Statistical inference with exchangeability and martingales.

In this paper, we start by reviewing exchangeability and its relevance to the Bayesian approach. We highlight the predictive nature of Bayesian models and the symmetry assumptions implied by beliefs of an underlying exchangeable sequence of observations. By taking a closer look at the Bayesian bootstrap, the parametric bootstrap of Efron and a version of Bayesian thinking about inference uncovered by Doob based on martingales, we introduce a parametric Bayesian bootstrap. Martingales play a fundamental role. Illustrations are presented as is the relevant theory. This article is part of the theme issue 'Bayesian inference: challenges, perspectives, and prospects'.

MEK1/2 activity modulates TREM2 cell surface recruitment.

Rare sequence variants in the microglial cell surface receptor TREM2 have been shown to increase the risk for Alzheimer's disease (AD). Disease-linked TREM2 mutations seem to confer a partial loss of function, and increasing TREM2 cell surface expression and thereby its function(s) might have therapeutic benefit in AD. However, druggable targets that could modulate microglial TREM2 surface expression are not known. To identify such targets, we conducted a screen of small molecule compounds with known pharmacology using human myeloid cells, searching for those that enhance TREM2 protein at the cell surface. Inhibitors of the kinases MEK1/2 displayed the strongest and most consistent increases in cell surface TREM2 protein, identifying a previously unreported pathway for TREM2 regulation. Unexpectedly, inhibitors of the downstream effector ERK kinases did not have the same effect, suggesting that noncanonical MEK signaling regulates TREM2 trafficking. In addition, siRNA knockdown experiments confirmed that decreased MEK1 and MEK2 were required for this recruitment. In iPSC-derived microglia, MEK inhibition increased cell surface TREM2 only modestly, so various cytokines were used to alter iPSC microglia phenotype, making cells more sensitive to MEK inhibitor-induced TREM2 recruitment. Of those tested, only IFN-gamma priming prior to MEK inhibitor treatment resulted in greater TREM2 recruitment. These data identify the first known mechanisms for increasing surface TREM2 protein and TREM2-regulated function in human myeloid cells and are the first to show a role for MEK1/MEK2 signaling in TREM2 activity.

Evaluation of different materials used for sealing of implant abutment access channel and the peri-implant sulcus microbiota: A 6-month, randomized controlled trial.

OBJECTIVE: Peri-implantitis has been attributed to a myriad of factors, including microleakage at the abutment-implant interface. Implant abutment access channel sealing materials (IACSM) are readily used in implant dentistry, with little evidence on their effect on microleakage. This study aims to evaluate the effect of IACSM on the microbial composition in the implant access channel and the peri-implant sulcus. METHODS: A total of n = 8 patients (64 implants) were included in this single-blinded, randomized controlled trial, whereas four different materials (cotton, polytetrafluoroethylene [PTFE], synthetic foam, or polyvinyl siloxane [PVS]) were randomly placed as an IACSM. Following 6 months, microbial analysis was completed on the IACSM and samples from the peri-implant sulci via PCR and high-throughput sequencing. Bacterial samples on the IACSM and in the peri-implant sulci were classified according to Socransky's microbial complexes. RESULTS: There was a preponderance of early colonizing bacteria within the IACSM, while the peri-implant sulci were dominated by Orange complex bacteria. The proportion of Red and Orange complex members on the IACSM was significantly less than in the peri-implant sulci. The proportion of Green, Yellow, and Blue complex members found on the IACSM was significantly greater than in the peri-implant sulci. Atopobium, a diverse species not included in the microbial complexes, was frequently detected in the peri-implant sulcus samples. CONCLUSIONS: No detectable effects of IACSM on the microbial community in the peri-implant sulcus or on the IACSM were identified. Variation of bacterial species was most dependent on the individual patient. No significant differences were found in the periodontal parameters between the different treatment groups.

An Objective Prior from a Scoring Rule.

In this paper, we introduce a novel objective prior distribution levering on the connections between information, divergence and scoring rules. In particular, we do so from the starting point of convex functions representing information in density functions. This provides a natural route to proper local scoring rules using Bregman divergence. Specifically, we determine the prior which solves setting the score function to be a constant. Although in itself this provides motivation for an objective prior, the prior also minimizes a corresponding information criterion.

Peptide-Based Inhibitors of Fimbrial Biogenesis in Porphyromonas gingivalis.

