Associations of tissue tumor mutational burden and mutational status with clinical outcomes in KEYNOTE-042: pembrolizumab versus chemotherapy for advanced PD-L1-positive NSCLC.

BACKGROUND: We evaluated whether tissue tumor mutational burden (tTMB) and STK11, KEAP1, and KRAS mutations have clinical utility as biomarkers for pembrolizumab monotherapy versus platinum-based chemotherapy in patients with programmed death ligand 1 (PD-L1)-positive (tumor proportion score ≥1%) advanced/metastatic non-small-cell lung cancer (NSCLC) without EGFR/ALK alterations in the phase III KEYNOTE-042 trial. PATIENTS AND METHODS: This retrospective exploratory analysis assessed prevalence of tTMB and STK11, KEAP1, and KRAS mutations determined by whole-exome sequencing of tumor tissue and matched normal DNA and their associations with outcomes in KEYNOTE-042. Clinical utility of tTMB was assessed using a prespecified cut point of 175 mutations/exome. RESULTS: Of 793 patients, 345 (43.5%) had tTMB ≥175 mutations/exome and 448 (56.5%) had tTMB <175 mutations/exome. No association was observed between PD-L1 expression and tTMB. Continuous tTMB score was associated with improved overall survival (OS) and progression-free survival among patients receiving pembrolizumab (Wald test, one-sided P < 0.001) but not those receiving chemotherapy (Wald test, two-sided P > 0.05). tTMB ≥175 mutations/exome was associated with improved outcomes for pembrolizumab versus chemotherapy, whereas tTMB <175 mutations/exome was not {OS: hazard ratio, 0.62 [95% confidence interval (CI) 0.48-0.80] and 1.09 (95% CI 0.88-1.36); progression-free survival: 0.75 (0.59-0.95) and 1.27 (1.04-1.55), respectively}. Improved OS [hazard ratio (95% CI)] for pembrolizumab versus chemotherapy was observed regardless of STK11 [STK11 mutant (n = 33): 0.37 (0.16-0.86), STK11 wild-type (n = 396): 0.83 (0.65-1.05)]; KEAP1 [KEAP1 mutant (n = 64): 0.75 (0.42-1.35), KEAP1 wild-type (n = 365): 0.78 (0.61-0.99)], or KRAS [KRAS mutant (n = 69): 0.42 (0.22-0.81); KRAS wild-type (n = 232): 0.86 (0.63-1.18)] mutation status. CONCLUSION: tTMB with a cut point of ≥175 mutations/exome is a potential predictive biomarker for pembrolizumab monotherapy for advanced/metastatic PD-L1 tumor proportion score ≥1% NSCLC. Pembrolizumab is a standard first-line treatment in this setting regardless of STK11, KEAP1, or KRAS mutation status.

Evaluation of the anxiolytic activity of dry extract of Patrinia scabiosifolia under acoustic stress.

OBJECTIVE: Patrinia scabiosifolia has been used in traditional medicine in East Asia, Africa, and South America for a variety of diseases for more than 2000 years. The purpose of the article is to evaluate the anxiolytic properties of dry extract of P. scabiosifolia. MATERIALS AND METHODS: In vivo experiments were performed on outbred white male mice. The psychotropic effect of P. scabiosifolia dry extract was assessed using behavioral test systems aimed at identifying changes in the psycho-emotional state of animals under the influence of acoustic stress. In addition, the preparation toxicity was also assessed. HPLC-MS analysis was carried out to confirm the presence of active components in local raw materials. RESULTS: The article describes the possibility of using dry extract of P. scabiosifolia as an anxiolytic and sedative for psycho-emotional stress in experimental animals. Based on comprehensive research results, the effectiveness and safety of the studied herbal preparation have been proven. CONCLUSIONS: In this study, a dry extract of P. scabiosifolia has been proposed as a novel means of combating neuropsychiatric disorders. P. scabiosifolia showed efficacy comparable to the reference drug (Mebicar), reducing sleep time and increasing sleep duration. The results obtained can subsequently serve as the basis for clinical trials.

An accessible pharmacodynamic transcriptional biomarker for notch target engagement.

γ-Secretase mediates amyloid production in Alzheimer's disease (AD) and oncogenic activity of Notch. γ-Secretase inhibitors (GSIs) are thus of interest for AD and oncology. A peripheral biomarker of Notch activity would aid determination of the therapeutic window and dosing regimen for GSIs, given toxicities associated with chronic Notch inhibition. This study examined the effects of GSI MK-0752 on blood and hair follicle transcriptomes in healthy volunteers. The effects of a structurally diverse GSI on rhesus blood and hair follicles were also compared. Significant dose-related effects of MK-0752 on transcription were observed in hair follicles, but not blood. The GSI biomarker identified in follicles exhibited 100% accuracy in a clinical test cohort, and was regulated in rhesus by a structurally diverse GSI. This study identified a translatable, accessible pharmacodynamic biomarker of GSI target engagement and provides proof of concept of hair follicle RNA as a translatable biomarker source.

