Phenotypic Impact Research Articles

Conformation of the Solute-Binding Protein AdcAII Influences Zinc Uptake in Streptococcus pneumoniae.

Streptococcus pneumoniae scavenges essential zinc ions from the host during colonization and infection. This is achieved by the ATP-binding cassette transporter, AdcCB, and two solute-binding proteins (SBPs), AdcA and AdcAII. It has been established that AdcAII serves a greater role during initial infection, but the molecular details of how the protein selectively acquires Zn(II) remain poorly understood. This can be attributed to the refractory nature of metal-free AdcAII to high-resolution structural determination techniques. Here, we overcome this issue by separately mutating the Zn(II)-coordinating residues and performing a combination of structural and biochemical analyses on the variant proteins. Structural analyses of Zn(II)-bound AdcAII variants revealed that specific regions within the protein underwent conformational changes via direct coupling to each of the metal-binding residues. Quantitative in vitro metal-binding assays combined with affinity determination and phenotypic growth assays revealed that each of the four Zn(II)-coordinating residues contributes to metal binding by AdcAII. Intriguingly, the phenotypic growth impact of the mutant adcAII alleles was, in general, independent of affinity, suggesting that the Zn(II)-bound conformation of the SBP is crucial for efficacious metal uptake. Collectively, these data highlight the intimate coupling of ligand affinity with protein conformational change in ligand-receptor proteins and provide a putative mechanism for AdcAII. These findings provide further mechanistic insight into the structural and functional diversity of SBPs that is broadly applicable to other prokaryotes.

Identification of BRCA2 Cis Double Heterozygous Breast Cancer Cases Using Whole Exome Sequencing: Phenotypic Expression and Impact on Personalized Oncology.

BRCA1 and BRCA2 are the most commonly mutated breast cancer susceptibility genes that convey a high risk of breast and ovarian cancer. Most BRCA1 or BRCA2 mutation carriers have inherited a single heterozygous mutation. In recent years, very rare cases with biallelic or trans double heterozygous mutations on BRCA1 and or BRCA2 have been identified and seem to be associated with distinctive phenotypes. Given that this genotype-phenotype correlation in cancer predisposing hereditary conditions is of relevance for oncological prevention and genetic testing, it is important to investigate these rare BRCA genotypes for better clinical management of BRCA mutation carriers. Here we present the first report on Cis double heterozygosity (Cis DH) on BRCA2 gene identified using Whole exome sequencing (WES) in a Tunisian family with two BRCA2 mutations namely: c.632-1G>A and c.1310_1313DelAAGA that are both reported as pathogenic in ClinVar database. Subsequent analysis in 300 high-risk Tunisian breast cancer families detected this Cis double heterozygous genotype in 8 additional individuals belonging to 5 families from the same geographic origin suggesting a founder effect. Moreover, the observed Cis DH seems to be associated with an early age of onset (mean age = 35.33 years) and severe phenotype of the disease with high breast cancer grade and multiple cancer cases in the family. The identification of unusual BRCA genotypes in this Tunisian cohort highlights the importance of performing genetic studies in under-investigated populations. This will also potentially help avoiding erroneous classifications of genetic variants in African population and therefore avoiding clinical misdiagnosis of BRCA related cancers. Our findings will also have an impact on the genetic testing and the clinical management of North African breast cancer patients as well as patients from different other ethnic groups in regard to several emerging target therapies such as PARP inhibitors.

O-161 Cumulative live birth rate after a freeze-all approach in women with polycystic ovaries: does the PCOS phenotype have an impact?

