High level soluble expression, one-step purification and characterization of HIV-1 p24 protein

<p>Abstract</p> <p>Background</p> <p>P24 protein is the major core protein of HIV virus particle and has been suggested as a specific target for antiviral strategies. Recombinant p24 protein with natural antigenic activity would be useful for various studies, such as diagnostic reagents and multi-component HIV vaccine development. The aim of this study was to express and purify the p24 protein in soluble form in <it>E.coli</it>.</p> <p>Results</p> <p>According to the sequence of the p24 gene, a pair of primers was designed, and the target sequence of 700 bp was amplified using PCR. The PCR product was cloned into pQE30 vector, generating the recombinant plasmid pQE30-p24. SDS-PAGE analysis showed that the His-tagged recombinant p24 protein was highly expressed in soluble form after induction in <it>E. coli </it>strain BL21. The recombinant protein was purified by nickel affinity chromatography and used to react with HIV infected sera. The results showed that the recombinant p24 protein could specifically react with the HIV infected sera. To study the immunogenicity of this soluble recombinant p24 protein, it was used to immunize mice for the preparation of polyclonal antibody. Subsequent ELISA and Western-Blot analysis demonstrated that the p24 protein had proper immunogenicity in inducing mice to produce HIV p24 specific antibodies.</p> <p>Conclusion</p> <p>In this work, we report the high level soluble expression of HIV-1 p24 protein in <it>E. coli</it>. This soluble recombinant p24 protein specifically react with HIV infected sera and elicit HIV p24 specific antibodies in mice, indicating this soluble recombinant p24 protein could be a promising reagent for HIV diagnosis.</p

Neuropathic Injury-Induced Plasticity of GABAergic System in Peripheral Sensory Ganglia

GABA is a major inhibitory neurotransmitter in the mammalian central nervous system (CNS). Inhibitory GABAA channel circuits in the dorsal spinal cord are the gatekeepers of the nociceptive input from the periphery to the CNS. Weakening of these spinal inhibitory mechanisms is a hallmark of chronic pain. Yet, recent studies have suggested the existence of an earlier GABAergic “gate” within the peripheral sensory ganglia. In this study, we performed systematic investigation of plastic changes of the GABA-related proteins in the dorsal root ganglion (DRG) in the process of neuropathic pain development. We found that chronic constriction injury (CCI) induced general downregulation of most GABAA channel subunits and the GABA-producing enzyme, glutamate decarboxylase, consistent with the weakening of the GABAergic inhibition at the periphery. Strikingly, the α5 GABAA subunit was consistently upregulated. Knock-down of the α5 subunit in vivo moderately alleviated neuropathic hyperalgesia. Our findings suggest that while the development of neuropathic pain is generally accompanied by weakening of the peripheral GABAergic system, the α5 GABAA subunit may have a unique pro-algesic role and, hence, might represent a new therapeutic target

Bacterial Microbiota and Metabolic Character of Traditional Sour Cream and Butter in Buryatia, Russia

Traditional sour cream and butter are widely popular fermented dairy products in Russia for their flavor and nutrition, and contain rich microbial biodiversity, particularly in terms of lactic acid bacteria (LAB). However, few studies have described the microbial communities and metabolic character of traditional sour cream and butter. The objective of this study was to determine the bacterial microbiota and metabolic character of eight samples collected from herdsmen in Buryatia, Russia. Using single-molecule real-time (SMRT) sequencing techniques, we identified a total of 294 species and/or subspecies in 169 bacterial genera, belonging to 14 phyla. The dominant phylum was Firmicutes (81.47%) and the dominant genus was Lactococcus (59.28%). There were differences between the bacterial compositions of the sour cream and butter samples. The relative abundances of Lactococcus lactis, Lactococcus raffinolactis, and Acetobacter cibinongensis were significantly higher in sour cream than in butter, and the abundance of Streptococcusthermophilus was significantly lower in sour cream than in butter. Using a pure culture method, 48 strains were isolated and identified to represent seven genera and 15 species and/or subspecies. Among these isolates, Lactococccus lactis subsp. lactis (22.50%) was the dominant LAB species. Ultra-performance liquid chromatography–quadrupole–time of flight mass spectrometry at elevated energy was used in combination with statistical methods to detect metabolite differences between traditional sour cream and butter. A total of 27,822 metabolites were detected in all samples, and Lys-Lys, isohexanal, palmitic acid, Leu-Val, and 2′-deoxycytidine were the most dominant metabolites found in all samples. In addition, 27 significantly different metabolites were detected between the sour cream and butter samples, including short peptides, organic acids, and amino acids. Based on correlation analyses between the most prevalent bacterial species and the main metabolites in sour cream, we conclude that there may be a connection between the dominant LAB species and these metabolites. This study combined omics techniques to analyze the bacterial diversity and metabolic character of traditional sour cream and butter, and we hope that our findings will enrich species resource libraries and provide valuable resources for further research on dairy product flavor

27-Hydroxycholesterol Contributes to Lysosomal Membrane Permeabilization-Mediated Pyroptosis in Co-cultured SH-SY5Y Cells and C6 Cells