Alterations in Tear Biochemistry Associated with Postanesthetic Chronic Dry Eye Syndrome.

Perioperative dry eye syndrome (DES) is a common ocular complication of long-term general anesthesia. Chronic DES can lead to permanent damage to the cornea and disturbance of visual function, up to total loss of vision. Here, a relationship between the duration of general anesthesia and the risk of chronic DES in patients was demonstrated. Using an experimental model of perioperative corneal abrasions in rabbits, it was found that introduction of animals to 3-h general anesthesia resulted in clinically significant chronic damage to the cornea in 50% of cases. The development of the complication was not associated with irreversible or long-term impairment of tear secretion, but it was accompanied by a decrease in tear film stability and growth of the total protein content as well as decrease in total antioxidant activity of the tear induced by low molecular weight antioxidants. In addition, anesthesia-induced changes in activity of tear antioxidant enzymes including superoxide dismutase and enzymes providing homeostasis of reduced glutathione (glutathione peroxidase, glutathione-S-transferase, glutathione reductase) were observed. All these alterations were protracted (up to 1-2 weeks) and therefore might account for transition of the perioperative DES into the chronic form. These findings can be useful in the development of novel approaches for the prevention and treatment of chronic forms of DES in the postanesthetic period.

[THE LEVEL OF LACTATE DEHYDROGENASE AS A MARKER OF RENAL DYSFUNCTION IN NEONATES WITH ASPHYXIA].

The article examines the possibility of determining the level of lactate dehydrogenase (LD) in biological fluids as a marker of renal dysfunction and energy supply in neonates with asphyxia. Investigation included 200 full-term newborns with disturbance kidney function: 100 infants who had severe asphyxia, and 100--with moderate asphyxia. LD activity was determined by kinetic spectrophotometric method. Determination of the activity of LD in the urine in the early neonatal period it is advisable to use as a non-invasive marker for the diagnosis of renal dysfunction in neonates with asphyxia. The content of LD in the blood serum can be used as one of the early markers of kidney damage in newborns with asphyxia.

Elevated serum osteoprotegerin is associated with decreased osteoclastic differentiation in stenotic aortic valves.

Calcific aortic valve stenosis (CAVS) is an actively regulated process that involves mechanisms of bone development, including the receptor activator of nuclear factor κB, its ligand, and osteoprotegerin (RANK/RANKL/OPG) regulatory system. The aim of this study was to investigate whether the levels of circulating OPG and RANKL can be correlated with some histopathological features of the stenotic valves. Serum levels of osteoprotegerin (OPG) and soluble RANKL (sRANKL) were assessed in 27 patients with CAVS prior to valve replacement surgery and in 12 control subjects. The removed valves were examined macroscopically and microscopically. Valve sections were stained with hematoxylin and eosin for general morphology, with Oil Red O for lipids and immunostained with antibodies against markers visualizing osteoclastic cells (tartrate-resistant acid phosphatase, TRAP), macrophages (CD68) and blood vessels (CD34). Patients with CAVS had elevated levels of OPG as compared to the control group (p=0.005). Within the CAVS group, patients with osteoclastic TRAP-positive cells in their valves had significantly lower serum levels of OPG (p=0.009) and lipid content (p=0.03) than those without such cells. Moreover, osteogenic metaplasia was observed exclusively in the valves containing TRAP-positive cells. Results of this study suggest that the circulating OPG can influence the processes occurring in the calcifying valves by inhibiting osteoclastic differentiation of cells involved in calcification and by preventing osteogenic metaplasia.

A fragmentation study of the Rhodamine 610 cation using visible-laser desorption ionization.

The fragmentation of a potential visible matrix-assisted laser desorption ionization: Rhodamine 610 was studied under 532 nm visible irradiation, as a function of anion counter ion. It was found that at a fixed fluence, the chloride salt produced fewer fragments than those formed with ClO(4)(-) or BF(4)(-). Evidence presented suggests that the degree of fragmentation is inversely proportional to the strength of the contact ion pair in the solid state; that is, more energy is deposited into the radical cation which can lead to fragmentation when less energy is required to separate the ion pair. Similar results were found for salts of Rhodamine 6G.

Biomarker discovery: identification of a growth factor gene signature.

Gene expression signatures can be developed as comprehensive pathway readouts and used as pharmacodynamic or patient-stratification biomarkers. While a consensus on the best practices for selecting gene expression signatures from microarray data is evolving, we have developed basic guidelines to ensure consistency and quality. Here we illustrate these guidelines through the identification of a growth factor gene expression signature that is responsive to phosphatidylinositol 3-kinase (PI3K) pathway perturbations in vitro and related to phosphatase and tensin homolog (PTEN) deregulation in vivo.

