Pentameric Form Research Articles

Understanding the biosynthesis of human IgM SAM-6 through a combinatorial expression of mutant subunits that affect product assembly and secretion.

Polymeric IgMs are secreted from plasma cells abundantly despite their structural complexity and intricate multimerization steps. To gain insights into IgM's assembly mechanics that underwrite such high-level secretion, we characterized the biosynthetic process of a natural human IgM, SAM-6, using a heterologous HEK293(6E) cell platform that allowed the production of IgMs both in hexameric and pentameric forms in a controlled fashion. By creating a series of mutant subunits that differentially disrupt secretion, folding, and specific inter-chain disulfide bond formation, we assessed their effects on various aspects of IgM biosynthesis in 57 different subunit chain combinations, both in hexameric and pentameric formats. The mutations caused a spectrum of changes in steady-state subcellular subunit distribution, ER-associated inclusion body formation, intracellular subunit detergent solubility, covalent assembly, secreted IgM product quality, and secretion output. Some mutations produced differential effects on product quality depending on whether the mutation was introduced to hexameric IgM or pentameric IgM. Through this systematic combinatorial approach, we consolidate diverse overlapping knowledge on IgM biosynthesis for both hexamers and pentamers, while unexpectedly revealing that the loss of certain inter-chain disulfide bonds, including the one between μHC and λLC, is tolerated in polymeric IgM assembly and secretion. The findings highlight the differential roles of underlying non-covalent protein-protein interactions in hexamers and pentamers when orchestrating the initial subunit interactions and maintaining the polymeric IgM product integrity during ER quality control steps, secretory pathway trafficking, and secretion.

Computational Insights into the Conformational Dynamics of HIV-1 Vpr in a Lipid Bilayer for Ion Channel Modeling.

HIV-1 Vpr is a multifunctional accessory protein consisting of 96 amino acids that play a critical role in viral pathogenesis. Among its diverse range of activities, Vpr can create a cation-selective ion channel within the plasma membrane. However, the oligomeric state of this channel has not yet been elucidated. In this study, we investigated the conformational dynamics of Vpr helices to model the ion channel topology. First, we employed a series of multiscale simulations to investigate the specific structure of monomeric Vpr in a membrane model. During the lipid bilayer self-assembly coarse grain simulation, the C-terminal helix (residues 56-77) effectively formed the transmembrane region, while the N-terminal helix exhibited an amphipathic nature by associating horizontally with a single leaflet. All-atom molecular dynamics (MD) simulations of full-length Vpr inside a phospholipid bilayer show that the C-terminal helix remains very stable inside the bilayer core in a vertical orientation. Subsequently, using the predicted C-terminal helix orientation and conformation, various oligomeric states (ranging from tetramer to heptamer) possibly forming the Vpr ion channel were built and further evaluated. Among these models, the pentameric form exhibited consistent stability in MD simulations and displayed a compatible conformation for a water-assisted ion transport mechanism. This study provides structural insights into the ion channel activity of the Vpr protein and the foundation for developing therapeutics against HIV-1 Vpr-related conditions.

C-reactive protein lowers the serum level of IL-17, but not TNF-α, and decreases the incidence of collagen-induced arthritis in mice.

The biosynthesis of C-reactive protein (CRP) in the liver is increased in inflammatory diseases including rheumatoid arthritis. Previously published data suggest a protective function of CRP in arthritis; however, the mechanism of action of CRP remains undefined. The aim of this study was to evaluate the effects of human CRP on the development of collagen-induced arthritis (CIA) in mice which is an animal model of autoimmune inflammatory arthritis. Two CRP species were employed: wild-type CRP which binds to aggregated IgG at acidic pH and a CRP mutant which binds to aggregated IgG at physiological pH. Ten CRP injections were given on alternate days during the development of CIA. Both wild-type and mutant CRP reduced the incidence of CIA, that is, reduced the number of mice developing CIA; however, CRP did not affect the severity of the disease in arthritic mice. The serum levels of IL-17, IL-6, TNF-α, IL-10, IL-2 and IL-1β were measured: both wild-type and mutant CRP decreased the level of IL-17 and IL-6 but not of TNF-α, IL-10, IL-2 and IL-1β. These data suggest that CRP recognizes and binds to immune complexes, although it was not clear whether CRP functioned in its native pentameric or in its structurally altered pentameric form in the CIA model. Consequently, ligand-complexed CRP, through an as-yet undefined mechanism, directly or indirectly, inhibits the production of IL-17 and eventually protects against the initiation of the development of arthritis. The data also suggest that IL-17, not TNF-α, is critical for the development of autoimmune inflammatory arthritis.

