Blood oxygen regulation via P2Y12R expressed in the carotid body.

BACKGROUND: Peripheral blood oxygen monitoring via chemoreceptors in the carotid body (CB) is an integral function of the autonomic cardiorespiratory regulation. The presence of the purinergic P2Y12 receptor (P2Y12R) has been implicated in CB; however, the exact role of the receptor in O2 sensing and signal transduction is unknown. METHODS: The presence of P2Y12R was established by immunoblotting, RT qPCR and immunohistochemistry. Primary glomus cells were used to assess P2Y12R function during hypoxia and hypercapnia, where monoamines were measured by HPLC; calcium signal was recorded utilizing OGB-1 and N-STORM Super-Resolution System. Ingravescent hypoxia model was tested in anaesthetized mice of mixed gender and cardiorespiratory parameters were recorded in control and receptor-deficient or drug-treated experimental animals. RESULTS: Initially, the expression of P2Y12R in adult murine CB was confirmed. Hypoxia induced a P2Y12R-dependent release of monoamine transmitters from isolated CB cells. Receptor activation with the endogenous ligand ADP promoted release of neurotransmitters under normoxic conditions, while blockade disrupted the amplitude and duration of the intracellular calcium concentration. In anaesthetised mice, blockade of P2Y12R expressed in the CB abrogated the initiation of compensatory cardiorespiratory changes in hypoxic environment, while centrally inhibited receptors (i.e. microglial receptors) or receptor-deficiency induced by platelet depletion had limited influence on the physiological adjustment to hypoxia. CONCLUSIONS: Peripheral P2Y12R inhibition interfere with the complex mechanisms of acute oxygen sensing by influencing the calcium signalling and the release of neurotransmitter molecules to evoke compensatory response to hypoxia. Prospectively, the irreversible blockade of glomic receptors by anti-platelet drugs targeting P2Y12Rs, propose a potential, formerly unrecognized side-effect to anti-platelet medications in patients with pulmonary morbidities.

European Respiratory Society statement on frailty in adults with chronic lung disease.

Frailty is a complex, multidimensional syndrome characterised by a loss of physiological reserves that increases a person's susceptibility to adverse health outcomes. Most knowledge regarding frailty originates from geriatric medicine; however, awareness of its importance as a treatable trait for people with chronic respiratory disease (including asthma, COPD and interstitial lung disease) is emerging. A clearer understanding of frailty and its impact in chronic respiratory disease is a prerequisite to optimise clinical management in the future. This unmet need underpins the rationale for undertaking the present work. This European Respiratory Society statement synthesises current evidence and clinical insights from international experts and people affected by chronic respiratory conditions regarding frailty in adults with chronic respiratory disease. The scope includes coverage of frailty within international respiratory guidelines, prevalence and risk factors, review of clinical management options (including comprehensive geriatric care, rehabilitation, nutrition, pharmacological and psychological therapies) and identification of evidence gaps to inform future priority areas of research. Frailty is underrepresented in international respiratory guidelines, despite being common and related to increased hospitalisation and mortality. Validated screening instruments can detect frailty to prompt comprehensive assessment and personalised clinical management. Clinical trials targeting people with chronic respiratory disease and frailty are needed.

Elevated Serum Gastrin Is Associated with Melanoma Progression: Putative Role in Increased Migration and Invasion of Melanoma Cells.

