Viral Metagenomics Analysis of Planktonic Viruses in East Lake,Wuhan,China

East Lake(Lake Donghu),located in Wuhan,China,is a typical city freshwater lake that has been experiencing eutrophic conditions and algal blooming during recent years.Marine and fresh water are considered to contain a large number of viruses.However,little is known about their genetic diversity because of the limited techniques for culturing viruses.In this study,we conducted a viral metagenomic analysis using a high-throughput sequencing technique with samples collected from East Lake in Spring,Summer,Autumn,and Winter.The libraries from four samples each generated 234,669,71,837,12,820,and 34,236 contigs(>90 bp each),respectively.The genetic structure of the viral community revealed a high genetic diversity covering 23 viral families,with the majority of contigs homologous to DNA viruses,including members of Myoviridae,Podoviridae,Siphoviridae,Phycodnaviridae,and Microviridae,which infect bacteria or algae,and members of Circoviridae,which infect invertebrates and vertebrates.The highest viral genetic diversity occurred in samples collected in August,then December and June,and the least diversity in March.Most contigs have low-sequence identities with known viruses.PCR detection targeting the conserved sequences of genes(g20,psbA,psbD,and DNApol)of cyanophages further confirmed that there are novel cyanophages in the East Lake.Our viral metagenomic data provide the first preliminary understanding of the virome in one freshwater lake in China and would be helpful for novel virus discovery and the control of algal blooming in the future.

Improving chemical and sensory quality of high-oleic peanut by application of foliar fertilizer

High-oleic peanut is favored by consumers because its a longer shelf life and benefits for cardiovascular health.Up to the high-oleic standard and with good taste is essential for peanut products in food processing.The aim of present study was to explore the effect of foliar fertilizers on chemical and eating quality of high-oleic peanuts.Three high-oleic peanut cultivars,namely Huayu 09C2,Huayu 9621,and Huayu 962,were used as materials.Huayu 09C2 and Huayu 9621 were planted in Fuxin,Liaoning,foliar sprayed with Fosforil at 2250 mL/ha,and Huayu 962 was sown in Jvnan,Shandong,foliar sprayed with Foliplus at 1500/3000 mL/ha.The chemical and sensory quality of peanut harvested from the treatment plots and controls was evaluated.Results showed that,Huayu 09C2 and Huayu 9621,foliar spraying with Fosforil in Fuxin,Huayu 962,foliar spraying with Foliplus in Jvnan,all still maintained high oleate content levels,and the Fosforil treatment was effective in raising the intensity of sweetness of roasted kernels.This study demonstrated the feasibility of improving chemical and sensory quality of high-oleic peanut through foliar fertilizer.

Improvement of the tissue culture technique for Melaleuca alternifolia

Tea tree oil is extracted from the leaves and twigs of Melaleuca alternifolia （Maiden ＆ Betche） Cheel, and it is widely used in medicines, food preservatives, cosmetics and health care products. Traditional propagation of M. alternifolia from seeds does not necessarily transfer the desired characteristics from their mother trees, the seedlings are not uniform, and the multiplication rate from cuttings is relatively low. For these reasons, it is necessary to develop tissue culture techniques for this species. This study showed that an efficient explant initiation medium for M. alternifolia was MS 1/2 ＋ BA 0.6mg L^-1 ＋NAA 0.1 mg L^-1＋sucrose 30g L-l, which yielded a 75.9 % initiation rate. An efficient multi- plication medium was MS ＋ BA 0.3 mg L^-1＋ NAA 0.15 mg L^-1 ＋ sucrose 30 g L^-1, which yielded a 4.3 multiplication rate and 3.2 cm shoot length. The rooting medium was MS 1/2 ＋ IBA 0.1-0.25 mg L^-1 ＋ sucrose 15 g L^-1, which yielded a 100 % rooting rate, 2.94-3.32 roots per individual and 1.36-1.44 cm root length. Local red-core soil was suitable as a transplant medium, and yielded 98 % survival. This study improved the tissue culture technique for mass-propagation of M. alternifolia, enabling the production of high quality plants for market.

