The Cys‐loop pentameric ligand‐gated ion channels comprise a dynamic group of proteins that have been extensively studied for decades, yielding a wealth of findings at both the structural and ...functional levels. The nicotinic acetylcholine receptor (nAChR) is no exception, as it is part of this large protein family involved in proper organismal function. Our efforts have successfully produced a highly pure nAChR in detergent complex (nAChR‐DC), enabling more robust studies to be conducted on it, including beginning to experiment with high‐throughput crystallization. Our homogeneous product has been identified and extensively characterized with 100% identity using Nano Lc MS/MS and MALDI ToF/ToF for each nAChR subunit. Additionally, the N‐linked glycans in the Torpedo californica‐nAChR (Tc‐nAChR) subunits have been identified. To study this, the Tc‐nAChR subunits were digested with PNGase F and the released glycans were analyzed by MALDI‐ToF. The MS results showed the presence of high‐mannose N‐glycan in all native Tc‐nAChR subunits. Specifically, the oligommanose population Man8‐9GlcNac2 with peaks at m/z 1742 and 1904 (M + Na+ ions) were observed.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Many pathogens, such as Pseudomonas aeruginosa and Escherichia coli bacteria can easily attach to surfaces and form stable biofilms. The formation of such biofilms in surfaces presents a problem in ...environmental, biomedical, and industrial processes, among many others. Aiming to provide a plausible solution to this issue, the anionic and hydrophobic peptide Maximin H5 C-terminally deaminated isoform (MH5C) has been modified with a cysteine in the C-terminal (MH5C-Cys) and coupled to polyethylene glycol (PEG) polymers of varying sizes (i.e., 2 kDa and 5 kDa) to serve as a surface protective coating. Briefly, the MH5C-Cys was bioconjugated to PEG and purified by size exclusion chromatography while the reaction was confirmed via SDS-PAGE and MALDI ToF. Moreover, the preventive antimicrobial activity of the MH5C-Cys-PEG conjugates was performed via the growth curves method, showing inhibition of bacterial growth after 24 h. The efficacy of these peptide–polymer conjugates was extensively characterized via scanning electron microscopy (SEM), minimum inhibition concentration (MIC), minimum biofilm inhibition concentration (MBIC), and minimum biofilm eradication concentration (MBEC) assays to evaluate their ability to eradicate and prevent the biofilms. Interestingly, this work demonstrated a critical PEG polymer weight of 5 kDa as ideal when coupled to the peptide to achieve inhibition and eradication of the biofilm formation in both bacteria strains. According to the MICs (40 μM) and MBICs (300 μM), we can conclude that this conjugate (MH5C-Cys-5 kDa) has an action that prevents/inhibits the formation of biofilms and the eradication of biofilms (MBEC 500 μM). In contrast, the MH5C-Cys peptide with PEG polymer of 2 kDa did not show inhibition or eradication of the biofilms.
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IJS, KILJ, NUK, PNG, UL, UM
AbstractLevee failure due to overtopping during floods is a common phenomena. An experimental study was conducted in the hydraulics laboratory, University of South Carolina, to develop an ...understanding of the effects of soil properties on the breaching process of earthen levees. A total of eight experiments were conducted. A new methodology to measure the subaqueous breach evolution has been developed and validated. Measurements of breach width evolution and headcut migration are shown to be related to soil properties. Inclusion of noncohesive, silt size crushed silica in the levee material (45% by weight) significantly reduced the peak discharge and breach evolution time compared to a levee constructed with sand alone. Increase in kaolin clay in the levee material led to an increase in unconfined compressive strength which in turn increased resistance to levee failure. Nondimensional equations relating the breach width evolution and headcut migration to the soil properties have been developed by performing a multivariate analysis of the experimental data.
•Vaccine production efforts require quick methods for titer determination in cultures.•Reversed phase HPLC can be used to quickly determine amount of vaccine in cultures.•The new reversed phase HPLC ...method showed to be linear with high protein recovery.•This new reported method is quicker and more reliable than antibody-based methods.
A reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the quantitative determination of recombinant HIV-1 gp145 produced in CHO-K1 cells, as measured directly from culture supernatants. Samples were diluted in 50% D-PBS and 50% PowerCHO-2 (PC2) spent medium, and resolved on a Zorbax 300SB-C8 Rapid Resolution (2.1 × 50 mm, 3.5 µm) column, fitted with a C8 guard column (Zorbax 300SB-C8, 2.1 × 12.5 mm, 5 µm), using 0.1% TFA and 2% n-propanol in LC-MS water as mobile phase A and 0.1% TFA, 70% isopropanol, and 20% acetonitrile in LC-MS water as mobile phase B. The column temperature was 80 °C, the flow rate was 0.4 mL/min and the absorbance was monitored at 280 nm. The procedures and capabilities of the method were evaluated against the criteria for linearity, limit of detection (LOD), accuracy, repeatability, and robustness as defined by the International Conference on Harmonization (ICH) 2005 Q2(R1) guidelines. Two different variants of the HIV-1 envelope protein (Env), CO6980v0c22 gp145 and SF162 gp140, were analyzed and their retention times were found to be different. The method showed good linearity (R2 = 0.9996), a lower LOD of 2.4 µg/mL, and an average recovery of 101%. The analysis includes measurements of accuracy, inter-user precision, and robustness. Overall, we present a RP-HPLC method that could be applied for the quantitation of cell culture titers for this and other variants of HIV Env following ICH guidelines.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We report here the versatility of Mn-doped ZnS quantum dots (ZnS:Mn QDs) synthesized in aqueous medium for generating reactive oxygen species and for detecting cells. Our experiments provide evidence ...leading to the elimination of Cd-based cores in CdSe/ZnS systems by substitution of Mn-doped ZnS. Advanced electron microscopy, X-ray diffraction, and optical spectroscopy were applied to elucidate the formation, morphology, and dispersion of the products. We study for the first time the ability of ZnS:Mn QDs to act as immobilizing agents for Tyrosinase (Tyr) enzyme. It was found that ZnS:Mn QDs show no deactivation of Tyr enzyme, which efficiently catalyzed the hydrogen peroxide (H
2
O
2
) oxidation and its eventual reduction (−0.063 V vs. Ag/AgCl) on the biosensor surface. The biosensor showed a linear response in the range of 12 μmol/L–0.1 mmol/L at low operation potential. Our observations are explained in terms of a catalase-cycled kinetic mechanism based on the binding of H
2
O
2
to the axial position of one of the active copper sites of the
oxy
-Tyr during the catalase cycle to produce
deoxy
-Tyr. A singlet oxygen quantum yield of 0.62 in buffer and 0.54 in water was found when ZnS:Mn QDs were employed as a photosensitizer in the presence of a chemical scavenger and a standard dye. These results are consistent with a chemical trapping energy transfer mechanism. Our results also indicate that ZnS:Mn QDs are well tolerated by HeLa Cells reaching cell viabilities as high as 88 % at 300 µg/mL of QDs for 24 h of incubation. The ability of ZnS:Mn QDs as luminescent nanoprobes for bioimaging is also discussed.
