Targeting Xist with compounds that disrupt RNA structure and X inactivation.

Although more than 98% of the human genome is non-coding1, nearly all of the drugs on the market target one of about 700 disease-related proteins. The historical reluctance to invest in non-coding RNA stems partly from requirements for drug targets to adopt a single stable conformation2. Most RNAs can adopt several conformations of similar stabilities. RNA structures also remain challenging to determine3. Nonetheless, an increasing number of diseases are now being attributed to non-coding RNA4 and the ability to target them would vastly expand the chemical space for drug development. Here we devise a screening strategy and identify small molecules that bind the non-coding RNA prototype Xist5. The X1 compound has drug-like properties and binds specifically the RepA motif6 of Xist in vitro and in vivo. Small-angle X-ray scattering analysis reveals that RepA can adopt multiple conformations but favours one structure in solution. X1 binding reduces the conformational space of RepA, displaces cognate interacting protein factors (PRC2 and SPEN), suppresses histone H3K27 trimethylation, and blocks initiation of X-chromosome inactivation. X1 inhibits cell differentiation and growth in a female-specific manner. Thus, RNA can be systematically targeted by drug-like compounds that disrupt RNA structure and epigenetic function.

Targeting RNA with small molecules: lessons learned from Xist RNA.

Although more than 98% of the human genome is noncoding, nearly all drugs on the market target one of about 700 disease-related proteins. However, an increasing number of diseases are now being attributed to noncoding RNA and the ability to target them would vastly expand the chemical space for drug development. We recently devised a screening strategy based upon affinity-selection mass spectrometry and succeeded in identifying bioactive compounds for the noncoding RNA prototype, Xist. One such compound, termed X1, has drug-like properties and binds specifically to the RepA motif of Xist in vitro and in vivo. Small-angle X-ray scattering analysis reveals that X1 changes the conformation of RepA in solution, thereby explaining the displacement of cognate interacting protein factors (PRC2 and SPEN) and inhibition of X-chromosome inactivation. In this Perspective, we discuss lessons learned from these proof-of-concept experiments and suggest that RNA can be systematically targeted by drug-like compounds to disrupt RNA structure and function.

Dihydrophenanthrene Open-Shell Singlet Diradicals and Their Roles in the Mallory Photocyclization Reaction.

A computational study (ωB97X-D/6-31G(d)) of the Mallory photocyclization reaction has revealed that the well-established dihydrophenanthrene (DHP) intermediates can adopt either closed-shell (CS) or open-shell-diradical (OS) singlet ground states. A detailed study of the properties of DHPs allowed their classifications as OS, borderline-OS, borderline-CS, or CS intermediates. The triplet electronic state and higher energy CS* isomer of all the OS singlet diradicals were computationally located, and the expected relationship between the diradical index, yo, and the triplet energy and the OS-CS* energy gaps was established. The importance of aromaticity in stabilizing the OS singlet diradicals was confirmed by using the Harmonic Oscillator Model of Aromaticity (HOMA). The thermal decompositions of DHPs by cycloreversions to regenerate the Mallory starting materials were also studied. The cycloreversion mechanism was described as a homolytic cleavage characterized by an anchimeric assistance continuum promoted by bis-ß-homolytic cleavage.

Pharmacological validation of dihydrofolate reductase as a drug target in Mycobacterium abscessus.

