Astrocyte-secreted lipocalin-2 elicits bioenergetic failure-induced neuronal death that is causally related to high fatality in a mouse model of hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neurological complication arising from acute liver failure with poor prognosis and high mortality; the underlying cellular mechanisms are still wanting. We previously found that neuronal death caused by mitochondrial dysfunction in rostral ventrolateral medulla (RVLM), which leads to baroreflex dysregulation, is related to high fatality in an animal model of HE. Lipocalin-2 (Lcn2) is a secreted glycoprotein mainly released by astrocytes in the brain. We noted the presence of Lcn2 receptor (Lcn2R) in RVLM neurons and a parallel increase of Lcn2 gene in astrocytes purified from RVLM during experimental HE. Therefore, our guiding hypothesis is that Lcn2 secreted by reactive astrocytes in RVLM may underpin high fatality during HE by eliciting bioenergetic failure-induced neuronal death in this neural substrate. In this study, we first established the role of astrocyte-secreted Lcn2 in a liver toxin model of HE induced by azoxymethane (100 µg/g, ip) in C57BL/6 mice, followed by mechanistic studies in primary astrocyte and neuron cultures prepared from postnatal day 1 mouse pups. In animal study, immunoneutralization of Lcn2 reduced apoptotic cell death in RVLM, reversed defunct baroreflex-mediated vasomotor tone and prolonged survival during experimental HE. In our primary cell culture experiments, Lcn2 produced by cultured astrocytes and released into the astrocyte-conditioned medium significantly reduced cell viability of cultured neurons. Recombinant Lcn2 protein reduced cell viability, mitochondrial ATP (mitoATP) production, and pyruvate dehydrogenase (PDH) activity but enhanced the expression of pyruvate dehydrogenase kinase (PDK) 1, PDK3 and phospho-PDHA1 (inactive PDH) through MAPK/ERK pathway in cultured neurons, with all cellular actions reversed by Lcn2R knockdown. Our results suggest that astrocyte-secreted Lcn2 upregulates PDKs through MAPK/ERK pathway, which leads to reduced PDH activity and mitoATP production; the reinforced neuronal death in RVLM is causally related to baroreflex dysregulation that underlies high fatality associated with HE.

DUSP8 induces TGF-ß-stimulated IL-9 transcription and Th9-mediated allergic inflammation by promoting nuclear export of Pur-α.

Dual-specificity phosphatase 8 (DUSP8) is a MAPK phosphatase that dephosphorylates and inactivates the kinase JNK. DUSP8 is highly expressed in T cells; however, the in vivo role of DUSP8 in T cells remains unclear. Using T cell-specific Dusp8 conditional KO (T-Dusp8 cKO) mice, mass spectrometry analysis, ChIP-Seq, and immune analysis, we found that DUSP8 interacted with Pur-α, stimulated interleukin-9 (IL-9) gene expression, and promoted Th9 differentiation. Mechanistically, DUSP8 dephosphorylated the transcriptional repressor Pur-α upon TGF-ß signaling, leading to the nuclear export of Pur-α and subsequent IL-9 transcriptional activation. Furthermore, Il-9 mRNA levels were induced in Pur-α-deficient T cells. In addition, T-Dusp8-cKO mice displayed reduction of IL-9 and Th9-mediated immune responses in the allergic asthma model. Reduction of Il-9 mRNA levels in T cells and allergic responses of T-Dusp8-cKO mice was reversed by Pur-α knockout. Remarkably, DUSP8 protein levels and the DUSP8-Pur-α interaction were indeed increased in the cytoplasm of T cells from people with asthma and patients with atopic dermatitis. Collectively, DUSP8 induces TGF-ß-stimulated IL-9 transcription and Th9-induced allergic responses by inhibiting the nuclear translocation of the transcriptional repressor Pur-α. DUSP8 may be a T-cell biomarker and therapeutic target for asthma and atopic dermatitis.

Dengue virus serotype did not contribute to clinical severity or mortality in Taiwan's largest dengue outbreak in 2015.