C60 secondary ion mass spectrometry with a hybrid-quadrupole orthogonal time-of-flight mass spectrometer.

A hybrid quadrupole orthogonal time-of-flight mass spectrometer optimized for matrix-assisted laser desorption ionization (MALDI) and electrospray ionization has been equipped with a C 60 cluster ion source. This configuration is shown to exhibit a number of characteristics that improve the performance of traditional time-of-flight secondary ion mass spectrometry (TOF-SIMS) experiments for the analysis of complex organic materials and, potentially, for chemical imaging. Specifically, the primary ion beam is operated as a continuous rather than a pulsed beam, resulting in up to 4 orders of magnitude greater ion fluence on the target. The secondary ions are extracted at very low voltage into 8 mTorr of N 2 gas introduced for collisional focusing and cooling purposes. This extraction configuration is shown to yield secondary ions that rapidly lose memory of the mechanism of their birth, yielding tandem mass spectra that are identical for SIMS and MALDI. With implementation of ion trapping, the extraction efficiency is shown to be equivalent to that found in traditional TOF-SIMS machines. Examples are given, for a variety of substrates that illustrate mass resolution of 12,000-15,600 with a mass range for inorganic compounds to m/ z 40,000. Preliminary chemical mapping experiments show that with added sensitivity, imaging in the MS/MS mode of operation is straightforward. In general, the combination of MALDI and SIMS is shown to add capabilities to each technique, providing a robust platform for TOF-SIMS experiments that already exists in a large number of laboratories.

[Metabolic syndrome: more questions than answers].

Metabolic syndrome has a close association with "diseases of civilization" and widespread in economically developed countries. By the end of XX century, unification in clusters of cardiovascular diseases risk factors showed, that simultaneous presence of obesity, type 2 diabetes mellitus, arterial hypertension, insulin resistance, hyperinsulinemia and hyperlipidemia contributed to significant increase in mortality.

Effect of ochratoxin A on redox-regulated transcription factors, antioxidant enzymes and glutathione-S-transferase in cultured kidney tubulus cells.

Ochratoxin A (OTA), a mycotoxin mostly produced by Aspergillus ochraceus and Penicillium verrucosum, is a worldwide contaminant of food and feedstuff. OTA is nephrotoxic and a renal carcinogen in rodents. The underlying molecular and cellular mechanisms by which OTA exhibits its toxicity have yet not been fully clarified. In the present study the effects of ochratoxin A on the activity of redox-regulated transcription factors, antioxidant enzymes, as well as glutathione-S-transferase (GST) have been studied in cultured kidney tubulus cells (LLC-PK1). Confluent LLC-PK1 cells were incubated with increasing concentrations of OTA for 24h. OTA decreased SOD activity and enhanced intracellular levels of reactive oxygen species (ROS) as measured by flow cytometry. Furthermore OTA resulted in a down-regulation of GST mRNA and activity levels. Lower GST levels were accompanied by a decreased transactivation of activator protein-1 (AP-1) and NF-E2-related factor-2 (Nrf2), which mediate GST gene transcription. Present data indicate that enhanced ROS production and an impairment of GST activity, possibly due to an AP-1 and Nrf2 dependent signal transduction pathway, may be centrally involved in OTA induced nephrotoxicity.

[On the problem of obesity in internal diseases].

A review of available data on epidemiology of obesity, its classification, physiological role and morphological structure of adipose tissue, effect of obesity on metabolic processes depending on fat distribution in the body. The influence of obesity on the function of cardio-vascular, respiratory, digestive, urinary, and endocrine systems is considered along with its role in the clinical picture and outcome of somatic diseases.

Heme oxygenase-1-dependent and -independent regulation of angiogenic genes expression: effect of cobalt protoporphyrin and cobalt chloride on VEGF and IL-8 synthesis in human microvascular endothelial cells.

Induction of heme oxygenase-1 (HO-1) expression can be achieved by stimulation with cobalt protoporphyrin (CoPPIX) or cobalt chloride (CoCl2). HO-1 has been recently implicated in regulation of angiogenesis and CoCl2 is known to potently activate hypoxia inducible factor-1 (HIF-1) transcription factor, a key regulator of angiogenic response in hypoxia. Here we determined the effect of CoPPIX and CoCl2 on the expression of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), the two major angiogenic mediators, in human microvascular endothelial cells (HMEC-1). CoPPIX induced HO-1 expression and strongly enhanced VEGF and IL-8 synthesis, through the activation of VEGF and IL-8 promoters. Inhibition of HO activity by SnPPIX decreased VEGF production, while, interestingly, it did not affect IL-8. CoCl2 activated hypoxia-responsive element (HRE) and consequently VEGF generation via the enhancement of production of reactive oxygen species (ROS). On the other hand, CoCl2 did not influence IL-8 expression, while CoPPIX did not induce ROS elevation neither it affected HRE activity in VEGF promoter. Our data show that although both CoCl2 and CoPPIX induce HO-1, the influence of CoCl2 on VEGF does not involve HO-1 and is HIF-1-dependent, while the effect of CoPPIX does not involve HIF-1 but relies on HO-1.

