Group II introns are mobile genetic elements that perform both self-splicing and intron mobility reactions. These ribozymes are comprised of a catalytic RNA core that binds to an intron-encoded protein (IEP) to form a ribonucleoprotein (RNP) complex. Splicing proceeds through two competing reactions: hydrolysis or branching. Group IIC intron ribozymes have a minimal RNA architecture, and splice almost exclusively through hydrolysis in ribozyme reactions. Addition of the IEP allows the splicing reaction to form branched lariat RNPs capable of intron mobility. Here we examine ribozyme splicing, IEP-dependent splicing, and mobility reactions of a group IIC intron from the thermophilic bacterium Thermoanerobacter italicus (Ta.it.I1). We show that Ta.it.I1 is highly active for ribozyme activity, forming linear hydrolytic intron products. Addition of purified IEP switches activity to the canonical lariat forming splicing reaction. We demonstrate that the Ta.it.I1 group IIC intron coordinates the progression of the forward splicing reaction through a –' interaction between intron domains II and VI. We further show that branched splicing is supported in the absence of the IEP when the –' interaction is mutated. We also investigated the regulation of the two steps of reverse splicing during intron mobility into DNA substrates. Using a fluorescent mobility assay that simultaneously visualizes all steps of intron integration into DNA, we show that completion of reverse splicing is tightly coupled to cDNA synthesis regardless of mutation of the –' interaction.

Hypoxia is a hallmark of solid cancers, supporting proliferation, angiogenesis, and escape from apoptosis. There is still limited understanding of how cancer cells adapt to hypoxic conditions and survive. We analyzed transcriptome changes of human lung and breast cancer cells under chronic hypoxia. Hypoxia induced highly concordant changes in transcript abundance, but divergent splicing responses, underlining the cell type-specificity of alternative splicing programs. While RNA-binding proteins were predominantly reduced, hypoxia specifically induced muscleblind-like protein 2 (MBNL2). Strikingly, MBNL2 induction was critical for hypoxia adaptation by controlling the transcript abundance of hypoxia response genes, such as vascular endothelial growth factor A (VEGFA). MBNL2 depletion reduced the proliferation and migration of cancer cells, demonstrating an important role of MBNL2 as cancer driver. Hypoxia control is specific for MBNL2 and not shared by its paralog MBNL1. Thus, our study revealed MBNL2 as central mediator of cancer cell responses to hypoxia, regulating the expression and alternative splicing of hypoxia-induced genes.

Many noncoding RNAs are known to play a role in the cell directly linked to their structure. Structure prediction based on the sole sequence is, however, a challenging task. On the other hand, thanks to the low cost of sequencing technologies, a very large number of homologous sequences are becoming available for many RNA families. In the protein community, the idea of exploiting the covariance of mutations within a family to predict the protein structure using the direct-coupling-analysis (DCA) method has emerged in the last decade. The application of DCA to RNA systems has been limited so far. We here perform an assessment of the DCA method on 17 riboswitch families, comparing it with the commonly used mutual information analysis and with state-of-the-art R-scape covariance method. We also compare different flavors of DCA, including mean-field, pseudolikelihood, and a proposed stochastic procedure (Boltzmann learning) for solving exactly the DCA inverse problem. Boltzmann learning outperforms the other methods in predicting contacts observed in high-resolution crystal structures.

Long noncoding RNA molecules (lncRNAs) are estimated to account for the majority of eukaryotic genomic transcripts, and have been associated with multiple diseases in humans. However, our understanding of their structure–function relationships is scarce, with structural evidence coming mostly from indirect biochemical approaches or computational predictions. Here we describe direct visualization of the lncRNA HOTAIR (HOx Transcript AntIsense RNA) using atomic force microscopy (AFM) in nucleus-like conditions at 37°. Our observations reveal that HOTAIR has a discernible, although flexible, shape. Fast AFM scanning enabled the quantification of the motion of HOTAIR, and provided visual evidence of physical interactions with genomic DNA segments. Our report provides a biologically plausible description of the anatomy and intrinsic properties of HOTAIR, and presents a framework for studying the structural biology of lncRNAs.

Functions of eukaryotic mRNAs are characterized by intramolecular interactions between their ends. We have addressed the question whether 5' and 3' ends meet by diffusion-controlled encounter "through solution" or by a mechanism involving the RNA backbone. For this purpose, we used a translation system derived from Drosophila embryos that displays two types of 5'–3' interactions: Cap-dependent translation initiation is stimulated by the poly(A) tail and inhibited by Smaug recognition elements (SREs) in the 3' UTR. Chimeric RNAs were made consisting of one RNA molecule carrying a luciferase coding sequence and a second molecule containing SREs and a poly(A) tail; the two were connected via a protein linker. The poly(A) tail stimulated translation of such chimeras even when disruption of the RNA backbone was combined with an inversion of the 5'–3' polarity between the open reading frame and poly(A) segment. Stimulation by the poly(A) tail also decreased with increasing RNA length. Both observations suggest that contacts between the poly(A) tail and the 5' end are established through solution, independently of the RNA backbone. In the same chimeric constructs, SRE-dependent inhibition of translation was also insensitive to disruption of the RNA backbone. Thus, tracking of the backbone is not involved in the repression of cap-dependent initiation. However, SRE-dependent repression was insensitive to mRNA length, suggesting that the contact between the SREs in the 3' UTR and the 5' end of the RNA might be established in a manner that differs from the contact between the poly(A) tail and the cap.

Axonal protein synthesis has been shown to play a role in developmental and regenerative growth, as well as in the maintenance of the axoplasm in a steady state. Recent studies have begun to identify the mRNAs localized in axons, which could be translated locally under different conditions. Despite that by now hundreds or thousands of mRNAs have been shown to be localized into the axonal compartment of cultured neurons in vitro, knowledge of which mRNAs are localized in mature myelinated axons is quite limited. With the purpose of characterizing the transcriptome of mature myelinated motor axons of peripheral nervous systems, we modified the axon microdissection method devised by Koenig, enabling the isolation of the axoplasm RNA to perform RNA-seq analysis. The transcriptome analysis indicates that the number of RNAs detected in mature axons is lower in comparison with in vitro data, depleted of glial markers, and enriched in neuronal markers. The mature myelinated axons are enriched for mRNAs related to cytoskeleton, translation, and oxidative phosphorylation. Moreover, it was possible to define core genes present in axons when comparing our data with transcriptomic data of axons grown in different conditions. This work provides evidence that axon microdissection is a valuable method to obtain genome-wide data from mature and myelinated axons of the peripheral nervous system, and could be especially useful for the study of axonal involvement in neurodegenerative pathologies of motor neurons such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophies (SMA).