BACKGROUND: Dengue virus serotype 2 (DENV-2) was the major serotype in the 2015 dengue outbreak in Taiwan, while DENV-1 and DENV-3 were dominant between 2005 and 2014. We aimed to investigate whether DENV-2 contributed to disease severity and mortality in the outbreak in Kaohsiung city, Taiwan. METHODS: We collected serum samples from dengue patients to detect the presence of DENV and determine the serotypes by using quantitative reverse transcription-polymerase chain reaction. Our cohorts comprised 105 DENV-1-infected cases and 1,550 DENV-2-infected cases. Demographic data, DENV serotype, and comorbidities were covariates for univariate and multivariate analyses to explore the association with severity and mortality. RESULTS: The results suggested that DENV-1 persisted and circulated, while DENV-2 was dominant during the dengue outbreak that occurred between September and December 2015. However, DENV-2 did not directly contribute to either severity or mortality. Aged patients and patients with diabetes mellitus (DM) or moderate to severe chronic kidney disease (CKD) had a higher risk of developing severe dengue. The mortality of dengue patients was related to a higher Charlson comorbidity index score and severe dengue. Among DENV-2-infected patients and older patients, preexisting anti-dengue IgG, DM, and moderate to severe CKD were associated with severe dengue. Moreover, female sex and severe dengue were associated with a significantly higher risk of death. CONCLUSIONS: Our findings highlight the importance of timely serological testing in elderly patients to identify potential secondary infections and focus on the meticulous management of elderly patients with DM or moderate to severe CKD to reduce dengue-related death.

Modified gefitinib conjugated Fe3O4 NPs for improved delivery of chemo drugs following an image-guided mechanistic study of inner vs. outer tumor uptake for the treatment of non-small cell lung cancer.

Gefitinib (GEF) is an FDA-approved anti-cancer drug for the first-line treatment of patients with metastatic non-small cell lung cancer (NSCLC). However, the efficacy of anticancer drugs is limited due to their non-specificity, lower accumulation at target sites, and systemic toxicity. Herein, we successfully synthesized a modified GEF (mGEF) drug and conjugated to Iron oxide nanoparticles (Fe3O4 NPs) for the treatment of NSCLC via magnetic resonance (MR) image-guided drug delivery. A traditional EDC coupling pathway uses mGEF to directly conjugate to Fe3O4 NPs to overcom the drug leakage issues. As a result, we found in vitro drug delivery on mGEF- Fe3O4 NPs exhibits excellent anticancer effects towards the PC9 cells selectively, with an estimated IC 50 value of 2.0 µM. Additionally, in vivo MRI and PET results demonstrate that the NPs could accumulate in tumor-specific regions with localized cell growth inhibition. Results also revealed that outer tumor region exhibiting a stronger contrast than the tinner tumor region which may due necrosis in inner tumor region. In vivo biodistribution further confirms Fe3O4 NPs are more biocompatible and are excreated after the treatment. Overall, we believe that this current strategy of drug modification combined with chemical conjugation on magnetic NPs will lead to improved cancer chemotherapy as well as understanding the tumor microenvironments for better therapeutic outcomes.

Short-chain fatty acids ameliorate allergic airway inflammation via sequential induction of PMN-MDSCs and Treg cells.

