Clinical and Histopathologic Features of a Feline SARS-CoV-2 Infection Model Are Analogous to Acute COVID-19 in Humans.

The emergence and ensuing dominance of COVID-19 on the world stage has emphasized the urgency of efficient animal models for the development of therapeutics for and assessment of immune responses to SARS-CoV-2 infection. Shortcomings of current animal models for SARS-CoV-2 include limited lower respiratory disease, divergence from clinical COVID-19 disease, and requirements for host genetic modifications to permit infection. In this study, n = 12 specific-pathogen-free domestic cats were infected intratracheally with SARS-CoV-2 to evaluate clinical disease, histopathologic lesions, and viral infection kinetics at 4 and 8 days post-inoculation; n = 6 sham-inoculated cats served as controls. Intratracheal inoculation of SARS-CoV-2 produced a significant degree of clinical disease (lethargy, fever, dyspnea, and dry cough) consistent with that observed in the early exudative phase of COVID-19. Pulmonary lesions such as diffuse alveolar damage, hyaline membrane formation, fibrin deposition, and proteinaceous exudates were also observed with SARS-CoV-2 infection, replicating lesions identified in people hospitalized with ARDS from COVID-19. A significant correlation was observed between the degree of clinical disease identified in infected cats and pulmonary lesions. Viral loads and ACE2 expression were also quantified in nasal turbinates, distal trachea, lungs, and other organs. Results of this study validate a feline model for SARS-CoV-2 infection that results in clinical disease and histopathologic lesions consistent with acute COVID-19 in humans, thus encouraging its use for future translational studies.

A Matrix Metalloproteinase Mediates Tracheal Development in Bombyx mori.

The trachea of insects is a tubular epithelia tissue that transports oxygen and other gases. It serves as a useful model for the studying of the cellular and molecular events involved in epithelial tube formation. Almost all of the extracellular matrix can be degraded by Matrix metalloproteinases (MMPs), which is closely related to the processes of development and regeneration. The regulation of trachea by MMPs is roughly known in previous studies, but the detailed regulation mechanism and involved gene function are not fully explored. In this article, we found MMP1 expressed highly during tracheal remodeling, and knocked out it makes the tracheal branch number reduced in Bombyx mori. In trachea of transgenic BmMMP1-KO silkworm, the space expanding of taenidium and epidermal cells and the structure of apical membrane were abnormal. To explore the underlying mechanism, we detected that DE-cadherin and Integrin ß1 were accumulated in trachea of transgenic BmMMP1-KO silkworm by immunohistochemistry. Moreover, 5-Bromo-2'-Deoxyuridine (BrdU) labeling showed that knockout of BmMMP1 in silkworm inhibited tracheal cell proliferation, and BmMMP1 also regulated the proliferation and migration of BmNS cells. All of the results demonstrated that BmMMP1 regulates the development of the tracheal tissue by expanding the space of tracheal cuticles and increases the number of tracheal branches by degrading DE-cadherin and Integrin ß1.

Obesity alters Ace2 and Tmprss2 expression in lung, trachea, and esophagus in a sex-dependent manner: Implications for COVID-19.

Obesity is a major risk factor for SARS-CoV-2 infection and COVID-19 severity. The underlying basis of this association is likely complex in nature. The host-cell receptor angiotensin converting enzyme 2 (ACE2) and the type II transmembrane serine protease (TMPRSS2) are important for viral cell entry. It is unclear whether obesity alters expression of Ace2 and Tmprss2 in the lower respiratory tract. Here, we show that: 1) Ace2 expression is elevated in the lung and trachea of diet-induced obese male mice and reduced in the esophagus of obese female mice relative to lean controls; 2) Tmprss2 expression is increased in the trachea of obese male mice but reduced in the lung and elevated in the trachea of obese female mice relative to lean controls; 3) in chow-fed lean mice, females have higher expression of Ace2 in the lung and esophagus as well as higher Tmprss2 expression in the lung but lower expression in the trachea compared to males; and 4) in diet-induced obese mice, males have higher expression of Ace2 in the trachea and higher expression of Tmprss2 in the lung compared to females, whereas females have higher expression of Tmprss2 in the trachea relative to males. Our data indicate diet- and sex-dependent modulation of Ace2 and Tmprss2 expression in the lower respiratory tract and esophagus. Given the high prevalence of obesity worldwide and a sex-biased mortality rate, we discuss the implications and relevance of our results for COVID-19.