Micro-environmental factors, including stromal and immune cells, cytokines, and circulating hormones are well recognized to determine cancer progression. Melanoma cell growth was recently shown to be suppressed by cholecystokinin/gastrin (CCK) receptor antagonists, and our preliminary data suggested that melanoma patients with Helicobacter gastritis (which is associated with elevated serum gastrin) might have an increased risk of cancer progression. Therefore, in the present study, we examined how gastrin may act on melanoma cells. In 89 melanoma patients, we found a statistically significant association between circulating gastrin concentrations and melanoma thickness and metastasis, which are known risk factors of melanoma progression and prognosis. Immunocytochemistry using a validated antibody confirmed weak to moderate CCK2R expression in both primary malignant melanoma cells and the melanoma cell lines SK-MEL-2 and G361. Furthermore, among the 219 tumors in the Skin Cutaneous Melanoma TCGA Pan-Cancer dataset showing gastrin receptor (CCKBR) expression, significantly higher CCKBR mRNA levels were linked to stage III-IV than stage I-II melanomas. In both cell lines, gastrin increased intracellular calcium levels and stimulated cell migration and invasion through mechanisms inhibited by a CCK2 receptor antagonist. Proteomic studies identified increased MMP-2 and reduced TIMP-3 levels in response to gastrin that were likely to contribute to the increased migration of both cell lines. However, the effects of gastrin on tumor cell invasion were relatively weak in the presence of the extracellular matrix. Nevertheless, dermal fibroblasts/myofibroblasts, known also to express CCK2R, increased gastrin-induced cancer cell invasion. Our data suggest that in a subset of melanoma patients, an elevated serum gastrin concentration is a risk factor for melanoma tumor progression, and that gastrin may act on both melanoma and adjacent stromal cells through CCK2 receptors to promote mechanisms of tumor migration and invasion.

Herpes Simplex Virus Infection Alters the Immunological Properties of Adipose-Tissue-Derived Mesenchymal-Stem Cells.

The proper functioning of mesenchymal stem cells (MSCs) is of paramount importance for the homeostasis of the body. Inflammation and infection can alter the function of MSCs, which can also affect the regenerative potential and immunological status of tissues. It is not known whether human herpes simplex viruses 1 and 2 (HSV1 and HSV2), well-known human pathogens that can cause lifelong infections, can induce changes in MSCs. In non-healing ulcers, HSV infection is known to affect deeper tissue layers. In addition, HSV infection can recur after initially successful cell therapies. Our aim was to study the response of adipose-derived MSCs (ADMSCs) to HSV infection in vitro. After confirming the phenotype and differentiation capacity of the isolated cells, we infected the cells in vitro with HSV1-KOS, HSV1-532 and HSV2 virus strains. Twenty-four hours after infection, we examined the gene expression of the cells via RNA-seq and RT-PCR; detected secreted cytokines via protein array; and determined autophagy via Western blot, transmission electron microscopy (TEM) and fluorescence microscopy. Infection with different HSV strains resulted in different gene-expression patterns. In addition to the activation of pathways characteristic of viral infections, distinct non-immunological pathways (autophagy, tissue regeneration and differentiation) were also activated according to analyses with QIAGEN Ingenuity Pathway Analysis, Kyoto Encyclopedia of Genes and Genome and Genome Ontology Enrichment. Viral infections increased autophagy, as confirmed via TEM image analysis, and also increased levels of the microtubule-associated protein light chain 3 (LC3B) II protein. We identified significantly altered accumulation for 16 cytokines involved in tissue regeneration and inflammation. Our studies demonstrated that HSV infection can alter the viability and immunological status of ADMSCs, which may have implications for ADMSC-based cell therapies. Alterations in autophagy can affect numerous processes in MSCs, including the inhibition of tissue regeneration as well as pathological differentiation.

Fatigue, sleepiness and sleep quality are SARS-CoV-2 variant independent in patients with long COVID symptoms.

Acute infections with SARS-CoV-2 variants of concerns (VOCs) differ in clinical presentation. Discrepancies in their long-term sequelae, commonly referred to as long COVID, however, remain to be explored. We retrospectively analyzed data of 287 patients presented at the post-COVID care of the Pulmonology Department, Semmelweis University, Budapest, Hungary, and infected with SARS-CoV-2 during a period of 3 major epidemic waves in Hungary (February-July 2021, VOC: B.1.1.7, Alpha, N = 135; August-December 2021, VOC: B.1.617.2, Delta, N = 89; and January-June 2022, VOC: B.1.1.529, Omicron; N = 63), > 4 weeks after acute COVID-19. Overall, the ratio of long COVID symptomatic (LC) and asymptomatic (NS) patients was 2:1. Self-reported questionnaires on fatigue (Fatigue Severity Scale, FSS), sleepiness (Epworth Sleepiness Scale, ESS) and sleep quality (Pittsburgh Sleep Quality Index, PSQI) showed higher scores for LC (4.79 ± 0.12, 7.45 ± 0.33 and 7.46 ± 0.27, respectively) than NS patients (2.85 ± 0.16, 5.23 ± 0.32 and 4.26 ± 0.29, respectively; p < 0.05 for all vs. LC). By comparing data of the three waves, mean FSS and PSQI scores of LC patients, but not ESS scores, exceeded the normal range in all, with no significant inter-wave differences. Considering FSS ≥ 4 and PSQI > 5 cutoff values, LC patients commonly exhibited problematic fatigue (≥ 70%) and poor sleep quality (> 60%) in all three waves. Comparative analysis of PSQI component scores of LC patients identified no significant differences between the three waves. Our findings highlight the importance of concerted efforts to manage both fatigue and sleep disturbances in long COVID patient care. This multifaceted approach should be followed in all cases infected with either VOCs of SARS-CoV-2.