Periodontitis is a progressive inflammatory disease that affects roughly half of American adults. Colonization of the oral cavity by the Gram-negative bacterial pathogen Porphyromonas gingivalis is a key event in the initiation and development of periodontal disease. Adhesive surface structures termed fimbriae (pili) mediate interactions of P. gingivalis with other bacteria and with host cells throughout the course of disease. The P. gingivalis fimbriae are assembled via a novel mechanism that involves proteolytic processing of lipidated precursor subunits and their subsequent polymerization on the bacterial surface. Given their extracellular assembly mechanism and central roles in pathogenesis, the P. gingivalis fimbriae are attractive targets for anti-infective therapeutics to prevent or treat periodontal disease. Here we confirm that conserved sequences in the N and C termini of the Mfa1 fimbrial subunit protein perform critical roles in subunit polymerization. We show that treatment of P. gingivalis with peptides corresponding to the conserved C-terminal region inhibits the extracellular assembly of Mfa fimbriae on the bacterial surface. We also show that peptide treatment interferes with the function of Mfa fimbriae by reducing P. gingivalis adhesion to Streptococcus gordonii in a dual-species biofilm model. Finally, we show that treatment of bacteria with similar peptides inhibits extracellular polymerization of the Fim fimbriae, which are also expressed by P. gingivalis These results support a donor strand-based assembly mechanism for the P. gingivalis fimbriae and demonstrate the feasibility of using extracellular peptides to disrupt the biogenesis and function of these critical periodontal disease virulence factors.

A Definition of Conditional Probability with Non-Stochastic Information.

The current definition of a conditional probability enables one to update probabilities only on the basis of stochastic information. This paper provides a definition for conditional probability with non-stochastic information. The definition is derived by a set of axioms, where the information is connected to the outcome of interest via a loss function. An illustration is presented.

Translating slow-binding inhibition kinetics into cellular and in vivo effects.

Many drug candidates fail in clinical trials owing to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug-target complex has a longer half-life than off-target-drug complexes. However, though slow-binding inhibition kinetics are a key feature of many marketed drugs, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters, including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection.

Exposure to TiO2 nanoparticles increases Staphylococcus aureus infection of HeLa cells.

BACKGROUND: Titanium dioxide (TiO2) is one of the most common nanoparticles found in industry ranging from food additives to energy generation. Approximately four million tons of TiO2 particles are produced worldwide each year with approximately 3000 tons being produced in nanoparticulate form, hence exposure to these particles is almost certain. RESULTS: Even though TiO2 is also used as an anti-bacterial agent in combination with UV, we have found that, in the absence of UV, exposure of HeLa cells to TiO2 nanoparticles significantly increased their risk of bacterial invasion. HeLa cells cultured with 0.1 mg/ml rutile and anatase TiO2 nanoparticles for 24 h prior to exposure to bacteria had 350 and 250 % respectively more bacteria per cell. The increase was attributed to bacterial polysaccharides absorption on TiO2 NPs, increased extracellular LDH, and changes in the mechanical response of the cell membrane. On the other hand, macrophages exposed to TiO2 particles ingested 40 % fewer bacteria, further increasing the risk of infection. CONCLUSIONS: In combination, these two factors raise serious concerns regarding the impact of exposure to TiO2 nanoparticles on the ability of organisms to resist bacterial infection.

Rational design of broad spectrum antibacterial activity based on a clinically relevant enoyl-acyl carrier protein (ACP) reductase inhibitor.

Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. CG400549, a Staphylococcus-specific 2-pyridone compound that inhibits the enoyl-acyl carrier protein reductase (FabI), has recently been shown to possess human efficacy for the treatment of methicillin-resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including CG400549. Based on these structures, we rationalize the 65-fold reduced affinity of CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4-pyridone-based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram-negative and mycobacterial organisms.

Screen for Modulation of Nucleocapsid Protein Condensation Identifies Small Molecules with Anti-Coronavirus Activity.

