Peramivir pharmacokinetics in a patient receiving continuous veno-venous hemodiafiltration during the 2009 H1N1 influenza A pandemic.

OBJECTIVE: Peramivir is a neuraminidase inhibitor having activity against various influenza A and B subtypes. The main route of elimination is the kidney and a dose reduction is justified for multiple-day therapy when the creatinine clearance is < 50 mL/min. Before the 2009 influenza pandemic, dosing guidelines did not exist for patients receiving continuous renal replacement therapy (CRRT). This case report provides data on the dialysis membrane saturation coefficient (SA) and pharmacokinetic parameters of peramivir in a 29-year-old female receiving continuous veno-venous hemodiafiltration (CVVHDF), a mode of CRRT. METHODS: Plasma and effluent samples were collected to calculate the saturation coefficient, plasma half-life, maximum and minimum plasma concentrations, and area under the plasma drug concentration-time curve (AUC) for peramivir. CVVHDF was performed using a Prisma pump and an AN69 filter. During peramivir sampling, the dialysate flow rate was 16.7 mL/min. The mean total ultrafiltrate produced was 14.2 mL/min. To calculate a saturation coefficient (SA), simultaneous sampling of blood and effluent was performed. Pre- and post-filter as well as effluent samples were obtained 4.5 and 8.5 hours following the 3rd dose of 480 mg. Plasma concentrations were also obtained at several time points and the AUC estimated from 0 to 24 hours (AUC0-24). RESULTS: The maximum plasma concentration (C30min) was 19,477 ng/mL, the minimum plasma concentration (Cmin) 2,750 ng/mL, and AUC0-24 196,166 ng x h/mL. The estimated plasma half-life was 8.2 hours with a log-linear decrease over the 24-hour period suggesting significant extracorporeal clearance. The calculated SA was 0.98, similar to an estimated SA of 1. CONCLUSION: Peramivir is readily cleared by CVVHDF having a calculated SA close to 1. The maximum and minimum plasma concentrations, AUC0-24, and plasma half-life was similar to those previously reported. These data will be useful in determining appropriate peramivir dosing regimens for severely ill influenza patients with acute renal impairment managed by CVVHDF.

Early clinical observations in prospectively followed patients with fungal meningitis related to contaminated epidural steroid injections.

UNLABELLED: Chinese translation BACKGROUND: Administration of epidural steroid injections (ESIs) with contaminated methylprednisolone resulted in an outbreak of fungal meningitis in many locations in the United States. OBJECTIVE: To characterize early clinical findings and initial response to treatment. DESIGN: Case series with standardized observation studied from 4 October to 31 October 2012. SETTING: An 800-bed hospital in Virginia. PATIENTS: 172 patients who presented to the hospital with exposure to contaminated ESI. INTERVENTION: Standardized approach to screening, case definition, treatment, and data collection. MEASUREMENTS: Clinical findings, cerebrospinal fluid (CSF) values, magnetic resonance imaging (MRI), serum and CSF voriconazole concentrations, and clinician assessment of response to therapy. RESULTS: Of 172 patients presenting to the hospital who had had ESI, 131 had lumbar puncture because of symptoms or signs consistent with central nervous system disease. Twenty-five (19%) had neutrophilic meningitis. All were started on voriconazole therapy alone. Three patients developed stroke during treatment. Ten patients had arachnoiditis, another had an epidural abscess, and 9 had urine retention. Fifteen continued to receive voriconazole, and 10 were switched to amphotericin B. Cerebrospinal fluid leukocyte counts began to decrease by day 13 of treatment. Findings on MRI included ventriculitis, leptomeningeal enhancement, infarction, hemorrhage, and arachnoiditis. Serum voriconazole levels varied, and CSF concentrations of voriconazole were approximately 50% those of serum. Exserohilum rostratum and Cladosporium species have been cultured. LIMITATIONS: This is an observational study of an evolving outbreak. Not all exposed patients presented for evaluation. Follow-up is too short to determine final outcomes. CONCLUSION: Meningitis after receipt of contaminated ESI has been diagnosed in many exposed patients presenting to 1 hospital. Most patients have improved on receipt of empirical voriconazole therapy. The full natural history and long-term sequelae of this infection are currently unknown. PRIMARY FUNDING SOURCE: None.

A customized scaffolds approach for the detection and phasing of complex variants by next-generation sequencing.