Vasopressin as a Possible Link between Sleep-Disturbances and Memory Problems.

Normal biological rhythms, including sleep, are very important for a healthy life and their disturbance may induce-among other issues-memory impairment, which is a key problem of many psychiatric pathologies. The major brain center of circadian regulation is the suprachiasmatic nucleus, and vasopressin (AVP), which is one of its main neurotransmitters, also plays a key role in memory formation. In this review paper, we aimed to summarize our knowledge on the vasopressinergic connection between sleep and memory with the help of the AVP-deficient Brattleboro rat strain. These animals have EEG disturbances with reduced sleep and impaired memory-boosting theta oscillation and show memory impairment in parallel. Based upon human and animal data measuring AVP levels, haplotypes, and the administration of AVP or its agonist or antagonist via different routes (subcutaneous, intraperitoneal, intracerebroventricular, or intranasal), V1a receptors (especially of hippocampal origin) were implicated in the sleep-memory interaction. All in all, the presented data confirm the possible connective role of AVP between biological rhythms and memory formation, thus, supporting the importance of AVP in several psychopathological conditions.

Evaluation of cardiopulmonary exercise test in the prediction of disease progression in systemic sclerosis.

OBJECTIVES: Cardiopulmonary exercise test (CPET) is a widely used examination to predict the prognosis of many chronic pulmonary diseases, and it has also been tested in systemic sclerosis (SSc) with a focus on the development of pulmonary hypertension. CPET is a highly informative non-invasive tool that provides a more complex information than conventional lung function tests to predict the course of cardiopulmonary diseases, as it provides a general overview of the aerobic metabolism, influenced by pulmonary, cardiovascular and peripheral muscle function. The purpose of this investigation was to assess if the progression and the development of poor overall disease outcome in SSc can be predicted by this method. METHODS: Twenty-nine SSc patients were investigated prospectively with standard follow-up plus CPET for a mean of 3.7 years to match the results of conventional evaluation modalities and CPET. A composite end-point of several serious outcomes reflecting SSc-related vascular and cardiopulmonary damage was set up, and the predictive value of and correlations between the CPET parameters and resting lung function and echocardiography variables were assessed. RESULTS: None of the clinical parameters, resting lung function or echocardiographic test results proved to be predictive of the development of the endpoint of poor prognosis in this cohort. In contrast, several CPET parameters were found to discriminate between SSc patients with or without adverse outcome. The detection of desaturation (at any CPET test) was associated with a higher risk of poor prognosis (OR:5.265). VO2 and VE/VCO2 at baseline correlated with the annual decrease in FVC, anaerobic threshold with the development of digital ulcers, and VE/VO2 with the increase in pulmonary arterial pressure. CONCLUSIONS: Several CPET parameters obtained at the beginning of follow-up are informative of the appearance of various adverse end-points. CPET is a feasible examination in the care of SSc patients and provides excess information to current standard follow-up examinations.

Meiotic DNA double-strand breaks and chromosome asynapsis in mice are monitored by distinct HORMAD2-independent and -dependent mechanisms.

