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Despite decades of research, studies investigating the physiological alterations caused by an acute bout of inflammation induced by exposing the lung to lipopolysaccharide have yielded inconsistent results. This can be attributed to small effects and/or a lack of fitted physiological testing. Herein, a comprehensive investigation of lung mechanics was conducted on 270 male C57BL/6 mice at 24, 48, or 96 h after an intranasal exposure to saline or lipopolysaccharide at either 1 or 3 mg/kg (30 mice per group). Traditional techniques that probe the lung using small-amplitude perturbations (i.e., oscillometry) were used, together with less conventional and new techniques that probe the lung using maneuvers of large amplitudes. The latter include a partial and a full-range pressure-volume maneuvers to measure quasi-static elastance, compliance, total lung volume, vital capacity, and residual volume. The results demonstrate that lung mechanics assessed by oscillometry was only slightly affected by lipopolysaccharide, confirming previous findings. In contradistinction, lipopolysaccharide markedly altered mechanics when the lung was probed with maneuvers of large amplitudes. With the dose of 3 mg/kg at the peak of inflammation (48 h postexposure), lipopolysaccharide increased quasi-static elastance by 26.7% ( P < 0.0001) and decreased compliance by 34.5% ( P < 0.0001). It also decreased lung volumes, including total lung capacity, vital capacity, and residual volume by 33.3%, 30.5%, and 43.3%, respectively (all P < 0.0001). These newly reported physiological alterations represent sensitive outcomes to efficiently evaluate countermeasures (e.g., drugs) in the context of several lung diseases.

New findings What is the central question of this study? Does endogenous testosterone modulate the consequences of IH in the lungs of male mice ? What is the main finding and its importance? Orchiectomized mice exposed to IH develop a pattern that is similar to emphysema or obstructive lung disease with elevated lung volumes, low pulmonary elastance during a methacholine challenge test and high counts of lymphocytes in bronchoalveolar lavages. Since low testosterone levels and other respiratory diseases are common in sleep apnea there is a clear clinical relevance to these results. Abstract We tested the hypothesis that low testosterone levels modulate the pulmonary responses to intermittent hypoxia (IH - used as a model of sleep apnoea) in male mice. We used intact (SHAM) or orchiectomised (ORX) mice exposed to IH for 14 days (12h/day, 10cycles/h, 6% oxygen) or to normoxia (Nx). We first measured ventilation and metabolic rates in freely behaving mice (whole body plethysmography) and then respiratory mechanics in tracheotomised mice (flexiVent). We assessed the respiratory system resistance (Rrs) and elastance (Ers), Newtonian resistance (Rn: resistance of the large airways), tissue damping (G) and tissue elastance (H) under baseline conditions and during a methacholine challenge test. We also measured the quasi-static compliance (Cst) and inspiratory capacity with partial pressure-volume loops. Finally, inflammatory cells were counted in the broncho-alveolar lavage (BAL) and we measured lung volume by water displacement. ORX-IH mice had higher tidal volume, inspiratory capacity, and lung volume compared to the other groups, but showed signs of low efficiency of O2 exchange rate relative to minute ventilation. During the methacholine challenge, orchiectomy decreased the values of most mechanical parameters and IH reduced Ers and H leading to very low values in ORX-IH mice. Finally, the total number of cells and the number of lymphocytes in BAL were both increased by IH in ORX mice. Since reduced lung elasticity, low O2 extraction, increased lung volumes, and inflammation are signs of emphysematous lung disease, we conclude that testosterone might prevent lung emphysema during IH exposures. This article is protected by copyright. All rights reserved.

