Evidence-Based Assessment of Genes in Dilated Cardiomyopathy.

BACKGROUND: Each of the cardiomyopathies, classically categorized as hypertrophic cardiomyopathy, dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy, has a signature genetic theme. Hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy are largely understood as genetic diseases of sarcomere or desmosome proteins, respectively. In contrast, >250 genes spanning >10 gene ontologies have been implicated in DCM, representing a complex and diverse genetic architecture. To clarify this, a systematic curation of evidence to establish the relationship of genes with DCM was conducted. METHODS: An international panel with clinical and scientific expertise in DCM genetics evaluated evidence supporting monogenic relationships of genes with idiopathic DCM. The panel used the Clinical Genome Resource semiquantitative gene-disease clinical validity classification framework with modifications for DCM genetics to classify genes into categories on the basis of the strength of currently available evidence. Representation of DCM genes on clinically available genetic testing panels was evaluated. RESULTS: Fifty-one genes with human genetic evidence were curated. Twelve genes (23%) from 8 gene ontologies were classified as having definitive (BAG3, DES, FLNC, LMNA, MYH7, PLN, RBM20, SCN5A, TNNC1, TNNT2, TTN) or strong (DSP) evidence. Seven genes (14%; ACTC1, ACTN2, JPH2, NEXN, TNNI3, TPM1, VCL) including 2 additional ontologies were classified as moderate evidence; these genes are likely to emerge as strong or definitive with additional evidence. Of these 19 genes, 6 were similarly classified for hypertrophic cardiomyopathy and 3 for arrhythmogenic right ventricular cardiomyopathy. Of the remaining 32 genes (63%), 25 (49%) had limited evidence, 4 (8%) were disputed, 2 (4%) had no disease relationship, and 1 (2%) was supported by animal model data only. Of the 16 evaluated clinical genetic testing panels, most definitive genes were included, but panels also included numerous genes with minimal human evidence. CONCLUSIONS: In the curation of 51 genes, 19 had high evidence (12 definitive/strong, 7 moderate). It is notable that these 19 genes explain only a minority of cases, leaving the remainder of DCM genetic architecture incompletely addressed. Clinical genetic testing panels include most high-evidence genes; however, genes lacking robust evidence are also commonly included. We recommend that high-evidence DCM genes be used for clinical practice and that caution be exercised in the interpretation of variants in variable-evidence DCM genes.

Meals and Room Temperature Storage do not Significantly Affect Feasibility of Direct RT-PCR Tests for SARS-CoV-2 Using Saliva.

BACKGROUND: Rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using saliva samples has emerged as a preferred technique since sample collection is easy and noninvasive. In addition, several commercial high-throughput PCR kits that do not require RNA extraction/purification have been developed and are now available for testing saliva samples. However, an optimal protocol for SARS-CoV-2 RT-PCR testing of saliva samples using the RNA extraction/purification-free kits has not yet been established. The aim of this study was to establish optimal preanalytical conditions, including saliva sample collection, storage, and dilution for RNA extraction/purification-free RT-PCR (direct RT-PCR). METHODS: Patients suspected with COVID-19 from March 02 to August 31, 2020, were enrolled in this study. A total of 248 samples, including 43 nasopharyngeal swabs and 205 saliva samples, were collected from 66 patients (37 outpatients and 29 inpatients) and tested using the 2019 Novel Coronavirus Detection Kit (nCoV-DK, Shimadzu Corporation, Kyoto, Japan). RESULTS: The detection results obtained using nasopharyngeal swabs and saliva samples matched 100%. The sampling time, i.e., either awakening time or post-breakfast, had no significant effect on the viral load of the saliva samples. Although saliva samples are routinely diluted to reduce viscosity, we observed that dilution negatively affected PCR sensitivity. Saliva samples could be stored at room temperature (25°C) for 24 hours or at 4°C for up to 48 hours. CONCLUSIONS: This study demonstrated the appropriate conditions of saliva sample collection, processing, and storage, and indicated that the nCoV-DK is applicable to saliva samples, making the diagnosis method simple and safe.

Modification of the Algorithm Used by Automated Hematology Analyzer XN-3000 Improves Specificity in the Detection of Schistocytes.

