Memory effects of prior subculture may impact the quality of multiomic perturbation profiles.

Mass spectrometry-based omics technologies are increasingly used in perturbation studies to map drug effects to biological pathways by identifying significant molecular events. Significance is influenced by fold change and variation of each molecular parameter, but also by multiple testing corrections. While the fold change is largely determined by the biological system, the variation is determined by experimental workflows. Here, it is shown that memory effects of prior subculture can influence the variation of perturbation profiles using the two colon carcinoma cell lines SW480 and HCT116. These memory effects are largely driven by differences in growth states that persist into the perturbation experiment. In SW480 cells, memory effects combined with moderate treatment effects amplify the variation in multiple omics levels, including eicosadomics, proteomics, and phosphoproteomics. With stronger treatment effects, the memory effect was less pronounced, as demonstrated in HCT116 cells. Subculture homogeneity was controlled by real-time monitoring of cell growth. Controlled homogeneous subculture resulted in a perturbation network of 321 causal conjectures based on combined proteomic and phosphoproteomic data, compared to only 58 causal conjectures without controlling subculture homogeneity in SW480 cells. Some cellular responses and regulatory events were identified that extend the mode of action of arsenic trioxide (ATO) only when accounting for these memory effects. Controlled prior subculture led to the finding of a synergistic combination treatment of ATO with the thioredoxin reductase 1 inhibitor auranofin, which may prove useful in the management of NRF2-mediated resistance mechanisms.

Plasma Instead of Serum Avoids Critical Confounding of Clinical Metabolomics Studies by Platelets.

Metabolomics is an emerging and powerful bioanalytical method supporting clinical investigations. Serum and plasma are commonly used without rational prioritization. Serum is collected after blood coagulation, a complex biochemical process involving active platelet metabolism. This may affect the metabolome and increase the variance, as platelet counts and function may vary substantially in individuals. A multiomics approach systematically investigating the suitability of serum and plasma for clinical studies demonstrated that metabolites correlated well (n = 461, R2 = 0.991), whereas lipid mediators (n = 83, R2 = 0.906) and proteins (n = 322, R2 = 0.860) differed substantially between specimen. Independently, analysis of platelet releasates identified most biomolecules significantly enriched in serum compared to plasma. A prospective, randomized, controlled parallel group metabolomics trial with acetylsalicylic acid administered for 7 days demonstrated that the apparent drug effects significantly differ depending on the analyzed specimen. Only serum analyses of healthy individuals suggested a significant downregulation of TXB2 and 12-HETE, which were specifically formed during coagulation in vitro. Plasma analyses reliably identified acetylsalicylic acid effects on metabolites and lipids occurring in vivo such as an increase in serotonin, 15-deoxy-PGJ2 and sphingosine-1-phosphate and a decrease in polyunsaturated fatty acids. The present data suggest that plasma should be preferred above serum for clinical metabolomics studies as the serum metabolome may be substantially confounded by platelets.

Gene expression markers in peripheral blood and outcome in patients with platinum-resistant ovarian cancer: A study of the European GANNET53 consortium.

Disease progression is a major problem in ovarian cancer. There are very few treatment options for patients with platinum-resistant ovarian cancer (PROC), and therefore, these patients have a particularly poor prognosis. The aim of the present study was to identify markers for monitoring the response of 123 PROC patients enrolled in the Phase I/II GANNET53 clinical trial, which evaluated the efficacy of Ganetespib in combination with standard chemotherapy versus standard chemotherapy alone. In total, 474 blood samples were collected, comprising baseline samples taken before the first administration of the study drugs and serial samples taken during treatment until further disease progression (PD). After microfluidic enrichment, 27 gene transcripts were analyzed using quantitative polymerase chain reaction and their utility for disease monitoring was evaluated. At baseline, ERCC1 was associated with an increased risk of PD (hazard ratio [HR] 1.75, 95% confidence interval [CI]: 1.20-2.55; p = 0.005), while baseline CDH1 and ESR1 may have a risk-reducing effect (CDH1 HR 0.66, 95% CI: 0.46-0.96; p = 0.024; ESR1 HR 0.58, 95% CI: 0.39-0.86; p = 0.002). ERCC1 was observed significantly more often (72.7% vs. 53.9%; p = 0.032) and ESR1 significantly less frequently (59.1% vs. 78.3%; p = 0.018) in blood samples taken at radiologically confirmed PD than at controlled disease. At any time during treatment, ERCC1-presence and ESR1-absence were associated with short PFS and with higher odds of PD within 6 months (odds ratio 12.77, 95% CI: 4.08-39.97; p < 0.001). Our study demonstrates the clinical relevance of ESR1 and ERCC1 and may encourage the analysis of liquid biopsy samples for the management of PROC patients.

