A platform for predicting mechanism of action based on bacterial transcriptional responses identifies an unusual DNA gyrase inhibitor.

In the search for much-needed new antibacterial chemical matter, a myriad of compounds have been reported in academic and pharmaceutical screening endeavors. Only a small fraction of these, however, are characterized with respect to mechanism of action (MOA). Here, we describe a pipeline that categorizes transcriptional responses to antibiotics and provides hypotheses for MOA. 3D-printed imaging hardware PFIboxes) profiles responses of Escherichia coli promoter-GFP fusions to more than 100 antibiotics. Notably, metergoline, a semi-synthetic ergot alkaloid, mimics a DNA replication inhibitor. In vitro supercoiling assays confirm this prediction, and a potent analog thereof (MLEB-1934) inhibits growth at 0.25 µg/mL and is highly active against quinolone-resistant strains of methicillin-resistant Staphylococcus aureus. Spontaneous suppressor mutants map to a seldom explored allosteric binding pocket, suggesting a mechanism distinct from DNA gyrase inhibitors used in the clinic. In all, the work highlights the potential of this platform to rapidly assess MOA of new antibacterial compounds.

Correlating transcription and protein expression profiles of immune biomarkers following lipopolysaccharide exposure in lung epithelial cells.

Universal and early recognition of pathogens occurs through recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) by innate immune receptors and the consequent secretion of cytokines and chemokines. The intrinsic complexity of innate immune signaling and associated signal transduction challenges our ability to obtain physiologically relevant, reproducible and accurate data from experimental systems. One of the reasons for the discrepancy in observed data is the choice of measurement strategy. Immune signaling is regulated by the interplay between pathogen-derived molecules with host cells resulting in cellular expression changes. However, these cellular processes are often studied by the independent assessment of either the transcriptome or the proteome. Correlation between transcription and protein analysis is lacking in a variety of studies. In order to methodically evaluate the correlation between transcription and protein expression profiles associated with innate immune signaling, we measured cytokine and chemokine levels following exposure of human cells to the PAMP lipopolysaccharide (LPS) from the Gram-negative pathogen Pseudomonas aeruginosa. Expression of 84 messenger RNA (mRNA) transcripts and 69 proteins, including 35 overlapping targets, were measured in human lung epithelial cells. We evaluated 50 biological replicates to determine reproducibility of outcomes. Following pairwise normalization, 16 mRNA transcripts and 6 proteins were significantly upregulated following LPS exposure, while only five (CCL2, CSF3, CXCL5, CXCL8/IL8, and IL6) were upregulated in both transcriptomic and proteomic analysis. This lack of correlation between transcription and protein expression data may contribute to the discrepancy in the immune profiles reported in various studies. The use of multiomic assessments to achieve a systems-level understanding of immune signaling processes can result in the identification of host biomarker profiles for a variety of infectious diseases and facilitate countermeasure design and development.

Transcription-replication conflicts underlie sensitivity to PARP inhibitors.

An important advance in cancer therapy has been the development of poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of homologous recombination (HR)-deficient cancers1-6. PARP inhibitors trap PARPs on DNA. The trapped PARPs are thought to block replisome progression, leading to formation of DNA double-strand breaks that require HR for repair7. Here we show that PARP1 functions together with TIMELESS and TIPIN to protect the replisome in early S phase from transcription-replication conflicts. Furthermore, the synthetic lethality of PARP inhibitors with HR deficiency is due to an inability to repair DNA damage caused by transcription-replication conflicts, rather than by trapped PARPs. Along these lines, inhibiting transcription elongation in early S phase rendered HR-deficient cells resistant to PARP inhibitors and depleting PARP1 by small-interfering RNA was synthetic lethal with HR deficiency. Thus, inhibiting PARP1 enzymatic activity may suffice for treatment efficacy in HR-deficient settings.

Genomic overview of INA-induced NPR1 targeting and transcriptional cascades in Arabidopsis.