Two Forms of C-Reactive Protein and Their Implication for Atherogenesis: Focus on Monomeric Form

In this review, we discuss the role of C-reactive protein (CRP), a biomarker of inflammation, in the development of atherosclerosis. CRP exists in two forms: the native pentameric form (nCRP) and the non-native form (mCRP), which is formed during inflammatory processes. The article explores the functions of both forms in atherosclerosis. nCRP binds to molecules with uncoated phosphocholine groups and activates the classical complement pathway. It can bind to enzymatically-modified LDL (E-LDL) and ox-LDL, affecting their pro-inflammatory properties and reducing LDL oxidation. nCRP also interacts with endothelial cells and reduces the absorption of acetylated LDL by these cells. In contrast, mCRP, formed in the presence of pathological conditions, exhibits distinct pro-inflammatory characteristics. While the role of nCRP in atherosclerosis remains unclear in animal models, mCRP has been associated with inflammation and is found in atherosclerotic lesions. The study suggests that nCRP may have atheroprotective effects, while mCRP may contribute to the progression of atherosclerosis. Further research is needed to fully understand the complex mechanisms of CRP in atherosclerosis.

FUNCTIONAL ACTIVITY OF MONOMERIC C-REACTIVE PROTEIN IN INTERVERTEBRAL DISC

Monomeric C reactive protein (mCRP) presents important proinflammatory effects in endothelial cells, leukocytes, or chondrocytes. However, CRP in its pentameric form exhibits weak anti-inflammatory activity. It is used as a biomarker to follow severity and progression in infectious or inflammatory diseases, such as intervertebral disc degeneration (IVDD). This work assesses for the first time the mCRP effects in human intervertebral disc cells, trying to verify the pathophysiological relevance and mechanism of action of mCRP in the etiology and progression of IVD degeneration.We demonstrated that mCRP induces the expression of multiple proinflammatory and catabolic factors, like nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and lipocalin 2 (LCN2), in human annulus fibrosus (AF) and nucleus pulposus (NP) cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signaling of mCRP.Our results indicate that the effect of mCRP is persistent and sustained, regardless of the proinflammatory environment, as it was similar in healthy and degenerative human primary AF cells. This is the first article that demonstrates the localization of mCRP in intravertebral disc cells of the AF and NP and that provides evidence for the functional activity of mCRP in healthy and degenerative human AF and NP disc cells.

Monomeric C‐reactive protein exacerbates choroidal neovascularization in vivo: Implications for age‐related macular degeneration

Aims/Purpose: Increased systemic levels of the acute phase reactant C‐reactive protein (CRP) have been linked to increased risk of age‐related macular degeneration (AMD). CRP undergoes conformational changes upon activation in inflammatory microenvironments between pentameric (pCRP) and monomeric forms (mCRP). CRP conformations present different phenotypes, as mCRP has more proinflammatory properties and has been shown to induce blood retinal barrier disruption in vitro. Thus, we aimed to evaluate the impact of CRP forms on choroidal neovascularization (CNV) in vivo.Methods: pCRP and mCRP forms were intravenously (iv) or intravitreally (ivt) injected in C57BL6/J mice (n = 12) previously subjected to CNV induction by laser and compared to controls. CNV lesions were monitored by fluorescein angiography and mice were euthanized 11 days after lesion induction. CNV areas were measured in lectin stained choroidal flatmounts. To evaluate the CRP dissociation, pCRP was coupled to a fluorescent molecule before injection. After tissue extraction mCRP was detected by immunofluorescence in posterior pole flatmounts.Results: Both pCRP and mCRP injected significantly increased CNV areas compared to control mice. Retinal severe edema was also observed close to the lesion in retinographies, and was more severe in intravitreally injected groups. Finally, pCRP was observed to dissociate into mCRP in intravitreally injected animals with pCRP.Conclusions: We confirmed that CRP increases CNV areas and severe edema in the retina, suggesting that CRP is activated in the CNV inflammatory environment. Additionally, the pCRP form dissociates in vivo into mCRP close to CNV damaged tissue, hypothesizing that mCRP is the CRP conformation that contributes to the exacerbation of the CNV process. Further studies are needed to investigate anti‐mCRP molecules as candidates for mono‐ or anti‐VEGF combined therapy for AMD treatments.