AIM OF THE STUDY The current gold standard to assess respiratory mechanics in mice is oscillometry, a technique from which several readouts of the respiratory system can be deduced, such as resistance and elastance. However, these readouts are often not altered in mouse models of asthma. This is in stark contrast with humans, where asthma is generally associated with alterations when assessed by either oscillometry or other techniques. In the present study, we have used double-chamber plethysmography (DCP) to evaluate the breathing pattern and the degree of airflow obstruction in a mouse model of asthma. MATERIALS AND METHODS Female C57BL/6 and BALB/c mice were studied at day 1 using DCP, as well as at day 11 using both DCP and oscillometry following a once-daily exposure to either house-dust mite (HDM) or saline for 10 consecutive days. RESULTS All DCP readouts used to describe either the breathing pattern (e.g., tidal volume and breathing frequency) or the degree of airflow obstruction (e.g., specific airway resistance) were different between mouse strains at day 1. Most of these strain differences persisted at day 11. Most oscillometric readouts (e.g., respiratory system resistance and elastance) were also different between strains. Changes caused by HDM were obvious with DCP, including decreases in tidal volume, minute ventilation, inspiratory time and mid-tidal expiratory flow and an increase in specific airway resistance. HDM also caused some strain specific alterations in breathing pattern, including increases in expiratory time and end inspiratory pause, which were only observed in C57BL/6 mice. Oscillometry also detected a small but significant increase in tissue elastance in HDM versus saline-exposed mice. CONCLUSIONS DCP successfully identified differences between C57BL/6 and BALB/c mice, as well as alterations in mice from both strains exposed to HDM. We conclude that, depending on the study purpose, DCP may sometimes outweigh oscillometry.

Cells respond to multiple signals from the environment simultaneously which often creates crosstalk between pathways affecting the capacity to adapt to the changing environment. Chaperones are an important component in the cellular integration of multiple responses to environmental signals, often implicated in negative feedback and inactivation mechanisms. These mechanisms include the stabilization of steroid hormone nuclear receptors in the cytoplasm in the absence of their ligand. Here we show using immunofluorescence, chromatin immunoprecipitation and nascent transcripts production that the HSP70 chaperone plays a central role in a new crosstalk mechanism between the steroid and heat shock response pathways. HSP70-dependent feedback mechanisms are required to inactivate the heat shock factor 1 (HSF1) after activation. Interestingly, a steroid stimulation leads to faster accumulation of HSF1 in inactive foci following heat shock. Our results further show that in the presence of estrogen, HSP70 accumulates at HSF1-regulated noncoding regions, leading to deactivation of HSF1 and the abrogation of the heat shock transcriptional response. Using an HSP70 inhibitor, we demonstrate that the crosstalk between both pathways is dependent on the chaperone activity. These results suggest that HSP70 availability is a key determinant in the transcriptional integration of multiple external signals. Overall, these results offer a better understanding of the crosstalk between the heat shock and steroid responses, which are salient in neurodegenerative disorders and cancers.

The airway smooth muscle undergoes an elastic transition during a sustained contraction, characterized by a gradual decrease in hysteresivity caused by a relatively greater rate of increase in elastance than resistance. We recently demonstrated that these mechanical changes are more likely to persist after a large strain when they are acquired in dynamic versus static conditions; as if the microstructural adaptations liable for the elastic transition are more flexible when they evolve in dynamic conditions. The extent of this flexibility is undefined. Herein, contracted ovine tracheal smooth muscle strips were kept in dynamic conditions simulating tidal breathing (sinusoidal length oscillations at 5% amplitude) and then subjected to simulated deep inspirations (DI). Each DI was straining the muscle by either 10%, 20%, or 30% and was imposed at either 2, 5, 10, or 30 min after the preceding DI. The goal was to assess whether and the extent by which the time-dependent decrease in hysteresivity is preserved following the DI. The results show that the time-dependent decrease in hysteresivity seen pre-DI was preserved after a strain of 10%, but not after a strain of 20% or 30%. This suggests that the microstructural adaptations liable for the elastic transition withstood a strain at least twofold greater than the oscillating strain that pertained during their evolution (10% vs. 5%). We propose that a muscle adapting in dynamic conditions forges microstructures exhibiting a substantial degree of flexibility.