The phytohormone salicylic acid (SA) triggers transcriptional reprogramming that leads to SA-induced immunity in plants. NPR1 is an SA receptor and master transcriptional regulator in SA-triggered transcriptional reprogramming. Despite the indispensable role of NPR1, genome-wide direct targets of NPR1 specific to SA signaling have not been identified. Here, we report INA (functional SA analog)-specific genome-wide targets of Arabidopsis NPR1 in plants expressing GFP-fused NPR1 under its native promoter. Analyses of NPR1-dependently expressed direct NPR1 targets revealed that NPR1 primarily activates genes encoding transcription factors upon INA treatment, triggering transcriptional cascades required for INA-induced transcriptional reprogramming and immunity. We identified genome-wide targets of a histone acetyltransferase, HAC1, including hundreds of co-targets shared with NPR1, and showed that NPR1 and HAC1 regulate INA-induced histone acetylation and expression of a subset of the co-targets. Genomic NPR1 targeting was principally mediated by TGACG-motif binding protein (TGA) transcription factors. Furthermore, a group of NPR1 targets mostly encoding transcriptional regulators was already bound to NPR1 in the basal state and showed more rapid and robust induction than other NPR1 targets upon SA signaling. Thus, our study unveils genome-wide NPR1 targeting, its role in transcriptional reprogramming, and the cooperativity between NPR1, HAC1, and TGAs in INA-induced immunity.

RNA G-quadruplexes inhibit translation of the PE/PPE transcripts in Mycobacterium tuberculosis.

The role of RNA G-quadruplexes (rG4s) in bacteria remains poorly understood. High G-quadruplex densities have been linked to organismal stress. Here we investigate rG4s in mycobacteria, which survive highly stressful conditions within the host. We show that rG4-enrichment is a unique feature exclusive to slow-growing pathogenic mycobacteria, and Mycobacterium tuberculosis (Mtb) transcripts contain an abundance of folded rG4s. Notably, the PE/PPE family of genes, unique to slow-growing pathogenic mycobacteria, contain over 50% of rG4s within Mtb transcripts. We found that RNA oligonucleotides of putative rG4s in PE/PPE genes form G-quadruplex structures in vitro, which are stabilized by the G-quadruplex ligand BRACO19. Furthermore, BRACO19 inhibits the transcription of PE/PPE genes and selectively suppresses the growth of Mtb but not Mycobacterium smegmatis or other rapidly growing bacteria. Importantly, the stabilization of rG4s inhibits the translation of Mtb PE/PPE genes (PPE56, PPE67, PPE68, PE_PGRS39, and PE_PGRS41) ectopically expressed in M. smegmatis or Escherichia coli. In addition, the rG4-mediated reduction in PE/PPE protein levels attenuates proinflammatory response upon infection of THP-1 cells. Our findings shed new light on the regulation of PE/PPE genes and highlight a pivotal role for rG4s in Mtb transcripts as regulators of post-transcriptional translational control. The rG4s in mycobacterial transcripts may represent potential drug targets for newer therapies.

High-resolution landscape of an antibiotic binding site.

Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering1 to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug-enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription-replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.

Platelet factors attenuate inflammation and rescue cognition in ageing.

Identifying therapeutics to delay, and potentially reverse, age-related cognitive decline is critical in light of the increased incidence of dementia-related disorders forecasted in the growing older population1. Here we show that platelet factors transfer the benefits of young blood to the ageing brain. Systemic exposure of aged male mice to a fraction of blood plasma from young mice containing platelets decreased neuroinflammation in the hippocampus at the transcriptional and cellular level and ameliorated hippocampal-dependent cognitive impairments. Circulating levels of the platelet-derived chemokine platelet factor 4 (PF4) (also known as CXCL4) were elevated in blood plasma preparations of young mice and humans relative to older individuals. Systemic administration of exogenous PF4 attenuated age-related hippocampal neuroinflammation, elicited synaptic-plasticity-related molecular changes and improved cognition in aged mice. We implicate decreased levels of circulating pro-ageing immune factors and restoration of the ageing peripheral immune system in the beneficial effects of systemic PF4 on the aged brain. Mechanistically, we identified CXCR3 as a chemokine receptor that, in part, mediates the cellular, molecular and cognitive benefits of systemic PF4 on the aged brain. Together, our data identify platelet-derived factors as potential therapeutic targets to abate inflammation and rescue cognition in old age.

