Structures of synthetic nanobody–SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction

Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16–RBD, Sb45–RBD, Sb14–RBD–Sb68, and Sb45–RBD–Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD–angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both “up” and “down” configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.     

Signaling through cAMP and cAMP-dependent protein kinase: diverse strategies for drug design

The catalytic subunit of cAMP-dependent protein kinase has served as a prototype for the protein kinase superfamily for many years while structures of the cAMP-bound regulatory subunits have defined the conserved cyclic nucleotide binding (CNB) motif. It is only structures of the holoenzymes, however, that enable us to appreciate the molecular features of inhibition by the regulatory subunits as well as activation by cAMP. These structures reveal for the first time the remarkable malleability of the regulatory subunits and the CNB domains. At the same time, they allow us to appreciate that the catalytic subunit is not only a catalyst but also a scaffold that mediates a wide variety of protein:protein interactions. The holoenzyme structures also provide a new paradigm for designing isoform-specific activators and inhibitors of PKA. In addition to binding to the catalytic subunits, the regulatory subunits also use their N-terminal dimerization/docking domain to bind with high affinity to A Kinase Anchoring Proteins using an amphipathic helical motif. This targeting mechanism, which localizes PKA near to its protein substrates, is also a target for therapeutic intervention of PKA signaling.     

Computational study of the fibril organization of polyglutamine repeats reveals a common motif identified in beta-helices

The formation of fibril aggregates by long polyglutamine sequences is assumed to play a major role in neurodegenerative diseases such as Huntington. Here, we model peptides rich in glutamine, through a series of molecular dynamics simulations. Starting from a rigid nanotube-like conformation, we have obtained a new conformational template that shares structural features of a tubular helix and of a beta-helix conformational organization. Our new model can be described as a super-helical arrangement of flat beta-sheet segments linked by planar turns or bends. Interestingly, our comprehensive analysis of the Protein Data Bank reveals that this is a common motif in beta-helices (termed beta-bend), although it has not been identified so far. The motif is based on the alternation of beta-sheet and helical conformation as the protein sequence is followed from the N to the C termini (beta-alpha(R)-beta-polyPro-beta). We further identify this motif in the ssNMR structure of the protofibril of the amyloidogenic peptide Abeta(1-40). The recurrence of the beta-bend suggests a general mode of connecting long parallel beta-sheet segments that would allow the growth of partially ordered fibril structures. The design allows the peptide backbone to change direction with a minimal loss of main chain hydrogen bonds. The identification of a coherent organization beyond that of the beta-sheet segments in different folds rich in parallel beta-sheets suggests a higher degree of ordered structure in protein fibrils, in agreement with their low solubility and dense molecular packing.     

Novel synthesis and structural characterization of fluorine and chlorine co-substituted hydroxyapatites

Fluorine and chlorine co-substituted hydroxyapatites (HAP) were prepared through aqueous precipitation method. Characterization results from X-ray diffraction (XRD) revealed that co-substitution of these anions resulted in the formation of pure HAP phase except for the small observed difference in the lattice parameters of the resultant apatites. The elemental analysis and FT-IR results have also confirmed the incorporation of substituted anions in the apatite structure. The calculated crystallite size for all anionic substituted apatite fall well below the range of 50 nm size that tend to coincide with the value of bone mineral crystallite size.     

Photoreactive cellulose membrane--A novel matrix for covalent immobilization of biomolecules

We report a simple and mild procedure for the preparation of a photoreactive cellulose membrane capable of forming a covalent bond with a biomolecule in presence of 365 nm UV light. Photoreactive cellulose membrane was prepared by the reaction of fluoro group of 1-fluoro-2-nitro-4-azidobenzene (FNAB) and hydroxyl group of the cellulose in an alkaline medium. X-ray photoelectron spectroscopy (XPS) of the photoreactive cellulose confirmed the incorporation of FNAB moiety. Azido group of the photoreactive membrane on exposure to UV light transforms into highly reactive nitrene which binds with a protein. The efficacy of the activated membrane was checked by immobilizing glucose oxidase (GOD) onto it in presence of light. Immobilized GOD was found to have improved thermal, pH and storage stability. Photoreactive cellulose membrane was successfully used in enzyme-linked immunosorbent assay (ELISA) technique. The antibody immobilized onto such support by UV irradiation in 30 min showed similar ELISA value than the antibody immobilized onto a polystyrene ELISA plate in 12h incubation at 4 degrees C by conventional method.     