Biomolecular condensates formed by liquid-liquid phase separation have been implicated in multiple diseases. Modulation of condensate dynamics by small molecules has therapeutic potential, but so far, few condensate modulators have been disclosed. The SARS-CoV-2 nucleocapsid (N) protein forms phase-separated condensates that are hypothesized to play critical roles in viral replication, transcription, and packaging, suggesting that N condensation modulators might have anti-coronavirus activity across multiple strains and species. Here, we show that N proteins from all seven human coronaviruses (HCoVs) vary in their tendency to undergo phase separation when expressed in human lung epithelial cells. We developed a cell-based high-content screening platform and identified small molecules that both promote and inhibit condensation of SARS-CoV-2 N. Interestingly, these host-targeted small molecules exhibited condensate-modulatory effects across all HCoV Ns. Some have also been reported to exhibit antiviral activity against SARS-CoV-2, HCoV-OC43, and HCoV-229E viral infections in cell culture. Our work reveals that the assembly dynamics of N condensates can be regulated by small molecules with therapeutic potential. Our approach allows for screening based on viral genome sequences alone and might enable rapid paths to drug discovery with value for confronting future pandemics.

Efficacy of hypochlorous acid (HOCl) fog in sanitizing surfaces against Enterococcus faecalis.

BACKGROUND: Fogging is an efficient method when disinfection of large areas is desired. METHODS: Two methods of ultrasonic fogging, pulsed and continuous, were compared on bacteria dried on either aluminum or polystyrene surfaces. We characterized commercial and home-made hypochlorous acid (HOCl) with respect to storage and means of production. RESULTS: We found that the initial chlorine concentration of the commercial solution was approximately 550 ppm, and when stored open under ambient conditions, the chlorine content decreased at a rate of 30% every 100 days. The HOCl produced using the home synthesizers had a maximum chlorine content of 257.6 ppm which decayed by 65% after 100 days. A second synthesizer produced a liquid with high chlorine content and pH, 750ppm and pH = 8.55. The anti-bacterial efficacy was probed using Enterococcus faecalis, a persistent source of infection in public and clinical spaces. Time course studies determined that E. faecalis could survive dry on surfaces for more than 12 weeks, but was easily eliminated in half the fogging time. CONCLUSIONS: The most effective mode of application was determined to be continuous fogging where a 6.59 log reduction was established in vertical geometry. The optimal pulsed fogging protocol produced a similar reduction, but required nearly 5 times as long. The home synthesized versions yielded much lower log bacterial reductions. No significant differences in outcome were determined between polymer or metal surfaces.

Heterobivalent Inhibitors of Acetyl-CoA Carboxylase: Drug Target Residence Time and Time-Dependent Antibacterial Activity.

The relationship between drug-target residence time and the post-antibiotic effect (PAE) provides insights into target vulnerability. To probe the vulnerability of bacterial acetyl-CoA carboxylase (ACC), a series of heterobivalent inhibitors were synthesized based on pyridopyrimidine 1 and moiramide B (3) which bind to the biotin carboxylase and carboxyltransferase ACC active sites, respectively. The heterobivalent compound 17, which has a linker of 50 Å, was a tight binding inhibitor of Escherichia coli ACC (Kiapp 0.2 nM) and could be displaced from ACC by a combination of both 1 and 3 but not just by 1. In agreement with the prolonged occupancy of ACC resulting from forced proximity binding, the heterobivalent inhibitors produced a PAE in E. coli of 1-4 h in contrast to 1 and 3 in combination or alone, indicating that ACC is a vulnerable target and highlighting the utility of kinetic, time-dependent effects in the drug mechanism of action.

Structure-Kinetic Relationship Studies for the Development of Long Residence Time LpxC Inhibitors.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a promising drug target in Gram-negative bacteria. Previously, we described a correlation between the residence time of inhibitors on Pseudomonas aeruginosa LpxC (paLpxC) and the post-antibiotic effect (PAE) caused by the inhibitors on the growth of P. aeruginosa. Given that drugs with prolonged activity following compound removal may have advantages in dosing regimens, we have explored the structure-kinetic relationship for paLpxC inhibition by analogues of the pyridone methylsulfone PF5081090 (1) originally developed by Pfizer. Several analogues have longer residence times on paLpxC than 1 (41 min) including PT913, which has a residence time of 124 min. PT913 also has a PAE of 4 h, extending the original correlation observed between residence time and PAE. Collectively, the studies provide a platform for the rational modulation of paLpxC inhibitor residence time and the potential development of antibacterial agents that cause prolonged suppression of bacterial growth.

The utility of clusters and a Hungarian clustering algorithm.

