A SARS-CoV-2 neutralizing antibody discovery by single cell sequencing and molecular modeling

Although vaccines have been developed,mutations of SARS-CoV-2,especially the dominant B.1.617.2(delta)and B.1.529(omicron)strains with more than 30 mutations on their spike protein,have caused a significant decline in prophylaxis,calling for the need for drug improvement.Antibodies are drugs preferentially used in infectious diseases and are easy to get from immunized organisms.The current study combined molecular modeling and single memory B cell sequencing to assess candidate sequences before experiments,providing a strategy for the fabrication of SARS-CoV-2 neutralizing antibodies.A total of 128 sequences were obtained after sequencing 196 memory B cells,and 42 sequences were left after merging extremely similar ones and discarding incomplete ones,followed by homology modeling of the antibody variable region.Thirteen candidate sequences were expressed,of which three were tested positive for receptor binding domain recognition but only one was confirmed as having broad neutralization against several SARS-CoV-2 variants.The current study successfully obtained a SARS-CoV-2 antibody with broad neutralizing abilities and provided a strategy for antibody development in emerging infectious diseases using single memory B cell BCR sequencing and computer assistance in antibody fabrication.

Radiofrequency ablation or microwave ablation combined with transcatheter arterial chemoembolization in treatment of hepatocellular carcinoma by comparing with radiofrequency ablation alone

Objective：To compare radiofrequency ablation （RFA） or microwave ablation （MWA） and transcatheter arterial chemoembolization （TACE） with RFA or MWA monotherapy in hepatocellular carcinoma （HCC）.Methods：A prospective,randomized,controlled trial was conducted on 94 patients with HCC ≤7 cm at a single tertiary referral center from June 2008 to June 2010 at the Department of Hepatobiliary Surgery,the Second Affiliated Hospital of Southeast University.The patients were randomly assigned into the TACERFA or TACE-MWA （combined treatment group） and the RFA-alone or MWA-alone groups （control group）.The primary end point was overall survival.The secondary end point was recurrence-free survival,and the tertiary end point was adverse effects.Results：Until the time of censor,17 patients in the TACE-RFA or TACE-MWA group had died.The median follow-up time of the patients who were still alive for the TACE-RFA or TACE-MWA group was 47.5±11.3 months （range,29 to 62 months）.The 1-,3-and 5-year overall survival for the TACE-RFA or TACE-MWA group was 93.6％,68.1％ and 61.7％,respectively.Twenty-five patients in the RFA or MWA group had died.The median follow-up time of the patients who were still alive for the RFA or MWA group was 47.0±12.9 months （range,28 to 62 months）.The 1-,3-and 5-year overall survival for the RFA or MWA group was 85.1％,59.6％ and 44.7％,respectively.The patients in the TACE-RFA or TACE-MWA group had better overall survival than the RFA or MWA group [hazard ratio （HR）,0.526; 95％ confidence interval （95％ CO,0.334-0.823; P=0.002],and showed better recurrence-free survival than the RFA or MWA group （HR,0.582; 95％ CI,0.368-0.895; P=0.008）.Conclusions：RFA or MWA combined with TACE in the treatment of HCC ≤7 cm was superior to RFA or MWA alone in improving survival by reducing arterial and portal blood flow due to TACE with iodized oil before RFA.

T cells expressing a LMP1-specific chimeric antigen receptor mediate antitumor effects against LMP1-positive nasopharyngeal carcinoma cells in vitro and in vivo

T cells modified with chimeric antigen receptor are an attractive strategy to treat Epstein-Barr virus（EBV） associated malignancies.The EBV latent membrane protein 1（LMP1） is a 66-KD integral membrane protein encoded by EBV that consists of transmembrane-spanning loops.Previously,we have identified a functional signal chain variable fragment（scFv） that specifically recognizes LMP1 through phage library screening.Here,we constructed a LMP1 specific chimeric antigen receptor containing anti-LMP1 scFv,the CD28 signalling domain,and the CD3ζchain（HELA/CAR）.We tested its functional ability to target LMP1 positive nasopharyngeal carcinoma cells.HELA/CAR cells were efficiently generated using lentivirus vector encoding the LMP1-specific chimeric antigen receptor to infect activated human CD3＋ T cells.The HELA/CAR T cells displayed LMP1 specific cytolytic action and produced IFN-γ and IL-2 in response to nasopharyngeal carcinoma cells overexpressing LMP1.To demonstrate in vivo anti-tumor activity,we tested the HELA/CAR T cells in a xenograft model using an LMP1 overexpressing tumor.Intratumoral injection of anti-LMP1 HELA/CAR-T cells significantly reduced tumor growth in vivo.These results show that targeting LMP1 using HELA/CAR cells could represent an alternative therapeutic approach for patients with EBV-positive cancers.