Effect of prehospital high-dose glucocorticoid on hemodynamics in patients resuscitated from out-of-hospital cardiac arrest: a sub-study of the STEROHCA trial.

BACKGROUND: Following resuscitated out-of-hospital cardiac arrest (OHCA), inflammatory markers are significantly elevated and associated with hemodynamic instability and organ dysfunction. Vasopressor support is recommended to maintain a mean arterial pressure (MAP) above 65 mmHg. Glucocorticoids have anti-inflammatory effects and may lower the need for vasopressors. This study aimed to assess the hemodynamic effects of prehospital high-dose glucocorticoid treatment in resuscitated comatose OHCA patients. METHODS: The STEROHCA trial was a randomized, placebo-controlled, phase 2 trial comparing one prehospital injection of methylprednisolone 250 mg with placebo immediately after resuscitated OHCA. In this sub-study, we included patients who remained comatose at admission and survived until intensive care unit (ICU) admission. The primary outcome was cumulated norepinephrine use from ICU admission until 48 h reported as mcg/kg/min. Secondary outcomes included hemodynamic status characterized by MAP, heart rate, vasoactive-inotropic score (VIS), and the VIS/MAP-ratio as well as cardiac function assessed by pulmonary artery catheter measurements. Linear mixed-model analyses were performed to evaluate mean differences between treatment groups at all follow-up times. RESULTS: A total of 114 comatose OHCA patients were included (glucocorticoid: n = 56, placebo: n = 58) in the sub-study. There were no differences in outcomes at ICU admission. From the time of ICU admission up to 48 h post-admission, patients in the glucocorticoid group cumulated a lower norepinephrine use (mean difference - 0.04 mcg/kg/min, 95% CI - 0.07 to - 0.01, p = 0.02). Moreover, after 12-24 h post-admission, the glucocorticoid group demonstrated a higher MAP with mean differences ranging from 6 to 7 mmHg (95% CIs from 1 to 12), a lower VIS (mean differences from - 4.2 to - 3.8, 95% CIs from - 8.1 to 0.3), and a lower VIS/MAP ratio (mean differences from - 0.10 to - 0.07, 95% CIs from - 0.16 to - 0.01), while there were no major differences in heart rate (mean differences from - 4 to - 3, 95% CIs from - 11 to 3). These treatment differences between groups were also present 30-48 h post-admission but to a smaller extent and with increased statistical uncertainty. No differences were found in pulmonary artery catheter measurements between groups. CONCLUSIONS: Prehospital treatment with high-dose glucocorticoid was associated with reduced norepinephrine use in resuscitated OHCA patients. TRIAL REGISTRATION: EudraCT number: 2020-000855-11; submitted March 30, 2020. URL: https://www. CLINICALTRIALS: gov ; Unique Identifier: NCT04624776.

The Proteome of Extracellular Vesicles Released from Pulmonary Microvascular Endothelium Reveals Impact of Oxygen Conditions on Biotrauma.

The lung can experience different oxygen concentrations, low as in hypoxia, high as under supplemental oxygen therapy, or oscillating during intermittent hypoxia as in obstructive sleep apnea or intermittent hypoxia/hyperoxia due to cyclic atelectasis in the ventilated patient. This study aimed to characterize the oxygen-condition-specific protein composition of extracellular vesicles (EVs) released from human pulmonary microvascular endothelial cells in vitro to decipher their potential role in biotrauma using quantitative proteomics with bioinformatic evaluation, transmission electron microscopy, flow cytometry, and non-activated thromboelastometry (NATEM). The release of vesicles enriched in markers CD9/CD63/CD81 was enhanced under intermittent hypoxia, strong hyperoxia and intermittent hypoxia/hyperoxia. Particles with exposed phosphatidylserine were increased under intermittent hypoxia. A small portion of vesicles were tissue factor-positive, which was enhanced under intermittent hypoxia and intermittent hypoxia/hyperoxia. EVs from treatment with intermittent hypoxia induced a significant reduction of Clotting Time in NATEM analysis compared to EVs isolated after normoxic exposure, while after intermittent hypoxia/hyperoxia, tissue factor in EVs seems to be inactive. Gene set enrichment analysis of differentially expressed genes revealed that EVs from individual oxygen conditions potentially induce different biological processes such as an inflammatory response under strong hyperoxia and intermittent hypoxia/hyperoxia and enhancement of tumor invasiveness under intermittent hypoxia.

