The mutational dynamics of the SARS-CoV-2 virus in serial passages in vitro

Since its outbreak in 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) keeps surprising the medical community by evolving diverse immune escape mutations in a rapid and effective manner. To gain deeper insight into mutation frequency and dynamics, we isolated ten ancestral strains of SARS-CoV-2 and performed consecutive serial incubation in ten replications in a suitable and common cell line and subsequently analysed them using RT-qPCR and whole genome sequencing. Along those lines we hoped to gain fundamental insights into the evolutionary capacity of SARS-CoV-2 in vitro. Our results identified a series of adaptive genetic changes, ranging from unique convergent substitutional mutations and hitherto undescribed insertions. The region coding for spike proved to be a mutational hotspot, evolving a number of mutational changes including the already known substitutions at positions S:484 and S:501. We discussed the evolution of all specific adaptations as well as possible reasons for the seemingly inhomogeneous potential of SARS-CoV-2 in the adaptation to cell culture. The combination of serial passage in vitro with whole genome sequencing uncovers the immense mutational potential of some SARS-CoV-2 strains. The observed genetic changes of SARS-CoV-2 in vitro could not be explained solely by selectively neutral mutations but possibly resulted from the action of directional selection accumulating favourable genetic changes in the evolving variants, along the path of increasing potency of the strain. Competition among a high number of quasi-species in the SARS-CoV-2 in vitro population gene pool may reinforce directional selection and boost the speed of evolutionary change.     

Prevalence of the genes encoding propionicin T1 and protease-activated antimicrobial peptide and their expression in classical propionibacteria

The purpose of this study was to investigate the frequency of production of the bacteriocin propionicin T1 and the protease-activated antimicrobial peptide (PAMP) and their corresponding genes in 64 isolates of classical propionibacteria. This study revealed that these genes are widespread in Propionibacterium jensenii and Propionibacterium thoenii but absent from the remaining species of classical propionibacteria that were studied. The pro-PAMP-encoding gene (pamA) was found in 63% of the P. jensenii strains and 61% of the P. thoenii strains, and all of these strains displayed PAMP activity. The propionicin T1-encoding gene (pctA) was present in 89% of the P. thoenii strains and 54% of the P. jensenii strains. All P. thoenii strains containing the pctA gene exhibited antimicrobial activity corresponding to propionicin T1 activity, whereas only 38% of the pctA-containing P. jensenii strains displayed this activity. Sequencing of the pctA genes revealed the existence of two allelic variants that differed in a single nucleotide in six strains of P. jensenii; in these strains the glycine at position 55 of propionicin T1 was replaced by an aspartate residue (A variant). No strains harboring the A variant showed any antimicrobial activity against propionicin T1-sensitive bacteria. An open reading frame (orf2) located immediately downstream from the pctA gene was absent in three strains containing the G variant of propionicin T1. Two of these strains showed low antimicrobial activity, while the third strain showed no antimicrobial activity at all. The protein encoded by orf2 showed strong homology to ABC transporters, and it has been proposed previously that this protein is involved in the producer immunity against propionicin T1. The limited antimicrobial activity exhibited by the strains lacking orf2 further suggests that this putative ABC transporter plays an important role in propionicin T1 activity.     

Ghrelin stimulates motility in the small intestine of rats through intrinsic cholinergic neurons

BACKGROUND AND PURPOSE: Ghrelin is a peptide discovered in endocrine cells of the stomach. Since ghrelin mRNA expression and plasma levels are elevated in the fasting state, we investigated the effects of ghrelin on the interdigestive migrating myoelectric complex (MMC) in the small intestine in vivo and compared with motor effects of ghrelin in vitro. Methods: Sprague-Dawley rats were supplied with a venous catheter and bipolar electrodes in the duodenum and jejunum for electromyography of small intestine in awake rats. In organ baths, isometric contractions of segments of rat jejunum were studied. RESULTS: Ghrelin dose-dependently shortened the MMC cycle length at all three recording points. At the duodenal site, the interval shortened from 17.2+/-2.0 to 9.9+/-0.8 min during infusion of ghrelin (1000 pmol kg(-1) min(-1)) and at the jejunal site from 17.5+/-2.2 to 10.5+/-0.8 min. Ghrelin contracted the muscle strips with a pD2 of 7.97+/-0.47. Atropine (10(-6) M) in vitro and (1 mg kg(-1)) in vivo blocked the effect of ghrelin. CONCLUSION: Ghrelin stimulates interdigestive motility through cholinergic neurons. Ghrelin also stimulates motility, in vitro, suggesting that ghrelin receptors are present in the intestinal neuromuscular tissue and mediate its effects via cholinergic mechanisms.     

