Reprogramming the maternal zebrafish genome after fertilization to match the paternal methylation pattern.

Early vertebrate embryos must achieve totipotency and prepare for zygotic genome activation (ZGA). To understand this process, we determined the DNA methylation (DNAme) profiles of zebrafish gametes, embryos at different stages, and somatic muscle and compared them to gene activity and histone modifications. Sperm chromatin patterns are virtually identical to those at ZGA. Unexpectedly, the DNA of many oocyte genes important for germline functions (i.e., piwil1) or early development (i.e., hox genes) is methylated, but the loci are demethylated during zygotic cleavage stages to precisely the state observed in sperm, even in parthenogenetic embryos lacking a replicating paternal genome. Furthermore, this cohort constitutes the genes and loci that acquire DNAme during development (i.e., ZGA to muscle). Finally, DNA methyltransferase inhibition experiments suggest that DNAme silences particular gene and chromatin cohorts at ZGA, preventing their precocious expression. Thus, zebrafish achieve a totipotent chromatin state at ZGA through paternal genome competency and maternal genome DNAme reprogramming.

Performance of Bayesian Area Under the Concentration-Time Curve-Based Pharmacokinetic Dosing Based on a One-Compartment Model and Trough-Only Monitoring for Vancomycin.

A novel Bayesian method was developed to interpret serum vancomycin concentrations (SVCs) following the administration of one or more vancomycin doses with potential varying doses and intervals based on superposition principles. The method was evaluated using retrospective data from 442 subjects from three hospitals. The patients were required to receive vancomycin for more than 3 days, have stable renal function (fluctuation in serum creatinine of ≤0.3 mg/dL), and have at least 2 trough concentrations reported. Pharmacokinetic parameters were predicted using the first SVC, and the fitted parameters were then used to predict subsequent SVCs. Using only covariate-adjusted population prior estimates, the first two SVC prediction errors were 47.3 to 54.7% for the scaled mean absolute error (sMAE) and 62.1 to 67.8% for the scaled root mean squared error (sRMSE). "Scaled" refers to the division of the MAE or RMSE by the mean value. The Bayesian method had minimal errors for the first SVC (by design), and for the second SVC, the sMAE was 8.95%, and the sRMSE was 36.5%. The predictive performance of the Bayesian method did degrade with subsequent SVCs, which we attributed to time-dependent pharmacokinetics. The 24-h area under the concentration-time curve (AUC) was determined from simulated concentrations before and after the first SVC was reported. Prior to the first SVC, 170 (38.4%) patients had a 24-h AUC of <400 mg · h/L, 186 (42.1%) had a 24-h AUC within the target range, and 86 (19.5%) had a 24-h AUC of >600 mg · h/L. After the first SVC was reported, 322 (72.9%) had a 24-h AUC within the target range, 68 (15.4%) had low values, and 52 (11.8%) had high values based on the model simulation. Target attainments were 38% before the first SVC and 73% after the first SVC. The hospitals had no policies or procedures in place for targeting 24-h AUCs, although the trough target was typically 13 to 17 mg/L. Our data provide evidence of time-dependent pharmacokinetics, which will require regular therapeutic drug monitoring regardless of the method used to interpret SVCs.

A preliminary study of the plasma concentrations of orally administered fluconazole in alpacas (Vicugna pacos).

Alpacas residing in the region endemic for Coccidioides spp. are susceptible to serious, disseminated coccidioidomycosis that may result in death. There is currently no oral antifungal dose recommendation for this species. We used a steady-state study design to assess the pharmacokinetics of oral generic fluconazole in alpacas dosed q 24 h for 14 days. Cohorts of 2-3 animals received fluconazole from 6 to 15 mg/kg/day, and pharmacokinetic analysis was performed after each group of animals in order to make dose adjustments for the next group. The final three animals were used as confirmation of our dose recommendation. The median Tmax was 7 h, and the median Cmax was 1.25 µg/ml per mg/kg dose. The mean dose-normalized 24-h AUC was 41.7 µg h/ml per mg/kg dose (CV = 72%). Based on these results, we recommend alpacas receive a starting dose of oral fluconazole at 10-15 mg/kg/day based on the fluconazole AUC in humans (313-625 µg h/ml). Testing to ascertain putative therapeutic plasma concentrations and monitoring of serum transaminases should be performed.