A2BAR Antagonism Decreases the Glomerular Expression and Secretion of Chemoattractants for Monocytes and the Pro-Fibrotic M2 Macrophages Polarization during Diabetic Nephropathy.

Some chemoattractants and leukocytes such as M1 and M2 macrophages are known to be involved in the development of glomerulosclerosis during diabetic nephropathy (DN). In the course of diabetes, an altered and defective cellular metabolism leads to the increase in adenosine levels, and thus to changes in the polarity (M1/M2) of macrophages. MRS1754, a selective antagonist of the A2B adenosine receptor (A2BAR), attenuated glomerulosclerosis and decreased macrophage-myofibroblast transition in DN rats. Therefore, we aimed to investigate the effect of MRS1754 on the glomerular expression/secretion of chemoattractants, the intraglomerular infiltration of leukocytes, and macrophage polarity in DN rats. Kidneys/glomeruli of non-diabetic, DN, and MRS1754-treated DN rats were processed for transcriptomic analysis, immunohistopathology, ELISA, and in vitro macrophage migration assays. The transcriptomic analysis identified an upregulation of transcripts and pathways related to the immune system in the glomeruli of DN rats, which was attenuated using MRS1754. The antagonism of the A2BAR decreased glomerular expression/secretion of chemoattractants (CCL2, CCL3, CCL6, and CCL21), the infiltration of macrophages, and their polarization to M2 in DN rats. The in vitro macrophages migration induced by conditioned-medium of DN glomeruli was significantly decreased using neutralizing antibodies against CCL2, CCL3, and CCL21. We concluded that the pharmacological blockade of the A2BAR decreases the transcriptional expression of genes/pathways related to the immune response, protein expression/secretion of chemoattractants, as well as the infiltration of macrophages and their polarization toward the M2 phenotype in the glomeruli of DN rats, suggesting a new mechanism implicated in the antifibrotic effect of MRS1754.

Identification of antimycin A as a c-Myc degradation accelerator via high-throughput screening.

c-Myc is a critical regulator of cell proliferation and growth. Elevated levels of c-Myc cause transcriptional amplification, leading to various types of cancers. Small molecules that specifically inhibit c-Myc-dependent regulation are potentially invaluable for anticancer therapy. Because c-Myc does not have enzymatic activity or targetable pockets, researchers have attempted to obtain small molecules that inhibit c-Myc cofactors, activate c-Myc repressors, or target epigenetic modifications to regulate the chromatin of c-Myc-addicted cancer without any clinical success. In this study, we screened for c-Myc inhibitors using a cell-dependent assay system in which the expression of c-Myc and its transcriptional activity can be inferred from monomeric Keima and enhanced GFP fluorescence, respectively. We identified one mitochondrial inhibitor, antimycin A, as a hit compound. The compound enhanced the c-Myc phosphorylation of threonine-58, consequently increasing the proteasome-mediated c-Myc degradation. The mechanistic analysis of antimycin A revealed that it enhanced the degradation of c-Myc protein through the activation of glycogen synthetic kinase 3 by reactive oxygen species (ROS) from damaged mitochondria. Furthermore, we found that the inhibition of cell growth by antimycin A was caused by both ROS-dependent and ROS-independent pathways. Interestingly, ROS-dependent growth inhibition occurred only in the presence of c-Myc, which may reflect the representative features of cancer cells. Consistently, the antimycin A sensitivity of cells was correlated to the endogenous c-Myc levels in various cancer cells. Overall, our study provides an effective strategy for identifying c-Myc inhibitors and proposes a novel concept for utilizing ROS inducers for cancer therapy.

Tire-Derived Transformation Product 6PPD-Quinone Induces Mortality and Transcriptionally Disrupts Vascular Permeability Pathways in Developing Coho Salmon.