Palmitoylation of phospholipid scramblase 1 controls its distribution between nucleus and plasma membrane

Phospholipid scramblase 1 (PLSCR1) is a Ca(2+)-binding, endofacial plasma membrane protein thought to contribute to the transbilayer movement of phosphatidylserine and other membrane phospholipids that is observed upon influx of calcium into the cytosol. Expression of PLSCR1 is markedly induced by interferon and other cytokines, and PLSCR1-/- bone marrow cells exhibit defective myeloid proliferation and differentiation in response to stimulation by select growth factors, implying that PLSCR1 also functions in cytokine signaling or response pathways. PLSCR1 is multiply palmitoylated and partitions into membrane lipid raft domains. We have now identified the Cys-rich sequence (184)CCCPCC(189) in PLSCR1 as required for palmitoylation of the polypeptide. Mutation of these five cysteines abrogates PLSCR1 trafficking to the plasma membrane and results in virtually all of the expressed protein localizing to the nucleus. Consistent with this observation, cell treatment with the palmitoylation inhibitor, 2-bromo-palmitate, results in a marked redistribution of endogenous PLSCR1 from plasma membrane to nucleus. In a small percentage of untreated cells, predominantly nuclear localization of PLSCR1 is also observed. Furthermore, PLSCR1 is also found in the nucleus following its cytokine-induced expression. These data suggest that under the circumstance of rapid biosynthesis in response to gene induction by cytokines, PLSCR1 traffics into the nucleus, implying a potential nuclear function for this protein.     

Deamidations in recombinant human phenylalanine hydroxylase. Identification of labile asparagine residues and functional characterization of Asn --> Asp mutant forms

Recombinant human phenylalanine hydroxylase (hPAH) expressed in Escherichia coli for 24 h at 28 degrees C has been found by two-dimensional electrophoresis to exist as a mixture of four to five molecular forms as a result of nonenzymatic deamidation of labile Asn residues. The multiple deamidations alter the functional properties of the enzyme including its affinity for l-phenylalanine and tetrahydrobiopterin, catalytic efficiency, and substrate inhibition and also result in enzyme forms more susceptible to limited tryptic proteolysis. Asn(32) in the regulatory domain deamidates very rapidly because of its nearest neighbor amino acid Gly(33) (Solstad, T., Carvalho, R. N., Andersen, O. A., Waidelich, D., and Flatmark, T. (2003) Eur. J. Biochem., in press). Matrix-assisted laser desorption/ionization time of flight-mass spectrometry of the tryptic peptides in the catalytic domain of a 24-h (28 degrees C) expressed enzyme has shown Asn(376) and Asn(133) to be labile residues. Site-directed mutagenesis of nine Asn residues revealed that the deamidations of Asn(32) and Asn(376) are the main determinants for the functional and regulatory differences observed between the 2- and 24-h-induced wild-type (wt) enzyme. The Asn(32) --> Asp, Asn(376) --> Asp, and the double mutant forms expressed for 2 h at 28 degrees C revealed qualitatively similar regulatory properties as the highly deamidated 24-h expressed wt-hPAH. Moreover, deamidation of Asn(32) in the wt-hPAH (24 h expression at 28 degrees C) and the Asn(32) --> Asp mutation both increase the initial rate of phosphorylation of Ser(16) by cAMP-dependent protein kinase (p < 0.005). By contrast, the substitution of Gly(33) with Ala or Val, both preventing the deamidation of Asn(32), resulted in enzyme forms that were phosphorylated at a similar rate as nondeamidated wt-hPAH, even on 24-h expression. The other Asn --> Asp substitutions (in the catalytic domain) revealed that Asn(207) and Asn(223) have an important stabilizing structural function. Finally, two recently reported phenylketonuria mutations at Asn residues in the catalytic domain were studied, i.e. Asn(167) --> Ile and Asn(207) --> Asp, and their phenotypes were characterized.     

Expression and distribution of a vacuolar aquaporin in young and mature leaf tissues of Brassica napus in relation to water fluxes

 Recently, it has been shown that water fluxes across biological membranes occur not only through the lipid bilayer but also through specialized water-conducting proteins, the so called aquaporins. In the present study, we investigated in young and mature leaves of Brassica napus L. the expression and localization of a vacuolar aquaporin homologous to radish γ-tonoplast intrinsic protein/vacuolar-membrane integral protein of 23 kDa (TIP/VM 23). In-situ hybridization showed that these tonoplast aquaporins are highly expressed not only in developing but also in mature leaves, which export photosynthates. No substantial differences could be observed between different tissues of young and mature leaves. However, independent of the developmental stage, an immunohistochemical approach revealed that the vacuolar membrane of bundle-sheath cells contained more protein cross-reacting with antibodies raised against radish γ-TIP/VM 23 than the mesophyll cells. The lowest labeling was detected in phloem cells. We compared these results with the distribution of plasma-membrane aquaporins cross-reacting with antibodies detecting a domain conserved among members of the plasma-membrane intrinsic protein 1 (PIP1) subfamily. We observed the same picture as for the vacuolar aquaporins. Furthermore, a high density of gold particles labeling proteins of the PIP1 group could be observed in plasmalemmasomes of the vascular parenchyma. Our results indicate that γ-TIP/VM 23 and PIP1 homologous proteins show a similar expression pattern. Based on these results it is tempting to speculate that bundle-sheath cells play an important role in facilitating water fluxes between the apoplastic and symplastic compartments in close proximity to the vascular tissue. Received: 23 December 1999 / Accepted: 3 June 2000     

