⁶⁸Ga-labeled urea-based inhibitors of the prostate-specific membrane antigen (PSMA), such as ⁶⁸Ga-PSMA-11, are promising small molecules for targeting prostate cancer (PCa). Although this radiopharmaceutical was produced mostly by means of manual synthesis and automated synthesis modules, a sterile cold kit was recently introduced. The aim of our study was to evaluate the image quality of ⁶⁸Ga-PSMA-11 PET/CT (PSMA-PET) in a population of PCa patients after the injection of comparable activities of ⁶⁸Ga-PSMA-11 obtained with the 2 different synthetic procedures. A secondary aim was to identify secondary factors that may have an impact on image quality and, thus, final interpretation. Methods: Two different groups of 100 consecutive PCa patients who underwent PSMA-PET were included in the study. The first group of patients was imaged with ⁶⁸Ga-PSMA-11 obtained using synthesis modules, whereas the second group’s tracer activity was synthesized using a sterile cold kit. All PET images were independently reviewed by 2 nuclear medicine diagnosticians with at least 2 y of experience in PSMA-based imaging and unaware of the patients’ clinical history. The 2 reviewers independently rated the quality of each PSMA-PET scan using a 3-point Likert-type scale. In cases of discordance, the operators together reviewed the images and reached a consensus. Performance was evaluated on the basis of the expected biodistribution, lesion detection rate, and physiologic background uptake. Results: Overall, 104 of 200 (52%) PSMA-PET scans were positive for PCa-related findings. No significant differences in image quality between cold kits and synthesis modules were found (P = 0.13), although a higher proportion of images was rated as excellent by the observers for kits than for modules (45% vs. 34%). Furthermore, after image quality had been dichotomized as excellent or not excellent, multivariate regression analysis found several factors to be significantly associated with a not-excellent quality: an increase in patient age (+5 y: odds ratio [OR], 1.40; 95% confidence interval [CI], 1.12–1.75), an increase in patient weight (+5 kg: OR, 1.89; 95% CI, 1.53–2.32), an increase in ⁶⁸Ga-PSMA-11 uptake time (+10 min: OR, 1.45; 95% CI, 1.08–1.96), and a decrease in injected activity (–10 MBq: OR, 1.28; 95% CI, 1.07–1.52). Conclusion: No significant differences were identified between the 2 groups of patients undergoing PSMA-PET; therefore, we were not able to ascertain any significant influences of tracer production methodology on final scan quality. However, increased patient age, increased patient weight, decreased injected activity, and increased ⁶⁸Ga-PSMA-11 uptake time were significantly associated with an overall poorer image quality.

Neuroendocrinelike transdifferentiation of prostate cancer adenocarcinomas correlates with serum levels of chromogranin A (CgA) and drives treatment resistance. The aim of this work was to evaluate whether CgA can serve as a response predictor for ¹⁷⁷Lu-prostate-specific membrane antigen 617 (PSMA) radioligand therapy (RLT) in comparison with the established tumor markers. Methods: One hundred consecutive patients with metastasized castration-resistant prostate cancer scheduled for PSMA RLT were evaluated for prostate-specific antigen (PSA), lactate dehydrogenase (LDH), and CgA at baseline and in follow-up of PSMA RLT. Tumor uptake of PSMA ligand, a known predictive marker for response, was assessed as a control variable. Results: From the 100 evaluated patients, 35 had partial remission, 16 stable disease, 15 mixed response, and 36 progression of disease. Tumor uptake above salivary gland uptake translated into partial remission, with an odds ratio (OR) of 60.265 (95% confidence interval [CI], 5.038–720.922). Elevated LDH implied a reduced chance for partial remission, with an OR of 0.094 (95% CI, 0.017–0.518), but increased the frequency of progressive disease (OR, 2.717; 95% CI, 1.391–5.304). All patients who achieved partial remission had a normal baseline LDH. Factor-2 elevation of CgA increased the risk for progression, with an OR of 3.089 (95% CI, 1.302–7.332). Baseline PSA had no prognostic value for response prediction. Conclusion: In our cohort, baseline PSA had no prognostic value for response prediction. LDH was the marker with the strongest prognostic value, and elevated LDH increased the risk for progression of disease under PSMA RLT. Elevated CgA demonstrated a moderate impact as a negative prognostic marker in general but was explicitly related to the presence of liver metastases. Well in line with the literature, sufficient tumor uptake is a prerequisite to achieve tumor response.

Prostate-specific membrane antigen (PSMA)–targeting α-radiation therapy (TAT) is an emerging treatment modality for metastatic castration-resistant prostate cancer. There is a subgroup of patients with poor response despite sufficient expression of PSMA in their tumors. The aim of this work was to characterize PSMA-TAT–nonresponding lesions by targeted next-generation sequencing. Methods: Of 60 patients treated with ²²⁵Ac-PSMA-617, we identified 10 patients who presented with a poor response despite sufficient tumor uptake in PSMA PET/CT. We were able to perform CT-guided biopsies with histologic validation of the nonresponding lesions in 7 of these nonresponding patients. Specimens were analyzed by targeted next-generation sequencing interrogating 37 DNA damage-repair–associated genes. Results: In the 7 tumor samples analyzed, we found a total of 15 whole-gene deletions, deleterious or presumably deleterious mutations affecting TP53 (n = 3), CHEK2 (n = 2), ATM (n = 2), and BRCA1, BRCA2, PALB2, MSH2, MSH6, NBN, FANCB, and PMS1 (n = 1 each). The average number of deleterious or presumably deleterious mutations was 2.2 (range, 0–6) per patient. In addition, several variants of unknown significance in ATM, BRCA1, MSH2, SLX4, ERCC, and various FANC genes were detected. Conclusion: Patients with resistance to PSMA-TAT despite PSMA positivity frequently harbor mutations in DNA damage-repair and checkpoint genes. Although the causal role of these alterations in the patient outcome remains to be determined, our findings encourage future studies combining PSMA-TAT and DNA damage-repair–targeting agents such as poly(ADP-ribose)-polymerase inhibitors.