BACKGROUND: This study investigated the feasibility and accuracy of an automated hematology analyzer in the detection of schistocytes. METHODS: In total, 1,026 peripheral blood samples were collected. Schistocytes were morphologically diagnosed by manual examination of digital microscopic red blood cell images captured by a Sysmex DI-60. Automated diagnoses were performed using a Sysmex XN-3000. RESULT: The accuracy of automated diagnosis using the XN-3000 with the default algorithm "fragments?" was determined through comparison with the findings of morphological examination. The comparison showed a sensitivity of 100% and a specificity of 41.6% for automated diagnosis. To improve the low specificity, a two-step analysis was performed. Use of the algorithm "fragments?" in XN-3000 followed by an off-line analysis using the cell parameter %FRC (percent fragmented red blood cells) yielded a sensitivity of 86.5% and a specificity of 70.3%. CONCLUSIONS: Our study indicated that combined use of the %FRC parameter with the default algorithm of the Sysmex XN-3000 automated hematology analyzer can improve the low specificity of the default algorithm in rapid screening for schistocytes.

Telethonin variants found in Brugada syndrome, J-wave pattern ECG, and ARVC reduce peak Nav 1.5 currents in HEK-293 cells.

BACKGROUND: Telethonin (TCAP) is a Z-disk protein that maintains cytoskeletal integrity and various signaling pathways in cardiomyocytes. TCAP is shown to modulate α-subunit of the human cardiac sodium channel (hNav 1.5) by direct interactions. Several TCAP variants are found in cardiomyopathies. We sought to investigate whether TCAP variants are associated with arrhythmia syndromes. METHODS: Mutational analyses for TCAP were performed in 303 Japanese patients with Brugada syndrome, arrhythmogenic right ventricular cardiomyopathy, and J-wave pattern ECG. Using patch-clamp techniques, electrophysiological characteristics of hNav 1.5 were studied in HEK-293 cells stably expressing hNav 1.5 and transiently transfected with wild-type (WT) or variant TCAP. RESULTS: We identified two TCAP variants, c.145G>A:p.E49K and c.458G>A:p.R153H, in four individuals. p.E49K was found in two patients with ARVC or BrS. p.R153H was found in two patients with BrS or J-wave pattern ECG. No patient had variant hNav 1.5. Patch-clamp experiments demonstrated that peak sodium currents were significantly reduced in cells expressing p.R153H and p.E49K compared with WT-TCAP (66%, p.R153H; 72%, p.E49K). Voltage dependency of peak IV curve was rightward-shifted by 5 mV in cells expressing p.E49K compared with WT-TCAP. Voltage dependency of activation was not leftward-shifted by p.R153H, while voltage dependency of steady-state inactivation was leftward-shifted by p.E49K. CONCLUSIONS: We found two TCAP variants in the patients with BrS, J-wave pattern ECG, and ARVC that can cause loss-of-function of the hNav 1.5 in heterologous expression systems. Our observation suggests that these variants might impair INa and be associated with the patients' electrophysiological phenotypes. Further studies linking our experimental data to clinical phenotypes are warranted.

Atrial fibrillation and electrophysiology in transgenic mice with cardiac-restricted overexpression of FKBP12.

Cardiomyocyte-restricted overexpression of FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice develop spontaneous atrial fibrillation (AF). The aim of the present study is to explore the mechanisms underlying the occurrence of AF in αMyHC-FKBP12 mice. Spontaneous AF was documented by telemetry in vivo and Langendorff-perfused hearts of αMyHC-FKBP12 and littermate control mice in vitro. Atrial conduction velocity was evaluated by optical mapping. The patch-clamp technique was applied to determine the potentially altered electrophysiology in atrial myocytes. Channel protein expression levels were evaluated by Western blot analyses. Spontaneous AF was recorded in four of seven αMyHC-FKBP12 mice but in none of eight nontransgenic (NTG) controls. Atrial conduction velocity was significantly reduced in αMyHC-FKBP12 hearts compared with NTG hearts. Interestingly, the mean action potential duration at 50% but not 90% was significantly prolonged in αMyHC-FKBP12 atrial myocytes compared with their NTG counterparts. Consistent with decreased conduction velocity, average peak Na+ current ( INa) density was dramatically reduced and the INa inactivation curve was shifted by approximately +7 mV in αMyHC-FKBP12 atrial myocytes, whereas the activation and recovery curves were unaltered. The Nav1.5 expression level was significantly reduced in αMyHC-FKBP12 atria. Furthermore, we found increases in atrial Cav1.2 protein levels and peak L-type Ca2+ current density and increased levels of fibrosis in αMyHC-FKBP12 atria. In summary, cardiomyocyte-restricted overexpression of FKBP12 reduces the atrial Nav1.5 expression level and mean peak INa, which is associated with increased peak L-type Ca2+ current and interstitial fibrosis in atria. The combined electrophysiological and structural changes facilitated the development of local conduction block and altered action potential duration and spontaneous AF. NEW & NOTEWORTHY This study addresses a long-standing riddle regarding the role of FK506-binding protein 12 in cardiac physiology. The work provides further evidence that FK506-binding protein 12 is a critical component for regulating voltage-gated sodium current and in so doing has an important role in arrhythmogenic physiology, such as atrial fibrillation.