c-Met-targeted chimeric antigen receptor T cells inhibit hepatocellular carcinoma cells in vitro and in vivo

c-Met is a hepatocyte growth factor receptor overexpressed in many tumors such as hepatocellular carcinoma(HCC).Therefore,c-Met may serve as a promising target for HCC immunotherapy.Modifying T cells to express c-Met-specific chimeric antigen receptor(CAR)is an attractive strategy in treating c-Met-positive HCC.This study aimed to systematically evaluate the inhibitory effects of 2^(nd)-and 3^(rd)-generation c-Met CAR-T cells on hepatocellular carcinoma(HCC)cells.Here,2^(nd)-and 3^(rd)-generation c-Met CARs containing an anti-c-Met singlechain variable fragment(scFv)as well as the CD28 signaling domain and CD3ζ(c-Met-28-3ζ),the CD137 signaling domain and CD3ζ(c-Met-137-3ζ),or the CD28 and CD137 signaling domains and CD3ζ(c-Met-28-137-3ζ)were constructed,and their abilities to target c-Met-positive HCC cells were evaluated in vitro and in vivo.All c-Met CARs were stably expressed on T cell membrane,and c-Met CAR-T cells aggregated around c-Met-positive HCC cells and specifically killed them in vitro.c-Met-28-137-3ζCAR-T cells secreted more interferon-gamma(IFN-γ)and interleukin 2(IL-2)than c-Met-28-3ζCAR-T cells and c-Met-137-3ζCAR-T cells.Compared with c-Met low-expressed cells,c-Met CAR-T cells secreted more cytokines when co-cultured with c-Met high-expressed cells.Moreover,c-Met-28-137-3ζCAR-T cells eradicated HCC more effectively in xenograft tumor models compared with the control groups.This study suggests that 3^(rd)-generation c-Met CAR-T cells are more effective in inhibiting c-Met-positive HCC cells than 2^(nd)-generation c-Met CAR-T cells,thereby providing a promising therapeutic intervention for c-Met-positive HCC.

Direct Trajectory Optimization and Costate Estimation of Infinite-horizon Optimal Control Problems Using Collocation at the Flipped Legendre-Gauss-Radau Points

A pseudospectral method is presented for direct trajectory optimization and costate estimation of infinite-horizon optimal control problems using global collocation at flipped Legendre-Gauss-Radau points which include the end point +1. A distinctive feature of the method is that it uses a new smooth, strictly monotonically decreasing transformation to map the scaled left half-open interval τ (-1, +1] to the descending time interval t (+∞, 0]. As a result, the singularity of collocation at point +1 associated with the commonly used transformation, which maps the scaled right half-open interval τ [-1, +1) to the increasing time interval [0,+∞), is avoided. The costate and constraint multiplier estimates for the proposed method are rigorously derived by comparing the discretized necessary optimality conditions of a finite-horizon optimal control problem with the Karush-Kuhn-Tucker conditions of the resulting nonlinear programming problem from collocation. Another key feature of the proposed method is that it provides highly accurate approximation to the state and costate on the entire horizon, including approximation at t = +∞, with good numerical stability. Numerical results show that the method presented in this paper leads to the ability to determine highly accurate solutions to infinite-horizon optimal control problems. © 2014 Chinese Association of Automation.