Urban stormwater runoff frequently contains the car tire transformation product 6PPD-quinone, which is highly toxic to juvenile and adult coho salmon (Onchorychus kisutch). However, it is currently unclear if embryonic stages are impacted. We addressed this by exposing developing coho salmon embryos starting at the eyed stage to three concentrations of 6PPD-quinone twice weekly until hatch. Impacts on survival and growth were assessed. Further, whole-transcriptome sequencing was performed on recently hatched alevin to address the potential mechanism of 6PPD-quinone-induced toxicity. Acute mortality was not elicited in developing coho salmon embryos at environmentally measured concentrations lethal to juveniles and adults, however, growth was inhibited. Immediately after hatching, coho salmon were sensitive to 6PPD-quinone mortality, implicating a large window of juvenile vulnerability prior to smoltification. Molecularly, 6PPD-quinone induced dose-dependent effects that implicated broad dysregulation of genomic pathways governing cell-cell contacts and endothelial permeability. These pathways are consistent with previous observations of macromolecule accumulation in the brains of coho salmon exposed to 6PPD-quinone, implicating blood-brain barrier disruption as a potential pathway for toxicity. Overall, our data suggests that developing coho salmon exposed to 6PPD-quinone are at risk for adverse health events upon hatching while indicating potential mechanism(s) of action for this highly toxic chemical.

Proteomic analysis reveals mechanisms underlying increased efficacy of bleomycin by photochemical internalization in bladder cancer cells.

Photochemical internalization (PCI) is a promising new technology for site-specific drug delivery, developed from photodynamic therapy (PDT). In PCI, light-induced activation of a photosensitizer trapped inside endosomes together with e.g. chemotherapeutics, nucleic acids or immunotoxins, allows cytosolic delivery and enhanced local therapeutic effect. Here we have evaluated the photosensitizer meso-tetraphenyl chlorine disulphonate (TPCS2a/fimaporfin) in a proteome analysis of AY-27 rat bladder cancer cells in combination with the chemotherapeutic drug bleomycin (BML). We find that BLMPCI attenuates oxidative stress responses induced by BLM alone, while concomitantly increasing transcriptional repression and DNA damage responses. BLMPCI also mediates downregulation of bleomycin hydrolase (Blmh), which is responsible for cellular degradation of BLM, as well as several factors known to be involved in fibrotic responses. PCI-mediated delivery might thus allow reduced dosage of BLM and alleviate unwanted side effects from treatment, including pulmonary fibrosis.

The U1 antisense morpholino oligonucleotide (AMO) disrupts U1 snRNP structure to promote intronic PCPA modification of pre-mRNAs.

Functional depletion of the U1 small nuclear ribonucleoprotein (snRNP) with a 25 nt U1 AMO (antisense morpholino oligonucleotide) may lead to intronic premature cleavage and polyadenylation of thousands of genes, a phenomenon known as U1 snRNP telescripting; however, the underlying mechanism remains elusive. In this study, we demonstrated that U1 AMO could disrupt U1 snRNP structure both in vitro and in vivo, thereby affecting the U1 snRNP-RNAP polymerase II interaction. By performing chromatin immunoprecipitation sequencing for phosphorylation of Ser2 and Ser5 of the C-terminal domain of RPB1, the largest subunit of RNAP polymerase II, we showed that transcription elongation was disturbed upon U1 AMO treatment, with a particular high phosphorylation of Ser2 signal at intronic cryptic polyadenylation sites (PASs). In addition, we showed that core 3'processing factors CPSF/CstF are involved in the processing of intronic cryptic PAS. Their recruitment accumulated toward cryptic PASs upon U1 AMO treatment, as indicated by chromatin immunoprecipitation sequencing and individual-nucleotide resolution CrossLinking and ImmunoPrecipitation sequencing analysis. Conclusively, our data suggest that disruption of U1 snRNP structure mediated by U1 AMO provides a key for understanding the U1 telescripting mechanism.

Neurog1-Derived Peptides RMNE1 and DualPep-Shine Penetrate the Skin and Inhibit Melanin Synthesis by Regulating MITF Transcription.

