Site-specific PEGylation of lidamycin and its antitumor activity

In this study,N-terminal site-specific mono-PEGylation of the recombinant lidamycin apoprotein(r LDP) of lidamycin(LDM) was prepared using a polyethyleneglycol(PEG) derivative(Mw20 k Da) through a reactive terminal aldehyde group under weak acidic conditions(p H 5.5).The biochemical properties of m PEG-r LDP-AE,an enediyne-integrated conjugate,were analyzed by SDSPAGE,RP-HPLC,SEC-HPLC and MALDI-TOF.Meanwhile,in vitro and in vivo antitumor activity of m PEG-r LDP-AE was evaluated by MTT assays and in xenograft model.The results indicated that m PEGr LDP-AE showed significant antitumor activity both in vitro and in vivo.After PEGylation,m PEG-r LDP still retained the binding capability to the enediyne AE and presented the physicochemical characteristics similar to that of native LDP.It is of interest that the PEGylation did not diminish the antitumor efficacy of LDM,implying the possibility that this derivative may function as a payload to deliver novel tumortargeted drugs.

Site-specific PEGylation of Human Growth Hormone by Mutated Sortase A

Human growth hormone（hGH）, a classic therapeutic protein, which promotes growth and wound healing, is released from the pituitary gland. As a protein drug, its short half-life is its main barrier to therapeutic efficacy. Various strategies have been designed to prolong its serum half-life, the most conunon of which is the conjugation with polyethylene glycol（PEG）, as this has been shown to significantly extend protein＇s serum half-life. However, PEGylation often results in random conjugation, which can lead to impaired protein function and hinder purification, characterization and evaluation of the PEGylated protein. Therefore, site specific PEGylation is a promising direction for PEG-protein conjugation. Here we took advantages of the mutated sortase A（7M） enzyme, which can enzymati- cally ligate the universal a-amino acids to a C-terminal tagged protein. This then allows specific modification of the C-terminal of hGH with PEG. This site-specific bound PEG-hGH has similar efficacy, receptor binding and cell proliferation as wild-type hGH; however, pharmacokinetic analysis demonstrates that its serum half-life is almost 24 times that of wild-type hGH. Herein, we provided a promising advancement in the development of site specific PEGylated therapeutic proteins.

Achieving structurally stable O3-type layered oxide cathodes through site-specific cation-anion co-substitution for sodium-ion batteries

O3-type layered oxides have garnered great attention as cathode materials for sodium-ion batteries because of their abundant reserves and high theoretical capacity.However,challenges persist in the form of uncontrollable phase transitions and intricate Na^(+)diffusion pathways during cycling,resulting in compromised structural stability and reduced capacity over cycles.This study introduces a special approach employing site-specific Ca/F co-substitution within the layered structure of O_(3)-NaNi_(0.5)Mn_(0.5)O_(2) to effectively address these issues.Herein,the strategically site-specific doping of Ca into Na sites and F into O sites not only expands the Na^(+)diffusion pathways but also orchestrates a mild phase transition by suppressing the Na^(+)/vacancy ordering and providing strong metal-oxygen bonding strength,respectively.The as-synthesized Na_(0.95)Ca_(0.05)Ni_(0.5)Mn_(0.5)O_(1.95)F_(0.05)(NNMO-CaF)exhibits a mild O3→O3+O'3→P3 phase transition with minimized interlayer distance variation,leading to enhanced structural integrity and stability over extended cycles.As a result,NNMO-CaF delivers a high specific capacity of 119.5 mA h g^(-1)at a current density of 120 mA g^(-1)with a capacity retention of 87.1%after 100 cycles.This study presents a promising strategy to mitigate the challenges posed by multiple phase transitions and augment Na^(+)diffusion kinetics,thus paving the way for high-performance layered cathode materials in sodium-ion batteries.