The Mycobacterium abscessus drug development pipeline is poorly populated, with particularly few validated target-lead couples to initiate de novo drug discovery. Trimethoprim, an inhibitor of dihydrofolate reductase (DHFR) used for the treatment of a range of bacterial infections, is not active against M. abscessus. Thus, evidence that M. abscessus DHFR is vulnerable to pharmacological intervention with a small molecule inhibitor is lacking. Here, we show that the pyrrolo-quinazoline PQD-1, previously identified as a DHFR inhibitor active against Mycobacterium tuberculosis, exerts whole cell activity against M. abscessus. Enzyme inhibition studies showed that PQD-1, in contrast to trimethoprim, is a potent inhibitor of M. abscessus DHFR and over-expression of DHFR causes resistance to PQD-1, providing biochemical and genetic evidence that DHFR is a vulnerable target and mediates PQD-1's growth inhibitory activity in M. abscessus. As observed in M. tuberculosis, PQD-1 resistant mutations mapped to the folate pathway enzyme thymidylate synthase (TYMS) ThyA. Like trimethoprim in other bacteria, PQD-1 synergizes with the dihydropteroate synthase (DHPS) inhibitor sulfamethoxazole (SMX), offering an opportunity to exploit the successful dual inhibition of the folate pathway and develop similarly potent combinations against M. abscessus. PQD-1 is active against subspecies of M. abscessus and a panel of clinical isolates, providing epidemiological validation of the target-lead couple. Leveraging a series of PQD-1 analogs, we have demonstrated a dynamic structure-activity relationship (SAR). Collectively, the results identify M. abscessus DHFR as an attractive target and PQD-1 as a chemical starting point for the discovery of novel drugs and drug combinations that target the folate pathway in M. abscessus.

Pathogen-microbiome interactions and the virulence of an entomopathogenic fungus.

Bacteria shape interactions between hosts and fungal pathogens. In some cases, bacteria associated with fungi are essential for pathogen virulence. In other systems, host-associated microbiomes confer resistance against fungal pathogens. We studied an aphid-specific entomopathogenic fungus called Pandora neoaphidis in the context of both host and pathogen microbiomes. Aphids host several species of heritable bacteria, some of which confer resistance against Pandora. We first found that spores that emerged from aphids that harbored protective bacteria were less virulent against subsequent hosts and did not grow on plate media. We then used 16S amplicon sequencing to study the bacterial microbiome of fungal mycelia and spores during plate culturing and host infection. We found that the bacterial community is remarkably stable in culture despite dramatic changes in pathogen virulence. Last, we used an experimentally transformed symbiont of aphids to show that Pandora can acquire host-associated bacteria during infection. Our results uncover new roles for bacteria in the dynamics of aphid-pathogen interactions and illustrate the importance of the broader microbiological context in studies of fungal pathogenesis. IMPORTANCE: Entomopathogenic fungi play important roles in the population dynamics of many insect species. Understanding the factors shaping entomopathogen virulence is critical for agricultural management and for the use of fungi in pest biocontrol. We show that heritable bacteria in aphids, which confer protection to their hosts against fungal entomopathogens, influence virulence against subsequent hosts. Aphids reproduce asexually and are typically surrounded by genetically identical offspring, and thus these effects likely shape the dynamics of fungal disease in aphid populations. Furthermore, fungal entomopathogens are known to rapidly lose virulence in lab culture, complicating their laboratory use. We show that this phenomenon is not driven by changes in the associated bacterial microbiome. These results contribute to our broader understanding of the aphid model system and shed light on the biology of the Entomophthorales-an important but understudied group of fungi.

Intraspecific variation in immune gene expression and heritable symbiont density.

Host genetic variation plays an important role in the structure and function of heritable microbial communities. Recent studies have shown that insects use immune mechanisms to regulate heritable symbionts. Here we test the hypothesis that variation in symbiont density among hosts is linked to intraspecific differences in the immune response to harboring symbionts. We show that pea aphids (Acyrthosiphon pisum) harboring the bacterial endosymbiont Regiella insecticola (but not all other species of symbionts) downregulate expression of key immune genes. We then functionally link immune expression with symbiont density using RNAi. The pea aphid species complex is comprised of multiple reproductively-isolated host plant-adapted populations. These 'biotypes' have distinct patterns of symbiont infections: for example, aphids from the Trifolium biotype are strongly associated with Regiella. Using RNAseq, we compare patterns of gene expression in response to Regiella in aphid genotypes from multiple biotypes, and we show that Trifolium aphids experience no downregulation of immune gene expression while hosting Regiella and harbor symbionts at lower densities. Using F1 hybrids between two biotypes, we find that symbiont density and immune gene expression are both intermediate in hybrids. We propose that in this system, Regiella symbionts are suppressing aphid immune mechanisms to increase their density, but that some hosts have adapted to prevent immune suppression in order to control symbiont numbers. This work therefore suggests that antagonistic coevolution can play a role in host-microbe interactions even when symbionts are transmitted vertically and provide a clear benefit to their hosts. The specific immune mechanisms that we find are downregulated in the presence of Regiella have been previously shown to combat pathogens in aphids, and thus this work also highlights the immune system's complex dual role in interacting with both beneficial and harmful microbes.

