Chest computed tomography analysis of lung sparing morphology: differentiation of COVID-19 pneumonia from influenza pneumonia and bacterial pneumonia using the arched bridge and vacuole signs.

INTRODUCTION: This study evaluated the arched bridge and vacuole signs, which constitute morphological patterns of lung sparing in coronavirus disease 2019 (COVID-19), then examined whether these signs could be used to differentiate COVID-19 pneumonia from influenza pneumonia or bacterial pneumonia. METHODS: In total, 187 patients were included: 66 patients with COVID-19 pneumonia, 50 patients with influenza pneumonia and positive computed tomography findings, and 71 patients with bacterial pneumonia and positive computed tomography findings. Images were independently reviewed by two radiologists. The incidences of the arched bridge sign and/or vacuole sign were compared among the COVID-19 pneumonia, influenza pneumonia, and bacterial pneumonia groups. RESULTS: The arched bridge sign was much more common among patients with COVID-19 pneumonia (42/66, 63.6%) than among patients with influenza pneumonia (4/50, 8.0%; P<0.001) or bacterial pneumonia (4/71, 5.6%; P<0.001). The vacuole sign was also much more common among patients with COVID-19 pneumonia (14/66, 21.2%) than among patients with influenza pneumonia (1/50, 2.0%; P=0.005) or bacterial pneumonia (1/71, 1.4%; P<0.001). The signs occurred together in 11 (16.7%) patients with COVID-19 pneumonia, but they did not occur together in patients with influenza pneumonia or bacterial pneumonia. The arched bridge and vacuole signs predicted COVID-19 pneumonia with respective specificities of 93.4% and 98.4%. CONCLUSION: The arched bridge and vacuole signs are much more common in patients with COVID-19 pneumonia and can help differentiate COVID-19 pneumonia from influenza and bacterial pneumonia.

Atrial fibrillation patients who sustained warfarin-associated intracerebral haemorrhage have poor neurological outcomes: results from a matched case series.

INTRODUCTION: Coagulopathy-associated intracerebral haemorrhage has become increasingly common because of the rising demand in the ageing population for anticoagulation for atrial fibrillation. This study compared the clinical features and neurological outcomes of intracerebral haemorrhage in patients with atrial fibrillation who were prescribed warfarin with those who were not. METHODS: This was a retrospective matched case series of patients with intracerebral haemorrhage from three tertiary hospitals in Hong Kong from 1 January 2006 to 31 December 2011. Patients who developed intracerebral haemorrhage and who were prescribed warfarin for atrial fibrillation (ICH-W group) were compared with those with intracerebral haemorrhage and not prescribed warfarin (ICH-C group); they were matched for age and gender in 1:1 ratio. Clinical features and neurological outcomes were compared, and the impact of coagulopathy on haematoma size was also studied. RESULTS: We identified 114 patients in the ICH-W group with a mean age of 75 years. Both ICH-W and ICH-C groups had a median intracerebral haemorrhage score of 2. There was a non-statistically significant trend of higher intracerebral haemorrhage volume in the ICH-W group (12.9 mL vs 10.5 mL). The median modified Rankin Scale and the proportion with good recovery (modified Rankin Scale score ≤3) at 6 months were comparable. Nonetheless, ICH-W patients had higher hospital mortality (51.8% vs 36.0%; P=0.02) and 6-month mortality (60.5% vs 43.0%; P=0.01) than ICH-C patients. Overall, 60% of ICH-W patients had their admission international normalised ratio within the therapeutic range during intracerebral haemorrhage, and 14% had a subtherapeutic admission international normalised ratio. International normalised ratio at admission was not associated with intracerebral haemorrhage volume or neurological outcome. CONCLUSION: Warfarin-associated intracerebral haemorrhage in patients with atrial fibrillation carried a higher stroke mortality than the non-warfarinised patients.

Relative contribution of individual oxidized components in ox-LDL to inhibition on endothelium-dependent relaxation in rat aorta.