Presence of Gastric Pepsinogen in the Trachea Is Associated with Altered Inflammation and Microbial Composition.

Gastroesophageal reflux is a common gastrointestinal issue that can lead to aspiration and contribute to respiratory problems. Little is known about how reflux can alter the respiratory microenvironment. We aimed to determine if the presence of gastric pepsinogen in the trachea was associated with changes in the microbial and inflammatory microenvironment. A pediatric cohort at high risk of reflux aspiration was prospectively recruited, and the tracheal microenvironment was examined. Pepsinogen A3 (PGA3) and cytokines were measured. The microbiome (bacterial and fungal) was profiled using 16S rRNA and internal transcribed spacer 2 (ITS2) amplicon sequencing. Increased bacterial richness and an altered composition driven by an enrichment of Prevotella correlated with high PGA3 levels. Fungal richness increased with PGA3, with higher Candida relative abundances observed in a subset of samples with high PGA3 levels. Source tracking of tracheal microbial taxa against taxa from matched oral and gastric samples revealed a significantly greater contribution of oral than of gastric taxa with higher PGA3 levels. Tracheal cytokines were differentially produced when stratified according to PGA3, with higher levels of interleukin-1 (IL-1)-related cytokines and IL-8 being associated with high PGA3 levels. Network analysis across cytokine and microbiome measures identified relationships between IL-1-related proteins and microbial taxa, with the presence of respiratory issues associated with higher levels of IL-1ß, IP-10, and Prevotella In conclusion, PGA3 levels in the trachea are correlated with increases in specific microbial taxa and inflammatory molecules, with an increase in oral microbes with increasing PGA3.

PTEN participates in airway remodeling of asthma by regulating CD38/Ca2+/CREB signaling.

Both phosphatase and tensin homologue deleted on chromosome ten (PTEN) and cluster of differentiation 38 (CD38) have been suggested to be key regulators of the pathogenesis of asthma. However, the precise role and molecular mechanisms by which PTEN and CD38 are involved in airway remodeling throughout asthma pathogenesis remains poorly understood. This study aimed to elucidate the role of PTEN and CD38 in airway remodeling of asthma. Exposure to tumor necrosis factor-α (TNF-α) in airway smooth muscle (ASM) cells markedly decreased PTEN expression, and increased expression of CD38. Overexpression of PTEN suppressed the expression of CD38 and downregulated proliferation and migration induced by TNF-α stimulation, which was partially reversed by CD38 overexpression. PTEN/CD38 axis regulated Ca2+ levels and cyclic AMP response-element binding protein (CREB) phosphorylation in TNF-α-stimulated ASM cells. The in vitro knockdown of CD38 or overexpression of PTEN remarkably restricted airway remodeling and decreased Ca2+ concentrations and CREB phosphorylation in asthmatic mice. CD38 overexpression abolished the inhibitory effects of PTEN overexpression on airway remodeling. These findings demonstrate that PTEN inhibits airway remodeling of asthma through the downregulation of CD38-mediated Ca2+/CREB signaling, highlighting a key role of PTEN/CD38/Ca2+/CREB signaling in the molecular pathogenesis of asthma.

Glycosylhydrolase genes control respiratory tubes sizes and airway stability.

