Clinical grade lentiviral vector purification and quality control requirements.

Lentiviral vectors have been proven to be a powerful tool in gene therapies that includes the ability to perform long-term gene editing in both dividing and non-dividing cells. In order to meet the rising demand for clinical-grade lentiviral vectors for future clinical trials and requirements by regulatory agencies, new methods and technologies were developed, including the rapid optimization of production and purification processes. However, gaps still exist in achieving ideal yields and recovery rates in large-scale manufacturing process steps. The downstream purification process is a critical step required to obtain a sufficient quantity and high-quality lentiviral vectors products, which is challenged by the low stability of the lentiviral vector particles and large production volumes associated with the manufacturing process. This review summarizes the most recent and promising technologies and enhancements used in the large-scale purification process step of lentiviral vector manufacturing and aims to provide a significant contribution towards the achievement of providing sufficient quantity and quality of lentiviral vectors in scalable processes.

A meta-analysis of combined generic-covered stent-graft with or without bare-metal stent for refractory variceal bleeding.

Objectives: The meta-analysis was conducted to systematically assess the efficacy and safety of generic stent-graft/bare-stent combination compared with Fluency stent alone in transjugular intrahepatic portosystemic shunt procedure for refractory variceal bleeding. Methods: PubMed, EMBASE, Scopus, Web of Science and the Cochrane Database were searched for relevant studies from January 1990 to September 2020; outcome measures studied were primary patency, hepatic encephalopathy, survival, re-bleeding and portal venous pressure. Results: Four studies (1 randomised controlled trial and 3 retrospective studies) with 449 subjects (157 patients in the combined stent group and 292 patients in the covered stent group) were included. No significant difference was observed in the incidence of mortality (hazard ratio [HR] = 1.069, 95% confidence interval [CI] [0.524, 2.178]), hepatic encephalopathy (odds ratio [OR] = 0.860, 95% CI [0.341, 2.169], P = 0.750) and re-bleeding (OR = 1.049, 95% CI [0.226, 4.881], P = 0.951). Compared with Fluency stent alone, combination therapy was associated with moderate decrease in outcomes on the post-operative portal venous pressure (standard mean difference [SMD] -0.210, 95% CI [-0.418, -0.001], P = 0.049) and was not associated with significant decrease in outcomes on the pre-operative portal venous pressure (SMD - 0.129, 95% CI [-0.336, 0.078], P = 0.223). The primary patency was significantly lower in the Fluency/bare-stent combination group (HR = 0.473, 95% CI [0.288, 0.776]). Conclusions: Generic stent-graft/bare-stent combination therapy was associated with significantly lower primary patency compared to Fluency stent alone.

Feasibility of peer support services among people with severe mental illness in China.

BACKGROUND: Peer-delivered services potentially provide broad, multifaceted benefits for persons suffering severe mental illness. Most studies to date have been conducted in countries with well-developed outpatient mental health systems. The objective of this study was to examine the feasibility for developing a community-based peer service in China. METHODS: Thirteen peer service providers and 54 consumers were recruited from four communities in Beijing. We initiated the program in two communities, followed by another two in order to verify and add to our understanding of potential scalable feasibility. Semi-structured face-to-face interviews were conducted 12 month after initiation at each site to measure satisfaction and perceived benefits from perspectives of peer service providers, and consumers and their caregivers. RESULTS: Key stakeholders reported that peer support services were satisfying and beneficial. Eleven of 13 peer service providers were willing to continue in their roles. Ten, 8, and 7 of them perceived improvements in working skills, social communication skills, and mood, respectively. Among consumers, 39 of 54 were satisfied with peer services. Improvements in mood, social communication skills, illness knowledge, and illness stability were detected among 23, 18, 13, and 13 consumers, respectively. For caregivers, 31 of 32 expressed a positive view regarding peer services. Caregivers reported improvement in their own mood, confidence in recovery of their family members, and reduction in caretaker burdens. CONCLUSIONS: The findings highlight that peer-delivered services have promise in China for benefiting persons with severe mental illness and their family caregivers, as well as the peer service providers themselves.

Atomic-level modulation of electron density in iron sulfides for enhancing sodium storage kinetics.

Iron sulfides (FeS2) are promising anode materials for sodium ion batteries (SIBs); however, their inferior electronic conductivity, large volume swelling, and sluggish sodium ion diffusion kinetics lead to unsatisfactory rate performance and cycling durability. Heteroatom doping plays a crucial role in modifying the physicochemical properties of FeS2 anodes to enhance its sodium storage. Herein, ultra-fine Ni-doped FeS2 nanocrystals derived from a metal-organic framework (MOF) and in-situ anchored on a nitrogen doped carbon skeleton (Ni-FeS2@NC) are proposed to enhance both structural stability and reaction kinetics. Material characterization, electrochemical performance, and kinetics analysis demonstrate the critical role of Ni doping in sodium storage, particularly in accelerating Na+ diffusion efficiency. The N-doped carbon derived from the MOF can buffer the volume expansion and enhance the structural stability of electrode materials during sodiation/desodiation processes. As expected, Ni-FeS2@NC exhibits a high reversible capacity of 656.6 ± 65.1 mAh g-1 at 1.0 A g-1 after 200 cycles, superior rate performance (308.8 ± 6.0 mAh g-1 at 10.0 A g-1), and long-term cycling durability over 2000 cycles at 1.0 A g-1. Overall, this study presents an effective approach for enhancing the sodium storage performance and kinetics of anode materials for high efficiency SIBs.