BACKGROUND AND AIM: Oxidized low-density lipoprotein (ox-LDL) causes atherosclerosis and endothelial dysfunction. No study up to the present date has examined the relative contribution of all the oxidized components in ox-LDL to inhibition on vascular function. Our aim was to investigate the effects of individual oxidized components at concentrations similar to those in ox-LDL on the impairment of endothelium-dependent relaxation in rat aorta. METHODS AND RESULTS: Rat thoracic aorta was pre-treated with lysophosphatidylcholine (LPC), cholesterol oxidized products (COPs), oxidized linoleic acid (ox-18:2) and oxidized linolenic acid (ox-18:3) at concentrations similar to those in human ox-LDL. Ox-LDL as a whole caused 61% inhibition while LPC, COPs and ox-18:2 at concentrations similar to those in ox-LDL caused 12%, 24% and 19% inhibition, respectively, on endothelium-dependent relaxation, suggesting that COPs produced the most adverse effect followed by ox-18:2 and LPC in an additional way. Three COPs including 7-ketocholesterol, 7α-hydroxycholesterol and 7ß-hydroxycholesterol showed inhibition on endothelium-dependent relaxation with E(max) being reduced to 79-87% compared with the control E(max) (95%). At Western blot analysis phosphorylation of eNOS at Ser1177 site and total eNOS were not altered by ox-LDL treatment, indicating that ox-LDL did not affect nitric oxide (NO) synthesis capacity. Ox-LDL might react directly with NO and lower NO bioavailability. CONCLUSION: The present study demonstrated the relative contribution of individual oxidized components in ox-LDL in the inhibition of endothelium-dependent relaxation in rat aorta. This inhibitory effect could be caused by the reduction of NO bioactivity.

Comparison of high-flow nasal cannula versus oxygen face mask for environmental bacterial contamination in critically ill pneumonia patients: a randomized controlled crossover trial.

Whereas high-flow nasal cannula use is gaining prevalence, its high gas flow raises concerns about aerosolization of infectious particles and spread of infection. This randomized controlled crossover non-inferiority trial (N = 20) evaluated the degree of environmental contamination by viable bacteria associated with the use of high-flow nasal cannula compared with conventional oxygen mask for critically ill patients with Gram-negative pneumonia. The results show that high-flow nasal cannula use was not associated with increased air or contact surface contamination by either Gram-negative bacteria or total bacteria, suggesting that additional infection control measures are not required.

Multicolor "DiOlistic" labeling of the nervous system using lipophilic dye combinations.

We describe a technique for rapid labeling of a large number of cells in the nervous system with many different colors. By delivering lipophilic dye-coated particles to neuronal preparations with a "gene gun," individual neurons and glia whose membranes are contacted by the particles are quickly labeled. Using particles that are each coated with different combinations of various lipophilic dyes, many cells within a complex neuronal network can be simultaneously labeled with a wide variety of colors. This approach is most effective in living material but also labels previously fixed material. In living material, labeled neurons continue to show normal synaptic responses and undergo dendritic remodeling. This technique is thus useful for studying structural plasticity of neuronal circuits in living preparations. In addition, the Golgi-like labeling of neurons with many different colors provides a novel way to study neuronal connectivity.

Changing specificity of neurotransmitter regulation of rapid dendritic remodeling during synaptogenesis.

Wiring the developing nervous system requires appropriate contact between presynaptic axons and postsynaptic dendrites. Rapid movements of filopodia-like structures on immature dendrites are thought to facilitate initial synaptogenic contact with axons. Here we show that not only can different forms of neurotransmission regulate dendritic filopodial motility, but they do so in a developmentally regulated manner, suggestive of a specific relationship between the action of a neurotransmitter and the corresponding type of synapse being formed.

eps15, a novel tyrosine kinase substrate, exhibits transforming activity.

An expression cloning method which allows direct isolation of cDNAs encoding substrates for tyrosine kinases was applied to the study of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway. A previously undescribed cDNA was isolated and designated eps15. The structural features of the predicted eps15 gene product allow its subdivision into three domains. Domain I contains signatures of a regulatory domain, including a candidate tyrosine phosphorylation site and EF-hand-type calcium-binding domains. Domain II presents the characteristic heptad repeats of coiled-coil rod-like proteins, and domain III displays a repeated aspartic acid-proline-phenylalanine motif similar to a consensus sequence of several methylases. Antibodies specific for the eps15 gene product recognize two proteins: a major species of 142 kDa and a minor component of 155 kDa, both of which are phosphorylated on tyrosine following EGFR activation by EGF in vivo. EGFR is also able to directly phosphorylate the eps15 product in vitro. In addition, phosphorylation of the eps15 gene product in vivo is relatively receptor specific, since the erbB-2 kinase phosphorylates it very inefficiently. Finally, overexpression of eps15 is sufficient to transform NIH 3T3 cells, thus suggesting that the eps15 gene product is involved in the regulation of mitogenic signals.

