Generation of HIY-resistant cells with a single-domain antibody:implications for HIV-1 gene therapy

The cure or functional cure of the"Berlin patient"and"London patient"indicates that infusion of HIV-resistant cells could be a viable treatment strategy.Very recently,we genetically linked a short-peptide fusion inhibitor with a glycosylphosphatidylinositol(GPI)attachment signal,rendering modified cells fully resistant to HIV infection.In this study,GPI-anchored m36.4,a single-domain antibody(nanobody)targeting the coreceptor-binding site of gp120,was constructed with a lentiviral vector.We verified that m36.4 was efficiently expressed on the plasma membrane of transduced TZM-bl cells and targeted lipid raft sites without affecting the expression of HIV receptors(CD4,CCR5,and CXCR4).Significantly,TZM-bl cells expressing GPI-m36.4 were highly resistant to infection with divergent HIV-1 subtypes and potently blocked HIV-1 envelope-mediated cell-cell fusion and cell-cell viral transmission.Furthermore,we showed that GPI-m36.4-modified human CEMss-CCR5 cells were nonpermissive to both CCR5-and CXCR4-tropic HIV-1 isolates and displayed a strong survival advantage over unmodified cells.It was found that GPI-m36.4 could also Impair HIV-1 Env processing and viral infectivity in transduced cells,underlying a multifaceted mechanism of antiviral action.In conclusion,our studies characterize m36.4 as a powerful nanobody that can generate HIV-resistant cells,offering a novel gene therapy approach that can be used alone or in combination.

Effects of Traditional Chinese Medicinal Plants on Antiinsulin Resistance Bioactivity of DXMS-Induced Insulin Resistant HepG2 Cells

Medicinal plants have a long history of use in China to treat diabetic symptoms.Ancient Chinese medical manuscripts and ethnobotanical surveys document plant remedies that continue to be actively used in China for the treatment of diabetic symptoms.Based on a systematic ancient Chinese medical manuscripts review in combination with ethnobotanical survey,16 medicinal plants for the traditional treatment of diabetic symptoms were identified for the evaluation of anti-insulin resistance bioactivity.The biological activity of 16 medicinal plants was tested on dexamethasone(DXMS)-induced insulin resistant HepG2 cells.The result shows that 11 of the 16 medicinal plants enhanced glucose uptake of DXMS-induced insulin resistant HepG2 cells,thereby demonstrating their ability to increase insulin sensitivity,other five medicinal plants including Astragalus membranaceus were found ineffective.The study shows that ancient Chinese medical manuscripts and ethnobotanical surveys on plants for the prevention and treatment of diabetic symptoms provide a promising knowledge base for drug discovery to mitigate the global diabetes epidemic.

Adriamycin Sensitizes Adriamycin-Resistant HL-60/ADR Cells to TRAIL-Mediated Apoptosis

OBJECTIVE To study whether an adriamycin-resistant cell line(HL-60/ADR) can be sensitized by adriamycin(ADR) to TRAIL-mediated apoptosis.METHODS The mRNA levels of the TRAIL receptor and apoptosis-related signaling molecules involved in the TRAIL-mediated apoptotic pathway were measured by RT-PCR.The protein levels of apoptotic-related signaling molecules involved in the TRAIL-mediated apoptotic pathway and processed caspase-3,caspase-9,and caspase-8 were measured by Western blots.Apoptosis was assessed by flow cytometry.Mitochondrial membrane potential was analyzed by DiOC6(3) staining.Cytotoxicity was determined by the colorimetric MTT viability/ proliferation assay.RESULTS Treatment with a combination of TRAIL and subtoxic concentrations of ADR resulted in synergistic cytotoxicity and apoptosis for both the parental HL-60 and the HL-60/ADR cells.For HL-60,there was a 5-fold potentiation and synergy in cytotoxicity for TRAIL and for HL-60/ADR,cytotoxicity to TRAIL was potentiated 6-fold with ADR.Adriamycin treatment modestly up-regulated TRAIL-R2(DR5),but had no effect on the expression of Fas-associated death domain,c-FLIP,Bcl-2,Bcl-xL,Bax,and IAP family members(cIAP-1,cIAP-2,XIAP,and survivin).The protein levels of pro-caspase-8 and pro-caspase-3 were not affected by ADR,whereas pro-caspase-9 and Apaf-1 were up-regulated.Combined treatment with TRAIL and ADR resulted in activation of caspase-9 and caspase-3,but there was no detectable processing of caspase-8 beyond the background levels.There was signif icant depolarization of the mitochondrial membrane by the combined treatment of both cell lines and it was more pronounced in the parental HL-60 cell line.The combined treatment with TRAIL and ADR resulted in 42.6% of the HL-60/ADR cells undergoing DNA fragmentation,whereas treatment with either ADR or TRAIL alone resulted in 5.46% and 21.3% DNA fragmented cells,respectively.Similar results were obtained with the HL-60 cells.CONCLUSION These fi ndings demonstrate that ADR can still signal ADR-resistant tumor cells,resulting in the modifi cation of the TRAIL-mediated signaling pathway and apoptosis.