Effect of the degree of milling on the physicochemical properties,pasting properties and in vitro digestibility of Simiao rice

Simiao rice is a variety of long-grain Indica rice popular in South China.However,researches on the milling technology of Simiao rice are limited.The effect of different degrees of milling(DOMs)of Simiao rice(0-12%)were evaluated in terms of the physicochemical properties and in vitro starch digestibility of the rice.The results showed that,as the DOM increased from 0 to 12%,the head rice yield(HRY)and the content of protein,lipid,dietary fiber,vitamin B1,vitamin E and niacin nonlinearly decreased,while the content of total starch and amylose and paste viscosity parameters nonlinearly increased.The differences in chemical constituents between the brown and milled Simiao rice with different DOMs lead to the variation in their pasting properties and in vitro digestibility.Because of lower content of slowly digestible starch(SDS)and resistant starch(RS),milled Simiao rice exhibited greater digestibility and a higher estimated glycaemic index(EGI).The EGI of Simiao rice ranged from 77.98 to 85.55,and remained almost constant(approximately 84)when the DOM was above 4%.Correlation analysis indicated that the EGI had strong negative correlations with lipid,protein,dietary fiber and RS,but a positive correlation with amylose.These results provide theoretical guidance for the production of Simiao rice with desired physicochemical properties and digestibility by adjusting the DOM.

Expression of human yrdC gene promotes proliferation of gastric carcinoma cells

Objective： The aim of the research was to study the function of human yrdC gene in the gastric carcinoma cells. Methods： Human yrdC gene was isolated from human spleen tissue by RT-PCR. Anti-human yrdC monoclonal antibody was prepared by hybridoma cell technique. Recombinant adenovirus Ad.yrdC carrying yrdC gene was constructed by using the AdEasy adenoviral vector system. Recombinant adenovirus Ad.yrdCshRNA mediated yrdCshRNA was prepared by RNA interference technology. Gastric adenocarcinoma BGC-823 cells of moderate differentiation were transfected and absorbance of the transfected cells was calculated at 490 nm by methyl thiazolyl tetrazolium （MTT） method. Results： A value of the transfected Ad.yrdC group was significantly greater than that of the non-transfected and transfected Ad.Null groups, and A value of Ad.yrdCshRNA group was significantly lower than that of the non-transfected and transfected Ad.Null groups. Conclusion： Expression of yrdC gene has a function of promoting the proliferation of gastric carcinoma cells.

Feridex-labeled bone marrow stromal cells for analysis of sciatic nerve defects in rabbits

BACKGROUND： Traumatic approaches, such as sacrifice and perfusion sampling, have been used to evaluate efficiency of stem cell transplantation. However, these methods are not applicable to human studies. Cell tracing, in combination with non-invasive imaging technology, can be utilized to trace cell survival following transplantation to evaluate the efficacy of cell transplantation therapy. OBJECTIVE： To explore feasibility of magnetic resonance imaging （MRI） to observe in vivo repair of injured sciatic nerves following feridex and polylysine （FE-PLL） complex-labeled bone marrow stromal cell （BMSC） transplantation. DESIGN, TIME AND SE＇I-rlNG： A randomized, controlled, animal experiment was performed at the Laboratory of the Department of Neurosurgery, Zhujiang Hospital from March to December 2008. MATERIALS： Feridex was purchased from Advanced Magnetic, USA, and polylysine was purchased from Sigma, USA. METHODS： BMSCs were harvested from adult rabbit femurs and were cultured in vitro with neural stem cell culture medium, leukemia inhibitory factor, and basic fibroblast growth factor. Bone marrow stromal cell-derived neural stem cells （BMSC-D-NSCs） were obtained and labeled with FE-PLL complex. The right sciatic nerve （0.8 mm） was excised from healthy, New Zealand rabbits, aged 1.5 months, and the epineuria of distal stumps underwent turnover and were anastomosed at the proximal ends. FE-PLL labeled BMSC-D-NSC suspension or culture medium was transplanted into the epineunal lumen using a microsyringe. The left sciatic nerve was left intact and sewed as the normal control. MAIN OUTCOME MEASURES： Cellular morphology, proliferation, and differentiation, as well as expression of nestin and neuron-specific enolase （NSE）, of BMSCs-D-NSCs were observed. Efficacy of FE-PLL labeling and effects on cells were measured. In addition, neural regeneration at 2, 8, and 16 weeks following transplantation was observed by MRI. Histopathology and mean number of regenerated nerve fibers in the proximodistal-injured sciatic nerve were evaluated by hematoxylin and eosin and Bielschowsky staining. RESULTS： Results demonstrated that BMSCs expanded, proliferated, and differentiated into neural-like cells with slim, long processes. The cells expressed nestin and NSE, as detected by immunocytochemistry. BMSC-D-NSCs were effectively labeled by FE-PLL, with a labeling efficiency of 98%. In addition, cell viability was not influenced by the FE-PLL complex. MRI results revealed low signals in the FE-labeled BMSC-D-NSC-implanted region of the sciatic nerve. A low-signal region was observed at 2 weeks, which was widely spread at 8-16 weeks after cell transplantation. The regenerated nerve fibers were orderly arranged in the cell transplantation group and exhibited no significant differences compared with the normal control side （P 〉 0.05）. CONCLUSION： BMSCs were successfully cultured in vitro, and the cells proliferated and trans-differentiated into neuronal-like cells, which expressed nestin and NSE. The FE-PLL complex effectively labeled rabbit BMSC-D-NSCs in vitro and did not affect peripheral neural regeneration following cell transplantation. Results demonstrated that MRI could be used to track FE-labeled BMSC-D-NSCs transplanted in the sciatic nerve.