Due to frequent and often severe lung affections caused by COVID-19, murine models of acute respiratory distress syndrome (ARDS) are increasingly used in experimental lung research. The one induced by a single lipopolysaccharide (LPS) exposure is practical. However, whether it is preferable to administer LPS intranasally or intratracheally remains an open question. Herein, female C57Bl/6 J mice were exposed intranasally or intratracheally to one dose of either saline or 3 mg/kg of LPS. They were studied 24 h later. The groups treated with LPS, either intranasally or intratracheally, exhibited a pronounced neutrophilic inflammation, signs of lung tissue damage and protein extravasation into the alveoli, and mild lung dysfunction. The magnitude of the response was generally not different between groups exposed intranasally versus intratracheally. However, the variability of some the responses was smaller in the LPS-treated groups exposed intranasally versus intratracheally. Notably, the saline-treated mice exposed intratracheally demonstrated a mild neutrophilic inflammation and alterations of the airway epithelium. We conclude that an intranasal exposure is as effective as an intratracheal exposure in a murine model of ARDS induced by LPS. Additionally, the groups exposed intranasally demonstrated less variability in the responses to LPS and less complications associated with the sham procedure.

Force adaptation of airway smooth muscle (ASM) is a process whereby the presence of tone (i.e., a sustained contraction) increases the contractile capacity. For example, tone has been shown to increase airway responsiveness in both healthy mice and humans. The goal of the present study is to elucidate the underlying molecular mechanisms. The maximal force generated by mouse tracheas was measured in response to 10−4M of methacholine following a 30-min period with or without tone elicited by the EC30of methacholine. To confirm the occurrence of force adaptation at the cellular level, traction force generated by cultured human ASM cells was also measured following a similar protocol. Different pharmacological inhibitors were used to investigate the role of Rho-associated coiled-coil containing protein kinase (ROCK), protein kinase C (PKC), myosin light chain kinase (MLCK), and actin polymerization in force adaptation. The phosphorylation level of the regulatory light chain (RLC) of myosin, the amount of actin filaments, and the activation level of the actin-severing protein cofilin were also quantified. Although ROCK, PKC, MLCK, and RLC phosphorylation was not implicated, force adaptation was prevented by inhibiting actin polymerization. Interestingly, the presence of tone blocked the activation of cofilin in addition to increasing the amount of actin filaments to a maximal level. We conclude that actin filamentogenesis induced by tone, resulting from both actin polymerization and the prevention of cofilin-mediated actin cleavage, is the main molecular mechanism underlying force adaptation.

Airway smooth muscle (ASM) is continuously strained during breathing at tidal volume. Whether this tidal strain influences the magnitude of the bronchodilator response to a deep inspiration (DI) is not clearly defined. The present in vitro study examines the effect of tidal strain on the bronchodilator effect of DIs. ASM strips from sheep tracheas were mounted in organ baths and then subjected to stretches (30% strain), simulating DIs at varying time intervals. In between simulated DIs, the strips were either held at a fixed length (isometric) or oscillated continuously by 6% (length oscillations) to simulate tidal strain. The contractile state of the strips was also controlled by adding either methacholine or isoproterenol to activate or relax ASM, respectively. Although the time-dependent gain in force caused by methacholine was attenuated by length oscillations, part of the acquired force in the oscillating condition was preserved postsimulated DIs, which was not the case in the isometric condition. Consequently, the bronchodilator effect of simulated DIs (i.e., the decline in force postsimulated versus presimulated DIs) was attenuated in oscillating versus isometric conditions. These findings suggest that an ASM operating in a dynamic environment acquired adaptations that make it refractory to the decline in contractility inflicted by a larger strain simulating a DI.

The shortening of airway smooth muscle (ASM) is greatly affected by time. This is because stimuli affecting ASM shortening, such as bronchoactive molecules or the strain inflicted by breathing maneuvers, not only alter quick biochemical processes regulating contraction but also slower processes that allow ASM to adapt to an ever-changing length. Little attention has been given to the effect of time on ASM shortening. The present study investigates the effect of changing the time interval between simulated deep inspirations (DIs) on ASM shortening and its responsiveness to simulated DIs. Excised tracheal strips from sheep were mounted in organ baths and either activated with methacholine or relaxed with isoproterenol. They were then subjected to simulated DIs by imposing swings in distending stress, emulating a transmural pressure from 5 to 30 cmH2O. The simulated DIs were intercalated by 2, 5, 10, or 30 min. In between simulated DIs, the distending stress was either fixed or oscillating to simulate tidal breathing. The results show that although shortening was increased by prolonging the interval between simulated DIs, the bronchodilator effect of simulated DIs (i.e., the elongation of the strip post- vs. pre-DI) was not affected, and the rate of re-shortening post-simulated DIs was decreased. As the frequency with which DIs are taken increases upon bronchoconstriction, our results may be relevant to typical alterations observed in asthma, such as an increased rate of re-narrowing post-DI. NEW & NOTEWORTHY The frequency with which patients with asthma take deep inspirations (DIs) increases during bronchoconstriction. This in vitro study investigated the effect of changing the time interval between simulated DIs on airway smooth muscle shortening. The results demonstrated that decreasing the interval between simulated DIs not only decreases shortening, which may be protective against excessive airway narrowing, but also increases the rate of re-shortening post-simulated DIs, which may contribute to the increased rate of re-narrowing post-DI observed in asthma.