Plant regeneration from protoplasts of hydroxyprolineresistant cell line in Onobrychis viciaefolia

An efficient protocol for plant regeneration from protoplasts of hydroxyproline(HYP)resistant cell line of Onoblychis viciaefolia was established. In SH medium supplemented with 1 mg/L 2, 4-dichlorophenoxy-acetic acid (2,4-D), 0.5 mg/L kinetin (KT) and 0.2 mg/L naphthalene acetic acid (NAA), the division frequency of protoplastderived cells reached uP to over 60 %, and microcalli were obtained in 5-6 wk. Upon transferring them on agar solidified MS medium plus 2 mg/L indole-3-acetic acid (IAA), shoots were induced. After cultivating them on MS medium with or without IAA, roots were regenerated.Chromosome number of all protoplast-regenerated plants examined were normal (2n=28). The protoplast-derived calli and plants grew vigorously on the medium containing 10 mmol/L HYP.

Calculated and Experimental Research of Sheet Resistances of Laser-Doped Silicon Solar Cells

The calculated and experimental research of sheet resistances of crystalline silicon solar cells by dry laser doping is investigated. The nonlinear numerical model on laser melting of crystalline silicon and liquid-phase diffusion of phosphorus atoms by dry laser doping is analyzed by the finite difference method implemented in MATLAB. The melting period and melting depth of crystalline silicon as a function of laser energy density is achieved. The effective liquid-phase diffusion of phosphorus atoms in melting silicon by dry laser doping is confirmed by the rapid decrease of sheet resistances in experimental measurement. The plateau of sheet resistances is reached at around 15 Ω/. The calculated sheet resistances as a function of laser energy density is obtained and the calculated results are in good agreement with the corresponding experimental measurement. Due to the successful verification by comparison between experimental measurement and calculated results, the simulation results could be used to optimize the virtual laser doping parameters.

The effective transfection of a low dose of negatively charged drug-loaded DNA-nanocarriers into cancer cells via scavenger receptors

DNA-nanotechnology-based nano-architecture scaffolds based on circular strands were designed in the form of DNA-nanowires(DNA-NWs) as a polymer of DNA-triangles. Circularizing a scaffold strand(84-NT) was the critical step followed by annealing with various staple strands to make stiff DNAtriangles. Atomic force microcopy(AFM), native polyacrylamide gel electrophoresis(PAGE), UVanalysis, MTT-assay, flow cytometry, and confocal imaging were performed to assess the formulated DNA-NWs and cisplatin(CPT) loading. The AFM and confocal microscopy images revealed a uniform shape and size distribution of the DNA-NWs, with lengths ranging from 2 to 4 mm and diameters ranging from 150 to 300 nm. One sharp band at the top of the lane(500 bp level) with the loss of electrophoretic mobility during the PAGE(native) gel analysis revealed the successful fabrication of DNA-NWs. The loading efficiency of CPT ranged from 66.85% to 97.35%. MTT and flow cytometry results showed biocompatibility of the blank DNA-NWs even at 95% concentration compared with the CPT-loaded DNANWs. The CPT-loaded DNA-NWs exhibited enhanced apoptosis(22%) compared to the apoptosis(7%)induced by the blank DNA-NWs. The release of CPT from the DNA-NWs was sustained at < 75% for 6 h in the presence of serum, demonstrating suitability for systemic applications. The IC_(50) of CPT@DNA-NWs was reduced to 12.8 nM CPT, as compared with the free CPT solution exhibiting an IC_(50) of 51.2 n M.Confocal imaging revealed the targetability, surface binding, and slow internalization of the DNA-NWs in the scavenger-receptor-rich cancer cell line(HepG2) compared with the control cell line.