PEGylation of Hirudin and Analysis of Its Antithrombin Activity in vitro

Hirudin is the most anticoagulant drug found in nature, but its short serum half-life significantly inhibits its clinical anpplication. The PEGvlation of hirudin, the most promising anticoagulant drug, was performed in this paper. The optimal reaction conditions for PEG ylated hirudin were investigated, wh.en the PEGylation react, on.wasconducted under 4℃ after 10h, in the borate buffer at pH 8.5 .with the molar ratio 230 ： 1 of PEG to hirudin, a higher modification extent was achieved. Finally, the bioactivity of PEGylated hirudin was measured in vitro.Compared with unmodified hirudin, 26% of anti-thrombin activity was retained.

Spatial variability of soil properties in red soil and its implications for site-specific fertilizer management

Assessing spatial variability and mapping of soil properties constitute important prerequisites for soil and crop management in agricultural areas. To explore the relationship between soil spatial variability and land management, 256 samples were randomly collected at two depths (surface layer 0–20 cm and subsurface layer 20–40 cm) under different land use types and soil parent materials in Yujiang County, Jiangxi Province, a red soil region of China. The pH, soil organic matter (SOM), total nitrogen (TN), cation exchange capacity (CEC), and base saturation (BS) of the soil samples were examined and mapped. The results indicated that soils in Yujiang were acidified, with an average pH of 4.87 (4.03–6.46) in the surface layer and 4.99 (4.03–6.24) in the subsurface layer. SOM and TN were significantly higher in the surface layer (27.6 and 1.50 g kg–1, respectively) than in the subsurface layer (12.1 and 0.70 g kg–1, respectively), while both CEC and BS were low (9.0 and 8.0 cmol kg–1, 29 and 38% for surface and subsurface layers, respectively). Paddy soil had higher pH (mean 4.99) than upland and forest soils, while soil derived from river alluvial deposits (RAD) had higher pH (mean 5.05) than the other three parent materials in both layers. Geostatistical analysis revealed that the best fit models were exponential for pH and TN, and spherical for BS in both layers, while spherical and Gaussian were the best fitted for SOM and CEC in the surface and subsurface layers. Spatial dependency varied from weak to strong for the different soil properties in both soil layers. The maps produced by selecting the best predictive variables showed that SOM, TN, and CEC had moderate levels in most parts of the study area. This study highlights the importance of site-specific agricultural management and suggests guidelines for appropriate land management decisions.

Effects of Site-Specific Nitrogen Management on Yield and Dry Matter Accumulation of Rice from Cold Areas of Northeastern China

The effects of yield increase and mechanism of site-specific nitrogen management （SSNM） in five rice varieties from cold areas of northeastern China were studied. Plot experiment for critical SPAD value and experiments of two fertilization methods, SSNM and farmer＇s fertilization practice （FFP） were conducted to study their effects on the quality and dry matter accumulation of rice population, as well as N uptake. Compared with FFP, SSNM significantly decreased the average N rate by 33.8%, significantly increased average ear-bearing tiller rate and LAI for grain-filling stage by 12.3% and 14.1-27.6%, correspondingly, improved dry matter weight and N uptake after heading period by 4.3-29.1% and 11.8-55.1% （P 〈 0.05）, and heightened recovery efficiency and agronomic efficiency by 38.5-133.4% （P 〈 0.05） and 39.8-194.3% （P 〈 0.05）, respectively, as well as increased the average yield by 9.8% in 2004 and 2005. The results indicated that the accumulation rate of dry matter and N increased the rice yield and N use efficiency, because of improving rice population quality and increasing LAI after heading period.

Effect of PEGylation on the physicochemical and pharmacokinetic characteristics of bovine serum albumin-encapsulated liposome

There is now little doubt that PEGylation is useful and is in widespread use because it provides a prolonged half-life,a higher stability and a lowerimmunogenicity[1].However,it is of concern that PEGylation may affect the physicochemical and pharmacokinetic characteristics of protein-encapsulated liposome.Thus,we prepared the bovine serum albumin(BSA)-encapsulated liposome(BSA-liposome)with or without PEG and then compared their physicochemical and pharmacokinetic characteristics(Fig.1).BSA-liposomes were prepared by the thin-film hydration method.