Parallel to the application of new PET radiopharmaceuticals for pheochromocytoma and paraganglioma (collectively named PPGLs) imaging, several studies have increased our understanding on their biology, genetics, metabolomics, and embryologic origin. In this review, we highlight the current relationship between genotypes and molecular imaging phenotypes. Additionally, we summarize the referral guidelines for imaging of PPGL patients with or without knowledge of their genetic background.

Management of cystic fibrosis has been revolutionised by the introduction of cystic fibrosis transmembrane conductance regulator (CFTR) modulators. These compounds treat the underlying molecular basis of the disease by increasing activity of defective CFTR channels, which improves many clinical parameters and enhances patient quality of life [1]. Next-generation modulators, also known as triple combination therapy, promise to be highly efficacious in up to 90% of patients [2] and will likely dramatically change the landscape of cystic fibrosis disease. Studies examining individuals before and after initiation of CFTR modulators have revealed novel functions of CFTR and shown that CFTR modulators do not reverse all disease manifestations [3–5]. Thus, knowledge of the post-modulator cystic fibrosis disease state is crucial for understanding what continued therapies will be needed for people with cystic fibrosis and what new challenges may arise.

Background

Positive serology for cytomegalovirus (CMV) has been associated with all-cause mortality risk but its role in COPD mortality is unknown. The objective of the present study was to assess the relationship between CMV serology and COPD mortality.

Electronic cigarettes (e-cigarettes) have been used in many countries for >10 years and in this time, there has been a division of opinions amongst both the general public and health professionals regarding the benefit or harms of e-cigarettes. Prior to the reporting of a new phenomenon known as vaping-associated pulmonary injury (VAPI), public opinion about the relative harm of e-cigarettes were increasing but they were perceived as less harmful than cigarettes by one third of people [1]. The recent cases of severe illness and death attributable to VAPI were first described in September 2019 [2]. VAPI appears to be related to either the addition of cannabis/cannabis derivates or vitamin E acetate [3], and as such has not caused radical swing away from the use of e-cigarettes without cannabis or cannabis derivates.

ABSTRACT

Bezlotoxumab is a human monoclonal antibody against Clostridium difficile toxin B, indicated to prevent recurrence of C. difficile infection (rCDI) in high-risk adults receiving antibacterial treatment for CDI. An exploratory genome-wide association study investigated whether human genetic variation influences bezlotoxumab response. DNA from 704 participants who achieved initial clinical cure in the phase 3 MODIFY I/II trials was genotyped. Single nucleotide polymorphisms (SNPs) and human leukocyte antigen (HLA) imputation were performed using IMPUTE2 and HIBAG, respectively. A joint test of genotype and genotype-by-treatment interaction in a logistic regression model was used to screen genetic variants associated with response to bezlotoxumab. The SNP rs2516513 and the HLA alleles HLA-DRB1*07:01 and HLA-DQA1*02:01, located in the extended major histocompatibility complex on chromosome 6, were associated with the reduction of rCDI in bezlotoxumab-treated participants. Carriage of a minor allele (homozygous or heterozygous) at any of the identified loci was related to a larger difference in the proportion of participants experiencing rCDI versus placebo; the effect was most prominent in the subgroup at high baseline risk for rCDI. Genotypes associated with an improved bezlotoxumab response showed no association with rCDI in the placebo cohort. These data suggest that a host-driven, immunological mechanism may impact bezlotoxumab response. Trial registration numbers are as follows: NCT01241552 (MODIFY I) and NCT01513239 (MODIFY II).

IMPORTANCE Clostridium difficile infection is associated with significant clinical morbidity and mortality; antibacterial treatments are effective, but recurrence of C. difficile infection is common. In this genome-wide association study, we explored whether host genetic variability affected treatment responses to bezlotoxumab, a human monoclonal antibody that binds C. difficile toxin B and is indicated for the prevention of recurrent C. difficile infection. Using data from the MODIFY I/II phase 3 clinical trials, we identified three genetic variants associated with reduced rates of C. difficile infection recurrence in bezlotoxumab-treated participants. The effects were most pronounced in participants at high risk of C. difficile infection recurrence. All three variants are located in the extended major histocompatibility complex on chromosome 6, suggesting the involvement of a host-driven immunological mechanism in the prevention of C. difficile infection recurrence.

The bioprocessing industry uses recombinant mammalian cell lines to generate therapeutic biologic drugs. To ensure consistent product quality of the therapeutic proteins, it is imperative to have a controlled production process. Regulatory agencies and the biotechnology industry consider cell line "clonal origin" an important aspect of maintaining process control. Demonstration of clonal origin of the cell substrate, or production cell line, has received considerable attention in the past few years, and the industry has improved methods and devised standards to increase the probability and/or assurance of clonal derivation. However, older production cell lines developed before the implementation of these methods, herein referred to as "legacy cell lines," may not meet current regulatory expectations for demonstration of clonal derivation. In this article, the members of the IQ Consortium Working Group on Clonality present our position that the demonstration of process consistency and product comparability of critical quality attributes throughout the development life cycle should be sufficient to approve a license application without additional genetic analysis to support clonal origin, even for legacy cell lines that may not meet current day clonal derivation standards. With this commentary, we discuss advantages and limitations of genetic testing methods to support clonal derivation of legacy cell lines and wish to promote a mutual understanding with the regulatory authorities regarding their optional use during early drug development, subsequent to Investigational New Drug (IND) application and before demonstration of product and process consistency at Biologics License Applications (BLA) submission.

