Pharmacokinetics of the Novel Nonsteroidal Mineralocorticoid Receptor Antagonist Ocedurenone (KBP-5074) in Individuals with Moderate Hepatic Impairment.

BACKGROUND AND OBJECTIVES: Ocedurenone (KBP-5074) is a novel nonsteroidal mineralocorticoid receptor antagonist that has demonstrated safety and efficacy in clinical trials in patients with uncontrolled hypertension and stage 3b/4 chronic kidney disease. The aim of this study was to assess the pharmacokinetics, safety, and tolerability of ocedurenone in individuals with moderate hepatic impairment. METHODS: This study was an open-label, nonrandomized, multi-center study investigating the pharmacokinetics, safety, and tolerability of a single dose of 0.5 mg ocedurenone administered orally in male and female subjects with moderate hepatic impairment (Child-Pugh B, score 7-9) compared with subjects with normal hepatic function. Serial blood samples were obtained from predose through 264 h postdose for analysis of ocedurenone concentrations using a validated liquid chromatography-tandem mass spectrometry method. Free ocedurenone concentrations in plasma were determined ex vivo using equilibrium dialysis. RESULTS: Following a single oral dose of 0.5 mg ocedurenone administered to subjects with moderate hepatic impairment and subjects with normal hepatic function, ocedurenone was steadily absorbed with median time to peak drug concentration (Tmax) values of 4 and 3 h, respectively. After reaching maximum plasma concentration (Cmax), the disposition of ocedurenone appeared to be biphasic. The geometric mean t1/2 values for the moderate hepatic impairment group and normal hepatic function group were 75.6 and 65.7 h, respectively. Ocedurenone systemic exposure, as assessed by area under the plasma concentration-time curve (AUC) was 23.5-26.6% lower in subjects with moderate hepatic impairment versus subjects with normal hepatic function, whereas Cmax was 41.2% lower. Ocedurenone was determined to be > 99.7% bound to total protein in plasma. Hepatic impairment appeared not to change plasma protein binding or the unbound free fraction. Ocedurenone was safe and well-tolerated in all participants. CONCLUSIONS: Considering the long half-life of ocedurenone and previously completed clinical studies using 0.25 mg and 0.5 mg doses demonstrating efficacy and safety, the observed decreases in AUC and Cmax do not warrant a dose adjustment in patients with moderate hepatic impairment. A single 0.5 mg dose of ocedurenone was safe and well-tolerated when administered to subjects with moderate hepatic impairment and subjects with normal hepatic function. CLINICAL TRIAL IDENTIFIER ( WWW. CLINICALTRIALS: GOV ): NCT04534699.

Epitope-Directed Antibody Elicitation by Genetically Encoded Chemical Cross-Linking Reactivity in the Antigen.

No current methods can selectively elicit an antibody response to a specific conformational epitope in a whole antigen in vivo. Here, we incorporated Nε-acryloyl-l-lysine (AcrK) or Nε-crotonyl-l-lysine (Kcr) with cross-linking activities into the specific epitopes of antigens and immunized mice to generate antibodies that can covalently cross-link with the antigens. By taking advantage of antibody clonal selection and evolution in vivo, an orthogonal antibody-antigen cross-linking reaction can be generated. With this mechanism, we developed a new approach for facile elicitation of antibodies binding to specific epitopes of the antigen in vivo. Antibody responses were directed and enriched to the target epitopes on protein antigens or peptide-KLH conjugates after mouse immunization with the AcrK or Kcr-incorporated immunogens. The effect is so prominent that the majority of selected hits bind to the target epitope. Furthermore, the epitope-specific antibodies effectively block IL-1ß from activating its receptor, indicating its potential for the development of protein subunit vaccines.

Pharmacokinetics and Drug-Drug Interaction of Ocedurenone (KBP-5074) in vitro and in vivo.