Effects of plant extracts on angiogenic activities of endothelial cells and keratinocytes.

Numerous bioactive chemical compounds of plant origin may influence the angiogenic activity of various cell types and may thus affect the formation of blood vessels. Here we present the angiogenic effects of extracts of edible plants collected in Crete, Southern Italy and Southern Spain. Extracts have been applied to cultured human microvascular endothelial cells (HMEC-1), human umbilical vein endothelial cells (HUVEC) and human keratinocytes (HaCaT). About half out of 96 extracts exerted an inhibitory effect on HMEC-1 proliferation. Additionally, we have noted the inhibitory effects of extracts on HUVEC differentiation on a Matrigel layer. None of the extracts showed a stimulatory activity. The extract of Thymus piperella exerted moderate inhibitory effect on cobalt-chloride induced VEGF synthesis, however, CoCl(2)-induced activation of hypoxia responsive element of VEGF promoter was significantly attenuated only by extract of Origanum heracleoticum. Our study indicates that extracts of local food plants, of potential value as nutraceuticals, contain chemical compounds which may inhibit angiogenesis. Demonstration of their real influence on human health requires, however, extensive animal studies and controlled clinical investigations.

A high-performance matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometer with collisional cooling.

A high-performance orthogonal time-of-flight (TOF) mass spectrometer was developed specifically for use in combination with a matrix-assisted laser desorption/ionization (MALDI) source. The MALDI source features an ionization region containing a buffer gas with variable pressure. The source is interfaced to the TOF section via a collisional focusing ion guide. The pressure in the source influences the rate of cooling and allows control of ion fragmentation. The instrument provides uniform resolution up to 18,000 FWHM (full width at half maximum). Mass accuracy routinely achieved with a single-point internal recalibration is below 2 ppm for protein digest samples. The instrument is also capable of recording spectra of samples containing compounds with a broad range of masses while using one set of experimental conditions and without compromising resolution or mass accuracy.

Charge state separation for protein applications using a quadrupole time-of-flight mass spectrometer.

A novel method for separating ions according to their charge state using a quadrupole time-of-flight mass spectrometer is presented. The benefits of charge state separation are particularly apparent in protein identification applications at low femtomole concentration levels, where in conventional TOF MS spectra peptide ions are often lost in a sea of chemical noise. When doubly and triply charged tryptic peptide ions need to be filtered from singly charged background ions, the latter are suppressed by two to three orders of magnitude, while from 10-50% of multiply charged ions remain. The suppression of chemical noise reduces the need for chromatography and can make this experimental approach the electrospray equivalent of conventional MALDI peptide maps. If unambiguous identification cannot be achieved, MS/MS experiments are performed on the precursor ions identified through charge separation, while the previously described Q2-trapping duty cycle enhancement is tuned for approximately 1.4 of the precursor m/z to enhance intensities of ions with m/z values above that of the precursor. The resulting product ion spectra contain few fragments of impurities and provide quick and unambiguous identification through database search. The multiple charge separation technique requires minimal tuning and may become a useful tool for analysis of complex mixtures.

Matrix-assisted laser desorption/ionization quadrupole time-of-flight mass spectrometry: an elegant tool for peptidomics.

A Matrix-assisted laser desorption/ionization hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer was employed to acquire neuropeptide mass spectra, directly from neuropeptide secreting tissue deposited on the sample target, in the presence of dihydroxybenzoic acid as matrix. The cockroach corpus cardiacum served as model neuroendocrine tissue. Twelve neuropeptide ion peaks, with mass-to-charge ratio values ranging between 800 and 3,000 Da were selected for tandem mass spectrometry. All peptides below 1,600 Da could be fully sequenced; tandem mass spectrometry analysis of the remaining (three) largest peptides resulted in (limited) sequence tags, which, also due to unavailability of an appropriate neuropeptide structure database, did not allow complete structure elucidation.

An introduction to quadrupole-time-of-flight mass spectrometry.

A brief introduction is presented to the basic principles and application of a quadrupole-time-of-flight (TOF) tandem mass spectrometer. The main features of reflecting TOF instruments with orthogonal injection of ions are discussed. Their operation and performance are compared with those of triple quadrupoles with electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) TOF mass spectrometers. Examples and recommendations are provided for all major operational modes: mass spectrometry (MS) and tandem MS (MS/MS), precursor ion scans and studies of non-covalent complexes. Basic algorithms for liquid chromatography/MS/MS automation are discussed and illustrated by two applications.