of IS. Loureirin C, a type of dihydrochalcone compound derived from the Chinese Dragon's blood (CDB). The effective components extracted from CDB have shown neuroprotective effects in ischemia reperfusion models. However, the role of Loureirin C in mice after IS is not well understood. Thus, it is worth to identify the effect and mechanism of Loureirin C on IS. PURPOSE: The present research aims to prove the existence of ferroptosis in IS and explore whether Loureirin C can inhibit ferroptosis by regulating nuclear factor E2 related factor 2 (Nrf2) pathway in mice and exert neuroprotective effects on IS models. METHODS: Middle cerebral artery occlusion and reperfusion (MCAO/R) model was established to evaluate the occurrence of ferroptosis and the potential Loureirin C brain-protective effect in vivo. The analysis of free iron, glutamate content, reactive oxygen species (ROS) and lipid peroxidation levels, along with transmission electron microscope (TEM) was applied to prove the existence of ferroptosis. The function of Loureirin C on Nrf2 nuclear translocation was verified by immunofluorescence staining. In vitro, primary neurons and SH-SY5Y cells were processed with Loureirin C after oxygen and glucose deprivation-reperfusion (OGD/R). ELISA kits, western blotting, co-immunoprecipitation (Co-IP) analysis, immunofluorescence, and quantitative real-time PCR were devoted to proving the neuroprotective effects of Loureirin C on IS via regulating ferroptosis and Nrf2 pathways. RESULTS: The results showed that Loureirin C not only dramatically alleviated brain injury and inhibited neurons ferroptosis in mice after MCAO/R, but also dose-dependently reduce ROS accumulation in ferroptosis after OGD/R. Further, Loureirin C inhibits ferroptosis by activating Nrf2 pathway, and promoting nuclear translocation of Nrf2. Besides, Loureirin C increases heme oxygenase 1 (HO-1), quinone oxidoreductase 1 (NQO1) and glutathione peroxidase 4 (GPX4) content after IS. Intriguingly, the anti-ferroptosis effect of Loureirin C is weakened by Nrf2 knockdown. CONCLUSION: Our discoveries first revealed that the inhibitory action of Loureirin C on ferroptosis may greatly depend on its adjusting effect on the Nrf2 pathway, suggesting that Loureirin C could act as a novel anti-ferroptosis candidate and play a therapeutic role in IS. These novel discoveries on the role of Loureirin C on IS models reveal an innovative method that may contribute to neuroprotection for the prevention of IS.

Manufacture and Characterization of Good Manufacturing Practice-Compliant SARS-COV-2 Cytotoxic T Lymphocytes.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 virus-specific cytotoxic T-cell lymphocytes (vCTLs) could provide a promising modality in COVID-19 treatment. We aimed to screen, manufacture, and characterize SARS-CoV-2-vCTLs generated from convalescent COVID-19 donors using the CliniMACS Cytokine Capture System (CCS). METHODS: Donor screening was done by stimulation of convalescent COVID-19 donor peripheral blood mononuclear cells with viral peptides and identification of interferonγ (IFN-γ)+ CD4 and CD8 T cells using flow cytometry. Clinical-grade SARS-CoV-2-vCTLs were manufactured using the CliniMACS CCS. The enriched SARS-CoV-2-vCTLs were characterized by T-cell receptor sequencing, mass cytometry, and transcriptome analysis. RESULTS: Of the convalescent donor blood samples, 93% passed the screening criteria for clinical manufacture. Three validation runs resulted in enriched T cells that were 79% (standard error of the mean 21%) IFN-γ+ T cells. SARS-CoV-2-vCTLs displayed a highly diverse T-cell receptor repertoire with enhancement of both memory CD8 and CD4 T cells, especially in CD8 TEM, CD4 TCM, and CD4 TEMRA cell subsets. SARS-CoV-2-vCTLs were polyfunctional with increased gene expression in T-cell function, interleukin, pathogen defense, and tumor necrosis factor superfamily pathways. CONCLUSIONS: Highly functional SARS-CoV-2-vCTLs can be rapidly generated by direct cytokine enrichment (12 hours) from convalescent donors. CLINICAL TRIALS REGISTRATION: NCT04896606.