VAR AND CTE BASED OPTIMAL REINSURANCE FROM A REINSURER'S PERSPECTIVE

In this article,we study optimal reinsurance design.By employing the increasing convex functions as the admissible ceded loss functions and the distortion premium principle,we study and obtain the optimal reinsurance treaty by minimizing the VaR(value at risk)of the reinsurer's total risk exposure.When the distortion premium principle is specified to be the expectation premium principle,we also obtain the optimal reinsurance treaty by minimizing the CTE(conditional tail expectation)of the reinsurer's total risk exposure.The present study can be considered as a complement of that of Cai et al.[5].

Polarized photoluminescence spectroscopy in WS_(2),WSe_(2) atomic layers and heterostructures by cylindrical vector beams

Due to the large exciton binding energy,two-dimensional(2D)transition metal dichalcogenides(TMDCs)provide an ideal platform for studying excitonic states and related photonics and optoelectronics.Polarization states lead to distinct light-matter interactions which are of great importance for device applications.In this work,we study polarized photoluminescence spectra from intralayer exciton and indirect exciton in WS_(2) and WSe_(2) atomic layers,and interlayer exciton in WS_(2)/WSe_(2) heterostructures by radially and azimuthally polarized cylindrical vector laser beams.We demonstrated the same in-plane and out-of-plane polarization behavior from the intralayer and indirect exciton.Moreover,with these two laser modes,we obtained interlayer exciton in WS_(2)/WSe_(2) heterostructures with stronger out-of-plane polarization,due to the formation of vertical electric dipole moment.

Pharmacokinetics of Arctigenin in Fructus Arctii Extract in Piglets

[Objectives]This study was conducted to investigate the pharmacokinetic characteristics of arctigenin from water extract of Fructus Arctii in piglets,understand its absorption,distribution,transformation and excretion in piglets,and provide theoretical reference for the development and clinical use of new veterinary drugs.[Methods]Six healthy piglets,weighing(30.0±5.0)kg,were selected and administered by gavage with a Fructus Arctii water extract at 1.0 g/kg·bw.Blood was collected from the anterior vena cava at different time points.The concentration of arctigenin in pig plasma was determined by HPLC.The main pharmacokinetic parameters were:absorption half-life(t1/2 ka)(0.098±0.006)h,distribution half-life(t1/2α)(0.208±0.009)h,elimination half-life(t1/2β)(23.816±2.151)h,apparent volume of distribution(Vd)(32.212±4.033)L/kg,apparent volume of distribution(Vd)(32.212±4.033)L/kg,clearance(CLb)(2.384±1.589)L/(h·kg),peak time(tmax)(0.251±0.011)h,peak concentration(Cmax)(0.560±0.063)μg/ml,and the area under the curve(AUC)at the time of drug administration(9.620±0.752)μg·h/ml.[Results]After oral administration of the water extract powder of Fructus Arctii,arctigenin showed a two-compartment model with absorption in piglets,with the characteristics of fast absorption,wide distribution and slow elimination.Arctigenin might have a hepatoenteral circulation in piglets,and the drug effect could last for a long time.[Conclusions]This study provides a theoretical basis and reference for the development and clinical use of new veterinary drugs.

Bcl-xL regulates radiation-induced ferroptosis through chaperone-mediated autophagy of GPX4 in tumor cells

Objective:To investigate the role and the molecular mechanisms of apoptotic signaling in ferroptosis to regulate tumor radiosensitivity.Methods:Reactive oxygen species(ROS)and lipid peroxide levels were detected in Mouse embryonic fibroblasts(MEFs)with Bcl-xL or Mcl-1 deficiency induced by erastin.Colony formation,ROS,lipid peroxidation and the transcription/translation levels of PTGS2 were measured in Bcl-xL knockdown tumor cells induced by 5 Gyγ-rays or co-treated with ferrostatin-1(Ferr-1).The protein levels of LPCAT3,ACSL4 and PEBP1 in Bcl-xL knockout MEF cells were evaluated in Bcl-xL knockout MEF cells post-radiation.Moreover,the interaction of heat shock protein 90(HSP90)with Bcl-xL,GPX4,or LAMP2A was detected by protein mass spectrometry and immunoprecipitation assays.Results:Manipulating Bcl-xL levels facilitated radiation-induced ferroptosis by augmenting the enzymatic oxidation of polyunsaturated fatty acids(PUFAs)and enhancing chaperone-mediated autophagy(CMA)of glutathione peroxidase 4(GPX4)(MEF cell line:t=4.540,P<0.01;A549 cell line:t=56.16,P<0.0001;t=4.885,P<0.01;HCT116 cell line:t=14.75,P<0.01;t=7.363,P<0.05).Downregulating Bcl-xL expression promoted the activity of acyl-CoA synthetase long-chain family member 4(ACSL4),thus increasing the enzymatic oxidation of PUFAs(t=4.258,P<0.01).Moreover,depletion of Bcl-xL expedited the CMA process targeting GPX4 by facilitating the association of GPX4 with heat shock protein 90(HSP90)and LAMP2A following radiation exposure.Subsequent degradation of GPX4 led to the accumulation of lipid peroxides,ultimately triggering ferroptosis.Conclusions:Our study provides initial insights into the regulatory role of Bcl-xL in ferroptosis and underscores the potential of targeting Bcl-xL as a promising therapeutic strategy for cancer by modulating both apoptotic and ferroptotic pathways.