A Novel Tri-band GPS/WLAN Antenna for Tablet with Full Metal Housing

A full metal housing antenna for tablet to cover the GPS and WLAN b/g/a bands(1565-1585,2412-2484 and 4920-5825 MHz) is presented. The cavity is created by the enclosed volume of PCB and metal housing of the tablet. The cavity is properly excited by a flat exciting element w ith dimensions of only 36×4mm^2 positioned at an open slot betw een PCB and metal housing at the tablet's top surface. Among many excited cavity mode resonances,the ones close to the target frequency bands w ere selected and properly adjusted by adding grounding vias and shorting strip based on the E field distributions. This unique cavity mode excited antenna has measured-6dB return loss bandw idth of 1500-1600MHz,2380-2500 MHz,4580-5960 MHz and the average efficiencies for all the three bands are over 50%. Therefore,it overcomes the efficiency degradation issue that traditional IFA or monopole antennas are facing at the full metal housing environment.

Fusion of clathrin and caveolae endocytic vesicles revealed by line-switching dual-color STED microscopy

Clathrin-and caveolae-mediated endocytosis are the most commonly used pathways for the internalization of cell membrane receptors.However,due to their dimensions are within the diffraction limit,traditional fluorescence microscopy cannot distinguish them and little is known about their interactions underneath cell membrane.In this study,we proposed the line-switching scanning imaging mode for dual-color triplet-state relaxation(T-Rex)stimulated emission depletion(STED)super-resolution microscopy.With this line-switching mode,the cross-talk between the two channels,the side effects from pulse picker and image drift in frame scanning mode can be effectively eliminated.The dual-color super-resolution imaging results in mixed fluorescent beads validated the excellent performance.With this super-resolution microscope,not only the ring-shaped structure of clathrin and caveolae endocytic vesicles,but also their semi-fused structures underneath the cell membrane were distinguished clearly.The resultant infor-mation will greatly facilitate the study of clathrin-and caveolae-mediated receptor endocytosis and signaling process and also our home-built dual-color T-Rex STED microscope with this line-switching imaging mode provides a precise and convenient way to study subcellular-scale protein interactions.

Clinical study on IL-8,TNF-α,T-cell subgroup in serum of patients with rectal cancer

Objective:The aim of our study was to investigate the immune status of patients with rectal cancer and its relationship with clinicopathological features.Methods:The serum levels of interleukin-8(IL-8),tumor necrosis factor(TNF-α) and T-cell subgroup contents were measured using a double-antibody sandwich assay of ELISA in 43 patients with rectal cancer,and compared with the normal health adults.Results:In patients with rectal cancer,the serum levels of CD4,CD4/CD8 of T-cell subgroup in peripheral blood were significantly lower than the control group(P < 0.01),which gradually decreased with increase of Dukes stage;but the levels of CD8,IL-8 and TNF-α were higher than the control group,which gradually increased with increase of Dukes stage.Conclusion:The immunocompromice exists in patients with rectal cancer,there is a correlation between the contents of T-cell subgroup,IL-8 and TNF-α in serum and the Dukes stage of rectal cancer.Therefore immunotherapy can be used in patients with rectal cancer.