BACKGROUND AND OBJECTIVES: Ocedurenone (KBP-5074) is a novel nonsteroidal mineralocorticoid receptor antagonist that has demonstrated safety and efficacy in clinical trials in patients with uncontrolled hypertension and stage 3b/4 chronic kidney disease. This study evaluated the involvement of cytochrome P450 (CYP) isozymes and drug transporters in the biotransformation of ocedurenone, and whether ocedurenone inhibited or induced CYP enzymes and transporters. Clinical pharmacokinetic drug-drug interaction (DDI) of ocedurenone with CYP3A inhibitor and inducer were investigated in healthy volunteers. METHODS: In vitro tests were conducted to determine which CYP enzymes were involved in ocedurenone's metabolism and whether ocedurenone inhibited or induced these CYP enzymes; ocedurenone substrate characteristics for efflux and uptake transporters and its inhibitory potential on major drug transporters were also assessed. A clinical DDI study was conducted in healthy volunteers to evaluate the effects of a strong CYP3A inhibitor (itraconazole) and inducer (rifampin) on ocedurenone's pharmacokinetics. RESULTS: The in vitro study showed that ocedurenone was primarily metabolized by CYP3A4 and that it did not inhibit CYP enzymes. Ocedurenone appeared to be a substrate of BCRP and P-gp efflux transporters and inhibited BCRP, BSEP, MDR1, MATE1 and 2-K, OATP1B1/3, and OCT1. The clinical DDI study showed that itraconazole reduced ocedurenone's oral clearance by 51% and increased area under the plasma concentration-time curve extrapolated to infinity (AUC0-inf) by 104%, while rifampin increased its oral clearance by 6.4-fold and decreased plasma AUC0-inf by 84%. CONCLUSION: Ocedurenone was shown to be a CYP3A substrate, with no inhibition potential on major drug metabolizing CYP enzymes and transporters at clinical efficacious doses. Ocedurenone did not induce CYP1A2 and 3A4 activity in cultured human primary hepatocytes. Clinical DDI study indicated ocedurenone was well tolerated when administered as a single 0.5-mg dose both alone and with itraconazole or rifampin, and while itraconazole had a weak effect on ocedurenone's pharmacokinetics, rifampin had a significant effect reducing systemic exposures.

Design, synthesis and biological evaluations of novel farnesoid X receptor (FXR) agonists.

As a member of the nuclear receptor superfamily, the farnesoid X receptor (FXR) is a bile acid activated transcription factor. FXR is involved in many important metabolic processes and serves as a promising therapeutic target for nonalcoholic steatohepatitis (NASH). Since discovered, the first non-steroidal FXR agonist GW4064 has been widely used to explore the biological functions of FXR, however, the low pharmacokinetic limited its further clinical application. In current study, we designed a series of substituted isothiazoles as new FXR agonists. Among them, five compounds exhibited better FXR agonistic activity than GW4064. Specially, the most potent compound S5 possessed better pharmacokinetic profile and in vivo potency than lead compound.

In Vitro Activity of KBP-7072 against 536 Acinetobacter baumannii Complex Isolates Collected in China.

Acinetobacter baumannii has emerged globally as a difficult-to-treat nosocomial pathogen and become resistant to carbapenems, resulting in limited treatment options. KBP-7072 is a novel semisynthetic aminomethylcycline, expanded spectrum tetracycline antibacterial agent with completed phase 1 clinical development studies. This study aimed to evaluate the in vitro activity of KBP-7072 and several comparators against clinical A. baumannii isolates collected from China. A collection of 536 A. baumannii clinical isolates were isolated from 20 hospitals across 13 provinces and cities in China between 2018 and 2019. Antimicrobial susceptibility testing of 12 antimicrobial agents was performed utilizing the broth microdilution method recommended by CLSI. KBP-7072 has shown active antibacterial activity against 536 A. baumannii isolates. It inhibited the growth of all isolates at 4 mg/liter, including 372 carbapenem-resistant isolates, 37 tigecycline MIC ≥ 4 mg/liter isolates, and 138 omadacycline MIC ≥ 4 mg/liter isolates. Compared with other expanded spectrum tetracyclines, KBP-7072 (MIC90, 1 mg/liter) outperformed 2-fold and 4-fold more active against 536 A. baumannii isolates than tigecycline (MIC90, 2 mg/liter) and omadacycline (MIC90, 4 mg/liter). KBP-7072 was as equally active as colistin (MIC90, 1 mg/liter, 99.4% susceptible). Doxycycline (33.4% susceptible), gentamicin (31.3% susceptible), meropenem (30.6%, susceptible), imipenem (30.2% susceptible), ceftazidime (27.8% susceptible), piperacillin-tazobactam (27.2% susceptible), and levofloxacin (27.2% susceptible) showed marginally poor antibacterial activity against tested isolates according to CLSI breakpoints, except for minocycline (73.7% susceptible). KBP-7072 is a potential alternative agent for the treatment of infection caused by A. baumannii, including carbapenem-resistant species. IMPORTANCE It is reported that A. baumannii has emerged as an intractable nosocomial pathogen in hospitals especially when it develops resistance to carbapenems and other antibiotics, which limits treatment options and leads to high mortality. In February 2017, the WHO published a list of ESKAPE pathogens designated "priority status" for which new antibiotics are urgently needed. Therefore, the epidemiological surveillance and new therapeutic development of A. baumannii must be strengthened to confront an emerging global epidemic. KBP-7072 is a novel, expanded spectrum tetracycline antibacterial and has demonstrated good in vitro activity against recent geographically diverse A. baumannii isolates collected from North America, Europe, Latin America, and Asia-Pacific. This study has shown excellent in vitro activity of KBP-7072 against clinical A. baumannii isolates collected from different regions of China, regarded as supplementary to KBP-7072 pharmacodynamics data, which is of great significance, as it is promising an alternative treatment in CRAB isolates infections in China.