Glass is the favorite material for parenteral packaging because of its physico-chemical properties. Type I borosilicate glass is worldwide use at this scope, but it may have some issues related to breakage, corrosion and delamination that might compromise the drug quality, safety and efficacy. These issues can be mitigated and avoided starting from the appropriate selection of the most suitable raw material at the early stage of the glass container design. In this study, Type I borosilicate glass vials manufactured using two glass tubes having different chemical compositions, were studied and compared in terms of their resistance to corrosion. Testing design was applied with the aim to select the best practice approach comparing different storage simulation conditions: ageing treatment through autoclaving and stability testing (real-time and accelerated). Clear differences were found between the different glass types in terms of hydrolytic and corrosion resistance that highlighted the relation between chemical composition and glass chemical durability. Non-negligible differences were also observed using different storage conditions.

Viruses and mobile genetic elements are molecular parasites or symbionts that coevolve with nearly all forms of cellular life. The route of virus replication and protein expression is determined by the viral genome type. Comparison of these routes led to the classification of viruses into seven "Baltimore classes" (BCs) that define the major features of virus reproduction. However, recent phylogenomic studies identified multiple evolutionary connections among viruses within each of the BCs as well as between different classes. Due to the modular organization of virus genomes, these relationships defy simple representation as lines of descent but rather form complex networks. Phylogenetic analyses of virus hallmark genes combined with analyses of gene-sharing networks show that replication modules of five BCs (three classes of RNA viruses and two classes of reverse-transcribing viruses) evolved from a common ancestor that encoded an RNA-directed RNA polymerase or a reverse transcriptase. Bona fide viruses evolved from this ancestor on multiple, independent occasions via the recruitment of distinct cellular proteins as capsid subunits and other structural components of virions. The single-stranded DNA (ssDNA) viruses are a polyphyletic class, with different groups evolving by recombination between rolling-circle-replicating plasmids, which contributed the replication protein, and positive-sense RNA viruses, which contributed the capsid protein. The double-stranded DNA (dsDNA) viruses are distributed among several large monophyletic groups and arose via the combination of distinct structural modules with equally diverse replication modules. Phylogenomic analyses reveal the finer structure of evolutionary connections among RNA viruses and reverse-transcribing viruses, ssDNA viruses, and large subsets of dsDNA viruses. Taken together, these analyses allow us to outline the global organization of the virus world. Here, we describe the key aspects of this organization and propose a comprehensive hierarchical taxonomy of viruses.

The type VI secretion system (T6SS) is a weapon for delivering effectors into target cells that is widespread in Gram-negative bacteria. The T6SS is a highly versatile machine, as it can target both eukaryotic and prokaryotic cells, and it has been proposed that T6SSs are adapted to the specific needs of each bacterium. The expression of T6SS gene clusters and the activation of the secretion apparatus are therefore tightly controlled. In enteroaggregative Escherichia coli (EAEC), the sci1 T6SS gene cluster is subject to a complex regulation involving both the ferric uptake regulator (Fur) and DNA adenine methylase (Dam)-dependent DNA methylation. In this study, an additional, internal, promoter was identified within the sci1 gene cluster using +1 transcriptional mapping. Further analyses demonstrated that this internal promoter is controlled by a mechanism strictly identical to that of the main promoter. The Fur binding box overlaps the –10 transcriptional element and a Dam methylation site, GATC-32. Hence, the expression of the distal sci1 genes is repressed and the GATC-32 site is protected from methylation in iron-rich conditions. The Fur-dependent protection of GATC-32 was confirmed by an in vitro methylation assay. In addition, the methylation of GATC-32 negatively impacted Fur binding. The expression of the sci1 internal promoter is therefore controlled by iron availability through Fur regulation, whereas Dam-dependent methylation maintains a stable ON expression in iron-limited conditions.

IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Its expression and activation therefore need to be tightly regulated. Here, we identified an internal promoter within the sci1 T6SS gene cluster in enteroaggregative E. coli. We show that this internal promoter is controlled by Fur and Dam-dependent methylation. We further demonstrate that Fur and Dam compete at the –10 transcriptional element to finely tune the expression of T6SS genes. We propose that this elegant regulatory mechanism allows the optimum production of the T6SS in conditions where enteroaggregative E. coli encounters competing species.

OBJECTIVE

Debate continues regarding the influence of dietary fats and sugars on the risk of developing metabolic diseases, including insulin resistance and nonalcoholic fatty liver disease (NAFLD). We investigated the effect of two eucaloric diets, one enriched with saturated fat (SFA) and the other enriched with free sugars (SUGAR), on intrahepatic triacylglycerol (IHTAG) content, hepatic de novo lipogenesis (DNL), and whole-body postprandial metabolism in overweight males.

OBJECTIVE

Hispanics/Latinos are the largest ethnic/racial group in the U.S., have the highest prevalence of diabetes, and are at increased risk for neurodegenerative disorders. Currently, little is known about the relationship between diabetes and cognitive decline and disorders among diverse Hispanics/Latinos. The purpose of this study is to clarify these relationships in diverse middle-aged and older Hispanics/Latinos.