Creatinine Assessment in Non-Steady-State Conditions: A Critical Review.

OBJECTIVES: To discuss methods for the assessment of creatinine clearance (Clcr) when serum creatinine (SCr) is not at steady state in order to estimate kidney function and apply the estimate to kidney function staging for clinical assessment or drug dosing. DATA SOURCES: A PubMed search was conducted from 1976 to mid-January 2021 with other articles identified through review of bibliographies of retrieved articles and citations in Scopus. STUDY SELECTION AND DATA EXTRACTION: Articles assessing Clcr under non-steady-state conditions and studies evaluating predictive equations were selected. DATA SYNTHESIS: When SCr is systematically changing (ie, trending up or down), kinetic methods to estimate Clcr are appropriate. Estimates from kinetic methods should be individual based and not indexed to body surface area, and careful monitoring is required to confirm predictions as the situation evolves. Standard methods intended for steady-state conditions should not be used to estimate Clcr in patients with unstable SCr. RELEVANCE TO PATIENT CARE AND CLINICAL PRACTICE: Creatinine continues to be a monitoring parameter of choice and is an important variable in all the commonly used equations for estimating Clcr and most important for estimating glomerular filtration rate. However, standard methods of estimating Clcr for medication dosing are not accurate under non-steady-state conditions. CONCLUSION: The methods for kinetic clearance estimation and standards methods for clearance estimation, such as the Cockcroft-Gault equation, are mutually exclusive. There are no benefits of using the kinetic method in patients with stable SCr concentrations, and standard equations are not appropriate with unstable SCr concentrations.

A Critical Evaluation of Newer ß-Lactam Antibiotics for Treatment of Pseudomonas aeruginosa Infections.

OBJECTIVE: This article critically evaluates common Pseudomonas aeruginosa resistance mechanisms and the properties newer ß-lactam antimicrobials possess to evade these mechanisms. DATA SOURCES: An extensive PubMed, Google Scholar, and ClinicalTrials.gov search was conducted (January 1995 to July 2020) to identify relevant literature on epidemiology, resistance mechanisms, antipseudomonal agents, newer ß-lactam agents, and clinical data available pertaining to P aeruginosa. STUDY SELECTION AND DATA EXTRACTION: Relevant published articles and package inserts were reviewed for inclusion. DATA SYNTHESIS: Therapeutic options to treat P aeruginosa infections are limited because of its intrinsic and acquired resistance mechanisms. The goal was to identify advances with newer ß-lactams and characterize improvements in therapeutic potential for P aeruginosa infections. RELEVANCE TO PATIENT CARE AND CLINICAL PRACTICE: Multidrug-resistant (MDR) P aeruginosa isolates are increasingly encountered from a variety of infections. This review highlights potential activity gains of newer ß-lactam antibacterial drugs and the current clinical data to support their use. Pharmacists will be asked to recommend or evaluate the use of these agents and need to be aware of information specific to P aeruginosa, which differs from experience derived from Enterobacterales infections. CONCLUSIONS: Newer agents, including ceftazidime-avibactam, ceftolozane-tazobactam, imipenem-relebactam, and cefiderocol, are useful for the treatment of MDR P aeruginosa infections. These agents offer improved efficacy and less toxicity compared with aminoglycosides and polymyxins and can be used for pathogens that are resistant to first-line antipseudomonal ß-lactams. Selection of one agent over another should consider availability, turnaround of susceptibility testing, and product cost. Efficacy data specific for pseudomonal infections are limited, and there are no direct comparisons between the newer agents.

Assessment of Kidney Function in Patients With Extreme Obesity: A Narrative Review.