Endogenous viral elements (EVEs) are found in many eukaryotic genomes. Despite considerable knowledge about genomic elements such as transposons (TEs) and retroviruses, we still lack information about nonretroviral EVEs. Aedes aegypti mosquitoes have a highly repetitive genome that is covered with EVEs. Here, we identified 129 nonretroviral EVEs in the AaegL5 version of the A. aegypti genome. These EVEs were significantly associated with TEs and preferentially located in repeat-rich clusters within intergenic regions. Genome-wide transcriptome analysis showed that most EVEs generated transcripts although only around 1.4% were sense RNAs. The majority of EVE transcription was antisense and correlated with the generation of EVE-derived small RNAs. A single genomic cluster of EVEs located in a 143 kb repetitive region in chromosome 2 contributed with 42% of antisense transcription and 45% of small RNAs derived from viral elements. This region was enriched for TE-EVE hybrids organized in the same coding strand. These generated a single long antisense transcript that correlated with the generation of phased primary PIWI-interacting RNAs (piRNAs). The putative promoter of this region had a conserved binding site for the transcription factor Cubitus interruptus, a key regulator of the flamenco locus in Drosophila melanogaster. Here, we have identified a single unidirectional piRNA cluster in the A. aegypti genome that is the major source of EVE transcription fueling the generation of antisense small RNAs in mosquitoes. We propose that this region is a flamenco-like locus in A. aegypti due to its relatedness to the major unidirectional piRNA cluster in Drosophila melanogaster.

Glycine riboswitches utilize both single- and tandem-aptamer architectures. In the tandem system, the relative contribution of each aptamer toward gene regulation is not well understood. To dissect these contributions, the effects of 684 single mutants of a tandem ON switch from Bacillus subtilis were characterized for the wild-type construct and binding site mutations that selectively restrict ligand binding to either the first or second aptamer. Despite the structural symmetry of tandem aptamers, the response to these mutations was frequently asymmetrical. Mutations in the first aptamer often significantly weakened the K_1/2, while several mutations in the second aptamer improved the amplitude. These results demonstrate that this ON switch favors ligand binding to the first aptamer. This is in contrast to the tandem OFF switch variant from Vibrio cholerae, which was previously shown to have preferential binding to its second aptamer. A bioinformatic analysis of tandem glycine riboswitches revealed that the two binding pockets are differentially conserved between ON and OFF switches. Altogether, this indicates that tandem ON switch variants preferentially utilize binding to the first aptamer to promote helical switching, while OFF switch variants favor binding to the second aptamer. The data set also revealed a cooperative glycine response when both binding pockets were maximally stabilized with three GC base pairs. This indicates a cooperative response may sometimes be obfuscated by a difference in the affinities of the two aptamers. This conditional cooperativity provides an additional layer of tunability to tandem glycine riboswitches that adds to their versatility as genetic switches.

Transposable elements (TEs) can damage genomes, thus organisms use a variety of mechanisms to repress TE expression. The PIWI–piRNA pathway is a small RNA pathway that represses TE expression in the germline of animals. Here we explore the function of the pathway in the somatic stem cells of Hydra, a long-lived freshwater cnidarian. Hydra have three stem cell populations, all of which express PIWI proteins; endodermal and ectodermal epithelial stem cells (ESCs) are somatic, whereas the interstitial stem cells have germline competence. To study somatic function of the pathway, we isolated piRNAs from Hydra that lack the interstitial lineage and found that these somatic piRNAs map predominantly to TE transcripts and display the conserved sequence signatures typical of germline piRNAs. Three lines of evidence suggest that the PIWI–piRNA pathway represses TEs in Hydra ESCs. First, epithelial knockdown of the Hydra piwi gene hywi resulted in up-regulation of TE expression. Second, degradome sequencing revealed evidence of PIWI-mediated cleavage of TE RNAs in epithelial cells using the ping-pong mechanism. Finally, we demonstrated a direct association between Hywi protein and TE transcripts in epithelial cells using RNA immunoprecipitation. Altogether, our data reveal that the PIWI–piRNA pathway represses TE expression in the somatic cell lineages of Hydra, which we propose contributes to the extreme longevity of the organism. Furthermore, our results, in combination with others, suggest that somatic TE repression is an ancestral function of the PIWI–piRNA pathway.

The PI3K/Akt/mTOR kinase pathway is extensively deregulated in human cancers. One critical node under regulation of this signaling axis is eukaryotic initiation factor (eIF) 4F, a complex involved in the control of translation initiation rates. eIF4F-dependent addictions arise during tumor initiation and maintenance due to increased eIF4F activity—generally in response to elevated PI3K/Akt/mTOR signaling flux. There is thus much interest in exploring eIF4F as a small molecule target for the development of new anticancer drugs. The DEAD-box RNA helicase, eIF4A, is an essential subunit of eIF4F, and several potent small molecules (rocaglates, hippuristanol, pateamine A) affecting its activity have been identified and shown to demonstrate anticancer activity in vitro and in vivo in preclinical models. Recently, a number of new small molecules have been reported as having the capacity to target and inhibit eIF4A. Here, we undertook a comparative analysis of their biological activity and specificity relative to the eIF4A inhibitor, hippuristanol.

The importance of noncoding RNA sequences has become increasingly clear over the past decade. New RNA families are often detected and analyzed using comparative methods based on multiple sequence alignments. Accordingly, a number of programs have been developed for aligning and deriving secondary structures from sets of RNA sequences. Yet, the best tools for these tasks remain unclear because existing benchmarks contain too few sequences belonging to only a small number of RNA families. RNAconTest (RNA consistency test) is a new benchmarking approach relying on the observation that secondary structure is often conserved across highly divergent RNA sequences from the same family. RNAconTest scores multiple sequence alignments based on the level of consistency among known secondary structures belonging to reference sequences in their output alignment. Similarly, consensus secondary structure predictions are scored according to their agreement with one or more known structures in a family. Comparing the performance of 10 popular alignment programs using RNAconTest revealed that DAFS, DECIPHER, LocARNA, and MAFFT created the most structurally consistent alignments. The best consensus secondary structure predictions were generated by DAFS and LocARNA (via RNAalifold). Many of the methods specific to noncoding RNAs exhibited poor scalability as the number or length of input sequences increased, and several programs displayed substantial declines in score as more sequences were aligned. Overall, RNAconTest provides a means of testing and improving tools for comparative RNA analysis, as well as highlighting the best available approaches. RNAconTest is available from the DECIPHER website (http://DECIPHER.codes/Downloads.html).