Abstract Study question Do cumulative live birth rates (CLBR) differ between PCOS phenotypes when a freeze-all strategy is used to prevent OHSS after ovarian stimulation (OS)? Summary answer When conventional-dose OS resulted in high response, a CLBR of ∼ 70% was observed after “freeze-all” in women with PCOS, irrespective of their phenotype. What is known already Previous observational studies have shown that CLBR in women with PCOS who undergo assisted reproductive technologies (ART) may depend on their phenotype. When OS was performed with caution to avoid ovarian hyperresponse, CLBR was lower in women with a hyperandrogenic PCOS phenotype. However, when women with PCOS do exhibit hyperresponse and a freeze-all strategy is used, the impact of the PCOS phenotype on the clinical outcome of the ART cycle is unclear. Study design, size, duration This is a single-centre, retrospective cohort study including 422 women with polycystic ovary syndrome (PCOS) as defined by Rotterdam criteria or PCO-like ovarian morphology-only (PCOM) in whom a freeze-all strategy was applied after GnRH agonist triggering in the context of hyperresponse defined as ³19 follicles of ³11mm in their first or second IVF-ICSI cycle between January 2015 and December 2019 in a tertiary referral hospital. Participants/materials, setting, methods PCOS phenotype was based on hyperandrogenism (H), ovulatory dysfunction (O) and PCO-like ovarian morphology (P). Ovarian stimulation was performed with rFSH or HPhMG, adjusted to BMI. The primary outcome was cumulative live birth rate (CLBR) resulting from the transfer of all cryopreserved embryos from the same IVF-ICSI cycle. Patient and cycle characteristics and laboratory and clinical data were analysed. Data were analysed by multivariate logistic regression adjusting for covariates. Main results and the role of chance In total, 91/422 (21.6%) patients had PCOS phenotype A (HOP); 33 (7.8%) had phenotype C (HP), 161/422 (38.2%) had phenotype D (OP) and 137/422 (32.5%) had PCOM (n = 137). BMI, AMH and AFC were significantly different between phenotype groups (p < 0.001), and highest in PCOS phenotype A. The type of gonadotropin used, as well as the mean daily and total stimulation dose were comparable for all groups. The mean number of retrieved oocytes was comparable among groups (22.4±10.8 for phenotype A, 21.4±7.1 for phenotype C, 20.4±7.8 for phenotype D and 22.2±9.7 for PCOM; p = 0.46). The mean number of embryos available for vitrification differed significantly (4.4±3.7, 5.7±3.4, 5.7±3.4 and 5.2±3.6, respectively; p = 0.005). Following the first frozen embryo transfer, LBR was comparable among groups (41.5%, 43.3%, 49.3% and 38.5%, respectively; p = 0.31). Unadjusted CLBR was also similar (69.2%, 69.7%, 79.5% and 67.9%, respectively; p = 0.11). The multivariate logistic regression model adjusting for age, BMI, number of oocytes and embryo stage (cleavage vs. blastocyst stage) confirmed that the PCOS/PCOM phenotype did not have any impact on CLBR (OR 0.80, CI 0.28-2.29 (phenotype C); OR 1.40, CI 0.67-2.90 (phenotype D); OR 0.65, CI 0.31-1.34 (PCOM); p = 0.1, with phenotype A as reference). Limitations, reasons for caution These data should be interpreted with caution as the retrospective nature of the study holds the possibility of unmeasured confounding factors. The results cannot be generalised to all ART cycles in women with polycystic ovaries as they pertain to those cycles where OS leads to hyperresponse. Wider implications of the findings In subfertile women with PCOS eligible for ART, hyperresponse after OS confers excellent cumulative live birth rates when a freeze-all strategy is used, eliminating unfavourable clinical outcomes that had previously been observed in hyperandrogenic PCOS women after mild OS targeting normal ovarian response and fresh embryo transfer. Trial registration number not applicable

Tepoxalin increases chemotherapy efficacy in drug-resistant breast cancer cells overexpressing the multidrug transporter gene ABCB1

Effective cancer chemotherapy treatment requires both therapy delivery and retention by malignant cells. Cancer cell overexpression of the multidrug transmembrane transporter gene ABCB1 (MDR1, multi-drug resistance protein 1) thwarts therapy retention, leading to a drug-resistant phenotype. We explored the phenotypic impact of ABCB1 overexpression in normal human mammary epithelial cells (HMECs) via acute adenoviral delivery and in breast cancer cell lines with stable integration of inducible ABCB1 expression. One hundred sixty-two genes were differentially expressed between ABCB1-expressing and GFP-expressing HMECs, including the gene encoding the cyclooxygenase-2 protein, PTGS2. Several breast cancer cell lines with inducible ABCB1 expression demonstrated sensitivity to the 5-lipoxygenase, cyclooxygenase-1/2 inhibitor tepoxalin in two-dimensional drug response assays, and combination treatment of tepoxalin either with chemotherapies or with histone deacetylase (HDAC) inhibitors improved therapeutic efficacy in these lines. Moreover, selection for the ABCB1-expressing cell population was reduced in three-dimensional co-cultures of ABCB1-expressing and GFP-expressing cells when chemotherapy was given in combination with tepoxalin. Further study is warranted to ascertain the clinical potential of tepoxalin, an FDA-approved therapeutic for use in domesticated mammals, to restore chemosensitivity and improve drug response in patients with ABCB1-overexpressing drug-resistant breast cancers.