Tight barriers are crucial for animals. Insect respiratory cells establish barriers through their extracellular matrices. These chitinous-matrices must be soft and flexible to provide ventilation, but also tight enough to allow oxygen flow and protection against dehydration, infections, and environmental stresses. However, genes that control soft, flexible chitin-matrices are poorly known. We investigated the genes of the chitinolytic glycosylhydrolase-family 18 in the tracheal system of Drosophila melanogaster. Our findings show that five chitinases and three chitinase-like genes organize the tracheal chitin-cuticles. Most of the chitinases degrade chitin from airway lumina to enable oxygen delivery. They further improve chitin-cuticles to enhance tube stability and integrity against stresses. Unexpectedly, some chitinases also support chitin assembly to expand the tube lumen properly. Moreover, Chitinase2 plays a decisive role in the chitin-cuticle formation that establishes taenidial folds to support tube stability. Chitinase2 is apically enriched on the surface of tracheal cells, where it controls the chitin-matrix architecture independently of other known cuticular proteins or chitinases. We suppose that the principle mechanisms of chitin-cuticle assembly and degradation require a set of critical glycosylhydrolases for flexible and not-flexible cuticles. The same glycosylhydrolases support thick laminar cuticle formation and are evolutionarily conserved among arthropods.

Airway smooth muscle tone increases actin filamentogenesis and contractile capacity.

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 10-4 M of methacholine following a 30-min period with or without tone elicited by the EC30 of 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.

Manduca sexta hemolymph protease-2 (HP2) activated by HP14 generates prophenoloxidase-activating protease-2 (PAP2) in wandering larvae and pupae.

Melanization is a universal defense mechanism of insects against microbial infection. During this response, phenoloxidase (PO) is activated from its precursor by prophenoloxidase activating protease (PAP), the terminal enzyme of a serine protease (SP) cascade. In the tobacco hornworm Manduca sexta, hemolymph protease-14 (HP14) is autoactivated from proHP14 to initiate the protease cascade after host proteins recognize invading pathogens. HP14, HP21, proHP1*, HP6, HP8, PAP1-3, and non-catalytic serine protease homologs (SPH1 and SPH2) constitute a portion of the extracellular SP-SPH system to mediate melanization and other immune responses. Here we report the expression, purification, and functional characterization of M. sexta HP2. The HP2 precursor is synthesized in hemocytes, fat body, integument, nerve and trachea. Its mRNA level is low in fat body of 5th instar larvae before wandering stage; abundance of the protein in hemolymph displays a similar pattern. HP2 exists as an active enzyme in plasma of the wandering larvae and pupae in the absence of an infection. HP14 cleaves proHP2 to yield active HP2. After incubating active HP2 with larval hemolymph, we detected higher levels of PO activity, i.e. an enhancement of proPO activation. HP2 cleaved proPAP2 (but not proPAP3 or proPAP1) to yield active PAP2, responsible for a major increase in IEARpNA hydrolysis. PAP2 activates proPOs in the presence of a cofactor of SPH1 and SPH2. In summary, we have identified a new member of the proPO activation system and reconstituted a pathway of HP14-HP2-PAP2-PO. Since high levels of HP2 mRNA were present in integument and active HP2 in plasma of wandering larvae, HP2 likely plays a role in cuticle melanization during pupation and protects host from microbial infection in a soil environment.

Phosphodiesterase 9 (PDE9) regulates bovine tracheal smooth muscle relaxation.

The present study was designed to clarify phosphodiesterase 9 (PDE9) expression in bovine tracheal smooth muscle tissue, and to elucidate that PDE9 may contribute to the regulation of airway relaxation. PDE9 mRNA expression was detected in bovine tracheal smooth muscle. Sodium nitroprusside (an NO donor) and BAY 73-6691 (a selective PDE9 inhibitor) reduced high K+- and carbachol-induced contraction. BAY 73-6691 relaxed tracheal tissue on the same level with vardenafil (a selective PDE5 inhibitor). These results support our hypothesis that PDE9 plays functional role in the tracheal smooth muscle relaxation. PDE9 inhibitors are expected to be a novel target of the add-on treatment of airway hyperresponsiveness.