Engineering immunosuppressive drug-resistant armored(IDRA)SARS-CoV-2 T cells for cell therapy

Solid organ transplant(SOT)recipients receive immunosuppressive drugs(ISDs)and are susceptible to developing severe COVID-19.Here,we analyze the Spike-specific T-cell response after 3 doses of mRNA vaccine in a group of SOT patients(n=136)treated with different ISDs.We demonstrate that a combination of a calcineurin inhibitor(CNI),mycophenolate mofetil(MMF),and prednisone(Pred)treatment regimen strongly suppressed the mRNA vaccine-induced Spike-specific cellular response.Such defects have clinical consequences because the magnitude of vaccine-induced Spike-specific T cells was directly proportional to the ability of SOT patients to rapidly clear SARS-CoV-2 after breakthrough infection.To then compensate for the T-cell defects induced by immunosuppressive treatment and to develop an alternative therapeutic strategy for SOT patients,we describe production of 6 distinct SARS-CoV-2 epitope-specific ISD-resistant T-cell receptor(TCR)-T cells engineered using the mRNA electroporation method with reactivity minimally affected by mutations occurring in Beta,Delta,Gamma,and Omicron variants.This strategy with transient expression characteristics marks an improvement in the immunotherapeutic field and provides an attractive and novel therapeutic possibility for immunosuppressed COVID-19 patients.

New Li-ion Battery Evaluation Research Based on Thermal Property and Heat Generation Behavior of Battery

We do a new Li-ion battery evaluation research on the effects of cell resistance and polariza- tion on the energy loss in batteries based on thermal property and heat generation behavior of battery. Series of 18650 cells with different capacities and electrode materials are evalu- ated by measuring input and output energy which change with charge-discharge time and current. Based on the results of these tests, we build a model of energy loss in cells＇ charge- discharge process, which include Joule heat and polarization heat impact factors. It was reported that Joule heat was caused by cell resistance, which included De-resistance and reaction resistance, and reaction resistance could not be easily obtained through routine test method. Using this new method, we can get the total resistance R and the polarization parameter U. The relationship between R, η, and temperature is also investigated in order to build a general model for series of different Li-ion batteries, and the research can be used in the performance evaluation, state of charge prediction and the measuring of consistency of the batteries.

Efficacy of pemetrexed combined with erlotinib/gefitinib in advanced non-small cell lung cancer patients during tyrosine kinase inhibitor treatment

Objective We aimed to evaluate the efficacy and safety of pemetrexed combined with erlotinib/gefitinib in advanced non-small cell lung cancer(NSCLC) patients during tyrosine kinase inhibitor(TKI) treatment. Methods Thirty-two patients with advanced NSCLC were divided into two groups. Patients in the control group received continuous daily epidermal growth factor receptor tyrosine kinase inhibitor(EGFRTKI) treatment, and patients in the experimental group received continuous daily EGFR-TKI along with pemetrexed treatment, which was administered on day 1 at 500 mg/m2. Erlotinib(150 mg) or gefitinib(250 mg) was administered daily from day 1 to day 21, with a cycle of every 21 days. Dexamethasone, folic acid, and vitamin B12 were also administered during the treatment. The endpoint of the primary study was the disease control rate. Results The objective response rate was 21.9%(95% CI: 7.6% to 36.3%) in the control group, whereas the disease control rate was 84.4%(95% CI: 71.8% to 97.0%) in the experimental group. The median progression-free survival was 6.2(95% CI: 2.4 to 10.0). Grades 3 or 4 adverse effects of leucopenia(15.6%), neutropenia(12.5%), anemia(3.1%), and nausea or vomiting(3.1%) were found in the experimental group.Conclusion The administration of pemetrexed combined with erlotinib or gefitinib showed a higher efficacy in TKI-resistant NSCLC patients. Further, the adverse effects of this drug combination were well tolerated by the patients. Pemetrexed combined with TKI treatment might provide a satisfactory therapeutic strategy for advanced NSCLC patients after TKI treatment.