Tracking of human cells in mice

Tracking and tracing of transplanted cells in mice is required in many fields of research. Examples are transplantation of stem cells into organs of mice to study their differentiation capacity and injection of tumor cells to examine metastatic behavior. In the present study we tested the lipid dye CM-DiI and red fluorescent nanoparticles Qdot655 for their applicability in tagging and tracing of human cells in mice. Labeling of different cell types, including MCF-7 human breast cancer cells, human cord blood derived cells, human NeoHep cells and human hepatopancreatic precursor cells, is technically easy and did not compromise further cell culture. After transplantation of CM-DiI or Qdot655 marked cells, red fluorescent structures could be detected already in unprocessed paraffin slices of the studied organs, namely liver, lung, pancreas, kidney, spleen and bone marrow. Next, we examined whether the red fluorescent structures represent the transplanted human cells. For this purpose, we established an in situ hybridization (ISH) technique that allows clear-cut differentiation between human and murine nuclei, based on simultaneous hybridization with human alu and mouse major satellite (mms) probes. We observed a high degree of coincidence between CM-DiI-marked cells and alu positive nuclei. However, also some mms positive cells contained CM-DiI, suggesting phagocytosis of the transplanted CM-DiI-marked cells. The degree of such CM-DiI-positive mouse cells depended on the cell type and route of administration. From a technical point of view it was important that CM-DiI-positive structures in paraffin slices remained fluorescent also after ISH. In contrast, Qdot655 positive structures faded during further staining procedures. In conclusion, marking of cells with CM-DiI or Qdot655 prior to transplantation facilitates recovery of human cells, since a high fraction of positive structures in the host's tissue originate from the transplanted cells. However, CM-DiI or Qdot655 positive staining of individual cells in transplanted tissues is not sufficient to prove their human origin. Additional procedures, such as ISH with alu-probes, are essential, when characterizing individual cells.     

Rapid acid treatment of <Emphasis Type="Italic">Escherichia coli</Emphasis>: transcriptomic response and recovery

Background  

Many E. coli genes show pH-dependent expression during logarithmic growth in acid (pH 5–6) or in base (pH 8–9). The effect of rapid pH change, however, has rarely been tested. Rapid acid treatment could distinguish between genes responding to external pH, and genes responding to cytoplasmic acidification, which occurs transiently following rapid external acidification. It could reveal previously unknown acid-stress genes whose effects are transient, as well as show which acid-stress genes have a delayed response.     

Carbimazole embryopathy-bilateral choanal atresia and patent vitello-intestinal duct: a case report and review of literature

BACKGROUND: In utero exposure to carbimazole for maternal hyperthyroidism has been reported to cause choanal atresia. There are case reports of patent vitello‐intestinal duct and Meckel's diverticulum in similar association. CASE: We report another such instance of an infant who was exposed to carbimazole in utero, presenting with bilateral choanal atresia and patent vitello‐intestinal duct. CONCLUSIONS: As there seems to be no reports of a possible association between propylthiouracil and congenital malformations, it may be safer to use propylthiouracil instead of carbimazole. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

Probiotics in aquaculture: importance and future perspectives

Aquaculture is one of the fastest developing growth sectors in the world and Asia presently contributes about 90% to the global production. However, disease outbreaks are constraint to aquaculture production thereby affects both economic development of the country and socio-economic status of the local people in many countries of Asia-Pacifi c region. Disease control in aquaculture industry has been achieved by following different methods using traditional ways, synthetic chemicals and antibiotics. However, the use of such expensive chemotherapeutants for controlling diseases has been widely criticized for their negative impacts like accumulation of residues, development of drug resistance, immunosuppressants and reduced consumer preference for aqua products treated with antibiotics and traditional methods are ineffective against controlling new diseases in large aquaculture systems. Therefore, alternative methods need to be developed to maintain a healthy microbial environment in the aquaculture systems there by to maintain the health of the cultured organisms. Use of probiotics is one of such method that is gaining importance in controlling potential pathogens. This review provides a summary of the criteria for the selection of the potential probiotics, their importance and future perspectives in aquaculture industry.     

A comprehensive analysis of the naturally occurring polymorphisms in HIV-1 Vpr: Potential impact on CTL epitopes

In this report we test the hypothesis that long-term virus-induced alterations in CYP occur from changes initiated by the virus that may not be related to the immune response. Enzyme activity, protein expression and mRNA of CYP3A2, a correlate of human CYP3A4, and CYP2C11, responsive to inflammatory mediators, were assessed 0.25, 1, 4, and 14 days after administration of several different recombinant adenoviruses at a dose of 5.7 × 10¹² virus particles (vp)/kg to male Sprague Dawley rats. Wild type adenovirus, containing all viral genes, suppressed CYP3A2 and 2C11 activity by 37% and 39%, respectively within six hours. Levels fell to 67% (CYP3A2) and 79% (CYP2C11) of control by 14 days (p ≤ 0.01). Helper-dependent adenovirus, with all viral genes removed, suppressed CYP3A2 (43%) and CYP2C11 (55%) within six hours. CYP3A2 remained significantly suppressed (47%, 14 days, p ≤ 0.01) while CYP2C11 returned to baseline at this time. CYP3A2 and 2C11 were reduced by 45 and 42% respectively 6 hours after treatment with PEGylated adenovirus, which has a low immunological profile (p ≤ 0.05). CYP3A2 remained suppressed (34%, p ≤ 0.05) for 14 days while CYP2C11 recovered. Inactivated virus suppressed CYP3A2 activity by 25–50% for 14 days (p ≤ 0.05). CYP2C11 was affected similar manner but recovered by day 14. Microarray and in vitro studies suggest that changes in cellular signaling pathways initiated early in virus infection contribute to changes in CYP.