Graphical Abstract
The envelope glycoprotein (Env) of the human immunodeficiency virus (HIV), has been the primary target for the development of a protective vaccine against infection. The extensive N-linked ...glycosylation on Env is an important consideration as it may affect efficacy, stability, and expression yields. The expression host has been shown to influence the extent and type of glycosylation that decorates the protein target. Here, we report the glycosylation profile of the candidate subtype C immunogen CO6980v0c22 gp145, which is currently in Phase I clinical trials, produced in two different host cells: CHO-K1 and Expi293F. The amino acid sequence for both glycoproteins was confirmed to be identical by peptide mass fingerprinting. However, the isoelectric point of the proteins differed; 4.5-5.5 and 6.0-7.0 for gp145 produced in CHO-K1 and Expi293F, respectively. These differences in pI were eliminated by enzymatic treatment with sialidase, indicating a large difference in the incorporation of sialic acid between hosts. This dramatic difference in the number of sialylated glycans between hosts was confirmed by analysis of PNGase F-released glycans using MALDI-ToF MS. These differences in glycosylation, however, did not greatly translate into differences in antibody recognition. Biosensor assays showed that gp145 produced in CHO-K1 had similar affinity toward the broadly neutralizing antibodies, 2G12 and PG16, as the gp145 produced in Expi293F. Additionally, both immunogens showed the same reactivity against plasma of HIV-infected patients. Taken together, these results support the notion that there are sizeable differences in the glycosylation of Env depending on the expression host. How these differences translate to vaccine efficacy remains unknown.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Amorphous Cr–SiO
2
materials were synthesized by the sol–gel method and hydrothermally treated at temperatures between 150 and 220 °C. These materials were used as catalysts for cyclohexanol ...oxidation with H
2
O
2
as oxidant and CH
3
CN as a solvent. They were responsible for the decomposition of H
2
O
2
, which triggers, by a free radical mechanism in the homogeneous phase, the oxidation or degradation of the hydrocarbon chain. Metal leaching causes a drop in catalytic activity when the material is recycled. Studies on the hydrothermal treatment effect on the leaching process have demonstrated that the higher the hydrothermal treatment temperature, the higher the metal/support interaction, leading to a diminution of the leaching process. Under mild reaction conditions, and using TBHP as oxidant, leaching was reduced, and improvements were obtained on the selectivity towards the formation of cyclohexanone. The use of these catalysts in the oxidation of verbenol, an allylic alcohol, showed a significant increase in the substrate conversion and in the selectivity to carbonyl derivative formation.
Graphical Abstract
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
GRB 221009A: The BOAT Burns, Eric; Svinkin, Dmitry; Fenimore, Edward ...
Astrophysical journal. Letters,
03/2023, Volume:
946, Issue:
1
Journal Article
Peer reviewed
Open access
Abstract GRB 221009A has been referred to as the brightest of all time (BOAT). We investigate the veracity of this statement by comparing it with a half century of prompt gamma-ray burst ...observations. This burst is the brightest ever detected by the measures of peak flux and fluence. Unexpectedly, GRB 221009A has the highest isotropic-equivalent total energy ever identified, while the peak luminosity is at the ∼99th percentile of the known distribution. We explore how such a burst can be powered and discuss potential implications for ultralong and high-redshift gamma-ray bursts. By geometric extrapolation of the total fluence and peak flux distributions, GRB 221009A appears to be a once-in-10,000-year event. Thus, it is almost certainly not the BOAT over all of cosmic history; it may be the brightest gamma-ray burst since human civilization began.
Stem cells sense and respond to the mechanical properties of the extracellular matrix. However, both the extent to which extracellular-matrix mechanics affect stem-cell fate in three-dimensional ...microenvironments and the underlying biophysical mechanisms are unclear. We demonstrate that the commitment of mesenchymal stem-cell populations changes in response to the rigidity of three-dimensional microenvironments, with osteogenesis occurring predominantly at 11-30 kPa. In contrast to previous two-dimensional work, however, cell fate was not correlated with morphology. Instead, matrix stiffness regulated integrin binding as well as reorganization of adhesion ligands on the nanoscale, both of which were traction dependent and correlated with osteogenic commitment of mesenchymal stem-cell populations. These findings suggest that cells interpret changes in the physical properties of adhesion substrates as changes in adhesion-ligand presentation, and that cells themselves can be harnessed as tools to mechanically process materials into structures that feed back to manipulate their fate.
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IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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