Complementary Sequential Circulating Tumor Cell (CTC) and Cell-Free Tumor DNA (ctDNA) Profiling Reveals Metastatic Heterogeneity and Genomic Changes in Lung Cancer and Breast Cancer.

IntroductionCirculating tumor cells (CTCs) and cell-free tumor DNA (ctDNA) are tumor components present in circulation. Due to the limited access to both CTC enrichment platforms and ctDNA sequencing in most laboratories, they are rarely analyzed together.MethodsConcurrent isolation of ctDNA and single CTCs were isolated from lung cancer and breast cancer patients using the combination of size-based and CD45-negative selection method via DropCell platform. We performed targeted amplicon sequencing to evaluate the genomic heterogeneity of CTCs and ctDNA in lung cancer and breast cancer patients.ResultsHigher degrees of genomic heterogeneity were observed in CTCs as compared to ctDNA. Several shared alterations present in CTCs and ctDNA were undetected in the primary tumor, highlighting the intra-tumoral heterogeneity of tumor components that were shed into systemic circulation. Accordingly, CTCs and ctDNA displayed higher degree of concordance with the metastatic tumor than the primary tumor. The alterations detected in circulation correlated with worse survival outcome for both lung and breast cancer patients emphasizing the impact of the metastatic phenotype. Notably, evolving genetic signatures were detected in the CTCs and ctDNA samples during the course of treatment and disease progression.ConclusionsA standardized sample processing and data analysis workflow for concurrent analysis of CTCs and ctDNA successfully dissected the heterogeneity of metastatic tumor in circulation as well as the progressive genomic changes that may potentially guide the selection of appropriate therapy against evolving tumor clonality.

Whole-exome imputation within UK Biobank powers rare coding variant association and fine-mapping analyses.

Exome association studies to date have generally been underpowered to systematically evaluate the phenotypic impact of very rare coding variants. We leveraged extensive haplotype sharing between 49,960 exome-sequenced UK Biobank participants and the remainder of the cohort (total N~500K) to impute exome-wide variants with accuracy (R2>0.5) down to minor allele frequency (MAF) ~0.00005. Association and fine-mapping analyses of 54 quantitative traits identified 1,189 significant associations (P<5 x 10−8) involving 675 distinct rare protein-altering variants (MAF<0.01) that passed stringent filters for likely causality. Across all traits, 49% of associations (578/1,189) occurred in genes with two or more hits; follow-up analyses of these genes identified allelic series containing up to 45 distinct likely-causal variants. Our results demonstrate the utility of within-cohort imputation in population-scale GWAS cohorts, provide a catalog of likely-causal, large-effect coding variant associations, and foreshadow the insights that will be revealed as genetic biobank studies continue to grow.

Shifting landscapes of human MTHFR missense-variant effects

SummaryMost rare clinical missense variants cannot currently be classified as pathogenic or benign. Deficiency in human 5,10-methylenetetrahydrofolate reductase (MTHFR), the most common inherited disorder of folate metabolism, is caused primarily by rare missense variants. Further complicating variant interpretation, variant impacts often depend on environment. An important example of this phenomenon is the MTHFR variant p.Ala222Val (c.665C>T), which is carried by half of all humans and has a phenotypic impact that depends on dietary folate. Here we describe the results of 98,336 variant functional-impact assays, covering nearly all possible MTHFR amino acid substitutions in four folinate environments, each in the presence and absence of p.Ala222Val. The resulting atlas of MTHFR variant effects reveals many complex dependencies on both folinate and p.Ala222Val. MTHFR atlas scores can distinguish pathogenic from benign variants and, among individuals with severe MTHFR deficiency, correlate with age of disease onset. Providing a powerful tool for understanding structure-function relationships, the atlas suggests a role for a disordered loop in retaining cofactor at the active site and identifies variants that enable escape of inhibition by S-adenosylmethionine. Thus, a model based on eight MTHFR variant effect maps illustrates how shifting landscapes of environment- and genetic-background-dependent missense variation can inform our clinical, structural, and functional understanding of MTHFR deficiency.

Impact of CYP3A5 phenotype on tacrolimus time in therapeutic range and clinical outcomes in pediatric renal and heart transplant recipients.