Constitutive phosphorylation of eps8 in tumor cell lines: relevance to malignant transformation.

eps8, a recently identified tyrosine kinase substrate, has been shown to augment epidermal growth factor (EGF) responsiveness, implicating it in EGF receptor (EGFR)-mediated mitogenic signaling. We investigated the status of eps8 phosphorylation in normal and transformed cells and the role of eps8 in transformation. In NIH 3T3 cells overexpressing EGFR (NIH-EGFR), eps8 becomes rapidly phosphorylated upon EGF stimulation. At receptor-saturating doses of EGF, approximately 30% of the eps8 pool is tyrosine phosphorylated. Under physiological conditions of activation (i.e., at low receptor occupancy), corresponding to the 50% effective dose of EGF for mitogenesis, approximately 3 to 4% of the eps8 contains phosphotyrosine. In human tumor cell lines, we detected constitutive tyrosine phosphorylation of eps8, with a stoichiometry (approximately 5%) similar to that associated with potent mitogenic response in NIH-EGFR cells. Overexpression of eps8 was able to transform NIH 3T3 cells under limiting conditions of activation of the EGFR pathway. Concomitant tyrosine phosphorylation of eps8 and shc, but not of rasGAP, phospholipase C-gamma, and eps15, was frequently detected in tumor cells. This suggested that eps8 and shc might be part of a pathway which is preferentially selected in some tumors. Cooperation between these two transducers was further indicated by the finding of their in vivo association. This association was, at least in part, dependent on recognition of shc by the SH3 domain of eps8. Our results indicate that eps8 is physiologically part of the EGFR-activated signaling and that its alterations can contribute to the malignant phenotype.

An epidermal growth factor receptor/ret chimera generates mitogenic and transforming signals: evidence for a ret-specific signaling pathway.

A chimeric expression vector which encoded for a molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) and the intracellular domain of the ret kinase (EGFR/ret chimera) was generated. Upon ectopic expression in mammalian cells, the EGFR/ret chimera was correctly synthesized and transported to the cell surface, where it was shown capable of binding EGF and transducing an EGF-dependent signal intracellularly. Thus, the EGFR/ret chimera allows us to study the biological effects and biochemical activities of the ret kinase under controlled conditions of activation. Comparative analysis of the growth-promoting activity of the EGFR/ret chimera expressed in fibroblastic or hematopoietic cells revealed a biological phenotype clearly distinguishable from that of the EGFR, indicating that the two kinases couple with mitogenic pathways which are different to some extent. Analysis of biochemical pathways implicated in the transduction of mitogenic signals also evidenced significant differences between the ret kinase and other receptor tyrosine kinases. Thus, the sum of our results indicates the existence of a ret-specific pathway of mitogenic signaling.

Rapid dendritic movements during synapse formation and rearrangement.

Major technical advances in the imaging of live cells have led to a recent flurry of studies demonstrating how dendrites remodel dynamically during development. Taken together with our current understanding of axonal development, these studies help provide a more unified picture of how neural circuits might be formed altered or maintained throughout life.

A randomized controlled trial of 2 protocols for weaning cardiac surgical patients receiving adaptive support ventilation.

PURPOSE: This study aims to compare the effectiveness of weaning with adaptive support ventilation (ASV) incorporating progressively reduced or constant target minute ventilation in the protocol in postoperative care after cardiac surgery. MATERIAL AND METHODS: A randomized controlled unblinded study of 52 patients after elective coronary artery bypass surgery was carried out to determine whether a protocol incorporating a decremental target minute ventilation (DTMV) results in more rapid weaning of patients ventilated in ASV mode compared to a protocol incorporating a constant target minute ventilation. RESULTS: Median duration of mechanical ventilation (145 vs 309 minutes; P = .001) and intubation (225 vs 423 minutes; P = .005) were significantly shorter in the DTMV group. There was no difference in adverse effects (42% vs 46%) or mortality (0% vs 0%) between the 2 groups. CONCLUSIONS: Use of a DTMV protocol for postoperative ventilation of cardiac surgical patients in ASV mode results in a shorter duration of ventilation and intubation without evidence of increased risk of adverse effects.

Isolation and characterization of e3B1, an eps8 binding protein that regulates cell growth.