OBJECTIVE

With rising health care costs and finite health care resources, understanding the population needs of different type 2 diabetes mellitus (T2DM) patient subgroups is important. Sparse data exist for the application of population segmentation on health care needs among Asian T2DM patients. We aimed to segment T2DM patients into distinct classes and evaluate their differential health care use, diabetes-related complications, and mortality patterns.

MYC translocations, a hallmark of Burkitt lymphoma, occur in 5-15% of diffuse large B-cell lymphoma, and have a negative prognostic impact. Numerical aberrations of MYC have also been detected in these patients, but their incidence and prognostic role are still controversial. We analyzed the clinical impact of MYC increased copy number on 385 patients with diffuse large B-cell lymphoma screened at diagnosis for MYC, BCL2, and BCL6 rearrangements. We enumerated the number of MYC copies, defining as amplified those cases with an uncountable number of extra-copies. The prevalence of MYC translocation, increased copy number and amplification was 8.8%, 15%, and 1%, respectively. Patients with 3 or 4 gene copies, accounting for more than 60% of patients with MYC copy number changes, had a more favorable outcome compared to patients with >4 copies or translocation of MYC, and were not influenced by the type of treatment received as first-line. Stratification according to the number of MYC extra-copies showed a negative correlation between an increasing number of copies and survival. Patients with >7 copies or the amplification of MYC had the poorest prognosis. Patients with >4 copies of MYC showed a similar, trending towards worse prognosis compared to patients with MYC translocation. The survival of patients with >4 copies, translocation or amplification of MYC seemed to be superior if intensive treatments were used. Our study underlines the importance of fluorescence in situ hybridization testing at diagnosis of diffuse large B-cell lymphoma to detect the rather frequent and clinically significant numerical aberrations of MYC.

Tlarge granular lymphocyte leukemia (T-LGLL) is characterized by the expansion of several large granular lymphocyte clones, among which a subset of large granular lymphocytes showing constitutively activated STAT3, a specific CD8⁺/CD4^– phenotype and the presence of neutropenia has been identified. Although STAT3 is an inducer of transcription of a large number of oncogenes, so far its relationship with miRNAs has not been evaluated in T-LGLL patients. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. The expression level of 756 mature miRNA was assessed on purified T large granular lymphocytes (T-LGLs) by using a TaqMan Human microRNA Array. Hierarchical Clustering Analysis of miRNA array data shows that the global miRNome clusters with CD8 T-LGLs. Remarkably, CD8 T-LGLs exhibit a selective and STAT3-dependent repression of miR-146b expression, that significantly correlated with the absolute neutrophil counts and inversely correlated with the expression of Fas ligand (FasL), that is regarded as the most relevant factor in the pathogenesis of neutropenia. Experimental evidence demonstrates that the STAT3-dependent reduction of miR-146b expression in CD8 T-LGLs occurs as a consequence of miR-146b promoter hypermethylation and results in the disruption of the HuR-mediated post-transcriptional machinery controlling FasL mRNA stabilization. Restoring miR-146b expression in CD8 T-LGLs lead to a reduction of HuR protein and, in turn, of FasL mRNA expression, thus providing mechanistic insights for the existence of a STAT3-miR146b-FasL axis and neutropenia in T-LGLL.

Megakaryoblastic leukemia 1 (MKL1) is a coactivator of serum response factor and together they regulate transcription of actin cytoskeleton genes. MKL1 is associated with hematologic malignancies and immunodeficiency, but its role in B cells is unexplored. Here we examined B cells from monozygotic triplets with an intronic deletion in MKL1, two of whom had been previously treated for Hodgkin lymphoma (HL). To investigate MKL1 and B-cell responses in the pathogenesis of HL, we generated Epstein-Barr virus-transformed lymphoblastoid cell lines from the triplets and two controls. While cells from the patients with treated HL had a phenotype close to that of the healthy controls, cells from the undiagnosed triplet had increased MKL1 mRNA, increased MKL1 protein, and elevated expression of MKL1-dependent genes. This profile was associated with elevated actin content, increased cell spreading, decreased expression of CD11a integrin molecules, and delayed aggregation. Moreover, cells from the undiagnosed triplet proliferated faster, displayed a higher proportion of cells with hyperploidy, and formed large tumors in vivo. This phenotype was reversible by inhibiting MKL1 activity. Interestingly, cells from the triplet treated for HL in 1985 contained two subpopulations: one with high expression of CD11a that behaved like control cells and the other with low expression of CD11a that formed large tumors in vivo similar to cells from the undiagnosed triplet. This implies that pre-malignant cells had re-emerged a long time after treatment. Together, these data suggest that dysregulated MKL1 activity participates in B-cell transformation and the pathogenesis of HL.