PFOA/PFOS Facilitated Intestinal Fatty Acid Absorption by Activating the PPARα Pathway:Insights from Organoids Model

Perfluorooctanoic acid(PFOA)and perfluorooctanesulfonate(PFOS)continue to be extensively present in the natural environment and seriously threaten human health.The intestinal tract is the primary organ of PFOA/PFOS exposure due to the consumption of contaminated food and drinking water.However,it remains unclear how PFOA/PFOS affects intestinal function and overall health.The aim of this study was to investigate the influence of PFOA/PFOS on the absorption of fatty acids in the intestine and the underlying mechanisms using three-dimensional(3D)intestinal organoids.Our results showed that PFOS,but not PFOA,could significantly enhance the fatty acid uptake capacity without obvious damage to the organoids.Furthermore,PFOS markedly reduced the protein levels of ChgA in enteroendocrine cells,but with no observed impact on aldolase B+enterocytes.Mechanistically,exposure to PFOS induced the activation of the peroxisome proliferator-activated receptor(PPAR)αpathway in intestinal organoids,with enhanced expression of PPARαtarget genes associated with fatty acid metabolism,such as Fabp1 and Cd36(fatty acid transporter genes),Acox1 and Pdk4(fatty acid oxidation genes),and Plin2 and Plin3(lipid droplet synthesis genes).These data suggest that PFOS have the potential to affect the absorption function of the intestinal epithelium through the PPARαpathway,and its effect is much stronger than that of PFOA.Our findings also highlight that organoids can be used as a valuable model for conducting toxicological research on environmental chemicals.

Modulating the zinc ion flux and electric field intensity by multifunctional metal-organic complex interface layer for highly stable Zn anode

The uncontrollable growth of Zn dendrites accompanied by side reactions severely impedes the industrialized process of zinc ion electrochemical energy storage devices.Herein,we propose a practical metalorganic complex interface layer to manipulate the zinc ion flux and electric field intensity,enabling highly homogeneous zinc electrodeposition.The zinc-terephthalic acid complex(ZnPTA)with lower adsorption energy for zinc ion(-1.3 eV)builds a zincophilic interface favoring the ordered nucleation and growth of Zn.Moreover,the ZnPTA layer can serve as physical barrier to protect the newly deposited Zn from corrosion in the aqueous electrolyte.The modified Zn anode with the ZnPTA layer(ZnPTA@Zn)demonstrates excellent cycling stability more than 3000 h at 1 mA/cm^(2).Besides,the zinc-ion battery and zinc-ion hybrid capacitor using the ZnPTA@Zn electrode deliver outstanding cycle performance(up to 5500 cycles with high residual capacity ratio of 77.9%).This work provides a promising metal-organic complex interface design on enhancing the performance of Zn metal anode.