Human monoclonal antibodies against Staphylococcus aureus A protein identified by high-throughput single-cell sequencing of phase I clinical volunteers' B cells

Methicillin-resistant Staphylococcus aureus, poses a significant threat through infections in both community and hospital settings. To address this challenge, we conducted a phase I clinical trial study involving a recombinant Staphylococcus aureus vaccine. Utilizing peripheral blood lymphocytes from 64 subjects, we isolated antigen-specific memory B cells for subsequent single-cell sequencing. Among the 676 identified antigen-binding IgG1+ clones, we selected the top 10 antibody strains for construction within expression vectors. Successful expression and purification of these monoclonal antibodies led to the discovery of a highly expressed human antibody, designated as IgG-6. This antibody specifically targets the pentameric form of the Staphylococcus aureus protein A (SpA5). In vivo assessments revealed that IgG-6 provided prophylactic protection against MRSA252 infection. This study underscores the potential of human antibodies as an innovative strategy against Staphylococcus aureus infections, offering a promising avenue for further research and clinical development.

C-Reactive Protein: Pathophysiology, Diagnosis, False Test Results and a Novel Diagnostic Algorithm for Clinicians.

The current literature provides a body of evidence on C-Reactive Protein (CRP) and its potential role in inflammation. However, most pieces of evidence are sparse and controversial. This critical state-of-the-art monography provides all the crucial data on the potential biochemical properties of the protein, along with further evidence on its potential pathobiology, both for its pentameric and monomeric forms, including information for its ligands as well as the possible function of autoantibodies against the protein. Furthermore, the current evidence on its potential utility as a biomarker of various diseases is presented, of all cardiovascular, respiratory, hepatobiliary, gastrointestinal, pancreatic, renal, gynecological, andrological, dental, oral, otorhinolaryngological, ophthalmological, dermatological, musculoskeletal, neurological, mental, splenic, thyroid conditions, as well as infections, autoimmune-supposed conditions and neoplasms, including other possible factors that have been linked with elevated concentrations of that protein. Moreover, data on molecular diagnostics on CRP are discussed, and possible etiologies of false test results are highlighted. Additionally, this review evaluates all current pieces of evidence on CRP and systemic inflammation, and highlights future goals. Finally, a novel diagnostic algorithm to carefully assess the CRP level for a precise diagnosis of a medical condition is illustrated.

Studies of Benzotriazole on and into the Copper Electrodeposited Layer by Cyclic Voltammetry, Time-of-Flight Secondary-Ion Mass Spectrometry, Atomic Force Microscopy, and Surface Enhanced Raman Spectroscopy.

Benzotriazole (BTA) is an important compound that demonstrates the strongest anticorrosion properties of copper and plays a role as a leveler and an additive to the electroplating bath for control of the roughness and corrosion resistance of the electrodeposited copper layer. In this paper, we combined cyclic voltammetry (CV), time-of-flight secondary-ion mass spectrometry (TOF-SIMS), surface enhanced Raman spectroscopy (SERS), and atomic force microscopy (AFM) to study the interaction of BTA with copper surfaces at varied concentrations with and without the presence of chloride ions. We identified the most relevant molecular copper and its complex forms with BTA on the copper electrodeposited layer. BTA is adsorbed and incorporated into the copper surface in monomeric, dimeric, trimeric, tetrameric, and pentameric forms, inhibiting the copper electrodeposition. The addition of chloride ions diminishes the inhibiting properties of BTA. The Cu-BTA-Cl complexes were identified in the forms C12H8N6Cu2Cl- and C6H4N3CuCl-. Coadsorption of chloride ions and BTA molecules depends on their concentration and applied potential. Chloride ions are replaced by BTA molecules. BTA and chloride ions, depending on their concentration and applied potential, control the copper nucleation processes at the micro- and nanoscales. We compared the abilities and limitations of TOF-SIMS and SERS for studies of the interactions of benzotriazole with copper and chloride ions at the molecular level.