Flies across the water: genetic differentiation and reproductive isolation in allopatric desert Drosophila

Between sister species of Drosophila, both pre- and postzygotic reproductive isolation commonly appear by the time a Nei's genetic distance of 0.5 is observed. The degree of genetic differentiation present when allopatric populations of the same Drosophila species exhibit incipient reproductive isolation has not been systematically investigated. Here we compare the relationship between genetic differentiation and pre- and postzygotic isolation among allopatric populations of three cactophilic desert Drosophila: D. mettleri, D. nigrospiracula, and D. mojavensis. The range of all three is interrupted by the Gulf of California, while two species, D. mettleri and D. mojavensis, have additional allopatric populations residing on distant Santa Catalina Island, off the coast of southern California. Significant population structure exists within all three species, but only for allopatric populations of D. mojavensis is significant isolation at the prezygotic level observed. The genetic distances for the relevant populations of D. mojavensis were in the range of 0.12, similar to that for D. mettleri whose greatest D = 0.11 was unassociated with any form of isolation. These observations suggest further investigations of Drosophila populations with genetic distances in this range be undertaken to identify any potential patterns in the relationship between degree of genetic differentiation and the appearance of pre- and/or postzygotic isolation.     

Characterization of recombinant soluble macrophage scavenger receptor MARCO

MARCO is a type II transmembrane protein of the class A scavenger receptor family. It has a short N-terminal cytoplasmic domain, a transmembrane domain, and a large extracellular part composed of a 75-residue long spacer domain, a 270-residue collagenous domain, and a 99-residue long scavenger receptor cysteine-rich (SRCR) domain. Previous studies have indicated a role for this receptor in anti-microbial host defense functions. In this work we have produced the extracellular part of MARCO as a recombinant protein, and analyzed its binding properties. The production of this protein, soluble MARCO (sMARCO), has made it possible for the first time to study MARCO and its binding properties in a cell-free system. Using circular dichroism analyses, a protease-sensitive assay, and rotary shadowing electron microscopy, sMARCO was shown to have a triple-helical collagenous structure. Rotary shadowing also demonstrated that the molecules often associate with each other via the globes. sMARCO was found to bind avidly both heat-killed and living bacteria. Lipopolysaccharide, an important component of the outer membrane of Gram-negative bacteria, was shown to be a ligand of MARCO. Studies with different bacterial strains indicated that the O-side chain of lipopolysaccharide is not needed for the bacterial recognition. Finally, the C-terminal SRCR domain was also produced as a recombinant protein, and its bacteria-binding capability was studied. Although the transfection experiments with transmembrane MARCO variants have indicated a crucial role for this domain in bacterial binding, the monomeric domain exhibited low, barely detectable bacteria-binding activity. Thus, it is possible that cooperation between the SRCR domain and the collagenous domain is needed for high-affinity bacterial binding, or that the SRCR domain has to be in a trimeric form to effectively bind to bacteria.     

Gene expression patterns in ovarian carcinomas

We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.     

What are we missing with only ground‐level mist nets? Using elevated nets at a migration stopover site

ABSTRACT Mist nets deployed in a standard ground‐level fashion capture birds approximately 0.5–2.6 m above the ground. In habitats where the vegetation extends above this height, standard mist net deployment may inadequately sample the targeted avian community and age‐ and sex‐classes within species. Such sampling biases may raise questions regarding studies based on data from mist‐net captures. To determine if birds were equally likely to be captured by mist nets at different heights, we constructed a series of paired ground‐level and elevated mist nets (hereafter “net rigs”) at a research station in western New York State. Net rigs were operated during 14 migration seasons from 2000 to 2006 (spring and fall each year), and 19,735 birds of 118 species were captured. Capture rates were significantly higher in ground‐level nets, but 12 species were only captured in elevated nets. Of 44 species with at least 50 captures, 25 species were more likely to be captured in the ground‐level nets and two species in the elevated nets. For four of 18 species, more birds were captured in the elevated nets during fall migration than during spring migration. We conclude that standard ground‐level net placement was more efficient in capturing birds in the secondary growth habitats that we sampled. However, ground‐level nets may not adequately sample the entire targeted community or all age‐ or sex‐classes within species.