Germline mutations in a G protein identify signaling cross-talk in T cells.

Humans with monogenic inborn errors responsible for extreme disease phenotypes can reveal essential physiological pathways. We investigated germline mutations in GNAI2, which encodes Gαi2, a key component in heterotrimeric G protein signal transduction usually thought to regulate adenylyl cyclase-mediated cyclic adenosine monophosphate (cAMP) production. Patients with activating Gαi2 mutations had clinical presentations that included impaired immunity. Mutant Gαi2 impaired cell migration and augmented responses to T cell receptor (TCR) stimulation. We found that mutant Gαi2 influenced TCR signaling by sequestering the guanosine triphosphatase (GTPase)-activating protein RASA2, thereby promoting RAS activation and increasing downstream extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-AKT S6 signaling to drive cellular growth and proliferation.

In vitro assessment of the anti-adenoviral activity of artemisinin and its derivatives.

Adenoviral infections, particularly in children, remain a significant public health issue with no approved targeted treatments. Artemisinin and its derivatives, well-known for their use in malaria treatment, have shown antiviral activities in recent studies. However, their efficacy against human adenovirus (HAdV) remains unexplored. This study aimed to assess the activity of artemisinin and its derivatives against HAdV infection in vitro using cell lines and primary cells. Our data revealed that artemisinin exhibited dose-dependent anti-HAdV activity with no apparent cytotoxicity over a wide concentration range. Mechanistically, artemisinin did not affect viral attachment or entry into target cells, nor the viral genome entry into cell nucleus. Instead, it inhibited HAdV through suppression of viral DNA replication. Comparative analysis with its derivatives, artesunate and artemisone, showed distinct cytotoxicity and anti-adenoviral profiles, with artemisone showing superior efficacy and lower toxicity. Further validation using a primary airway epithelial cell model confirmed the anti-adenoviral activity of both artemisinin and artemisone against different virus strains. Together, our findings suggest that artemisinin and its derivatives may be promising candidates for anti-HAdV treatment.

Quantitative Monitoring of GPCR-Mediated Spatiotemporal IP3 Dynamics Using Confocal Fluorescence Microscopy.

Activation of G protein-coupled receptors upon chemoattractant stimulation induces activation of multiple signaling pathways. To fully understand how these signaling pathway coordinates to achieve directional migration of neutrophils, it is essential to determine the dynamics of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we describe a detailed methodology for monitoring and quantitatively analyzing the spatiotemporal dynamics of 1,4,5-inositol trisphosphate (IP3) in neutrophil-like HL60 cells in response to various chemoattractant fields by applying Förster resonance energy transfer (FRET) fluorescence microscopy.

Analysis of the Risk Factors for Plastic Bronchitis in Children with Severe Adenovirus Pneumonia: A Retrospective Study.

Purpose: Plastic bronchitis (PB), a rare complication of respiratory infection characterized by the formation of casts in the tracheobronchial tree, can lead to airway obstruction and severe condition. Adenovirus is one of the common pathogens of PB caused by infection. This study aimed to evaluate the clinical features and risk factors for PB in children with severe adenovirus pneumonia. Methods: A retrospective study of children with severe adenovirus pneumonia with bronchoscopy results at Guangzhou Women and Children's Hospital between January 2018 and January 2020 was performed. Based on bronchoscopy, we divided children with severe adenovirus pneumonia into two groups: PB and non-PB. Binary logistic regression analysis was used to identify independent risk factors for PB in patients with severe adenovirus pneumonia after univariate analysis. Results: Our study examined 156 patients with severe adenovirus pneumonia with bronchoscopy results in hospital. Among them, 18 developed PB and 138 did not. On multivariate analysis, the independent risk factors of PB in children with severe adenovirus pneumonia were history of allergies (OR 10.147, 95% CI 1.727-59.612; P=0.010), diminished breath sounds (OR 12.856, 95% CI 3.259-50.713; P=0.001), and increased proportion of neutrophils (>70%; OR 8.074, 95% CI 1.991-32.735; P=0.003). Conclusion: Children with severe adenovirus pneumonia with a history of allergies, diminished breath sounds, and increased the proportion of neutrophils >70% may show higher risk of PB.