Background: Reinforcement of the immune-regulatory pathway is a feasible strategy for prevention and therapy of allergic asthma. The short-chain fatty acids (SCFAs) acetate, propionate, and butyrate are pleiotropic microbial fermentation products known to induce regulatory T (Treg) cells and exert an immune-regulatory effect. The cellular mechanism underlying SCFA immune regulation in asthma is not fully understood. Objective: We investigated the role of myeloid-derived suppressor cells (MDSCs) and Treg cells, the immune-regulatory cells of innate and adaptive origin, respectively, in SCFA-elicited protection against allergic airway inflammation. Methods: BALB/c mice were given SCFA-containing drinking water before being rendered asthmatic in response to ovalbumen. When indicated, mice were given a GR1-depleting antibody to investigate the function of MDSCs in allergic inflammation of the airways. MDSCs were sorted to examine their immunosuppressive function and interaction with T cells. Results: The mice receiving SCFAs developed less severe asthma that was accompanied by expansion of PMN-MDSCs and Treg cells. Mice depleted of PMN-MDSCs exhibited aggravated asthma, and the protective effect of SCFAs was abrogated after PMN-MDSC depletion. SCFAs were able to directly induce T-cell differentiation toward Treg cells. Additionally, we found that PMN-MDSCs enhanced Treg cell expansion in a cell contact-dependent manner. Whilst membrane-bound TGF-ß has been shown to induce Treg cell differentiation, we found that MDSCs upregulated surface expression of TGF-ß after coculture with T-cells and that MDSC-induced Treg cell differentiation was partially inhibited by TGF-ß blockage. Conclusions: Although previous studies revealed Treg cells as the effector mechanism of SCFA immune regulation, we found that SCFAs ameliorate allergic airway inflammation by relaying immune regulation, with sequential induction of PMN-MDSCs and Treg cells.

Epidemiology and analysis of SARS-CoV-2 Omicron subvariants BA.1 and 2 in Taiwan.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first detected in October 2021, possessed many mutations compared to previous variants. We aimed to identify and analyze SARS-CoV-2 Omicron subvariants among coronavirus disease 2019 (COVID-19) patients between January 2022 and September 2022 in Taiwan. The results revealed that BA.2.3.7, featuring K97E and G1251V in the spike protein compared with BA.2, emerged in March 2022 and persistently dominated between April 2022 and August 2022, resulting in the largest COVID-19 outbreak since 2020. The accumulation of amino acid (AA) variations, mainly AA substitution, in the spike protein was accompanied by increasing severity in Omicron-related COVID-19 between April 2022 and January 2023. Older patients were more likely to have severe COVID-19, and comorbidity was a risk factor for COVID-19-related mortality. The accumulated case fatality rate (CFR) dropped drastically after Omicron variants, mainly BA.2.3.7, entered Taiwan after April 2022, and the CFR was 0.16% in Taiwan, which was lower than that worldwide (0.31%) between April 2021 and January 2023. The relatively low CFR in Omicron-related COVID-19 patients can be attributed to adjustments to public health policies, promotion of vaccination programs, effective antiviral drugs, and the lower severity of the Omicron variant.

Corrigendum: Identification and analysis of SARS-CoV-2 alpha variants in the largest Taiwan COVID-19 outbreak in 2021.

[This corrects the article DOI: 10.3389/fmed.2022.869818.].

Comparing the performance of dengue virus IgG and IgG-capture enzyme-linked immunosorbent assays in seroprevalence study.

BACKGROUND: Dengue virus (DENV) is the leading cause of arboviral diseases in humans worldwide. Currently Dengvaxia, the first dengue vaccine licensed in 20 countries, was recommended for DENV seropositive individuals aged 9-45 years. Studying dengue seroprevalence can improve our understanding of the epidemiology and transmission dynamics of DENV, and facilitate future intervention strategies and assessment of vaccine efficacy. Several DENV envelope protein-based serological tests including IgG and IgG-capture enzyme-linked immunosorbent assays (ELISAs) have been employed in seroprevalence studies. Previously DENV IgG-capture ELISA was reported to distinguish primary and secondary DENV infections during early convalescence, however, its performance over time and in seroprevalence study remains understudied. METHODS: In this study, we used well-documented neutralization test- or reverse-transcription-polymerase-chain reaction-confirmed serum/plasma samples including DENV-naïve, primary and secondary DENV, primary West Nile virus, primary Zika virus, and Zika with previous DENV infection panels to compare the performance of three ELISAs. RESULTS: The sensitivity of the InBios IgG ELISA was higher than that of InBios IgG-capture and SD IgG-capture ELISAs. The sensitivity of IgG-capture ELISAs was higher for secondary than primary DENV infection panel. Within the secondary DENV infection panel, the sensitivity of InBios IgG-capture ELISA decreased from 77.8% at < 6 months to 41.7% at 1-1.5 years, 28.6% at 2-15 years and 0% at > 20 years (p < 0.001, Cochran-Armitage test for trend), whereas that of IgG ELISA remains 100%. A similar trend was observed for SD IgG-capture ELISA. CONCLUSIONS: Our findings demonstrate higher sensitivity of DENV IgG ELISA than IgG-capture ELISA in seroprevalence study and interpretation of DENV IgG-capture ELISA should take sampling time and primary or secondary DENV infection into consideration.