Long non-coding RNA MALAT1 promotes cell proliferation, migration and invasion by targeting miR-590-3p in osteosarcoma.

Osteosarcoma (OS) is a common malignant bone cancer and commonly occurs in adolescents and children. Long non-coding RNAs (lncRNAs) play major roles in cancer cell proliferation and metastasis. The present study aimed to investigate the potential molecular mechanism of lncRNA MALAT1 in OS. The levels of lncRNA MALAT1 and microRNA-590-3p were detected by reverse transcription-quantitative PCR in OS tissues and cells. Cell Counting Kit-8 and flow cytometry assays were conducted to assess cell proliferation and apoptosis. Cell migration and invasion were examined by Transwell assay. The levels of E-cadherin, N-Cadherin, Vimentin and Snail were measured by western blotting. The target of MALAT1 was predicted using online software and confirmed by luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. The results indicated that MALAT1 was highly expressed in OS tissues and cell lines. MALAT1 knockdown promoted apoptosis and suppressed proliferation, migration, invasion and epithelial- mesenchymal transition (EMT) of OS cells. Overexpression of miR-590-3p increased cell apoptosis and hampered cell proliferation, migration, invasion and EMT in OS cells. In addition, MALAT1 knockdown upregulated the expression of miR-590-3p in OS cells. In conclusion, MALAT1 was demonstrated to suppress cell apoptosis and induce cell proliferation, migration, invasion and EMT by inhibiting miR-590-3p in OS, which indicated that MALAT1 has potential value in the diagnosis and treatment of OS.

Effect and mechanism of a novel Mg-Nd-Gd-Sr alloy on osteogenic differentiation of bone marrow mesenchymal stem cells.

We investigated the effect and mechanism of a novel Mg-3Nd-1Gd-0.3Sr-0.2Zn-0.4Zr (abbreviated to Mg-Nd-Gd-Sr) alloy on the osteogenic differentiation of bone marrow mesenchymal stem cells extracted from Sprague-Dawley rats. Cultured cells were divided into five groups: a control group cultured in osteogenic induction medium alone without Mg-Nd-Gd-Sr alloy extract, and four experimental groups cultured in the same medium with 25%, 50%, 75%, and 100% Mg-Nd-Gd-Sr alloy extracts, respectively. After 14 days of culture, ALP activity was determined and expressions of osteogenesis-related factors Runx2, OCN, and OPN at the mRNA level and Runx2, OCN, and OPN at the protein level were detected by RT-PCR and western blot, respectively. After 21 days of culture, mineralized nodules were detected by alizarin red staining. The results showed that bone marrow mesenchymal stem cells from Sprague-Dawley rats were successfully isolated in vitro using the whole bone marrow adherence method. Flow cytometry revealed that the cells expressed high levels of CD44 and CD90, but low levels of CD31 and CD45. Alizarin red staining indicated the formation of mineralized nodules in all five groups. Compared with the control group, the number of mineralized nodules was increased significantly in the four experimental groups (p < 0.05). The ALP activity in each group was significantly higher on day 14 than on day 7, and was significantly higher in the four experimental groups compared with the control group (p < 0.05). Moreover, the ALP activity was highest when the concentration of Mg-Nd-Gd-Sr alloy extract was 75% (p < 0.05). RT-PCR results showed that, compared with the control group, the mRNA expression of Runx2, OPN, and OCN was significantly higher in the four experimental groups (p < 0.05), and the highest mRNA expression of Runx2, OPN, and OCN was observed in the 75% experimental group (p < 0.05). Western blotting showed that Mg-Nd-Gd-Sr alloy extract significantly increased the protein expression of Runx2, OCN, and OPN compared with the control group (p < 0.05). Our data indicate that the novel Mg-Nd-Gd-Sr alloy can promotes the osteogenic differentiation of bone marrow mesenchymal stem cells isolated from Sprague-Dawley rats. During this process, there is an increase in the expressions of Runx2, OPN, and OCN mRNAs and Runx2, OCN, and OPN proteins.

circ-RANGAP1/MicroRNA-542-3p/Myosin Regulatory Light Chain Interacting Protein Axis Modulates the Osteosarcoma Cell Progression.