Development of a new choroidal metastasis in resistance to crizotinib therapy in anaplastic lymphoma kinaserearranged non-small cell lung cancer

INTRODUCTION Non-small cell lung cancer（NSCLC）is a common malignant disease with an extremely poor prognosis.Lung cancer has been reported to metastasize to the eye in 0.2%to7%of patients based on clinical studies,and in 6%to 7%of patients based on postmortem histopathologic studies.

Proliferation-Inhibiting and Apoptosis-lnducing Effects of Ursolic Acid and Oleanolic Acid on Multi-Drug Resistance Cancer Cells in Vitro

Objective：To investigate the proliferation-inhibiting and apoptosis-inducing effects of ursolic acid（UA） and oleanolic acid（OA） on multi-drug resistance（MDR） cancer cells in vitro.Methods：UA and OA in different concentrations（0-100μmol/L） were added separately to cultures of different cancer cell lines, including the human colon cancer cell lines SW480 and SW620,human acute myelocytic leukemia cancer cell lines HL60 and HL60/ADR,human chronic myelogenous leukemia cell lines K562 and K562/ADR,and the human breast cancer cell lines MCF-7 and MCF-7/ADR.Effects of UA and OA on cell proliferation were detected by 3-（4,5-dimethyl-2-thiazole）-2-5-biphenly-tetrazole bromide（MTT） method and effects on cell apoptosis were tested by flow cytometry（FCM） and Western blot at 24,48,and 72 h after treatment.Results：Both UA and OA showed significant inhibition on parent and MDR cell lines in a time- and concentration-dependent manner;the drug-resistant multiple of them on K562 and K562/ADR as well as on HL60 and HL60/ADR was 1;the effects of UA were better than those of OA in inhibiting cell growth of solid colonic cancer and breast cancer.After SW480 cells were treated by UA at the concentrations of 0-40μmol/L for 48 h,FCM showed that annexin V （AV） positive cells and hypodiploid peak ratio increased along with the increase in the drug＇s concentrations; and Western blot found that expressions of Bcl-2,Bcl-xL and survivin decreased in a concentration-dependent manner.Conclusions：Both UA and OA have antitumor effects on cancer cells with MDR,and the optimal effect is shown by UA on colonic cancer cells.Also,UA shows cell apoptosis-inducing effect on SW480,possibly by way of down-regulating the expressions of apoptosis antagonistic proteins,Bcl-2,Bcl-xL,and survivin.

Near infrared light triggered nitric oxide releasing platform based on upconversion nanoparticles for synergistic therapy of cancer stem-like cells

Near infrared(NIR) light-driven nitric oxide(NO) release nano-platform based on upconversion nanoparticles(UCNPs) and light sensitive NO precursor Roussin's black salt(RBS) was fabricated to generate NO upon 808 nm irradiation. The application of 808 nm laser as the excitation source could achieve better penetration depth and avoid overheating problem. The combination of UCNPs and RBS could realize the on-demand release of NO at desired time and location by simply controlling the output of NIR laser.Cellular uptake results showed that more nanoparticles were internalized in cancer stem-like cells(CSCs)rather than non-CSCs. Therefore, a synergistic cancer therapy strategy to eradicate both CSCs and nonCSCs simultaneously was developed. Traditional chemo-drug could inhibit non-CSCs but has low killing efficiency in CSCs. However, we found that the combination of NO and chemotherapy could efficiently inhibit CSCs in bulk cells, including inhibiting mammosphere formation ability, decreasing CD44^+/CD24^- subpopulation and reducing tumorigenic ability. The mechanism studies confirmed that NO could not only induce apoptosis but also increase drug sensitivity by declining drug efflux in CSCs. This UCNPsbased platform may provide a new combinatorial strategy of NO and chemotherapy to improve cancer treatment.