The identification and phylogenetic analysis of SARS-CoV-2 delta variants in Taiwan.

In Taiwan, coronavirus disease 2019 (COVID-19) involving the delta variant occurred after that involving the alpha variant in 2021. In this study, we aimed to analyze the Delta variant. A total of 318 patients in Taiwan infected with delta variants were identified. The case fatality rate (CFR) of patients infected with delta variants was 0.94% in Taiwan compared with that of those infected with alpha variants (5.95%). The possible reasons for the low CFR might be hybrid immunity due to infection and rapid promotion of the COVID-19 vaccination program during the alpha variant outbreak. We identified three 21J delta variants. Two long gene deletions were detected in these severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) isolates: ORF7aΔ91 in KMUH-8 and SpikeΔ30 in KMUH-9. Protein structure prediction indicates that ORF7aΔ91 results in malfunction of NS7a as an interferon antagonist and that SpikeΔ30 results in a truncated spike protein (N679-A688del), resulting in a lower infection rate compared with the delta variant without these deletions. The impact of these two deletions on SARS-CoV-2-associated pathogenesis deserves further investigation. Delta variants still exist in many regions in the omicron era, and the backbone of the delta variant genome possibly spread worldwide in the form of delta-omicron hybrids (deltacron; e.g., XBC.1 and XAY.2), which casts a potential threat to public health. Our study further highlighted the importance of more understanding of the delta variants.

Cisplatin and Albumin-Based Gold-Cisplatin Nanoparticles Enhance Ablative Radiation Therapy-Induced Antitumor Immunity in Local and Distant Tumor Microenvironment.

PURPOSE: Ablative radiation therapy (RT) is an important strategy to eliminate primary tumor and can potentially induce the abscopal effect. Human serum albumin nanoparticle (NP) was used for controlled release of cisplatin to decrease cisplatin's systemic toxicity, and gold (Au) was added to increase RT-induced immunogenic cell death and potentiate the abscopal antitumor immunity. METHODS AND MATERIALS: The designed albumin-based cisplatin-conjugated AuNPs were administered concurrently with ablative RT. C57BL/6 mice implanted with syngeneic murine Lewis lung carcinoma or murine MB49 tumor models were treated with ablative RT (12 Gy per fraction for 2 fractions, total 24 Gy), cisplatin, or Au-cisplatin NPs. RESULTS: Combining ablative RT with cisplatin or Au-cisplatin NPs both destroyed the primary tumor effectively and elicited immunogenic cell death accompanied by release of danger-associated molecular patterns. This enhanced recruitment of effector tumor-infiltrating immune cells, including natural killer T cells and CD8+ T cells, and elicited an increased percentage of professional antigen-presenting CD11c+ dendritic cells. Transient weight loss, accompanying hepatotoxicity, nephrotoxicity, and hematopoietic suppression, was observed as a systemic adverse event in the cisplatin but not the Au-cisplatin NPs group. Cisplatin and Au-cisplatin NPs both showed equivalent ability to reduce metastatic potential when combined with ablative RT, confirmed by suppressed unirradiated flank tumor growth and decreased metastatic lung tumor burden, which translated to improved survival. Mobilization and abundance of effector tumor-infiltrating immune cells including CD8+ T cells and dendritic cells were observed in the distant lung tumor microenvironment after ablative RT with cisplatin or Au-cisplatin NPs, demonstrating increased antitumor immunotherapeutic activity as an abscopal effect. CONCLUSIONS: Compared with cisplatin, the albumin-based Au-cisplatin NPs exhibited equivalent but no superior antitumor immunotherapeutic activity while reducing systemic adverse events and can be safely administered concurrently with ablative RT. Alternative NP formulations may be designed to further improve anticancer outcomes.