Objective: This study is aimed at exploring the influence of circular RNA- (circRNA-) RANGAP1 targeting microRNA- (miR-) 542-3p/myosin regulatory light chain interacting protein (MYLIP) on the biological function of osteosarcoma (OS) cells. Methods: Tumor tissues and normal tissues were collected from OS patients and circ-RANGAP1, miR-542-3p, and MYLIP expression was tested by RT-qPCR. The correlation between the clinicopathology/prognosis of patients with OS and circ-RANGAP1 expression was observed. Human OS cell line MG-63 was screened to determine the influences of circ-RANGAP1 and miR-542-3p on OS cell progression. The targeting relation of circ-RANGAP1, miR-542-3p, and MYLIP was probed. Results: circ-RANGAP1 expression was elevated in tumor tissues from OS patients, which was correlated to the poor clinicopathology. circ-RANGAP1 expression was augmented in males or patients younger than 20 years old or patients with advanced OS. Higher circ-RANGAP1 expression indicated a poor prognosis in OS patients. After silencing circ-RANGAP1 or elevating miR-542-3p in MG63 cells, cell progression was limited. miR-542-3p downregulation reduced the therapeutic efficacy of silenced circ-RANGAP1. circ-RANGAP1 bound with miR-542-3p to target MYLIP. Conclusion: Silenced circ-RANGAP1 boosts MYLIP expression via competitive binding of miR-542-3p to facilitate OS cell progression.

Unrelated donor α/ß T cell- and B cell-depleted HSCT for the treatment of pediatric acute leukemia.

Unrelated donor (URD) hematopoietic stem cell transplant (HSCT) is associated with an increased risk of severe graft-versus-host disease (GVHD). TCRαß/CD19 depletion may reduce this risk, whereas maintaining graft-versus-leukemia. Outcome data with TCRαß/CD19 depletion generally describe haploidentical donors, with relatively few URDs. We hypothesized that TCRαß/CD19-depletion would attenuate the risks of GVHD and relapse for URD HSCT. Sixty pediatric and young adult (YA) patients with hematologic malignancies who lacked a matched-related donor were enrolled at 2 large pediatric transplantation centers between October 2014 and September 2019. All patients with acute leukemia had minimal residual disease testing, and DP typing was available for 77%. All patients received myeloablative total body irradiation- or busulfan-based conditioning with no posttransplant immune suppression. Engraftment occurred in 98%. Four-year overall survival was 69% (95% confidence interval [CI], 52%-81%), and leukemia-free survival was 64% (95% CI, 48%-76%), with no difference between lymphoid and myeloid malignancies (P = .6297 and P = .5441, respectively). One patient (1.7%) experienced primary graft failure. Relapse occurred in 11 patients (3-year cumulative incidence, 21%; 95% CI, 11-34), and 8 patients (cumulative incidence, 15%; 95% CI, 6.7-26) experienced nonrelapse mortality. Grade III to IV acute GVHD was seen in 8 patients (13%), and 14 patients (26%) developed chronic GVHD, of which 6 (11%) had extensive disease. Nonpermissive DP mismatch was associated with higher likelihood of acute GVHD (odds ratio, 16.50; 95% CI, 1.67-163.42; P = .0166) but not with the development of chronic GVHD. URD TCRαß/CD19-depleted peripheral HSCT is a safe and effective approach to transplantation for children/YAs with leukemia. This trial was registered at www.clinicaltrials.gov as #NCT02323867.

Increased expression of lncRNA SNHG7 promotes the cell viability, migration, and invasion in pre-eclampsia via modulating the miR-214-5p/TWIST1 axis.

PURPOSE: This study is aimed to elucidate the molecular mechanism of lncRNA SNHG7 on pre-eclampsia (PE). METHODS: The expression of SNHG7, miR-214-5p and TWIST1 in PE placental tissues was detected by qRT-PCR. The regulatory mechanism of SNHG7/miR-214-5p/TWIST1 axis on PE was determined using MTT, wound healing, transwell invasion, and western blot assays. RESULTS: In PE pregnancies, SNHG7 and TWIST1 were decreased, while miR-214-5p was increased.The elevated miR-214-5p and decreased TWIST1 partly eliminated the promoting effects of SNHG7 up-regulation on the viability and metastasis of JEG-3 cells. CONCLUSIONS: Up-regulated SNHG7 protects against PE through modulating the miR-214-5p/TWIST1 axi.