Endoplasmic Reticulum Stress Mediates Palmitic Acid-induced Insulin Resistance in Mouse Skeletal Muscle Cells

Increasing evidence shows that pathological elevation of plasma fatty acids, especially long-chain saturated forms, which ordinarily occurs in obesity patients, increases the risk

LMM5.1 and LMM5.4,two eukaryotic translation elongation factor 1Alike gene family members,negatively affect cell death and disease resistance in rice

Lesion mimic mutant（LMM） genes, stimulating lesion formation in the absence of pathogens, play significant roles in immune response. In this study, we characterized a rice lesion mimic mutant, lmm5,which displayed light-dependent spontaneous lesions. Additionally, lmm5 plants exhibited enhanced resistance to all of the tested races of Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae（Xoo） by increasing the expression of defense-related genes and the accumulation of hydrogen peroxide. Genetic analysis showed that the lesion mimic phenotype of lmm5 was controlled by two genes, lmm5.1 and lmm5.4, which were isolated with a map-based cloning strategy. Remarkably, LMM5.1 and LMM5.4 share a 97.4% amino acid sequence identity, and they each encode a eukaryotic translation elongation factor 1A（e EF1A）-like protein. Besides, LMM5.1 and LMM5.4 were expressed in a tissue-specific and an indicaspecific manner, respectively. In addition, high-throughput m RNA sequencing analysis confirmed that the basal immunity was constitutively activated in the lmm5 mutant. Taken together, these results suggest that the homologous e EF1A-like genes, LMM5.1 and LMM5.4, negatively affect cell death and disease resistance in rice.

Human stem cell model to study signal transduction and molecular regulation mechanisms in CML

Abstract:Objective To develop a primary human hematopoietic stem/progenitor cell model for chronic myeloid leukemia (CML) and study signal transduction and molecular regulation mechanisms in CML. Methods We developed a human model of p210BCR/ABL positive CML by transducing normal human umbilical cord blood CD34+ cells with a retroviral vector containing the b3a2 bcr/abl cDNA. We also examined whether this model recreated the cellular phenotype of CML by assessing cell adhesion, cell migration, cell proliferation and cell survival. Results We found that significantly more myeloid colony forming units grew from p210BCR/ABL expressing cells, adhesion of p210BCR/ABL expressing CD34+ cells to fibronectin was decreased but migration over fibronectin was enhanced compared with mock transduced CD34+ cells. In this model, we showed that the presence of p210BCR/ABL leads to elevated levels of p27kip in p210BCR/ABL expressing CD34+ cells. We also showed that multidrug resistance-1 (MDR-1) Pgp was upregulated in the p210BCR/ABL expressing cells which correlates with the expression of p210BCR/ABL. Conclusion This primary human CML model recreates most of the features of CML and provides a useful tool to study signal transduction and downstream molecular regulation drived by the p210BCR/ABL oncogene in normal CD34+ cells.

Resistive switching characteristics of Ni/HfO_2/Pt ReRAM

This study investigated the resistive switching characteristics of the Ni/HfCVPt structure for nonvolatile memory application.The Ni/HfO_2/Pt device showed bipolar resistive switching（RS） without a forming process, and the formation and rupture of conducting filaments are responsible for the resistive switching phenomenon.In addition,the device showed some excellent memory performances,including a large on/off ratio（〉 3×10~5）,very good data retention（〉 10~3 s @ 200℃） and uniformity of switching parameters.Considering these results,the Ni/HfO_2/Pt device has the potential for nonvolatile memory applications.