Alpha-tocopherol succinate increases cyclooxygenase-2 activity: Tissue-specific action in pregnant rat uterus in vitro.

AIMS: Lipid soluble vitamin E plays a role in several physiological mechanisms, however, the mechanism of this action is controversial. We investigated how tocopherol (α-tocopherol acid succinate) influences the effects of cyclooxygenase inhibitors (COXi) in the smooth muscles. MAIN METHODS: The contractility of the samples from 22-day-pregnant myometrium and non-pregnant myometrium and trachea was determined in an isolated organ bath in vitro. The activity of cyclooxygenase enzymes (COX) was also measured in the tissues. KEY FINDINGS: Diclofenac (10-9-10-5M) and rofecoxib (10-10-10-5M) decreased the contractions in non-pregnant and 22-day-pregnant uteri. Tocopherol (10-7M) increased the relaxant effect only in pregnant uteri. Both diclofenac (10-9-10-5M) and rofecoxib (10-10-10-5M) reduced the tracheal tones, while they were slightly intensified by pretreatment with tocopherol (10-7M). Tocopherol enhanced the contractions of pregnant uteri. Tocopherol (10-7M) itself can induce the cyclooxygenase activity and shift the COX-1 and COX-2 ratio to COX-2. The lowest COX activity was found in non-pregnant uteri, while the highest one was in the trachea. SIGNIFICANCE: The COX enzymes, especially COX-2, play an important role in the contraction of pregnant uteri in rat. Tocopherol has a tissue specific COX-2 activity increasing effect in pregnant rat uterus but has no such action in non-pregnant uteri or tracheal tissue. Hereby, tocopherol may intensify selectively the uterine relaxing effect of COX-2 inhibitors in preterm contractions. However, tocopherol can enhance the contractile response of pregnant uterus that may increase the risk of premature contractions.

Elastase alters contractility and promotes an inflammatory synthetic phenotype in airway smooth muscle tissues.

Neutrophil elastase is secreted by inflammatory cells during airway inflammation and can elicit airway hyperreactivity in vivo. Elastase can degrade multiple components of the extracellular matrix. We hypothesized that elastase might disrupt the connections between airway smooth muscle (ASM) cells and the extracellular matrix and that this might have direct effects on ASM tissue responsiveness and inflammation. The effect of elastase treatment on ASM contractility was assessed in vitro in isolated strips of canine tracheal smooth muscle by stimulation of tissues with cumulatively increasing concentrations of acetylcholine (ACh) and measurement of contractile force. Elastase treatment potentiated contractile responses to ACh at low concentrations but suppressed the maximal contractile force generated by the tissues without affecting the phosphorylation of myosin regulatory light chain (RLC). Elastase also promoted the secretion of eotaxin and the activation of Akt in ASM tissues and decreased expression of smooth muscle myosin heavy chain, consistent with promotion of a synthetic inflammatory phenotype. As the degradation of matrix proteins can alter integrin engagement, we evaluated the effect of elastase on the assembly and activation of integrin-associated adhesion junction complexes in ASM tissues. Elastase led to talin cleavage, reduced talin binding to vinculin, and suppressed activation of the adhesome proteins paxillin, focal adhesion kinase, and vinculin, indicating that elastase causes the disassembly of adhesion junction complexes and the inactivation of adhesome signaling proteins. We conclude that elastase promotes an inflammatory phenotype and increased sensitivity to ACh in ASM tissues by disrupting signaling pathways mediated by integrin-associated adhesion complexes.

Predictive value of α-amylase in tracheal aspirates for ventilator-associated pneumonia in elderly patients.