Treatment of post-meniscectomy knee symptoms with medial meniscus replacement results in greater pain reduction and functional improvement than non-surgical care.

PURPOSE: Partial meniscectomy is a common orthopedic procedure intended to improve knee pain and function in patients with irreparable meniscal tears. However, 6-25% of partial meniscectomy patients experience persistent knee pain after surgery. In this randomized controlled trial (RCT) involving subjects with knee pain following partial meniscectomy, it was hypothesized that treatment with a synthetic medial meniscus replacement (MMR) implant provides significantly greater improvements in knee pain and function compared to non-surgical care alone. METHODS: In this prospective, multicenter RCT, subjects with persistent knee pain following one or more previous partial meniscectomies were randomized to receive either MMR or non-surgical care. This analysis evaluated the 1-year outcomes of this 2-year clinical trial. Patient-reported knee pain, function, and quality of life were measured using nine separate patient-reported outcomes. The primary outcomes were the pain subscale of the Knee injury and Osteoarthritis Outcome Score (KOOS) and the average of all five KOOS subscales (KOOS Overall). Treatment cessation was defined as permanent device removal in the MMR group and any surgical procedure to the index knee in the non-surgical care group. RESULTS: Treated subjects had a median age of 52 years old (range 30-69 years) and one or more previous partial meniscectomies at a median of 34 months (range 5-430 months) before trial entry. Among 127 subjects treated with either MMR (n = 61) or non-surgical care (n = 66), 11 withdrew from the trial or were lost to follow-up (MMR, n = 0; non-surgical care, n = 11). The magnitude of improvement from baseline to 1 year was significantly greater in subjects who received MMR in both primary outcomes of KOOS Pain (P = 0.013) and KOOS Overall (P = 0.027). Treatment cessation was reported in 14.5% of non-surgical care subjects and only 4.9% of MMR subjects (n.s.). CONCLUSION: Treatment with the synthetic MMR implant resulted in significantly greater improvements in knee pain, function, and quality of life at 1 year of follow-up compared to treatment with non-surgical care alone. LEVEL OF EVIDENCE: I.

SAR towards indoline and 3-azaindoline classes of IDO1 inhibitors.

A novel series of IDO1 inhibitors have been identified with good IDO1 Hela cell and human whole blood activity. These inhibitors contain an indoline or a 3-azaindoline scaffold. Their structure-activity-relationship studies have been explored. Compounds 37 and 41 stood out as leads due to their good potency in IDO1 Hela assay, good IDO1 unbound hWB IC50s, reasonable unbound clearance, and good MRT in rat and dog PK studies.

RNA-ALIS: Methodology for screening soluble RNAs as small molecule targets using ALIS affinity-selection mass spectrometry.

Recent advances resulting from the completion of the human genome have shown that RNA has the promise to be a target for small molecule drugs, and therefore represents a previously unexploited class of targets for novel human therapeutics. We recently reported the adaptation of an affinity selection mass spectrometry screening technique, termed ALIS (Automatic Ligand Identification System), to screen and characterize a variety of RNA species from both prokaryotic and eukaryotic sources. We demonstrated that the ALIS technique, which had previously been used for protein targets, was also compatible for screening, ranking and characterizing small molecule ligands for RNA targets. We present here a detailed description of the use of ALIS for screening and characterizing ligands for RNA and discuss issues of validating and testing RNA for use in the ALIS system. We have also further elaborated on issues of RNA stability and testing in the ALIS system and demonstrate that the affinity-selection screening system has the potential to be a general solution for label-free screening and characterization of small molecule drug candidates for RNA targets.