There are renewed interests in using the parameter K of Salazar–Knowles’ equation to assess lung tissue compliance. K either decreases or increases when the lung’s parenchyma stiffens or loosens, respectively. However, whether K is affected by other common features of respiratory diseases, such as inflammation and airway smooth muscle (ASM) contraction, is unknown. Herein, male C57BL/6 mice were treated intranasally with either saline or lipopolysaccharide (LPS) at 1 mg/kg to induce pulmonary inflammation. They were then subjected to either a multiple or a single-dose challenge with methacholine to activate ASM to different degrees. A quasi-static pressure-driven partial pressure-volume (P-V) maneuver was performed before and after methacholine. The Salazar–Knowles’ equation was then fitted to the deflation limb of the P-V loop to obtain K, as well as the parameter A, an estimate of lung volume (inspiratory capacity). The fitted curve was also used to derive the quasi-static elastance (Est) at 5 cmH2O. The results demonstrate that LPS and both methacholine challenges increased Est. LPS also decreased A, but did not affect K. In contradistinction, methacholine decreased both A and K in the multiple-dose challenge, whereas it decreased K but not A in the single-dose challenge. These results suggest that LPS increases Est by reducing the open lung volume (A) and without affecting tissue compliance (K), whereas methacholine increases Est by decreasing tissue compliance with or without affecting lung volume. We conclude that lung tissue compliance, assessed using the parameter K of Salazar–Knowles’ equation, is insensitive to inflammation but sensitive to ASM contraction.

The current gold standard to assess respiratory mechanics in mice is oscillometry, a technique from which several readouts of the respiratory system can be deduced, such as resistance and elastance. However, these readouts are often not altered in mouse models of asthma. This is in stark contrast with humans, where asthma is generally associated with alterations when assessed by either oscillometry or other techniques. In the present study, we have used double-chamber plethysmography (DCP) to evaluate the breathing pattern and the degree of airflow obstruction in a mouse model of asthma.Female C57BL/6 and BALB/c mice were studied at day 1 using DCP, as well as at day 11 using both DCP and oscillometry following a once-daily exposure to either house-dust mite (HDM) or saline for 10 consecutive days.All DCP readouts used to describe either the breathing pattern (e.g., tidal volume and breathing frequency) or the degree of airflow obstruction (e.g., specific airway resistance) were different between mouse strains at day 1. Most of these strain differences persisted at day 11. Most oscillometric readouts (e.g., respiratory system resistance and elastance) were also different between strains. Changes caused by HDM were obvious with DCP, including decreases in tidal volume, minute ventilation, inspiratory time and mid-tidal expiratory flow and an increase in specific airway resistance. HDM also caused some strain specific alterations in breathing pattern, including increases in expiratory time and end inspiratory pause, which were only observed in C57BL/6 mice. Oscillometry also detected a small but significant increase in tissue elastance in HDMDCP successfully identified differences between C57BL/6 and BALB/c mice, as well as alterations in mice from both strains exposed to HDM. We conclude that, depending on the study purpose, DCP may sometimes outweigh oscillometry.