结节性多动脉炎诊疗规范

结节性多动脉炎(PAN)是主要累及中小动脉的一种少见的血管炎,好发于血管的分叉处,导致微动脉瘤、血栓形成、动脉瘤破裂出血及器官梗死。约1/3的患者与乙型肝炎病毒(HBV)感染相关。全身各组织器官均可受累,以皮肤、关节、外周神经最常见。病理改变急性期为血管壁纤维蛋白样坏死,炎性细胞浸润和管腔内血栓形成,慢性期为血管壁纤维性增生。近几十年来,PAN的总体预后得到了改善,主要反映了早期诊断和更有效的治疗方法。PAN治疗主要是糖皮质激素和环磷酰胺,而HBV相关PAN患者应接受抗病毒治疗和血浆置换。中华医学会风湿病学分会组织有关专家,在借鉴国内外诊治经验和指南的基础上,制定了本规范,旨在规范PAN的诊断和治疗,以降低致死率及严重并发症的发生率,更好地控制病情,改善患者预后。

Chinese registry of rheumatoid arthritis(CREDIT)V:sex impacts rheumatoid arthritis in Chinese patients

Background: The impact of sex on the clinical manifestations of rheumatoid arthritis (RA) were diversely reported in the literature. The Chinese Registry of rhEumatoiD arthrITis provides a platform for the investigation of this issue in Chinese patients.Methods: Demographic and clinical parameters were collected from all enrolled patients with RA and from patients with early RA (disease duration ≤6 months). The differences in data regarding disease activity, comorbidities, and medications for RA were compared between men and women. The proportions of patients who achieved remission and low disease activity were compared at enrollment and during 3-, 6-, and 12-month follow-up visits.Results: A total of 11,564 patients were enrolled, 83.6% of whom were female. In all the enrolled patients and patients with early RA, C-reactive protein (CRP, 12.0vs. 6.7 mg/L), pain visual analogue scale (4.8vs. 4.5), patient’s and physician’s global assessment (4.9vs. 4.5 and 4.9vs. 4.5), 28-joint disease activity score using DAS28-CRP (4.3vs. 4.0) simplified disease activity index (21.9vs. 19.9), and clinical disease activity index (19.3vs. 18.0) were significantly higher in men than in women. Additionally, the swollen joint count/tender joint count and DAS28 using erythrocyte sedimentation rate were higher in male patients than in female patients with early RA. More female patients with early RA reached the treatment target at baseline than male patients (23.4%vs. 18.2%, assessed by CDAI). At 3 months, 6 months, and 12 months, the proportion of remission and treatment target achievement was similar in both sexes. Coronary artery disease (CAD) and stroke were more frequent in men than in women.Conclusions: In Chinese patients with RA, men were found to have more active disease, as well as more cases of CAD and stroke. Therefore, sex should be carefully considered during the personalization of RA treatment.

B1-cell-produced anti-phosphatidylserine antibodies contribute to lupus nephritis development via TLR-mediated Syk activation

Autoantibodies produced by B cells play a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). However, both the cellular source of antiphospholipid antibodies and their contributions to the development of lupus nephritis (LN) remain largely unclear. Here, we report a pathogenic role of anti-phosphatidylserine (PS) autoantibodies in the development of LN. Elevated serum PS-specific IgG levels were measured in model mice and SLE patients, especially in those with LN. PS-specific IgG accumulation was found in the kidney biopsies of LN patients. Both transfer of SLE PS-specific IgG and PS immunization triggered lupus-like glomerular immune complex deposition in recipient mice. ELISPOT analysis identified B1a cells as the main cell type that secretes PS-specific IgG in both lupus model mice and patients. Adoptive transfer of PS-specific B1a cells accelerated the PS-specific autoimmune response and renal damage in recipient lupus model mice, whereas depletion of B1a cells attenuated lupus progression. In culture, PS-specific B1a cells were significantly expanded upon treatment with chromatin components, while blockade of TLR signal cascades by DNase I digestion and inhibitory ODN 2088 or R406 treatment profoundly abrogated chromatin-induced PS-specific IgG secretion by lupus B1a cells. Thus, our study has demonstrated that the anti-PS autoantibodies produced by B1 cells contribute to lupus nephritis development. Our findings that blockade of the TLR/Syk signaling cascade inhibits PS-specific B1-cell expansion provide new insights into lupus pathogenesis and may facilitate the development of novel therapeutic targets for the treatment of LN in SLE.

