Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

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Assessment of two flexible and compatible SNP genotyping platforms: TaqMan SNP Genotyping Assays and the SNPlex Genotyping System

In this review we describe the principles, protocols, and applications of two commercially available SNP genotyping platforms, the TaqMan SNP Genotyping Assays and the SNPlex Genotyping System. Combined, these two technologies meet the requirements of multiple SNP applications in genetics research and pharmacogenetics. We also describe a set of SNP selection tools and validated assay resources which we developed to accelerate the cycle of experimentation on these platforms. Criteria for selecting the more appropriate of these two genotyping technologies are presented: the genetic architecture of the trait of interest, the throughput required, and the number of SNPs and samples needed for a successful study. Overall, the TaqMan assay format is suitable for low- to mid-throughput applications in which a high assay conversion rate, simple assay workflow, and low cost of automation are desirable. The SNPlex Genotyping System, on the other hand, is well suited for SNP applications in which throughput and cost-efficiency are essential, e.g., applications requiring either the testing of large numbers of SNPs and samples, or the flexibility to select various SNP subsets.     

Selective precipitation and purification of monovalent proteins using oligovalent ligands and ammonium sulfate

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins--for which appropriate oligovalent ligands can be synthesized--and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.     

Protein kinase CK2 promotes cancer cell viability via up-regulation of cyclooxygenase-2 expression and enhanced prostaglandin E2 production

Augmented expression of protein kinase CK2 is associated with hyperproliferation and resistance to apoptosis in cancer cells. Effects of CK2 are at least partially linked to signaling via the Wnt/β-catenin pathway, which is dramatically enhanced in colon cancer. Cyclooxygenase-2 (COX-2), a Wnt/β-catenin target gene, has been associated with enhanced cancer progression and metastasis. However, the possibility that a connection may exist between CK2 and COX-2 has not been explored previously. Here we investigated changes in COX-2 expression and activity upon CK2 modulation and evaluated how these changes affected cell viability. COX-2 expression and cell viability decreased upon selective inhibition of COX-2 with SC-791 or CK2 with 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), both in human colon (HT29-ATCC, HT29-US, DLD-1) and breast (ZR-75) cancer cells, as well as in human embryonic kidney (HEK-293T) cells. On the other hand, ectopic CK2α expression promoted up-regulation of COX-2 by activating the Wnt/β-catenin pathway in HEK-293T cells. Noteworthy, over-expression of either CK2α, β-catenin or COX-2, as well as supplementation of the medium with prostaglandin E2 (PGE2), all were individually sufficient to overcome limitations in cell viability triggered by CK2 inhibition either upon addition of DMAT or over-expression of a dominant negative CK2α variant. Altogether, these findings provide new insight to the role of CK2 in cancer by up-regulating COX-2 expression and thereby PGE2 production.     

Posttranslational regulation of AMPA receptor trafficking and function

In the mammalian central nervous system, the majority of fast excitatory synaptic transmission is mediated by glutamate acting on AMPA-type ionotropic glutamate receptors. The abundance of AMPA receptors at the synapse can be modulated through receptor trafficking, which dynamically regulates many fundamental brain functions, including learning and memory. Reversible posttranslational modifications, including phosphorylation, palmitoylation and ubiquitination of AMPA receptor subunits are important regulatory mechanisms for controlling synaptic AMPA receptor expression and function. In this review, we highlight recent advances in the study of AMPA receptor posttranslational modifications and discuss how these modifications regulate AMPA receptor trafficking and function at synapses.     

Human METTL16 is a N6‐methyladenosine (m6A) methyltransferase that targets pre‐mRNAs and various non‐coding RNAs

N⁶‐methyladenosine (m⁶A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m⁶A modifications are installed by the METTL3–METTL14 complex, others appear to be introduced independently, implying that additional human m⁶A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m⁶A “writer” protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre‐mRNAs. We demonstrate that METTL16 is responsible for N⁶‐methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5′ splice sites of pre‐mRNAs during splicing, suggesting that METTL16‐mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m⁶A methyltransferase in human cells expands our understanding of the mechanisms by which the m⁶A landscape is installed on cellular RNAs.     