Structural basis for the ubiquitination of G protein βγ subunits by KCTD5/Cullin3 E3 ligase.

G protein-coupled receptor (GPCR) signaling is precisely controlled to avoid overstimulation that results in detrimental consequences. Gβγ signaling is negatively regulated by a Cullin3 (Cul3)-dependent E3 ligase, KCTD5, which triggers ubiquitination and degradation of free Gβγ. Here, we report the cryo-electron microscopy structures of the KCTD5-Gβγ fusion complex and the KCTD7-Cul3 complex. KCTD5 in pentameric form engages symmetrically with five copies of Gβγ through its C-terminal domain. The unique pentameric assembly of the KCTD5/Cul3 E3 ligase places the ubiquitin-conjugating enzyme (E2) and the modification sites of Gβγ in close proximity and allows simultaneous transfer of ubiquitin from E2 to five Gβγ subunits. Moreover, we show that ubiquitination of Gβγ by KCTD5 is important for fine-tuning cyclic adenosine 3´,5´-monophosphate signaling of GPCRs. Our studies provide unprecedented insights into mechanisms of substrate recognition by unusual pentameric E3 ligases and highlight the KCTD family as emerging regulators of GPCR signaling.

Monomeric CRP regulates inflammatory responses in human intervertebral disc cells.

CRP is an acute-phase protein that is used as a biomarker to follow severity and progression in infectious and inflammatory diseases. Its pathophysiological mechanisms of action are still poorly defined. CRP in its pentameric form exhibits weak anti-inflammatory activity. The monomeric isoform (mCRP) exerts potent proinflammatory properties in chondrocytes, endothelial cells, and leucocytes. No data exist regarding mCRP effects in human intervertebral disc (IVD) cells. This work aimed to verify the pathophysiological relevance of mCRP in the aetiology and/or progression of IVD degeneration. We investigated the effects of mCRP and the signalling pathways that are involved in cultured human primary annulus fibrosus (AF) cells and in the human nucleus pulposus (NP) immortalized cell line HNPSV-1. We determined messenger RNA (mRNA) and protein levels of relevant factors involved in inflammatory responses, by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. We also studied the presence of mCRP in human AF and NP tissues by immunohistochemistry. We demonstrated that mCRP increases nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and Lipocalin 2 (LCN2) expression in human AF and NP cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signalling of mCRP. Finally, we demonstrated the presence of mCRP in human AF and NP tissues. Our results indicate, for the first time, that mCRP can be localized in IVD tissues, where it triggers a proinflammatory and catabolic state in degenerative and healthy IVD cells, and that NF-κβ signalling may be implicated in the mediation of this mCRP-induced state.

In Silico Screening of Drugs That Target Different Forms of E Protein for Potential Treatment of COVID-19.

Recently the E protein of SARS-CoV-2 has become a very important target in the potential treatment of COVID-19 since it is known to regulate different stages of the viral cycle. There is biochemical evidence that E protein exists in two forms, as monomer and homopentamer. An in silico screening analysis was carried out employing 5852 ligands (from Zinc databases), and performing an ADMET analysis, remaining a set of 2155 compounds. Furthermore, docking analysis was performed on specific sites and different forms of the E protein. From this study we could identify that the following ligands showed the highest binding affinity: nilotinib, dutasteride, irinotecan, saquinavir and alectinib. We carried out some molecular dynamics simulations and free energy MM-PBSA calculations of the protein-ligand complexes (with the mentioned ligands). Of worthy interest is that saquinavir, nilotinib and alectinib are also considered as a promising multitarget ligand because it seems to inhibit three targets, which play an important role in the viral cycle. On the other side, saquinavir was shown to be able to bind to E protein both in its monomeric as well as pentameric forms. Finally, further experimental assays are needed to probe our hypothesis derived from in silico studies.