Evolving landscape of treatments targeting the microenvironment of liver metastases in non-small cell lung cancer

Liver metastases(LMs)are common in lung cancer.Despite substantial advances in diagnosis and treatment,the survival rate of patients with LM remains low as the immune-suppressive microenvironment of the liver allows tumor cells to evade the immune system.The impact of LMs on the outcomes of immune checkpoint inhibitors in patients with solid tumors has been the main focus of recent translational and clinical research.Growing evidence indicates that the hepatic microenvironment delivers paracrine and autocrine signals from non-parenchymal and parenchymal cells.Overall,these microenvironments create pre-and post-metastatic conditions for the progression of LMs.Herein,we reviewed the epidemiology,physiology,pathology and immunology,of LMs associated with non-small cell lung cancer and the role and potential targets of the liver microenvironment in LM in each phase of metastasis.Additionally,we reviewed the current treatment strategies and challenges that should be overcome in preclinical and clinical investigations.These approaches target liver elements as the basis for future clinical trials,including combinatorial interventions reported to resolve hepatic immune suppression,such as immunotherapy plus chemotherapy,immunotherapy plus radiotherapy,immunotherapy plus anti-angiogenesis therapy,and surgical resection.

Immune cell membrane-based biomimetic nanomedicine for treating cancer metastasis

Metastasis is the leading cause of cancer-related death.Despite extensive treatment,the prognosis for patients with metastatic cancer remains poor.In addition to conventional surgical resection,radiotherapy,immunotherapy,chemotherapy,and targeted therapy,various nanobiomaterials have attracted attention for their enhanced antitumor performance and low off-target effects.However,nanomedicines exhibit certain limitations in clinical applications,such as rapid clearance from the body,low biological stability,and poor targeting ability.Biomimetic methods utilize the natural biomembrane to mimic or hybridize nanoparticles and circumvent some of these limitations.Considering the involvement of immune cells in the tumor microenvironment of the metastatic cascade,biomimetic methods using immune cell membranes have been proposed with unique tumor-homing ability and high biocompatibility.In this review,we explore the impact of immune cells on various processes of tumor metastasis.Furthermore,we summarize the synthesis and applications of immune cell membrane-based nanocarriers increasing therapeutic efficacy against cancer metastases via immune evasion,prolonged circulation,enhanced tumor accumulation,and immunosuppression of the tumor microenvironment.Moreover,we describe the prospects and existing challenges in clinical translation.

重组小麦静息巯基氧化酶的表达、酶学特性及其对面包品质的影响

为发展新型面粉改良酶制剂,利用大肠杆菌Escherichia coli原核表达了小麦静息巯基氧化酶(Wheat quiescin sulfhydryl oxidase,wQSOX)。将合成的wqsox基因构建至pMAL-c5x载体,并在大肠杆菌中进行表达,优化蛋白表达条件后对重组蛋白进行分离纯化及融合标签切除,获得的重组wQSOX蛋白用于酶学性质探究以及面包品质改良。结果表明,合成的截短wqsox基因包含1359 bp,编码453个氨基酸,理论蛋白分子量51 kDa;构建的pMAL-c5x-wqsox重组质粒在E.coli Rosetta gamiB(DE3)中可溶表达了重组蛋白MBP-wQSOX,其最佳表达条件为:诱导温度25℃,诱导剂IPTG浓度0.3 mmol/L,诱导时间6 h;利用Xa因子蛋白酶切除了MBP融合标签,亲和层析纯化得到了wQSOX;wQSOX可催化DTT、GSH和Cys氧化,并伴随着H2O2的生成,其中对DTT表现出最高的底物特异性;酶学性质研究发现,wQSOX最适反应温度和pH分别为50℃和10.0,在高温和碱性环境条件下表现出较好的稳定性;每克面粉中添加1.1 U wQSOX能够显著(P<0.05)提高26.4%的面包比容,降低20.5%的面包芯硬度和24.8%的咀嚼性,表现出了较好的改良面包加工品质能力。研究结果对丰富新型面粉改良酶制剂种类以及推动wQSOX在焙烤行业的应用奠定了理论基础。

MicroRNA-663 induces immune dysregulation by inhibiting TGF-β1 production in bone marrow-derived mesenchymal stem cells in patients with systemic lupus erythematosus

