In vivo evolution of resistant subpopulations of KPC-producing Klebsiella pneumoniae during ceftazidime/avibactam treatment.

Objectives: KPC-producing Klebsiella pneumoniae (KPC-Kp) represent a serious problem worldwide. Herein, we describe the evolution of ceftazidime/avibactam resistance by sequencing longitudinal clinical isolates from a patient with KPC-Kp bloodstream infection undergoing ceftazidime/avibactam treatment. Methods: WGS was performed on one ceftazidime/avibactam-susceptible KPC-Kp (BOT-CA-S) and two phenotypically different ceftazidime/avibactam-resistant KPC-Kp with low (BOT-CA-R) and high (BOT-EMO) carbapenem MICs. The population diversity was assessed by the frequency of allele mutations and population analysis profiles (PAPs). Results: Phylogenetic analysis demonstrated clonal relatedness of the KPC-Kp isolates, all belonging to the clone ST1519. The D179Y mutation in blaKPC-3 was detected in both of the ceftazidime/avibactam-resistant KPC-Kp, whereas it was absent in the ceftazidime/avibactam-susceptible isolate. The mutation emerged independently in the two ceftazidime/avibactam-resistant isolates and was associated with a significant reduction in carbapenem MICs in BOT-CA-R, but not in BOT-EMO. WGS analysis revealed that the frequency of the D179Y mutation was 96.32% and 51.05% in BOT-CA-R and BOT-EMO, respectively. PAP results demonstrated that carbapenem resistance in BOT-EMO was due to the coexistence of mixed subpopulations harbouring WT and mutated blaKPC-3. A bacterial subpopulation with high ceftazidime/avibactam resistance for BOT-EMO KPC-Kp showed low carbapenem MICs, whereas a subpopulation with high meropenem resistance had a low MIC of ceftazidime/avibactam. Conclusions: Our analysis indicates that mixed subpopulations of ceftazidime/avibactam-resistant KPC-Kp emerge after ceftazidime/avibactam treatment. The evolution of different subpopulations that are highly resistant to ceftazidime/avibactam likely contributes to treatment failure, thereby highlighting the need for combination treatment strategies to limit selection of ceftazidime/avibactam-resistant KPC-Kp subpopulations.

p63 isoforms regulate metabolism of cancer stem cells.

p63 is an important regulator of epithelial development expressed in different variants containing (TA) or lacking (ΔN) the N-terminal transactivation domain. The different isoforms regulate stem-cell renewal and differentiation as well as cell senescence. Several studies indicate that p63 isoforms also play a role in cancer development; however, very little is known about the role played by p63 in regulating the cancer stem phenotype. Here we investigate the cellular signals regulated by TAp63 and ΔNp63 in a model of epithelial cancer stem cells. To this end, we used colon cancer stem cells, overexpressing either TAp63 or ΔNp63 isoforms, to carry out a proteomic study by chemical-labeling approach coupled to network analysis. Our results indicate that p63 is implicated in a wide range of biological processes, including metabolism. This was further investigated by a targeted strategy at both protein and metabolite levels. The overall data show that TAp63 overexpressing cells are more glycolytic-active than ΔNp63 cells, indicating that the two isoforms may regulate the key steps of glycolysis in an opposite manner. The mass-spectrometry proteomics data of the study have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with data set identifiers PXD000769 and PXD000768.

Persistent cAMP signaling by internalized TSH receptors occurs in thyroid but not in HEK293 cells.

G-protein-coupled receptors (GPCRs) have long been believed to activate G proteins only on the cell surface. However, we have recently shown that, in thyroid cells, the GPCR for the thyroid-stimulating hormone (TSH) can continue stimulating cAMP production after cointernalization with TSH. cAMP signaling by internalized TSH receptors (TSHRs) was persistent, whereas that by cell-surface TSHRs was apparently transient, but the reasons for the transient signaling by cell-surface TSHRs were not investigated. Here, we developed and used fluorescence resonance energy transfer (FRET)-based methods to precisely compare the kinetics of TSH binding and dissociation from cell-surface TSHRs with those of the subsequent termination of cAMP signaling directly in living cells. Our results indicate that both TSH binding to human TSHRs expressed in a human embryonic kidney cell line (HEK 293) and the ensuing cAMP signals are rapidly and fully reversible (t(1/2,off)=2.96±1.04 and 2.70±0.73 min, respectively). The FRET measurement of TSH binding was specific, as shown by the lack of a detectable interaction between TSH and the ß(2)-adrenergic receptor expressed in control cells. Enhancing TSHR internalization by ß-arrestin 2 overexpression did not modify the reversibility of TSHR-cAMP signaling. These findings strengthen the view that the cointernalization of TSH-TSHR complexes to a signaling compartment present in thyroid, but not in HEK 293 cells, is responsible for persistent cAMP signaling.