“E-cigarettes” are a class of consumer devices designed to deliver drugs, primarily nicotine or marijuana oils, to the lung by vaporization. Regulation of the devices in the United States is relatively minimal, and research on both epidemiology and potential toxicity has focused on nicotine devices. In 2019, an outbreak of an acute respiratory illness in the United States was traced back to the contamination of e-cigarette fluids with vitamin E acetate, which had been used to disguise the dilution of marijuana oils. The outbreak, termed “e-cigarette or vaping associated lung injury” by the U.S. Centers for Disease Control, was characterized by pulmonary infiltrates and hypoxia, which usually required hospitalization and, often, admission to ICUs. The syndrome sickened >2,600 people, mostly young men, and killed >50 people before it began to abate 6 months later. No current regulations exist to prevent a similar event with the same or different chemical contaminants. Absent such regulation, respiratory practitioners should be prepared to evaluate, identify, and treat future cases of acute lung toxicity from e-cigarettes.

Relevant publications related to medicinal and toxic aerosols are discussed in this review. Treatment of COPD includes a combination of long-acting bronchodilators and long-acting muscarinic antagonists. A combination of aclidinium bromide and formoterol fumarate was approved in the United States. The combination was superior to its components alone, as well as tiotropium and a salmeterol-fluticasone combination. Increased risk of an asthma exacerbation was reported in children exposed to electronic nicotine delivery systems. A smart inhaler capable of recording inspiratory flow was approved in the United States. The use of as-needed budesonide-formoterol was reported to be superior to scheduled budesonide and as-needed terbutaline for the treatment of adults with mild-to-moderate asthma. A survey among teens with asthma and their caregivers revealed a disagreement in the number of inhaled controller medications the teen was taking. Treatment with inhaled hypertonic saline resulted in a decreased lung clearance index in infants and preschool children with cystic fibrosis. Surgical masks were well tolerated and significantly decreased the burden of aerosolized bacteria generated by coughing in adults with cystic fibrosis. Inhaled liposomal amikacin in addition to guideline-based therapy was reported to be superior to guideline-based therapy alone in achieving negative sputum cultures in adult subjects with Mycobacterium avium complex pulmonary disease. During 2019, lung injury associated to e-cigarette or vaping was reported, including 60 casualties.

Respiratory support of the critically ill neonate has steadily shifted from invasive to noninvasive forms of support. There have recently been a number of important advances in our understanding of the changes to neonatal resuscitation practices as they pertain to clinically important outcomes, mechanisms of gas exchange for high-flow nasal cannula, and best use of noninvasive ventilation and predicting response. Although the proportion of infants requiring intubation and mechanical ventilation has decreased, the most severely ill often still require intubation and ventilation. Recently, volume-targeted ventilation, high-frequency ventilation, and different methods of assessing weaning and extubation have been investigated. This review summarizes a number of important advances that have been made in the management of prematurity and neonatal respiratory distress syndrome.

BACKGROUND:Radiotherapy for breast cancer has been implicated in the development of bronchiolitis obliterans organizing pneumonia (BOOP). Patients may be asymptomatic or may have pulmonary and constitutional symptoms that are moderate or severe. Postradiotherapy BOOP usually develops during the 12 months after completion of radiotherapy and is characterized by ground-glass opacities in the radiation-exposed lung and frequently in the non-irradiated lung.METHODS:An updated literature search and review was performed to update the systematic review we conducted in 2014. Ten new publications were identified: 2 Japanese epidemiological studies, 1 Japanese case series study, 6 case reports, and 1 review article.RESULTS:The incidence of postradiotherapy BOOP was 1.4% in both Japanese epidemiological studies. Risk factors included increasing age, cigarette smoking, and increasing central lung distance. The case reports included 7 women who had breast cancer postradiation BOOP and 1 woman who had an ataxia telangiectasia mutated (ATM) gene mutation, which may increase radiation sensitivity.CONCLUSION:Postradiotherapy BOOP in women with breast cancer occurs at a rate of 1.0–3.0% and may occur in women with immune system dysfunction and genetic mutations.

BACKGROUND:Ventilator-associated pneumonia (VAP) is a common and serious complication of mechanical ventilation. We conducted a meta-analysis of published randomized controlled trials to evaluate the efficacy and safety of probiotics for VAP prevention in patients who received mechanical ventilation.METHODS:We searched a number of medical literature databases to identify randomized controlled trials that compared probiotics with controls for VAP prevention. The results were expressed as odds ratios (OR) or mean differences with accompanying 95% CIs. Study-level data were pooled by using a random-effects model. Data syntheses were accomplished by using statistical software.RESULTS:Fourteen studies that involved 1,975 subjects met our inclusion criteria. Probiotic administration was associated with a reduction in VAP incidence among all 13 studies included in the meta-analysis (OR 0.62, 95% CI 0.45–0.85; P = .003; I2 = 43%) but not among the 6 double-blinded studies (OR 0.72, 95% CI 0.44–1.19; P = .20; I2 = 55%). We found a shorter duration of antibiotic use for VAP (mean difference −1.44, 95% CI −2.88 to −0.01; P = .048, I2 = 30%) in the probiotics group than in the control group, and the finding comes from just 2 studies. No statistically significant differences were found between the groups in terms of ICU mortality (OR 0.95, 95% CI 0.67–1.34; P = .77; I2 = 0%), ICU stay (mean difference –0.77, 95% CI –2.58 to 1.04; P = .40; I2 = 43%), duration of mechanical ventilation (mean difference –0.91, 95% CI –2.20 to 0.38; P = .17; I2 = 25%), or occurrence of diarrhea (OR 0.72, 95% CI 0.45–1.15; P = .17; I2 = 41%).CONCLUSIONS:The meta-analysis results indicated that the administration of probiotics significantly reduced the incidence of VAP. Furthermore, our findings need to be verified in large-scale, well-designed, randomized, multi-center trials.

BACKGROUND:In this study, we aimed to validate the agreement between pulmonary function measurements obtained with a portable spirometer and measurements obtained with conventional spirometry in Chinese pediatric and adult populations.METHODS:Pulmonary function testing was performed to evaluate subjects enrolled at Shanghai Zhongshan Hospital (n = 104) and Shanghai Children's Medical Center (n = 103). The portable spirometers and conventional devices were applied to each subject with a 20-min quiescent period between each measurement. Pulmonary function parameters of FVC, FEV1, peak expiratory flow, maximum expiratory flow at 25%, 50%, and 75% of FVC (MEF25, MEF50, and MEF75, respectively), and FEV1/FVC% were compared with intraclass correlation and Bland-Altman methods.RESULTS:A satisfactory concordance of pulmonary function was observed between spirometry measurements obtained with portable versus conventional spirometers. Intraclass correlation indicated excellent reliability (>0.75) for all pulmonary function indicators in pediatric and adult subjects. Significant positive correlations of all variables measured with different spirometers were observed (all P < .001). No significant bias was observed in either group, although limits of agreement varied. Funnel effects were observed for peak expiratory flow in pediatric subjects and for FVC, FEV1, MEF50, and MEF25 in adult subjects.CONCLUSIONS:The portable spirometer is an alternative to the conventional device for the measurement of pulmonary function. Compared with the conventional device, the portable spirometer is expected to provide convenient, operational, and financial advantages.