Therapeutic efficacy of cyclin-dependent kinase inhibition in combination with ionizing radiation for lung cancer.

PURPOSE: To evaluate the therapeutic efficacy of cyclin-dependent kinase (CDK) inhibition in combination with ionizing radiation for lung cancer. MATERIALS AND METHODS: Human lung adenocarcinoma (A549) and squamous cell carcinoma (H520) cells were used to evaluate the therapeutic efficacy of CDK inhibition in combination with ionizing radiation in vitro using colony formation assay, γH2AX immunofluorescence staining, western blotting, and cell cycle phase analysis. We also performed in vivo evaluations of ectopic tumor growth. RESULTS: In vitro pretreatment with the CDK inhibitor, seliciclib, before irradiation significantly decreased the survival of A549 and H520 cells in a dose-dependent manner. Although CDK inhibition alone did not increase the intensity of γH2AX foci, its combination with ionizing radiation increased DNA double-strand breaks, as shown by γH2AX immunofluorescence staining and western blotting. The combination of CDK inhibition and ionizing radiation-induced G2/M arrest and increased apoptosis, as evidenced by the increased proportion of cells in G2/M arrest, subG1 apoptotic population, and expression of apoptotic markers (cleaved PARP-1 and cleaved caspase-3). Mechanistic studies showed reduced expression of cyclin A with combined treatment, indicating cell cycle shifting effects. An in vivo xenograft model showed that the combination of CDK inhibition and ionizing radiation delayed xenograft tumor growth, and increased the proportion of cleaved PARP-1- and cleaved caspase-3-positive cells, compared to either treatment alone. CONCLUSIONS: We provide preclinical tumoricidal evidence that the combination of CDK inhibition and ionizing radiation is an efficacious treatment for lung cancer.

MAP4K3/GLK inhibits Treg differentiation by direct phosphorylating IKKß and inducing IKKß-mediated FoxO1 nuclear export and Foxp3 downregulation.

Rationale: GLK (MAP4K3) activates PKCθ-IKKß axis in T-cell activation and induces IL-17A-mediated autoimmune diseases. Attenuation of Treg differentiation and function by GLK could also contribute to autoimmune diseases. Methods: We analyzed the roles of GLK and IKKß in Treg differentiation and function using T-cell-specific GLK transgenic mice and IKKß conditional knockout mice. The mechanism of GLK/IKKß-mediated attenuation of Treg differentiation/function was studied by chromatin-immunoprecipitation, reporter assays, in vitro kinase assays, protein-protein interaction assays, mass spectrometry, confocal microscopy, flow cytometry, and single-cell RNA sequencing (scRNA-seq) analysis. Results: We found that GLK signaling inhibited Foxp3 transcription by blocking the function of the transcription factor FoxO1. Mechanistically, GLK directly phosphorylated and activated IKKß at Ser733 in a PKCθ-independent manner. The phospho-IKKß Ser733 induced FoxO1 Ser319 phosphorylation and nuclear export, leading to Foxp3 downregulation. Consistently, scRNA-seq analyses showed that Foxp3 mRNA levels were inversely correlated with FoxO1 mRNA levels in GLK transgenic CD4+ T cells. Conclusions: GLK-IKKß-FoxO1 signaling axis inhibits Foxp3 transcription, leading to reduction of Treg differentiation and suppressive activity, as well as induction of autoimmune disease.

QTc Interval is Associated with Atrial Fibrillation in Individuals with Metabolic Syndrome Phenotype.