One-dimensional N-doped carbon nanofibers produced by pre-oxide treatment for effective lithium storage.

Amorphous carbon materials have been confirmed as attractive anode materials for lithium-ion batteries. Herein, an effective strategy to fabricate amorphous carbon materials at low temperature under air atmosphere is proposed. As demonstrated, one-dimensional nitrogen-doping carbon nanofibers were obtained through simple electrospinning technology, following low-temperature heat treatment. Meanwhile, the nitrogen-doping concentration can be regulated by the heating temperature, which can further introduce different levels of adsorption sites on the surface of carbon and enhance the electronic conductivity. Based on experimental investigation, carbon nanofibers with a high nitrogen doping concentration of 18.1 at% achieved an outstanding cycling durability (194.0 mA h g-1 at 2.0 A g-1 after 2000 cycles).

The thermal expansion exfoliation technology and lithium promoter assistant afford CuOx/graphene as a high-performance anode for lithium-ion batteries.

The ability to rationally design a copper oxide anode with superior rate performance that possesses an ultra-small particle size is highly desirable for lithium-ion batteries (LIBs). Herein, the rapid and effective thermal expansion exfoliation technology was employed to synthesize ultra-small CuOx nanoparticles (∼2.7 nm) uniformly dispersed on graphite oxide after popping (CuOx/Li-PGO), in which the addition of lithium promoted the exfoliation process to obtain an enlarged specific surface area and efficient transfer ability of PGO. The CuOx/Li-PGO electrode achieved a reversible capacity as high as 512.1 mA h g-1 under 2.0 A g-1 after 1000 cycles, demonstrating superior rate performance and cycling stability.

Fabrication of Few-Layer Graphene-Supported Copper Catalysts Using a Lithium-Promoted Thermal Exfoliation Method for Methanol Oxidative Carbonylation.

Exfoliation of graphene oxide (GO) via thermal expansion is regarded as the most promising approach to obtain few-layer graphene (FLG) in bulk. Herein, we introduce an efficient strategy for improving the exfoliation process by adding a tiny amount of lithium nitrate in the precursors, which significantly enhances the removal of oxygen-containing functional groups and produces 1-2 layer graphene. FLG-supported highly dispersed Cu nanoparticles (NPs, ≈4.2 nm) can be further synthesized through exfoliating the mixture of GO, lithium nitrate, and copper(II) nitrate, which displayed superior catalytic activity and stability in the synthesis of dimethyl carbonate (DMC) using liquid methanol oxidative carbonylation. The characterization results demonstrate that during the thermal expansion process, lithium nitrate was decomposed to Li2O and immediately reacted with CO2 released by the decomposition of GO to form stable Li2CO3, which promotes efficient charge transfer and produces Cuδ+ (0 < δ < 1) species in the Cu/Li-PGO catalyst. Density functional theory calculations prove that the presence of Cuδ+ markedly facilitates CO adsorption over the resulting catalyst and causes a decrease of the energy barrier of the rate-limiting step for DMC formation (CO insertion). These findings give a theoretical explanation of the enhanced catalytic performance of the Cu/Li-PGO catalyst. The present work provides a simple and practical avenue to the exfoliation of graphene and the dispersions of metal NPs on graphene sheets.

Multiple binding modes for dicationic Hoechst 33258 to DNA.

The binding of dicationic Hoechst 33258 (ligand) to DNA was characterized by means of the fluorescence spectra, fluorescence intensity titration, time-resolved fluorescence decay, light scattering, circular dichroism, and fluorescence thermal denaturation measurements, and two binding modes were distinguished by the experimental results. Type 1 binding has the stoichiometry of one ligand to more than 12 base pairs, and it is defined as quasi-minor groove binding which has the typical prolonged fluorescence lifetime of about 4.4 ns. In type 1 binding, planar conformation of the ligand is favorable. Type 2 binding with phosphate to ligand ratio (P/L) < 2.5 has the stoichiometry of one ligand to two phosphates. It is defined as a highly dense and orderly stacked binding with DNA backbone as the template. Electrostatic interactions between doubly protonated ligands and negatively charged DNA backbone play a predominant role in the type 2 binding mode. The characteristics of this type of binding result in a twisted conformation of the ligand that has a fluorescence lifetime of less than 1 ns. The results also indicate that the binding is in a cooperative manner primarily by stacking of the aromatic rings of the neighboring ligands. Type 1 binding is only observed for double-stranded DNA (dsDNA) with affinity constant of 1.83 x 10(7) M-1. In the type 2 binding mode, the binding affinity constants are 4.9 x 10(6) and 4.3 x 10(6) M-1 for dsDNA and single-stranded DNA (ssDNA), respectively. The type 2 binding is base pair independent while the type 1 binding is base pair related. The experiments described in this paper revealed that the dication bindings are different from the monocation bindings reported by previous study. The dication binding leads to stronger aggregation at low ligand concentration and results in orderly arrangements of the ligands along DNA chains. Furthermore the dication binding is demonstrated to be beneficial for enhancing the DNA's stability.