Mesenchymal stem cells(MSCs)are critical for immune regulation.Although several microRNAs(miRNAs)have been shown to participate in autoimmune pathogenesis by affecting lymphocyte development and function,the roles of miRNAs in MSC dysfunction in autoimmune diseases remain unclear.Here,we show that patients with systemic lupus erythematosus(SLE)display a unique miRNA signature in bone marrow-derived MSCs(BMSCs)compared with normal controls,among which miR-663 is closely associated with SLE disease activity.MiR-663 inhibits the proliferation and migration of BMSCs and impairs BMSC-mediated downregulation of follicular T helper(Tfh)cells and upregulation of regulatory T(Treg)cells by targeting transforming growth factorβ1(TGF-β1).MiR-663 overexpression weakens the therapeutic effect of BMSCs,while miR-663 inhibition improves the remission of lupus disease in MRL/lpr mice.Thus,miR-663 is a key mediator of SLE BMSC regulation and may serve as a new therapeutic target for the treatment of lupus.

A design of a surface-doped Yb：YAG slab laser with high power and high efficiency

We demonstrate a high-efficiency and high-power quasi-three-level laser based on a trapezoidal composite slab architecture with a 270 μm-thick Yb-doping surface. The design of a surface-doped slab architecture,temperature effects, laser oscillator model, and laser oscillator experiments with a surface-doped slab as a laser host medium have been presented. By theoretical calculation, the temperature rise in the surface-doped slab is only one seventh of that in the bulk-doped slab at the same maximum pump power of 30 kW. Finally, in the laser oscillator experiments, an output energy of 21.6 J is obtained when the pump energy is 48 J with a repetition rate of 5 Hz and a pulse width of 1 ms. The optical-optical efficiency is 45%.

Space-time evolution rules study on acoustic emission location in rock under cyclic loading

An acoustic emission(AE)location experiment was performed on sandstone using an advanced AE test system.The space-time evolution rule regarding damage was analyzed under cyclic loading as well as AE.The results show that AE on static loading process is consistent with the damage evolution rule of compression and the elastic-plastic deformation phase;at the beginning of cyclic loading with low duration time and energy,AE events came from a small crack.The location result showed that most events occurred in the core zone forming at the static loading process,and the location points changed slowly.AE energy changed little during the metaphase of cyclic process.There was a modest increase of location points in every cycle.The tendency of steady development could be predicted from the AE location events.At the end of each cyclic loading,the quantity of AE events and energy increased quite rapidly,reaching a maximum at the last cycle.AE events had high energy and duration time.Location events changed quite rapidly and assembled and linked continuously in the core zone.At the same time,they expanded to the top of specimen.A macroscopic crack finally formed.In the postfailure process,some AE events still existed due to fracturing of gliding friction.Owing to the inner stress balance of rock even after loading stopped,minor AE events still occurred.

MSCs relieve SLE by modulation of Th17 cells through MMPs–CCL2–CCR2–IL-17 pathway

Objective:To explore the modulation of mesenchymal stem cells(MSCs)on T helper 17(Th17)cells in systemic lupus erythematosus(SLE)and underlying mechanism.Methods:The concentration of matrix metalloproteinases(MMPs),CC chemokine ligand-2(CCL2),and interleukin-17(IL-17)in the serum of SLE patients and mice were detected by enzyme-linked immunosorbent assay.The expression of CCR2 and IL-17 of T lymphocytes were determined by flow cytometry.The effects of MSCs on Th17 cells were analyzed in lupus mice and coculture system in vitro.Results:The levels of MMPs,CCL2,IL-17,CCR2,and percentages of Th17 cells were significantly increased in SLE patients.These molecules and numbers of Th17 cells were downregulated by umbilical cord-derived MSCs(UC-MSCs)which relieve SLE disease.CCL2 neutralizing antibody blocked the effects of MSCs on Th17 cells.MMPs reversed the function of CCL2.Conclusion:The beneficial effects of MSCs on SLE patients rely on secreting MMPs,which reverse the activity of CCL2 to inhibit Th17 cells,suggesting the crucial MSCs–MMP–CCL2–CCR2–Th17–IL-17 pathway in SLE.