A dilated cardiomyopathy mutation of phospholamban, R14del, increases phospholamban pentamer stability.

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca2+ stores in cardiac muscle. Phospholamban (PLB), a micropeptide inhibitor of SERCA, forms avid homo-pentamers, but it is the monomeric form that regulates the pump. The dynamic exchange of monomeric PLB between pentamers and the regulatory complex with SERCA is an important determinant of cardiac responsiveness to exercise. Here, we investigated two naturally occurring pathogenic mutations of PLB, a cysteine substitution of arginine 9 (R9C) and an in-frame deletion of arginine 14 (R14del). Both of these mutations are associated with dilated cardiomyopathy. We previously showed that the R9C mutation causes disulfide crosslinking and hyperstabilization of pentamers. While the pathogenic mechanism of R14del is unclear, we hypothesized that this mutation may also alter PLB homo-oligomerization and disrupt the PLB-SERCA regulatory interaction. SDS-PAGE revealed a significantly increased pentamer:monomer ratio for R14del-PLB (87.2±3.2% pentamers) when compared to wild-type PLB (79.5±0.8% pentamers). In addition, we quantified homo-oligomerization and SERCA-binding in live cells using fluorescence resonance energy transfer (FRET) microscopy. R14del-PLB showed an increased affinity for homo-oligomerization and decreased binding affinity for SERCA compared to WT, suggesting that, like R9C, the R14del mutation traps PLB in its pentameric form, decreasing its ability to regulate SERCA. Moreover, the R14del mutation reduces the rate of PLB unbinding from the pentamer after a transient Ca2+ elevation, limiting the rate of re-binding to SERCA. A computational model predicted that hyperstabilization of R14del-PLB pentamers impairs the ability of cardiac Ca2+ handling to respond to changing heart rates between rest and exercise. We theorize that reduced responsiveness may contribute to arrhythmogenesis in human carriers of the R14del mutation.

C-reactive protein, immunothrombosis and venous thromboembolism.

C-reactive protein (CRP) is a member of the highly conserved pentraxin superfamily of proteins and is often used in clinical practice as a marker of infection and inflammation. There is now increasing evidence that CRP is not only a marker of inflammation, but also that destabilized isoforms of CRP possess pro-inflammatory and pro-thrombotic properties. CRP circulates as a functionally inert pentameric form (pCRP), which relaxes its conformation to pCRP* after binding to phosphocholine-enriched membranes and then dissociates to monomeric CRP (mCRP). with the latter two being destabilized isoforms possessing highly pro-inflammatory features. pCRP* and mCRP have significant biological effects in regulating many of the aspects central to pathogenesis of atherothrombosis and venous thromboembolism (VTE), by directly activating platelets and triggering the classical complement pathway. Importantly, it is now well appreciated that VTE is a consequence of thromboinflammation. Accordingly, acute VTE is known to be associated with classical inflammatory responses and elevations of CRP, and indeed VTE risk is elevated in conditions associated with inflammation, such as inflammatory bowel disease, COVID-19 and sepsis. Although the clinical data regarding the utility of CRP as a biomarker in predicting VTE remains modest, and in some cases conflicting, the clinical utility of CRP appears to be improved in subsets of the population such as in predicting VTE recurrence, in cancer-associated thrombosis and in those with COVID-19. Therefore, given the known biological function of CRP in amplifying inflammation and tissue damage, this raises the prospect that CRP may play a role in promoting VTE formation in the context of concurrent inflammation. However, further investigation is required to unravel whether CRP plays a direct role in the pathogenesis of VTE, the utility of which will be in developing novel prophylactic or therapeutic strategies to target thromboinflammation.

The Genetic Stability, Replication Kinetics and Cytopathogenicity of Recombinant Avian Coronaviruses with a T16A or an A26F Mutation within the E Protein Is Cell-Type Dependent.