Anti-pigmentation peptides have been developed as alternative skin-lightening agents to replace conventional chemicals that have adverse effects on the skin. However, the maximum size of these peptides is often limited by their low skin and cell penetration. To address this issue, we used our intra-dermal delivery technology (IDDT) platform to identify peptides with hypo-pigmenting and high cell-penetrating activity. Using our cell-penetrating peptides (CPPs) from the IDDT platform, we identified RMNE1 and its derivative RMNE3, "DualPep-Shine", which showed levels of α-Melanocyte stimulating hormone (α-MSH)-induced melanin inhibition comparable to the conventional tyrosinase inhibitor, Kojic acid. In addition, DualPep-Shine was delivered into the nucleus and regulated the gene expression levels of melanogenic enzymes by inhibiting the promoter activity of microphthalmia-associated transcription factor-M (MITF-M). Using a 3D human skin model, we found that DualPep-Shine penetrated the lower region of the epidermis and reduced the melanin content in a dose-dependent manner. Furthermore, DualPep-Shine showed high safety with little immunogenicity, indicating its potential as a novel cosmeceutical ingredient and anti-pigmentation therapeutic agent.

Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control.

Host-derived cellular pathways can provide an unfavorable environment for virus replication. These pathways have been a subject of interest for herpesviruses, including the betaherpesvirus human cytomegalovirus (HCMV). Here, we demonstrate that a compound, ARP101, induces the noncanonical sequestosome 1 (SQSTM1)/p62-Keap1-Nrf2 pathway for HCMV suppression. ARP101 increased the levels of both LC3 II and SQSTM1/p62 and induced phosphorylation of p62 at the C-terminal domain, resulting in its increased affinity for Keap1. ARP101 treatment resulted in Nrf2 stabilization and translocation into the nucleus, binding to specific promoter sites and transcription of antioxidant enzymes under the antioxidant response element (ARE), and HCMV suppression. Knockdown of Nrf2 recovered HCMV replication following ARP101 treatment, indicating the role of the Keap1-Nrf2 axis in HCMV inhibition by ARP101. SQSTM1/p62 phosphorylation was not modulated by the mTOR kinase or casein kinase 1 or 2, indicating ARP101 engages other kinases. Together, the data uncover a novel antiviral strategy for SQSTM1/p62 through the noncanonical Keap1-Nrf2 axis. This pathway could be further exploited, including the identification of the responsible kinases, to define the biological events during HCMV replication. IMPORTANCE Antiviral treatment for human cytomegalovirus (HCMV) is limited and suffers from the selection of drug-resistant viruses. Several cellular pathways have been shown to modulate HCMV replication. The autophagy receptor sequestosome 1 (SQSTM1)/p62 has been reported to interact with several HCMV proteins, particularly with components of HCMV capsid, suggesting it plays a role in viral replication. Here, we report on a new and unexpected role for SQSTM1/p62, in HCMV suppression. Using a small-molecule probe, ARP101, we show SQSTM1/p62 phosphorylation at its C terminus domain initiates the noncanonical Keap1-Nrf2 axis, leading to transcription of genes under the antioxidant response element, resulting in HCMV inhibition in vitro. Our study highlights the dynamic nature of SQSTM1/p62 during HCMV infection and how its phosphorylation activates a new pathway that can be exploited for antiviral intervention.

Formin-mediated nuclear actin at androgen receptors promotes transcription.

Steroid hormone receptors are ligand-binding transcription factors essential for mammalian physiology. The androgen receptor (AR) binds androgens mediating gene expression for sexual, somatic and behavioural functions, and is involved in various conditions including androgen insensitivity syndrome and prostate cancer1. Here we identified functional mutations in the formin and actin nucleator DAAM2 in patients with androgen insensitivity syndrome. DAAM2 was enriched in the nucleus, where its localization correlated with that of the AR to form actin-dependent transcriptional droplets in response to dihydrotestosterone. DAAM2 AR droplets ranged from 0.02 to 0.06 µm3 in size and associated with active RNA polymerase II. DAAM2 polymerized actin directly at the AR to promote droplet coalescence in a highly dynamic manner, and nuclear actin polymerization is required for prostate-specific antigen expression in cancer cells. Our data uncover signal-regulated nuclear actin assembly at a steroid hormone receptor necessary for transcription.

Dynamic Covalent Template-Guided Screen for Nucleic Acid-Targeting Agents.