TAK-242 protects against oxygen-glucose deprivation and reoxygenation-induced injury in brain microvascular endothelial cells and alters the expression pattern of lncRNAs.

Background: Deep hypothermic circulatory arrest (DHCA) is a technique used during the surgical treatment of aneurysms of the thoracic aorta in adult patients, and complex congenital heart disease in neonates. And brain microvascular endothelial cells (BMECs) are essential components of the cerebrovascular network and participate in maintaining the blood-brain barrier (BBB) and brain function. In our previous study, we found that oxygen-glucose deprivation and reoxygenation (OGD/R) activated Toll-like receptor 4 (TLR4) signaling in BMECs, and induced pyroptosis and inflammation. In this study, we further investigated the potential mechanism of ethyl(6R)-6-[N-(2-Chloro-4-fluorophenyl) sulfamoyl] cyclohex-1-ene-1-carboxylate (TAK-242) on BMECs under OGD/R, as in patients with sepsis, the TAK-242 was tested in clinical trials. Methods: To confirm the function of TAK-242 on BMECs under OGD/R, cell viability, inflammatory factors, inflammation-associated pyroptosis, and nuclear factor-κB (NF-κB) signaling were determined using Cell Counting Kit-8 (CCK-8) assay, enzyme-linked immunosorbent assay (ELISA), and western blotting, respectively. To investigate the lncRNAs associated with TLR4 during OGD/R, long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) expression patterns were profiled with RNA deep sequencing. Moreover, to confirm whether lncRNA-encoded short peptides, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used. Results: Relative control group, OGD/R inhibited the cell viability, increased the section of inflammatory factors secretion, including IL-1ß, IL-6, and TNF-α, and promoted the pathways of TLR4/NLRP3/Caspase-1 and TLR4/NF-κB. However, TAK-242 + OGD/R group promoted OGD/R cell viability, decreased OGD/R-induced inflammatory factors secretion, and inhibited the pathways of TLR4/NLRP3/Caspase-1 and TLR4/NF-κB. In addition, AABR07000411.1, AABR070006957.1, and AABR070008256.1 were decreased in OGD/R cells compared with controls, but TAK-242 restored their expression under OGD/R condition. AABR07000473.1, AC130862.4, and LOC10254972.6 were induced by OGD/R, but were suppressed in TAK-242 + OGD/R cells compared with OGD/R. Moreover, AABR07049961.1, AC127076.2, AABR07066020.1, and AABR07025303.1-encoded short peptides were dysregulated in OGD/R cells, and TAK-242 attenuated the dysregulation of AABR07049961.1, AC127076.2, and AABR07066020.1-encoded short peptides. Conclusions: TAK-242 alters the expression pattern of lncRNAs in OGD/R cells, and differently expressed lncRNAs may exert a protective effect against OGD/R injury through a mechanism of competing endogenous RNA (ceRNA) and encoding short peptides. These findings maybe provide a new theory basis for the treatment of DHCA.

Human adenovirus infection induces pulmonary inflammatory damage by triggering noncanonical inflammasomes activation and macrophage pyroptosis.