Purpose: Manifestations of metabolic syndrome (MetS) carry risks for atrial fibrillation (AF). The study determined whether any electrocardiographic parameter can reflect increased AF risk in individuals with MetS. Patients and Methods: From our University Hospital database, we examined the presence of AF and its correlation with MetS manifestations, renal function, lipid profiles, and electrocardiographic parameters (P wave duration, PR interval, QRS width, and QTc intervals). Between January 2008 and December 2015, data from 4479 adults (women 41.6% vs men 58.4%) were identified. Results: The overall prevalence of AF was 12.4%, without sex differences (women, 12.8% vs men, 12.1%). Patients with AF were older (age 73.9 ± 11.8 vs non-AF 67 ± 13.5 years), with lower lipid levels (TG, total cholesterol, and LDL-cholesterol, all p < 0.0001), and at lower eGFR level (64.1 ± 30.9 vs non-AF 68.8 ± 41.4 mL/min/1.73m2, p < 0.0001). Besides, sex differences were present in all electrocardiographic parameters (all p < 0.05). Hypertension had the highest odds ratio (1.33; p = 0.026) for AF. Comparing AF to non-AF, the QTc of quartiles was significantly different (p < 0.0089). The shortest and longest QTc quartiles had an increased incidence of AF. Conclusion: AF risk in patients with MetS phenotypes can be reflected by QTc quartiles.

SARS-CoV-2 spike protein enhances MAP4K3/GLK-induced ACE2 stability in COVID-19.

ACE2 on epithelial cells is the SARS-CoV-2 entry receptor. Single-cell RNA-sequencing data derived from two COVID-19 cohorts revealed that MAP4K3/GLK-positive epithelial cells were increased in patients. SARS-CoV-2-induced GLK overexpression in epithelial cells was correlated with COVID-19 severity and vesicle secretion. GLK overexpression induced the epithelial cell-derived exosomes containing ACE2; the GLK-induced exosomes transported ACE2 proteins to recipient cells, facilitating pseudovirus infection. Consistently, ACE2 proteins were increased in the serum exosomes from another COVID-19 cohort. Remarkably, SARS-CoV-2 spike protein-stimulated GLK, and GLK stabilized ACE2 in epithelial cells. Mechanistically, GLK phosphorylated ACE2 at two serine residues (Ser776, Ser783), leading to the dissociation of ACE2 from its E3 ligase UBR4. Reduction in UBR4-induced Lys48-linked ubiquitination at three lysine residues (Lys26, Lys112, Lys114) of ACE2 prevented its degradation. Furthermore, SARS-CoV-2 pseudovirus or live virus infection in humanized ACE2 mice induced GLK and ACE2 protein levels, and ACE2-containing exosomes. Collectively, ACE2 stabilization by SARS-CoV-2-induced MAP4K3/GLK may contribute to the pathogenesis of COVID-19.

Protective role of VEGF/VEGFR2 signaling against high fatality associated with hepatic encephalopathy via sustaining mitochondrial bioenergetics functions.

BACKGROUND: The lack of better understanding of the pathophysiology and cellular mechanisms associated with high mortality seen in hepatic encephalopathy (HE), a neurological complication arising from acute hepatic failure, remains a challenging medical issue. Clinical reports showed that the degree of baroreflex dysregulation is related to the severity of HE. Furthermore, mitochondrial dysfunction in the rostral ventrolateral medulla (RVLM), a key component of the baroreflex loop that maintains blood pressure and sympathetic vasomotor tone, is known to underpin impairment of baroreflex. Realizing that in addition to angiogenic and vasculogenic effects, by acting on its key receptor (VEGFR2), vascular endothelial growth factor (VEGF) elicits neuroprotection via maintenance of mitochondrial function, the guiding hypothesis of the present study is that the VEGF/VEGFR2 signaling plays a protective role against mitochondrial dysfunction in the RVLM to ameliorate baroreflex dysregulation that underpins the high fatality associated with HE. METHODS: Physiological, pharmacological and biochemical investigations were carried out in proof-of-concept experiments using an in vitro model of HE that involved incubation of cultured mouse hippocampal neurons with ammonium chloride. This was followed by corroboratory experiments employing a mouse model of HE, in which adult male C57BL/6 mice and VEGFR2 wild-type and heterozygous mice received an intraperitoneal injection of azoxymethane, a toxin used to induce acute hepatic failure. RESULTS: We demonstrated that VEGFR2 is present in cultured neurons, and observed that whereas recombinant VEGF protein maintained cell viability, gene-knockdown of vegfr2 enhanced the reduction of cell viability in our in vitro model of HE. In our in vivo model of HE, we found that VEGFR2 heterozygous mice exhibited shorter survival rate and time when compared to wild-type mice. In C57BL/6 mice, there was a progressive reduction in VEGFR2 mRNA and protein expression, mitochondrial membrane potential and ATP levels, alongside augmentation of apoptotic cell death in the RVLM, accompanied by a decrease in baroreflex-mediated sympathetic vasomotor tone and hypotension. Immunoneutralization of VEGF exacerbated all those biochemical and physiological events. CONCLUSIONS: Our results suggest that, acting via VEGFR2, the endogenous VEGF plays a protective role against high fatality associated with HE by amelioration of the dysregulated baroreflex-mediated sympathetic vasomotor tone through sustaining mitochondrial bioenergetics functions and eliciting antiapoptotic action in the RVLM.