The envelope (E) protein of the avian coronavirus infectious bronchitis virus (IBV) is a small-membrane protein present in two forms during infection: a monomer and a pentameric ion channel. Each form has an independent role during replication; the monomer disrupts the secretory pathway, and the pentamer facilitates virion production. The presence of a T16A or A26F mutation within E exclusively generates the pentameric or monomeric form, respectively. We generated two recombinant IBVs (rIBVs) based on the apathogenic molecular clone Beau-R, containing either a T16A or A26F mutation, denoted as BeauR-T16A and BeauR-A26F. The replication and genetic stability of the rIBVs were assessed in several different cell types, including primary and continuous cells, ex vivo tracheal organ cultures (TOCs) and in ovo. Different replication profiles were observed between cell cultures of different origins. BeauR-A26F replicated to a lower level than Beau-R in Vero cells and in ovo but not in DF1, primary chicken kidney (CK) cells or TOCs. Genetic stability and cytopathic effects were found to differ depending on the cell system. The effect of the T16A and A26F mutations appear to be cell-type dependent, which, therefore, highlights the importance of cell type in the investigation of the IBV E protein.

The monomeric C-reactive protein level is associated with the increase in carotid plaque number in patients with subclinical carotid atherosclerosis.

The high-sensitivity C-reactive protein (hsCRP) assay measures the level of the pentameric form of CRP in blood. Currently, there are no available assays measuring the level of the monomeric form of CRP (mCRP), produced at sites of local inflammation. We developed an assay measuring the mCRP level in blood plasma with functional beads for flow cytometry. The assay was used to measure the mCRP level in 80 middle-aged individuals with initially moderate cardiovascular SCORE risk. By the time of the mCRP measurement, the patients have been followed up for subclinical carotid atherosclerosis progression for 7 years. Ultrasound markers of subclinical atherosclerosis, which included plaque number (PN) and total plaque height (PH), were measured at baseline and at the 7th-year follow-up survey. Inflammatory biomarkers, including mCRP, hsCRP, inteleukin-6 (IL-6) and von Willebrand factor (VWF) level, were measured at the 7th-year follow-up survey. The median level of mCRP was 5.2 (3.3; 7.1) μg/L, hsCRP 1.05 (0.7; 2.1) mg/L, IL-6 0.0 (0.0; 2.8) pg/mL, VWF 106 (77; 151) IU/dL. In the patients with the mCRP level below median vs. the patients with the median mCRP level or higher, change from baseline in PN was 0.0 (0.0; 1.0) vs. 1.0 (1.0; 2.0) and PH 0.22 (−0.24; 1.91) mm vs. 1.97 (1.14; 3.14) mm, respectively (p < 0.05). The adjusted odds ratio for the formation of new carotid atherosclerotic plaques was 4.7 (95% CI 1.7; 13.2) for the patients with the median mCRP level or higher. The higher mCRP level is associated with the more pronounced increase in PN and PH in patients with normal level of traditional inflammatory biomarkers and initially moderate cardiovascular SCORE risk.

MCRP as a Biomarker of Adult-Onset Still's Disease: Quantification of mCRP by ELISA.

BackgroundC-reactive protein (CRP) is a dynamic protein that undergoes conformational changes between circulating native pentameric CRP (pCRP), pentameric symmetrical forms (pCRP*) and monomeric (or modified) CRP (mCRP) forms. mCRP exhibits strong pro-inflammatory activity and activates platelets, leukocytes, and endothelial cells. Abundant deposition of mCRP in inflamed tissues plays a role in several disease conditions, such as ischemia/reperfusion injury, Alzheimer’s disease, and cardiovascular disease. Although pCRP is typically quantified rather than mCRP for clinical purposes, mCRP may be a more appropriate disease marker of inflammatory diseases. Therefore, simple methods for quantifying mCRP are needed.MethodsWe developed a specific enzyme-linked immunosorbent assay (ELISA) to measure plasma levels of mCRP. Plasma mCRP concentration was measured in patients with adult-onset Still’s disease (AOSD) (n=20), polymyalgia rheumatica (PMR) (n=20), rheumatoid arthritis (RA) (n=30), infection (n=50), and in control subjects (n=30) using the developed ELISA.ResultsWe demonstrated that mCRP is elevated in some inflammatory autoimmune diseases, particularly AOSD. The mCRP concentration was also significantly higher among AOSD patients than RA, PMR patients and controls (477 ng/ml, 77 ng/ml, 186 ng/ml, and 1.2 ng/ml, respectively). Also, the mCRP (×1,000)/pCRP ratio was significantly higher among AOSD patients than RA, PMR, and infection patients (3.5, 0.6, 1,6, and 2.0, respectively).ConclusionThe plasma mCRP levels are elevated in some autoimmune diseases, particularly AOSD. The plasma mCRP levels may therefore be a potentially useful biomarker for AOSD.