Eps8, a substrate of receptor tyrosine kinases, is an SH3 domain containing protein that plays an important role in mitogenic signaling. To determine the cellular function of eps8, we used the SH3 domain of eps8 to screen a human fibroblast M426 expression library and identified, a full-length cDNA clone of 3.2 kb. We designated this clone e3B1 for eps8 SH3 domain binding protein 1. Northern analysis revealed that expression of e3B1 mRNA was ubiquitous in human tissues. The e3B1 gene encodes a SH3 domain containing protein. We show that anti-e3B1 antibodies detect three cytosolic protein species of 65, 68 and 72 kDa in cell lysate isolated from asynchronously growing NIH3T3 cells. E3B1 binds to the SH3 domain of eps8 and Abl in vitro. We also demonstrated that e3B1 associates with eps8 in vivo. Phosphatase digestion and phosphoamino acid analysis revealed that p65e3B1 is a phosphoserine containing protein and p72e3B1 and p68e3B1 are hyperserine-phosphorylated form of p65e3B1. We further determined that the p65e3B1 was the most abundant in serum-starved NIH/EGFR cells. Time course studies initiated by the addition of epidermal growth factor (EGF) revealed that the p72e3B1 started to accumulate at 4 h, peaked at 8 h and remained high until 24 h. Finally, we demonstrate that NIH/EGFR fibroblasts overexpressing e3B1 grow more slowly relative to matched controls.

The ezrin-like family of tyrosine kinase substrates: receptor-specific pattern of tyrosine phosphorylation and relationship to malignant transformation.

A method for the isolation of tyrosine kinases substrates was developed. The method takes advantage of immuno-affinity purification of an entire set of proteins phosphorylated by tyrosine kinases, followed by generation of antisera against the purified protein pool and immunological screening of bacterial expression libraries with these antisera. By applying this methodology to the study of the phosphorylation events triggered by activation of the epidermal growth factor receptors, we have isolated several cDNAs encoding novel putative tyrosine kinase substrates. One of these cDNAs encodes radixin, a protein belonging to the band 4.1 family of proteins and highly related to ezrin and moesin. We demonstrated that, despite a high degree of relatedness, these three proteins exhibit a distinct receptor-specific pattern of phosphorylation, raising the possibility that they might mediate receptor-specific cellular changes. In addition the generation of antibodies specific for either radixin, ezrin or moesin allowed us to show that a previously described tumor transplantation antigen is indeed ezrin, thus implicating this protein in the determination of the biological phenotype of certain tumors.

The high transforming potency of erbB-2 and ret is associated with phosphorylation of paxillin and a 23 kDa protein.

Two-dimensional gel maps of proteins phosphorylated by the epidermal growth factor receptor (EGFR) and erbB-2 kinases were obtained, to investigate the molecular basis of the different biological properties of these two molecules. Several proteins were phosphorylated by EGFR or erbB-2 with different stoichiometry. Differences were either quantitative or qualitative. In NIH3T3 cells, erbB-2 is 100-fold more transforming than EGFR. In the same cell line several proteins were preferentially phosphorylated by erbB-2, as compared to EGFR. To identify which of these substrates might be directly involved in mitogenic signaling, we obtained two-dimensional maps of proteins phosphorylated on tyrosine by EGFR/ret and an EGFR/erbB-2TK chimeric receptors. Both these chimerae behaved indistinguishably from erbB-2 in a number of bioassays and potently transformed NIH3T3 cells. Paxillin and a 23 kDa substrate were invariably phosphorylated to higher stoichiometry whenever potent mitogenic and transforming signals were activated. We propose that paxillin and the 23 kDa substrate are important elements in the erbB-2 and ret-activated mitogenic and transforming signaling.

RN-tre specifically binds to the SH3 domain of eps8 with high affinity and confers growth advantage to NIH3T3 upon carboxy-terminal truncation.

We isolated a cDNA encoding a protein, RN-tre, which shows homology to the N-terminus of the tre oncogene product and has SH3-binding ability as well as an evolutionarily conserved domain, termed TrH, with protein-binding ability in vitro. In the present study, we identify the product of the RN-tre gene as a 97-100 kDa protein. We demonstrate stable association in vivo and in vitro between RN-tre and eps8, mediated by the SH3 domain of the latter. In vitro, RN-tre displayed remarkable preference for binding to the eps8-SH3, as compared to eight other SH3s. The Kd for the in vitro interaction between RN-tre and eps8-SH3 was between 10(-8) and 10(-7) M. A role for RN-tre in cell proliferation was suggested by the finding that a C-terminal truncated mutant was able to confer proliferative advantage and reduced serum-requirement to NIH3T3 fibroblasts. Finally, comparison of the structure and biological activities of RN-tre and of the tre oncogene product, provided insight into the mechanism of oncogenic activation of tre.