Development of a radiolabeled site-specific single-domain antibody positron emission tomography probe for monitoring PD-L1 expression in cancer

Despite advances in immunotherapy for the treatment of cancers,not all patients can benefit from programmed cell death ligand 1(PD-L1)immune checkpoint blockade therapy.Anti-PD-L1 therapeutic effects reportedly correlate with the PD-L1 expression level;hence,accurate detection of PD-L1 expression can guide immunotherapy to achieve better therapeutic effects.Therefore,based on the high affinity antibody Nb109,a new site-specifically radiolabeled tracer,^(68)Ga-NODA-cysteine,aspartic acid,and valine(CDV)-Nb109,was designed and synthesized to accurately monitor PD-L1 expression.The tracer ^(68)Ga-NODA-CDV-Nb109 was obtained using a site-specific conjugation strategy with a radiochemical yield of about 95%and radiochemical purity of 97%.It showed high affinity for PD-L1 with a dissociation constant of 12.34±1.65 nM.Both the cell uptake assay and positron emission tomography(PET)imaging revealed higher tracer uptake in PD-L1-positive A375-hPD-L1 and U87 tumor cells than in PD-L1-negative A375 tumor cells.Meanwhile,dynamic PET imaging of a NCI-H1299 xenograft indicated that doxorubicin could upregulate PD-L1 expression,allowing timely interventional immunotherapy.In conclusion,this tracer could sensitively and dynamically monitor changes in PD-L1 expression levels in different cancers and help screen patients who can benefit from anti-PD-L1 immunotherapy.

Construction of Marker-Free GFP Transgenic Tobacco by Cre/lox Site-Specific Recombination System

Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation. The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene retransformation. After removal of the Cre-NPT Ⅱ locus by genetic segregation through self-cross, plants that incorporated only the GFP transgene were obtained. Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained, which resisted herbicide Basta and GFP expressed well, then the Cre gene was subsequently introduced into 5 plants of them, respectively, by retransformation. The leaf disks from Cre transgenic plants were used to test the resistance to Basta on the medium with 8 mg L-1 of PPT. The results showed that few discs were able to regenerate normally, and the excision at 76-100% efficiency depended on individual retransformation events. Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basta sensitive plants. The result indicated that the excision event in the recombination sites was precise and conservative, without loss or alteration of any submarginal nucleotides of the recombination sites. Bar gene excised plants were selfpollinated to allow segregation of the GFP gene from the Cre-NPT Ⅱ locus. The progenies from self-pollinated plants were scored for Kan senstivity, then the segregation of GFP gene from Cre-NPT Ⅱ locus in the Kan senstive plants were confirmed by PCR analysis subsequently. Hence, constructing marker-free transgenic tobacco plants by Cre/lox sitespecific recombination system was reliable, and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.

The Site-Specific Hydrolysis of l-β-Hydroxybaccatin Ⅱ by Aspergillus Niger

The site-specific microbiological hydrolysis of a natural 1β-hydroxybaccatin I, with the culture of Aspergillus niger. is described.

Application of site-specific biomass models to quantify spatial distribution of stocks and historical emissions from deforestation in a tropical forest ecosystem

Allometric equations developed for the Lama forest, located in southern Benin, West Africa, were applied to estimate carbon stocks of three vegetation types：undisturbed forest, degraded forest, and fallow. Carbon stock of the undisturbed forest was 2.7 times higher than that in the degraded forest and 3.4 times higher than that in fallow. The structure of the forest suggests that the individual species were generally concentrated in lower diameter classes. Carbon stock was positively correlated to basal area and negatively related to tree density, suggesting that trees in higher diameter classes contributed significantly to the total carbon stock. The study demonstrated that large trees constitute an important component to include in the sampling approach to achieve accurate carbon quantification in forestry. Historical emissions from deforestation that converted more than 30% of the Lama forest into cropland between the years 1946 and 1987 amounted to 260,563.17 tons of carbon per year（t CO2/year） for the biomass pool only. The study explained the application of biomass models and ground truth data to estimate reference carbon stock of forests.