Activity of the Novel Aminomethylcycline KBP-7072 and Comparators against 1,057 Geographically Diverse Recent Clinical Isolates from the SENTRY Surveillance Program, 2019.

KBP-7072 is a novel broad-spectrum tetracycline (aminomethylcycline) antibacterial in clinical development (oral and intravenous formulations) for the treatment of acute bacterial skin and skin structure infections, community-acquired bacterial pneumonia, and complicated intra-abdominal infections. KBP-7072 is active against many of the World Health Organization priority pathogens. In this study, KBP-7072 and tetracycline class comparators were susceptibility tested against 1,057 geographically diverse surveillance isolates from 2019 according to Clinical and Laboratory Standards Institute (CLSI) guidelines. KBP-7072 demonstrated potent in vitro activity against Gram-positive and Gram-negative bacterial pathogens. KBP-7072 was active against Staphylococcus aureus (MIC50/90, 0.06/0.12 mg/liter), methicillin-resistant S. aureus (MIC50/90, 0.06/0.12 mg/liter), S. lugdunensis (MIC50/90, 0.03/0.03 mg/liter), and other coagulase-negative staphylococci (MIC50/90, 0.06/0.25 mg/liter). KBP-7072 was active against Enterococcus faecalis (MIC50/90, 0.03/0.06 mg/liter) and vancomycin-susceptible and -nonsusceptible E. faecium (MIC50/90, 0.03/0.03 mg/liter); Streptococcus pneumoniae (MIC50/90, ≤0.015/0.03 mg/liter), including penicillin- and tetracycline-resistant strains; S. agalactiae (MIC50/90, 0.03/0.06 mg/liter), including macrolide-resistant strains; S. pyogenes (MIC50/90, 0.03/0.03 mg/liter); and viridans group streptococci, including S. anginosus group (MIC50/90, ≤0.015/0.03 mg/liter) isolates. KBP-7072 inhibited 90.2% (MIC50/90, 0.25/2 mg/liter) of all Enterobacterales isolates, including expanded-spectrum ß-lactamase-phenotype strains at ≤2 mg/liter. KBP-7072 demonstrated potent activity against Acinetobacter baumannii-calcoaceticus species complex and Stenotrophomonas maltophilia isolates (MIC50/90 values, 0.5/1 mg/liter), Haemophilus influenzae (MIC50/90, 0.12/0.25 mg/liter; 100.0% inhibited at ≤0.25 mg/liter), and Moraxella catarrhalis (MIC50/90, 0.06/0.06 mg/liter). Based on MIC90 values, KBP-7072 in vitro activity was generally superior to that the other tetracycline class comparators tested. The potent activity of KBP-7072, including resistant organism groups, merits further clinical investigation in infections where these organisms are likely to occur.

Site-Specific Conjugation of a Selenopolypeptide to Alpha-1-antitrypsin Enhances Oxidation Resistance and Pharmacological Properties.