OBJECTIVES: To discuss the evidence and caveats associated with estimated and measured creatinine clearance (eClCr and mClCr) and glomerular filtration rate (eGFR and mGFR) assessments of kidney function in patients with more extreme forms of obesity. DATA SOURCES: PubMed (1976 to mid-May 2020) was used, with bibliographies of retrieved articles searched for additional articles. STUDY SELECTION AND DATA EXTRACTION: Articles using gold standard mGFR to evaluate eClCr, mClCr, and eGFR assessments of kidney function in patients with more extreme forms of obesity were included. DATA SYNTHESIS: The overestimation of GFR by mClCr is well established, but mClCr is an alternative to mGFR assessments for determining medication dosing in patients with extremes of body size or muscle mass, or in patients receiving narrow therapeutic index medications when eGFR is likely to be inaccurate. The vast majority of studies comparing eGFR assessments with gold standard indicators of kidney function were attempts to validate eGFR equations for diagnosing and staging chronic kidney disease (CKD). RELEVANCE TO PATIENT CARE AND CLINICAL PRACTICE: For dosing medications in patients with stable kidney function and extreme obesity, a deindexed 4-variable Modification of Diet in Renal Disease or CKD Epidemiology Collaboration equation is an alternative to Cockcroft-Gault. Consistent use of the same equation by provider and between providers within any given setting is of paramount importance. CONCLUSIONS: In patients with extreme obesity and stable kidney function, eClCr or eGFR using deindexed values provides estimates of function for dosing adjustments of medications with elimination by the kidneys, but more research is needed with respect to the best size descriptor to use with estimating equations.

Endotracheal tube mucus as a source of airway mucus for rheological study.

Muco-obstructive lung diseases (MOLDs), like cystic fibrosis and chronic obstructive pulmonary disease, affect a spectrum of subjects globally. In MOLDs, the airway mucus becomes hyperconcentrated, increasing osmotic and viscoelastic moduli and impairing mucus clearance. MOLD research requires relevant sources of healthy airway mucus for experimental manipulation and analysis. Mucus collected from endotracheal tubes (ETT) may represent such a source with benefits, e.g., in vivo production, over canonical sample types such as sputum or human bronchial epithelial (HBE) mucus. Ionic and biochemical compositions of ETT mucus from healthy human subjects were characterized and a stock of pooled ETT samples generated. Pooled ETT mucus exhibited concentration-dependent rheologic properties that agreed across spatial scales with reported individual ETT samples and HBE mucus. We suggest that the practical benefits compared with other sample types make ETT mucus potentially useful for MOLD research.

Current Concepts and Future Directions in the Pharmacology and Treatment of Coccidioidomycosis.

Coccidioidomycosis remains a significant clinical problem with substantial morbidity and mortality. The vast majority of infections are asymptomatic and the need for early primary therapy remains controversial. The use of triazole antifungals has improved tolerability of therapy but concerns about acute and long-term toxicities among available agents limit their use. In addition, recent findings of decreased in vitro fluconazole susceptibility to as many as 37% of Coccidioides spp. isolates raises concerns regarding optimal therapy for these infections as fluconazole is commonly used for therapy including central nervous system disease. Thus, new agents from novel antifungal classes are currently in preclinical and clinical development aimed at reducing toxicity and improving outcomes of these serious infections.

Cefazolin Monotherapy Versus Cefazolin Plus Aminoglycosides for Antimicrobial Prophylaxis of Type III Open Fractures.

BACKGROUND: There are conflicting recommendations between organizations regarding aminoglycoside use for the prophylaxis of type III open fractures. STUDY QUESTION: To compare cefazolin monotherapy versus cefazolin plus aminoglycoside therapy for prophylaxis of type III open fractures in trauma patients. STUDY DESIGN: This was a multicenter, retrospective, cohort study conducted in 3 academic medical centers in the United States. Consecutive adult trauma patients with type III open fractures between January 2014 and September 2016 were included. Patients were divided into 2 groups: (1) cefazolin monotherapy versus (2) cefazolin plus aminoglycoside. MEASURES AND OUTCOMES: The primary outcome measure was the occurrence of infection at the open fracture site. The secondary outcome measure was the occurrence of acute kidney injury. RESULTS: There were 134 patients included in the study cohort. Of these, 39 received cefazolin monotherapy and 95 received cefazolin plus aminoglycoside. Overall, the mean age was 39 ± 15 years, 105 (78%) were male, and the most common fracture location was tibia/fibula (n = 74, 56%). Infection at the open fracture site occurred in 6 of 39 patients (15%) in the cefazolin monotherapy group and 15 of 95 patients (16%) in the cefazolin plus aminoglycoside group (P = 1.000). Acute kidney injury occurred in 0 of 39 (0%) in the cefazolin monotherapy group and 1 of 95 (1%) in the cefazolin plus aminoglycoside group (P = 1.000). CONCLUSIONS: Cefazolin monotherapy may be appropriate for antimicrobial prophylaxis of type III open fractures in trauma patients.