Study on Site-specific Nutrient Management in Cotton Field

The study on the characteristics of spatial variability of soil nutrients and fertilizer recommendations in cotton field under certain conditions of agricultural management was conducted with GIS and systematic approach for soil nutrient constrains. The results showed that of the spatial variability of soil nutrient was greatly related to the management condition of previous crops. Grid sampling and variable rate application technology (VRAT) were the tools that would hopefully increase fertilizer efficiency. The fertilizers were applied where they were needed and at proper rate. Balance fertilization demonstration showed that fertilizer recommendations according to the available nutrient level in soil could decrease fertilizer cost with 657.4 yuan / ha and increase seed cotton yield by 19.8%. A net profit of the balanced fertilization was 5314.9 yuan / ha higher than that of local fertilization practice.

Dynamic micelles with detachable PEGylation at tumoral extracellular pH for enhanced chemotherapy

Although surface PEGylation of nanomedicines can decrease serum protein adsorption in vivo,it also blocks uptake by tumor cells.This dilemma could be overcome by employing detachably PEGylated strategy at tumoral extracellular microenvironment to achieve improved cellular uptake while prolonged circulation times.Herein,the amphiphilic graft copolymers with p H-sensitive ortho ester-linked m PEG in side chains and polyurethanes in backbone,can self-assemble into the free and doxorubicin(DOX)-loaded micelles.The p H-sensitive micelles could undergo several characteristics as follows:(i)PEGylated shells for stability in sodium dodecyl sulfonate(SDS)solution;(ii)De PEGylation via degradation of ortho ester linkages at tumoral extracellular pH(6.5)for gradually dynamic size changes and effective release of DOX;and(iii)enhanced cellular uptake and cytotoxicity via positive DOX.Moreover,the dynamic micelles with detachable PEGylation could quickly penetrate the centers of SH-SY5 Y multicellular spheroids(MCs)and strongly inhibit tumor growth in vitro and in vivo,and might be considered as promising and effective drug carriers in tumor therapy.

Development of rabbit monoclonal and polyclonal antibodies for detection of site-specific histone modifications and their application in analyzing overall modification levels

In addition to DNA sequence information, site-specific histone modifications are another important determinant of gene expression in a eukaryotic organism. We selected four modification sites in common histones that are known to significantly impact chromatin function and generated monoclonal or polyclonal antibodies that recognize each of those site-specific modifications. We used these antibodies to demonstrate that the site-specific histone modification levels remain relatively constant in different organs of the same organism. We also compared the levels of selected histone modifications among several representative organisms and found that site-specific modifications are highly variable among different organisms, providing new insight into the evolutionary divergence of specific histone modifications.

Effects of Long-term Site-specific Fertilization on Soil Physical and Chemical Properties and Nutrients in Dry Farmland

In order to investigate the variation in soil physical and chemical properties and nutrients in the mountainous areas in southern Ningxia, and to provide a theoretical basis for fertilization management in local farmland, the soil p H, total salt content,crop root length, root weight, soil organic matter, available nitrogen, total nitrogen, total phosphorous and total potassium in different fertilization treatments were measured from 2010 to 2016. Multiple comparisons of the data were performed using Duncan's new multiple range test. The results indicated that in the 0-20 cm soil layer, soil p H value and total salt content changed in different patterns, and varied greatly from 2010 to 2016(P<0.05). The changes of both root length and root weight of millet over time fitted S-shaped curves. The root length and root weight in the four fertilization treatments(Treatment 2 to Treatment5) increased faster than those in the control(Treatment 1). The soil organic matter content in all the five treatments gradually increased from 2010 to 2016. The content of alkaline hydrolyzable nitrogen in soil rapidly increased in the first two to three years of the experiment, followed by a slow increase or decrease in 2013, and then raised rapidly again from 2014 to 2016.The soil total nitrogen content varied significantly from 2010 to 2016. The total phosphorus content in soil changed in a different pattern from that of total nitrogen content. The seven-year field trails revealed that soil p H, total salt content, root length, root weight and soil nutrient all changed with the increase of fertilizer level, and that long-term fertilization is of significance for maintaining soil fertility, improving soil quality and reducing soil salinization.