Meiotic crossover formation involves the repair of programmed DNA double-strand breaks (DSBs) and synaptonemal complex (SC) formation. Completion of these processes must precede the meiotic divisions in order to avoid chromosome abnormalities in gametes. Enduring key questions in meiosis have been how meiotic progression and crossover formation are coordinated, whether inappropriate asynapsis is monitored, and whether asynapsis elicits prophase arrest via mechanisms that are distinct from the surveillance of unrepaired DNA DSBs. We disrupted the meiosis-specific mouse HORMAD2 (Hop1, Rev7, and Mad2 domain 2) protein, which preferentially associates with unsynapsed chromosome axes. We show that HORMAD2 is required for the accumulation of the checkpoint kinase ATR along unsynapsed axes, but not at DNA DSBs or on DNA DSB-associated chromatin loops. Consistent with the hypothesis that ATR activity on chromatin plays important roles in the quality control of meiotic prophase, HORMAD2 is required for the elimination of the asynaptic Spo11(-/-), but not the asynaptic and DSB repair-defective Dmc1(-/-) oocytes. Our observations strongly suggest that HORMAD2-dependent recruitment of ATR to unsynapsed chromosome axes constitutes a mechanism for the surveillance of asynapsis. Thus, we provide convincing evidence for the existence of a distinct asynapsis surveillance mechanism that safeguards the ploidy of the mammalian germline.

Molecular Characteristics and Treatment of Endothelial Dysfunction in Patients with COPD: A Review Article.

Patients with chronic obstructive pulmonary disease (COPD) show systemic consequences, such as chronic systemic inflammation leading to changes in the airway, airway penetrability, and endothelial function. Endothelial dysfunction is characterized by a list of alterations of endothelium towards reduced vasodilation, proinflammatory state, detachment and apoptosis of endothelial cells, and development of atherosclerosis. COPD-induced endothelial dysfunction is associated with elevated cardiovascular risk. The increment of physical activities such as pulmonary rehabilitation (PR) training have a significant effect on COPD, thus, PR can be an integrative part of COPD treatment. In this narrative review the focus is on the function of endothelial inflammatory mediators [cytokines, chemokines, and cellular proteases] and pulmonary endothelial cells and endothelial dysfunction in COPD as well as the effects of dysfunction of the endothelium may play in COPD-related pulmonary hypertension. The relationship between smoking and endothelial dysfunction is also discussed. The connection between different pulmonary rehabilitation programs, arterial stiffness and pulse wave velocity (PWV) is presented. Endothelial dysfunction is a significant prognostic factor of COPD, which can be characterized by PWV. We discuss future considerations, like training programs, as an important part of the treatment that has a favorable impact on the endothelial function.

Structural assembly of the signaling competent ERK2-RSK1 heterodimeric protein kinase complex.

Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase-kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 "docking" groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they "readjust," whereas generic kinase domain surface contacts bring them into a catalytically competent state.

Assessment of the Anti-Aging Klotho Protein in Patients with COPD Undergoing Pulmonary Rehabilitation.

Chronic obstructive pulmonary disease (COPD) is associated with the accelerated aging of the lung. The protein klotho has been implicated in longevity, and there is some evidence that it might be involved in the pathomechanism of chronic respiratory diseases. Therefore, we aimed to examine whether the clinical condition of COPD patients is reflected in plasma klotho concentration. As plasma concentration of the protein is modulated by physiological factors that are generally improved during pulmonary rehabilitation, we hypothesized that a complex rehabilitation program may alter plasma klotho concentration. Blood samples were taken from 31 stable COPD patients. Clinical parameters such as respiratory function, 6-minute walking distance (6MWD), impact of disease (CAT), dyspnea, grip strength, chest expansion and breath holding time, smoking history, and body mass index (BMI) were evaluated. 19 patients who participated in a 3-week inpatient rehabilitation program had blood sample collection on the first, third, and last days of the program and had the above functional measurements before and after rehabilitation. Plasma klotho concentration was assessed by enzyme-linked immunosorbent assay. Klotho levels showed no correlation with clinical parameters (FEV1%, 6MWD, grip strength, CAT, smoking history, p > 0.05). Coefficient of variation of klotho measurements was 4.5% between Day 1 and Day 3. Although the rehabilitation resulted in significant improvements in 6MWD, CAT, grip strength, and chest expansion, klotho levels did not change significantly (510.1 ± 149.9 vs. 504.2 ± 139.8 pg/ml, p > 0.05). Plasma klotho concentration can be reliably measured in stable COPD; however, its levels are not correlated with clinical parameters of patients. Despite functional improvement, klotho level remains unchanged during the rehabilitation program.