Management of recurrent implantation failure by gonadotropin-releasing hormone agonist and aromatase inhibitor suppression, in women without evidence of endometriosis.

Endometriosis is common among those with infertility, although many cases go undiagnosed. This study was performed to determine whether empiric treatment with two months of depo-leuprolide 3.75 mg monthly (dep-GnRH-ag) and letrozole 5 mg daily improves pregnancy outcomes in patients with at least two unexplained failed embryo transfers (ETs) but without a previous diagnosis of endometriosis. A retrospective cohort study was performed with subjects who failed at least two good quality ET. The study excluded women with a known history of endometriosis or ovarian cysts (possible endometriomas). Subjects (N = 38) were treated with dep-GnRH-ag and letrozole pre-cycle. Matched women (N = 37) who did not receive either pretreatment served as a control group. Data were compared by non-paired T-tests and multivariate logistic regression to control for confounding effects. Demographic data, hormonal profiles, and ovarian reserve parameters were similar between the two groups. The treated group had failed more embryo transfers (3.5 ± 1.7 vs. 2.0 ± 1.3, p = .01) than the controls. When adjusting for the number of MII oocytes collected, number of blastocysts developed and number of blastocysts transferred, there were more pregnancies (24/38 vs. 11/37, p = .02) and ongoing pregnancies (18/38 vs. 9/37, p = .03) in the treated group. Patients with multiple unexplained failed ET may have undiagnosed endometriosis and may benefit from pretreatment with dep-GnRH-ag and letrozole. These results would benefit from being subjected to a randomized prospective study.

High prevalence of Clostridium difficile on retail root vegetables, Western Australia.

AIMS: The incidence of community-associated Clostridium difficile infection (CA-CDI) in Australia has increased since mid-2011. With reports of clinically important C. difficile strains being isolated from retail foods in Europe and North America, a foodborne source of C. difficile in cases of CA-CDI is a possibility. This study represents the first to investigate the prevalence and genotypes of C. difficile in Australian retail vegetables. METHODS AND RESULTS: A total of 300 root vegetables grown in Western Australia (WA) were collected from retail stores and farmers' markets. Three vegetables of the same kind bought from the same store/market were treated as one sample. Selective enrichment culture, toxin profiling and PCR ribotyping were performed. Clostridium difficile was isolated from 30% (30/100) of pooled vegetable samples, 55·6% of organic potatoes, 50% of nonorganic potatoes, 22·2% of organic beetroots, 5·6% of organic onions and 5·3% of organic carrots. Over half (51·2%, 22/43) the isolates were toxigenic. Many of the ribotypes of C. difficile isolated were common among human and Australian animals. CONCLUSIONS: Clostridium difficile could be found commonly on retail root vegetables of WA. This may be potential sources for CA-CDI. SIGNIFICANCE AND IMPACT OF THE STUDY: This study enhances knowledge of possible sources of C. difficile in the Australian community, outside the hospital setting.

Tridentate Phosphine Ligands Bearing Aza-Crown Ether Lariats.

Crown ethers are useful macrocycles that act as size-selective binding sites for alkali metals. These frameworks have been incorporated into a number of macromolecular assemblies that use simple cations as reporters and/or activity triggers. Incorporating crown ethers into secondary coordination sphere ligand frameworks for transition metal chemistry will lead to new potential methods for controlling bond formation steps, and routes that couple traditional ligand frameworks with these moieties are highly desirable. Herein we report the syntheses of a family of tridentate phosphine complexes bearing tethered aza-crown ethers (lariats) designed to modularize the variation of aza-crown size, lariat length, and distal phosphine substituents, followed by the synthesis and solid-state structures of Mo(III) complexes bearing cations in the pendent crown ethers.