Radical new paradigm for heme degradation in Escherichia coli O157:H7.

All of the heme-degrading enzymes that have been characterized to date require molecular oxygen as a cosubstrate. Escherichia coli O157:H7 has been shown to express heme uptake and transport proteins, as well as use heme as an iron source. This enteric pathogen colonizes the anaerobic space of the lower intestine in mammals, yet no mechanism for anaerobic heme degradation has been reported. Herein we provide evidence for an oxygen-independent heme-degradation pathway. Specifically, we demonstrate that ChuW is a radical S-adenosylmethionine methyltransferase that catalyzes a radical-mediated mechanism facilitating iron liberation and the production of the tetrapyrrole product we termed "anaerobilin." We further demonstrate that anaerobilin can be used as a substrate by ChuY, an enzyme that is coexpressed with ChuW in vivo along with the heme uptake machinery. Our findings are discussed in terms of the competitive advantage this system provides for enteric bacteria, particularly those that inhabit an anaerobic niche in the intestines.

Sialic acid-binding immunoglobulin-like lectin 8 (Siglec-8) is an activating receptor mediating ß2-integrin-dependent function in human eosinophils.

BACKGROUND: Siglec-8 is a CD33 subfamily cell-surface receptor selectively expressed on human eosinophils. After cytokine priming, Siglec-8 mAb or glycan ligand binding causes eosinophil apoptosis associated with reactive oxygen species (ROS) production. Most CD33-related Siglecs function as inhibitory receptors, but the ability of Siglec-8 to stimulate eosinophil ROS production and apoptosis suggests that Siglec-8 might instead function as an activating receptor. OBJECTIVE: We sought to determine the role of IL-5 priming and identify the signaling molecules involved in Siglec-8 function for human eosinophils. METHODS: We used an mAb and/or a multimeric synthetic sulfated sialoglycan ligand recognizing Siglec-8 in combination with integrin blocking antibodies, pharmacologic inhibitors, phosphoproteomics, and Western blot analysis to define the necessity of various proteins involved in Siglec-8 function for human eosinophils. RESULTS: Cytokine priming was required to elicit the unanticipated finding that Siglec-8 engagement promotes rapid ß2-integrin-dependent eosinophil adhesion. Also novel was the finding that this adhesion was necessary for subsequent ROS production and apoptosis. Siglec-8-mediated ROS was generated through reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation because pretreatment of eosinophils with catalase (an extracellular superoxide scavenger) or NSC 23766 (a Rac GTPase inhibitor) completely inhibited Siglec-8-mediated eosinophil apoptosis. Finally, engagement of Siglec-8 on IL-5-primed eosinophils resulted in increased phosphorylation of Akt, p38, and c-Jun N-terminal kinase 1 that was also ß2-integrin dependent; pharmacologic inhibition of these kinases completely prevented Siglec-8-mediated eosinophil apoptosis. CONCLUSIONS: These data demonstrate that Siglec-8 functions uniquely as an activating receptor on IL-5-primed eosinophils through a novel pathway involving regulation of ß2-integrin-dependent adhesion, NADPH oxidase, and a subset of protein kinases.

Impact of requiring re-authorization of restricted antibiotics on day 3 of therapy.

Background: Pre-authorization of restricted antibiotics is a core component of an antibiotic stewardship programme (ASP). On day 3, information about culture results and clinical status is typically available. Our objective was to compare an ASP that requires initial authorization alone with one requiring initial authorization and re-authorization on day 3 of therapy. Methods: A single-centre, retrospective, before and after study was conducted. Randomly selected adults were eligible if receiving a restricted antibiotic for ≥3 days during April to June in 2012 (pre-intervention) and during the same months in 2013 (post-intervention). The target sample size was 166 patients. The intervention required re-authorization of restricted antibiotics that were continuing on day 3. The days of therapy of restricted antibiotic(s), length of hospital stay (LOS) and hospital mortality were compared between pre- and post-intervention periods. Results: The ASP intervention was associated with a decrease in median days of therapy from 5 (4-9) to 4 (3-5) days (P < 0.001) for all restricted agents, from 5 (3-6) to 3 (3-5) days for broad-spectrum Gram-negative agents (P < 0.001) and from 6.5 (6-7) to 3 (3-4.5) days for oral vancomycin. The proportion of subjects receiving restricted agents for >4 days decreased from 57.8% to 30.1% (P < 0.001). LOS decreased from 8 (5-17) to 6 (5-9) days (P = 0.005) without a significant change in hospital mortality. Conclusions: Requiring re-authorization of restricted antibiotics on day 3 of therapy in addition to initial authorization was associated with reduction in overall consumption of restricted antibiotics and LOS without adversely affecting hospital mortality.