Reasoning about knowledge, belief and certainty in hierarchical multi-agent systems

Multi-agent systems （MAS） have received exten- sive studies in the last decade. However, little attention is paid to investigation on reasoning about logics in MAS with hier- archical structures. This paper proposes a complete quantified temporal KBC （knowledge, belief and certainty） logic and corresponding reasoning in hierarchical multi-agent systems （HMAS）. The key point is that internal beliefs and certainty, and external belief and certainty are considered in our logic. The internal beliefs and certainty show every agent is au- tonomous, while the external belief and certainty indicate the mutual influence of mental attitudes between two different agents on different layers in HMAS. To interpret this logic, we propose four classes of corresponding quantified interpreted systems, and define first-order KBC axiomatisations over HMAS, which are sound and complete with respect to the corresponding semantical classes. Finally, we give a case study to show the advantages in terms of expressiveness of our logic.

Role of nitric oxide in the genotoxic response to chronic microcystin-LR exposure in human–hamster hybrid cells

Microcystin-LR （MC-LR） is the most abundant and toxic microcystin congener and has been classified as a potential human carcinogen （Group 2B） by the International Agency for Research on Cancer. However, the mechanisms underlying the genotoxic effects of MC-LR during chronic exposure are still poorly understood. In the present study, human-hamster hybrid （AL） cells were exposed to MC-LR for varying lengths of time to investigate the role of nitrogen radicals in MC-LR-induced genotoxicity. The mutagenic potential at the CD59 locus was more than 2-fold higher （p 〈 0.01） in AL ceUs exposed to a cytotoxic concentration （1 μmol/L） of MC-LR for 30 days than in untreated control ceils, which was consistent with the formation of micronucleus. MC-LR caused a dose-dependent increase in nitric oxide （NO） production in treated cells. Moreover, this was blocked by concurrent treatment with the NO synthase inhibitor NC-methyl-L-arginine （L-NMMA）, which suppressed MC-LR- induced mutations as well. The survival of mitochondrial DNA-depleted （pO） AL ceils was markedly decreased by MC-LR treatment compared to that in AL cells, while the CD59 mutant fraction was unaltered. These results provided clear evidence that the genotoxicity associated with chronic MC-LR exposure in mammalian cells was mediated by NO and might be considered as a basis for the development of therapeutics that prevent carcinogenesis.

Avian Influenza A(H7N9) Virus in a Wild Land Bird in Central China,Late 2015

Dear Editor,In early 2013,the first case of a human infection with avian influenza A(H7N9)virus was reported in the Yangtze River Delta region of China(Liu et al.2013).Since early2013,a total of 1533 laboratory-confirmed human infections with avian H7N9 viruses have been reported to

Assessment of the cytotoxic and mutagenic potential of the Jialu River and adjacent groundwater using human-hamster hybrid cells

The Jialu River in China has been seriously polluted by the direct discharge of industrial and domestic wastewater. The predominant contaminants of the Jialu River and its adjacent groundwater were recently investigated. However, the potential genotoxic impact of polluted water on human health remains to be clarified. Here, we used human–hamster hybrid（AL） cells, which are sensitive for detecting environmental mutagens. We found that the cytotoxicity and mutagenicity of the groundwater in the Jialu River basin were influenced by the infiltration of the Jialu River. Hydrological periods significantly affected the cytotoxicity, but not the mutagenic potential, of surface and groundwater. Further, the mutagenic potential of groundwater samples located 〈 1 km from the Jialu River（S（M-2） water samples） was detected earlier than that of groundwater samples located approximately 20 km from the Jialu River（SN water samples）. Because of high cytotoxicity, the mutagenic potential of water samples from the Jialu River（S（M-1） water samples） was not significantly enhanced compared with that of untreated controls. To further assess the mutagenic dispersion potential, an artificial neural network model was adopted. The results showed that the highest mutagenic potential of groundwater was observed approximately 10 km from the Jialu River. Although further investigation of mutagenic spatial dispersion is required, our data are significant for advancing our understanding of the origin, dispersion,and biological effects of water samples from polluted areas.