OBJECTIVE: This study aims to investigate the correlation between α-amylase in tracheal aspirates and risk factors of aspiration, as well as ventilator-associated pneumonia (VAP), in elderly patients undergoing mechanical ventilation and explore the clinical value of α-amylase for predicting VAP. METHODS: Tracheal aspirates were collected from elderly patients within 2 weeks after tracheal intubation in mechanical ventilation, and α-amylase was detected. Patients were grouped according to the presence of VAP. The correlation between α-amylase and risk factors of aspiration before intubation, as well as VAP, were analyzed. RESULTS: The sample of this study comprised 147 patients. The average age of these patients was 86.9 years. The incidence of VAP was 21% during the study period. Tracheal aspirate α-amylase level increased with the increase in the number of risk factors for aspiration before intubation, α-amylase level was significantly higher in the VAP group than in the non-VAP group, the area under the receiver operating characteristic curve (ROC) of the diagnostic value of α-amylase for VAP was 0.813 (95% CI: 0.721-0.896), threshold value was 4,681.5 U/L, sensitivity was 0.801 and specificity was 0.793. Logistic multivariate analysis revealed the following risk factors for VAP: a number of risk factors before intubation of ≥3, a Glasgow score of <8 points, the absence of continuous aspiration of subglottic secretion and a tracheal aspirate α-amylase level of >4681.5 U/L. CONCLUSION: Tracheal aspirate α-amylase can serve as a biomarker for predicting VAP in elderly patients undergoing mechanical ventilation.

The impact of bilateral vagotomy on the physostigmine-induced airway constriction in ferrets.

Vagal innervations have a great role in the respiratory function and are the main route of signal transmission from respiratory neural centers into the trachea and others conducting airways. We have investigated the role of central mechanisms related to vagal neural pathways and the cholinergic outflow in tracheobronchial smooth muscle tone and lung mechanics parameters. Parameters of lung mechanics such as lung resistance (RL), dynamic compliance (Cdyn) and pressure in bypassed tracheal segment (Ptseg) were measured before and after vagotomy and asphyxia test. Before vagotomy (BV), the control measurements were obtained and physostigmine was administered systemically, in increasing dose 10, 40 and 100µg/kg body weight (bw) with 15min interval between doses. After vagotomy (AV), administration of physostigmine with the same doses as BV has been done and the asphyxia challenge was conducted as per study protocol. The values of Ptseg and RL after physostigmine administration, BV vs. AV, respectively, at maximal dose of 100µg/kg bw were 32.5±3.3cm H2O, and 10.6±1.5cm H2O (p<0.0001); 0.16±0.04cm H2O/mL/s, and 0.067±0.006cm H2O/mL/s AV (P<0.05). The Cydn values were affected after physostigmine administration only at the lowest dose of 10µg/kg bw, and BV was 0.75±0.05mL/cm H2O vs. 0.53±0.04mL/cm H2O AV (P<0.004). Cholinergic outflow produced increases in tracheal tone, lung resistance and a decrease in dynamic compliance before, but not after vagotomy. Our results show the high impact of central neuronal mechanism in parameters of lung mechanics and respiration. This study indicates that vagal nerves have a crucial role, in the transmission of impulses initiated from central nervous system, in regulating the respiration by contraction or relaxation of airway smooth muscle tone.

Vimentin dephosphorylation at ser-56 is regulated by type 1 protein phosphatase in smooth muscle.