Purpose: Emerging evidence suggests that 27-Hydroxycholesterol (27-OHC) causes neurodegenerative diseases through the induction of cytotoxicity and cholesterol metabolism disorder. The objective of this study is to determine the impacts of 27-OHC on lysosomal membrane permeabilization (LMP) and pyroptosis in neurons in the development of neural degenerative diseases.Methods: In this study, SH-SY5Y cells and C6 cells were co-cultured in vitro to investigate the influence of 27-OHC on the function of lysosome, LMP and pyroptosis related factors in neuron. Lyso Tracker Red (LTR) was used to detect the changes of lysosome pH, volume and number. Acridine orange (AO) staining was also used to detect the LMP in neurons. Then the morphological changes of cells were observed by a scanning electron microscope (SEM). The content of lysosome function associated proteins [including Cathepsin B (CTSB), Cathepsin D (CTSD), lysosomal-associated membraneprotein-1 (LAMP-1), LAMP-2] and the pyroptosis associated proteins [including nod-like recepto P3 (NLRP3), gasdermin D (GSDMD), caspase-1 and interleukin (IL)-1β] were detected through Western blot.Results: Results showed higher levels of lysosome function associated proteins, such as CTSB (p &lt; 0.05), CTSD (p &lt; 0.05), LAMP-1 (p &lt; 0.01), LAMP-2; p &lt; 0.01) in 27-OHC treated group than that in the control group. AO staining and LTR staining showed that 27-OHC induced lysosome dysfunction with LMP. Content of pyroptosis related factor proteins, such as GSDMD (p &lt; 0.01), NLRP3 (p &lt; 0.001), caspase-1 (p &lt; 0.01) and IL-1β (p &lt; 0.01) were increased in 27-OHC treated neurons. Additionally, CTSB was leaked through LMP into the cytosol and induced pyroptosis. Results from the present study also suggested that the CTSB is involved in activation of pyroptosis.Conclusion: Our data indicate that 27-OHC contributes to the pathogenesis of cell death by inducing LMP and pyroptosis in neurons

The transmembrane channel-like 6 (TMC6) in primary sensory neurons involving thermal sensation via modulating M channels

Introduction: The transmembrane channel-like (TMC) protein family contains eight members, TMC1–TMC8. Among these members, only TMC1 and TMC2 have been intensively studied. They are expressed in cochlear hair cells and are crucial for auditory sensations. TMC6 and TMC8 contribute to epidermodysplasia verruciformis, and predispose individuals to human papilloma virus. However, the impact of TMC on peripheral sensation pain has not been previously investigated.Methods: RNAscope was employed to detect the distribution of TMC6 mRNA in DRG neurons. Electrophysiological recordings were conducted to investigate the effects of TMC6 on neuronal characteristics and M channel activity. Zn2+ indicators were utilized to detect the zinc concentration in DRG tissues and dissociated neurons. A series of behavioural tests were performed to assess thermal and mechanical sensation in mice under both physiological and pathological conditions.Results and Discussion: We demonstrated that TMC6 is mainly expressed in small and medium dorsal root ganglion (DRG) neurons and is involved in peripheral heat nociception. Deletion of TMC6 in DRG neurons hyperpolarizes the resting membrane potential and inhibits neuronal excitability. Additionally, the function of the M channel is enhanced in TMC6 deletion DRG neurons owing to the increased quantity of free zinc in neurons. Indeed, heat and mechanical hyperalgesia in chronic pain are alleviated in TMC6 knockout mice, particularly in the case of heat hyperalgesia. This suggests that TMC6 in the small and medium DRG neurons may be a potential target for chronic pain treatment

Diagnostic Yields of Trio-WES Accompanied by CNVseq for Rare Neurodevelopmental Disorders

ObjectiveThis study is to investigate the diagnostic yield of the combination of trio whole exome sequencing (Trio-WES) and copy number variation sequencing (CNVseq) for rare neurodevelopmental disorders (NDDs).MethodsClinical data from consecutive pediatric patients who were diagnosed with rare NDDs that were suspected to be monogenic disorders, who were admitted to our hospital from April 2017 to March 2019, and who underwent next generation sequencing (NGS) were extracted from the medical records. Patients for whom Trio-WES and CNVseq data were available were enrolled in this study. Sanger sequencing was applied for the validation of the variants identified by Trio-WES. Sequence alignment and structural modeling were conducted for analyzing the possibility of the variants in the onset of the NDDs.ResultsIn total, 54 patients were enrolled in this study, with the median age of 15 (8–26) months. A total of 242 phenotypic abnormalities belonging to 20 different systems were identified in the cohort. Twenty-four patients were diagnosed by Trio-WES, eight patients were diagnosed by CNVseq, and one case was identified by both WES and CNVseq. Compared with Trio-WES, the diagnosis rate of Trio-WES accompanied by CNVseq was significantly higher (P = 0.016). Trio-WES identified 36 variants in 26 different genes, among which 27 variants were novel. CNVseq detected four duplications and eight deletions, ranging from 310 kb to 23.27 Mb. Our case examples demonstrated the high heterogeneity of NDDs and showed the challenges of rare NDDs for physicians.ConclusionThe significantly higher diagnosis rate of Trio-WES accompanied by CNVseq makes this strategy a potential alternative to the most widely used approaches for pediatric children with rare and undiagnosed NDDs