Combined genotyping strategy reveals structural differences between Aspergillus flavus lineages from different habitats impacting human health.

Aspergillus flavus is a filamentous fungus which is widespread on agricultural products and also able to cause various human diseases. This species is frequently isolated from indoor air as well, furthermore, it is known as a common causal agent of keratomycosis, particularly in subtropical and tropical areas. It is also able to produce aflatoxins, one of the most carcinogenic mycotoxins which are harmful to animals and humans. In this study, 59 A. flavus isolates from four different habitats and 1 A. minisclerotigenes isolate were investigated. The isolates were identified and confirmed at the species level by the sequence analysis of a part of their calmodulin gene. Applying a combined analysis of UP-PCR, microsatellite, and calmodulin sequence data, the four group of isolates formed separate clusters on the phylogenetic tree. Examining the distribution of mating type genes MAT1-1 and MAT1-2, a ratio of approximately 3:1 was determined, and no correlation was found between the carried mating type gene and the aflatoxin production capability. HPLC analysis revealed that none of the examined isolates collected from indoor air or maize in Central Europe were able to produce aflatoxins, while about half of the isolates from India produced these mycotoxins under the test conditions.

Biodiversity of species of Aspergillus section Fumigati in semi-desert soils in Argentina.

The distribution of Aspergillus species in soil has been widely studied all over the world. The aim of this study was the phenotypic and genotypic characterization of species Aspergillus belonging to section Fumigati present in soils from two Argentinian semi-desert areas having different geological conditions. Altogether, 23 isolates belonging to Aspergillus section Fumigati were recovered and identified using a polyphasic approach including phenotypic and molecular identifications. Aspergillus fumigatus sensu stricto and Aspergillus fumigatiaffinis had the highest frequency, of occurrence while isolates closely related to Aspergillus udagawae and Aspergillus felis were rarely observed. A. fumigatiaffinis and isolates closer to A. udagawae were isolated for the first time from Argentinian soils and this is the first report on the occurrence of species belonging to the A. felis clade in South America. Recent scientific interests in biodiversity, as well as the increasing importance of aspergilli as causative agents of human and animal diseases increase the need to understand the diversity and occurrence of these fungi in nature.

[Risk stratification before thoracic surgery, perioperative pulmonary rehabilitation]. / A mellkassebészeti mutéti teherbíró képesség megítélése, perioperatív légzésrehabilitáció.

INTRODUCTION: Besides the oncology and operative surgical technics, functional aspects influence the operability of lung cancer. Preoperative risk stratification, evaluation of postoperative complications needs to be considered. AIM: To review international literature and experiences of our institute. METHOD: We focused the literature of risk stratification of thoracic surgery. Lung function, lung mechanics, chest kinematics, exercise physiology were considered. Effectiveness of pulmonary rehabilitation for cardiovascular system, lung mechanics, muscles, exercise capacity and quality of life were evaluated. Laboratory parameters, comorbidities, obesity, cachexia, smoking cessation were considered. RESULTS: Elevated blood sugar, kidney function, reduced albumin level increased the risk. COPD, sleep apnoea, heart failure, obesity and cachexia influences the outcome. Smoking cessation may reduce postoperative complications. Controlled breathing technics, chest wall mobilization, training have favourable effects. Psychosocial support and dietetics are important. CONCLUSIONS: Risk stratification is supported by laboratory parameters, lung function, oxygen uptake and comorbidities. Pulmonary rehabilitation can improve functionality and quality of life. Orv Hetil. 2017; 158(50): 1989-1997.

Dual-Stage Consumable-Free Thermal Modulator for the Hyphenation of Thermal Analysis, Gas Chromatography, and Mass Spectrometry.