Potential role for snoRNAs in PKR activation during metabolic stress.

Protein kinase RNA-activated (PKR) has long been known to be activated by viral double-stranded RNA (dsRNA) as part of the mammalian immune response. However, in mice PKR is also activated by metabolic stress in the absence of viral infection, and this requires a functional kinase domain, as well as a functional dsRNA-binding domain. The endogenous cellular RNA that potentially leads to PKR activation during metabolic stress is unknown. We investigated this question using mouse embryonic fibroblast cells expressing wild-type PKR (PKRWT) or PKR with a point mutation in each dsRNA-binding motif (PKRRM). Using this system, we identified endogenous RNA that interacts with PKR after induction of metabolic stress by palmitic acid (PA) treatment. Specifically, RIP-Seq analyses showed that the majority of enriched RNAs that interacted with WT PKR (≥twofold, false discovery rate ≤ 5%) were small nucleolar RNAs (snoRNAs). Immunoprecipitation of PKR in extracts of UV-cross-linked cells, followed by RT-qPCR, confirmed that snoRNAs were enriched in PKRWT samples after PA treatment, but not in the PKRRM samples. We also demonstrated that a subset of identified snoRNAs bind and activate PKR in vitro; the presence of a 5'-triphosphate enhanced PKR activity compared with the activity with a 5'-monophosphate, for some, but not all, snoRNAs. Finally, we demonstrated PKR activation in cells upon snoRNA transfection, supporting our hypothesis that endogenous snoRNAs can activate PKR. Our results suggest an unprecedented and unexpected model whereby snoRNAs play a role in the activation of PKR under metabolic stress.

Anaerobic Heme Degradation: ChuY Is an Anaerobilin Reductase That Exhibits Kinetic Cooperativity.

Heme catabolism is an important biochemical process that many bacterial pathogens utilize to acquire iron. However, tetrapyrrole catabolites can be reactive and often require further processing for transport out of the cell or conversion to another useful cofactor. In previous work, we presented in vitro evidence of an anaerobic heme degradation pathway in Escherichia coli O157:H7. Consistent with reactions that have been reported for other radical S-adenosyl-l-methionine methyltransferases, ChuW transfers a methyl group to heme by a radical-mediated mechanism and catalyzes the ß-scission of the porphyrin macrocycle. This facilitates iron release and the production of a new linear tetrapyrrole termed "anaerobilin". In this work, we describe the structure and function of ChuY, an enzyme expressed downstream from chuW within the same heme utilization operon. ChuY is structurally similar to biliverdin reductase and forms a dimeric complex in solution that reduces anaerobilin to the product we have termed anaerorubin. Steady state analysis of ChuY exhibits kinetic cooperativity that is best explained by a random addition mechanism with a kinetically preferred path for initial reduced nicotinamide adenine dinucleotide phosphate binding.

Genome-wide profiling of the C. elegans dsRNAome.