Comparison of the Analytical Performance Between cobas EGFR Assay and PCR-Clamp Method in the Detection of EGFR Mutations in Japanese Non-Small Cell Lung Cancer Patients.

BACKGROUND: EGFR, a tyrosine-kinase, plays an important role in the progression of lung cancer. Since genetic abnormality of EGFR alters the effects of tyrosine-kinase inhibitors targeting EGFR, molecular analyses of EGFR have recently gained more attention in the treatment of lung cancer. However, several different techniques are available and which method is superior has not been determined. In this study, we compared two recently developed PCR-based techniques, PCR-clamp method and cobas EGFR assay. METHODS: Ninety-four surgical samples and 58 biopsy samples from patients suffering from non-small cell lung cancers (NSCLCs) were included in the study. Samples with positive and negative genetic abnormalities, 66 and 28 respectively, were chosen for PCR-Clamp methods. Those same samples were reanalyzed with cobas EGFR assay. RESULTS: The concordance between PCR-Clamp and cobas EGFR methods was 95.7%. PCR-Clamp failed to detect four mutations that were detected with cobas EGFR assay. These two methods were further tested by analyzing 58 random biopsy samples. The concordance for the biopsy samples was 93.1%, and PCR-Clamp, again, failed to detect three mutations that were detected with cobas EGFR assay. CONCLUSIONS: Our results showed both methods detected most of the known EGFR mutations and the concordance was similar to those previously reported in different ethnicities. However, in our study, PCR-Clamp method failed to detect a total of seven mutations that were detected with cobas EGFR assay. Thus, we concluded that cobas EGFR assay is an easier and more accurate screening assay than PCR-Clamp method in detecting EGFR genetic abnormalities.

Small-conductance calcium-activated potassium channel and recurrent ventricular fibrillation in failing rabbit ventricles.

RATIONALE: Fibrillation/defibrillation episodes in failing ventricles may be followed by action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (SVF). OBJECTIVE: We hypothesized that activation of apamin-sensitive small-conductance Ca(2+)-activated K(+) (SK) channels is responsible for the postshock APD shortening in failing ventricles. METHODS AND RESULTS: A rabbit model of tachycardia-induced heart failure was used. Simultaneous optical mapping of intracellular Ca(2+) and membrane potential (V(m)) was performed in failing and nonfailing ventricles. Three failing ventricles developed SVF (SVF group); 9 did not (no-SVF group). None of the 10 nonfailing ventricles developed SVF. Increased pacing rate and duration augmented the magnitude of APD shortening. Apamin (1 µmol/L) eliminated recurrent SVF and increased postshock APD(80) in the SVF group from 126±5 to 153±4 ms (P<0.05) and from 147±2 to 162±3 ms (P<0.05) in the no-SVF group but did not change APD(80) in nonfailing group. Whole cell patch-clamp studies at 36°C showed that the apamin-sensitive K(+) current (I(KAS)) density was significantly larger in the failing than in the normal ventricular epicardial myocytes, and epicardial I(KAS) density was significantly higher than midmyocardial and endocardial myocytes. Steady-state Ca(2+) response of I(KAS) was leftward-shifted in the failing cells compared with the normal control cells, indicating increased Ca(2+) sensitivity of I(KAS) in failing ventricles. The K(d) was 232±5 nmol/L for failing myocytes and 553±78 nmol/L for normal myocytes (P=0.002). CONCLUSIONS: Heart failure heterogeneously increases the sensitivity of I(KAS) to intracellular Ca(2+), leading to upregulation of I(KAS), postshock APD shortening, and recurrent SVF.