NDRG1 regulates osteosarcoma cells via mediating the mitochondrial function and CSCs differentiation.

BACKGROUND: Cancer stem cells (CSCs) are mainly contributed to malignancy metastatic potential and resistant therapy of osteosarcoma (OS). The mitochondria-related apoptosis was generally accepted as the target of tumor therapy. However, the effect of N-myc downstream-regulated gene 1 (NDRG1) on CSCs and mitochondrial health in OS is still unknown. METHODS: In OS cells, MG63 and U2OS, the siRNA of NDRG1 were conducted. Transwell, western blot, RT-qPCR, and mitochondria isolation were used to identify the effect of NDRG on OS cells and mitochondria. Moreover, the differentiation-related factors of CSCs were determined. RESULTS: After downregulation of NDRG1, the cell viability, invasion ability decreased whereas cell apoptosis increased. The expressions profiles of fibronectin, vimentin, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP) 2, MMP9, and MMP13 were downregulated, but E-cadherin expression level was upregulated by NDRG1 siRNA. At the same time, cytochrome (Cyt) C levels were increased in cytosol with the decreasing in mitochondria after siRNA treatment. The mitochondrial membrane potential (MMPs) was declined, and the function of mitochondria was impeded. The expressions of uncoupling proteins (UCP) 2, voltage dependent anion channel (VDAC), peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α, and cyclooxygenase (COX) 2 were downregulated by NDRG1 silencing. Moreover, NDRG performed its function primarily through the Wnt pathway and could regulate the differentiation of osteosarcoma stem cells. CONCLUSION: Silencing of NDRG1 could damage the function of mitochondria, promote the CSCs differentiation, alleviating OS progression.

Immune Reconstitution Following TCRαß/CD19-Depleted Hematopoietic Cell Transplantation for Hematologic Malignancy in Pediatric Patients.

TCRαß/CD19-depleted HCT has been used with excellent outcomes in pediatric patients with hematologic malignancies, and several studies have demonstrated rapid immune reconstitution in the nonmalignant setting. However, immune recovery following TCRαß/CD19-depleted hematopoietic cell transplantation (HCT) for malignancy remains incompletely elucidated. Furthermore, the majority of studies to date have used haploidentical and matched unrelated donors. Here we report results of immune reconstitution following TCRαß/CD19-depleted HCT for hematologic malignancy in 51 pediatric patients with hematologic malignancies, the majority of whom received grafts from unrelated donors. Grafts were from matched unrelated (n = 20), mismatched unrelated (n = 20), and haploidentical (n = 11) donors. The median CD34+ cell dose was 10.2 × 106/kg (range, 4.54 to 20 × 106/kg), and the median TCRαß+ cell dose was 2.53 × 104/kg (range, 0 to 44.9 × 104/kg). Conditioning was myeloablative with either busulfan or total body irradiation, cyclophosphamide, and thiotepa. Thirty-three patients also received rabbit antithymocyte globulin. No prophylactic post-transplantation immune suppression was routinely given. Forty-three patients received rituximab on day +1 for recipient positive Epstein-Barr virus serology. Forty-nine patients (96%) engrafted with a median time to neutrophil recovery of 13 days (range, 8 to 30 days). Thirty-seven patients (73%) are alive at a median follow-up of 25 months (range, 6 to 50 months). Nine patients (18%) developed grade II-IV acute graft-versus-host disease (GVHD), and 5 patients (11%) developed extensive chronic GVHD. Twenty-six patients (51%) experienced viral reactivation. T cell reconstitution was rapid with significant numbers of CD3+, CD4+, and CD8+ T cells present on first assessment at 4 months post-HCT, and significant numbers of naïve CD4+ T cells were present by 8 months post-HCT. Chronic GVHD was associated with delayed T cell recovery; however, T cell reconstitution was not affected by underlying diagnosis, donor source, TCRαß+ T cell dose, conditioning regimen, or use of antithymocyte globulin. B cell recovery mirrored T cell recovery, and i.v. Ig was discontinued at a median of 8 months (range, 4 to 22 months) post-HCT in patients alive and relapse-free at last follow-up. Immune reconstitution is rapid following TCRαß/CD19-depleted HCT in pediatric patients with hematologic malignancies. Donor graft source, haploidentical or unrelated, did not affect immune reconstitution. Viral reactivation is common in the first 100 days post-HCT, indicating that improved T cell defense is needed in the early post-HCT period.