Introduction: Human adenovirus (HAdV) is a common respiratory virus, which can lead to severe pneumonia in children and immunocompromised persons, and canonical inflammasomes are reported to be involved in anti-HAdV defense. However, whether HAdV induced noncanonical inflammasome activation has not been explored. This study aims to explore the broad roles of noncanonical inflammasomes during HAdV infection to investigate the regulatory mechanism of HAdV-induced pulmonary inflammatory damage. Methods: We mined available data on GEO database and collected clinical samples from adenovirus pneumonia pediatric patients to investigate the expression of noncanonical inflammasome and its clinical relevance. An in vitro cell model was employed to investigate the roles of noncanonical inflammasomes in macrophages in response to HAdV infection. Results: Bioinformatics analysis showed that inflammasome-related genes, including caspase-4 and caspase-5, were enriched in adenovirus pneumonia. Moreover, caspase-4 and caspase-5 expression levels were significantly increased in the cells isolated from peripheral blood and broncho-alveolar lavage fluid (BALF) of pediatric patients with adenovirus pneumonia, and positively correlated with clinical parameters of inflammatory damage. In vitro experiments revealed that HAdV infection promoted caspase-4/5 expression, activation and pyroptosis in differentiated THP-1 (dTHP-1) human macrophages via NF-κB, rather than STING signaling pathway. Interestingly, silencing of caspase-4 and caspase-5 in dTHP-1 cells suppressed HAdV-induced noncanonical inflammasome activation and macrophage pyroptosis, and dramatically decreased the HAdV titer in cell supernatants, by influencing virus release rather than other stages of virus life cycle. Discussion: In conclusion, our study demonstrated that HAdV infection induced macrophage pyroptosis by triggering noncanonical inflammasome activation via a NF-kB-dependent manner, which may explore new perspectives on the pathogenesis of HAdV-induced inflammatory damage. And high expression levels of caspase-4 and caspase-5 may be a biomarker for predicting the severity of adenovirus pneumonia.

G protein-coupled receptor-mediated membrane targeting of PLCγ2 is essential for neutrophil chemotaxis.

The current dogma is that chemoattractants G protein-coupled receptors activate ß phospholipase C while receptor tyrosine kinases activate γ phospholipase C. Here, we show that chemoattractant/G protein-coupled receptor-mediated membrane recruitment of γ2 phospholipase C constitutes G protein-coupled receptor-mediated phospholipase C signaling and is essential for neutrophil polarization and migration during chemotaxis. In response to a chemoattractant stimulation, cells lacking γ2 phospholipase C (plcg2kd) displayed altered dynamics of diacylglycerol production and calcium response, increased Ras/PI3K/Akt activation, elevated GSK3 phosphorylation and cofilin activation, impaired dynamics of actin polymerization, and, consequently, defects in cell polarization and migration during chemotaxis. The study reveals a molecular mechanism of membrane targeting of γ2 phospholipase C and the signaling pathways by which γ2 phospholipase C plays an essential role in neutrophil chemotaxis.

Analysis of mortality risk factors in children with severe adenovirus pneumonia: A single-center retrospective study.

BACKGROUND: Human adenovirus (HAdV) is one of the most common viruses causing respiratory infections among young children. Most adenovirus infections are mild and self-limited; however, these infections may occasionally cause severe pneumonia and even death. The mortality risk factors for severe adenovirus pneumonia are not completely clear. This study aimed to evaluate the mortality risk factors in children with severe adenovirus pneumonia. METHODS: A retrospective study of children with severe adenovirus pneumonia hospitalized in Guangzhou Women and Children's Hospital between July 2018 and January 2020 was performed. Binary logistic regression analysis was used to identify independent mortality risk factors for severe adenovirus pneumonia after univariate analysis. RESULTS: Our study included 189 patients (123 males and 66 females). Among them, 13 patients did not survive with a mortality of 6.88%. In multivariate analysis, the independent mortality risk factors in children with severe adenovirus pneumonia were age less than 1 year (OR = 18.513, 95% CI: 2.157-158.883, p = 0.008), hypoxia (OR = 62.335, 95% CI: 2.385-1629.433, p = 0.013), and thrombocytopenia (platelet <100∗10ˆ9/L) (OR = 13.324, 95% CI: 1.232-144.075, p = 0.033). CONCLUSIONS: In children with severe adenovirus pneumonia who are younger than one year old, hypoxia and platelet counts less than 100∗10ˆ9/L represent mortality risk factors.

C2GAP2 is a common regulator of Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.