BACKGROUND:The N95 filtering facepiece respirator (FFR) is the most popular individual protective device to reduce exposure to particulate matter. However, concerns have been raised with regard to its use because it can increase respiratory resistance and dead space. Therefore, this study assessed the safety of N95 use in patients with COPD and air-flow limitation.METHODS:This prospective study was performed at a tertiary hospital and enrolled 97 subjects with COPD. The subjects were monitored for symptoms and physiologic variables during a 10-min rest period and 6-min walking test while wearing an N95.RESULTS:Of the 97 subjects, 7 with COPD did not wear the N95 for the entire test duration. This mask-failure group showed higher British modified Medical Research Council dyspnea scale scores and lower FEV1 percent of predicted values than did the successful mask use group. A modified Medical Research Council dyspnea scale score ≥ 3 (odds ratio 167, 95% CI 8.4 to >999.9; P = .008) or a FEV1 < 30% predicted (odds ratio 163, 95% CI 7.4 to >999.9; P = .001) was associated with a risk of failure to wear the N95. Breathing frequency, blood oxygen saturation, and exhaled carbon dioxide levels also showed significant differences before and after N95 use.CONCLUSIONS:This study demonstrated that subjects with COPD who had modified Medical Research Council dyspnea scale scores ≥ 3 or FEV1 < 30% predicted wear N95s only with care.

BACKGROUND:Most children are exposed to human metapneumovirus (HMPV) by the age of 5 y. This study aimed to describe the morbidity associated with HMPV infections in a cohort of children in the Midwest of the United States.METHODS:This was a retrospective 2-center cohort study including children (0–17 y old) hospitalized with HMPV infections at 2 tertiary care pediatric hospitals from 2009 to 2013. Demographics, chronic medical conditions, viral coinfections, and hospitalization characteristics, including the need for respiratory support, high-flow nasal cannula, CPAP, bi-level positive airway pressure, invasive mechanical ventilation, pediatric ICU admission, acute kidney injury (AKI), use of extracorporeal membrane oxygenation, and length of stay, were collected.RESULTS:In total, 131 subjects were included. Those with one or more comorbidities were older than their otherwise healthy counterparts, with a median age of 2.8 y (interquartile range [IQR] 1.1–7.0) compared to 1.3 y (IQR 0.6–2.0, P < .001), respectively. Ninety-nine (75.6%) subjects required respiratory support; 72 (55.0%) subjects required nasal cannula, simple face mask, or tracheostomy mask as their maximum support. Additionally, 1 (0.8%) subject required high-flow nasal cannula, 1 (0.8%) subject required CPAP, 2 (1.5%) subjects required bi-level positive airway pressure, 15 (11.5%) subjects required invasive mechanical ventilation, 4 (3.1%) subjects required high-frequency oscillatory or jet ventilation, and 4 (3.1%) subjects required extracorporeal membrane oxygenation. Fifty-one (38.9%) subjects required pediatric ICU admission, and 16 (12.2%) subjects developed AKI. Subjects with AKI were significantly older than those without AKI at 5.4 y old (IQR 1.6–11.7) versus 1.9 y old (IQR 0.7–3.5, P = .003). After controlling for the presence of at least one comorbidity and cystic fibrosis, each year increase in age led to a 16% increase in the odds of AKI (P = .01). The median length of stay for the entire cohort was 4.0 d (IQR 2.7–7.0).CONCLUSIONS:Children hospitalized with HMPV may be at risk for AKI. Risk of HMPV-associated AKI appears to increase with age regardless of severity of respiratory illness or presence of comorbidities.

BACKGROUND:Patients with cystic fibrosis develop decreased exercise capacity. However, the main factors responsible for this decline are still unclear. Thus, the objective of this study was to evaluate the factors influencing exercise capacity assessed with the modified shuttle test (MST) in individuals with cystic fibrosis.METHODS:A cross-sectional study was carried out in subjects with a diagnosis of cystic fibrosis who were 6–26 y old and were regularly monitored at 2 cystic fibrosis reference centers in Brazil. Individuals who were unable to perform the tests or who exhibited hemodynamic instability and exacerbation of respiratory symptoms were excluded. Anthropometric, clinical, and genotype data were collected. In addition, lung function and exercise capacity were evaluated with the MST.RESULTS:73 subjects (mean age 12.2 ± 4.9 y and FEV1 76.8 ± 23.3%) were included. The mean distance achieved in the MST was 765 ± 258 m (71.6% of predicted). The distance achieved on the MST correlated significantly with age (r = 0.49, P < .001), body mass index (r = 0.41, P < .001), resting heart rate (r = −0.51, P < .001), and FEV1 (r = 0.24, P = .042). Subjects with FEV1 > 67% of predicted (P = .02) and those with resting heart rate < 100 beats/min (P = .01) had a greater exercise capacity. Resting heart rate, age, and FEV1 (%) were found as significant variables to explain the distance achieved on the MST (R2 = 0.48, standard error = 191.0 m).CONCLUSIONS:The main determinants of exercise capacity assessed with the MST in individuals with cystic fibrosis were resting heart rate, age, and lung function.

BACKGROUND:Prolonged ventilatory weaning may expose patients to unnecessary discomfort, increase the risk of complications, and raise the costs of hospital treatment. In this scenario, indexes that reliably predict successful liberation can be helpful.OBJECTIVE:To evaluate the intra- and interobserver reproducibility of the timed inspiratory effort index as a weaning predictor.METHODS:This prospective observational study included subjects judged as able to start liberation from mechanical ventilation. For the intra-observer analysis, the same investigator performed 2 measurements in each selected patient with an interval of 30 min a rest. For interobserver analysis, 2 measurements were obtained in another sample of subjects, also with an interval of 30 min rest, but each of one performed by a different investigator. The Bland-Altman diagram, the coefficient concordance of kappa, and the Pearson correlation coefficient were used to compare the measurements. The performance of the timed inspiratory effort index was assessed by receiver operating characteristic curves. Values of P < .05 were considered significant.RESULTS:We selected 113 subjects (43 males; mean ± SD age, 77 ± 14 y). Fifty-six (49.6%) achieved successful liberation, and 33 (29%) died in the ICU. The mean ± SD duration of mechanical ventilation was 14.4 ± 6.7 d. The Bland-Altman diagrams that addressed intra- and interobservers agreement showed low variability between measurements. Values of the concordance coefficients of kappa were 0.82 (0.68–0.95) and 0.80 (0.65–0.94), and of the linear correlation coefficients, 0.86 (0.77–0.91) and 0.89 (0.82–0.93) for the intra- and interobservers measurements, respectively. The mean ± SD values for the area under the curve for each pair of the intra- and interobserver measurements were 0.96 ± 0.07 versus 0.94 ± 0.07 (P = .41) and 0.94 ± 0.05 versus 0.90 ± 0.07 (P = .14), respectively.CONCLUSIONS:The variability of the measurement of the timed inspiratory effort index by intra- and interobservers showed very high reproducibility, which reinforced the index as a sensible, accurate, and reliable outcome predictor of liberation from mechanical ventilation.