Aggressive diffuse large B-cell lymphoma manifested by splenic rupture progressed 2 months after transverse myelitis: an autopsy case report.

Background: Splenic rupture by diffuse large B-cell lymphoma (DLBCL), which usually progresses insidiously, is extremely rare. Case Presentation: A 60-year-old man presented with paralysis in his lower left extremity. A magnetic resonance imaging suggested transverse myelitis. No lymphadenopathy or organomegaly was noted. Two months after remission, he was referred to the emergency department complaining of presyncope. He was in preshock due to splenic rupture, and underwent laparotomy after attempts of transcatheter arterial embolization. Splenomegaly, hepatomegaly, and disseminated enlarged lymph nodes were observed. Histological examinations of the resected spleen showed DLBCL. He died of multiple organ failure associated with intractable bleeding. His autopsy revealed diffuse systemic invasions of lymphoma cells except for the brain and spinal cord. Microscopically, the spinal cord showed macular incomplete necrosis and histiocytic infiltration, suggestive of hemophagocytic syndrome. Conclusion: The progression of DLBCL in our case is drastically rapid. Undiagnosed transverse myelitis preceded the onset.

Performance evaluation of the Ortho VITROS SARS-CoV-2 Spike-Specific Quantitative IgG test by comparison with the surrogate virus neutralizing antibody test and clinical assessment.

BACKGROUND: Despite the worldwide campaigns of COVID-19 vaccinations, the pandemic is still a major medical and social problem. The Ortho VITROS SARS-CoV-2 spike-specific quantitative IgG (VITROS S-IgG) assay has been developed to assess neutralizing antibody (NT antibody) against SARS-CoV-2 spike (S) antibodies. However, it has not been evaluated in Japan, where the total cases and death toll are lower than the rest of the world. METHODS: The clinical performance of VITROS S-IgG was evaluated by comparing with the NT antibody levels measured by the surrogate virus neutralizing antibody test (sVNT). A total of 332 serum samples from 188 individuals were used. Of these, 219 samples were from 75 COVID-19 patients: 96 samples from 20 severe/critical cases (Group S), and 123 samples from 55 mild/moderate cases (Group M). The remaining 113 samples were from 113 healthcare workers who had received 2 doses of the BNT162b2 vaccine. RESULTS: VITROS S-IgG showed good correlation with the cPass sVNT assay (Spearman rho = 0.91). Both VITROS S-IgG and cPass sVNT showed significantly higher plateau levels of antibodies in Group S compared to Group M. Regarding the humoral immune responses after BNT162b2 vaccination, individuals who were negative for SARS-CoV-2 nucleocapsid (N)-specific antibodies had statistically lower titers of both S-IgG and sVNT compared to individuals with a history of COVID-19 and individuals who were positive for N-specific antibodies without history of COVID-19. In individuals who were positive for N-specific antibodies, S-IgG and sVNT titers were similar to individuals with a history of COVID-19. CONCLUSIONS: Although the automated quantitative immunoassay VITROS S-IgG showed a reasonable correlation with sVNT antibodies, there is some discrepancy between Vitros S-IgG and cPass sVNT in milder cases. Thus, VITROS S-IgG can be a useful diagnostic tool in assessing the immune responses to vaccination and herd immunity. However, careful analysis is necessary to interpret the results.

Evaluation of bone marrow aspirates using the automated hematology analyzer Sysmex XN-3000.