BACKGROUND: The intermediate filament protein vimentin undergoes reversible phosphorylation and dephosphorylation at Ser-56, which plays an important role in regulating the contraction-relaxation cycles of smooth muscle. The protein phosphatases that mediate vimentin dephosphorylation in smooth muscle have not been previously investigated. METHODS: The associations of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) with vimentin in mouse tracheal rings was evaluated by co-immunoprecipitation. Lentivirus-mediated shRNA against PP1 was used to assess the role of PP1 in vimentin dephosphorylation and the vimentin-associated process in smooth muscle. RESULTS: Co-immunoprecipitation analysis showed that vimentin interacted with PP1, but barely with PP2A, in airway smooth muscle. Knockdown of PP1 by lentivirus-mediated shRNA increased the acetylcholine-induced vimentin phosphorylation and smooth muscle contraction. Because vimentin phosphorylation is able to modulate p130 Crk-associated substrate (p130CAS) and actin polymerization, we also evaluated the role of PP1 in the biological processes. Silencing of PP1 also enhanced the agonist-induced the dissociation of p130CAS from vimentin and F/G-actin ratios (an index of actin polymerization). However, PP1 knockdown did not affect c-Abl tyrosine phosphorylation, an important molecule that controls actin dynamics. CONCLUSIONS: Taken together, these findings suggest that PP1 is a key protein serine/threonine phosphatase that controls vimentin Ser-56 dephosphorylation in smooth muscle. PP1 regulates actin polymerization by modulating the dissociation of p130CAS from vimentin, but not by affecting c-Abl tyrosine kinase.

Alkaline phosphatase activity in airway fluid obtained by tracheal wash from adult horses.

BACKGROUND: Collection of fluid from the lower respiratory tract (LRT) plays an important role in both the pathophysiological investigation and diagnosis of respiratory tract disease. Enzymes such as ALP are, among others, indicators of cell damage or death, type II pneumocyte proliferation, and neutrophil invasion, and have been useful as biomarkers of respiratory disease in other species. OBJECTIVES: The purpose of this study was to determine and compare tracheal wash (TW) ALP activity in healthy horses and horses with LRT inflammation (LRTI) determined by TW cytology profile. METHODS: Tracheal washes were collected from asymptomatic adult geldings to measure ALP activity. The horses were allocated to the healthy group or the group with LRT inflammation based on differential leukocyte counts of TW preparations. Horses with > 20% neutrophils and > 1% eosinophils were allocated to the LRTI group, the horses with < 20% neutrophils and < 1% eosinophils were the controls. RESULTS: Tracheal wash ALP activity, measured using a semiautomatic chemistry analyzer, was statistically significantly higher in 18 horses with LRTI (18.9 ± 11.2 × 10(3) U/L) than in healthy horses (10.3 ± 5.9 × 10(3) U/L) (P = .021). CONCLUSIONS: Determining tracheal wash ALP activity is a simple, inexpensive and safe technique that can be used to facilitate the early diagnosis of equine respiratory disease, since it is higher in asymptomatic adult horses with a TW cytology profile consistent with LRT inflammation than in healthy adult horses with a normal TW cytology profile.

Transtracheal puncture: a forgotten procedure

Transtracheal puncture has long been known as a safe, low-cost procedure. However, with the advent of bronchoscopy, it has largely been forgotten. Two researchers have suggested the use of α-amylase activity to diagnose salivary aspiration, but the normal values of this enzyme in tracheobronchial secretions are unknown. We aimed to define the normal values of α-amylase activity in tracheobronchial secretions and verify the rate of major complications of transtracheal puncture. From October 2009 to June 2011, we prospectively evaluated 118 patients without clinical or radiological signs of salivary aspiration who underwent transtracheal puncture before bronchoscopy. The patients were sedated with a solution of lidocaine and diazepam until they reached a Ramsay sedation score of 2 or 3. We then cleaned the cervical region and anesthetized the superficial planes with lidocaine. Next, we injected 10 mL of 2% lidocaine into the tracheobronchial tree. Finally, we injected 10 mL of normal saline into the tracheobronchial tree and immediately aspirated the saline with maximum vacuum pressure to collect samples for measurement of the α-amylase level. The α-amylase level mean ± SE, median, and range were 1914 ± 240, 1056, and 24-10,000 IU/L, respectively. No major complications (peripheral desaturation, subcutaneous emphysema, cardiac arrhythmia, or hemoptysis) occurred among 118 patients who underwent this procedure. Transtracheal aspiration is a safe, low-cost procedure. We herein define for the first time the normal α-amylase levels in the tracheobronchial secretions of humans.