BACKGROUND:Pneumoperitoneum and Trendelenburg position affect respiratory system mechanics and oxygenation during elective pelvic robotic surgery. The primary aim of this randomized pilot study was to compare the effects of a conventional low tidal volume ventilation with PEEP guided by gas exchange (VGas-guided) versus low tidal volume ventilation tailoring PEEP according to esophageal pressure (VPes-guided) on oxygenation and respiratory mechanics during elective pelvic robotic surgery.METHODS:This study was conducted in a single-center tertiary hospital between September 2017 and January 2019. Forty-nine adult patients scheduled for elective pelvic robotic surgery were screened; 28 subjects completed the full analysis. Exclusion criteria were American Society of Anesthesiologists physical status ≥ 3, contraindications to nasogastric catheter placement, and pregnancy. After dedicated naso/orogastric catheter insertion, subjects were randomly assigned to VGas-guided (FIO2 and PEEP set to achieve SpO2 > 94%) or VPes-guided (PEEP tailored to equalize end-expiratory transpulmonary pressure). Oxygenation (PaO2/FIO2) was evaluated (1) at randomization, after pneumoperitoneum and Trendelenburg application; (2) at 60 min; (3) at 120 min following randomization; and (4) at end of surgery. Respiratory mechanics were assessed during the duration of the study.RESULTS:Compared to VGas-guided, oxygenation was higher with VPes-guided at 60 min (388 ± 90 vs 308 ± 95 mm Hg, P = .02), at 120 min after randomization (400 ± 90 vs 308 ± 81 mm Hg, P = .008), and at the end of surgery (402 ± 95 vs 312 ± 95 mm Hg, P = .009). Respiratory system elastance was lower with VPes-guided compared to VGas-guided at 20 min (24.2 ± 7.3 vs 33.4 ± 10.7 cm H2O/L, P = .001) and 60 min (24.1 ± 5.4 vs 31.9 ± 8.5 cm H2O/L, P = .006) from randomization.CONCLUSIONS:Oxygenation and respiratory system mechanics were improved when applying a ventilatory strategy tailoring PEEP to equalize expiratory transpulmonary pressure in subjects undergoing pelvic robotic surgery compared to a VGas-guided approach. (ClinicalTrials.gov registration NCT03153592).

BACKGROUND:Field walking tests are commonly used in patients with chronic pulmonary diseases for assessment of functional capacity. However, the physiological demands and magnitude of desaturation on 6-min walk test (6MWT), incremental shuttle walk test (ISWT), and endurance shuttle walk test (ESWT) have not been investigated in patients with bronchiectasis. The objective of this study was to compare the physiological responses and the magnitude of desaturation of subjects with bronchiectasis when performing the 6MWT, ISWT, and ESWT.METHODS:Thirty-two subjects underwent the 6MWT, ISWT, and ESWT on 3 different days. Pulmonary gas exchange, heart rate, and SpO2 were measured in all tests.RESULTS:There were no differences in the peak rate of oxygen uptake, ventilation, dyspnea, and leg fatigue between the tests. Equivalent cardiac demand (ie, heart rate at peak) was observed with the 6MWT (137 ± 21 beats/min) and the ESWT (142 ± 21 beats/min), but this was lower in the ISWT (135 ± 19 beats/min) compared to ESWT (P < .05). Most subjects achieved a vigorous exercise intensity (heart rate of 70–90% of predicted) in all tests. There was no difference in desaturation among the tests (6MWT: −6.8 ± 6.6%, ISWT: −6.1 ± 6.0%, and ESWT: −7.0 ± 5.4%).CONCLUSIONS:The 6MWT, ISWT, and ESWT induced similar physiological responses at the peak of exercise, eliciting a vigorous exercise intensity. The magnitude of desaturation was similar across tests. This means these tests can be used interchangeably for evaluation of exercise-induced desaturation.

BACKGROUND:High-flow nasal cannula (HFNC) therapy may reduce the re-intubation rate compared with conventional oxygen therapy. However, HFNC has not been sufficiently compared with conventional oxygen therapy with a heated humidifier, even though heated humidification is beneficial for facilitating airway clearance.METHODS:This study was a single-center, open-label, randomized controlled trial. We randomized subjects with respiratory failure after extubation to either HFNC group or to a large-volume humidified nebulization-based nebulizer. The primary end point was the re-intubation rate within 7 d after extubation.RESULTS:We could not recruit enough subjects for the sample size we designed, therefore, we analyzed 69 subjects (HFNC group, 30 subjects; nebulizer group, 39 subjects). The re-intubation rate within 7 d was not significantly different between the HFNC and nebulizer groups (5/30 subjects [17%] and 6/39 subjects [15%], respectively; P > .99). PaO2/set FIO2 at 24 h after extubation was also not significantly different between the respective groups (264 ± 105 mm Hg in the HFNC group vs 224 ± 53 mm Hg in the nebulizer group; P = .07).CONCLUSIONS:Compared with a large-volume nebulization-based humidifier, HFNC may not reduce the re-intubation rate within 7 d. However, because of insufficient statistical power, further studies are needed to reach a conclusion.

BACKGROUND:The threshold of the lower limit of the normal range of lung function has been suggested to be more accurate than the 0.7 fixed ratio (FEV1/FVC < 0.7) for a diagnosis of COPD. We aimed to explore the health status and risk factors of patients overdiagnosed with COPD when using the lower limit of the normal range as a diagnostic reference.METHODS:Subjects with COPD diagnosed by a pulmonologist according to guidelines of the Global Initiative for Chronic Obstructive Lung Disease were recruited from October 2016 to April 2018. Overdiagnosed COPD was defined as FEV1/FVC that meets the criterion of the 0.7 fixed ratio but not the the lower limit of the normal range criterion. Spirometry and questionnaires were performed by eligible subjects.RESULTS:Of the 513 subjects included in the final analysis, 20 (3.9%) were overdiagnosed when using the lower limit of the normal range as the diagnostic reference. The subjects who were overdiagnosed were older, weighed more, had better lung function, lower modified Medical British Research Council scores, and higher St. George's Respiratory Questionnaire and 36-item Short Form Survey scores than the subjects who were correctly diagnosed. Older age, heavier weight, exposure to cooking oil fumes, or a new-built or newly renovated home were associated with an increased risk of overdiagnosis of COPD (age adjusted odds ratio (OR) 1.17, 95% CI 1.09–1.26; weight adjusted OR 1.08, 95% CI 1.03–1.13; exposure to cooking oil fumes adjusted OR 3.00, 95% CI, 1.04–8.68; exposure to new-built or newly renovated home adjusted OR 10.88, 95% CI 1.46–80.87.CONCLUSIONS:The subjects with overdiagnosed COPD had a better health status and lung function than the subjects who were correctly diagnosed. Older age, heavier weight, and exposure to cooking oil fumes or a new-built or newly renovated home were factors associated with the overdiagnosis of COPD. These findings may help reduce overdiagnosis of COPD.