Human alpha-1-antitrypsin (A1AT), a native serine-protease inhibitor that protects tissue damage from excessive protease activities, is used as an augmentation therapy to treat A1AT-deficienct patients. However, A1AT is sensitive to oxidation-mediated deactivation and has a short circulating half-life. Currently, there is no method that can effectively protect therapeutic proteins from oxidative damage in vivo. Here we developed a novel biocompatible selenopolypeptide and site-specifically conjugated it with A1AT. The conjugated A1AT fully retained its inhibitory activity on neutrophil elastase, enhanced oxidation resistance, extended the serum half-life, and afforded long-lasting protective efficacy in a mouse model of acute lung injury. These results demonstrated that conjugating A1AT with the designed selenopolymer is a viable strategy to improve its pharmacological properties, which could potentially further be applied to a variety of oxidation sensitive biotherapeutics.

In vitro activity of a novel aminomethylcycline antibacterial (KBP-7072), a third-generation tetracycline, against clinical isolates with molecularly characterized tetracycline resistance mechanisms.

OBJECTIVES: This study evaluated the in vitro activity of KBP-7072 against 413 contemporary surveillance isolates, including subsets with known tetracycline resistance genes. MATERIALS: In total, 105 Klebsiella pneumoniae (51 tetracycline resistant), 103 Escherichia coli (52 tetracycline resistant), 103 Staphylococcus aureus (51 tetracycline resistant) and 102 Streptococcus pneumoniae (51 tetracycline resistant) isolates were included. These isolates were tested by broth microdilution using fresh media. CLSI/EUCAST breakpoints were applied, except for tigecycline and omadacycline, which used FDA criteria. RESULTS: KBP-7072 (MIC50, 0.06 mg/L), tigecycline (MIC50, 0.12 and 0.25 mg/L) and omadacycline (MIC50, 0.12 and 0.5 mg/L) showed similar MIC50s for tetracycline-susceptible and -resistant S. aureus. Other tetracycline comparators had their MIC50 increased 64- to 256-fold by tet. For S. pneumoniae, KBP-7072 (MIC50/90, ≤0.015/0.03 mg/L) showed the lowest MICs, which remained unchanged for tetracycline-susceptible or -resistant isolates [mostly tet(M)]. Similar MICs were observed for omadacycline (MIC50/90, 0.03-0.06/0.06 mg/L) and tigecycline (MIC50/90, 0.03/0.03 mg/L) in the S. pneumoniae population. Tetracycline-susceptible and -resistant E. coli [94.2% tet(A)/tet(B)], KBP-7072 (MIC90, 0.25 and 1 mg/L, respectively) and tigecycline (MIC90, 0.25 and 0.5 mg/L) showed similar MIC90s. KBP-7072 (MIC50/90, 0.25/0.5 mg/L) and tigecycline (MIC50/90, 0.5/0.5 mg/L) had the lowest MIC for tetracycline-susceptible K. pneumoniae. The MIC for KBP-7072 (MIC50/90, 1/4 mg/L) and tigecycline (MIC50/90, 1/2 mg/L) increased 2- to 8-fold for tetracycline-resistant K. pneumoniae, which mostly produced Tet(A). CONCLUSIONS: KBP-7072 activity was minimally affected by the presence of acquired tetracycline genes.

The Non-Steroidal Mineralocorticoid Receptor Antagonist KBP-5074 Limits Albuminuria and has Improved Therapeutic Index Compared With Eplerenone in a Rat Model With Mineralocorticoid-Induced Renal Injury.