Cytogenetic risk stratification at diagnosis has long been one of the most useful tools to assess prognosis in acute lymphoblastic leukemia (ALL). To examine the prognostic impact of cytogenetic abnormalities on outcomes after allogeneic hematopoietic cell transplantation, we studied 1731 adults with Philadelphia-negative ALL in complete remission who underwent myeloablative or reduced intensity/non-myeloablative conditioning transplant from unrelated or matched sibling donors reported to the Center for International Blood and Marrow Transplant Research. A total of 632 patients had abnormal conventional metaphase cytogenetics. The leukemia-free survival and overall survival rates at 5 years after transplantation in patients with abnormal cytogenetics were 40% and 42%, respectively, which were similar to those in patients with a normal karyotype. Of the previously established cytogenetic risk classifications, modified Medical Research Council-Eastern Cooperative Oncology Group score was the only independent prognosticator of leukemia-free survival (P=0.03). In the multivariable analysis, monosomy 7 predicted post-transplant relapse [hazard ratio (HR)=2.11; 95% confidence interval (95% CI): 1.04-4.27] and treatment failure (HR=1.97; 95% CI: 1.20-3.24). Complex karyotype was prognostic for relapse (HR=1.69; 95% CI: 1.06-2.69), whereas t(8;14) predicted treatment failure (HR=2.85; 95% CI: 1.35-6.02) and overall mortality (HR=3.03; 95% CI: 1.44-6.41). This large study suggested a novel transplant-specific cytogenetic scheme with adverse [monosomy 7, complex karyotype, del(7q), t(8;14), t(11;19), del(11q), tetraploidy/near triploidy], intermediate (normal karyotype and all other abnormalities), and favorable (high hyperdiploidy) risks to prognosticate leukemia-free survival (P=0.02). Although some previously established high-risk Philadelphia-negative cytogenetic abnormalities in ALL can be overcome by transplantation, monosomy 7, complex karyotype, and t(8;14) continue to pose significant risks and yield inferior outcomes.

An aging global population combined with sedentary lifestyles and unhealthy diets has contributed to an increasing incidence of obesity and type 2 diabetes. These metabolic disorders are associated with perturbations to nitric oxide (NO) signaling and impaired glucose metabolism. Dietary inorganic nitrate, found in high concentration in green leafy vegetables, can be converted to NO in vivo and demonstrates antidiabetic and antiobesity properties in rodents. Alongside tissues including skeletal muscle and liver, white adipose tissue is also an important physiological site of glucose disposal. However, the distinct molecular mechanisms governing the effect of nitrate on adipose tissue glucose metabolism and the contribution of this tissue to the glucose-tolerant phenotype remain to be determined. Using a metabolomic and stable-isotope labeling approach, combined with transcriptional analysis, we found that nitrate increases glucose uptake and oxidative catabolism in primary adipocytes and white adipose tissue of nitrate-treated rats. Mechanistically, we determined that nitrate induces these phenotypic changes in primary adipocytes through the xanthine oxidoreductase–catalyzed reduction of nitrate to NO and independently of peroxisome proliferator–activated receptor-α. The nitrate-mediated enhancement of glucose uptake and catabolism in white adipose tissue may be a key contributor to the antidiabetic effects of this anion.

Subjects with low serum HDL cholesterol levels are reported to be susceptible to diabetes, with insulin resistance believed to be the underlying pathological mechanism. Apolipoprotein M (apoM) is a carrier of sphingosine-1-phosphate (S1P), a multifunctional lipid mediator, on HDL, and the pleiotropic effects of HDL are believed to be mediated by S1P. In the current study, we attempted to investigate the potential association between apoM/S1P and insulin resistance. We observed that the serum levels of apoM were lower in patients with type 2 diabetes and that they were negatively correlated with BMI and the insulin resistance index. While deletion of apoM in mice was associated with worsening of insulin resistance, overexpression of apoM was associated with improvement of insulin resistance. Presumably, apoM/S1P exerts its protective effect against insulin resistance by activating insulin signaling pathways, such as the AKT and AMPK pathways, and also by improving the mitochondrial functions through upregulation of SIRT1 protein levels. These actions of apoM/S1P appear to be mediated via activation of S1P1 and/or S1P3. These results suggest that apoM/S1P exerts protective roles against the development of insulin resistance.

Nayeli G. Reyes-Nava, Hung-Chun Yu, Curtis R. Coughlin II, Tamim H. Shaikh, and Anita M. Quintana

We used whole-exome sequencing (WES) to determine the genetic etiology of a patient with a multi-system disorder characterized by a seizure phenotype. WES identified a heterozygous de novo missense mutation in the GABRA1 gene (c.875C>T). GABRA1 encodes the alpha subunit of the gamma-aminobutyric acid receptor A (GABA_AR). The GABA_AR is a ligand gated ion channel that mediates the fast inhibitory signals of the nervous system, and mutations in the subunits that compose the GABA_AR have been previously associated with human disease. To understand the mechanisms by which GABRA1 regulates brain development, we developed a zebrafish model of gabra1 deficiency. gabra1 expression is restricted to the nervous system and behavioral analysis of morpholino injected larvae suggests that the knockdown of gabra1 results in hypoactivity and defects in the expression of other subunits of the GABA_AR. Expression of the human GABRA1 protein in morphants partially restored the hypomotility phenotype. In contrast, the expression of the c.875C>T variant did not restore these behavioral deficits. Collectively, these results represent a functional approach to understand the mechanisms by which loss-of-function alleles cause disease.

PURPOSE

We aimed to evaluate the efficacy and safety of use of the Fasting Algorithm for Singaporeans with Type 2 Diabetes (FAST) during Ramadan.

PURPOSE

Online programs may help to engage patients in advance care planning in outpatient settings. We sought to implement an online advance care planning program, PREPARE (Prepare for Your Care; http://www.prepareforyourcare.org), at home and evaluate the changes in advance care planning engagement among patients attending outpatient clinics.