Chemotactic Activity of Site-Specific Multivalent Conjugates of Stromal Cell-Derived Factor 1<i>α</i>on Branched Nanoparticles

Stromal cell-derived factor 1α (SDF1α) is a potent chemokine for the recruitment of stem cells. A challenge is to maintain its activity and control its release. In this study, we engineered a recombinant cysteine-SDF1α (cysSDF1α) protein, and performed multivalent conjugation of cysSDF1 through the maleimide functional group to two forms of branched nanoparticles: multi-arm poly (ethylene glycol) (MA-PEG) and hyaluronic acid (HA). We characterized the chemotactic activity of the conjugates, and determined how the molecular weight (MW) of MA-PEG and HA affected the chemotactic activity. CysSDF1α had similar efficiency to wild-type SDF1α in cell recruitment. Multivalent conjugation of cysSDF1α to low MW MA-PEG (~18 nm) did not significantly affect the chemotactic activity, while the conjugation of cysSDF1α to high MW MA-PEG (~72 nm) lowered the efficiency, possibly due to the larger spacing between conjugated SDF1α molecules. HA has a linear backbone and a high density of multivalent binding sites;however, the chemotactic activity of HA-linked cys-SDF1α was much lower, which further decreased with the increase of HA MW from 200 kDa (~0.78 μm) to 700 kDa (~2.7 μm). Digestion of HA into smaller fragments using hyaluronidase partially recovered the chemotactic activity of cysSDF1α, suggesting that high MW HA might exert steric hindrance for SDF1α binding to its receptors on cell surface and that HA could be used as a depot for SDF1α storage and release. These results demonstrate that multivalent conjugates of SDF1α to nanoparticles may be used to engineer SDF1α delivery for cell recruitment and tissue regeneration.

Site-specific recombination in Escherichia coli mediated by actinomyces phage R4 integrase

The purpose of this study was to demonstrate that actinomyces phage R4 integrase Sre protein efficiently mediate site-specific recombination in Escherichia coll. An intramolecular recombination assay system in E. coli was constructed. The plasmid pBZP contains attB and attP sites in direct orientation flanking a lacZ gene. When pBZP was introduced into E. coli cells, in which the plasmid pSREA containing sre gene was resident, Sre protein catalyzed integration of attP into attB site, resulting in excision of the lacZ gene. This integration changed bacteria colonies from blue to white on agar plates containing X-Gal, which showed that the lacZ was removed. The integrant DNAs were identified by enzyme digestion, PCR and DNA sequencing. The minimal sizes of attB and attP were 50 bp and 47 bp for 100% recombination efficiency. The phage recombinase Sre efficiently integrated attP into attB site to create attR and attL in E. coli host environment without Streptomyces specific cofactors. This intrmolecular assay system is a simple and efficient system for Sre-mediated recombination in E. coll.

聚乙二醇化多柔比星脂质体在乳腺癌中的应用进展

蒽环类药物是乳腺癌化疗的常用药物,但较明显的不良反应限制了其临床使用。新型蒽环类药物聚乙二醇化多柔比星脂质体(PLD)具有特殊的结构,其用于治疗乳腺癌的效果与传统蒽环类药物相当,且不良反应发生率较低,主要不良反应为手足综合征和口腔黏膜炎,但一般不影响化疗进程。然而,目前PLD在乳腺癌辅助化疗和新辅助化疗中应用的循证医学证据尚不足,故仍主要推荐使用传统蒽环类药物。未来,期待开展更多有关PLD用于乳腺癌各期治疗的高质量临床试验,以为乳腺癌合理用药提供临床依据。

C-X-C chemokine receptor type 5+CD8+T cells as immune regulators in hepatitis Be antigen-positive chronic hepatitis B under interferonalpha treatment