The therapeutic indices (TIs) and efficacy of the non-steroidal mineralocorticoid receptor antagonist (MRA) KBP-5074 and steroidal MRA eplerenone were evaluated in a uninephrectomized Sprague Dawley rat model of aldosterone-mediated renal disease. In two parallel studies, rats were placed on a high-salt diet and received aldosterone by osmotic mini-pump infusion over the course of 27 days. The urinary albumin-to-creatinine ratio (UACR) was evaluated after 7, 14, and 26 days of treatment. Serum K+ was evaluated after 14 and 27 days of treatment. Urinary Na+, urinary K+, and urinary Na+/K+ ratio were evaluated after 7, 14, and 26 days of treatment. The TI was calculated for each drug as the ratio of the concentration of drug producing 50% of maximum effect (EC50) for increasing serum K+ to the EC50 for lowering UACR. The TIs were 24.5 for KBP-5074 and 0.620 for eplerenone, resulting in a 39-fold improved TI for KBP-5074 compared with eplerenone. Aldosterone treatment increased UACR, decreased serum K+, and decreased urinary Na+ relative to sham-operated controls that did not receive aldosterone infusion in both studies, validating the aldosterone/salt renal injury model. KBP-5074 prevented the increase in UACR at 0.5, 1.5, and 5 mg/kg BID while eplerenone did so only at the two highest doses of 50 and 450 mg/kg BID. Both KBP-5074 and eplerenone blunted the reduction in serum K+ seen in the aldosterone treatment group, with significant increases in serum K+ at the high doses only (5 mg/kg and 450 mg/kg BID, respectively). Additionally, the urinary Na+ and Na+/K+ ratio significantly increased at the middle and high doses of KBP-5074, but only at the highest dose of eplerenone. These results showed increased TI and efficacy for KBP-5074 compared with eplerenone over a wider therapeutic window.

In vitro activity of KHP-3757 (a novel LpxC inhibitor) and comparator agents against recent and molecularly characterized Pseudomonas aeruginosa isolates from a global surveillance program (2017-2018).

This study evaluated the in vitro activity of KHP-3757 (a novel LpxC inhibitor) and comparator agents against recent, geographically diverse, Pseudomonas aeruginosa isolates from a global surveillance program as well as molecularly characterized extended-spectrum-ß-lactamase-positive, metallo-ß-lactamase-positive, and colistin-resistant strains. KHP-3757 (MIC50/90, 0.25/0.5 mg/L; 97.4% inhibited at ≤0.5 mg/L) demonstrated potent in vitro activity based on MIC90 values against P. aeruginosa isolates including extended-spectrum-ß-lactamase-positive, metallo-ß-lactamase-positive, and colistin-resistant strains outperforming other comparator agents.

Nonclinical Pharmacokinetics, Protein Binding, and Elimination of KBP-7072, an Aminomethylcycline Antibiotic, in Animal Models.

KBP-7072 is a semisynthetic aminomethylcycline with broad-spectrum activity against Gram-positive and Gram-negative pathogens, including multidrug-resistant bacterial strains. The pharmacokinetics (PK) of KBP-7072 after oral and intravenous (i.v.) administrations of single and multiple doses were investigated in animal models, including during fed and fasted states, and the protein binding and excretion characteristics were also evaluated. In Sprague-Dawley (SD) rats, beagle dogs, and CD-1 mice, KBP-7072 demonstrated a linear PK profile after the administration of single oral and i.v. and multiple oral doses. The oral bioavailability ranged from 12% to 32%. The mean time to maximum concentration (Tmax) ranged from 0.5 to 4 h, and the mean half-life ranged from approximately 6 to 11 h. The administration of oral doses in the fed state resulted in marked reductions in the maximum plasma concentration (Cmax) and the area under the concentration-time curve (AUC) compared with dosing in fasted animals. The mean bound fractions of KBP-7072 were 77.5%, 69.8%, 64.5%, 69.3%, and 69.2% in mouse, rat, dog, monkey, and human plasma, respectively. Following a single 22.5-mg/kg oral dose of KBP-7072 in SD rats, the cumulative excretion in feces was 64% and that in urine was 2.5% of the administered dose. The PK results in animal models are consistent with single- and multiple-ascending-dose studies in healthy volunteers and confirm the suitability of KBP-7072 for once-daily oral and i.v. administration in clinical studies.

In Vitro Activity of KBP-7072, a Novel Third-Generation Tetracycline, against 531 Recent Geographically Diverse and Molecularly Characterized Acinetobacter baumannii Species Complex Isolates.