Sex differences in systemic lupus erythematosus(SLE):an inception cohort of the Chinese SLE Treatment and Research Group(CSTAR)registry XVII

Background:The onset and clinical presentation of systemic lupus erythematosus(SLE)are sex-related.Few studies have investigated the distinctions in clinical characteristics and treatment preferences in male and female SLE patients in the initial cohort.This study aimed to improve the understanding of Chinese SLE patients by characterizing the different sexes of SLE patients in the inception cohort.Methods:Based on the initial patient cohort established by the Chinese SLE Treatment and Research Group,a total of 8713 patients(795 men and 7918 women)with newly diagnosed SLE were enrolled between April 2009 and March 2021.Of these,2900 patients(347 men and 2553 women)were eligible for lupus nephritis(LN).A cross-sectional analysis of the baseline demographic characteristics,clinical manifestations,laboratory parameters,organ damage,initial treatment regimens,and renal pathology classification was performed according to sex.Results:In the SLE group,as compared to female patients,male patients had a later age of onset(male vs.female:37.0±15.8 years vs.35.1±13.7 years,P=0.006)and a higher SLE International Collaborative Clinic/American College of Rheumatology damage index score(male vs.female:0.47±1.13 vs.0.34±0.81,P=0.015),LN(male vs.female:43.6%vs.32.2%,P<0.001),fever(male vs.female:18.0%vs.14.6%,P=0.010),thrombocytopenia(male vs.female:21.4%vs.18.5%,P=0.050),serositis(male vs.female:14.7%vs.11.7%,P=0.013),renal damage(male vs.female:11.1%vs.7.4%,P<0.001),and treatment with cyclophosphamide(CYC)(P<0.001).The frequency of leukopenia(male vs.female:20.5%vs.25.4%,P=0.002)and arthritis(male vs.female:22.0%vs.29.9%,P<0.001)was less in male patients with SLE.In LN,no differences were observed in disease duration,SLE Disease Activity Index score,renal biopsy pathological typing,or 24-h urine protein quantification among the sexes.In comparisons with female patients with LN,male patients had later onset ages(P=0.026),high serum creatinine(P<0.001),higher end-stage renal failure rates(P=0.002),musculoskeletal damage(P=0.023),cardiovascular impairment(P=0.009),and CYC use(P=0.001);while leukopenia(P=0.017),arthritis(P=0.014),and mycophenolate usage(P=0.013)rates were lower.Conclusions:Male SLE patients had more severe organ damage and a higher LN incidence compared with female SLE patients;therefore,they may require more aggressive initial treatment compared to female patients.

The 2018 new asset management regulation and LGFV bonds in China

While the local government debts and local government financing vehicle(LGFV)bonds underwent reconstruction,in 2018 the new asset management regulation introduced some tighter measures to restrain shadow banking business and to mitigate the moral hazard issue that led to an ongoing boom of the LGFV market.This paper examines the impacts of the 2018 new asset management regulation on the credit spread of newly issued LGFV bonds and explores how the new asset management regulation affects the implicit guarantee of local governments in the LGFV market that has piled up systemic risks.We find that the release of the new asset management regulation has raised the credit spread of LGFV bonds at issuance.More importantly,the credit spread becomes increasingly sensitive to the local governments’financial capacity and credibility right after the introduction of the new asset management regulation.Therefore,investors are compensated by more risk premiums for holding LGFV bonds issued by the local governments with a weaker balance sheet.Consequently,the implicit guarantee problem in the LGFV bond market has worsened.

Fabrication of layered structure VS4 anchor in 3D graphene aerogels as a new cathode material for lithium ion batteries

VS4 has gained more and more attention for its high theoretical capacity (449 mAh/g with 3e^- transfer) in lithium ion batteries (LIBs). Herein, a layered structure VS4 anchored in graphene aerogels is prepared and first reported as cathode material for LIBs. VS4@GAs composite exhibits an exceptional high initial reversible capacity (511 mAh/g), an excellent high-rate capability (191 mAh/g at the 5 C), and an excellent cyclic stability (239 mAh/g after 15 cycles).

Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells

Activation-induced cytidine deaminase(AID)is required for the generation of antibody diversity through initiat-ing both somatic hypermutation(SHM)and class switch recombination.A few research groups have success-fully used the feature of AID for generating mutant li-braries in directed evolution of target proteins in B cells in vitro.B cells,cultured in suspension,are not con-venient for transfection and cloning.In this study,we established an AID-based mutant accumulation and sorting system in adherent human cells.Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells,and a stable cell clone(H1299-AID)was selected.Afterwards,anti-hTNF-αscFv(ATscFv)was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells.By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha,two ATscFv mutant gene clones were isolated.Compared with the wild type ATscFv,the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha.The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells,which makes directed evolution in mammalian cells easier and more efficient.