Trinucleotide repeat diseases such as myotonic dystrophy type 1 (DM1) and Huntington's disease (HD) are caused by expanded DNA repeats that can be used as templates to synthesize their own inhibitors. Because it would be particularly advantageous to reversibly assemble multivalent nucleic acid-targeting agents in situ, we sought to develop a target-guided screen that uses dynamic covalent chemistry to identify multitarget inhibitors. We report the synthesis of a library of amine- or aldehyde-containing fragments. The assembly of these fragments led to a diverse set of hit combinations that was confirmed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in the presence of DM1 and HD repeat sequences. Of interest for both diseases, the resulting hit combinations inhibited transcription selectively and in a cooperative manner in vitro, with inhibitory concentration (IC50) values in the micromolar range. This dynamic covalent library and screening approach could be applied to identify compounds that reversibly assemble on other nucleic acid targets.

Development of an improved inhibitor of Lats kinases to promote regeneration of mammalian organs.

The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular signals can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily postmitotic and aids regeneration in mammals. In the present study, we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of Yap target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies.

Effect of 17ß-estradiol on the daily pattern of ACE2, ADAM17, TMPRSS2 and estradiol receptor transcription in the lungs and colon of male rats.

Covid-19 progression shows sex-dependent features. It is hypothesized that a better Covid-19 survival rate in females can be attributed to the presence of higher 17ß-estradiol (E2) levels in women than in men. Virus SARS-CoV-2 is enabled to enter the cell with the use of angiotensin converting enzyme 2 (ACE2). The expression of several renin-angiotensin system components has been shown to exert a rhythmic pattern, and a role of the circadian system in their regulation has been implicated. Therefore, the aim of the study is to elucidate possible interference between E2 signalling and the circadian system in the regulation of the expression of ACE2 mRNA and functionally related molecules. E2 was administered at a dosage of 40 µg/kg/day for 7 days to male Wistar rats, and sampling of the lungs and colon was performed during a 24-h cycle. The daily pattern of expression of molecules facilitating SARS-CoV-2 entry into the cell, clock genes and E2 receptors was analysed. As a consequence of E2 administration, a rhythm in ACE2 and TMPRSS2 mRNA expression was observed in the lungs but not in the colon. ADAM17 mRNA expression showed a pronounced rhythmic pattern in both tissues that was not influenced by E2 treatment. ESR1 mRNA expression exerted a rhythmic pattern, which was diminished by E2 treatment. The influence of E2 administration on ESR2 and GPER1 mRNA expression was greater in the lungs than in the colon as a significant rhythm in ESR2 and GPER1 mRNA expression appeared only in the lungs after E2 treatment. E2 administration also increased the amplitude of bmal1 expression in the lungs, which implicates altered functioning of peripheral oscillators in response to E2 treatment. The daily pattern of components of the SARS-CoV-2 entrance pathway and their responsiveness to E2 should be considered in the timing of pharmacological therapy for Covid-19.

[Metformin and lipopolysaccharide regulate transcription of NFATc2 gene via the transcription factor RUNX2].

OBJECTIVE: To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene. METHODS: The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription. RESULTS: We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively. CONCLUSION: pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.

Strain-level fitness in the gut microbiome is an emergent property of glycans and a single metabolite.

The human gut microbiota resides within a diverse chemical environment challenging our ability to understand the forces shaping this ecosystem. Here, we reveal that fitness of the Bacteroidales, the dominant order of bacteria in the human gut, is an emergent property of glycans and one specific metabolite, butyrate. Distinct sugars serve as strain-variable fitness switches activating context-dependent inhibitory functions of butyrate. Differential fitness effects of butyrate within the Bacteroides are mediated by species-level variation in Acyl-CoA thioesterase activity and nucleotide polymorphisms regulating an Acyl-CoA transferase. Using in vivo multi-omic profiles, we demonstrate Bacteroides fitness in the human gut is associated together, but not independently, with Acyl-CoA transferase expression and butyrate. Our data reveal that each strain of the Bacteroides exists within a unique fitness landscape based on the interaction of chemical components unpredictable by the effect of each part alone mediated by flexibility in the core genome.