BALB/c mice from both sexes underwent one of two nebulized methacholine challenges that were preceded by a period of 20 min either with or without tone induced by repeated contractions of the airway smooth muscle. Impedance was monitored throughout and the constant phase model was used to dissociate the impact of tone on conducting airways (R

The contractility of airway smooth muscle (ASM) is labile. Although this feature can greatly modulate the degree of airway responsiveness in vivo, the extent by which ASM’s contractility is affected by pulmonary allergic inflammation has never been compared between strains of mice exhibiting a different susceptibility to develop airway hyperresponsiveness (AHR). Herein, female C57BL/6 and BALB/c mice were treated intranasally with either saline or house dust mite (HDM) once daily for 10 consecutive days to induce pulmonary allergic inflammation. The doses of HDM were twice greater in the less susceptible C57BL/6 strain. All outcomes, including ASM contractility, were measured 24 h after the last HDM exposure. As expected, while BALB/c mice exposed to HDM became hyperresponsive to a nebulized challenge with methacholine in vivo, C57BL/6 mice remained normoresponsive. The lack of AHR in C57BL/6 mice occurred despite exhibiting more than twice as much inflammation than BALB/c mice in bronchoalveolar lavages, as well as similar degrees of inflammatory cell infiltrates within the lung tissue, goblet cell hyperplasia and thickening of the epithelium. There was no enlargement of ASM caused by HDM exposure in either strain. Unexpectedly, however, excised tracheas derived from C57BL/6 mice exposed to HDM demonstrated a decreased contractility in response to both methacholine and potassium chloride, while tracheas from BALB/c mice remained normocontractile following HDM exposure. These results suggest that the lack of AHR in C57BL/6 mice, at least in an acute model of HDM-induced pulmonary allergic inflammation, is due to an acquired ASM hypocontractility.

Colorectal cancer (CRC), the second leading cause of cancer mortality, constitutes a significant global health burden. An accurate, noninvasive detection method for CRC as complement to colonoscopy could improve the effectiveness of treatment. In the present study, SureSelectXT Methyl-Seq was performed on cancerous and normal colon tissues and CLDN1, INHBA and SLC30A10 were found as candidate methylated genes. MethyLight assay was run on formalin-fixed paraffin-embedded (FFPE) and fresh case and control tissues to validate the methylation of the selected gene. The methylation was significantly different (p-values

Background: Autosomal dominant polycystic kidney disease (ADPKD) is the predominant type of inherited kidney disorder, which occurs due to PKD1 and PKD2 gene mutations. ADPKD diagnosis is made primarily by kidney imaging; however, molecular genetic analysis needs to be implicated to confirm the diagnosis. It is critical to perform a molecular genetic analysis when the diagnosis is uncertain, particularly in simplex cases (i.e., a single occurrence in a family), in people with remarkably mild symptoms, or in individuals with atypical presentations. The main aim of this study is to determine the likelihood of PKD1 gene mutations in Iranian patients with ADPKD diagnosis. Methods: Genomic DNA was isolated from blood samples from 26 ADPKD patients, who were referred to the Qaem Hospital in Mashhad, Iran. By using suitable primers, 16 end exons of PKD1 gene that are regional hotspots, were replicated with PCR. Then, PCR products were subjected to DNA directional Sanger sequencing.Results: The results of DNA sequencing in the patients showed that exons 35, 36 and 37 were non- polymorphic, while most mutations had occurred in exons 44 and 45. Only in two patients, exon-intron boundary mutation had occurred in intron 44. Most of the variants were missense and non-synonymous types. Conclusion: In this study, we present nine novel mutations/polymorphisms in PKD1. These data will contribute to an improved diagnostic and genetic counseling in clinical settings. Keywords: Autosomal dominant polycystic kidney disease; PKD1; mutational analysis; Iranian