Identification and Analysis of SARS-CoV-2 Alpha Variants in the Largest Taiwan COVID-19 Outbreak in 2021.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is believed to have originated in Wuhan City, Hubei Province, China, in December 2019. Infection with this highly dangerous human-infecting coronavirus via inhalation of respiratory droplets from SARS-CoV-2 carriers results in coronavirus disease 2019 (COVID-19), which features clinical symptoms such as fever, dry cough, shortness of breath, and life-threatening pneumonia. Several COVID-19 waves arose in Taiwan from January 2020 to March 2021, with the largest outbreak ever having a high case fatality rate (CFR) (5.95%) between May and June 2021. In this study, we identified five 20I (alpha, V1)/B.1.1.7/GR SARS-CoV-2 (KMUH-3 to 7) lineage viruses from COVID-19 patients in this largest COVID-19 outbreak. Sequence placement analysis using the existing SARS-CoV-2 phylogenetic tree revealed that KMUH-3 originated from Japan and that KMUH-4 to KMUH-7 possibly originated via local transmission. Spike mutations M1237I and D614G were identified in KMUH-4 to KMUH-7 as well as in 43 other alpha/B.1.1.7 sequences of 48 alpha/B.1.1.7 sequences deposited in GISAID derived from clinical samples collected in Taiwan between 20 April and July. However, M1237I mutation was not observed in the other 12 alpha/B.1.1.7 sequences collected between 26 December 2020, and 12 April 2021. We conclude that the largest COVID-19 outbreak in Taiwan between May and June 2021 was initially caused by the alpha/B.1.1.7 variant harboring spike D614G + M1237I mutations, which was introduced to Taiwan by China Airlines cargo crew members. To our knowledge, this is the first documented COVID-19 outbreak caused by alpha/B.1.1.7 variant harboring spike M1237I mutation thus far. The largest COVID-19 outbreak in Taiwan resulted in 13,795 cases and 820 deaths, with a high CFR, at 5.95%, accounting for 80.90% of all cases and 96.47% of all deaths during the first 2 years. The high CFR caused by SARS-CoV-2 alpha variants in Taiwan can be attributable to comorbidities and low herd immunity. We also suggest that timely SARS-CoV-2 isolation and/or sequencing are of importance in real-time epidemiological investigations and in epidemic prevention. The impact of D614G + M1237I mutations in the spike gene on the SARS-CoV-2 virus spreading as well as on high CFR remains to be elucidated.

Exploiting exercise electrocardiography to improve early diagnosis of atrial fibrillation with deep learning neural networks.