Phagocytosis, macropinocytosis, and G protein coupled receptor-mediated chemotaxis are Ras-regulated and actin-driven processes. The common regulator for Ras activity in these three processes remains unknown. Here, we show that C2GAP2, a Ras GTPase activating protein, highly expressed in the vegetative growth state in model organism Dictyostelium. C2GAP2 localizes at the leading edge of chemotaxing cells, phagosomes during phagocytosis, and macropinosomes during micropinocytosis. c2gapB- cells lacking C2GAP2 displayed increased Ras activation upon folic acid stimulation and subsequent impaired chemotaxis in the folic acid gradient. In addition, c2gaB- cells have elevated phagocytosis and macropinocytosis, which subsequently results in faster cell growth. C2GAP2 binds multiple phospholipids on the plasma membrane and the membrane recruitment of C2GAP2 requires calcium. Taken together, we show a shared negative regulator of Ras signaling that mediates Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.

Ras inhibitors gate chemoattractant concentration range for chemotaxis through controlling GPCR-mediated adaptation and cell sensitivity.

Chemotaxis plays an essential role in recruitment of leukocytes to sites of inflammation. Eukaryotic cells sense chemoattractant with G protein-coupled receptors (GPCRs) and chemotax toward gradients with an enormous concentration range through adaptation. Cells in adaptation no longer respond to the present stimulus but remain sensitive to stronger stimuli. Thus, adaptation provides a fundamental strategy for eukaryotic cells to chemotax through a gradient. Ras activation is the first step in the chemosensing GPCR signaling pathways that displays a transient activation behavior in both model organism Dictyostelium discoideum and mammalian neutrophils. Recently, it has been revealed that C2GAP1 and CAPRI control the GPCR-mediated adaptation in D. discoideum and human neutrophils, respectively. More importantly, both Ras inhibitors regulate the sensitivity of the cells. These findings suggest an evolutionarily conserved molecular mechanism by which eukaryotic cells gate concentration range of chemoattractants for chemotaxis.

Validation of a Classification Model Using Complete Blood Count to Predict Severe Human Adenovirus Lower Respiratory Tract Infections in Pediatric Cases.

Background: Human adenovirus (HAdV) lower respiratory tract infections (LRTIs) are prone to severe cases and even cause death in children. Here, we aimed to develop a classification model to predict severity in pediatric patients with HAdV LRTIs using complete blood count (CBC). Methods: The CBC parameters from pediatric patients with a diagnosis of HAdV LRTIs from 2013 to 2019 were collected during the disease's course. The data were analyzed as potential predictors for severe cases and were selected using a random forest model. Results: We enrolled 1,652 CBC specimens from 1,069 pediatric patients with HAdV LRTIs in the present study. Four hundred and seventy-four patients from 2017 to 2019 were used as the discovery cohort, and 470 patients from 2013 to 2016 were used as the validation cohort. The monocyte ratio (MONO%) was the most obvious difference between the mild and severe groups at onset, and could be used as a marker for the early accurate prediction of the severity [area under the subject operating characteristic curve (AUROC): 0.843]. Four risk factors [MONO%, hematocrit (HCT), red blood cell count (RBC), and platelet count (PLT)] were derived to construct a classification model of severe and mild cases using a random forest model (AUROC: 0.931 vs. 0.903). Conclusion: Monocyte ratio can be used as an individual predictor of severe cases in the early stages of HAdV LRTIs. The four risk factors model is a simple and accurate risk assessment tool that can predict severe cases in the early stages of HAdV LRTIs.

A systems approach to investigate GPCR-mediated Ras signaling network in chemoattractant sensing.

A GPCR-mediated signaling network enables a chemotactic cell to generate adaptative Ras signaling in response to a large range of concentrations of a chemoattractant. To explore potential regulatory mechanisms of GPCR-controlled Ras signaling in chemosensing, we applied a software package, Simmune, to construct detailed spatiotemporal models simulating responses of the cAR1-mediated Ras signaling network. We first determined the dynamics of G-protein activation and Ras signaling in Dictyostelium cells in response to cAMP stimulations using live-cell imaging and then constructed computation models by incorporating potential mechanisms. Using simulations, we validated the dynamics of signaling events and predicted the dynamic profiles of those events in the cAR1-mediated Ras signaling networks with defective Ras inhibitory mechanisms, such as without RasGAP, with RasGAP overexpression, or with RasGAP hyperactivation. We describe a method of using Simmune to construct spatiotemporal models of a signaling network and run computational simulations without writing mathematical equations. This approach will help biologists to develop and analyze computational models that parallel live-cell experiments.