RiboScreenTM Technology Delivers a Ribosomal Target and a Small-Molecule Ligand for Ribosome Editing to Boost the Production Levels of Tropoelastin, the Monomeric Unit of Elastin.

Elastin, a key structural protein essential for the elasticity of the skin and elastogenic tissues, degrades with age. Replenishing elastin holds promise for anti-aging cosmetics and the supplementation of elastic activities of the cardiovascular system. We employed RiboScreenTM, a technology for identifying molecules that enhance the production of specific proteins, to target the production of tropoelastin. We make use of RiboScreenTM in two crucial steps: first, to pinpoint a target ribosomal protein (TRP), which acts as a switch to increase the production of the protein of interest (POI), and second, to identify small molecules that activate this ribosomal protein switch. Using RiboScreenTM, we identified ribosomal protein L40, henceforth eL40, as a TRP switch to boost tropoelastin production. Drug discovery identified a small-molecule hit that binds to eL40. In-cell treatment demonstrated activity of the eL40 ligand and delivered increased tropoelastin production levels in a dose-dependent manner. Thus, we demonstrate that RiboScreenTM can successfully identify a small-molecule hit capable of selectively enhancing tropoelastin production. This compound has the potential to be developed for topical or systemic applications to promote skin rejuvenation and to supplement elastic functionality within the cardiovascular system.

Multidrug-Resistant Profiles in Non-Small Cell Lung Carcinoma Patient-Derived Cells: Implications for Personalized Approaches with Tyrosine Kinase Inhibitors.

The impact of tyrosine kinase inhibitors (TKIs) on multidrug resistance (MDR) in non-small cell lung carcinoma (NSCLC) is a critical aspect of cancer therapy. While TKIs effectively target specific signaling pathways of cancer cells, they can also act as substrates for ABC transporters, potentially triggering MDR. The aim of our study was to evaluate the response of 17 patient-derived NSCLC cultures to 10 commonly prescribed TKIs and to correlate these responses with patient mutational profiles. Using an ex vivo immunofluorescence assay, we analyzed the expression of the MDR markers ABCB1, ABCC1, and ABCG2, and correlated these data with the genetic profiles of patients for a functional diagnostic approach. NSCLC cultures responded differently to TKIs, with erlotinib showing good efficacy regardless of mutation burden or EGFR status. However, the modulation of MDR mechanisms by erlotinib, such as increased ABCG2 expression, highlights the challenges associated with erlotinib treatment. Other TKIs showed limited efficacy, highlighting the variability of response in NSCLC. Genetic alterations in signaling pathways associated with drug resistance and sensitivity, including TP53 mutations, likely contributed to the variable responses to TKIs. The relationships between ABC transporter expression, gene alterations, and response to TKIs did not show consistent patterns. Our results suggest that in addition to mutational status, performing functional sensitivity screening is critical for identifying appropriate treatment strategies with TKIs. These results underscore the importance of considering drug sensitivity, off-target effects, MDR risks, and patient-specific genetic profiles when optimizing NSCLC treatment and highlight the potential for personalized approaches, especially in early stages.

TFEB controls sensitivity to chemotherapy and immuno-killing in non-small cell lung cancer.