Recent studies hint that endogenous dsRNA plays an unexpected role in cellular signaling. However, a complete understanding of endogenous dsRNA signaling is hindered by an incomplete annotation of dsRNA-producing genes. To identify dsRNAs expressed in Caenorhabditis elegans, we developed a bioinformatics pipeline that identifies dsRNA by detecting clustered RNA editing sites, which are strictly limited to long dsRNA substrates of Adenosine Deaminases that act on RNA (ADAR). We compared two alignment algorithms for mapping both unique and repetitive reads and detected as many as 664 editing-enriched regions (EERs) indicative of dsRNA loci. EERs are visually enriched on the distal arms of autosomes and are predicted to possess strong internal secondary structures as well as sequence complementarity with other EERs, indicative of both intramolecular and intermolecular duplexes. Most EERs were associated with protein-coding genes, with ∼1.7% of all C. elegans mRNAs containing an EER, located primarily in very long introns and in annotated, as well as unannotated, 3' UTRs. In addition to numerous EERs associated with coding genes, we identified a population of prospective noncoding EERs that were distant from protein-coding genes and that had little or no coding potential. Finally, subsets of EERs are differentially expressed during development as well as during starvation and infection with bacterial or fungal pathogens. By combining RNA-seq with freely available bioinformatics tools, our workflow provides an easily accessible approach for the identification of dsRNAs, and more importantly, a catalog of the C. elegans dsRNAome.

Clinical failure with and without empiric atypical bacteria coverage in hospitalized adults with community-acquired pneumonia: a systematic review and meta-analysis.

BACKGROUND: Both typical and atypical bacteria can cause community-acquired pneumonia (CAP); however, the need for empiric atypical coverage remains controversial. Our objective was to evaluate the impact of antibiotic regimens with atypical coverage (a fluoroquinolone or combination of a macrolide/doxycycline with a ß-lactam) to a regimen without atypical antibiotic coverage (ß-lactam monotherapy) on rates of clinical failure (primary endpoint), mortality, bacteriologic failure, and adverse events, (secondary endpoints). METHODS: We searched the PubMed, EMBASE and Cochrane Library databases for relevant RCTs of hospitalized CAP adults. We estimated risk ratios (RRs) with 95% confidence intervals (CIs) using a fixed-effect model, but used a random-effects model if significant heterogeneity (I 2 ) was observed. RESULTS: Five RCTs with a total of 2011 patients were retained. A statistically significant lower clinical failure rate was observed with empiric atypical coverage (RR, 0.851 [95% CI, 0.732-0.99; P = 0.037]; I 2  = 0%). The secondary outcomes did not differ between the two study groups: mortality (RR = 0.549 [95% CI, 0.259-1.165, P = 0.118], I 2  = 61.434%) bacteriologic failure (RR = 0.816 [95% CI, 0.523-1.272, P = 0.369], I 2  = 0%), diarrhea (RR = 0.746 [95% CI, 0.311-1.790, P = 0.512], I 2  = 65.048%), and adverse events requiring antibiotic discontinuation (RR = 0.83 [95% CI, 0.542-1.270, P = 0.39], I 2  = 0%). CONCLUSIONS: Empiric atypical coverage was associated with a significant reduction in clinical failure in hospitalized adults with CAP. Reduction in mortality, bacterial failure, diarrhea, and discontinuation due to adverse effects were not significantly different between groups, but all estimates favored atypical coverage. Our findings provide support for the current guidelines recommendations to include empiric atypical coverage.

Adding In Silico Assessment of Potential Splice Aberration to the Integrated Evaluation of BRCA Gene Unclassified Variants.

Clinical mutation screening of the cancer susceptibility genes BRCA1 and BRCA2 generates many unclassified variants (UVs). Most of these UVs are either rare missense substitutions or nucleotide substitutions near the splice junctions of the protein coding exons. Previously, we developed a quantitative method for evaluation of BRCA gene UVs-the "integrated evaluation"-that combines a sequence analysis-based prior probability of pathogenicity with patient and/or tumor observational data to arrive at a posterior probability of pathogenicity. One limitation of the sequence analysis-based prior has been that it evaluates UVs from the perspective of missense substitution severity but not probability to disrupt normal mRNA splicing. Here, we calibrated output from the splice-site fitness program MaxEntScan to generate spliceogenicity-based prior probabilities of pathogenicity for BRCA gene variants; these range from 0.97 for variants with high probability to damage a donor or acceptor to 0.02 for exonic variants that do not impact a splice junction and are unlikely to create a de novo donor. We created a database http://priors.hci.utah.edu/PRIORS/ that provides the combined missense substitution severity and spliceogenicity-based probability of pathogenicity for BRCA gene single-nucleotide substitutions. We also updated the BRCA gene Ex-UV LOVD, available at http://hci-exlovd.hci.utah.edu, with 77 re-evaluable variants.