BACKGROUND:Assisted coughing via mechanical in-exsufflation (MI-E) is a first-line treatment for secretion management in patients with amyotrophic lateral sclerosis (ALS) with unassisted CPF < 4.25 L/s. Some devices enable oscillations to be added to MI-E (MI-E+O). We sought to determine whether adding oscillations to MI-E enables a reduction in the use of invasive secretion management procedures (ie, bronchoscopy or tracheostomy) in subjects with ALS.METHODS:We conducted a 12-month, prospective, randomized follow-up study of subjects with ALS for whom assisted coughing techniques were indicated. One group was treated with oscillations in addition to MI-E (MI-E+O), and the other group was treated with conventional MI-E.RESULTS:29 subjects were included in the MI-E group and 27 subjects were included in the MI-E+O group. Five subjects (8.9%) required invasive techniques for secretion management (3 in the MI-E group and 2 in the MI-E+O group, P = .70). Treatment with MI-E+O did not alter the risk of invasive procedures (odds ratio 0.69, 95% CI 0.10–4.50, P = .70). The mean number of respiratory infections was 0.58 ± 0.16 in the MI-E group and 0.025 ± 0.08 in the MI-E+O group (P = .10). Survival was 8.96 ± 0.18 months in the MI-E group and 7.70 ± 0.70 months in the MI-E+O group (P = .10).CONCLUSION:Adding oscillations to MI-E did not enable a reduction in the need to perform invasive procedures for secretion management in subjects with ALS.

BACKGROUND:Electrical impedance tomography (EIT) is a noninvasive, portable lung imaging technique that provides functional distribution of ventilation. We aimed to describe the relationship between the distribution of ventilation by mode of ventilation and level of oxygenation impairment in children who are critically ill. We also aimed to describe the safety of EIT application.METHODS:A prospective observational study of EIT images obtained from subjects in the pediatric ICU. Images were categorized by whether the subjects were on intermittent mandatory ventilation (IMV), continuous spontaneous ventilation, or no positive-pressure ventilation. Images were categorized by the level of oxygenation impairment when using SpO2/FIO2. Distribution of ventilation is described by the center of ventilation.RESULTS:Sixty-four images were obtained from 25 subjects. Forty-two images obtained during IMV with a mean ± SD center of ventilation of 55 ± 6%, 14 images during continuous spontaneous ventilation with a mean ± SD center of ventilation of 48.1 ± 11%, and 8 images during no positive-pressure ventilation with a mean ± SD center of ventilation of 47.5 ± 10%. Seventeen images obtained from subjects with moderate oxygenation impairment with a mean ± SD center of ventilation of 59.3 ± 1.9%, 12 with mild oxygenation impairment with a mean ± SD center of ventilation of 52.6 ± 2.3%, and 4 without oxygenation impairment with a mean ± SD center of ventilation of 48.3 ± 4%. There was more ventral distribution of ventilation with IMV versus continuous spontaneous ventilation (P = .009), with IMV versus no positive-pressure ventilation (P = .01) cohorts, and with moderate oxygenation impairment versus cohorts without oxygenation impairment (P = .009). There were no adverse events related to the placement and use of EIT in our study.CONCLUSIONS:Children who had worse oxygen impairment or who received controlled modes of ventilation had more ventral distribution of ventilation than those without oxygen impairment or the subjects who were spontaneously breathing. The ability of EIT to detect changes in the distribution of ventilation in real time may allow for distribution-targeted mechanical ventilation strategies to be deployed proactively; however, future studies are needed to determine the effectiveness of such a strategy.

BACKGROUND:Observational studies report that lower driving pressure (ie, the difference between plateau pressure and PEEP) is associated with improved survival in patients with ARDS and may be a key mediator of lung-protective ventilation strategies. The primary objective of this study was to characterize reductions in driving pressure that could be achieved through changes in PEEP.METHODS:In this prospective physiological pilot study, 10 subjects with ARDS were placed on PEEP according to the ARDS Network Lower PEEP/FIO2 Table. PEEP was adjusted in small increments and decrements above and below this initial PEEP, and driving pressure was measured at each PEEP level. Subsequently, PEEP was set at the level resulting in the lowest driving pressure, and driving pressure was measured after 1, 5, 15, and 30 min to assess stability over time at constant PEEP.RESULTS:All subjects had ARDS with a median (interquartile range [IQR]) PaO2/FIO2 of 116 (98–132) at enrollment. Median (IQR) driving pressure at baseline was 14 (13–17) cm H2O. After PEEP titration, median driving pressure decreased to 13 (12–14) cm H2O. The largest reduction in driving pressure was 4 cm H2O. Two subjects had no change in driving pressure at multiple PEEP levels. To achieve the lowest driving pressure, final PEEP was increased in 6 subjects and decreased in 4 subjects from the baseline PEEP prescribed by the ARDS Network Lower PEEP/FIO2 Table. Driving pressure reached equilibrium within 1–5 min and remained stable for 30 min following PEEP titration.CONCLUSIONS:PEEP titration had a variable effect in changing driving pressure across this small sample of ARDS subjects. In some subjects, PEEP was decreased from values given in the ARDS Network Lower PEEP/FIO2 Table to minimize driving pressure. Changes in driving pressure stabilized within a few minutes of PEEP titration.

The organic anion transporting polypeptide (OATP)2B1 is localized on the basolateral membrane of hepatocytes and is expressed in enterocytes. Based on its distribution pattern and functional similarity to OATP1B-type transporters, OATP2B1 might have a role in the absorption and disposition of a range of xenobiotics. Although several prescription drugs, including hydroxymethylglutaryl-coenzyme A-CoA reductase inhibitors (statins) such as fluvastatin, are OATP2B1 substrates in vitro, evidence supporting the in vivo relevance of this transporter remains limited, and most has relied on substrate-inhibitor interactions resulting in altered pharmacokinetic properties of the victim drugs. To address this knowledge deficit, we developed and characterized an Oatp2b1-deficient mouse model and evaluated the impact of this transporter on the absorption and disposition of fluvastatin. Consistent with the intestinal localization of Oatp2b1, we found that the genetic deletion or pharmacological inhibition of Oatp2b1 was associated with decreased absorption of fluvastatin by 2- to 3-fold. The availability of a viable Oatp2b1-deficient mouse model provides an opportunity to unequivocally determine the contribution of this transporter to the absorption and drug-drug interaction potential of drugs.