Transtracheal puncture: a forgotten procedure.

Transtracheal puncture has long been known as a safe, low-cost procedure. However, with the advent of bronchoscopy, it has largely been forgotten. Two researchers have suggested the use of α-amylase activity to diagnose salivary aspiration, but the normal values of this enzyme in tracheobronchial secretions are unknown. We aimed to define the normal values of α-amylase activity in tracheobronchial secretions and verify the rate of major complications of transtracheal puncture. From October 2009 to June 2011, we prospectively evaluated 118 patients without clinical or radiological signs of salivary aspiration who underwent transtracheal puncture before bronchoscopy. The patients were sedated with a solution of lidocaine and diazepam until they reached a Ramsay sedation score of 2 or 3. We then cleaned the cervical region and anesthetized the superficial planes with lidocaine. Next, we injected 10 mL of 2% lidocaine into the tracheobronchial tree. Finally, we injected 10 mL of normal saline into the tracheobronchial tree and immediately aspirated the saline with maximum vacuum pressure to collect samples for measurement of the α-amylase level. The α-amylase level mean ± SE, median, and range were 1914 ± 240, 1056, and 24-10,000 IU/L, respectively. No major complications (peripheral desaturation, subcutaneous emphysema, cardiac arrhythmia, or hemoptysis) occurred among 118 patients who underwent this procedure. Transtracheal aspiration is a safe, low-cost procedure. We herein define for the first time the normal α-amylase levels in the tracheobronchial secretions of humans.

PCSK5 mutation in a patient with the VACTERL association.

BACKGROUND: The VACTERL association is a typically sporadic, non-random collection of congenital anomalies that includes vertebral defects, anal atresia, cardiac defects, tracheoesophageal fistula with esophageal atresia, renal anomalies, and limb abnormalities. Although several chromosomal aberrations and gene mutations have been reported as disease-causative, these findings have been sparsely replicated to date. CASE PRESENTATION: In the present study, whole exome sequencing of a case with the VACTERL association uncovered a novel frameshift mutation in the PCSK5 gene, which has been reported as one of the causative genes for the VACTERL association. Although this mutation appears potentially pathogenic in its functional aspects, it was also carried by the healthy father. Furthermore, a database survey revealed several other deleterious variants in the PCSK5 gene in the general population. CONCLUSIONS: Further studies are necessary to clarify the etiological role of the PCSK5 mutation in the VACTERL association.

Deciphering the genetic programme triggering timely and spatially-regulated chitin deposition.

Organ and tissue formation requires a finely tuned temporal and spatial regulation of differentiation programmes. This is necessary to balance sufficient plasticity to undergo morphogenesis with the acquisition of the mature traits needed for physiological activity. Here we addressed this issue by analysing the deposition of the chitinous extracellular matrix of Drosophila, an essential element of the cuticle (skin) and respiratory system (tracheae) in this insect. Chitin deposition requires the activity of the chitin synthase Krotzkopf verkehrt (Kkv). Our data demonstrate that this process equally requires the activity of two other genes, namely expansion (exp) and rebuf (reb). We found that Exp and Reb have interchangeable functions, and in their absence no chitin is produced, in spite of the presence of Kkv. Conversely, when Kkv and Exp/Reb are co-expressed in the ectoderm, they promote chitin deposition, even in tissues normally devoid of this polysaccharide. Therefore, our results indicate that both functions are not only required but also sufficient to trigger chitin accumulation. We show that this mechanism is highly regulated in time and space, ensuring chitin accumulation in the correct tissues and developmental stages. Accordingly, we observed that unregulated chitin deposition disturbs morphogenesis, thus highlighting the need for tight regulation of this process. In summary, here we identify the genetic programme that triggers the timely and spatially regulated deposition of chitin and thus provide new insights into the extracellular matrix maturation required for physiological activity.