The Stenotrophomonas maltophilia complex (Smc) comprises opportunistic environmental Gram-negative bacilli responsible for a variety of infections in both humans and animals. Beyond its large genetic diversity, its genetic organization in genogroups was recently confirmed through the whole-genome sequencing of human and environmental strains. As they are poorly represented in these analyses, we sequenced the whole genomes of 93 animal strains to determine their genetic background and characteristics. Combining these data with 81 newly sequenced human strains and the genomes available from RefSeq, we performed a genomic analysis that included 375 nonduplicated genomes with various origins (animal, 104; human, 226; environment, 30; unknown, 15). Phylogenetic analysis and clustering based on genome-wide average nucleotide identity confirmed and specified the genetic organization of Smc in at least 20 genogroups. Two new genogroups were identified, and two previously described groups were further divided into two subgroups each. Comparing the strains isolated from different host types and their genogroup affiliation, we observed a clear disequilibrium in certain groups. Surprisingly, some antimicrobial resistance genes, integrons, and/or clusters of attC sites lacking integron-integrase (CALIN) sequences targeting antimicrobial compounds extensively used in animals were mainly identified in animal strains. We also identified genes commonly found in animal strains coding for efflux systems. The result of a large whole-genome analysis performed by us supports the hypothesis of the putative contribution of animals as a reservoir of Stenotrophomonas maltophilia complex strains and/or resistance genes for strains in humans.

IMPORTANCE Given its naturally large antimicrobial resistance profile, the Stenotrophomonas maltophilia complex (Smc) is a set of emerging pathogens of immunosuppressed and cystic fibrosis patients. As it is group of environmental microorganisms, this adaptation to humans is an opportunity to understand the genetic and metabolic selective mechanisms involved in this process. The previously reported genomic organization was incomplete, as data from animal strains were underrepresented. We added the missing piece of the puzzle with whole-genome sequencing of 93 strains of animal origin. Beyond describing the phylogenetic organization, we confirmed the genetic diversity of the Smc, which could not be estimated through routine phenotype- or matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF)-based laboratory tests. Animals strains seem to play a key role in the diversity of Smc and could act as a reservoir for mobile resistance genes. Some genogroups seem to be associated with particular hosts; the genetic support of this association and the role of the determinants/corresponding genes need to be explored.

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia coli fadD mutant JW1794. RpfB activates long-chain fatty acids (n-C_16:0 and n-C_18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes. Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2. In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenes.

IMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes. Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes. Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes. Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

To better understand how associated microorganisms ("microbiota") influence organismal aging, we focused on the model organism Drosophila melanogaster. We conducted a metagenome-wide association (MGWA) as a screen to identify bacterial genes associated with variation in the D. melanogaster life span. The results of the MGWA predicted that bacterial cysteine and methionine metabolism genes influence fruit fly longevity. A mutant analysis, in which flies were inoculated with Escherichia coli strains bearing mutations in various methionine cycle genes, confirmed a role for some methionine cycle genes in extending or shortening fruit fly life span. Initially, we predicted these genes might influence longevity by mimicking or opposing methionine restriction, an established mechanism for life span extension in fruit flies. However, follow-up transcriptome sequencing (RNA-seq) and metabolomic experiments were generally inconsistent with this conclusion and instead implicated glucose and vitamin B₆ metabolism in these influences. We then tested if bacteria could influence life span through methionine restriction using a different set of bacterial strains. Flies reared with a bacterial strain that ectopically expressed bacterial transsulfuration genes and lowered the methionine content of the fly diet also extended female D. melanogaster life span. Taken together, the microbial influences shown here overlap with established host genetic mechanisms for aging and therefore suggest overlapping roles for host and microbial metabolism genes in organismal aging.

IMPORTANCE Associated microorganisms ("microbiota") are intimately connected to the behavior and physiology of their animal hosts, and defining the mechanisms of these interactions is an urgent imperative. This study focuses on how microorganisms influence the life span of a model host, the fruit fly Drosophila melanogaster. First, we performed a screen that suggested a strong influence of bacterial methionine metabolism on host life span. Follow-up analyses of gene expression and metabolite abundance identified stronger roles for vitamin B₆ and glucose than methionine metabolism among the tested mutants, possibly suggesting a more limited role for bacterial methionine metabolism genes in host life span effects. In a parallel set of experiments, we created a distinct bacterial strain that expressed life span-extending methionine metabolism genes and showed that this strain can extend fly life span. Therefore, this work identifies specific bacterial genes that influence host life span, including in ways that are consistent with the expectations of methionine restriction.

This study showed that treatment with a therapeutic monoclonal immunoglobulin-G1 antibody against phosphorylcholine on oxidized phospholipids preserves coronary flow reserve and attenuates atherosclerotic inflammation as determined by the uptake of ¹⁸F-fluorodeoxyglucose in atherosclerotic mice. The noninvasive imaging techniques represent translational tools to assess the efficacy of phosphorylcholine-targeted therapy on coronary artery function and atherosclerosis in clinical studies.

Chronic obstructive pulmonary disease (COPD) is characterised by persistent respiratory symptoms and airflow limitation, which is caused by small airway disease (bronchiolitis) and alveolar destruction (emphysema) [1]. Patients primarily suffering from severe emphysema are often limited in exercise capacity due to the consequences of hyperinflation [2].