INTRODUCTION: This study evaluated the feasibility of the Sysmex XN-3000 automated hematology analyzer for the assessment of total nucleated cells (TNC) and bone marrow (BM) cell density in routine bone marrow aspiration (BMA) samples. METHODS: A total of 54 BMA samples from 39 hematological patients were evaluated. The number of megakaryocytes was calculated by a specific gating algorithm using the body fluid mode of the WBC differential (WDF) channel. Lipid contents were calculated through a newly developed algorithm utilizing the WDF channel. The ratio of lipid particles over TNCs by the WNR channel was compared with the BM cellularity assessed by the BM biopsy. The myeloid/erythroid (M/E) ratio was calculated by measuring the number of myeloid cells in the WDF channel and the number of nucleated red blood cells (NRBCs) in the WNR channel. RESULTS: XN-3000 counts and microscopic results showed a linear correlation in TNC (R2  = .98, p < .001), megakaryocytes (R2  = .59, p = .002), NRBC (R2  = .84, p < .001), and M/E ratio (R2  = .59, p < .001). There were significant differences in the lipid/TNC ratios of hypercellular, normocellular, and hypocellular BMs measured by XN-3000 (p < .001). Receiver-operating characteristic analysis detected cut-off values of the lipid/TNC ratio of >0.4054 for hypoplasia and <0.157 for hyperplasia. The sensitivity and specificity for hypoplasia were 100% and 88%, and for hyperplasia were 89% and 86%, respectively. CONCLUSION: XN-3000 provides a quantitative assessment of BM cellularity, supporting the qualitative assessment by myelogram and BM biopsy.

Assessment of antibody dynamics and neutralizing activity using serological assay after SARS-CoV-2 infection and vaccination.

The COVID-19 antibody test was developed to investigate the humoral immune response to SARS-CoV-2 infection. In this study, we examined whether S antibody titers measured using the anti-SARS-CoV-2 IgG II Quant assay (S-IgG), a high-throughput test method, reflects the neutralizing capacity acquired after SARS-CoV-2 infection or vaccination. To assess the antibody dynamics and neutralizing potency, we utilized a total of 457 serum samples from 253 individuals: 325 samples from 128 COVID-19 patients including 136 samples from 29 severe/critical cases (Group S), 155 samples from 71 mild/moderate cases (Group M), and 132 samples from 132 health care workers (HCWs) who have received 2 doses of the BNT162b2 vaccinations. The authentic virus neutralization assay, the surrogate virus neutralizing antibody test (sVNT), and the Anti-N SARS-CoV-2 IgG assay (N-IgG) have been performed along with the S-IgG. The S-IgG correlated well with the neutralizing activity detected by the authentic virus neutralization assay (0.8904. of Spearman's rho value, p < 0.0001) and sVNT (0.9206. of Spearman's rho value, p < 0.0001). However, 4 samples (2.3%) of S-IgG and 8 samples (4.5%) of sVNT were inconsistent with negative results for neutralizing activity of the authentic virus neutralization assay. The kinetics of the SARS-CoV-2 neutralizing antibodies and anti-S IgG in severe cases were faster than the mild cases. All the HCWs elicited anti-S IgG titer after the second vaccination. However, the HCWs with history of COVID-19 or positive N-IgG elicited higher anti-S IgG titers than those who did not have it previously. Furthermore, it is difficult to predict the risk of breakthrough infection from anti-S IgG or sVNT antibody titers in HCWs after the second vaccination. Our data shows that the use of anti-S IgG titers as direct quantitative markers of neutralizing capacity is limited. Thus, antibody tests should be carefully interpreted when used as serological markers for diagnosis, treatment, and prophylaxis of COVID-19.

Imaging arrhythmogenic calcium signaling in intact hearts.

Protein complex of the cardiac junctional sarcoplasmic reticulum (SR) membrane formed by type 2 ryanodine receptor, junction, triadin, and calsequestrin is responsible for controlling SR calcium (Ca) release. Increased intracellular calcium (Ca(i)) activates the electrogenic sodium-Ca exchanger current, which is known to be important in afterdepolarization and triggered activities (TAs). Using optical-mapping techniques, it is possible to simultaneously map membrane potential (V (m)) and Ca(i) transient in Langendorff-perfused rabbit ventricles to better define the mechanisms by which V (m) and Ca(i) interactions cause early afterdepolarizations (EADs). Phase 3 EAD is dependent on heterogeneously prolonged action potential duration (APD). Electrotonic currents that flow between a persistently depolarized region and its recovered neighbors underlies the mechanisms of phase 3 EADs and TAs. In contrast, "late phase-3 EAD" is induced by APD shortening, not APD prolongation. In failing ventricles, upregulation of apamin-sensitive Ca-activated potassium (K) channels (I(KAS)) causes APD shortening after fibrillation-defibrillation episodes. Shortened APD in the presence of large Ca(i) transients generates late-phase 3 EADs and recurrent spontaneous ventricular fibrillation. The latter findings suggest that I (KAS) may be a novel antiarrhythmic targets in patients with heart failure and electrical storms.