KBP-7072 is a novel third-generation tetracycline (aminomethylcycline) antibacterial that overcomes common efflux and ribosomal protection resistance mechanisms that cause resistance in older-generation tetracyclines. KBP-7072 completed phase 1 clinical development studies for safety, tolerability, and pharmacokinetics (ClinicalTrials.gov identifier NCT02454361) and multiple ascending doses in healthy subjects (ClinicalTrials.gov identifier NCT02654626) in December 2015. Both oral and intravenous formulations of KBP-7072 are being developed. In this study, we evaluated the in vitro activities of KBP-7072 and comparator agents by CLSI document M07 (2018) broth microdilution against 531 recent geographically diverse and/or molecularly characterized Acinetobacter baumannii-A. calcoaceticus species complex (A. baumannii) isolates from the United States, Europe, Asia-Pacific (excluding China), and Latin America. A. baumannii isolates included carbapenem-resistant, colistin-resistant, tetracycline-resistant, and extended-spectrum-ß-lactamase (ESBL)- and metallo-ß-lactamase (MBL)-producing isolates. Overall, KBP-7072 (MIC50/90, 0.25/1 mg/liter) was comparable in activity to colistin (92.8%/92.8% susceptible [S] [CLSI/EUCAST]) against A. baumannii isolates, inhibiting 99.2% of isolates at ≤2 mg/liter and 97.6% of isolates at ≤1 mg/liter. KBP-7072 was equally active against A. baumannii isolates, including carbapenem-resistant, colistin-resistant, and tetracycline-resistant isolates, regardless of geographic location, and maintained activity against ESBL- and MBL-producing isolates. KBP-7072 outperformed comparator agents, including ceftazidime (40.3% S [CLSI]), gentamicin (48.2%/48.2% S [CLSI/EUCAST]), levofloxacin (39.5%/37.9% S [CLSI/EUCAST]), meropenem (42.0%/42.0% S [CLSI/EUCAST]), piperacillin-tazobactam (33.3% S [CLSI]), and all tetracycline-class comparator agents, which include doxycycline (67.3% S [CLSI]), minocycline (73.8% S [CLSI]), tetracycline (37.2% S [CLSI]), and tigecycline (79.5% inhibited by ≤2 mg/liter). The potent in vitro activity of KBP-7072 against recent geographically diverse, molecularly characterized, and drug-resistant A. baumannii isolates supports continued clinical development for the treatment of serious infections, including those caused by A. baumannii.

A novel role for the actin-binding protein drebrin in regulating opiate addiction.

Persistent transcriptional and morphological events in the nucleus accumbens (NAc) and other brain reward regions contribute to the long-lasting behavioral adaptations that characterize drug addiction. Opiate exposure reduces the density of dendritic spines on medium spiny neurons of the NAc; however, the underlying transcriptional and cellular events mediating this remain unknown. We show that heroin self-administration negatively regulates the actin-binding protein drebrin in the NAc. Using virus-mediated gene transfer, we show that drebrin overexpression in the NAc is sufficient to decrease drug seeking and increase dendritic spine density, whereas drebrin knockdown potentiates these effects. We demonstrate that drebrin is transcriptionally repressed by the histone modifier HDAC2, which is relieved by pharmacological inhibition of histone deacetylases. Importantly, we demonstrate that heroin-induced adaptations occur only in the D1+ subset of medium spiny neurons. These findings establish an essential role for drebrin, and upstream transcriptional regulator HDAC2, in opiate-induced plasticity in the NAc.

Favorable prognosis and high discrepancy of genetic features in surgical patients with multiple primary lung cancers.

OBJECTIVE: Multiple primary lung cancers are detected with increasing frequency, but the ideal strategy for diagnosis and treatment remains disputable. This study evaluated both clinical characteristics and genetic alterations to investigate the appropriate strategy for patients with multiple primary lung cancer. METHODS: A total of 96 patients in our practice were diagnosed with multiple primary lung cancer over 7 years by clinical-pathologic criteria. According to consolidation/tumor ratio, they were classified into 3 groups: group A (multiple ground-glass opacity-dominant nodules, consolidation/tumor ratio ≤0.5), group B (1 solid-dominant nodule, consolidation/tumor ratio >0.5 with other ground-glass opacity-dominant nodules), and group C (2 solid-dominant nodules). A series of somatic genetic mutations and fusions were analyzed in a portion of the patients. RESULTS: There were 24, 35, and 37 patients in groups A, B, and C, respectively. During follow-up, 23 patients had recurrence. The 5-year recurrence-free survival was 100% in patients with multiple ground-glass opacity, 68% in those with 1 solid lesion, and 51.4% in those with 2 solid tumors (P = .001). Eighteen patients died of lung cancer. The 5-year overall survival was 100% in group A, 80.5% in group B, and 59.9% in group C (P = .002). A total of 77 driver mutations were detected in 61 of the 82 lesions. A high rate of discordance of genetic alterations (89.7%) was found between cancers within individual patients. Two patients in group C had concordant driver mutations between the 2 lesions, and both of them harbored tumor recurrence. CONCLUSIONS: A high discordance of driver mutations between tumors in individual patients and a favorable prognosis were identified in patients with multiple primary lung cancers diagnosed by clinical-pathologic criteria, which support different strategies from those with metastatic disease.