Atrial fibrillation (AF) is the most common type of sustained arrhythmia. It results from abnormal irregularities in the electrical performance of the atria, and may cause heart thrombosis, stroke, arterial disease, thromboembolism, and heart failure. Prior to the onset of atrial fibrillation, most people experience atrial cardiomyopathy which, if effectively managed, can be prevented from progressing to atrial fibrillation. Electrocardiogram (ECG) can show changes in the heartbeats, and is a common and painless tool to detect heart problems. P-waves in exercise ECGs change more drastically than those in regular ECG, and are more effective in the detection of atrial myocardial diseases. In this paper, we propose a deep learning system to help clinicians to early detect if a patient has atrial enlargement or fibrillation. Firstly, a Convolutional Recurrent Neural Network is employed to locate the P-waves in the patient's exercise ECGs taken in the exercise ECG test process. Relevant parameters are then calculated from the located P-waves. Then a Parallel Bi-directional Long Short-Term Memory Network is applied to analyze the obtained parameters and make a diagnosis for the patient. With our proposed deep learning system, the changes of P-waves collected in different phases in the exercise ECG test can be analyzed simultaneously to get more stable and accurate results. The system can take data of different length as input, and is also applicable to any number of ECG collections. We conduct various experiments to show the effectiveness of our proposed system. We also show that the more ECG data collected in the exercise phase are involved, the more effective our system is in diagnosis of the diseases.

Different methods of detaching adherent cells and their effects on the cell surface expression of Fas receptor and Fas ligand.

In cell culture environment, some cells adhere firmly to the culture plates and may be vulnerable to cell detachment during passage. Therefore, it is important to harvest cells with a proper detaching method to maintain the viability of cells after detachment. Trypsinization is frequently used for cellular dissociation and detachment. However, most surface proteins and the extracellular matrix are degraded by enzymatic digestion. A mild cell detachment buffer, accutase, is recommended for the replacement of trypsin to dissociate adherent cells and thereby avoid cellular damage. In this study, we demonstrated that use of accutase for cellular detachment may compromise some surface proteins. Compared with ethylenediaminetetraacetic acid (EDTA)-based nonenzymatic cell dissociation buffers, accutase was associated with significant decreases in the surface Fas ligands and Fas receptors. Moreover, we found that accutase may be able to cleave surface Fas ligands into pieces. Our results also illustrated that surface proteins required 20 h to recover after accutase treatment. We demonstrated that using accutase to dissociate adherent cells compromised the expression of Fas ligands and Fas receptors on the cell surface. These findings indicate that it is important to choose suitable cell detachment buffers and allow cells to recover after detachment before experiments.

Induction of Interferon-γ and Tissue Inflammation by Overexpression of Eosinophil Cationic Protein in T Cells and Exosomes.

OBJECTIVE: T cells play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). Serum-derived exosomes are increased in SLE patients and are correlated with disease severity. This study was undertaken to investigate whether T cell-derived exosomal proteins play a role in SLE pathogenesis. METHODS: We characterized proteins in T cell-derived exosomes from SLE patients and healthy controls by MACSPlex exosome analysis and proteomics. To study the potential pathogenic functions of the exosomal protein identified, we generated and characterized T cell-specific transgenic mice that overexpressed that protein in T cells. RESULTS: We identified eosinophil cationic protein (ECP, also called human RNase III) as overexpressed in SLE T cell-derived exosomes. T cell-specific ECP-transgenic mice (n = 5 per group) displayed early induction of serum interferon-γ (IFNγ) levels (P = 0.062) and inflammation of multiple tissue types. Older T cell-specific ECP-transgenic mice (n = 3 per group) also displayed an increase in follicular helper T cell and plasma B cell numbers, and in autoantibody levels (P < 0.01). Single-cell RNA sequencing showed the induction of IFNγ messenger RNA (P = 2.2 × 10-13 ) and inflammatory pathways in ECP-transgenic mouse T cells. Notably, adoptively transferred ECP-containing exosomes stimulated serum autoantibody levels (P < 0.01) and tissue IFNγ levels in the recipient mice (n = 3 per group). The transferred exosomes infiltrated into multiple tissues of the recipient mice, resulting in hepatitis, nephritis, and arthritis. CONCLUSION: Our findings indicate that ECP overexpression in T cells or T cell-derived exosomes may be a biomarker and pathogenic factor for nephritis, hepatitis, and arthritis associated with SLE.