Background Autosomal dominant polycystic kidney disease (ADPKD), the predominant type of inherited kidney disorder, occurs due to PKD1 and PKD2 gene mutations. ADPKD diagnosis is made primarily by kidney imaging. However, molecular genetic analysis is required to confirm the diagnosis. It is critical to perform a molecular genetic analysis when the imaging diagnosis is uncertain, particularly in simplex cases (i.e. a single occurrence in a family), in people with remarkably mild symptoms, or in individuals with atypical presentations. The main aim of this study is to determine the frequency of PKD1 gene mutations in Iranian patients with ADPKD diagnosis. Methods Genomic DNA was extracted from blood samples from 22 ADPKD patients, who were referred to the Qaem Hospital in Mashhad, Iran. By using appropriate primers, 16 end exons of PKD1 gene that are regional hotspots, were replicated with PCR. Then, PCR products were subjected to DNA directional Sanger sequencing. Results The DNA sequencing in the patients has shown that exons 35, 36 and 37 were non- polymorphic, and that most mutations had occurred in exons 44 and 45. In two patients, an exon-intron boundary mutation had occurred in intron 44. Most of the variants were missense and synonymous types. Conclusion In the present study, we have shown the occurrence of nine novel missense or synonymous variants in PKD1 gene. These data could contribute to an improved diagnostic and genetic counseling in clinical settings.

The molecular mechanisms governing the contraction of airway smooth muscle have always been at the forefront of asthma research. New extracellular molecules affecting the contraction of airway smooth muscle are steadily being discovered. Although interesting, this is disconcerting for researchers trying to find a mend for the significant part of asthma symptoms caused by contraction. Additional efforts are being deployed to understand the intracellular signaling pathways leading to contraction. The goal being to find common pathways that are essential to convey the contractile signal emanating from any single or combination of extracellular molecules. Not only these pathways exist and their details are being slowly unveiled, but some carry the signal inside-out to interact back with extracellular molecules. These latter represent targets with promising therapeutic potential, not only because they are molecules downstream of pathways essential for contraction but also because their extracellular location makes them readily accessible by inhaled drugs.

α-Tocopherol is the only tocopherol that has been shown to prevent the human deficiency disease Ataxia with Isolated Vitamin E Deficiency (AVED), and thus it is the only one that, for humans, can be called vitamin E. Vitamin E in addition to preventing AVED has documented immune boosting properties and an activity against nonalcoholic hepatosteatosis and low-grade inflammation. Epidemiological studies indicating that vitamin E could prevent cardiovascular events, neurodegenerative disease, macular degeneration, and cancer were in general not confirmed by clinical intervention studies. Vitamin E and some of its metabolites modulate cell signaling and gene transcription. Future research is needed to achieve a better understanding of the molecular events leading to gene regulation by vitamin E, especially in its phosphorylated form. Isolation and characterization of the vitamin E kinase and vitamin E phosphate phosphatase will help in the understanding of cell regulation processes modulated by vitamin E. A clarification of the pathogenesis of AVED remains an important goal to be achieved. © 2018 IUBMB Life, 71(4):411-415, 2019.

Background Although different methods have been established to detect Helicobacter pylori (H. pylori) infection, identifying infected patients is an ongoing challenge. The aim of this meta-analysis was to provide pooled diagnostic accuracy measures for stool PCR test in the diagnosis of H. pylori infection. Methods In this study, a systematic review and meta-analysis were carried out on various sources, including MEDLINE, Web of Sciences, and the Cochrane Library from April 1, 1999, to May 1, 2016. This meta-analysis adheres to the guidelines provided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses report (PRISMA Statement). The clinical value of DNA stool PCR test was based on the pooled false positive, false negative, true positive, and true negative of different genes. Results Twenty-six of 328 studies identified met the eligibility criteria. Stool PCR test had a performance of 71% (95% CI: 68-73) sensitivity, 96% (95% CI: 94-97) specificity, and 65.6 (95% CI: 30.2-142.5) diagnostic odds ratio (DOR) in diagnosis of H. pylori. The DOR of genes which showed the highest performance of stool PCR tests was as follows: 23S rRNA 152.5 (95% CI: 55.5-418.9), 16S rRNA 67.9 (95%CI: 6.4-714.3), and glmM 68.1 (95%CI: 20.1-231.7). Conclusions The sensitivity and specificity of stool PCR test are relatively in the same spectrum of other diagnostic methods for the detection of H. pylori infection. In descending order of significance, the most diagnostic candidate genes using PCR detection were 23S rRNA, 16S rRNA, and glmM. PCR for 23S rRNA gene which has the highest performance could be applicable to detect H. pylori infection.