Differential Mobility Spectrometry Coupled with Multiple Ion Monitoring in Regulated LC-MS/MS Bioanalysis of a Therapeutic Cyclic Peptide in Human Plasma.

A differential mobility spectrometry (DMS) in combination with a multiple ion monitoring (MIM) method was developed and validated for quantitative LC-MS/MS bioanalysis of pasireotide (SOM230) in human plasma. Pasireotide, a therapeutic cyclic peptide, exhibits poor collision-induced dissociation (CID) efficiency for multiple reaction monitoring (MRM) detection. Therefore, in an effort to increase the overall sensitivity of the assay, a DMS-MIM approach was explored. By selecting the most abundant doubly charged precursor ion in both the Q1 and Q3 of the mass analyzer in MIM and combining the DMS capability to significantly reduce the high matrix/chemical background noise, this new LC-DMS-MIM method overcomes the sensitivity challenge in the typical MRM method due to poor CID fragmentation of the analyte. Human plasma was spiked with pasireotide with concentrations in the range 0.01-50 ng/mL. Weak cation-exchange solid-phase extraction was employed for sample preparation. The sample extracts were analyzed with a SCIEX QTRAP 6500 system equipped with an ESI source and DMS device. The separation voltage and compensation voltage of the DMS and other parameters of the MS system were optimized to maximize signal responses. The performance of the LC-DMS-MIM assay for quantitative analysis of pasireotide in human plasma was evaluated and compared to those obtained via LC-MRM and LC-MIM without DMS. Overall, the assay sensitivity with DMS-MIM was approximately 5-fold better than that observed in MRM or MIM without DMS. The assay was validated with accuracy (% bias) and precision (% CV) of the QC results at eight concentration levels (0.01, 0.02, 0.05, 0.15, 0.3, 1.5, 15, and 37.5 ng/mL) evaluated ranging from -4.8 to 5.0% bias and 0.7 to 8.6% CV for the intraday and interday runs. The current LC-DMS-MIM workflow can be expanded to quantitative analysis of other molecules that have poor fragmentation efficiency in CID.

Emergence and spread of predominantly community-onset Clostridium difficile PCR ribotype 244 infection in Australia, 2010 to 2012.

We describe an Australia-wide Clostridium difficile outbreak in 2011 and 2012 involving the previously uncommon ribotype 244. In Western Australia, 14 of 25 cases were community-associated, 11 were detected in patients younger than 65 years, 14 presented to emergency/outpatient departments, and 14 to non-tertiary/community hospitals. Using whole genome sequencing, we confirm ribotype 244 is from the same C. difficile clade as the epidemic ribotype 027. Like ribotype 027, it produces toxins A, B, and binary toxin, however it is fluoroquinolone-susceptible and thousands of single nucleotide variants distinct from ribotype 027. Fifteen outbreak isolates from across Australia were sequenced. Despite their geographic separation, all were genetically highly related without evidence of geographic clustering, consistent with a point source, for example affecting the national food chain. Comparison with reference laboratory strains revealed the outbreak clone shared a common ancestor with isolates from the United States and United Kingdom (UK). A strain obtained in the UK was phylogenetically related to our outbreak. Follow-up of that case revealed the patient had recently returned from Australia. Our data demonstrate new C. difficile strains are an on-going threat, with potential for rapid spread. Active surveillance is needed to identify and control emerging lineages.

Synaptic regulation of microtubule dynamics in dendritic spines by calcium, F-actin, and drebrin.