This study investigated the effect of CYP3A5 phenotype on time in therapeutic range (TTR) of tacrolimus post-transplant in pediatric patients. This retrospective study assessed medical records of pediatric kidney and heart recipients with available CYP3A5 genotype for tacrolimus dosing, troughs, and the clinical events (biopsy-proven acute rejection [BPAR] and de novo donor-specific antibodies [dnDSA]). The primary outcome, mean TTR in the first 90days post-transplant, was 9.0% (95% CI: -16.1, -1.9) lower in CYP3A5 expressers (p=0.014) when adjusting for time to therapeutic concentration and organ type. There was no difference between CYP3A5 phenotypes in time to the first clinical event using TTR during the first 90days. When applying TTR over the first year, there was a significant difference in event-free survival (EFS) which was 50.0% for CYP3A5 expressers/TTR<35%, 45.5% for expressers/TTR≥35%, 38.1% for nonexpressers/TTR<35%, and 72.9% for nonexpressers/TTR≥35% (log-rank p=0.03). A post hoc analysis of EFS identified CYP3A5 expressers had lower EFS compared to nonexpressers in patients with TTR≥35% (p=0.04) but no difference among patients with TTR<35% (p=0.6). The relationship between TTR and CYP3A5 phenotype suggests that achieving a TTR≥35% during the first year may be a modifiable factor to attenuate the risk of BPAR and dnDSA.

Patterns and Impact of Electronic Health Records-Defined Depression Phenotypes in Spine Surgery.

Preoperative depression is a risk factor for poor outcomes after spine surgery. To understand effects of depression on spine surgery outcomes and healthcare resource utilization. Using IBM's MarketScan Database, we identified 52 480 patients who underwent spinal fusion. Retained patients were classified into 6 depression phenotype groups based on International Classification of Disease, 9th/10th Revision (ICD-9/10) codes and use/nonuse of antidepressant medications: major depressive disorder (MDD), other depression (OthDep), antidepressants for other psychiatric condition (PsychRx), antidepressants for physical (nonpsychiatric) condition (NoPsychRx), psychiatric condition only (PsychOnly), and no depression (NoDep). We analyzed baseline demographics, comorbidities, healthcare utilization/payments, and chronic opioid use. Breakdown of groups in our cohort: MDD (15%), OthDep (12%), PsychRx (13%), NonPsychRx (15%), PsychOnly (12%), and NoDep (33%). Postsurgery: increased outpatient resource utilization, admissions, and medication refills at 1, 2, and 5 yr in the NoDep, PsychOnly, NonPsychRx, PsychRx, and OthDep groups, and highest in MDD. Postoperative opioid usage rates remained unchanged in MDD (44%) and OthDep (36%), and reduced in PsychRx (40%), NonPsychRx (31%), and PsychOnly (20%), with greatest reduction in NoDep (13%). Reoperation rates: 1 yr after index procedure, MDD, OthDep, PsychRx, NonPsychRx, and PsychOnly had more reoperations compared to NoDep, and same at 2 and 5 yr. In NoDep patients, 45% developed new depressive phenotype postsurgery. EHR-defined classification allowed us to study in depth the effects of depression in spine surgery. This increased understanding of the interplay of mental health will help providers identify cohorts at risk for high complication rates, and health care utilization.

Impact of CYP2C19 Phenotype and Drug-Drug Interactions on Voriconazole Concentration in Pediatric Patients.

Voriconazole (VRC), a first-line agent for the treatment of invasive fungal infections, is mainly metabolized by human cytochrome P450 (CYP) 2C19. In this study, a retrospective analysis was performed to investigate the key factors that influence the plasma trough concentration (Cmin) of VRC, and an appropriate dosing regimen for pediatric patients was drafted subsequently. Overall, factors such as age, CYP2C19 phenotype, and combination medication with proton pump inhibitors accounted for 23.4% of variability in dose-normalized Cmin values of VRC by a multiple linear regression analysis. Dose-normalized Cmin values in the poor metabolizers (PMs) and intermediate metabolizers (IMs) were significantly higher than those in extensive metabolizers (EMs) (P < 0.001). To achieve therapeutic Cmin for CYP2C19 ultrarapid metabolizers (UMs) or EMs, patients aged no more than 12 and more than 12 years required doses of 6.53 ± 2.08 and 3.95 ± 0.85 mg/kg of body weight twice daily (P = 0.007). For CYP2C19 PMs or IMs, patients aged under 12 and over 12 years required doses of 5.75 ± 1.73 and 4.23 ± 0.76 mg/kg twice daily, respectively (P = 0.019). Furthermore, coadministration of rifamycin sodium or omeprazole exhibited significant effects on VRC Cmin. Taken together, it is necessary to pay attention to the impact of CYP2C19 phenotype and drug-drug interactions to achieve optimal therapy.