The design of the so-called "Peltier modulator" is presented. It is a new dual-stage consumable-free thermal modulator for thermal analysis-gas chromatography-mass spectrometry (TA-GC-MS). It requires only electrical power for operation as it facilitates thermo-electric coolers instead of cryogenics for trapping and resistive on-column heating for reinjection. Trapping and desorption temperatures as well as modulation cycles are freely adjustable. The stationary phase for the trapping region can be selected to suit the specific application, since common fused silica capillary is used. The Peltier modulator's performance is demonstrated with a broad range of different standard substances and with heavy crude oil as a complex real life sample. Successful modulation from n-pentane to pyrene (boiling points = 36/394 °C) is presented. The produced peaks show the narrowest bandwidths ever reported for a consumable-free thermal modulator, i.e., 12.8 ± 1.2 ms for n-pentadecane. The Peltier modulator is rugged, cost-effective, requires low maintenance, and decreases security issues significantly, compared to commercial available solutions using liquid N2/CO2.

The CYLD p.R758X worldwide recurrent nonsense mutation detected in patients with multiple familial trichoepithelioma type 1, Brooke-Spiegler syndrome and familial cylindromatosis represents a mutational hotspot in the gene.

BACKGROUND: Multiple familial trichoepithelioma type 1 (MFT1; MIM 601606), a rare monogenic skin disease with autosomal dominant inheritance, is characterized by the development of multiple skin-colored papules on the central area of the face, frequently occurring in the nasolabial area. The disease is associated with various mutations in the cylindromatosis (CYLD; MIM 605018) gene that are also responsible for familial cylindromatosis (FC) and Brooke-Spiegler syndrome (BSS). METHODS: Recently we have identified a Spanish MFT1 pedigree with two affected family members (father and daughter). Direct sequencing of the CYLD gene revealed a worldwide recurrent heterozygous nonsense mutation (c.2272C/T, p.R758X) in the patients. RESULTS: This mutation has already been detected in patients with all three clinical variants - BSS, FC and MFT1 - of the CYLD-mutation spectrum. Haplotype analysis was performed for the Spanish patients with MFT1, Dutch patients with FC and an Austrian patient with BSS, all of whom carry the same heterozygous nonsense p.R758X CYLD mutation. CONCLUSIONS: Our results indicate that this position is a mutational hotspot on the gene and that patients carrying the mutation exhibit high phenotypic diversity.

[Interdisciplinary rehabilitation in patients with ankylosing spondylitis]. / Az interdiszciplináris rehabilitáció hatása spondylitis ankylopoeticában szenvedo betegekben.

INTRODUCTION: Complex pulmonary assessment related to respiratory manifestation in patients with ankylosing spondylitis may contribute to adaptation of an appropriate rehabilitation program. AIM: To examine the relationship between lung function, exercise physiological variables and change in quality of life after rehabilitation in patients with ankylosing spondylitis. METHOD: 5 patients in Seyfried's Stage 2 and 11 patients in Stage 3 underwent spinal physiotherapy, ultrasound, massage and paraffin Pack, 15 times each, followed by a high-intensity cycling 3 times a week for 8 weeks. The Bath Ankylosing Spondylitis Disease Activity and Bath Ankylosing Spondylitis Functional Indexes were recorded before and after rehabilitation. Lung function with exercise physiological variables were examined after rehabilitation. RESULTS: Both indexes showed a post-treatment significant improvement compared to the initial scores (p<0.05). These functional indices correlated with lung function at rest and physiological variables during exercise (p<0.05). CONCLUSIONS: Based on this study, it is possible that an initial pulmonary assessment may serve to develop a more effective program for ankylosing spondylitis. Orv. Hetil., 2016, 157(28), 1126-1132.

Optically heated ultra-fast-cycling gas chromatography module for separation of direct sampling and online monitoring applications.

This work describes an ultrafast-cycling gas chromatography module (fast-GC module) for direct-sampling gas chromatography/mass spectrometry (GC-MS). The sample can be introduced into the fast-GC module using a common GC injector or any GC × GC modulator. The new fast-GC module offers the possibility to conduct a complete temperature cycle within 30 s. Its thermal mass is minimized by using a specially developed home-built fused silica capillary column stack and a halogen lamp for heat generation, both placed inside a gold-coated quartz glass cylinder. A high airflow blower enables rapid cooling. The new device is highly flexible concerning the used separation column, the applied temperature program, and the integration into existing systems. An application of the fast-GC module is shown in this work by thermal analysis coupled to gas chromatography-mass spectrometry (TA-GC-MS). The continuously evolving gases of the TA are modulated by a liquid CO2 modulator. Because of the rapid cycling of the fast-GC module, it is possible to obtain the best separation while maintaining the online character of the TA. Restrictions in separation and retention time shifting, known from isothermal and normal ramped fast-GC systems, are overcome.