The objectives of the present study were to characterize GNE-947 for its phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitory activities, in vitro anti–cell migration activity in human umbilical vein endothelial cells (HUVECs), in vivo antineovascularization activity in laser-induced rat choroidal neovascular (CNV) eyes, pharmacokinetics in rabbit plasma and eyes, and ocular distribution using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) and autoradioluminography. Its PI3K and mTOR K_i were 0.0005 and 0.045 µM, respectively, and its HUVEC IC₅₀ was 0.093 µM. GNE-947 prevented neovascularization in the rat CNV model at 50 or 100 µg per eye with repeat dosing. After a single intravenous injection at 2.5 and 500 μg/kg in rabbits, its plasma terminal half-lives (t_1/2) were 9.11 and 9.59 hours, respectively. After a single intravitreal injection of a solution at 2.5 μg per eye in rabbits, its apparent t_1/2 values were 14.4, 16.3, and 23.2 hours in the plasma, vitreous humor, and aqueous humor, respectively. After a single intravitreal injection of a suspension at 33.5, 100, 200 μg per eye in rabbits, the t_1/2 were 29, 74, and 219 days in the plasma and 46, 143, and 191 days in the eyes, respectively. MALDI-IMS and autoradioluminography images show that GNE-947 did not homogenously distribute in the vitreous humor and aggregated at the injection sites after injection of the suspension, which was responsible for the long t_1/2 of the suspension because of the slow dissolution process. This hypothesis was supported by pharmacokinetic modeling analyses. In conclusion, the PI3K/mTOR inhibitor GNE-947 prevented neovascularization in a rat CNV model, with t_1/2 up to approximately 6 months after a single intravitreal injection of the suspension in rabbit eyes.

Dependence of drug metabolism on dosing time has long been recognized. However, only recently are the underlying mechanisms for circadian drug metabolism being clarified. Diurnal rhythmicity in expression of drug-metabolizing enzymes is believed to be a key factor determining circadian metabolism. Supporting the notion that biological rhythms are generated and maintained by the circadian clock, a number of diurnal enzymes are under the control of the circadian clock. In general, circadian clock genes generate and regulate diurnal rhythmicity in drug-metabolizing enzymes via transcriptional actions on one or two of three cis-elements (i.e., E-box, D-box, and Rev-erb response element or RAR-related orphan receptor response element). Additionally, cycling or clock-controlled nuclear receptors such as hepatocyte nuclear factor 4α and peroxisome proliferator–activated receptor are contributors to diurnal enzyme expression. These newly discovered mechanisms for each of the rhythmic enzymes are reviewed in this article. We also discuss how the rhythms of enzymes are translated to circadian pharmacokinetics and drug chronotoxicity, which has direct implications for chronotherapeutics. Our discussion is also extended to two diurnal transporters (P-glycoprotein and multidrug resistance-associated protein 2) that have an important role in drug absorption. Although the experimental evidence is lacking in metabolism-based chronoefficacy, circadian genes (e.g., Rev-erbα) as drug targets are shown to account for diurnal variability in drug efficacy.

Paclitaxel has been considered to cause OATP1B-mediated drug-drug interactions at therapeutic doses; however, its clinical relevance has not been demonstrated. This study aimed to elucidate in vivo inhibition potency of paclitaxel against OATP1B1 and OATP1B3 using endogenous OATP1B biomarkers. Paclitaxel is an inhibitor of OATP1B1 and OATP1B3, with K_i of 0.579 ± 0.107 and 5.29 ± 3.87 μM, respectively. Preincubation potentiated its inhibitory effect on both OATP1B1 and OATP1B3, with K_i of 0.154 ± 0.031 and 0.624 ± 0.183 μM, respectively. Ten patients with non–small cell lung cancer who received 200 mg/m² of paclitaxel by a 3-hour infusion were recruited. Plasma concentrations of 10 endogenous OATP1B biomarkers—namely, coproporphyrin I, coproporphyrin III, glycochenodeoxycholate-3-sulfate, glycochenodeoxycholate-3-glucuronide, glycodeoxycholate-3-sulfate, glycodeoxycholate-3-glucuronide, lithocholate-3-sulfate, glycolithocholate-3-sulfate, taurolithocholate-3-sulfate, and chenodeoxycholate-24-glucuronide—were determined in the patients with non–small cell lung cancer on the day before paclitaxel administration and after the end of paclitaxel infusion for 7 hours. Paclitaxel increased the area under the plasma concentration-time curve (AUC) of the endogenous biomarkers 2- to 4-fold, although a few patients did not show any increment in the AUC ratios of lithocholate-3-sulfate, glycolithocholate-3-sulfate, and taurolithocholate-3-sulfate. Therapeutic doses of paclitaxel for the treatment of non–small cell lung cancer (200 mg/m²) will cause significant OATP1B1 inhibition during and at the end of the infusion. This is the first demonstration that endogenous OATP1B biomarkers could serve as surrogate biomarkers in patients.

Taurine is one of the most abundant amino acids in mammalian tissues. It is obtained from the diet and by de novo synthesis from cysteic acid or hypotaurine. Despite the discovery in 1954 that the oxygenation of hypotaurine produces taurine, the identification of an enzyme catalyzing this reaction has remained elusive. In large part, this is due to the incorrect assignment, in 1962, of the enzyme as an NAD-dependent hypotaurine dehydrogenase. For more than 55 years, the literature has continued to refer to this enzyme as such. Here we show, both in vivo and in vitro, that the enzyme that oxygenates hypotaurine to produce taurine is flavin-containing monooxygenase (FMO) 1. Metabolite analysis of the urine of Fmo1-null mice by ¹H NMR spectroscopy revealed a buildup of hypotaurine and a deficit of taurine in comparison with the concentrations of these compounds in the urine of wild-type mice. In vitro assays confirmed that human FMO1 catalyzes the conversion of hypotaurine to taurine, utilizing either NADPH or NADH as cofactor. FMO1 has a wide substrate range and is best known as a xenobiotic- or drug-metabolizing enzyme. The identification that the endogenous molecule hypotaurine is a substrate for the FMO1-catalyzed production of taurine resolves a long-standing mystery. This finding should help establish the role FMO1 plays in a range of biologic processes in which taurine or its deficiency is implicated, including conjugation of bile acids, neurotransmitter, antioxidant and anti-inflammatory functions, and the pathogenesis of obesity and skeletal muscle disorders.