Influence of the timing of bronchoscopic alveolar lavage on children with adenovirus pneumonia: a comparative study.

BACKGROUND: Adenovirus pneumonia is prone to severe clinical and imaging manifestations in children. Bronchoscopic alveolar lavage (BAL) is an important adjunctive therapy for patients with severe imaging findings. The study aimed to evaluate the effect of the timing on the efficacy of bronchoalveolar lavage in children with adenovirus pneumonia. METHODS: This study included 134 patients with adenovirus pneumonia treated with BAL at Guangzhou Women and Children's Medical Center from January 2019 to January 2020.They were classified into the severe and mild groups. Based on the timing of BAL, each group was divided into the early BAL layer (received BAL within 1-9 days of the illness course) and the late BAL layer (received BAL within 10-14 days of the illness course). The clinical data of patients with different BAL timings were analyzed in two groups. RESULTS: Among the 134 patients, 70 were categorized into the mild group and 64 were categorized into the severe group. Of the 134 patients, 42 patients received BAL early (mild group: n = 21 and severe group: n = 21) and 92 patients received BAL later (mild group: n = 49 and severe group: n = 43). In the mild group, the fever and hospital duration were shorter in patients who received BAL early than in those who received BAL later (p < 0.05). However, in the severe group, there were no statistically significant differences in the fever and hospital duration between patients who received BAL early and those who received BAL later. However, the need for mechanical ventilation and the incidence of BAL complications, such as new need for oxygen, were higher in patients who received BAL early than in those who received BAL later in the severe group (p < 0.05). CONCLUSION: For mild adenovirus pneumonia, early BAL may shorten the fever and hospital duration. However, early BAL in severe cases might not shorten the course of the disease or improve prognosis and may even increase the risks of mechanical ventilation and BAL complications.

Prestimulation of CD2 confers resistance to HIV-1 latent infection in blood resting CD4 T cells.

HIV-1 infects blood CD4 T cells through the use of CD4 and CXCR4 or CCR5 receptors, which can be targeted through blocking viral binding to CD4/CXCR4/CCR5 or virus-cell fusion. Here we describe a novel mechanism by which HIV-1 nuclear entry can also be blocked through targeting a non-entry receptor, CD2. Cluster of differentiation 2 (CD2) is an adhesion molecule highly expressed on human blood CD4, particularly, memory CD4 T cells. We found that CD2 ligation with its cell-free ligand LFA-3 or anti-CD2 antibodies rendered blood resting CD4 T cells highly resistant to HIV-1 infection. We further demonstrate that mechanistically, CD2 binding initiates competitive signaling leading to cofilin activation and localized actin polymerization around CD2, which spatially inhibits HIV-1-initiated local actin polymerization needed for viral nuclear migration. Our study identifies CD2 as a novel target to block HIV-1 infection of blood resting T cells.

Mortality risk factors among hospitalized children with severe pertussis.

BACKGROUND: Some children hospitalized for severe pertussis need intensive care; moreover, some children die because of deterioration alone or in combination with other complications. The purpose of this study was to identify the mortality risk factors among hospitalized children with severe pertussis. METHODS: This study evaluated the medical records of 144 hospitalized children with severe pertussis at the Guangzhou Women and Children's Medical Centre between January 2016 and December 2019. RESULTS: The median age of patients was 2 months (IQR 1-4 months), with 90.3% of the patients aged < 6 months and 56.9% of the patients aged < 3 months. A total of 38 patients were admitted to intensive care unit (ICU), 13 patients died, and the mortality of severe pertussis was 34.2%, with patients younger than 6 weeks accounting for 76.9% of the deaths. On the multivariate analysis, the independent risk factors for death were WBC > 70.0 × 109/L (odds ratio [OR], 230.66; 95% confidence interval [CI], 5.16-10,319.09 P = 0.005) and pulmonary hypertension (PH) (OR 323.29; 95% CI 16.01-6529.42; P < 0.001). CONCLUSION: Severe pertussis mainly occurred in children aged < 3 months. The mortality of severe pertussis was 34.2%, with patients younger than 6 weeks accounting for the majority of the deaths. We recommend the first dose of diphtheria-tetanus-pertussis (DTP) should be advanced to the age of 2 months or even 6 weeks. The presence of a WBC > 70.0 × 109/L and PH were the prognostic independent variables associated with death.