Fabrication of Yolk-Shell Cu@C Nanocomposites as High-Performance Catalysts in Oxidative Carbonylation of Methanol to Dimethyl Carbonate.

A facile way was developed to fabricate yolk-shell composites with tunable Cu cores encapsulated within hollow carbon spheres (Cu@C) with an average diameter about 210 nm and cavity size about 80 nm. During pyrolysis, the confined nanospace of hollow cavity ensures that the nucleation-and-growth process of Cu nanocrystals take place exclusively inside the cavities. The size of Cu cores can be easily tuned from 30 to 55 nm by varying the copper salt concentration. By deliberately creating shell porosity through KOH chemical activation, at an optimized KOH/HCS mass ratio of 1/4, the catalytic performance for the oxidative carbonylation of methanol to dimethyl carbonate (DMC) of the activated sample is enhanced remarkably with TOF up to 8.6 h-1 at methanol conversion of 17.1%. The activated yolk-shell catalyst shows promising catalytic properties involving the reusability with slight loss of catalytic activity and negligible leaching of activated components even after seven recycles, which is beneficial to the implementation of clean production for the eco-friendly chemical DMC thoroughly.

Effect of interactions between LEPR polymorphisms and smoking on coronary artery disease susceptibility.

OBJECTIVE: This study was designed with the purpose of analyzing the relationship between the interactions of leptin receptor (LEPR) rs1137101, rs1137100 polymorphisms with smoking and the susceptibility to coronary artery disease (CAD). METHODS: The subjects of this study consisted of 101 CAD patients and 110 healthy people frequency-matched with the former in age and sex. The genotyping of LEPR polymorphisms were performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The distribution differences of genotype and allele between two groups was estimated by χ2 test. The relative risk of CAD was showed by odds ratio (OR) with 95% confidence interval (95% CI). Crossover analysis was applied in the studying of gene-environment interactions. RESULTS: LEPR rs1137101 polymorphism showed significant difference between CAD patients and healthy controls neither genotypes nor alleles in this study population (P>0.05). Fortunately, we detected that AA genotype of LEPR rs1137100 polymorphism had frequency difference between two study groups with a significant level, compared with the common genotype GG (P=0.03), which showed that this polymorphism had an independent association with CAD (OR=3.07, 95% CI=1.08-8.73). So was A allele of rs1137100 (OR=1.58, 95% CI=1.04-2.39). Furthermore, we observed the interaction of smoking and rs1137100 polymorphism in CAD occurrence (OR=2.69, P=0.01). CONCLUSIONS: Not only is LEPR rs1137100 polymorphism an independent risk factor for CAD, but it exists the interaction with smoking in the onset of CAD in this Chinese population.

Enzyme-linked immunosorbent assays for insulin-like growth factor-I using six-histidine tag fused proteins.

The fusion proteins of insulin-like growth factor-I (IGF-I) and six-histidine tag (IGF-I-6H, 6H-IGF-I-6H) were cloned, expressed, purified and renatured, with their immunoreaction properties and biological activities intact. The binding kinetics between these fusion proteins and anti-IGF-I antibody or anti-6H antibody were studied using surface plasmon resonance (SPR). Two enzyme-linked immunosorbent assay (ELISA) modes, which proved feasible in the measurement of human serum samples, were used to detect IGF-I with the help of the six-histidine tagged proteins. Furthermore, combining the production technique of the six-histidine tagged fusion protein with the competitive sandwich ELISA mode, using an enzyme labeled anti-6H antibody as a tracer, can be a universal immunochemical method to quantitate other polypeptides or proteins.

Imidazole as a catalyst for in vitro refolding of enhanced green fluorescent protein.

Imidazole is a reagent widely used in protein purifying processes. Here, we reveal a novel chaperone-like activity for imidazole using enhanced green fluorescent protein (EGFP) as a model protein. Experimental results showed that imidazole acted as an effective catalyst for refolding of the chemically denatured EGFP and suppressor for the heat-induced aggregation of EGFP. The refolding kinetics was determined in real time. Both the recovering yield and refolding rate of denatured EGFP in the presence of imidazole were increased. The studies on elucidating the mechanism show that imidazole may catalyze the prolyl cis/trans isomerization and the possible mechanism was discussed. To our knowledge, there are no data on the effect of imidazole on protein folding. Considering the prolyl isomerization is the rate-limited step for refolding of most proteins and aggregation is a universal serious problem for biotechnology, imidazole thus represents a previous unknown type of protein-folding catalyst.

Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay.

The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 x 10(-8) to 2.0 x 10(-6) M with a detection limit of 1.6 x 10(-8) M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications.