Conjugation of Multiple Copies of Polyethylene Glycol to Hemoglobin Facilitated Through Thiolation: Influence on Hemoglobin Structure and Function

PEGylation induced changes in molecular volume and solution properties of HbA have been implicated as potential modulators of its vasoconstrictive activity. However, our recent studies with PEGylated Hbs carrying two PEG chains/Hb, have demonstrated that the modulation of the vasoconstrictive activity of Hb is not a direct correlate of the molecular volume and solution properties of the PEGylated Hb and implicated a role for the surface charge and/or the pattern of surface decoration of Hb with PEG. HbA has now been modified by thiolation mediated maleimide chemistry based PEGylation that does not alter its surface charge and conjugates multiple copies of PEG5K chains. This protocol has been optimized to generate a PEGylated Hb, (SP-PEG5K)₆-Hb, that carries ~six PEG5K chains/Hb – HexaPEGylated Hb. PEGylation increased the O₂ affinity of Hb and desensitized the molecule for the influence of ionic strength, pH, and allosteric effectors, presumably a consequence of the hydrated PEG-shell generated around the protein. The total PEG mass in (SP-PEG5K)₆-Hb, its molecular volume, O₂ affinity and solution properties are similar to that of another PEGylated Hb, (SP-PEG20K)₂-Hb, that carries two PEG20K chains/Hb. However, (SP-PEG5K)₆-Hb exhibited significantly reduced vasoconstriction mediated response than (SP-PEG20K)₂-Hb. These results demonstrate that the enhanced molecular size and solution properties achieved through the conjugation of multiple copies of small PEG chains to Hb is more effective in decreasing its vasoconstrictive activity than that achieved through the conjugation of a comparable PEG mass using a small number of large PEG chains.     

深松对乌拉尔甘草根际土壤养分以及微生物群落功能多样性的影响

通过大田试验和室内分析相结合,研究了深松对乌拉尔甘草根际土壤养分和微生物群落功能多样性的影响,以期为乌拉尔甘草人工种植地土壤耕作措施优化和土壤环境改良提供依据。结果表明,与未深松(常规耕作)处理相比,深松处理对乌拉尔甘草根际土壤0—20 cm耕层土壤养分含量无显著性影响,可显著提高乌拉尔甘草根际土壤20—40 cm耕层有机质、全氮、全磷和全钾的含量,分别提高了60.8%、65.3%、48.9%和86.8%;明显增加了0—20 cm和20—40 cm耕层细菌、真菌和放线菌的数量(P0.05),3种类型的微生物数量均呈现出上层大于下层,深松大于未深松的变化趋势。在156 h的微生物温育期内,深松处理下不同土层的平均颜色变化率(AWCD)均显著高于未深松处理,并显著提高了AWCD的利用率(72 h,P0.05),较未深松分别提高了35.5%和130.8%。与未深松处理相比,深松处理显著提高了土壤微生物的多样性指数(H、S、D)。主成分分析(PCA)表明,深松优化了乌拉尔甘草根际土壤微生物的群落组成;聚合物、羧酸类化合物、氨基酸和碳水化合物是深松处理下根际土壤微生物利用的主要碳源。总而言之,深松处理显著提高乌拉尔甘草根际土壤养分含量、微生物数量和微生物多样性指数,改变了微生物群落功能多样性,造成这种差异的主要原因可能是深松改变了土壤耕层结构,改善了微生物的生存环境。因此,深松对乌拉尔甘草人工种植地土壤质量的改良有积极作用。     

Can long range mechanical interaction between drugs and membrane proteins define the notion of molecular promiscuity? Application to P-glycoprotein-mediated multidrug resistance (MDR)

Background

Failure of treatment in over 90% of patients with metastatic cancer is due to acquired MDR. P-glycoprotein (Pgp) remains the archetypal drug membrane transporter expressed in many MDR cancer cells. Albeit the ATPase activity of Pgp is triggered by the presence of drug in the membrane, it is commonly assumed that when two drug molecules meet the same Pgp the protein cannot handle them efficiently due to steric effects and as a result the ATPase activity drops. However it is also possible that drug accumulating in the lipid-phase may affect the membrane in such a way that it imposes the mechanical closure of transporters by opposing the force mediated by ATP consumption. In this context, long range interactions between drug and membrane proteins could exist.