Integrated analysis of deregulation microRNA expression in head and neck squamous cell carcinoma.

ABSTRACT: MicroRNAs (miRNAs) play critical roles in carcinogenesis and development of cancers. In this study, we analyzed the eccentrically expressed miRNAs in head and neck squamous cell carcinoma (HNSCC) tissues based on the miRNA-Seq data of HNSCC patients available in the Cancer Genome Atlas database. Aberrant expression of 2589 miRNAs was detected in HNSCC tissues (1128 downregulated and 1461 upregulated). The differential expression levels of the miRNAs were further validated by analysis of 25 HNSCC samples and paired control tissues and compared with the Gene Expression Omnibus database to determine the candidate miRNAs. Quantitative reverse transcription polymerase chain reaction was used to compare the expression of these candidate miRNAs between 22 fresh HNSCC tissue samples and 11 control samples. In addition, the relationship between the expression of these candidate miRNAs and Tumor, Node, Metastases staging of HNSCC was analyzed. Compared with the expression in control tissues, the levels of hsa-miR-410-3p, hsa-miR-411-5p, hsa-miR-125b-2-3p, and hsa-miR-99a-3p were significantly lower in HNSCC. According to the Cancer Genome Atlas dataset analyzed, all 4 miRNAs were shown to inhibit tumor progression (T stage), positive lymph node metastasis (N stage), and distant metastasis (M stage) in HNSCC. Kyoto Encyclopedia of Genes and Genomes analysis showed that genes regulated by these 4 miRNAs were enriched in certain pathways, including the transforming growth factor-ß signaling pathway and the Hippo pathway. Enriched gene ontology terms mainly included regulation of transcription, cell proliferation, and apoptosis, which are well-characterized functions of miRNAs. Moreover, all 4 miRNAs inhibited the progression of primary tumors (T stage) and metastasis of regional lymph nodes (N stage). The top 4 aberrantly expressed miRNAs identified in this study have great clinical value in developing strategies for early diagnosis and treatment of HNSCC. More intensive studies are required to elucidate the mechanism underlying the roles of these miRNAs in HNSCC.

Non-Invasive microRNA Profiling in Saliva can Serve as a Biomarker of Alcohol Exposure and Its Effects in Humans.

Alcohol Use Disorder (AUD) is one of the most prevalent mental disorders worldwide. Considering the widespread occurrence of AUD, a reliable, cheap, non-invasive biomarker of alcohol consumption is desired by healthcare providers, clinicians, researchers, public health and criminal justice officials. microRNAs could serve as such biomarkers. They are easily detectable in saliva, which can be sampled from individuals in a non-invasive manner. Moreover, microRNAs expression is dynamically regulated by environmental factors, including alcohol. Since excessive alcohol consumption is a hallmark of alcohol abuse, we have profiled microRNA expression in the saliva of chronic, heavy alcohol abusers using microRNA microarrays. We observed significant changes in salivary microRNA expression caused by excessive alcohol consumption. These changes fell into three categories: downregulated microRNAs, upregulated microRNAs, and microRNAs upregulated de novo. Analysis of these combinatorial changes in microRNA expression suggests dysregulation of specific biological pathways leading to impairment of the immune system and development of several types of epithelial cancer. Moreover, some of the altered microRNAs are also modulators of inflammation, suggesting their contribution to pro-inflammatory mechanisms of alcohol actions. Establishment of the cellular source of microRNAs in saliva corroborated these results. We determined that most of the microRNAs in saliva come from two types of cells: leukocytes involved in immune responses and inflammation, and buccal cells, involved in development of epithelial, oral cancers. In summary, we propose that microRNA profiling in saliva can be a useful, non-invasive biomarker allowing the monitoring of alcohol abuse, as well as alcohol-related inflammation and early detection of cancer.