BACKGROUND C-X-C chemokine receptor type 5(CXCR5)^(+)CD8^(+)T cells represent a unique immune subset with dual roles,functioning as cytotoxic cells in persistent viral infections while promoting B cell responses.Despite their importance,the specific role of CXCR5^(+)CD8^(+)T cells in chronic hepatitis B(CHB),particularly during interferon-alpha(IFN-α)treatment,is not fully understood.This study aims to elucidate the relationship between CXCR5^(+)CD8^(+)T cells and sustained serologic response(SR)in patients undergoing 48 weeks of pegylated IFN-α(peg-IFN-α)treatment for CHB.AIM To elucidate the relationship between CXCR5^(+)CD8^(+)T cells and sustained SR in patients undergoing 48 weeks of peg-IFN-αtreatment for CHB.METHODS This study enrolled 60 patients with hepatitis Be antigen(HBeAg)-positive CHB undergoing 48 weeks of peg-IFN-αtreatment.Participants were assessed for eligibility based on criteria such as persistent HBsAg-positive status for at least six months,HBeAb-negative,hepatitis B virus DNA levels exceeding 2×10^(4) copies/mL,and alanine aminotransferase(ALT)levels between 2 and 10 times the upper limit of normal.Blood samples were collected at baseline and at weeks 12,24,48,and a 24-week treatment-free follow-up(week 72)to measure serum interleukin(IL)-21 concentration via ELISA and to analyze CXCR5 and programmed death-ligand 1(PD-L1)expression on CD8^(+)T cells by flow cytometry,CXCR5 is a chemokine receptor that directs immune cells to specific tissues,while PD-L1 is a protein that regulates immune responses by inhibiting T cell activity.RESULTS Patients with CHB exhibited significantly lower levels of circulating CXCR5^(+)CD8^(+)T cells compared to healthy controls(P<0.01).Notably,CXCR5^(+)CD8^(+)T cells were prominently expressed in patients who achieved sustained SR compared to non-SR(NSR).A significant correlation was observed between CXCR5 and PD-L1 expression(r=-0.189,P=0.002).However,there was no significant correlation between serum IL-21 levels and CXCR5+CD8+lymphocytes(r=-0.03,P=0.625)or serum ALT levels(r=0.026,P=0.678).CONCLUSION The enhanced expression of CXCR5^(+)CD8^(+)T cells in patients achieving HBeAg seroconversion during IFN-αtreatment suggests that these cells play a crucial role in antiviral immune responses against hepatitis B.This study highlights the potential of CXCR5^(+)CD8^(+)T cells as immune regulators in CHB,which may inform future therapeutic strategies to optimize antiviral treatments.

低水平慢性乙型肝炎患者应用恩替卡韦与聚乙二醇干扰素α-2b联合治疗的效果研究

目的评估恩替卡韦与聚乙二醇干扰素α-2b联合应用于治疗慢性乙型肝炎病毒(HBV)载量较低患者的治疗效果。方法选取2021年4月至2022年12月在茂名市人民医院感染科门诊就诊的124例低水平乙型肝炎患者,根据治疗方法分为两组,对照组(83例)接受恩替卡韦治疗,干预组(41例)采用恩替卡韦联合皮下注射聚乙二醇干扰素α-2b的方法进行治疗。比较两组干预后的血清丙氨酸氨基转移酶(ALT)、HBsAg、HBeAg水平以及甲胎蛋白(AFP)含量。同时,比较HBV-DNA载量低于15 U/ml、HBeAg血清学转换以及ALT水平恢复正常患者在两组中的分布差异。结果治疗后,两组ALT、HBeAb、HBeAb、AFP和HBV-DNA<15 U/ml的例数比较无差异(P>0.05);干预组HbsAg和HbeAg水平均低于对照组(P<0.05);干预组HbsAb、HBeAg血清学转换率和ALT复常率均高于对照组(P<0.05)。治疗后,干预组HBsAg水平中位数为19.07 kU/L,低于治疗前的91.85 kU/L(P<0.05);干预组HBsAb水平中位数为0.98 kU/L,高于治疗前的0.37 kU/L(P<0.05)。但ALT、HBeAg、HBeAb、AFP及HBV-DNA低于15 U/ml的例数在联合治疗前后比较,差异均无统计学意义(P>0.05)。结论恩替卡韦联合聚乙二醇干扰素α-2b治疗可降低低水平慢性乙型肝炎患者HbsAg和HbeAg水平,相较于单独使用恩替卡韦治疗效果更优。