Dendritic spines are actin-rich compartments that protrude from the microtubule-rich dendritic shafts of principal neurons. Spines contain receptors and postsynaptic machinery for receiving the majority of glutamatergic inputs. Recent studies have shown that microtubules polymerize from dendritic shafts into spines and that signaling through synaptic NMDA receptors regulates this process. However, the mechanisms regulating microtubule dynamics in dendrites and spines remain unclear. Here we show that in hippocampal neurons from male and female mice, the majority of microtubules enter spines from highly localized sites at the base of spines. These entries occur in response to synapse-specific calcium transients that promote microtubule entry into active spines. We further document that spine calcium transients promote local actin polymerization, and that F-actin is both necessary and sufficient for microtubule entry. Finally, we show that drebrin, a protein known to mediate interactions between F-actin and microtubules, acts as a positive regulator of microtubule entry into spines. Together these results establish for the first time the essential mechanisms regulating microtubule entry into spines and contribute importantly to our understanding of the role of microtubules in synaptic function and plasticity.

Measurement of mercury species in human blood using triple spike isotope dilution with SPME-GC-ICP-DRC-MS.

The measurement of different mercury compounds in human blood can provide valuable information about the type of mercury exposure. To this end, our laboratory developed a biomonitoring method for the quantification of inorganic (iHg), methyl (MeHg), and ethyl (EtHg) mercury in whole blood using a triple-spike isotope dilution (TSID) quantification method employing capillary gas chromatography (GC) and inductively coupled dynamic reaction cell mass spectrometry (ICP-DRC-MS). We used a robotic CombiPAL(®) sample handling station featuring twin fiber-based solid-phase microextraction (SPME) injector heads. The use of two SPME fibers significantly reduces sample analysis cycle times making this method very suitable for high sample throughput, which is a requirement for large public health biomonitoring studies. Our sample preparation procedure involved solubilization of blood samples with tetramethylammonium hydroxide (TMAH) followed by the derivatization with sodium tetra(n-propyl)borate (NaBPr(4)) to promote volatility of mercury species. We thoroughly investigated mercury species stability in the blood matrix during the course of sample treatment and analysis. The method accuracy for quantifying iHg, MeHg, and EtHg was validated using NIST standard reference materials (SRM 955c level 3) and the Centre de Toxicologie du Québec (CTQ) proficiency testing (PT) samples. The limit of detection (LOD) for iHg, MeHg, and EtHg in human blood was determined to be 0.27, 0.12, and 0.16 µg/L, respectively.

Rapid, reversible heterolytic cleavage of bound H2.

Heterolytic cleavage of dihydrogen into a proton and a hydride ion is a fundamentally important step in many reactions, including the oxidation of hydrogen by hydrogenase enzymes and ionic hydrogenation of organic compounds. We report the facile, reversible heterolytic cleavage of H2 in a manganese complex bearing a pendant amine, leading to the formation of a manganese hydride and a protonated amine that undergo H(+)/H(-) exchange at an estimated rate of >10(7) s(-1) at 25 °C.

An oral spleen tyrosine kinase (Syk) inhibitor for rheumatoid arthritis.

BACKGROUND: Spleen tyrosine kinase (Syk) is an important modulator of immune signaling. The objective of this phase 2 study was to evaluate the efficacy and safety of R788, an oral inhibitor of Syk, in patients with active rheumatoid arthritis despite methotrexate therapy. METHODS: We enrolled 457 patients who had active rheumatoid arthritis despite long-term methotrexate therapy in a 6-month, double-blind, placebo-controlled trial. The primary outcome was the American College of Rheumatology (ACR) 20 response (which indicates at least a 20% reduction in the number of both tender and swollen joints and improvement in at least three of five other criteria) at month 6. RESULTS: R788, at a dose of 100 mg twice daily and at a dose of 150 mg once daily, was significantly superior to placebo at month 6 (ACR 20 response rates of 67% and 57%, respectively, vs. 35%; P<0.001 for the comparison of both doses with placebo). It was also significantly superior with respect to ACR 50, which indicates at least a 50% improvement (43% and 32% vs. 19%; P<0.001 for the comparison of the 100-mg dose with placebo, P=0.007 for the comparison of the 150-mg dose with placebo) and ACR 70 (28% and 14% vs. 10%; P<0.001 for the comparison of the 100-mg dose with placebo, P=0.34 for the comparison of the 150-mg dose with placebo). A clinically significant effect was noted by the end of the first week of treatment. Adverse effects included diarrhea (in 19% of subjects taking the 100-mg dose of R788 vs. 3% of those taking placebo), upper respiratory infections (14% vs. 7%), and neutropenia (6% vs. 1%). R788 was associated with an increase in systolic blood pressure of approximately 3 mm Hg between baseline and month 1, as compared with a decrease of 2 mm Hg with placebo; 23% of the patients taking R788 vs. 7% of the patients receiving placebo required the initiation of or a change in antihypertensive therapy. CONCLUSIONS: In this phase 2 study, a Syk inhibitor reduced disease activity in patients with rheumatoid arthritis; adverse events included diarrhea, hypertension, and neutropenia. Additional studies will be needed to further assess the safety and efficacy of Syk-inhibition therapy in patients with rheumatoid arthritis. (Funded by Rigel; ClinicalTrials.gov number, NCT00665925.)

Pharmacokinetics of fostamatinib, a spleen tyrosine kinase (SYK) inhibitor, in healthy human subjects following single and multiple oral dosing in three phase I studies.

AIM: Fostamatinib (R788) is an orally dosed prodrug designed to deliver the active metabolite R940406 (R406), a spleen tyrosine kinase (SYK) inhibitor, for the treatment of rheumatoid arthritis. The objectives were to evaluate the human pharmacokinetic properties of fostamatinib and R406. METHOD: Three clinical studies were conducted in healthy subjects: (A) A single ascending dose study for R406 with doses ranging from 80-600 mg, (B) a single- and multiple-dose study of fostamatinib in aqueous suspension, with single doses ranging from 80-400 mg and multiple doses at 160 mg twice daily and (C) a study comparing suspension and tablet of fostamatinib, with the latter tested in both fed and fasted states. RESULTS: These studies demonstrated that when administered as a solution, R406 was rapidly absorbed. Increases in exposure were observed with doses up to 400 mg. A terminal half-life of 12-21 h was observed. Similar R406 exposure could be achieved with fostamatinib suspension and steady-state was achieved after 3-4 days following twice daily administration. Fostamatinib tablet and suspension exhibited similar R406 exposure. Upon co-administration with food, a delay in peak time and lower peak concentrations of R406 were observed but at the same time the overall exposure did not change. CONCLUSION: Fostamatinib demonstrates rapid and extensive conversion to R406, an inhibitor of SYK. Solid dosage forms of fostamatinib overcome the challenge of low aqueous solubility of R406. The PK profile of R406 could potentially allow once daily or twice daily oral administration of fostamatinib.

Variation in density, immune gene suppression, and coinfection outcomes among strains of the aphid endosymbiont Regiella insecticola.

Many insects harbor heritable microbes that influence host phenotypes. Symbiont strains establish at different densities within hosts. This variation is important evolutionarily because within-host density has been linked to the costs and benefits of the symbiosis for both partners. Studying the factors shaping within-host density is important to our broader understanding of host-microbe coevolution. Here we focused on different strains of Regiella insecticola, a facultative symbiont of aphids. We first showed that strains of Regiella establish in pea aphids at drastically different densities. We then found that variation in density is correlated with the expression levels of two key insect immune system genes (phenoloxidase and hemocytin), with the suppression of immune gene expression correlating with higher Regiella density. We then performed an experiment where we established coinfections of a higher- and a lower-density Regiella strain, and we showed that the higher-density strain is better able to persist in coinfections than the lower-density strain. Together, our results point to a potential mechanism that contributes to strain-level variation in symbiont density in this system, and our data suggest that symbiont fitness may be increased by establishing at higher density within hosts. Our work highlights the importance of within-host dynamics shaping symbiont evolution.