Prediction of Fluoroquinolone Susceptibility Directly from Whole-Genome Sequence Data by Using Liquid Chromatography-Tandem Mass Spectrometry To Identify Mutant Genotypes.

Fluoroquinolone resistance in Gram-negative bacteria is multifactorial, involving target site mutations, reductions in fluoroquinolone entry due to reduced porin production, increased fluoroquinolone efflux, enzymes that modify fluoroquinolones, and Qnr, a DNA mimic that protects the drug target from fluoroquinolone binding. Here we report a comprehensive analysis, using transformation and in vitro mutant selection, of the relative importance of each of these mechanisms for fluoroquinolone nonsusceptibility using Klebsiella pneumoniae as a model system. Our improved biological understanding was then used to generate 47 rules that can predict fluoroquinolone susceptibility in K. pneumoniae clinical isolates. Key to the success of this predictive process was the use of liquid chromatography-tandem mass spectrometry to measure the abundance of proteins in extracts of cultured bacteria, identifying which sequence variants seen in the whole-genome sequence data were functionally important in the context of fluoroquinolone susceptibility.

Comparison of plasma to tissue DNA mutations in surgical patients with non-small cell lung cancer.

OBJECTIVE: Noninvasive liquid biopsies of circulating tumor DNA (ctDNA) can be used to assess non-small cell lung cancer (NSCLC), but previous work focused on patients with advanced-stage cancer. Thus, we evaluated the feasibility and their potential clinical application of circulating tumor DNA approached for surgical patients with NSCLC. METHOD: Consecutive patients with suspected lung cancer who underwent curative-intent lung resection were enrolled prospectively in this study. Targeted DNA sequencing with a next-generation sequencing platform was used to identify a series of somatic mutations in matched tumor tissue DNA (tDNA) and plasma ctDNA samples. Plasma was collected before, during, and after surgery. Concordance was defined as matched tDNA and ctDNA with the same identified mutations or with no mutations. RESULTS: In the enrolled 76 patients with lung cancer who were included, 31 had concordant mutations and 21 had no mutation in both ctDNA and tDNA, yielding an overall concordance of 68.4%. ctDNA samples obtained before and during surgery had the same mutations with a low variance in mutation frequency (1.2%) that was reduced to an average of 0.28% after surgery (P < .001). More patients were positive as assayed by ctDNA (48; 63.2%) than with serum tumor protein markers (36; 49.3%). The area under the curve was greater in ctDNA (0.887, 95% confidence interval [CI], 0.788-0.986) than for the 2 prediction models (0.803, 95% CI, 0.647-0.959; 0.69, 95% CI, 0.512-0.869) for estimating malignancy of solitary pulmonary nodules. CONCLUSION: ctDNA mutation analysis for stage I-III surgical patients with NSCLC is feasible. More studies are needed to investigate its clinical application.

Asymmetric transcript discovery by RNA-seq in C. elegans blastomeres identifies neg-1, a gene important for anterior morphogenesis.