Intrathecal administration of anti-infectives is indicated in central nervous system infections by multiresistant pathogens when drugs that can reach adequate cerebrospinal fluid (CSF) concentrations by systemic therapy are not available. Antibiotics that readily pass the blood-brain and blood-CSF barriers and/or that have low toxicity allowing an increase in the daily dosage should not be used for intrathecal therapy. Intrathecal therapy is accompanied by systemic treatment. Antibacterials indispensable for intrathecal therapy include aminoglycosides, colistin, daptomycin, tigecycline, and vancomycin. Limited experience suggests the utility of the antifungals amphotericin B and caspofungin. Intraventricular administration ensures distribution throughout the CSF compartment, whereas intralumbar dosing often fails to attain adequate antibiotic concentrations in the ventricles. The individual dose is determined by the estimated size of the CSF space and by the estimated clearance from CSF. For moderately lipophilic anti-infectives with a molecular weight above approximately 1,000 g/mol, as well as for hydrophilic drugs with a molecular weight above approximately 400 g/mol, one daily dose is normally adequate. The ventricular drain should be clamped for 15 to 120 min to facilitate the distribution of the anti-infective in the CSF space. Therapeutic drug monitoring of the trough levels is necessary only in cases of therapeutic failure.

Although not as ubiquitous as antibacterial susceptibility testing, antifungal susceptibility testing (AFST) is a tool of increasing importance in clinical microbiology laboratories. The goal of AFST is to reliably produce MIC values that may be used to guide patient therapy, inform epidemiological studies, and track rates of antifungal drug resistance. There are three methods that have been standardized by standards development organizations: broth dilution, disk diffusion, and azole agar screening for Aspergillus. Other commonly used methods include gradient diffusion and the use of rapid automated instruments. Novel methodologies for susceptibility testing are in development. It is important for laboratories to consider not only the method of testing but also the interpretation (or lack thereof) of in vitro data.

Purpose:

Gastrointestinal cancers remain areas of high unmet need despite advances in targeted and immunotherapies. Here, we demonstrate potent, tumor-selective efficacy with PF-07062119, a T-cell engaging CD3 bispecific targeting tumors expressing Guanylyl Cyclase C (GUCY2C), which is expressed widely across colorectal cancer and other gastrointestinal malignancies. In addition, to address immune evasion mechanisms, we explore combinations with immune checkpoint blockade agents and with antiangiogenesis therapy.

Purpose:

Preclinical data demonstrating androgen receptor (AR)–positive (AR⁺) triple-negative breast cancer (TNBC) cells are sensitive to AR antagonists, and PI3K inhibition catalyzed an investigator-initiated, multi-institutional phase Ib/II study TBCRC032. The trial investigated the safety and efficacy of the AR-antagonist enzalutamide alone or in combination with the PI3K inhibitor taselisib in patients with metastatic AR⁺ (≥10%) breast cancer.

Purpose:

Radium-223 is approved for metastatic castration-resistant prostate cancer (mCRPC) based on improved overall survival, and delay in skeletal related events. However, it is not associated with PSA or radiographic response, which poses a challenge in real-time assessment of its efficacy. Surrogate markers of treatment outcomes may facilitate tailoring treatment duration with radium-223, by limiting the duration of therapy with radium-223 in these patients. Here, we sought to investigate the utility of bone metabolic markers (BMMs) as surrogate markers of response to radium-223 in mCRPC.

Quinolones, such as the antimalarial atovaquone, are inhibitors of the malarial mitochondrial cytochrome bc₁ complex, a target critical to the survival of both liver- and blood-stage parasites, making these drugs useful as both prophylaxis and treatment. Recently, several derivatives of endochin have been optimized to produce novel quinolones that are active in vitro and in animal models. While these quinolones exhibit potent ex vivo activity against Plasmodium falciparum and Plasmodium vivax, their activity against the zoonotic agent Plasmodium knowlesi is unknown. We screened several of these novel endochin-like quinolones (ELQs) for their activity against P. knowlesi in vitro and compared this with their activity against P. falciparum tested under identical conditions. We demonstrated that ELQs are potent against P. knowlesi (50% effective concentration, <117 nM) and equally effective against P. falciparum. We then screened selected quinolones and partner drugs using a longer exposure (2.5 life cycles) and found that proguanil is 10-fold less potent against P. knowlesi than P. falciparum, while the quinolones demonstrate similar potency. Finally, we used isobologram analysis to compare combinations of the ELQs with either proguanil or atovaquone. We show that all quinolone combinations with proguanil are synergistic against P. falciparum. However, against P. knowlesi, no evidence of synergy between proguanil and the quinolones was found. Importantly, the combination of the novel quinolone ELQ-300 with atovaquone was synergistic against both species. Our data identify potentially important species differences in proguanil susceptibility and in the interaction of proguanil with quinolones and support the ongoing development of novel quinolones as potent antimalarials that target multiple species.

New drugs or therapeutic combinations are urgently needed against Mycobacterium abscessus. Previously, we demonstrated the potent activity of indole-2-carboxamides 6 and 12 against M. abscessus. We show here that these compounds act synergistically with imipenem and cefoxitin in vitro and increase the bactericidal activity of the β-lactams against M. abscessus. In addition, compound 12 also displays synergism with imipenem and cefoxitin within infected macrophages. The clinical potential of these new drug combinations requires further evaluation.