Implicit in the k-means algorithm is a way to assign a value, or utility, to a cluster of points. It works by taking the centroid of the points and the value of the cluster is the sum of distances from the centroid to each point in the cluster. The aim in this paper is to introduce an alternative way to assign a value to a cluster. Motivation is provided. Moreover, whereas the k-means algorithm does not have a natural way to determine k if it is unknown, we can use our method of evaluating a cluster to find good clusters in a sequential manner. The idea uses optimizations over permutations and clusters are set by the cyclic groups; generated by the Hungarian algorithm.

Impact of Target Turnover on the Translation of Drug-Target Residence Time to Time-Dependent Antibacterial Activity.

The translation of time-dependent drug-target occupancy to extended pharmacological activity at low drug concentration depends on factors such as target vulnerability and the rate of target turnover. Previously, we demonstrated that the postantibiotic effect (PAE) caused by inhibitors of bacterial drug targets could be used to assess target vulnerability, and that high levels of target vulnerability coupled with relatively low rates of target resynthesis resulted in a strong correlation between drug-target residence time and the PAE following compound washout. Although the residence time of inhibitors on UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) in Pseudomonas aeruginosa (paLpxC) results in significant PAE, inhibitors of the equivalent enzyme in Escherichia coli (ecLpxC) do not cause a PAE. Hyperactivity of the fatty acid biosynthesis enzyme FabZ or the inclusion of sub-MIC levels of azithromycin lead to the observation of a PAE for three inhibitors of ecLpxC. FabZ hyperactivity has been shown to stabilize ecLpxC, and using mass spectrometry, we demonstrate that the appearance of a PAE can be directly linked to a 3-fold increase in the stability of ecLpxC. These studies substantiate the importance of target turnover in time-dependent drug activity.

The impact of TiO2 nanoparticle exposure on transmembrane cholesterol transport and enhanced bacterial infectivity in HeLa cells.

We have previously shown that exposure to TiO2 nanoparticles (NPs) reduces the resistance of HeLa cells to bacterial infection. Here we demonstrate that the increased infectivity is associated with enhanced asymmetry in the cholesterol distribution. We applied a live cell imaging method which uses tunable orthogonal cholesterol sensors to visualize and quantify in-situ cholesterol distribution between the two leaflets of the plasma membrane (PM). In the control culture, we found marked transbilayer asymmetry of cholesterol, with the concentration in the outer plasma membrane (OPM) being 13 ± 2-fold higher than that in the inner plasma membrane (IPM). Exposure of the culture to 0.1 mg/mL of rutile TiO2 NPs increased the asymmetry such that the concentration in the OPM was 51 ± 10 times higher, while the total cholesterol content increased only 21 ± 2%. This change in cholesterol gradient may explain the increase in bacterial infectivity in HeLa cells exposed to TiO2 NPs since many pathogens, including Staphylococcus aureus used in the present study, require cholesterol for proper membrane attachment and virulence. RT-PCR indicated that exposure to TiO2 was responsible for upregulation of the ABCA1 and ABCG1 mRNAs, which are responsible for the production of the cholesterol transporter proteins that facilitate cholesterol transport across cellular membranes. This was confirmed by the observation of an overall decrease in bacterial infection in ABCA1 knockout or methyl-ß-cyclodextrin-treated HeLa cells, as regardless of TiO2 NP exposure. Hence rather than preventing bacterial infection, TiO2 nanoparticles upregulate genes associated with membrane cholesterol production and distribution, hence increasing infectivity. STATEMENT OF SIGNIFICANCE: A great deal of work has been done regarding the toxicology of the particles, especially focusing on detrimental outcomes associated with reactive oxygen species (ROS) production. In this paper we show unambiguously a very surprising result, namely the ability of these particles to enhance bacterial infection even at very small exposure levels, where none of the deleterious effects of ROS products can yet be detected. Using a new imaging technique, we are able to demonstrate, in operando, the effect of the particles on cholesterol generation and distribution in live HeLa cells. This paper also represents the first in a series where we explore other consequences of increased membrane cholesterol, due to particle exposure, which are known to have multiple other consequences on human tissue function and development.

Correlating Drug-Target Residence Time and Post-antibiotic Effect: Insight into Target Vulnerability.