Quantitative analysis of Grb2-Sos1 interaction: the N-terminal SH3 domain of Grb2 mediates affinity.

Grb2 is an adaptor protein that links receptor and cytoplasmic tyrosine kinases to the Ras signalling pathway by binding the Ras-specific guanine nucleotide exchange factor, Sos1, through its SH3 domains. The Grb2-SH3 domain binding has been localized to the carboxy-terminal two hundred amino acids of Sos1 (Sos1-c). By using real time biospecific interaction analysis (BIAcore), we studied the kinetic parameters and binding affinity of the Grb2-Sos1-c interaction. The binding of Grb2 to Sos1-c is a high affinity interaction with a moderate association rate (9.45 x 10(4) per M per s), a slow dissociation rate (13.8 x 10(-5) s), and an affinity constant of 1.48 nM. BIAcore measurements on isolated N-terminal and C-terminal SH3 domains (NSH3 and CSH3) further indicate that the high affinity Grb2-Sos1-c interaction is primarily mediated through the NSH3 domain (Kd = 1.68 nM). The CSH3 domain shows substantially reduced binding to Sos1-c in these measurements. Inhibition studies with BIAcore using proline rich peptides derived from the C-terminus of Sos1 show that there is a single major binding site for Grb2 in Sos1. This binding site is contained within the peptide N20, which corresponds to amino acids 1143-1162 of Sos1. This peptide completely blocks the Grb2-Sos1-c and NSH3-Sos1-c interactions with IC50 values of 8 microM and 4 microM respectively. The discrete interaction between the NSH3 domain and the N20 peptide may be amenable for drug discovery through screening or peptidomimetic approaches.

Expression of the receptor tyrosine kinase substrate genes eps8 and eps15 during mouse development.

Receptor tyrosine kinases (RTKs) control proliferation and differentiation through their ability to bind and/or phosphorylate intracellular substrates. The repertoire of substrates recruited by different RTK is largely overlapping. It is not clear, therefore, how a cell distinguishes among signals originating from different RTKs. One possibility is that selective availability of substrates participates in the regulation of this process. To gain insight into this issue, we studied the expression pattern, during mouse embryogenesis, of the eps8 and eps15 genes, which encode two recently identified RTK substrates. Both genes are expressed from E 10 in a restricted fashion. eps8 is first expressed in frontonasal neural crest-derived cells, in the mesenchyme of branchial arches and in the liver primordium. At E 12.5-E 14, eps8 is additionally expressed in the central nervous system (CNS) in a regional restricted pattern at the met-mesencephalic transition area and in the developing submandibular salivary glands. eps15 is expressed at E 10 in the liver primordium, in the spinal ganglia and in the encephalic ganglia derived from the hindbrain neural crest. In addition, at E 12.5-E 14, eps15 is expressed, along all the CNS, in the ventricular zone where undifferentiated neuroblasts are located. The regional pattern of developmental expression of these two substrates sharply contrasts with their ubiquitous expression in adults, raising the possibility that their expression during embryogenesis is linked to selective proliferative and/or differentiative responses of specific neuroectodermal regions and body organs.

Direct binding of eps8 to the juxtamembrane domain of EGFR is phosphotyrosine- and SH2-independent.

Several signal transducers bind through their SH2 domains to phosphotyrosine-containing motifs present in receptor tyrosine kinases (RTKs). However, the juxtamembrane regions of the epidermal growth factor receptor (EGFR) and of the related erbB-2 protein, while important in mitogenic signaling, lack demonstrable tyrosine phosphorylation sites, suggesting that other modalities of receptor-transducer interactions exist. A candidate for investigating this type of association is p97eps8, a recently described substrate for RTKs. p97eps8 is phosphorylated by several RTKs, associates with EGFR in vivo and, upon overexpression, enhances the transduction of EGFR-mediated mitogenic signals. Here we report that eps8 binds directly to the juxtamembrane region of EGFR through a domain that does not bear resemblance to SH2 domains and by a mechanism that does not require the presence of phosphotyrosine residues. Thus, the physical association between EGFR and eps8 represents a novel interaction between RTKs and their substrates.