Impact of CYP2D6 Genetic Variation on Radical Cure of Plasmodium vivax Malaria.

Plasmodium vivax (P. vivax) is the most widespread human malaria parasite, with 2.5 billion people at risk of infection worldwide. P. vivax forms liver hypnozoites, which trigger further symptomatic episodes (relapses) weeks or months after the initial episode. Radical cure of vivax malaria requires hypnozoitocide therapy to prevent relapses. The two US Food and Drug Administration (FDA)-approved hypnozoiticides for human use, primaquine, and tafenoquine, are pro-drugs, that require in vivo conversion into metabolites with redox activity. This mini-review focuses on the association between CYP2D6-mediated hydroxylation and hypnozoitocide efficacy of primaquine and tafenoquine. Studies in murine models show that the antimalarial activity of primaquine and tafenoquine is abolished by CYP2D knock-out and partially restored by knock-in of humanized CYP2D6. Human studies explored the impact of CYP2D6 genetic variation and genotype-inferred CYP2D6 phenotype on anti-relapse efficacy. Most, but not all, studies with primaquine report higher rates of relapse in patients with decreased CYP2D6 activity (activity scores (AS) ≤1) compared to normal activity (AS≥1.5). Potential factors for discordance among studies include risk of reinfection in endemic areas, adherence to primaquine-treatment, assignment of CYP2D6 phenotypes based on CYP2D6 polymorphism and choice of AS values for dichotomizing the study cohorts. Tafenoquine anti-relapse efficacy did not differ between patients with AS<1 vs. AS≥1.5 in 2 studies. Absence/small number of poor CYP2D6 metabolizers in AS≤1 groups, combined with lesser dependence of tafenoquine on CYP2D6-mediated conversion into active redox metabolites may account for this result. Additional tafenoquine studies with larger representation of poor CYP2D6 metabolizers are warranted.

Impact of frailty metabolic phenotypes on the management of older people with type 2 diabetes mellitus.

To provide a pathophysiological basis for distinguishing metabolic variants of the frailty phenotype in older adults with type 2 diabetes. We have made an in-depth review of the possible mechanisms in diabetes, ageing and frailty that will alter allow us to describe phenotypic changes which might assist in predicting responses to particular glucose-lowering therapy. Our review has enable us to describe with some confidence a sarcopenic obese phenotype and an anorexic malnourished phenotype. By identifying these two phenotypes we can predict which would be most responsive to certain classes of therapy and where therapies may be ill-advised. This represents the first novel approach in this area. Further work is being planned to develop this hypothesis. Geriatr Gerontol Int 2021; 21: 614-622.

Impact of Morbid Obesity and Obesity Phenotype on Outcomes After Transcatheter Aortic Valve Replacement.

BackgroundThere is a paucity of outcome data on patients who are morbidly obese (MO) undergoing transcatheter aortic valve replacement. We aimed to determine their periprocedural and midterm outcomes and investigate the impact of obesity phenotype.Methods and ResultsConsecutive patients who are MO (body mass index, ≥40 kg/m2, or ≥35 kg/m2 with obesity‐related comorbidities; n=910) with severe aortic stenosis who underwent transcatheter aortic valve replacement in 18 tertiary hospitals were compared with a nonobese cohort (body mass index, 18.5–29.9 kg/m2, n=2264). Propensity‐score matching resulted in 770 pairs. Pre–transcatheter aortic valve replacement computed tomography scans were centrally analyzed to assess adipose tissue distribution; epicardial, abdominal visceral and subcutaneous fat. Major vascular complications were more common (6.6% versus 4.3%; P=0.043) and device success was less frequent (84.4% versus 88.1%; P=0.038) in the MO group. Freedom from all‐cause and cardiovascular mortality were similar at 2 years (79.4 versus 80.6%, P=0.731; and 88.7 versus 87.4%, P=0.699; MO and nonobese, respectively). Multivariable analysis identified baseline glomerular filtration rate and nontransfemoral access as independent predictors of 2‐year mortality in the MO group. An adverse MO phenotype with an abdominal visceral adipose tissue:subcutaneous adipose tissue ratio ≥1 (VAT:SAT) was associated with increased 2‐year all‐cause (hazard ratio [HR], 3.06; 95% CI, 1.20–7.77; P=0.019) and cardiovascular (hazard ratio, 4.11; 95% CI, 1.06–15.90; P=0.041) mortality, and readmissions (HR, 1.81; 95% CI, 1.07–3.07; P=0.027). After multivariable analysis, a (VAT:SAT) ratio ≥1 remained a strong predictor of 2‐year mortality (hazard ratio, 2.78; P=0.035).ConclusionsTranscatheter aortic valve replacement in patients who are MO has similar short‐ and midterm outcomes to nonobese patients, despite higher major vascular complications and lower device success. An abdominal VAT:SAT ratio ≥1 identifies an obesity phenotype at higher risk of adverse clinical outcomes.