Target vulnerability correlates the level of drug-target engagement required to generate a pharmacological response. High vulnerability targets are those that require only a relatively small fraction of occupancy to achieve the desired pharmacological outcome, whereas low vulnerability targets require high levels of engagement. Here, we demonstrate that the slope of the correlation between drug-target residence time and the post-antibiotic effect (PAE) can be used to define the vulnerability of bacterial targets. For macrolides, a steep slope is observed between residence time on the E. coli ribosome and the PAE, indicating that the ribosome is a highly vulnerable drug target. The analysis of the residence time-PAE data for erythromycin, azithromycin, spiramycin, and telithromycin using a mechanistic pharmacokinetic-pharmacodynamic model that integrates drug-target kinetics into predictions of drug activity lead to the successful prediction of the cellular PAE for tylosin, which has the longest residence time (7.1 h) and PAE (5.8 h). Although the macrolide data support a connection between residence time, PAE, and bactericidality, many bactericidal ß-lactam antibiotics do not give a PAE, illustrating the role of factors such as protein resynthesis in the expression of target vulnerability.

Structural Basis for the Regulation of Biofilm Formation and Iron Uptake in A. baumannii by the Blue-Light-Using Photoreceptor, BlsA.

The opportunistic human pathogen, A. baumannii, senses and responds to light using the blue light sensing A (BlsA) photoreceptor protein. BlsA is a blue-light-using flavin adenine dinucleotide (BLUF) protein that is known to regulate a wide variety of cellular functions through interactions with different binding partners. Using immunoprecipitation of tagged BlsA in A. baumannii lysates, we observed a number of proteins that interact with BlsA, including several transcription factors. In addition to a known binding partner, the iron uptake regulator Fur, we identified the biofilm response regulator BfmR as a putative BlsA-binding partner. Using microscale thermophoresis, we determined that both BfmR and Fur bind to BlsA with nanomolar binding constants. To better understand how BlsA interacts with and regulates these transcription factors, we solved the X-ray crystal structures of BlsA in both a ground (dark) state and a photoactivated light state. Comparison of the light- and dark-state structures revealed that, upon photoactivation, the two α-helices comprising the variable domain of BlsA undergo a distinct conformational change. The flavin-binding site, however, remains largely unchanged from dark to light. These structures, along with docking studies of BlsA and Fur, reveal key mechanistic details about how BlsA propagates the photoactivation signal between protein domains and on to its binding partner. Taken together, our structural and biophysical data provide important insights into how BlsA controls signal transduction in A. baumannii and provides a likely mechanism for blue-light-dependent modulation of biofilm formation and iron uptake.

Positron Emission Tomography Imaging of Staphylococcus aureus Infection Using a Nitro-Prodrug Analogue of 2-[18F]F-p-Aminobenzoic Acid.

Deep-seated bacterial infections caused by pathogens such as Staphylococcus aureus are difficult to diagnose and treat and are thus a major threat to human health. In previous work we demonstrated that positron emission tomography (PET) imaging with 2-[18F]F-p-aminobenzoic acid (2-[18F]F-PABA) could noninvasively identify, localize, and monitor S. aureus infection with excellent sensitivity and specificity in a rodent soft tissue infection model. However, 2-[18F]F-PABA is rapidly N-acetylated and eliminated, and in an attempt to improve radiotracer accumulation in bacteria we adopted a prodrug strategy in which the acid was protected by an ester and the amine was replaced with a nitro group. Metabolite analysis indicated that the nitro group of ethyl 2-[18F]fluoro-4-nitrobenzoate (2-[18F]F-ENB) is converted to the corresponding amine by bacteria-specific nitroreductases while the ester is hydrolyzed in vivo into the acid. PET/CT imaging of 2-[18F]F-ENB and the corresponding acid 2-[18F]F-NB in a rat soft tissue infection model demonstrated colocalization of the radiotracer with the bioluminescent signal arising from S. aureus Xen29, and demonstrated that the tracer could differentiate S. aureus infection from sterile inflammation. Significantly, the accumulation of both 2-[18F]F-ENB and 2-[18F]F-NB at the site of infection was 17-fold higher than at the site of sterile inflammation compared to 8-fold difference observed for 2-[18F]F-PABA, supporting the proposal that the active radiotracer in vivo is 2-[18F]F-NB. Collectively, these data suggest that 2-[18F]F-ENB and 2-[18F]F-NB have the potential for translation to humans as a rapid, noninvasive diagnostic tool to identify and localize S. aureus infections.

Chemical genetics reveals an RGS/G-protein role in the action of a compound.

We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-alphaq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-alphaq signaling complex, and define new mutations in both RGS and G-alphaq, including a unique hypo-adapation allele of G-alphaq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation.