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There is considerable interest in combining echinocandin and triazole antifungal agents for treatment of invasive fungal infections; however, information is needed regarding the tolerability and potential for pharmacokinetic interactions. Anidulafungin is a semisynthetic echinocandin, and voriconazole is an extended-spectrum triazole. In a random sequence, 17 subjects received anidulafungin with placebo, voriconazole with placebo, and anidulafungin with voriconazole. Anidulafungin was administered intravenously: 200 mg on day 1, then 100 mg/d on days 2 through 4. Voriconazole was administered orally: 400 mg every 12 hours on day 1, then 200 mg every 12 hours on days 2 to 4. No dose-limiting toxicities or serious adverse events occurred, and all adverse events were mild and consistent with the known safety profiles of both drugs. Pharmacokinetic parameters were not affected by coadministration. The geometric mean ratio (90% confidence interval) of the combination/drug alone for AUC(SS) was 97.4% (94.9-99.9), 97.4% (92.1-103.0), and 94.4% (87.0-102.5) for anidulafungin, voriconazole, and the voriconazole metabolite, respectively. [ABSTRACT FROM AUTHOR]

Dalbavancin is a lipoglycopeptide antibiotic in clinical development as a once-weekly treatment for serious infections. A total of 532 patients, consisting of 502 patients with skin and soft tissue infections requiring parenteral therapy and 30 patients with catheter-related bloodstream infections, was available for population pharmacokinetic analysis. The majority of patients (78.4%) received dalbavancin intravenously as a 1000-mg dose on day 1 and a single 500-mg dose on day 8. A 2-compartment model with first-order elimination provided the best fit to the data. The clearance of dalbavancin was influenced by body surface area and creatinine clearance, but together they described less than 25% of the interpatient variability. Body surface area was determined to be a predictor of the central volume of distribution. There was no evidence that the presence of metabolic substrates, inhibitors, or inducers of cytochrome P450 or selected concomitant medications influenced the clearance of dalbavancin. [ABSTRACT FROM AUTHOR]

Anidulafungin is a novel antifungal agent of the echinocandin class that is intended for the treatment of invasive fungal disease. It is likely that anidulafungin will be coadministered with cyclosporine. In vitro studies and clinical studies were performed to evaluate the effect of anidulafungin on cyclosporine metabolism and to investigate the safety and pharmacokinetics of anidulafungin when concomitantly administered with cyclosporine. The potential for anidulafungin to inhibit the metabolism of cyclosporine was evaluated by pooled human hepatic microsomal protein fractions in vitro, incubating 3H-cyclosporine with different concentrations of anidulafungin. The safety of coadministration and the effects of cyclosporine on the pharmacokinetics of anidulafungin were assessed in a multiple-dose, open-label clinical study of 12 healthy volunteers. Subjects received a 200-mg intravenous loading dose of anidulafungin, followed by a daily 100-mg intravenous maintenance dose on days 2 through 8. An oral solution of cyclosporine (Neoral oral solution; 100 mg/mL) 1.25 mg/kg was also administered to subjects twice daily on days 5 through 8. In the in vitro study, the addition of anidulafungin had no effect on cyclosporine metabolism by human hepatic microsomal protein fractions. In the clinical study, no dose-limiting toxicities or serious adverse events occurred. A small increase in anidulafungin concentrations and drug exposure (22%) was observed after 4 days of dosing with cyclosporine and was not considered to be clinically relevant. The results support the concomitant use of anidulafungin and cyclosporine without the need for dosage adjustments of either drug. [ABSTRACT FROM AUTHOR]

Gemtuzumab ozogamicin is currently approved to treat CD33-positive acute myeloid leukemia (AML) in first relapse in patients older than age 60 years. The objective of this study was to characterize the pharmacokinetics of gemtuzumab ozogamicin in pediatric patients with relapsed or refractory AML. The study population comprised 29 subjects younger than age 18 with AML in first relapse. Dosages of 6, 7.5, and 9 mg/m(2) were administered during the study. Pharmacokinetic parameters were determined following each dose for hP67.6, total calicheamicin derivatives, and unconjugated calicheamicin derivatives. hP67.6 pharmacokinetic parameters had a consistent and statistically significant change between the first and second doses. Increases in AUC and decreases in both CL and V(ss) from the first dose to the second dose were consistent with those of the adult population. Changes between dose periods for total calicheamicin derivatives and unconjugated calicheamicin derivatives were consistent with those of hP67.6. Changes in pharmacokinetic parameters between dose periods are attributed to saturation of CD33 binding sites and diminished clearance resulting from a lower peripheral blast burden and antigen. Children receiving 9 mg/m(2) had the following hP67.6 pharmacokinetic parameters: C(max), 3.47+/-1.04 mg/L; AUC, 136 +/- 107 mg x h/L; CL, 0.12 +/- 0.15 L/h/m(2); V(ss), 6.5 +/- 5.5 L; and t(1/2), 64 +/- 44 h after their first dose. Mean pharmacokinetic values are similar to values reported in adults. Individual children demonstrated large intersubject variability, similar to adults. The pharmacokinetics of gemtuzumab ozogamicin in pediatric patients closely follow the profile and variability of adult patients. [ABSTRACT FROM AUTHOR]

The objective of this analysis was to describe the pharmacokinetic characteristics of anidulafungin in patients with serious fungal disease based on pharmacokinetic data collected during four recently completed or ongoing Phase II/III clinical studies. A total of 600 anidulafungin plasma samples from 225 patients across the four studies were available for analysis. Patients received daily intravenous infusions of 50, 75, or 100 mg anidulafungin, preceded by a loading dose that was twice the daily dose. The analysis population consisted of 129 patients with esophageal candidiasis, 87 with invasive candidiasis, 7 with invasive aspergillosis, and 2 with azole refractory mucosal candidiasis. A population analysis approach was used to develop a steady-state pharmacokinetic model for anidulafungin, assess the significance of possible covariates, and determine the amount of intersubject and random residual variability. A two-compartment model with first-order elimination provided the best fit to the data. The clearance of anidulafungin was influenced by weight and gender, and subjects in the invasive candidiasis study had a typical clearance that was approximately 30% higher than subjects from other studies. Weight was determined to be a predictor of the central volume of distribution. The covariates on clearance accounted for less than 20% of the intersubject variability and therefore are deemed to be of little clinical relevance. There was no evidence that the presence of rifampin or metabolic substrates, inhibitors, or inducers of cytochrome p450 influenced the clearance of anidulafungin. This indicates that dosing adjustments are not necessary when anidulafungin is administered in the presence of medications falling into these classifications. [ABSTRACT FROM AUTHOR]

The purpose of this study was to characterize the pharmacokinetics of gemtuzumab ozogamicin (Mylotarg; Wyeth-Ayerst Laboratories, St. Davids, PA) in patients with acute myeloid leukemia (AML) in first relapse. Gemtuzumab ozogamicin is an antibody-chemotherapeutic conjugate characterized as antibody-targeted chemotherapy, consisting of an engineered human anti-CD33 antibody (hP67.6) linked to a potent cytotoxic agent, N-acetyl-gamma calicheamicin DMH. The pharmacokinetics of gemtuzumab ozogamicin was evaluated in 59 adult AML patients in first relapse, enrolled in a phase II study. Plasma was collected following each dose at specified times, and the pharmacokinetics was characterized by measures of hP67.6, total calicheamicin derivatives, and unconjugated calicheamicin derivatives. After administration of the first 9 mg/m2 dose of gemtuzumab ozogamicin, the pharmacokinetic parameters (mean +/- SD) of hP67.6 following the first dose were as follows: peak plasma concentration, 2.86 +/- 1.35 mg/L; AUC, 123 +/- 105 mg x h/L; t 1/2, 72.4 +/- 42.0 hours; and clearance, 0.265 +/- 0.229L/h. Increased concentrations were observed after the second dose and are believed to be due to a decrease in clearance by CD33-positive blast cells, a result of the reduced tumor burden following the first dose. The concentration profiles of calicheamicin followed the same time course as hP67.6, evidence that calicheamicin remained conjugated to the antibody and delivered to leukemic cells. No relationship was found between plasma concentration and response at the recommended dose. The pharmacokinetics of gemtuzumab ozogamicin has been characterized in AML patients receiving doses at the proposed therapeutic level. [ABSTRACT FROM AUTHOR]

A study was performed to further investigate the apparent instability of tobramycin when coadministered with piperacillin/tazobactam in subjects with renal impairment. Twenty-six otherwise healthy volunteers between 23 and 74 years of age were studied. Eight subjects had moderate renal impairment, 10 had mild renal impairment, and 8 had normal renal function. Each subject received single doses of piperacillin/tazobactam and tobramycin alone as well as combined doses in a randomized, three-way crossover design. The subjects with normal renal function also received combined doses of piperacillin and tobramycin. Considerable care was taken to protect against in vitro inactivation of plasma and urine samples after collection. No systematic changes in pharmacokinetic parameters were observed. It is concluded that piperacillin, either alone or with tazobactam, did not change the pharmacokinetics of tobramycin in subjects with renal impairment. The apparent in vivo inactivation of tobramycin in the presence of piperacillin or piperacillin/tazobactam reported by others may be an artifact of ex vivo inactivation. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that affects neurons in the brain and spinal cord, causing loss of muscle control, and eventually leads to death. Phosphorylated transactive response DNA binding protein-43 (TDP-43) is the major pathological protein in both sporadic and familial ALS, forming cytoplasmic aggregates in over 95% of cases. Of the 10-15% of ALS cases that are familial, mutations in TDP-43 represent about 5% of those with a family history. We have developed an in vitro overexpression model by introducing three familial ALS mutations (A315T, M337V, and S379P) in the TDP-43 (TARDBP) gene which we define as 3X-TDP-43. This overexpression model TDP-43 shows deficits in autophagy flux and colocalization of TDP-43 with stress granules. We also observe a progressive shift of TDP-43 to the cytoplasm in this model. This overexpression model shows a reduction in solubility of phosphorylated TDP-43 from RIPA to urea soluble. Four glycolytic enzymes, phosphoglycerate kinase one (PGK1), aldolase A (ALDOA), enolase 1 (ENO1), and pyruvate dehydrogenase kinase 1 (PDK1) show significant time-dependent decreases in 3X-TDP-43 expressing cells. Shotgun proteomic analysis shows global changes in the importin subunit alpha-1 (KPNA2), heat shock 70 kDa protein 1A (HSPA1A), and protein disulfide-isomerase A3 (PDIA3) expression levels and coimmunoprecipitation reveals that these proteins complex with TDP-43. Overall, these results suggest that the 3X-TDP-43 model may provide new insights into pathophysiology and an avenue for drug screening in vitro for those suffering from ALS and related TDP-43 proteinopathies., (© 2025 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Premise: The use of hybrid breeding systems to increase crop yields has been the cornerstone of modern agriculture and is exemplified in the breeding and improvement of cultivated sunflower (Helianthus annuus). However, it is poorly understood what effect supporting separate breeding pools in such systems, combined with continued selection for yield, may have on leaf ecophysiology and specialized metabolite variation., Methods: We analyzed 288 lines of cultivated H. annuus to examine the genomic basis of several specialized metabolites and agronomically important traits across major heterotic groups., Results: Heterotic group identity supports phenotypic divergences between fertility restoring and cytoplasmic male-sterility maintainer lines in leaf ecophysiology and specialized metabolism. However, the divergence is not associated with physical linkage to nuclear genes that support current hybrid breeding practices in cultivated H. annuus. Additionally, we identified four genomic regions associated with leaf ecophysiology and specialized metabolism that colocalize with previously identified QTLs for quantitative self-compatibility traits and with S-protein homolog (SPH) proteins, a recently discovered family of proteins associated with self-incompatibility and self/nonself recognition in Papaver rhoeas (common poppy) with suggested conserved downstream mechanisms among eudicots., Conclusions: Further work is necessary to confirm the self-incompatibility mechanisms in cultivated H. annuus and their relationship to the integrative and polygenic architecture of leaf ecophysiology and specialized metabolism in cultivated sunflower. However, because self-compatibility is a derived quantitative trait in cultivated H. annuus, trait linkage to divergent phenotypic traits may have partially arisen as a potential unintended consequence of historical breeding practices and selection for yield., (© 2024 Botanical Society of America.)

Premise: The explosion of available genomic data poses significant opportunities and challenges for genome-wide association studies. Current approaches via linear mixed models (LMM) are straightforward but prevent flexible assumptions of an a priori genomic architecture, while Bayesian sparse LMMs (BSLMMs) allow this flexibility. Complex traits, such as specialized metabolites, are subject to various hierarchical effects, including gene regulation, enzyme efficiency, and the availability of reactants., Methods: To identify alternative genetic architectures, we examined the genetic architecture underlying the carotenoid content of an association mapping panel of Helianthus annuus individuals using multiple BSLMM and LMM frameworks., Results: The LMMs of genome-wide single-nucleotide polymorphisms (SNPs) identified a single transcription factor responsible for the observed variations in the carotenoid content; however, a BSLMM of the SNPs with the bottom 1% of effect sizes from the results of the LMM identified multiple biologically relevant quantitative trait loci (QTLs) for carotenoid content external to the known (annotated) carotenoid pathway. A candidate pathway analysis (CPA) suggested a β-carotene isomerase to be the enzyme with the highest impact on the observed carotenoid content within the carotenoid pathway., Discussion: While traditional LMM approaches suggested a single unknown transcription factor associated with carotenoid content variation in sunflower petals, BSLMM proposed several QTLs with interpretable biological relevance to this trait. In addition, the CPA allowed for the dissection of the regulatory vs. biosynthetic genetic architectures underlying this metabolic trait., (© 2023 The Authors. Applications in Plant Sciences published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Agricultural crop productivity relies on the application of chemical pesticides to reduce pest and pathogen damage. However, chemical pesticides also pose a range of ecological, environmental and economic penalties. This includes the development of pesticide resistance by insect pests and pathogens, rendering pesticides less effective. Alternative sustainable crop protection tools should therefore be considered. Semiochemicals are signalling molecules produced by organisms, including plants, microbes, and animals, which cause behavioural or developmental changes in receiving organisms. Manipulating semiochemicals could provide a more sustainable approach to the management of insect pests and pathogens across crops. Here, we review the role of semiochemicals in the interaction between plants, insects and microbes, including examples of how they have been applied to agricultural systems. We highlight future research priorities to be considered for semiochemicals to be credible alternatives to the application of chemical pesticides.

Premise: As plant lineages diversify across environmental gradients, species are predicted to encounter divergent biotic pressures. This study investigated the evolution of volatile secondary metabolism across species of Helianthus., Methods: Leaves and petals of 40 species of wild Helianthus were analyzed via gas chromatography-mass spectrometry to determine volatile secondary metabolite profiles., Results: Across all species, 500 compounds were identified; 40% were sesquiterpenes, 18% monoterpenes, 3% diterpenes, 4% fatty acid derivatives, and 35% other compounds such as phenolics and small organic molecules. Qualitatively, annuals and species from more arid western climates had leaf compositions with a higher proportion of total monoterpenes, while erect perennials and species from more mesic eastern habitats contained a higher proportion of total sesquiterpenes. Among species, mass-based leaf monoterpene and sesquiterpene abundance were identified as largely orthogonal axes of variation by principal component analysis. Profiles for leaves were not strongly correlated with those of petals., Conclusions: Volatile metabolites were highly diverse among wild Helianthus, indicating the value of this genus as a model system and rich genetic resource. The independence of leaf and petal volatile profiles indicates a low level of phenotypic integration between vegetative and reproductive structures, implying vegetative defense and reproductive defense or pollinator attraction functions mediated by terpene profiles in these two organs can evolve without major trade-offs. The major biosynthetic pathways for the major terpenes in wild Helianthus are already well described, providing a road map to deeper inquiry into the drivers of this diversity., (© 2022 Botanical Society of America.)

Taniborbactam, an investigational β-lactamase inhibitor that is active against both serine- and metallo-β-lactamases, is being developed in combination with cefepime to treat serious infections caused by multidrug-resistant Gram-negative bacteria. Anticipating the use of cefepime-taniborbactam in patients with impaired renal function, an open-label, single-dose clinical study was performed to examine the pharmacokinetics of both drugs in subjects with various degrees of renal function. Hemodialysis-dependent subjects were also studied to examine the amounts of cefepime and taniborbactam dialyzed. Single intravenous infusions of 2 g cefepime and 0.5 g taniborbactam coadministered over 2 h were examined, with hemodialysis-dependent subjects receiving doses both on- and off-dialysis. No subjects experienced serious adverse events or discontinued treatment due to adverse events. The majority of adverse events observed were mild in severity, and there were no trends in the safety of cefepime-taniborbactam related to declining renal function or the timing of hemodialysis. Clinically significant and similar decreases in drug clearance with declining renal function were observed for both cefepime and taniborbactam. The respective decreases in geometric mean clearance for subjects with mild, moderate, and severe renal impairment compared to subjects with normal renal function were 18%, 63%, and 78% for cefepime and 15%, 63%, and 81% for taniborbactam, respectively. Decreases in clearance were similar for both drugs and were shown to be proportional to decreases in renal function. Both cefepime and taniborbactam were dialyzable, with similar amounts removed during 4 h of hemodialysis. This study is registered at ClinicalTrials.gov as NCT03690362.

Histone post-translational modifications (PTMs) play important roles in many biological processes, including gene regulation and chromatin dynamics, and are thus of high interest across many fields of biological research. Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) is a powerful tool to profile histone PTMs in vivo. This method, however, is largely dependent on the specificity and availability of suitable commercial antibodies. While mass spectrometry (MS)-based proteomic approaches to quantitatively measure histone PTMs have been developed in mammals and several other model organisms, such methods are currently not readily available in plants. One major challenge for the implementation of such methods in plants has been the difficulty in isolating sufficient amounts of pure, high-quality histones, a step rendered difficult by the presence of the cell wall. Here, we developed a high-yielding histone extraction and purification method optimized for Arabidopsis thaliana that can be used to obtain high-quality histones for MS. In contrast to other methods used in plants, this approach is relatively simple, and does not require membranes or additional specialized steps, such as gel excision or chromatography, to extract highly purified histones. We also describe methods for producing MS-ready histone peptides through chemical labeling and digestion. Finally, we describe an optimized method to quantify and analyze the resulting histone PTM data using a modified version of EpiProfile 2.0 for Arabidopsis. In all, the workflow described here can be used to measure changes to histone PTMs resulting from various treatments, stresses, and time courses, as well as in different mutant lines. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Nuclear isolation and histone acid extraction Basic Protocol 2: Peptide labeling, digestion, and desalting Basic Protocol 3: Histone HPLC-MS/MS and data analysis., (© 2022 Wiley Periodicals LLC.)

Plant symbiosis with arbuscular mycorrhizal (AM) fungi provides many benefits, including increased nutrient uptake, drought tolerance, and belowground pathogen resistance. To develop a better understanding of the genetic architecture of mycorrhizal symbiosis, we conducted a genome-wide association study (GWAS) of this plant-fungal interaction in cultivated sunflower. A diversity panel of cultivated sunflower (Helianthus annuus L.) was phenotyped for root colonization under inoculation with the AM fungus Rhizophagus intraradices. Using a mixed linear model approach with a high-density genetic map, we identified genomic regions that are likely associated with R. intraradices colonization in sunflower. Additionally, we used a set of twelve diverse lines to assess the effect that inoculation with R. intraradices has on dried shoot biomass and macronutrient uptake. Colonization among lines in the mapping panel ranged from 0-70% and was not correlated with mycorrhizal growth response, shoot phosphorus response, or shoot potassium response among the Core 12 lines. Association mapping yielded three single-nucleotide polymorphisms (SNPs) that were significantly associated with R. intraradices colonization. This is the first study to use GWAS to identify genomic regions associated with AM colonization in an Asterid eudicot species. Three genes of interest identified from the regions containing these SNPs are likely related to plant defense., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Taniborbactam (formerly VNRX-5133), an investigational β-lactamase inhibitor active against both serine- and metallo-β-lactamases, is being developed in combination with cefepime to treat serious infections caused by multidrug-resistant Gram-negative bacteria. This first-in-human study evaluated the safety and pharmacokinetics of single and multiple doses of taniborbactam in healthy adult subjects. Single doses of 62.5 to 1,500 mg taniborbactam and multiple doses of 250 to 750 mg taniborbactam every 8 h (q8h) for 10 days were examined; all taniborbactam doses were administered as a 2-h intravenous infusion. No subjects experienced serious adverse events or discontinued treatment due to adverse events. The most common adverse event in both placebo- and taniborbactam-treated subjects was headache. The pharmacokinetics of taniborbactam were similar to the pharmacokinetics reported for cefepime. Taniborbactam demonstrated dose-proportional pharmacokinetics with low intersubject variability. Following single doses and with extended sampling, the mean terminal elimination half-life ranged from 3.4 to 5.8 h; however, the majority of exposure was characterized by an earlier phase with a half-life of about 2 h. Following multiple dosing, there was minimal accumulation of taniborbactam in plasma. At steady-state, approximately 90% of the administered dose of taniborbactam was recovered in urine as intact drug. There was no appreciable metabolism observed in either plasma or urine samples. (This study is registered at clinicaltrials.gov under registration number NCT02955459.).

Study Objective: Lefamulin is a novel IV and oral pleuromutilin recently approved for the treatment of community-acquired bacterial pneumonia (CABP). Given that renal comorbidities are common in patients admitted for CABP, understanding the pharmacokinetics of lefamulin in the face of severe renal impairment, including those requiring hemodialysis, is needed., Design: Open-label, Phase-1 pharmacokinetic study., Setting: Research Study Center., Patients: Twenty-three matched subjects were included, seven with "Normal" renal function (creatinine clearance >90 ml/min), eight with "Severe" renal impairment (glomerular filtration rate <30 ml/min/1.73 m 2 ), and eight subjects requiring hemodialysis., Measurements and Main Results: Subjects were administered a single dose of lefamulin IV 150 mg as a 1-h infusion. Subjects in the hemodialysis group started hemodialysis within 1 h after lefamulin infusion (On dialysis), as well as, on a non-dialysis day (Off dialysis). Plasma, urine, and dialysate fluid were collected for 36 h and analyzed for lefamulin and its major metabolite, BC-8041. Lefamulin was primarily excreted non-renally across groups. Statistical analyses revealed lefamulin and BC-8041 pharmacokinetics were similar between Normal and Severe groups, except for renal clearance, which decreased in Severe subjects (mean 1.3 L/h Normal vs. 0.4 L/h Severe). Likewise, lefamulin pharmacokinetics during on and off dialysis were unchanged, with lefamulin not measurably filtered in dialysate fluid. Two, three, and three subjects reported drug-related treatment-emergent adverse events (TEAE) in Normal, Severe, and Hemodialysis groups, respectively. All TEAEs were mild, except one (infusion-site reaction) that was classified as moderate., Conclusion: No dosage adjustment is required for patients with renal impairment, and lefamulin can be administered without regard to hemodialysis timing., (© 2021 Pharmacotherapy Publications, Inc.)

Study Objective: Lefamulin is a novel pleuromutilin recently approved by the FDA for the treatment of community-acquired bacterial pneumonia. Given that, lefamulin is primarily metabolized by CYP450 Phase-1 reactions, this study evaluated the pharmacokinetics of IV lefamulin in subjects with various degrees of hepatic impairment as compared with matched healthy subjects., Design: Open-label, Phase-1 clinical pharmacokinetic study., Setting: Research Study Center., Patients: Twenty-seven subjects; comprised of 11 individuals with normal hepatic function and eight each with moderate or severe hepatic impairment were included, as classified by Child-Pugh scores., Measurements and Main Results: Subjects were administered a single dose of IV lefamulin 150 mg over 1 h. Plasma was collected for 48 h and analyzed for lefamulin and its major metabolite, BC-8041, concentrations in addition to assessing lefamulin plasma protein binding. Pharmacokinetics were evaluated by noncompartmental analysis. Pharmacokinetic parameters were compared using least square geometric mean ratios. Lefamulin was well tolerated in all hepatic function groups. Statistical analyses revealed reductions in C max and increases in renal clearance for Moderate and Severe groups, as well as, the increased volume of distribution for the Severe group. Lefamulin plasma AUC mean (SD) was similar across groups at 7615 (1554), 8233 (2286), and 8938 (1640) h.ng/mL for Normal, Moderate, and Severe groups, respectively, despite decreased clearance observed primarily during terminal elimination phases. Decreased plasma-protein binding was seen in hepatically-impaired versus normal subjects., Conclusion: Lefamulin was generally well tolerated. Differences in lefamulin and BC-8041 pharmacokinetics were small, relative to the overall variability, and any changes appear to be compensated by increases in renal clearance and decreased protein binding., (© 2021 Pharmacotherapy Publications, Inc.)

Previous benchmarking studies have demonstrated the importance of instrument acquisition methodology and statistical analysis on quantitative performance in label-free proteomics. However, the effects of these parameters in combination with replicate number and false discovery rate (FDR) corrections are not known. Using a benchmarking standard, we systematically evaluated the combined impact of acquisition methodology, replicate number, statistical approach, and FDR corrections. These analyses reveal a complex interaction between these parameters that greatly impacts the quantitative fidelity of protein- and peptide-level quantification. At a high replicate number ( n = 8), both data-dependent acquisition (DDA) and data-independent acquisition (DIA) methodologies yield accurate protein quantification across statistical approaches. However, at a low replicate number (n = 4), only DIA in combination with linear models for microarrays (LIMMA) and reproducibility-optimized test statistic (ROTS) produced a high level of quantitative fidelity. Quantitative accuracy at low replicates is also greatly impacted by FDR corrections, with Benjamini-Hochberg and Storey corrections yielding variable true positive rates for DDA workflows. For peptide quantification, replicate number and acquisition methodology are even more critical. A higher number of replicates in combination with DIA and LIMMA produce high quantitative fidelity, while DDA performs poorly regardless of replicate number or statistical approach. These results underscore the importance of pairing instrument acquisition methodology with the appropriate replicate number and statistical approach for optimal quantification performance., Competing Interests: The authors declare no competing financial interest., (Not subject to U.S. Copyright. Published 2021 by American Chemical Society.)

Premise: Defense investment in plant reproductive structures is relatively understudied compared to the defense of vegetative organs. Here the evolution of chemical defenses in reproductive structures is examined in light of the optimal defense, apparency, and resource availability hypotheses within the genus Cornus using a phylogenetic comparative approach in relation to phenology and native habitat environmental data., Methods: Individuals representing 25 Cornus species were tracked for reproductive phenology over a full growing season at the Arnold Arboretum of Harvard University. Floral, fruit, and leaf tissue was sampled to quantify defensive chemistry as well as fruit nutritional traits relevant to bird dispersal. Native habitat environmental characteristics were estimated using locality data from digitized herbarium records coupled with global soil and climate data sets., Results: The evolution of later flowering was correlated with increased floral tannins, and the evolution of later fruiting was correlated with increased total phenolics. Leaves were found to contain the highest tannin activity, while inflorescences contained the highest total flavonoids. Multiple aspects of fruit defensive chemistry were correlated with fruit nutritional traits. Floral and fruit defensive chemistry were evolutionarily correlated with aspects of native habitat temperature, precipitation, and soil characteristics., Conclusions: Results provide tentative support for the apparency hypothesis with respect to both flower and fruit phenology, while relative concentrations of secondary metabolites across organs provide mixed support for the optimal defense hypothesis. The evolution of reproductive defense with native habitat provides, at best, mixed support for the resource availability hypothesis., (© 2020 Botanical Society of America.)

Introduction: Communication among health care providers, caregivers, and children with asthma is challenging and sometimes may exclude the child. This may result in delay in recognizing and responding appropriately to asthma symptoms. The purpose was to conduct focus groups among Puerto Rican children with asthma on communication with their health care provider about their asthma symptoms., Method: The qualitative arm (focus groups) of a mixed-method explanatory sequential study that was used to view communication with their health care provider through the lens of a group of Puerto Rican children. The sample included Puerto Rican children ages 8 to 12 years with asthma ( N = 23). The goal was to develop a child illness representation questionnaire., Results: The perspective of children with asthma provided enriched information to influence the development of instrument subscale on communication. The children were often not heard during a clinical visit. Most of the children express fear of their health care provider., Conclusions: Although this was a small sample, there were indications that children would like to opportunity to tell the story about their experience with having asthma. Further research will lead to the next step toward developing and computing a reliable measure that includes the child in a discussion during a clinical visit.

Floral morphology and pigmentation are both charismatic and economically relevant traits associated with cultivated sunflower (Helianthus annuus L.). Recent work has linked floral morphology and pigmentation to pollinator efficiency and seed yield. Understanding the genetic architecture of such traits is essential for crop improvement, and gives insight into the role of genetic constraints in shaping floral diversity. A diversity panel of 288 sunflower genotypes was phenotyped for a variety of morphological, phenological, and color traits in both a greenhouse and a field setting. Association mapping was performed using 5788 SNP markers using a mixed linear model approach. Several dozen markers across 10 linkage groups were significantly associated with variation in morphological and color trait variation. Substantial trait plasticity was observed between greenhouse and field phenotyping, and associations differed between environments. Color traits mapped more strongly than morphology in both settings, with markers together explaining 16% of petal carotenoid content in the greenhouse, and 17% and 24% of variation in disc anthocyanin presence in the field and greenhouse, respectively. Morphological traits like disc size mapped more strongly in the field, with markers together explaining up to 19% of disc size variation. Loci identified here through association mapping within cultivated germplasm differ from those identified through biparental crosses between modern cultivated sunflower and either its wild progenitor or domesticated landraces. Several loci lie within genomic regions involved in domestication. Differences between phenotype expression under greenhouse and field conditions highlight the importance of plasticity in determining floral morphology and pigmentation., (© The American Genetic Association 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Posttranslational modifications of proteins control many complex biological processes, including genome expression, chromatin dynamics, metabolism, and cell division through a language of chemical modifications. Improvements in mass spectrometry-based proteomics have demonstrated protein acetylation is a widespread and dynamic modification in the cell; however, many questions remain on the regulation and downstream effects, and an assessment of the overall acetylation stoichiometry is needed. In this chapter, we describe the determination of acetylation stoichiometry using data-independent acquisition mass spectrometry to expand the number of acetylation sites quantified. However, the increased depth of data-independent acquisition is limited by the spectral library used to deconvolute fragmentation spectra. We describe a powerful approach of subcellular fractionation in conjunction with offline prefractionation to increase the depth of the spectral library. This deep interrogation of subcellular compartments provides essential insights into the compartment-specific regulation and downstream functions of protein acetylation.

Transactive response DNA-binding protein of 43 kDa (TDP-43) functions as a heterogeneous nuclear ribonucleoprotein and is the major pathological protein in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis/motor neuron disease (ALS/MND). TDP-43 pathology may also be present as a comorbidity in approximately 20-50% of sporadic Alzheimer's disease cases. In a mouse model of MND, full-length TDP-43 increases association with the mitochondria and blocking the TDP-43/mitochondria interaction ameliorates motor dysfunction. Utilizing a proteomics screen, several mitochondrial TDP-43-interacting partners were identified, including voltage-gated anion channel 1 (VDAC1) and prohibitin 2 (PHB2), a crucial mitophagy receptor. Overexpression of TDP-43 led to an increase in PHB2 whereas TDP-43 knockdown reduced PHB2 expression in cells treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inducer of mitophagy. These results suggest that TDP-43 expression contributes to metabolism and mitochondrial function however we show no change in bioenergetics when TDP-43 is overexpressed and knocked down in HEK293T cells. Furthermore, the fusion protein mitofusin 2 (MFN2) interacts in complex with TDP-43 and selective expression of human TDP-43 in the hippocampus and cortex induced an age-dependent change in Mfn2 expression. Mitochondria morphology is altered in 9-month-old mice selectively expressing TDP-43 in an APP/PS1 background compared with APP/PS1 littermates. We further confirmed TDP-43 localization to the mitochondria using immunogold labeled TDP-43 transmission electron microscopy (TEM) and mitochondrial isolation methods There was no increase in full-length TDP-43 localized to the mitochondria in APP/PS1 mice compared to wild-type (littermates); however, using C- and N-terminal-specific TDP-43 antibodies, the N-terminal (27 kDa, N27) and C-terminal (30 kDa, C30) fragments of TDP-43 are greatly enriched in mitochondrial fractions. In addition, when the mitochondrial peptidase (PMPCA) is overexpressed there is an increase in the N-terminal fragment (N27). These results suggest that TDP-43 processing may contribute to metabolism and mitochondrial function., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Although the main focus in Alzheimer's disease (AD) has been an investigation of mechanisms causing Aβ plaque deposition and tau tangle formation, recent studies have shown that phosphorylated TDP-43 pathology is present in up to 50% of sporadic cases. Furthermore, elevated phosphorylated TDP-43 has been associated with more severe AD pathology. Therefore, we hypothesized that TDP-43 may regulate amyloid-beta precursor protein (APP) trafficking and tau phosphorylation/aggregation. In order to examine the role of TDP-43 in AD, we developed a transgenic mouse that overexpresses hippocampal and cortical neuronal TDP-43 in a mouse expressing familial mutations (K595N and M596L) in APP and presenilin 1 (PSEN1ΔE9). In our model, increased TDP-43 was related to increased tau aggregation as evidenced by thioflavin S-positive phosphorylated tau, which may implicate TDP-43 expression in pre-tangle formation. In addition, there was increased endosomal/lysosomal localization of APP and reduced Aβ plaque formation with increased TDP-43. Furthermore, there was decreased calcineurin with elevated TDP-43 expression. Since calcineurin is a phosphatase for TDP-43, the decreased calcineurin expression may be one mechanism leading to an increase in accumulation of diffuse phosphorylated TDP-43 in the hippocampus and cortex. We further show that when TDP-43 is knocked down there is an increase in calcineurin. In our model of selective TDP-43 overexpression in an APP/PSEN1 background, we show that TDP-43 decreases Aβ plaque deposition while increasing abnormal tau aggregation. These observations indicate that TDP-43 may play a role in regulating APP trafficking and tau aggregation. Our data suggest that TDP-43 could be a putative target for therapeutic intervention in AD affecting both Aβ plaque formation and tauopathy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Epigenetic modifications play critical roles in diverse biological processes. Histone Lys-to-Met (K-to-M) mutations act as gain-of-function mutations to inhibit a wide range of histone methyltransferases and are thought to promote tumorigenesis. However, it is largely unknown whether K-to-M mutations impact organismal development. Using Arabidopsis ( Arabidopsis thaliana ) as a model system, we discovered that a transgene exogenously expressing histone 3 Lys-36 to Met mutation (K36M) acts in a dominant-negative manner to cause global reduction of H3K36 methylation. Remarkably, this dominant repressive activity is dosage-dependent and causes strong developmental perturbations including extreme branching and early flowering by affecting the expression of genes involved in developmental and metabolic processes. Besides the established pathological roles of K-to-M mutations in tumor cells, we demonstrate a physiological outcome for K-to-M induced H3K36 hypomethylation. This study provides evidence for a conserved dominant-negative inhibitory role of histone K-to-M mutation across the plant and animal kingdoms. We also highlight the unique ability of K36M mutations to alter plant developmental processes leading to severe pleiotropic phenotypes. Finally, our data suggests K-to-M mutations may provide a useful strategy for altering epigenetic landscapes in organisms where histone methyltransferases are uncharacterized., (© 2017 The author(s). All Rights Reserved.)

This article reviews the literature on the effects of living in a disenfranchised community for low-income African American children who have asthma. The review focuses on social integration, social network, interactions with parents, and limited cultural resources, which lead to negative health outcomes among these children.

Mitochondria are essential organelles that adapt to stress and environmental changes. Among the nutrient signals that affect mitochondrial form and function is iron, whose depletion initiates a rapid and reversible decrease in mitochondrial biogenesis through unclear means. Here we demonstrate that, unlike the canonical iron-induced alterations to transcript stability, loss of iron dampens the transcription of genes encoding mitochondrial proteins with no change to transcript half-life. Using mass spectrometry, we demonstrate that these transcriptional changes are accompanied by dynamic alterations to histone acetylation and methylation levels that are largely reversible upon readministration of iron. Moreover, histone deacetylase inhibition abrogates the decreased histone acetylation observed upon iron deprivation and restores normal transcript levels at genes encoding mitochondrial proteins. Collectively, we demonstrate that deprivation of an essential nutrient induces transcriptional repression of organellar biogenesis involving epigenetic alterations., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The import of acetyl-CoA into the lumen of the endoplasmic reticulum (ER) by AT-1/SLC33A1 regulates Nε-lysine acetylation of ER-resident and -transiting proteins. Specifically, lysine acetylation within the ER appears to influence the efficiency of the secretory pathway by affecting ER-mediated quality control. Mutations or duplications in AT-1/SLC33A1 have been linked to diseases such as familial spastic paraplegia, developmental delay with premature death, and autism spectrum disorder with intellectual disability. In this study, we generated an AT-1 Tg mouse model that selectively overexpresses human AT-1 in neurons. These animals demonstrate cognitive deficits, autistic-like social behavior, aberrations in synaptic plasticity, an increased number of dendritic spines and branches, and widespread proteomic changes. We also found that AT-1 activity regulates acetyl-CoA flux, causing epigenetic modulation of the histone epitope H3K27 and mitochondrial adaptation. In conclusion, our results indicate that increased expression of AT-1 can cause an autistic-like phenotype by affecting key neuronal metabolic pathways., (© 2016 Hullinger et al.)

Unlabelled: The purpose of this proposed study was to explore experiences, needs and functioning of low-income, African American caregivers of children with asthma in order to better understand symptom management., Method: A qualitative, descriptive design was used. A purposive sampling technique was used to screen 32 caregivers of children with asthma, and 15 caregivers agreed to participate., Results: Three main themes were identified: managing the symptoms; cultural beliefs and values; and culturally competent health care provider., Conclusion: For the caregivers culture disparity is not just about access to health services but instead the healthcare providers' understanding of the complexity of symptoms management., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Histone post-translational modifications (PTMs) are important regulators of chromatin structure and gene expression. Quantitative analysis of histone PTMs by mass spectrometry remains extremely challenging due to the complex and combinatorial nature of histone PTMs. The most commonly used mass spectrometry-based method for high-throughput histone PTM analysis is data-dependent acquisition (DDA). However, stochastic precursor selection and dependence on MS1 ions for quantification impede comprehensive interrogation of histone PTM states using DDA methods. To overcome these limitations, we utilized a data-independent acquisition (DIA) workflow that provides superior run-to-run consistency and postacquisition flexibility in comparison to DDA methods. In addition, we developed a novel DIA-based methodology to quantify isobaric, co-eluting histone peptides that lack unique MS2 transitions. Our method enabled deconvolution and quantification of histone PTMs that are otherwise refractory to quantitation, including the heavily acetylated tail of histone H4. Using this workflow, we investigated the effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) on the global histone PTM state of human breast cancer MCF7 cells. A total of 62 unique histone PTMs were quantified, revealing novel SAHA-induced changes in acetylation and methylation of histones H3 and H4.

Acetylation of lysine ϵ-amino groups influences many cellular processes and has been mapped to thousands of sites across many organisms. Stoichiometric information of acetylation is essential to accurately interpret biological significance. Here, we developed and employed a novel method for directly quantifying stoichiometry of site-specific acetylation in the entire proteome of Escherichia coli. By coupling isotopic labeling and a novel pairing algorithm, our approach performs an in silico enrichment of acetyl peptides, circumventing the need for immunoenrichment. We investigated the function of the sole NAD(+)-dependent protein deacetylase, CobB, on both site-specific and global acetylation. We quantified 2206 peptides from 899 proteins and observed a wide distribution of acetyl stoichiometry, ranging from less than 1% up to 98%. Bioinformatic analysis revealed that metabolic enzymes, which either utilize or generate acetyl-CoA, and proteins involved in transcriptional and translational processes displayed the highest degree of acetylation. Loss of CobB led to increased global acetylation at low stoichiometry sites and induced site-specific changes at high stoichiometry sites, and biochemical analysis revealed altered acetyl-CoA metabolism. Thus, this study demonstrates that sirtuin deacetylase deficiency leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Fragile X syndrome (FXS) is caused by the loss of functional fragile X mental retardation protein (FMRP). Loss of FMRP results in an elevated basal protein expression profile of FMRP targeted mRNAs, a loss of local metabotropic glutamate receptor (mGluR)-regulated protein synthesis, exaggerated long-term depression and corresponding learning and behavioral deficits. Evidence shows that blocking mGluR signaling in FXS models ameliorates these deficits. Therefore, understanding the signaling mechanisms downstream of mGluR stimulation may provide additional therapeutic targets for FXS. Kinase cascades are an integral mechanism regulating mGluR-dependent protein translation. The c-Jun N-terminal kinase (JNK) pathway has been shown to regulate mGluR-dependent nuclear transcription; however, the involvement of JNK in local, synaptic signaling has not been explored. Here, we show that JNK is both necessary and sufficient for mGluR-dependent expression of a subset of FMRP target proteins. In addition, JNK activity is basally elevated in fmr1 knockout mouse synapses, and blocking JNK activity reduces the over-expression of post-synaptic proteins in these mice. Together, these data suggest that JNK may be an important signaling mechanism downstream of mGluR stimulation, regulating FMRP-dependent protein synthesis. Furthermore, local, post-synaptic dysregulation of JNK activity may provide a viable target to ameliorate the deficits involved in FXS. Expression of many FMRP target proteins is enhanced in FXS. Here, we evaluated the role of JNKs in FXS. We found that JNK signaling is activated upon mGluR stimulation in wild-type neurons. Conversely, JNK activity is basally elevated in fmr1 knockout. Inhibiting JNK reduced the expression of FMRP target proteins and driving JNK activity increased the expression of these proteins., (© 2013 The Authors Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

The Nrf2 (NF-E2 related factor 2)-ARE (antioxidant response element) pathway controls a powerful array of endogenous cellular antioxidant systems and is an important pathway in the detoxification of reactive oxygen species (ROS) in the brain. Using a combination of quantitative proteomics and siRNA screening, we have identified novel protective mechanisms of the Nrf2-ARE pathway against oxidative stress in astrocytes. Studies from our lab and others have shown Nrf2 overexpression protects astrocytes from oxidative stress. However, the exact mechanisms by which Nrf2 elicits these effects are unknown. In this study, we show that induction of Nrf2 reduces levels of reactive oxygen species (ROS) produced by various oxidative stressors and results in robust cytoprotection. To identify the enzymes responsible for these effects, we used stable isotope labeling by amino acids in cell culture (SILAC) and quantitative shotgun proteomics to identify 72 Nrf2-regulated proteins in astrocytes. We hypothesized a subset of these proteins might play a critical role in Nrf2 protection. In order to identify these critical proteins, we used bioinformatics to narrow our target list of proteins and then systematically screened each candidate with siRNA to assess the role of each in Nrf2 protection. We screened each target against H2O2, tert-butyl hydroperoxide, and 4-hydroxynonenal and subsequently identified three enzymes-catalase, prostaglandin reductase-1, and peroxiredoxin-6-that are critical for Nrf2-mediated protection in astrocytes.

Due to the complexity of the mammalian central nervous system, neuropeptidomic studies in mammals often yield very complicated mass spectra that make data analysis difficult. Careful sample preparation and extraction protocols must be employed in order to minimize spectral complexity and enable extraction of useful information on neuropeptides from a given sample. Controlling post-mortem protease activity is essential to simplifying mass spectra and to identifying low-abundance neuropeptides in tissue samples. Post-mortem microwave-irradiation coupled with cryostat dissection has proven to be effective in arresting protease activity to allow detection of endogenous neuropeptides instead of protein degradation products.

Astrocytes are important regulators of normal brain function in mammals, including roles in synaptic signaling, synapse formation, and neuronal health and survival. Many of these functions are executed via secreted proteins. To analyze the astrocyte secretome, a combination of shotgun proteomics and bioinformatics was employed to analyze conditioned media from primary murine astrocyte cultures. Both two- and one-dimensional LC-MS/MS were used to analyze astrocyte secreted proteins, resulting in the identification of over 420 proteins. To refine our results, the intracellular protein contaminants were removed in silico using a cytoplasmic control. In additional rounds of refinement, putative secreted proteins were subjected to analysis by SignalP, SecretomeP, and gene ontology analysis, yielding a refined list of 187 secreted proteins. In conclusion, the use of shotgun proteomics combined with multiple rounds of data refinement produced a high quality catalog of astrocyte secreted proteins.

Three open-label studies assessed the safety, tolerability, and pharmacokinetics of intravenous dalbavancin in patients with hepatic or renal impairment, including patients with end-stage renal disease (ESRD) receiving dialysis. In each study, 4 to 10 patients with mild, moderate, or severe impairment and age-, sex-, and weight-matched controls were administered either a single dose (500 or 1000 mg) or 2 doses (1000 mg followed by 500 mg 1 week apart) of dalbavancin. Dalbavancin exposures were not increased due to mild renal impairment. The mean area under the concentration-time curve from time 0 to infinity (AUC0-infinity) values were approximately 50% higher in patients with moderate renal impairment or ESRD and 100% higher in patients with severe renal impairment. Dose adjustment is not considered necessary in patients with mild or moderate renal impairment or for patients with ESRD receiving hemodialysis; however, a lower dose of dalbavancin (750 mg followed 1 week later by 375 mg) may be considered for patients with severe renal impairment (creatinine clearance<30 mL/min). AUC0-infinity values were similar in patients with mild hepatic impairment and were about 27% to 36% lower in patients with moderate to severe hepatic impairment compared with controls. No dosage adjustment is recommended in patients with any degree of hepatic impairment. Dalbavancin was well tolerated in all impairment groups.

Anidulafungin is a novel semisynthetic echinocandin with potent activity against Candida (including azole-resistant isolates) and Aspergillus spp. and is used for serious systemic fungal infections. The purpose of these studies was to characterize the clearance mechanism and potential for drug interactions of anidulafungin. Experiments included in vitro degradation of anidulafungin in buffer and human plasma, a bioassay for antifungal activity, in vitro human cytochrome P450 inhibition studies, in vitro incubation with rat and human hepatocytes, and mass balance studies in rats and humans. Clearance of anidulafungin appeared to be primarily due to slow chemical degradation, with no evidence of hepatic-mediated metabolism (phase 1 or 2). Under physiological conditions, further degradation of the primary degradant appears to take place. The primary degradation product does not retain antifungal activity. Anidulafungin was not an inhibitor of cytochrome P450 enzymes commonly involved in drug metabolism. Mass balance studies showed that anidulafungin was eliminated in the feces predominantly as degradation products, with only a small fraction (10%) eliminated as unchanged drug; fecal elimination likely occurred via biliary excretion. Only negligible renal involvement in the drug's elimination was observed. In conclusion, the primary biotransformation of anidulafungin is mediated by slow chemical degradation, with no evidence for hepatic enzymatic metabolism or renal elimination.

Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to induce expression of a variety of cytoprotective and detoxification genes. Several of the genes commonly regulated by Nrf2 have been implicated in protection from neurodegenerative conditions. Work from several laboratories has uncovered the potential for Nrf2-mediated transcription to protect from neurodegeneration resulting from mechanisms involving oxidative stress. For this reason, Nrf2 may be considered a therapeutic target for conditions that are known to involve free radical damage. Because common mechanisms of neurodegeneration, such as mitochondrial dysfunction and build-up of reactive oxygen species, are currently being uncovered, targeting Nrf2 may be valuable in combating conditions with variable causes and etiologies. Most effectively to target this protein in neurodegenerative conditions, a description of the involvement of Nrf2 and potential for neuroprotection must come from laboratory models. Herein, we review the current literature that suggests that Nrf2 may be a valuable therapeutic target for neurodegenerative disease, as well as experiments that illustrate potential mechanisms of protection.

Because of their great biological significance, neuropeptides are the subject of intensive research. Mass spectrometry (MS) is a highly informative and sensitive method used for detecting and characterizing these compounds. Successful MS analysis of neuropeptides is dependent on careful sample preparation. Herein, we present two common sample preparation strategies: direct tissue analysis and pooled tissue extraction coupled with fractionation.

Dalbavancin is a novel glycopeptide with a 2-dose, once-weekly dosing regimen that is being developed for the treatment of complicated skin and skin structure infections caused by gram-positive bacteria. Monte Carlo simulations were performed for dalbavancin using population pharmacokinetic data and minimum inhibitory concentrations (MICs) for clinical trial isolates. The time-dependent target was the maintenance of free drug concentrations above the MIC for 14 days (t>MIC). The concentration-dependent target was an area under the concentration-time curve (AUC)/MIC ratio of approximately 1000 for Staphylococcus aureus and 100 for Streptococcus sp. These targets were used to estimate susceptibility breakpoints for dalbavancin. For S aureus, the estimated susceptibility breakpoint was MIC. For Streptococcus sp, the estimated susceptibility breakpoint was at least 2 mug/mL. Because dalbavancin MIC(90)s for these species are well below these values, the analysis supports the use of once-weekly dosing regimens of dalbavancin in the treatment of complicated skin and skin structure infections.

Two-dimensional (2D) fractionation is a commonly used tool to increase dynamic range and proteome coverage for bottom-up, shotgun proteomics. However, there are few reports comparing the relative separation efficiencies of 2D methodologies using low-microgram sample quantities. In order to systematically evaluate 2D separation techniques, we fractionated microgram quantities of E. coli protein extract by seven different methods. The first dimension of separation was performed with either reversed-phase high-pressure liquid chromatography (RP-HPLC), gel electrophoresis (SDS-PAGE), or strong cation exchange (SCX-HPLC). The second dimension consisted of a standard reversed-phase capillary HPLC coupled to an electrospray ionization quadrupole time-of-flight mass spectrometer for tandem mass spectrometric analysis. The overall performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The protein-level RP-HPLC and the high-pH RP-HPLC peptide-level separations performed the best, identifying 281 and 266 proteins, respectively. The online pH variance SCX and the SDS-PAGE returned modest performances with 178 and 139 proteins identified, respectively. The offline SCX had the worst performance with 81 proteins identified. We also examined various chromatographic factors that contribute to separation efficiency, including resolving power, orthogonality, and sample loss.

This analysis quantifies the population pharmacokinetics of subcutaneous and intravenous epoetin delta, an epoetin produced in a human cell line, in pediatric patients with chronic kidney disease and estimates the effects of covariate factors on epoetin delta and epoetin alfa pharmacokinetic parameters. Erythropoietin serum concentration data, taken from a phase III study conducted in 60 patients aged 1 to 17 years, were best described by a 1-compartment model with first-order absorption and elimination. The typical point estimates were clearance (0.268 L/h), central volume of distribution (1.03 L), absorption rate constant (0.0554 h(-1)), and bioavailability (0.708) for a 35-kg male < or = 10 years who was predialysis and on subcutaneous epoetin delta treatment. Erythropoietin pharmacokinetic parameters were similar in pediatric patients as compared with adults when scaled by weight. The subcutaneous administration of epoetin alfa exhibited lower systemic bioavailability than subcutaneous administration of epoetin delta.

Objectives: Dalbavancin is a novel lipoglycopeptide antibiotic in development for the treatment of complicated skin and skin structure infections (cSSSIs) caused by Gram-positive bacteria. The aim of the present study was to assess the penetration of dalbavancin into skin blister fluid., Methods: Nine healthy subjects (five males; ranging in age from 26 to 57 years) were administered a single 30 min intravenous infusion of dalbavancin at a dose of 1000 mg. Skin blisters were induced by application of cantharidin ointment. Plasma and blister fluid samples were collected over 7 days post-dose, and concentrations of dalbavancin were assessed by a validated LC/MS/MS assay. Pharmacokinetics were determined by non-compartmental methods, and drug penetration was assessed based on the ratio of area under the curve (AUC) in the blister fluid versus plasma for each subject., Results: The mean (SD) peak concentration of dalbavancin in plasma and blister fluid was 285 (31.1) and 67.3 (18.2) mg/L, respectively; the corresponding AUC(Day 7) values were 10 806 (1926) and 6438 (1238) mg . h/L, respectively. The mean (SD) penetration of dalbavancin into blister fluid was 59.6% (6.3%). By Day 7, the mean concentration of dalbavancin in plasma and blister fluid was 46.5 and 30.3 mg/L, respectively., Conclusions: Dalbavancin concentrations in blister fluid remained well above the MIC90 values for pathogens commonly implicated in cSSSIs such as Staphylococcus aureus, including methicillin-resistant S. aureus (MIC90 = 0.06 mg/L) and beta-haemolytic streptococci (MIC90 = 0.03 mg/L) through Day 7. These pharmacokinetic data support the use of dalbavancin in the treatment of cSSSIs caused by susceptible Gram-positive pathogens.

Penetration of dalbavancin into noninfected bone and joint tissues was assessed after an intravenous dose of 20 mg/kg (of body weight) [(14)C]dalbavancin given to rabbits. Drug-derived radioactivity, determined over 14 days by either liquid scintillation counting or autoradiography, remained above the MIC for common gram-positive pathogens that cause bone and joint infections.

Background: Epoetin delta, unlike recombinant erythropoietins, is produced in a human cell line and therefore has a human-type glycosylation profile., Objectives: The pharmacokinetics of epoetin delta were examined in 2 studies in healthy volunteers and 2 studies in patients with chronic kidney disease., Methods: In study 1, 21 healthy men were randomized to receive epoetin delta 15, 40, or 100 IU/kg IV tiw or placebo for 4 weeks. In study 2, an open-label, cross-over study, 32 healthy volunteers were randomized to receive single doses of epoetin delta 75 IU/kg IV or SC. In study 3, 40 patients receiving hemodialysis were withdrawn from epoetin alfa and randomized to receive epoetin delta or epoetin alfa 50 or 100 IU/kg tiw for 4 weeks. Study 4 was a single-dose study comparing epoetin delta 150 and 300 IU/kg IV or SC in 28 hemodialysis patients., Results: In study 1, after repeated dosing (day 24) in healthy men, mean C(max) values ranged from 219.9 to 1793.0 enzyme-linked immunosorbent assay units (EU)/L; AUC from 827 to 9318 h x EU/L; C1 from 0.014 to 0.024 L/h per kg; Vd from 0.067 to 0.076 L/kg; and t(1/2) from 2.23 to 3.35 hours. There was evidence of a dose-dependent effect of epoetin delta on hemoglobin levels and hematocrit, with doses of 40 and 100 IU/kg associated with significant increases compared with 15 IU/kg (P < 0.001 for dose trend). The only adverse event occurring in > or = 10% of healthy individuals in study 1 was headache (1 [20.0%] in the epoetin delta 15 IU-kg group, 3 [60.0%] in the epoetin delta 100-IU/kg group, 2 [33.3%] in the placebo group). In study 2 in healthy volunteers, mean values for epoetin delta 75 IU/kg IV were 1771 EU/L for C(max), 10,632 h x EU/L for AUC, 0.010 L/h per kg for Cl, 0.074 L/kg for Vd, and 5.12 hours for t(1/2); the corresponding values for epoetin delta 75 IU/kg SC were 113 EU/L, 3231 h x EU/L, 0.035 L/h per kg, 0.760 L/kg, and 14.90 hours. The serum epoetin delta concentration peaked after 10.9 hours with subcutaneous administration. The most common adverse event in study 2 was back pain (10 [31.3%] individuals). In study 3 in patients receiving hemodialysis, mean values for C(max) and AUC with a single dose of epoetin delta 50 IU/kg were 1103 EU/L and 10,896 h x EU/L, respectively, and with the corresponding dose of epoetin alfa were 1354 EU/L and 9957 h x EU/L. Values for the 100-IU/kg doses were approximately double those for the 50-IU/kg doses. Values for Cl, Vd, and t(1/2) were numerically similar for epoetin delta and epoietin alfa across doses. Epoetin delta 100 IU/kg was associated with a numerically greater rate of increase in hemoglobin compared with the 50-IU/kg dose (mean, 0.025 vs -0.004, respectively); the results were similar for epoetin alfa (0.029 vs -0.001). The difference between epoetin alfa and epoetin delta was not statistically significant. The most common adverse events were related to edema (peripheral edema: 60%/50% for epoetin delta 50/100 IU/kg and 60%/60% for epoetin alfa 50/100 IU/kg; facial edema: 30%/30% and 50%/70%, respectively; generalized edema: 50%/30% and 40%/40%). In study 4 in patients receiving hemodialysis, mean C(max) values with epoetin delta 150 and 300 IU/kg IV were 3257 and 4770 EU/L, respectively; the corresponding mean values were 36,208 and 77,736 h x EU/L for AUC, 0.007 and 0.005 L/h per kg for Cl; 0.097 L/kg for Vd in both groups; and 9.9 and 13.2 hours for t(1/2). With epoetin delta 150 and 300 IU/kg SC, the respective values were 162.2 and 467.7 EU/L, 9547 and 27,888 h x EU/L, 0.026 and 0.020 L/h per kg, 1.28 and 0.78 L/kg, and 33.1 and 27.8 hours. The only adverse event occurring in > or = 10% of subjects was headache (2 [40.0%] in the epoetin delta 150-IU/kg IV group, 3 [50.0%] in the epoetin delta 300-IU/kg SC group). No neutralizing anti-erythropoietin antibodies were detected in any individual. The bioavailability of subcutaneous epoetin delta is approximately 30%, and concentrations peak later and decline more slowly than with intravenous injection. Pharmacokinetic parameters in hemodialysis patients were similar to those in healthy individuals, although AUC and t(1/2) were numerically higher (by 49% and 34%, respectively)., Conclusions: These studies in healthy volunteers and patients with chronic kidney disease indicate that the pharmacokinetics of epoetin delta are dose dependent but nonlinear, leading to dose-dependent increases in hemoglobin levels. The pharmacodynamic response to epoetin delta appeared to be as expected for an epoetin.

Two open-label studies assessed the effects of hepatic and renal impairment on anidulafungin pharmacokinetics. A single 50-mg dose was administered intravenously to subjects with varying degrees of hepatic or renal insufficiency or with end-stage renal disease; all were matched to normal healthy controls. Anidulafungin was well tolerated. AUC, CL, C(max), t(max), t(1/2), and V(ss) between renally impaired subjects and controls were not significantly different (P>.05), and no measurable amounts of drug were found in dialysate. The same pharmacokinetic parameters were also not affected (P>.05) by mild or moderate hepatic insufficiency, with respective mean AUCs of 50.6 +/- 11.7 microg x h/mL and 68.6 +/- 14.5 microg x h/mL, compared to 70.0 +/- 13.4 microg x h/mL in controls. Statistically significant decreases (P<05) of AUC (33% change) and C(max) (36% change) in severely hepatically impaired subjects compared to controls--most likely secondary to ascites and edema--were not clinically relevant. Anidulafungin can be safely administered to patients with any degree of hepatic or renal impairment without dosage adjustment and without regard to hemodialysis schedules.

The safety and pharmacokinetics of anidulafungin coadministered with tacrolimus were investigated using a single-sequence, open-label design. Healthy volunteers received 5 mg tacrolimus orally on days 1 and 13 of the study. Anidulafungin (200 mg) was administered intravenously on day 4, followed by 100-mg doses on days 5 through 13. Key pharmacokinetic parameters, including C(max), AUC, t((1/2)), CL, and V(ss), were derived from concentration-time data. The 90% confidence intervals (CIs) of the ratios of mean pharmacokinetic parameters of anidulafungin plus tacrolimus to each drug alone were well within the 80% to 125% bioequivalence range, indicating no pharmacokinetic interaction. This ratio was 101.6 (90% CI: 92.77-111.22) for tacrolimus AUC(0-infinity) and 107.2 (90% CI: 105.1-109.4) for anidulafungin AUC(ss). The 2 drugs were well tolerated, and no drug-related serious adverse events were reported. Because of its lack of pharmacokinetic interaction with key immunosuppressive agents, anidulafungin is an important option for the prevention and treatment of invasive fungal infections in transplant recipients.