Methods

Recent data concerning Pgp structure have allowed us to formalize this hypothesis and we present a physico-mathematical model that is not based on predictive QSAR or other empirical methods applied to experimental data.

Results

Long range mechanical interactions between Pgp and drugs are predicted to occur at an external concentration of drug ~ 10–100 μM as previously determined experimentally at which concentration ~ 50% of transporters should be rendered inactive.

Conclusion

Distance interaction(s) between Pgp and drugs exist explaining an ill-defined effect concerning the ability of any drug to inhibit Pgp once a threshold concentration in the membrane has been reached.

General significance

Potential application of the theory in the field of pharmacology concentrating on the notion of molecular promiscuity and toxicity in drug discovery prediction is discussed.     

Selective toxicity of wyerone and other phytoalexins to gram-positive bacteria

The antibacterial activities of isoflavonoid (kievitone and phaseollin), flavonoid (hydroxyflavans), furanoacetylenic (wyerone), and sesquiterpenoid (capsidiol and rishitin), phytoalexins against eight Gram-negative and six Gram-positive bacteria were examined using the paper-disc antibiotic assay method. With the exception of capsidiol, which was inactive at the highest concentration tested (200,μg/disc) all of the phytoalexins were selectively toxic towards Gram-positive species. Wyerone and kievitone were generally more toxic than other phytoalexins; rishitin was the least active inhibitor.     

Synthesis,Antimicrobial Activity and Molecular Docking Study of Novel α‐(Diphenylphosphoryl)‐ and α‐(Diphenylphosphorothioyl)cycloalkanone Oximes

A series of novel α‐(diphenylphosphoryl)‐ and α‐(diphenylphosphorothioyl)cycloalkanone oximes have been synthesized in search for novel bioactive molecules. Their structures were characterized by various spectroscopic methods including IR, NMR (¹H, ³¹P, ¹³C), mass spectrometry and single crystal X‐ray diffraction. The newly synthesized phosphorus‐containing oximes were screened for their in vitro antimicrobial activity against Gram‐positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram‐negative bacteria (Escherichia coli and Salmonella typhimurium) and fungal strains (Candida albicans and Candida glabrata). The biological assays showed that all the studied compounds exhibited high antibacterial and antifungal activities at only 0.1–2.1 μg/mL. In silico molecular docking studies in FabH enzyme active site were performed in order to predict the possible interaction modes and binding energies of the drug candidates at the molecular level.     

Direct Recycling of Spent NCM Cathodes through Ionothermal Lithiation

Ionic liquids (ILs) are a family of nonconventional molten salts that offer many advantages, such as negligible vapor pressures, negligible flammability, wide liquidus ranges, good thermal stability, and much synthesis flexibility. The unique solvation environment of these ILs provides new reaction or flux media for controlling formation of solid‐state materials with a minimum perturbation of morphologies. A successful lithiation via ionothermal synthesis using a cost‐effective Li halide as Li source and recyclable ILs as solvents is reported here for the direct recycling of LiNi_1/3Co_1/3Mn_1/3O₂ (NCM 111) cathodes. In addition, the ionic liquids can be readily recycled and reused after ionothermal lithiation. The lithiation of spent cathodes can enable the direct recycling of spent cathode materials in lithium‐ion batteries.     

Prediction of methionine oxidation risk in monoclonal antibodies using a machine learning method

ABSTRACT

Monoclonal antibodies (mAbs) have become a major class of protein therapeutics that target a spectrum of diseases ranging from cancers to infectious diseases. Similar to any protein molecule, mAbs are susceptible to chemical modifications during the manufacturing process, long-term storage, and in vivo circulation that can impair their potency. One such modification is the oxidation of methionine residues. Chemical modifications that occur in the complementarity-determining regions (CDRs) of mAbs can lead to the abrogation of antigen binding and reduce the drug’s potency and efficacy. Thus, it is highly desirable to identify and eliminate any chemically unstable residues in the CDRs during the therapeutic antibody discovery process. To provide increased throughput over experimental methods, we extracted features from the mAbs’ sequences, structures, and dynamics, used random forests to identify important features and develop a quantitative and highly predictive in silico methionine oxidation model.