Phenotyping COVID-19 Patients by Ventilation Therapy: Data Quality Challenges and Cohort Characterization.

The COVID-19 pandemic introduced unique challenges for treating acute respiratory failure patients and highlighted the need for reliable phenotyping of patients using retrospective electronic health record data. In this study, we applied a rule-based phenotyping algorithm to classify COVID-19 patients requiring ventilatory support. We analyzed patient outcomes of the different phenotypes based on type and sequence of ventilation therapy. Invasive mechanical ventilation, noninvasive positive pressure ventilation, and high flow nasal insufflation were three therapies used to phenotype patients leading to a total of seven subgroups; patients treated with a single therapy (3), patients treated with either form of noninvasive ventilation and subsequently requiring intubation (2), and patients initially intubated and then weaned onto a noninvasive therapy (2). In addition to summary statistics for each phenotype, we highlight data quality challenges and importance of mapping to standard terminologies. This work illustrates potential impact of accurate phenotyping on patient-level and system-level outcomes including appropriate resource allocation under resource constrained circumstances.

Impact on outcomes in Europe: a cluster analysis from the ESC-EHRA EORP AF general long-term registry

Abstract Funding Acknowledgements Type of funding sources: None. OnBehalf ESC-EHRA EORP AF General Long-Term Registry Investigators Introduction Data derived from recent observational studies in atrial fibrillation (AF) show how the complexity of the clinical phenotype, beyond baseline thromboembolic risk, can increase risk of major adverse outcomes. Importantly, risk factors tend to occur in clusters, rather than occur individually in isolation. Aims To describe AF patients’ clinical phenotypes among a large contemporary European AF cohort and to analyse the differential impact of these clinical phenotypes on the occurrence of major adverse outcomes. Methods We performed a hierarchical cluster analysis based on Ward’s Method and using Squared Euclidean Distance using 22 clinical covariates. All variables were considered as binary. Examining the distances between cluster coefficients and by visual inspection of the dendrogram produced we identified the optimal number of clusters. Patients with data available for all 22 variables were included. We considered occurrence of cardiovascular events and all-cause death. Results Among the original 11096 patients included, 9363 (84.4%) were available for this analysis. The cluster analysis identified three clusters: Cluster 1 (n = 3634; 38.8%) characterized by older patients with prevalent noncardiac comorbidities; Cluster 2 (n = 2774; 29.6%) characterized by younger patients mainly admitted for first detected and paroxysmal AF with low prevalence of concomitant conditions; Cluster 3 (n = 2955; 31.6%) included patients with high prevalence of permanent AF, cardiac risk factors and comorbidities. Thromboembolic and bleeding risks were higher in Cluster 3 and progressively lower in Cluster 1 and Cluster 2 (both p &amp;lt; 0.001). Use of oral anticoagulant was significantly lower for Cluster 2 (83.2% vs. 86.5% and 86.7% in Cluster 1 and Cluster 3, respectively; p &amp;lt; 0.001). Over a mean follow-up of 22.5 (SD5.5) months, Cluster 3 had the highest rate of both cardiovascular events (10.0%) and all-cause death (13.2%), compared with Cluster 1 (6.6% and 9.4%, respectively) and Cluster 2 (3.7% and 3.8%, respectively) (both p &amp;lt; 0.001). Kaplan-Meier curves (Figure) show that Cluster 2 (green line) had the lowest cumulative risk of outcomes; risk was progressively higher in Cluster 1 (orange line) and Cluster 3 (yellow line). A Cox multivariable regression analysis, adjusted for type of AF, symptomatic status, CHA2DS2-VASc score and use of oral anticoagulants, showed that both Cluster 3 and Cluster 1 were associated with a significantly increased risk of cardiovascular events (HR: 1.80, 95%CI: 1.39-2.33 and HR: 1.40, 95%CI: 1.09-1.80, respectively) and all-cause death (HR: 1.80, 95%CI: 1.40-2.30 and HR: 1.66, 95%CI: 1.30-2.11) compared to Cluster 2. Conclusions In European AF patients, three main clinical clusters were identified, those with non-cardiac comorbidities, low risk and cardiac comorbidities. Both non-cardiac and cardiac comorbidities clusters were found to be associated with an increased risk of cardiovascular events and all-cause death. Abstract Figure. Kaplan-Meier Curves for Outcomes