Serum anion gap is associated with mortality in intensive care unit patients with diastolic heart failure

Abstract Serum anion gap (AG) is closely related to mortality in critically ill patients with several diseases. We aimed to determine the relationship between serum AG levels and 28-day intensive care unit (ICU) mortality in patients with diastolic heart failure (DHF). This cohort study enrolled critically ill patients with DHF from the Medical Information Mart for Intensive Care IV (MIMIC-IV) database. Serum AG levels were calculated using the traditional and albumin-adjusted methods. Multivariate Cox proportional hazards regression and restricted cubic spline curves were used to determine the correlation between serum AG levels and 28-day ICU mortality. We used receiver operating characteristic (ROC) curves and area under the curve (AUC) to compare the ability of traditional and albumin-adjusted AG to predict mortality. Overall, 3290 patients were included. Multivariate analysis showed an association of high levels of traditional (hazard ratio [HR], 1.48; 95% confidence interval [CI], 1.1–1.98, p = 0.009) and albumin-adjusted AG (HR, 1.36; 95% CI, 1.02–1.79, p = 0.033) with higher risk of 28-day ICU mortality. Restricted cubic spline curves indicated a linear relationship between AG level and 28-day ICU mortality. Comparison of the ROC curves revealed that albumin-adjusted AG had a greater ability to predict 28-day ICU mortality compared with traditional AG (AUCs of 0.569 [95% CI, 0.536–0.601] and 0.619 [95% CI, 0.588–0.649], respectively). In ICU patients with DHF, higher levels of traditional and albumin-adjusted AG were associated with higher 28-day ICU mortality. Albumin-adjusted AG exhibited greater predictive ability for mortality compared with traditional AG

Band gap characteristics of radial wave in a two-dimensional cylindrical shell with radial and circumferential periodicities

A two-dimensional cylindrical shell (2DCS) structure with radial and circumferential periodicities is proposed in this paper. The band gap characteristics of radial wave propagating radically from the inner circle of the structure are investigated numerically. According to the calculation and discussion of wave transmission behavior, radial wave in the structure is found to show distinct attenuation in certain frequency region, i.e. wave band gap. The internal mechanism of the band gap is then investigated and the Bragg scattering effect is illustrated. Furthermore, the detailed behaviors of radial wave within the band gap are studied, especially for the transmission peaks. Special localization phenomenon is observed where the mechanical energy localizes strongly in a certain circle of scatterers. Consistency is then found by comparing these localization modes of the whole 2DCS with those of corresponding single scatterers, which indicates that the localization phenomenon is essentially resulted from the local resonance behavior of certain scatterers

Grain boundaries induce significant decrease in lattice thermal conductivity of CdTe

Semiconductors are promising in photoelectric and thermoelectric devices, for which the thermal transport properties are of particular interest. However, they have not been fully understood, especially when crystalline imperfections are present. Here, using cadmium telluride (CdTe) as an example, we illustrate how grain boundaries (GBs) affect the thermal transport properties of semiconductors. We develop a machine-learning force field from density functional theory calculations for predicting the lattice thermal conductivity (LTC) via equilibrium molecular dynamics simulations. The LTC of crystalline CdTe decreases with the relationship of κL∼1/T in the simulation temperature range of 300 – 900 K, in which the isotropic LTC decreases from 3.34 to 0.23 W/(m⋅K) due to the enhanced anharmonicity. More important, after introducing GBs, the LTC is suppressed in all directions, especially in the direction normal to the GB planes. More severe LTC suppression occurs in CdTe with Σ9 GB than that with Σ3 GB at 300 K, decreasing by 92.8% and 61.4% along the direction normal to the GB planes compared to the isotropic LTC of the crystalline CdTe, respectively. The decreased LTC is consistent with the weaker bonding near GB planes and lower shear modulus of the defective material. The analyses of the phonon dispersion curves, vibrational density of states, and phonon participation ratio indicate that the decreased LTC mainly arises from phonon scattering at GBs. Overall, our work highlights that GBs can greatly influence the LTC of semiconductors, thus providing a promising approach for thermal property design

Template-Free Electrochemical Preparation of Hexagonal CuSn Prism-Structural Electrode for Lithium-Ion Batteries

A hexagonal prism CuSn alloy was prepared at room temperature from 1-ethyl-3-methylimidazolium dicyanamide ([Emim][DCA]) by the direct template-free electrodeposition method with different concentrations of Cu(I) and Sn(II) at a low current density of 0.04 A dm−2. Moreover, the electrodeposition time was also investigated, and the results indicated that the composition of the CuSn alloy became complex and the structure turned unstable with expanding time. The cycling performance of the hexagonal prism-structural CuSn electrode was investigated, with the first discharge capacity of 345 mAh g−1 and a discharge capacity of about 210 mAh g−1 after 10 cycles