BACKGROUND: In non-small cell lung cancer (NSCLC) the efficacy of chemo-immunotherapy is affected by the high expression of drug efflux transporters as ABCC1 and by the low expression of ABCA1, mediating the isopentenyl pyrophosphate (IPP)-dependent anti-tumor activation of Vγ9Vδ2 T-lymphocytes. In endothelial cells ABCA1 is a predicted target of the transcription factor EB (TFEB), but no data exists on the correlation between TFEB and ABC transporters involved in the chemo-immuno-resistance in NSCLC. METHODS: The impact of TFEB/ABCC1/ABCA1 expression on NSCLC patients' survival was analyzed in the TCGA-LUAD cohort and in a retrospective cohort of our institution. Human NSCLC cells silenced for TFEB (shTFEB) were analyzed for ABC transporter expression, chemosensitivity and immuno-killing. The chemo-immuno-sensitizing effects of nanoparticles encapsulating zoledronic acid (NZ) on shTFEB tumors and on tumor immune-microenvironment were evaluated in Hu-CD34+ mice by single-cell RNA-sequencing. RESULTS: TFEBlowABCA1lowABCC1high and TFEBhighABCA1highABCC1low NSCLC patients had the worst and the best prognosis, respectively, in the TCGA-LUAD cohort and in a retrospective cohort of patients receiving platinum-based chemotherapy or immunotherapy as first-line treatment. By silencing shTFEB in NSCLC cells, we demonstrated that TFEB was a transcriptional inducer of ABCA1 and a repressor of ABCC1. shTFEB cells had also a decreased activity of ERK1/2/SREBP2 axis, implying reduced synthesis and efflux via ABCA1 of cholesterol and its intermediate IPP. Moreover, TFEB silencing reduced cholesterol incorporation in mitochondria: this event increased the efficiency of OXPHOS and the fueling of ABCC1 by mitochondrial ATP. Accordingly, shTFEB cells were less immuno-killed by the Vγ9Vδ2 T-lymphocytes activated by IPP and more resistant to cisplatin. NZ, which increased IPP efflux but not OXPHOS and ATP production, sensitized shTFEB immuno-xenografts, by reducing intratumor proliferation and increasing apoptosis in response to cisplatin, and by increasing the variety of anti-tumor infiltrating cells (Vγ9Vδ2 T-lymphocytes, CD8+T-lymphocytes, NK cells). CONCLUSIONS: This work suggests that TFEB is a gatekeeper of the sensitivity to chemotherapy and immuno-killing in NSCLC, and that the TFEBlowABCA1lowABCC1high phenotype can be predictive of poor response to chemotherapy and immunotherapy. By reshaping both cancer metabolism and tumor immune-microenvironment, zoledronic acid can re-sensitize TFEBlow NSCLCs, highly resistant to chemo- and immunotherapy.

A Transformative Leadership Journey: Experiences of Graduates of the American Physical Therapy Association Fellowship in Education Leadership.

INTRODUCTION: Physical therapy education has an acute need for effective leaders due to multiple factors, including increasing numbers of programs and retirement of experienced leaders. Recognizing the need for formal leadership education, the American Physical Therapy Association (APTA) developed a fellowship in physical therapy educational leadership in 2011. This study aimed to understand participants' perceptions of their leadership experience and the role of the APTA fellowship. LITERATURE REVIEW: A commonly reported approach to evaluating leadership education programs was designed by Kirkpatrick. PARTICIPANTS: Twenty-five fellowship graduates from 2012 to 2019 participated. METHODS: We used a qualitative methodological approach incorporating a heuristic technique to gain an understanding of fellowship graduates' long-term perspectives. Semi-structured interviews were conducted via Zoom. Data were analyzed using thematic analysis. RESULTS: Three themes were constructed using a journey metaphor: embarking on the journey, engaging in the journey, and realizing the effects of the journey. 1) Embarking on the journey included 2 subthemes: a) participants' personal aspirations as career planners, reluctant leaders, or explorers; and b) institutions' goals for a change in leadership or intentional succession planning. 2) Engaging in the journey included 3 subthemes: a) the fellowship learning environment builds a sense of community; b) consideration of leadership frames influences participants' thinking about leadership; and c) participants' institutional environment influences their learning. 3) Effects of the journey included 3 subthemes: a) personal growth as a leader including personal awareness and confidence; b) institutions' benefits from participants' leadership projects and increased engagement in institutional leadership; and c) physical therapy professions' gains from participants' seeking leadership positions in professional organizations. DISCUSSION/CONCLUSIONS: The APTA Fellowship in Educational Leadership results in transformative change of participants as leaders. Findings offer guidance to individuals considering embarking on a leadership journey and institutions considering how to prepare and support emerging leaders in physical therapy education.