TLR2- and TLR3-activated microglia induce different levels of neuronal network dysfunction in a context-dependent manner

Recognition of pathogen- or damage-associated molecular patterns (PAMPs, DAMPs) by innate Toll-like receptors (TLRs) is central to the activation of microglia (brain macrophages) in many CNS diseases. Notably, TLR-mediated microglial activation is complex and modulated by additional exogenous and endogenous immunological signals. The impact of different microglial reactive phenotypes on electrical activity and neurotransmission is widely unknown, however. We explored the effects of TLR ligands on microglia and neuronal network function in rat organotypic hippocampal slice cultures (in situ), i.e., postnatal cortical tissue lacking adaptive immunity. Single exposure of slice cultures to TLR2 or TLR3 ligands [PGN, poly(I:C)] for 2–3 days induced moderate microglial activation featuring IL-6 and TNF-α release and only mild alterations of fast neuronal gamma band oscillations (30–70 Hz) that are fundamental to higher cognitive functions, such as perception, memory and behavior. Paired exposure to TLR3/TLR2 or TLR3/TLR4 ligands (LPS) induced nitric oxide (NO) release, enhanced TNF-α release, and associated with advanced network dysfunction, including slowing to the beta frequency band (12–30 Hz) and neural bursts (hyperexcitability). Paired exposure to a TLR ligand and the leukocyte cytokine IFN-γ enhanced NO release and associated with severe network dysfunction, albeit sensitive parvalbumin- and somatostatin-positive inhibitory interneurons were preserved. Notably, the neuronal disturbance was prevented by either microglial depletion or pharmacological inhibition of oxidant-producing enzymes, inducible NO synthase (iNOS) and NADPH oxidase. In conclusion, TLR-activated microglia can induce different levels of neuronal network dysfunction, in which severe dysfunction is mainly caused by reactive oxygen and nitrogen species rather than proinflammatory cytokines. Our findings provide a mechanistic insight into microglial activation and functional neuronal network impairment, with relevance to neuroinflammation and neurodegeneration observed in, e.g., meningoencephalitis, multiple sclerosis and Alzheimer’s disease.

The Pharmacokinetics of Gefitinib in a Chinese Cancer Population Group: A Virtual Clinical Trials Population Study.

Gefitinib, a selective inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, is used to treat non-small-cell lung cancer (NSCLC). Lung cancer rates are high in China and are expected to increase over the next decade. CYP 2D6 intermediate metaboliser (IM) phenotypes are more prevalent in the Chinese population compared to Caucasians; the increased risk of drug-drug interactions (DDI) with chemotherapy polypharmacy may lead to different clinical pharmacokinetics outcomes for Chinese patients. This study developed and validated a virtual Chinese cancer population for the pragmatic assessment of gefitinib DDI as a victim drug in Chinese and Caucasian cancer populations. When assessing the impact of 2D6 phenotypes on bupropion mediated CYP 2D6 DDI in Chinese cancer population, we found that AUC increased by at least 60% in extensive metabolizers (EM) and 30% in IM. As a result, fmCYP2D6 was reduced by 15% in IM in the presence of bupropion, translating into > 70% of EM subjects and > 48% of IM subjects with trough concentrations at steady state (Ctrough,ss) below the gefitinib target trough level. The PBPK model predicted that a 500mg once daily dose in both EM and IM subjects successfully reduced the percent of subjects below the Ctrough,ss. Such changes in Ctrough,ss warrant further investigation and highlight the ability of pharmacokinetic modelling to investigate populations that may be difficult to recruit for traditional clinical studies.

Quercetin mediated inhibition of Staphylococcus aureus biofilms and the impact of the isolate phenotype and genotype