After fertilization but prior to the onset of zygotic transcription, the C. elegans zygote cleaves asymmetrically to create the anterior AB and posterior P1 blastomeres, each of which goes on to generate distinct cell lineages. To understand how patterns of RNA inheritance and abundance arise after this first asymmetric cell division, we pooled hand-dissected AB and P1 blastomeres and performed RNA-seq. Our approach identified over 200 asymmetrically abundant mRNA transcripts. We confirmed symmetric or asymmetric abundance patterns for a subset of these transcripts using smFISH. smFISH also revealed heterogeneous subcellular patterning of the P1-enriched transcripts chs-1 and bpl-1. We screened transcripts enriched in a given blastomere for embryonic defects using RNAi. The gene neg-1 (F32D1.6) encoded an AB-enriched (anterior) transcript and was required for proper morphology of anterior tissues. In addition, analysis of the asymmetric transcripts yielded clues regarding the post-transcriptional mechanisms that control cellular mRNA abundance during asymmetric cell divisions, which are common in developing organisms.

Transcriptome Analysis Reveals Novel Entry Mechanisms and a Central Role of SRC in Host Defense during High Multiplicity Mycobacterial Infection.

Mycobacterium tuberculosis (MTB) infects an estimated one-third of the global population and is one of the main causes of mortality from an infectious agent. The characteristics of macrophages challenged by MTB with a high multiplicity of infection (MOI), which mimics both clinical disseminated infection and granuloma formation, are distinct from macrophages challenged with a low MOI. To better understand the cross talk between macrophage host cells and mycobacteria, we compared the transcription patterns of mouse macrophages infected with bacille Calmette-Guérin, H37Ra and M. smegmatis. Attention was focused on the changes in the abundance of transcripts related to immune system function. From the results of a transcriptome profiling study with a high mycobacterial MOI, we defined a pathogen-specific host gene expression pattern. The present study suggests that two integrins, ITGA5 and ITGAV, are novel cell surface receptors mediating mycobacterium entry into macrophages challenged with high MOI. Our results indicate that SRC likely plays a central role in regulating multiple unique signaling pathways activated by MTB infection. The integrated results increase our understanding of the molecular networks behind the host innate immune response and identify important targets that might be useful for the development of tuberculosis therapy.

An in vivo screen of secreted proteins identifies adiponectin as a regulator of murine cutaneous wound healing.

Skin wounds comprise a serious medical issue for which few pharmacological interventions are available. Moreover, the inflammatory, angiogenic, and proliferative facets of a typical response to a wound each have broader relevance in other pathological conditions. Here we describe a genomics-driven approach to identify secreted proteins that modulate wound healing in a mouse ear punch model. We show that adiponectin, when injected into the wound edge, accelerates wound healing. Notably, adiponectin injection causes upregulation of keratin gene transcripts within hours of treatment, and subsequently promotes collagen organization, formation of pilosebaceous units, and proliferation of cells in the basal epithelial cell layer and pilosebaceous units of healing tissue. The globular domain of adiponectin is sufficient to mediate accelerated dorsal skin wound closure, and the effects are lost in mice that are homozygous null for the adiponectin receptor 1 gene. These findings extend recent observations of a protective role of adiponectin in other tissue injury settings, suggest modulation of AdipoR1 for the clinical management of wounds, and demonstrate a new approach to the identification of regulators of a wound healing response.

Regenerative phenotype in mice with a point mutation in transforming growth factor beta type I receptor (TGFBR1).

Regeneration of peripheral differentiated tissue in mammals is rare, and regulators of this process are largely unknown. We carried out a forward genetic screen in mice using N-ethyl-N-nitrosourea mutagenesis to identify genetic mutations that affect regenerative healing in vivo. More than 400 pedigrees were screened for closure of a through-and-through punch wound in the mouse ear. This led to the identification of a single pedigree with a heritable, fast, and regenerative wound-healing phenotype. Within 5 wk after ear-punch, a threefold decrease in the diameter of the wound was observed in the mutant mice compared with the wild-type mice. At 22 wk, new cartilage, hair follicles, and sebaceous glands were observed in the newly generated tissue. This trait was mapped to a point mutation in a receptor for TGF-ß, TGFBR1. Mouse embryonic fibroblasts from the affected mice had increased expression of a subset of TGF-ß target genes, suggesting that the mutation caused partial activation of the receptor. Further, bone marrow stromal cells from the mutant mice more readily differentiated to chondrogenic precursors, providing a plausible explanation for the enhanced development of cartilage islands in the regenerated ears. This mutant mouse strain provides a unique model to further explore regeneration in mammals and, in particular, the role of TGFBR1 in chondrogenesis and regenerative wound healing.