Next-generation sequencing (NGS) is widely used in genetic testing for the highly sensitive detection of single nucleotide changes and small insertions or deletions. However, detection and phasing of structural variants, especially in repetitive or homologous regions, can be problematic due to uneven read coverage or genome reference bias, resulting in false calls. To circumvent this challenge, a computational approach utilizing customized scaffolds as supplementary reference sequences for read alignment was developed, and its effectiveness demonstrated with two CBS gene variants: NM_000071.2:c.833T>C and NM_000071.2:c.[833T>C; 844_845ins68]. Variant c.833T>C is a known causative mutation for homocystinuria, but is not pathogenic when in cis with the insertion, c.844_845ins68, because of alternative splicing. Using simulated reads, the custom scaffolds method resolved all possible combinations with 100% accuracy and, based on > 60,000 clinical specimens, exceeded the performance of current approaches that only align reads to GRCh37/hg19 for the detection of c.833T>C alone or in cis with c.844_845ins68. Furthermore, analysis of two 1000 Genomes Project trios revealed that the c.[833T>C; 844_845ins68] complex variant had previously been undetected in these datasets, likely due to the alignment method used. This approach can be configured for existing workflows to detect other challenging and potentially underrepresented variants, thereby augmenting accurate variant calling in clinical NGS testing.

FXR1 splicing is important for muscle development and biomolecular condensates in muscle cells.

Fragile-X mental retardation autosomal homologue-1 (FXR1) is a muscle-enriched RNA-binding protein. FXR1 depletion is perinatally lethal in mice, Xenopus, and zebrafish; however, the mechanisms driving these phenotypes remain unclear. The FXR1 gene undergoes alternative splicing, producing multiple protein isoforms and mis-splicing has been implicated in disease. Furthermore, mutations that cause frameshifts in muscle-specific isoforms result in congenital multi-minicore myopathy. We observed that FXR1 alternative splicing is pronounced in the serine- and arginine-rich intrinsically disordered domain; these domains are known to promote biomolecular condensation. Here, we show that tissue-specific splicing of fxr1 is required for Xenopus development and alters the disordered domain of FXR1. FXR1 isoforms vary in the formation of RNA-dependent biomolecular condensates in cells and in vitro. This work shows that regulation of tissue-specific splicing can influence FXR1 condensates in muscle development and how mis-splicing promotes disease.

An electrophoretic capture method for efficient recovery of rare sequences from heterogeneous DNA.

It is difficult to isolate rare, PCR-quality DNA from specimens containing large quantities of nonspecific DNA from multiple sources (heterogeneous DNA). Extracting human DNA from stool for colorectal cancer (CRC) screening tests exemplifies this technically challenging sample preparation problem. The stool matrix is complex, the DNA composition heterogeneous, and CRC-associated mutated DNA is rare. This report describes a novel solid phase DNA sequence-specific hybrid capture sample preparation method: the reversible electrophoretic capture affinity protocol (RECAP). We show that RECAP, compared with other methods, is capable of extracting linearly increasing amounts of human DNA from increasing amounts of total stool DNA in a manner that avoids co-purifying PCR inhibitors. RECAP thereby increases the yield of rare mutated DNA molecules and thus increases the detection sensitivity for CRC-associated mutations.

mRNA structure determines specificity of a polyQ-driven phase separation.

RNA promotes liquid-liquid phase separation (LLPS) to build membraneless compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here, we report that secondary structure allows messenger RNAs (mRNAs) to self-associate and determines whether an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.

Lessons from Yeast on How to Avoid Stress Eating.

Cellular survival during periods of acute environmental stress is essential for single-celled organisms. Reporting in Nature Cell Biology, Saad et al. (2017) identify reversible aggregation of the metabolic enzyme pyruvate kinase under environmental stress and propose a method to protect and store proteins for immediate use post-stress.

Membrane curvature directs the localization of Cdc42p to novel foci required for cell-cell fusion.