Ras inhibitor CAPRI enables neutrophil-like cells to chemotax through a higher-concentration range of gradients.

Neutrophils sense and migrate through an enormous range of chemoattractant gradients through adaptation. Here, we reveal that in human neutrophils, calcium-promoted Ras inactivator (CAPRI) locally controls the GPCR-stimulated Ras adaptation. Human neutrophils lacking CAPRI (caprikd ) exhibit chemoattractant-induced, nonadaptive Ras activation; significantly increased phosphorylation of AKT, GSK-3α/3ß, and cofilin; and excessive actin polymerization. caprikd cells display defective chemotaxis in response to high-concentration gradients but exhibit improved chemotaxis in low- or subsensitive-concentration gradients of various chemoattractants, as a result of their enhanced sensitivity. Taken together, our data reveal that CAPRI controls GPCR activation-mediated Ras adaptation and lowers the sensitivity of human neutrophils so that they are able to chemotax through a higher-concentration range of chemoattractant gradients.

Quantitative Assessment of Abiotic Stress on the Main Functional Phytochemicals and Antioxidant Capacity of Wheatgrass at Different Seedling Age.

The wheat seedlings of 6 days old were daily subjected to ultraviolet irradiation (irradiating for 5, 10, 20, 40, and 60 min/day, respectively), Polyethylene glycol 6000 (5, 10, 15, 20, 25% in 1/2 Hoagland solution, respectively), and salinity solution (10, 25, 50, 100, 200 mM in 1/2 Hoagland solution, respectively), while the control group (CK) was supplied only with the Hoagland solution. The wheatgrass was harvested regularly seven times and the total soluble polysaccharides, ascorbic acid, chlorophyll, total polyphenol, total triterpene, total flavonoid, and proanthocyanins content were tested. The antioxidant capacity was evaluated through 2,2'-azino-bis (3-ethylbenzthia-zoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging ability, and ferric ion reducing power. Technique for order preference by similarity to ideal solution (TOPSIS) mathematical model was adopted to comprehensively assess the functional phytochemicals of the different treatments. The results showed that the accumulation patterns of phytochemicals under abiotic stress were complex and not always upregulated or downregulated. The antioxidant activity and functional phytochemicals content of wheatgrass were significantly affected by both the stress treatments and seedling age, while the latter affected the chemicals more efficiently. The top five highest functional phytochemicals were observed in the 200 mM NaCl treated group on the 21st and 27th day, 25% PEG treated group on the 24th day, 200 mM NaCl treated group on the 24th day, and the group of 40 min/day ultraviolet exposure on 27th day.

How Phagocytes Acquired the Capability of Hunting and Removing Pathogens From a Human Body: Lessons Learned From Chemotaxis and Phagocytosis of Dictyostelium discoideum (Review).

How phagocytes find invading microorganisms and eliminate pathogenic ones from human bodies is a fundamental question in the study of infectious diseases. About 2.5 billion years ago, eukaryotic unicellular organisms-protozoans-appeared and started to interact with various bacteria. Less than 1 billion years ago, multicellular animals-metazoans-appeared and acquired the ability to distinguish self from non-self and to remove harmful organisms from their bodies. Since then, animals have developed innate immunity in which specialized white-blood cells phagocytes- patrol the body to kill pathogenic bacteria. The social amoebae Dictyostelium discoideum are prototypical phagocytes that chase various bacteria via chemotaxis and consume them as food via phagocytosis. Studies of this genetically amendable organism have revealed evolutionarily conserved mechanisms underlying chemotaxis and phagocytosis and shed light on studies of phagocytes in mammals. In this review, we briefly summarize important studies that contribute to our current understanding of how phagocytes effectively find and kill pathogens via chemotaxis and phagocytosis.