Anxiogenic role of vasopressin during the early postnatal period: maternal separation-induced ultrasound vocalization in vasopressin-deficient Brattleboro rats.

Both animal and human studies suggest that in adulthood, plasma vasopressin level correlates well with anxiety. Little is known about the mood regulation during the perinatal period. Here, we aim to investigate the influence of vasopressin on anxiety during the early postnatal age. As a sign of distress, rat pups emit ultrasonic vocalizations (USVs) when they are separated from their mother. This USV was detected in 7- to 8-day-old vasopressin-deficient Brattleboro pups, and they were compared to their heterozygote littermates and wild-type pups. The results were confirmed by V1b antagonist treatment (SSR149415 10 mg/kg ip 30 min before test) in wild-types. Chlordiazepoxide (3 mg/kg ip 30 min before test)-an anxiolytic-was used to test the interaction with the GABAergic system. At the end of the test, stress-hormone levels were measured by radioimmunoassay. Vasopressin-deficient pups vocalized substantially less than non-deficient counterparts. Treatment with V1b antagonist resulted in similar effect. Chlordiazepoxide reduced the frequency and duration of the vocalization only in wild-types. Reduced vocalization was accompanied by smaller adrenocorticotropin levels but the level of corticosterone was variable. Our results indicate that the anxiolytic effect of vasopressin deficiency (both genetic and pharmacological) exists already during the early postnatal age. Vasopressin interacts with the GABAergic system. As mood regulation does not go parallel with glucocorticoid levels, we suggest that vasopressin might have a direct effect on special brain areas.

Cytotoxic and genotoxic potencies of single and combined spore extracts of airborne OTA-producing and OTA-non-producing Aspergilli in Human lung A549 cells.

Aspergillus sclerotiorum (AS) is a well-known producer of ochratoxin A (OTA) while Aspergillus pseudoglaucus (AP) produces a wide range of extrolites with poorly investigated toxicity. These species are frequently co-occur in grain mill aeromycota. The aim of this study was to determine OTA levels in spore extracts using HPLC and immunoaffinity columns, and to examine the cytotoxicity of pure OTA, OTA-positive (AS-OTA(+)) and OTA-negative (AS-OTA(-)) spore extracts, as well as of AP spore extract, on human lung adenocarcinoma cells A549, individually and in combination, using a colorimetric MTT test (540nm). To establish which type of cell death predominated after treatments, a quantitative fluorescent assay with ethidium bromide and acridine orange was used, and the level of primary DNA damage in A549 cells was evaluated using the alkaline comet assay. OTA was detected in spore extracts (0.3-28µg/mL) of 3/6 of the AS strains, while none of the tested AP strains were able to produce OTA. Taking into account the maximum detected concentration of OTA in the spores, the daily intake of OTA by inhalation was calculated to be 1ng/kg body weight (b.w.), which is below the tolerable daily intake for OTA (17ng/kg b.w.). Using the MTT test, the following IC50 values were obtained: single OTA (53µg/mL); AS-OTA(+) (mass concentration 934µg/mL corresponds to 10.5µg/mL of OTA in spore extract); and 2126µg/mL for AP. The highest applied concentration of AS-OTA(-) spore extract (4940µg/mL) decreased cell viability by 30% and IC50 for the extract could not be determined. Single OTA and AS-OTA(+) and combinations (AP+AS-OTA(+) and AP+AS-OTA(-)) in subtoxic concentrations provoked significant primary DNA damage, apoptosis, and to a lesser extent, necrosis in A549 cells. Mixture of AP+AS-OTA(+) and AP+AS-OTA(-) in subtoxic concentrations showed dominant additive interactions. Despite the low calculated daily intake of OTA by inhalation, our results suggest that chronic exposure to high levels of OTA-producing airborne fungi in combination with other more or less toxic moulds pose a significant threat to human health due to their possible additive and/or synergistic interactions.