In vitro interaction of ceftazidime-avibactam in combination with different antimicrobials against KPC-producing Klebsiella pneumoniae clinical isolates.

OBJECTIVES: Combination therapy has been recommended when using ceftazidime-avibactam (CAZ-AVI) for the treatment of KPC-producing Klebsiella pneumoniae (KPC-Kp), but the optimal combination is unknown. Six common antimicrobial agents (ertapenem, imipenem, meropenem, gentamicin, tigecycline, and ciprofloxacin) were evaluated for synergy with the recently approved cephalosporin-ß-lactamase inhibitor combination CAZ-AVI in this study. METHODS: Different antimicrobial combinations were tested against 13 KPC-Kp, including CAZ-AVI-susceptible (n=11) and resistant (n=2) clinical isolates. In vitro interactions of CAZ-AVI with different antimicrobials were tested using the gradient synergy test. Changes in the minimum inhibitory concentration (MIC) value were interpreted using the fractional inhibitory concentration (FIC) index and susceptible breakpoint index (SBPI). RESULTS: The combination of CAZ-AVI with gentamicin or ciprofloxacin displayed no synergism against any of the KPC-Kp isolates, whereas synergistic activity was observed with imipenem and meropenem against all KPC-Kp isolates. Notably, CAZ-AVI reduced MICs for meropenem and imipenem below the resistance breakpoints against all strains. The SBPI analysis showed that CAZ-AVI in combination with imipenem achieved higher SBPI values than other CAZ-AVI-based combinations. CONCLUSIONS: These data suggest that combinations of CAZ-AVI with imipenem may be considered a useful therapeutic option for the treatment of KPC-Kp infections.

Persistent cAMP Signaling by Internalized LH Receptors in Ovarian Follicles.

A crucial event in female reproduction occurs at midcycle, when a LH peak induces the final maturation of ovarian follicles. LH signals via a G protein-coupled receptor selectively expressed in the outermost follicular cell layers. However, how LH signals are relayed inside these cells and finally to the oocyte is incompletely understood. Here, we monitored LH signaling in intact ovarian follicles of transgenic mice expressing a fluorescent cAMP sensor. We found that LH stimulation induces 2 phases of cAMP signaling in all cell layers surrounding the oocyte. Interfering with LH receptor internalization abolished the second, persistent cAMP phase and partially inhibited oocyte meiosis resumption. These data suggest that persistent cAMP signals from internalized LH receptors contribute to transmitting LH effects inside follicle cells and ultimately to the oocyte. Thus, this study indicates that the recently proposed paradigm of cAMP signaling by internalized G protein-coupled receptors is implicated in receptor function and is physiologically relevant.

Cognitive recovery after delayed carbon monoxide encephalopathy.

We report on a patient with delayed carbon monoxide encephalopathy who presented with severe cognitive impairment associated with MRI findings of extensive demyelination of the cerebral white matter after a silent period of three weeks from acute intoxication. Despite the severe signs of structural and functional cerebral impairment in the sub-acute stage, the clinical course was favorable but for residual mild dysfunction of the frontal lobes.

ErbB-3 activation by NRG-1ß sustains growth and promotes vemurafenib resistance in BRAF-V600E colon cancer stem cells (CSCs).

Approximately 5-10% of metastatic colorectal cancers harbor a BRAF-V600E mutation, which is correlated with resistance to EGFR-targeted therapies and worse clinical outcome. Vice versa, targeted inhibition of BRAF-V600E with the selective inhibitor PLX 4032 (Vemurafenib) is severely limited due to feedback re-activation of EGFR in these tumors. Mounting evidence indicates that upregulation of the ErbB-3 signaling axis may occur in response to several targeted therapeutics, including Vemurafenib, and NRG-1ß-dependent re-activation of the PI3K/AKT survival pathway has been associated with therapy resistance.Here we show that colon CSCs express, next to EGFR and ErbB-2, also significant amounts of ErbB-3 on their membrane. This expression is functional as NRG-1ß strongly induces AKT/PKB and ERK phosphorylation, cell proliferation, clonogenic growth and promotes resistance to Vemurafenib in BRAF-V600E mutant colon CSCs. This resistance was completely dependent on ErbB-3 expression, as evidenced by knockdown of ErbB-3. More importantly, resistance could be alleviated with therapeutic antibody blocking ErbB-3 activation, which impaired NRG-1ß-driven AKT/PKB and ERK activation, clonogenic growth in vitro and tumor growth in xenograft models. In conclusion, our findings suggest that targeting ErbB-3 receptors could represent an effective therapeutic approach in BRAF-V600E mutant colon cancer.

CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis.

Cancer stem cells drive tumor formation and metastasis, but how they acquire metastatic traits is not well understood. Here, we show that all colorectal cancer stem cells (CR-CSCs) express CD44v6, which is required for their migration and generation of metastatic tumors. CD44v6 expression is low in primary tumors but demarcated clonogenic CR-CSC populations. Cytokines hepatocyte growth factor (HGF), osteopontin (OPN), and stromal-derived factor 1α (SDF-1), secreted from tumor associated cells, increase CD44v6 expression in CR-CSCs by activating the Wnt/ß-catenin pathway, which promotes migration and metastasis. CD44v6(-) progenitor cells do not give rise to metastatic lesions but, when treated with cytokines, acquire CD44v6 expression and metastatic capacity. Importantly, phosphatidylinositol 3-kinase (PI3K) inhibition selectively killed CD44v6 CR-CSCs and reduced metastatic growth. In patient cohorts, low levels of CD44v6 predict increased probability of survival. Thus, the metastatic process in colorectal cancer is initiated by CSCs through the expression of CD44v6, which is both a functional biomarker and therapeutic target.

CCR2 acts as scavenger for CCL2 during monocyte chemotaxis.

BACKGROUND: Leukocyte migration is essential for effective host defense against invading pathogens and during immune homeostasis. A hallmark of the regulation of this process is the presentation of chemokines in gradients stimulating leukocyte chemotaxis via cognate chemokine receptors. For efficient migration, receptor responsiveness must be maintained whilst the cells crawl on cell surfaces or on matrices along the attracting gradient towards increasing concentrations of agonist. On the other hand agonist-induced desensitization and internalization is a general paradigm for chemokine receptors which is inconsistent with the prolonged migratory capacity. METHODOLOGY/PRINCIPAL FINDINGS: Chemotaxis of monocytes was monitored in response to fluorescent CCL2-mCherry by time-lapse video microscopy. Uptake of the fluorescent agonist was used as indirect measure to follow the endogenous receptor CCR2 expressed on primary human monocytes. During chemotaxis CCL2-mCherry becomes endocytosed as cargo of CCR2, however, the internalization of CCR2 is not accompanied by reduced responsiveness of the cells due to desensitization. CONCLUSIONS/SIGNIFICANCE: During chemotaxis CCR2 expressed on monocytes internalizes with the bound chemoattractant, but cycles rapidly back to the plasma membrane to maintain high responsiveness. Moreover, following relocation of the source of attractant, monocytes can rapidly reverse their polarization axis organizing a new leading edge along the newly formed gradient, suggesting a uniform distribution of highly receptive CCR2 on the plasma membrane. The present observations further indicate that during chemotaxis CCR2 acts as scavenger consuming the chemokine forming the attracting cue.

Altered handwriting suggests cognitive impairment and may be relevant to posthumous evaluation.

Judging the validity of a disputed will is complex; however, one of the main issues is what the mental status of the testator was at the time of the will. If the will is handwritten, a handwriting analysis can provide information on the mental status of the testator. We tested how two writing parameters (the "writing score," a novel evaluation scale that we previously described, and the percentage of spelling mistakes) are capable to identify cognitively impaired persons. These parameters are especially helpful because they can be used to evaluate the mental status of a deceased person. We found a significant correlation between either parameter and established scales of neuropsychological evaluation (Mini Mental State Examination and Milan Overall Dementia Assessment scale). Specifically, a poor score on either parameter reliably identified a compromised cognitive status. These may represent helpful additions to existing techniques in posthumously identifying persons with severe cognitive impairment.

Polarization of migrating monocytic cells is independent of PI 3-kinase activity.

BACKGROUND: Migration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization. METHODOLOGY/PRINCIPAL FINDINGS: We present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the alpha(2A)-adrenoceptor (alpha(2A)AR) display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homologue UK 14'304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microscopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET) of alpha(2A)AR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinase activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes--in contrast to neutrophils--rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation. CONCLUSIONS/SIGNIFICANCE: Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the attractant cue. Polarized monocytes, which display typical amoeboid like motility, can rapidly develop a new leading edge facing the highest chemoattractant concentration at any site of the plasma membrane, including the uropod. The process appears to be independent of PI 3-kinase activity.