Aim: This study was designed to assess the antibiofilm activity of quercetin on characterized S. aureus isolates. Methodology: This study evaluated 36 S. aureus isolates, each of which was identified using Gram staining, culture, biochemical, and PCR assays. Isolates were cultured and their biofilm production was evaluated using Congo red agar (CRA) plates, microtiter plate tests and PCR, and the effects of quercetin were examined. Results: The CRA results revealed that eight (22.3%) S. aureus isolates were strongly positive for biofilm production and an additional 18 isolates (50%) showed moderate biofilm capacity. The remaining 10 isolates were negative (27.7%) for biofilm production. S. aureus isolates were divided into strong positive, intermediate, and negative groups, 27.8%, 44.5%, and 27.7%, respectively. Scanning electron microscopy showed that the biofilm-producing isolates appeared as aggregates of cells within a heavy matrix. In addition, PCR assay identified IcaA and IcaD (66.6% for both) biofilm production genes in most isolates and IcaC (61.1%), IcaB, FnbB (33.3% for both), and Fib (22.2%) in several other strains. Quercetin significantly inhibited biofilm activity in biofilm producing S. aureus isolates in a dose-dependent manner, with an inhibition rate of 29.6-87.7%. Interpretation: Biofilm production is dependent on Ica gene phenotype and strains with an IcaABCD or IcaABD phenotype produce more biofilm than strains with IcaAD phenotype. Quercetin significantly inhibited S. aureus biofilm production, irrespective of Ica phenotype.

Role of thin gingival phenotype and inadequate keratinized mucosa width(

There is growing evidence on the impact of thin gingival phenotype (TnP) and inadequate keratinized mucosa width (KMW<2mm) around dental implants on peri-implant health. This study investigated the role of TnP and inadequate KMW(<2mm) as risk indicators for peri-implantitis and mucositis and on dental patient-reported outcomes. Sixty-three patients with 193 implants (mean follow-up of 6.9 ± 3.7 years) were given a clinical and radiographic examination and a questionnaire to assess patient awareness of food impaction and pain/discomfort. Chi-squared tests and regression analysis for clustered data were used to compare outcomes. Implants with TnP had a statistically higher prevalence of peri-implantitis (27.1%versus 11.3%; PR, 3.32; 95% confidence interval (CI), 1.64-6.72; P=0.001) peri-implant mucositis (42.7%versus 33%; PR, 1.8; 95% CI, 1.12-2.9; P=0.016) and pain/discomfort during oral hygiene (25%versus 5%; PR, 3.7; 95% CI, 1.06-12.96; P=0.044) than thick phenotype. Implants with inadequate KMW had a statistically higher prevalence of peri-implantitis (24.1%versus 17%; PR, 1.87; 95% CI, 1.07-3.25; P=0.027) and peri-implant mucositis (46.6%versus 34.1%; PR, 1.53; 95% CI, 1-2.33; P=0.05) and pain/discomfort during oral hygiene (28%versus 10%; PR, 2.37; 95% CI, 1.1-5.1; P=0.027) than the adequate KMW. TnP was strongly associated with inadequate KMW (PR=3.18; 95% CI, 1.69-6.04; P<0.001). TnP and inadequate KMW(<2mm) may be significant risk indicators for peri-implant disease and pain/discomfort during brushing.

Silent spread of mobile colistin resistance gene mcr-9.1 on IncHI2 ‘superplasmids’ in clinical carbapenem-resistant Enterobacterales

Objectivesmcr-9.1 is a newly described mobile colistin resistance gene. We have noted its presence in multiple species of carbapenem-resistant Enterobacterales (CRE) from our institution. We aimed to determine the clinical features, genomic context and phenotypic impact of mcr-9.1 carriage in a series of patients between 2010 and 2019. MethodsWe identified 32 patients with mcr-9.1-carrying CRE isolates (mCRE) and collected demographic, antimicrobial exposure and infection data. Whole-genome sequencing (including short and long reads) was performed on 32 isolates. We assessed sequence similarity of mcr-9.1-harbouring plasmids, then compared our findings with plasmids for which sequence data were publicly available. ResultsThere was no colistin exposure in patients prior to isolation of mCRE. mcr-9.1 was identified on IncHI2 plasmids across four different bacterial species and was co-located with blaIMP-4 in 23/30 plasmids studied. mCRE isolates did not demonstrate phenotypic colistin resistance, either at baseline or following sublethal colistin exposure, thus showing that mcr-9.1 alone is not sufficient for resistance. Publicly available sequence data indicated the presence of carbapenemase genes in 236/619 mcr-9.1-carrying genomes (38%). IncHI2 plasmids carrying mcr-9.1 and carbapenemase genes were detected in genomes from North America, Europe, North Africa, Asia and Oceania. ConclusionsSpread of mcr-9.1 in CRE from our institution was driven by IncHI2 ‘superplasmids’, so termed because of their large size and their prolific carriage of resistance determinants. These were also detected in global CRE genomes. Phenotypic colistin resistance was not detected in our isolates but remains to be determined from global mCRE.