Bromodomain and extraterminal inhibitors block the Epstein-Barr virus lytic cycle at two distinct steps.

Lytic infection by the Epstein-Barr virus (EBV) poses numerous health risks, such as infectious mononucleosis and lymphoproliferative disorder. Proteins in the bromodomain and extraterminal (BET) family regulate multiple stages of viral life cycles and provide promising intervention targets. Synthetic small molecules can bind to the bromodomains and disrupt function by preventing recognition of acetylated lysine substrates. We demonstrate that JQ1 and other BET inhibitors block two different steps in the sequential cascade of the EBV lytic cycle. BET inhibitors prevent expression of the viral immediate-early protein BZLF1. JQ1 alters transcription of genes controlled by the host protein BACH1, and BACH1 knockdown reduces BZLF1 expression. BET proteins also localize to the lytic origin of replication (OriLyt) genetic elements, and BET inhibitors prevent viral late gene expression. There JQ1 reduces BRD4 recruitment during reactivation to preclude replication initiation. This represents a rarely observed dual mode of action for drugs.

Acid Evolution of Escherichia coli K-12 Eliminates Amino Acid Decarboxylases and Reregulates Catabolism.

Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932-1941, 2015, https://doi.org/10.1128/AEM.03494-14). Revised genomic analysis of these strains revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS5 insertion or IS-mediated deletion in cadC, while population B11 had a point mutation affecting the arginine activator adiY The cadC and adiY mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator ariR, yhiM, and Gad). Other strains showed downregulation of H2 consumption mediated by hydrogenases (hya and hyb) which release acid. Strains F9-2 and F9-3 had a deletion of fnr and showed downregulation of FNR-dependent genes (dmsABC, frdABCD, hybABO, nikABCDE, and nrfAC). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of catabolism.IMPORTANCE Experimental evolution of an enteric bacterium under a narrow buffered range of acid pH leads to loss of genes that enhance fitness above or below the buffered pH range, including loss of enzymes that may raise external pH in the absence of buffer. Prominent modes of evolutionary change involve IS-mediated insertions and deletions that knock out key regulators. Over generations of acid stress, catabolism undergoes reregulation in ways that differ for each evolving strain.

Benzoate- and Salicylate-Tolerant Strains of Escherichia coli K-12 Lose Antibiotic Resistance during Laboratory Evolution.

Escherichia coli K-12 W3110 grows in the presence of membrane-permeant organic acids that can depress cytoplasmic pH and accumulate in the cytoplasm. We conducted experimental evolution by daily diluting cultures in increasing concentrations of benzoic acid (up to 20 mM) buffered at external pH 6.5, a pH at which permeant acids concentrate in the cytoplasm. By 2,000 generations, clones isolated from evolving populations showed increasing tolerance to benzoate but were sensitive to chloramphenicol and tetracycline. Sixteen clones grew to stationary phase in 20 mM benzoate, whereas the ancestral strain W3110 peaked and declined. Similar growth occurred in 10 mM salicylate. Benzoate-evolved strains grew like W3110 in the absence of benzoate, in media buffered at pH 4.8, pH 7.0, or pH 9.0, or in 20 mM acetate or sorbate at pH 6.5. Genomes of 16 strains revealed over 100 mutations, including single-nucleotide polymorphisms (SNPs), large deletions, and insertion knockouts. Most strains acquired deletions in the benzoate-induced multiple antibiotic resistance (Mar) regulon or in associated regulators such as rob and cpxA, as well as the multidrug resistance (MDR) efflux pumps emrA, emrY, and mdtA Strains also lost or downregulated the Gad acid fitness regulon. In 5 mM benzoate or in 2 mM salicylate (2-hydroxybenzoate), most strains showed increased sensitivity to the antibiotics chloramphenicol and tetracycline; some strains were more sensitive than a marA knockout strain. Thus, our benzoate-evolved strains may reveal additional unknown drug resistance components. Benzoate or salicylate selection pressure may cause general loss of MDR genes and regulators. IMPORTANCE: Benzoate is a common food preservative, and salicylate is the primary active metabolite of aspirin. In the gut microbiome, genetic adaptation to salicylate may involve loss or downregulation of inducible multidrug resistance systems. This discovery implies that aspirin therapy may modulate the human gut microbiome to favor salicylate tolerance at the expense of drug resistance. Similar aspirin-associated loss of drug resistance might occur in bacterial pathogens found in arterial plaques.

Acid-adapted strains of Escherichia coli K-12 obtained by experimental evolution.