Mechanism of lipid bilayer disruption by the human antimicrobial peptide,LL-37

LL-37 is an amphipathic, alpha-helical, antimicrobial peptide. (15)N chemical shift and (15)N dipolar-shift spectroscopy of site-specifically labeled LL-37 in oriented lipid bilayers indicate that the amphipathic helix is oriented parallel to the surface of the bilayer. This surface orientation is maintained in both anionic and zwitterionic bilayers and at different temperatures and peptide concentrations, ruling out a barrel-stave mechanism for bilayer disruption by LL-37. In contrast, electrostatic factors, the type of lipid, and the presence of cholesterol do affect the extent to which LL-37 perturbs the lipids in the bilayer as observed with (31)P NMR. The (31)P spectra also show that micelles or other small, rapidly tumbling membrane fragments are not formed in the presence of LL-37, excluding a detergent-like mechanism. LL-37 does increase the lamellar to inverted hexagonal phase transition temperature of both PE model lipid systems and Escherichia coli lipids, demonstrating that it induces positive curvature strain in these environments. These results support a toroidal pore mechanism of lipid bilayer disruption by LL-37.     

Learned helplessness: unique features and translational value of a cognitive depression model

The concept of learned helplessness defines an escape or avoidance deficit after uncontrollable stress and is regarded as a depression-like coping deficit in aversive but avoidable situations. Based on a psychological construct, it ideally complements other stress-induced or genetic animal models for major depression. Because of excellent face, construct, and predictive validity, it has contributed to the elaboration of several pathophysiological concepts and has brought forward new treatment targets. Whereas learned helplessness can be modeled not only in a broad variety of mammals, but also in fish and Drosophila, we will focus here on the use of this model in rats and mice, which are today the most common species for preclinical in vivo research in psychiatry.     

Relationships among wood‐boring beetles,fungi, and the decomposition of forest biomass

A prevailing paradigm in forest ecology is that wood‐boring beetles facilitate wood decay and carbon cycling, but empirical tests have yielded mixed results. We experimentally determined the effects of wood borers on fungal community assembly and wood decay within pine trunks in the southeastern United States. Pine trunks were made either beetle‐accessible or inaccessible. Fungal communities were compared using culturing and high‐throughput amplicon sequencing (HTAS) of DNA and RNA. Prior to beetle infestation, living pines had diverse fungal endophyte communities. Endophytes were displaced by beetle‐associated fungi in beetle‐accessible trees, whereas some endophytes persisted as saprotrophs in beetle‐excluded trees. Beetles increased fungal diversity several fold. Over forty taxa of Ascomycota were significantly associated with beetles, but beetles were not consistently associated with any known wood‐decaying fungi. Instead, increasing ambrosia beetle infestations caused reduced decay, consistent with previous in vitro experiments that showed beetle‐associated fungi reduce decay rates by competing with decay fungi. No effect of bark‐inhabiting beetles on decay was detected. Platypodines carried significantly more fungal taxa than scolytines. Molecular results were validated by synthetic and biological mock communities and were consistent across methodologies. RNA sequencing confirmed that beetle‐associated fungi were biologically active in the wood. Metabarcode sequencing of the LSU/28S marker recovered important fungal symbionts that were missed by ITS2, though community‐level effects were similar between markers. In contrast to the current paradigm, our results indicate ambrosia beetles introduce diverse fungal communities that do not extensively decay wood, but instead reduce decay rates by competing with wood decay fungi.     

Impact of emotion on consciousness: positive stimuli enhance conscious reportability

Emotion and reward have been proposed to be closely linked to conscious experience, but empirical data are lacking. The anterior cingulate cortex (ACC) plays a central role in the hedonic dimension of conscious experience; thus potentially a key region in interactions between emotion and consciousness. Here we tested the impact of emotion on conscious experience, and directly investigated the role of the ACC. We used a masked paradigm that measures conscious reportability in terms of subjective confidence and objective accuracy in identifying the briefly presented stimulus in a forced-choice test. By manipulating the emotional valence (positive, neutral, negative) and the presentation time (16 ms, 32 ms, 80 ms) we measured the impact of these variables on conscious and subliminal (i.e. below threshold) processing. First, we tested normal participants using face and word stimuli. Results showed that participants were more confident and accurate when consciously seeing happy versus sad/neutral faces and words. When stimuli were presented subliminally, we found no effect of emotion. To investigate the neural basis of this impact of emotion, we recorded local field potentials (LFPs) directly in the ACC in a chronic pain patient. Behavioural findings were replicated: the patient was more confident and accurate when (consciously) seeing happy versus sad faces, while no effect was seen in subliminal trials. Mirroring behavioural findings, we found significant differences in the LFPs after around 500 ms (lasting 30 ms) in conscious trials between happy and sad faces, while no effect was found in subliminal trials. We thus demonstrate a striking impact of emotion on conscious experience, with positive emotional stimuli enhancing conscious reportability. In line with previous studies, the data indicate a key role of the ACC, but goes beyond earlier work by providing the first direct evidence of interaction between emotion and conscious experience in the human ACC.