The heat-labile toxin B subunit of E. coli fused with VP6 from GCRV (Grass carp reovirus) was expressed and folded into an active protein in rice calli

Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngon idellus) production in China. VP6 could be suitable for developing vaccine for the control of GCRV. Transgenic plants are an attractive bioreactor for their safety and ability to make economical vaccines. The B subunit of Escherichia coli heat-labile enterotoxin (LTB) fused to VP6 (LTB-VP6) was transformed into rice calli by Agrobacterium tumefaciens-mediated gene transformation. Transgenic rice calli was confirmed by PCR analysis separately. The copy numbers of LTB-VP6 inserted into the rice genome are between 1 and 2. The expression level of LTB-VP6 in rice calli was 0.0005–0.0019%, an average of 0.0011% of the TSP(total soluble proteins). LTB-VP6 was folded and assembled into a pentameric form of approximately 305 kDa capable of binding monosialoganglioside (GM1). The suitable concentration of LTB-VP6 in TSP was 0.4 μg/μl. LTB-VP6 is stable and highly active at room temperature. LTB-VP6 binding to GM1 is affected with different affinities under different temperatures. LTB-VP6 had a strong binding affinity at 25 °C and pH 8.4. Our results showed that LTB-VP6 is capable of forming an active pentameric form protein. It provides an ideal alternative to plant-based vaccines against GCRV in aquaculture.

Monomeric-to-pentameric aggregation of molecular Cs4PbBr6 halide perovskite: a first-principles investigation

The zero-dimensional halide perovskite, including the molecular state of the halide perovskite represented by A4BX6, has received much attention for their optoelectronic applications. In this manuscript, we employ first-principles calculations to systematically investigate the aggregation of the molecular Cs4PbBr6 halide perovskite. The monomeric, dimeric, trimeric, tetrameric and pentameric forms of Cs4PbBr6 are constructed. The structures of the aggregates are stabilized via the halogen bonds and the sharing of the A-site cations, and the charge transfer features from the halogen to the cesium species are observed upon the light excitation. Monotonic electronic band gap reductions and systematic red-shifts in the UV–vis absorption spectra are observed as the aggregation shifts to the higher degree. The detailed structural and electronic properties of the aggregates are further examined via the calculation of the Fukui function, electrostatic potential and frontier orbital energy levels. The present study facilitates the fundamental understanding of the higher-degree aggregates of the molecular halide perovskites and their structure-properties relationships for the optoelectronic applications such as the solar cells and photodetectors where the band gaps and the light-absorbing properties are essential.

Structure and dynamics of the SARS-CoV-2 envelope protein monomer.

Coronaviruses, especially severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), present an ongoing threat to human wellbeing. Consequently, elucidation of molecular determinants of their function and interaction with the host is an important task. Whereas some of the coronaviral proteins are extensively characterized, others remain understudied. Here, we use molecular dynamics simulations to analyze the structure and dynamics of the SARS-CoV-2 envelope (E) protein (a viroporin) in the monomeric form. The protein consists of the hydrophobic α-helical transmembrane domain (TMD) and amphiphilic α-helices H2 and H3, connected by flexible linkers. We show that TMD has a preferable orientation in the membrane, while H2 and H3 reside at the membrane surface. Orientation of H2 is strongly influenced by palmitoylation of cysteines Cys40, Cys43, and Cys44. Glycosylation of Asn66 affects the orientation of H3. We also observe that the monomeric E protein both generates and senses the membrane curvature, preferably localizing with the C-terminus at the convex regions of the membrane; the protein in the pentameric form displays these properties as well. Localization to curved regions may be favorable for assembly of the E protein oligomers, whereas induction of curvature may facilitate the budding of the viral particles. The presented results may be helpful for a better understanding of the function of the coronaviral E protein and viroporins in general, and for overcoming the ongoing SARS-CoV-2 pandemic.