Cell fusion is ubiquitous in eukaryotic fertilization and development. The highly conserved Rho-GTPase Cdc42p promotes yeast fusion through interaction with Fus2p, a pheromone-induced amphiphysin-like protein. We show that in prezygotes, Cdc42p forms a novel Fus2p-dependent focus at the center of the zone of cell fusion (ZCF) and remains associated with remnant cell walls after initial fusion. At the ZCF and during fusion, Cdc42p and Fus2p colocalized. In contrast, in shmoos, both proteins were near the cortex but spatially separate. Cdc42p focus formation depends on ZCF membrane curvature: mutant analysis showed that Cdc42p localization is negatively affected by shmoo-like positive ZCF curvature, consistent with the flattening of the ZCF during fusion. BAR-domain proteins such as the fusion proteins Fus2p and Rvs161p are known to recognize membrane curvature. We find that mutations that disrupt binding of the Fus2p/Rvs161p heterodimer to membranes affect Cdc42p ZCF localization. We propose that Fus2p localizes Cdc42p to the flat ZCF to promote cell wall degradation.

Kel1p Mediates Yeast Cell Fusion Through a Fus2p- and Cdc42p-Dependent Mechanism.

Cell fusion is ubiquitous among eukaryotes. Although little is known about the molecular mechanism, several proteins required for cell fusion in the yeast Saccharomyces cerevisiae have been identified. Fus2p, a key regulator of cell fusion, localizes to the shmoo tip in a highly regulated manner. C-terminal truncations of Fus2p cause mislocalization and fusion defects, which are suppressed by overexpression of Kel1p, a kelch-domain protein of unknown function previously implicated in cell fusion. We hypothesize that Fus2p mislocalization is caused by auto-inhibition, which is alleviated by Kel1p overexpression. Previous work showed that Fus2p localization is mediated by both Fus1p- and actin-dependent pathways. We show that the C-terminal mutations mainly affect the actin-dependent pathway. Suppression of the Fus2p localization defect by Kel1p is dependent upon Fus1p, showing that suppression does not bypass the normal pathway. Kel1p and a homolog, Kel2p, are required for efficient Fus2p localization, acting through the actin-dependent pathway. Although Kel1p overexpression can weakly suppress the mating defect of a FUS2 deletion, the magnitude of suppression is allele specific. Therefore, Kel1p augments, but does not bypass, Fus2p function. Fus2p mediates cell fusion by binding activated Cdc42p Although Kel1p overexpression suppresses a Cdc42p mutant that is defective for Fus2p binding, cell fusion remains dependent upon Fus2p These data suggest that Fus2p, Cdc42p, and Kel1p form a ternary complex, which is stabilized by Kel1p Supporting this hypothesis, Kel1p interacts with two domains of Fus2p, partially dependent on Cdc42p We conclude that Kel1p enhances the activity of Fus2p/Cdc42p in cell fusion.

An Amphiphysin-Like Domain in Fus2p Is Required for Rvs161p Interaction and Cortical Localization.

Cell-cell fusion fulfils essential roles in fertilization, development and tissue repair. In the budding yeast, Saccharomyces cerevisiae, fusion between two haploid cells of opposite mating type generates the diploid zygote. Fus2p is a pheromone-induced protein that regulates cell wall removal during mating. Fus2p shuttles from the nucleus to localize at the shmoo tip, bound to Rvs161p, an amphiphysin. However, Rvs161p independently binds a second amphiphysin, Rvs167p, playing an essential role in endocytosis. To understand the basis of the Fus2p-Rvs161p interaction, we analyzed Fus2p structural domains. A previously described N-terminal domain (NTD) is necessary and sufficient to regulate nuclear/cytoplasmic trafficking of Fus2p. The Dbl homology domain (DBH) binds GTP-bound Cdc42p; binding is required for cell fusion, but not localization. We identified an approximately 200 amino acid region of Fus2p that is both necessary and sufficient for Rvs161p binding. The Rvs161p binding domain (RBD) contains three predicted alpha-helices; structural modeling suggests that the RBD adopts an amphiphysin-like structure. The RBD contains a 13-amino-acid region, conserved with Rvs161p and other amphiphysins, which is essential for binding. Mutations in the RBD, predicted to affect membrane binding, abolish cell fusion without affecting Rvs161p binding. We propose that Fus2p/Rvs161p form a novel heterodimeric amphiphysin required for cell fusion. Rvs161p binding is required but not sufficient for Fus2p localization. Mutations in the C-terminal domain (CTD) of Fus2p block localization, but not Rvs161p binding, causing a significant defect in cell fusion. We conclude that the Fus2p CTD mediates an additional, Rvs161p-independent interaction at the shmoo tip.