Omadacycline is a broad-spectrum aminomethylcycline approved in October 2018 by the U.S. Food and Drug Administration for treating acute bacterial skin and skin structure infections and community-acquired pneumonia as both an oral and intravenous once-daily formulation. In this report, the activities of omadacycline and comparators were tested against 49,000 nonduplicate bacterial isolates collected prospectively during 2016 to 2018 from medical centers in Europe (24,500 isolates, 40 medical centers [19 countries]) and the United States (24,500 isolates, 33 medical centers [23 states and all 9 U.S. census divisions]). Omadacycline was tested by broth microdilution following the methods in Clinical and Laboratory Standards Institute document M07 (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, 11th ed., 2018). Omadacycline (MIC_50/90, 0.12/0.25 mg/liter) inhibited 98.6% of Staphylococcus aureus isolates at ≤0.5 mg/liter, including 96.3% of methicillin-resistant S. aureus isolates and 99.8% of methicillin-susceptible S. aureus isolates. Omadacycline potency was comparable for Streptococcus pneumoniae (MIC_50/90, 0.06/0.12 mg/liter), viridans group streptococci (MIC_50/90, 0.06/0.12 mg/liter), and beta-hemolytic streptococci (MIC_50/90, 0.12/0.25 mg/liter), regardless of species and susceptibility to penicillin, macrolides, or tetracycline. Omadacycline was active against all Enterobacterales tested (MIC_50/90, 1/8 mg/liter; 87.5% of isolates were inhibited at ≤4 mg/liter) except Proteus mirabilis (MIC_50/90, 16/>32 mg/liter) and indole-positive Proteus spp. (MIC_50/90, 8/32 mg/liter) and was most active against Escherichia coli (MIC_50/90, 0.5/2 mg/liter), Klebsiella oxytoca (MIC_50/90, 1/2 mg/liter), and Citrobacter spp. (MIC_50/90, 1/4 mg/liter). Omadacycline inhibited 92.4% of Enterobacter cloacae species complex and 88.5% of Klebsiella pneumoniae isolates at ≤4 mg/liter. Omadacycline was active against Haemophilus influenzae (MIC_50/90, 0.5/1 mg/liter), regardless of β-lactamase status, and against Moraxella catarrhalis (MIC_50/90, ≤0.12/0.25 mg/liter). The potent activity of omadacycline against Gram-positive and -negative bacteria indicates that omadacycline merits further study in serious infections in which multidrug resistance and mixed Gram-positive and Gram-negative bacterial infections may be a concern.

Plazomicin was active against 97.0% of 8,783 Enterobacterales isolates collected in the United States (2016 and 2017), and only 6 isolates carried 16S rRNA methyltransferases conferring resistance to virtually all aminoglycosides. Plazomicin (89.2% to 95.9% susceptible) displayed greater activity than amikacin (72.5% to 78.6%), gentamicin (30.4% to 45.9%), and tobramycin (7.8% to 22.4%) against carbapenem-resistant and extensively drug-resistant isolates. The discrepancies among the susceptibility rates for these agents was greater when applying breakpoints generated using the same stringent contemporary methods applied to determine plazomicin breakpoints.

Imipenem-relebactam (I-R) is a recently developed carbapenem–beta-lactamase inhibitor combination agent that can overcome carbapenem resistance, which has now emerged in Escherichia coli, including sequence type 131 (ST131) and its fluoroquinolone-resistant H30R subclone, the leading cause of extraintestinal E. coli infections globally. To clarify the likely utility of I-R for carbapenem-resistant (CR) E. coli infections in the United States, we characterized 203 recent CR clinical E. coli isolates from across the United States (years 2002 to 2017) for phylogroup, clonal group (including ST131, H30R, and the CTX-M-15-associated H30Rx subset within H30R), relevant beta-lactamase genes, and broth microdilution MICs for I-R and 11 comparator agents. Overall, I-R was highly active (89% susceptible), more so than all comparators except tigecycline and colistin (both 99% susceptible). I-R’s activity varied significantly in relation to phylogroup, clonal background, resistance genotype, and region. It was greatest among phylogroup B2, ST131-H30R, H30Rx, Klebsiella pneumoniae carbapenemase (KPC)-positive, and northeast U.S. isolates and lowest among phylogroup C, New Delhi metallo-β-lactamase (NDM)-positive, and southeast U.S. isolates. Relebactam improved imipenem’s activity against CR isolates within each phylogroup—especially groups A, B1, and B2—and particularly against isolates containing KPC. I-R remained substantially active against isolates coresistant to comparator agents, albeit somewhat less so than against the corresponding susceptible isolates. These findings suggest that I-R should be useful for treating most CR E. coli infections in the United States, largely independent of coresistance, although this likely will vary in relation to the local prevalence of specific E. coli lineages and carbapenem resistance mechanisms.

Bacterial persisters are phenotypic variants that temporarily demonstrate an extraordinary tolerance toward antibiotics. Persisters have been linked to the recalcitrance of biofilm-related infections; hence, a complete understanding of their physiology can lead to improvement of therapeutic strategies for such infections. Mechanisms pertaining to persister formation are thought to be associated with stress response pathways triggered by intra- or extracellular stress factors. Unfortunately, studies demonstrating the effects of osmolyte- and/or pH-induced stresses on bacterial persistence are largely missing. To fill this knowledge gap within the field, we studied the effects of various osmolytes and pH conditions on Escherichia coli persistence with the use of phenotype microarrays and antibiotic tolerance assays. Although we found that a number of chemicals and pH environments, including urea, sodium nitrite, and acidic pH, significantly reduced persister formation in E. coli compared to no-osmolyte/no-buffer controls, this reduction in persister levels was less pronounced in late-stationary-phase cultures. Our results further demonstrated a positive correlation between cell growth and persister formation, which challenges the general notion in the field that slow-growing cultures have more persister cells than fast-growing cultures.

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-β-lactamase (ESBL)- and metallo-β-lactamase (MBL)-producing isolates. Overall, KBP-7072 (MIC_50/90, 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.