Antibody-drug conjugates (ADCs) employ overexpressed cell surface antigens to deliver cytotoxic payloads inside cancer cells. However, the relationship between target expression and ADC efficacy remains ambiguous. In this manuscript, we have addressed a part of this ambiguity by quantitatively investigating the effect of antigen expression levels on ADC exposure within cancer cells. Trastuzumab-valine-citrulline-monomethyl auristatin E was used as a model ADC, and four different cell lines with diverse levels of human epidermal growth factor receptor 2 (HER2) expression were used as model cells. The pharmacokinetics (PK) of total trastuzumab, released monomethyl auristatin E (MMAE), and total MMAE were measured inside the cells and in the cell culture media following incubation with two different concentrations of ADC. In addition, target expression levels, target internalization rate, and cathepsin B and MDR1 protein concentrations were determined for each cell line. All the PK data were mathematically characterized using a cell-level systems PK model for ADC. It was found that SKBR-3, MDA-MB-453, MCF-7, and MDA-MB-468 cells had ~800,000, ~250,000, ~50,000, and ~10,000 HER2 receptors per cell, respectively. A strong linear relationship (R² > 0.9) was observed between HER2 receptor count and released MMAE exposure inside the cancer cells. There was an inverse relationship found between HER2 expression level and internalization rate, and cathepsin B and multidrug resistance protein 1 (MDR1) expression level varied slightly among the cell lines. The PK model was able to simultaneously capture all the PK profiles reasonably well while estimating only two parameters. Our results demonstrate a strong quantitative relationship between antigen expression level and intracellular exposure of ADCs in cancer cells.

The kidneys play an important role in many processes, including urine formation, water conservation, acid-base equilibrium, and elimination of waste. The anatomic and functional development of the kidney has different maturation time points in humans versus animals, with critical differences between species in maturation before and after birth. Absorption, distribution, metabolism, and excretion (ADME) of drugs vary depending on age and maturation, which will lead to differences in toxicity and efficacy. When neonate/juvenile laboratory animal studies are designed, a thorough knowledge of the differences in kidney development between newborns/children and laboratory animals is essential. The human and laboratory animal data must be combined to obtain a more complete picture of the development in the kidneys around the neonatal period and the complexity of ADME in newborns and children. This review examines the ontogeny and cross-species differences in ADME processes in the developing kidney in preterm and term laboratory animals and children. It provides an overview of insights into ADME functionality in the kidney by identifying what is currently known and which gaps still exist. Currently important renal function properties such as glomerular filtration rate, renal blood flow, and ability to concentrate are generally well known, while detailed knowledge about transporter and metabolism maturation is growing but is still lacking. Preclinical data in those properties is limited to rodents and generally covers only the expression levels of transporter or enzyme-encoding genes. More knowledge on a functional level is needed to predict the kinetics and toxicity in neonate/juvenile toxicity and efficacy studies.

Doxophylline (DOXO) and theophylline are widely used as bronchodilators for treating asthma and chronic obstructive pulmonary disease, and DOXO has a better safety profile than theophylline. How DOXO’s metabolism and disposition affect its antiasthmatic efficacy and safety remains to be explored. In this study, the metabolites of DOXO were characterized. A total of nine metabolites of DOXO were identified in vitro using liver microsomes from human and four other animal species. Among them, six metabolites were reported for the first time. The top three metabolites were theophylline acetaldehyde (M1), theophylline-7-acetic acid (M2), and etophylline (M4). A comparative analysis of DOXO metabolism in human using liver microsomes, S9 fraction, and plasma samples demonstrated the following: 1) The metabolism of DOXO began with a cytochrome P450 (P450)–mediated, rate-limiting step at the C ring and produced M1, the most abundant metabolite in human liver microsomes. However, in human plasma, the M1 production was rather low. 2) M1 was further converted to M2 and M4, the end products of DOXO metabolism in vivo, by non-P450 dismutase in the cytosol. This dismutation process also relied on the ratio of NADP⁺/NADPH in the cell. These findings for the first time elucidated the metabolic sites and routes of DOXO metabolism in human.

Sulfotransferase (SULT) 4A1 is a brain-selective sulfotransferase-like protein that has recently been shown to be essential for normal neuronal development in mice. In the present study, SULT4A1 was found to colocalize with SULT1A1/3 in human brain neurons. Using immunoprecipitation, SULT4A1 was shown to interact with both SULT1A1 and SULT1A3 when expressed in human cells. Mutation of the conserved dimerization motif located in the C terminus of the sulfotransferases prevented this interaction. Both ectopically expressed and endogenous SULT4A1 decreased SULT1A1/3 protein levels in neuronal cells, and this was also prevented by mutation of the dimerization motif. During differentiation of neuronal SH-SY5Y cells, there was a loss in SULT1A1/3 protein but an increase in SULT4A1 protein. This resulted in an increase in the toxicity of dopamine, a substrate for SULT1A3. Inhibition of SULT4A1 using small interference RNA abrogated the loss in SULT1A1/3 and reversed dopamine toxicity. These results show a reciprocal relationship between SULT4A1 and the other sulfotransferases, suggesting that it may act as a chaperone to control the expression of SULT1A1/3 in neuronal cells.

Drug-induced liver injury (DILI) is a global medical problem. The risk of DILI is often related to expression and activities of drug-metabolizing enzymes, especially cytochrome P450s (P450s). However, changes on expression and activities of P450s after DILI have not been determined. The aim of this study is to fill this knowledge gap. Acetaminophen (APAP) was used as a model drug to induce DILI in C57BL/6J mice at different ages of days 10 (infant), 22 (child), and 60 (adult). DILI was assessed by levels of alanine aminotransferase and aspartate aminotransferase in plasma with a confirmation by H&E staining on liver tissue sections. The expression of selected P450s at mRNA and protein levels was measured by real-time polymerase chain reaction and liquid chromatography–tandem mass spectrometry, respectively. The activities of these P450s were determined by the formation of metabolites from probe drugs for each P450 using ultraperformance liquid chromatography–quadrupole time of flight mass spectrometry. DILI was induced at mild to severe levels in a dose-dependent manner in 200, 300, and 400 mg/kg APAP-treated groups at child and adult ages, but not at the infant age. Significantly decreased expression at mRNA and protein levels as well as enzymatic activities of CYP2E1, 3A11, 1A2, and 2C29 were found at child and adult ages. Adult male mice were more susceptible to APAP-induced liver injury than female mice with more decreased expression of P450s. These results suggest that altered levels of P450s in livers severely injured by drugs may affect the therapeutic efficacy of drugs, which are metabolized by P450s, more particularly for males.

Clinical and Vaccine Immunology (CVI) will merge with the American Society for Microbiology (ASM) open-access journal mSphere in January 2018. We commemorate this transition by exploring the history of CVI and that of its predecessor, Clinical and Diagnostic Laboratory Immunology (CDLI), and by acknowledging their contributors. Research on vaccines, clinical immunology, and clinical diagnostic immunology published through mSphere will be available without restrictions to an ever-larger audience, which will expedite progress in the field. ASM remains committed to supporting its members and the research community by facilitating the dissemination of scientific knowledge in these important areas.