Enteric bacteria encounter a wide range of pHs throughout the human intestinal tract. We conducted experimental evolution of Escherichia coli K-12 to isolate clones with increased fitness during growth under acidic conditions (pH 4.5 to 4.8). Twenty-four independent populations of E. coli K-12 W3110 were evolved in LBK medium (10 g/liter tryptone, 5 g/liter yeast extract, 7.45 g/liter KCl) buffered with homopiperazine-N,N'-bis-2-(ethanosulfonic acid) and malate at pH 4.8. At generation 730, the pH was decreased to 4.6 with HCl. By 2,000 generations, all populations had achieved higher endpoint growth than the ancestor at pH 4.6 but not at pH 7.0. All evolving populations showed a progressive loss of activity of lysine decarboxylase (CadA), a major acid stress enzyme. This finding suggests a surprising association between acid adaptation and moderation of an acid stress response. At generation 2,000, eight clones were isolated from four populations, and their genomes were sequenced. Each clone showed between three and eight missense mutations, including one in a subunit of the RNA polymerase holoenzyme (rpoB, rpoC, or rpoD). Missense mutations were found in adiY, the activator of the acid-inducible arginine decarboxylase (adiA), and in gcvP (glycine decarboxylase), a possible acid stress component. For tests of fitness relative to that of the ancestor, lacZ::kan was transduced into each strain. All acid-evolved clones showed a high fitness advantage at pH 4.6. With the cytoplasmic pH depressed by benzoate (at external pH 6.5), acid-evolved clones showed decreased fitness; thus, there was no adaptation to cytoplasmic pH depression. At pH 9.0, acid-evolved clones showed no fitness advantage. Thus, our acid-evolved clones showed a fitness increase specific to low external pH.

Health Care Use and Spending Among Need-Based Subgroups of Medicare Beneficiaries With Full Medicaid Benefits.

Importance: Beneficiaries dual eligible for Medicare and Medicaid account for a disproportionate share of expenditures due to their complex care needs. Lack of coordination between payment programs creates misaligned incentives, resulting in higher costs, fragmented care, and poor health outcomes. Objective: To inform the design of integrated programs by describing the health care use and spending for need-based subgroups in North Carolina's full benefit, dual-eligible population. Design, Setting, and Participants: This cross-sectional study using Medicare and North Carolina Medicaid 100% claims data (2014-2017) linked at the individual level included Medicare beneficiaries with full North Carolina Medicaid benefits. Data were analyzed between 2021 and 2022. Exposure: Need-based subgroups: community well, home- and community-based services (HCBS) users, nursing home (NH) residents, and intensive behavioral health (BH) users. Measures: Medicare and Medicaid utilization and spending per person-year (PPY). Results: The cohort (n = 333 240) comprised subgroups of community well (64.1%, n = 213 667), HCBS users (15.0%, n = 50 095), BH users (15.2%, n = 50 509), and NH residents (7.5%, n = 24 927). Overall, 61.1% reported female sex. The most common racial identities included Asian (1.8%), Black (36.1%), and White (58.7%). Combined spending for Medicare and Medicaid was $26 874 PPY, and the funding of care was split evenly between Medicare and Medicaid. Among need-based subgroups, combined spending was lowest among community well at $19 734 PPY with the lowest portion (38.5%) of spending contributed by Medicaid ($7605). Among NH residents, overall spending ($68 359) was highest, and the highest portion of spending contributed by Medicaid (70.1%). Key components of spending among HCBS users' combined total of $40 069 PPY were clinician services on carrier claims ($14 523) and outpatient facility services ($9012). Conclusions and relevance: Federal and state policy makers and administrators are developing strategies to integrate Medicare- and Medicaid-funded health care services to provide better care to the people enrolled in both programs. Substantial use of both Medicare- and Medicaid-funded services was found across all need-based subgroups, and the services contributing a high proportion of the total spending differed across subgroups. The diversity of health care use suggests a tailored approach to integration strategies with comprehensive set benefits that comprises Medicare and Medicaid services, including long-term services and supports, BH, palliative care, and social services.

Rheological characterization of poly-dimethyl siloxane formulations with tunable viscoelastic properties.

Studies from the past two decades have demonstrated convincingly that cells are able to sense the mechanical properties of their surroundings. Cells make major decisions in response to this mechanosensation, including decisions regarding cell migration, proliferation, survival, and differentiation. The vast majority of these studies have focused on the cellular mechanoresponse to changing substrate stiffness (or elastic modulus) and have been conducted on purely elastic substrates. In contrast, most soft tissues in the human body exhibit viscoelastic behavior; that is, they generate responsive force proportional to both the magnitude and rate of strain. While several recent studies have demonstrated that viscous effects of an underlying substrate affect cellular mechanoresponse, there is not a straightforward experimental method to probe this, particularly for investigators with little background in biomaterial fabrication. In the current work, we demonstrate that polymers comprised of differing polydimethylsiloxane (PDMS) formulations can be generated that allow for control over both the strain-dependent storage modulus and the strain rate-dependent loss modulus. These substrates requires no background in biomaterial fabrication to fabricate, are shelf-stable, and exhibit repeatable mechanical properties. Here we demonstrate that these substrates are biocompatible and exhibit similar protein adsorption characteristics regardless of mechanical properties. Finally, we develop a set of empirical equations that predicts the storage and loss modulus for a given blend of PDMS formulations, allowing users to tailor substrate mechanical properties to their specific needs.