Antibody response and seroprevalence in healthcare workers after the BNT162b2 vaccination in a University Hospital at Tokyo.

In 2020, we reported a low seroprevalence of N-specific antibodies in 4147 health care workers (HCWs) at a frontline hospital in Tokyo, Japan. In Japan, a vaccine campaign was launched in early 2021. We re-evaluated seroprevalences of N- and S-specific antibodies in 2202 HCWs who took two doses of the BNT162b2 vaccine. In 2021, N-specific seroprevalence remains as low as 1.59%. The seroprevalences were comparable among all HCWs regardless of exposure levels. Almost all of the HCWs elicited S-specific antibodies after vaccination. However, the HCWs who had COVID-19 elicited higher S-specific antibody titers than those who did not have COVID-19. In the HCWs without a history of COVID-19, 1.1% (23 out of 2185) were seropositive with N-specific antibodies, indicating the existence of asymptomatic infections. Also, S-specific antibody titers were higher in females and younger HCWs, and in those who had severe side effects. However, S-specific antibody titers were lower depending on the number of days after the second dose of vaccination specifically in elderly individuals. In conclusion, this study indicates N-specific seroprevalence remains low in HCWs at a frontline hospital in Tokyo. The mRNA vaccine elicited S-specific antibody in HCWs, however, the titers decreased as the days proceeded.

Increased CaV1.2 late current by a CACNA1C p.R412M variant causes an atypical Timothy syndrome without syndactyly.

Timothy syndrome (TS) is a rare pleiotropic disorder associated with long QT syndrome, syndactyly, dysmorphic features, and neurological symptoms. Several variants in exon 8 or 8a of CACNA1C, a gene encoding the α-subunit of voltage-gated Ca2+ channels (Cav1.2), are known to cause classical TS. We identified a p.R412M (exon 9) variant in an atypical TS case. The aim of this study was to examine the functional effects of CACNA1C p.R412M on CaV1.2 in comparison with those of p.G406R. The index patient was a 2-month-old female infant who suffered from a cardio-pulmonary arrest in association with prolonged QT intervals. She showed dysmorphic facial features and developmental delay, but not syndactyly. Interestingly, she also presented recurrent seizures from 4 months. Genetic tests identified a novel heterozygous CACNA1C variant, p.R412M. Using heterologous expression system with HEK-293 cells, analyses with whole-cell patch-clamp technique revealed that p.R412M caused late Ca2+ currents by significantly delaying CaV1.2 channel inactivation, consistent with the underlying mechanisms of classical TS. A novel CACNA1C variant, p.R412M, was found to be associated with atypical TS through the same mechanism as p.G406R, the variant responsible for classical TS.

Performance evaluation of the Roche Elecsys® Anti-SARS-CoV-2 immunoassays by comparison with neutralizing antibodies and clinical assessment.

Quantitative measurement of SARS-CoV-2 neutralizing antibodies is highly expected to evaluate immune status, vaccine response, and antiviral therapy. The Elecsys® Anti-SARS-CoV-2 S (Elecsys® anti-S) was developed to measure anti-SARS-CoV-2 S proteins. We sought to investigate whether Elecsys® anti-S can be used to predict neutralizing activities in patients' serums using an authentic virus neutralization assay. One hundred forty-six serum samples were obtained from 59 patients with COVID-19 at multiple time points. Of the 59 patients, 44 cases were included in Group M (mild 23, moderate 21) and produced 84 samples (mild 35, moderate 49), while 15 cases were included in Group S (severe 11, critical 4) and produced 62 samples (severe 43, critical 19). The neutralization assay detected 73% positive cases, and Elecsys® anti-S and Elecsys® Anti-SARS-CoV-2 (Elecsys® anti-N) showed 72% and 66% positive cases, respectively. A linear correlation between the Elecsys® anti-S assay and the neutralization assay were highly correlated (r = 0.7253, r2 = 0.5261) than a linear correlation between the Elecsys® anti-N and neutralization assay (r = 0.5824, r2 = 0.3392). The levels of Elecsys® anti-S antibody and neutralizing activities were significantly higher in Group S than in Group M after 6 weeks from onset of symptoms (p < 0.05). Conversely, the levels of Elecsys® anti-N were comparable in both groups. Three immunosuppressed patients, including cancer patients, showed low levels of anti-S and anti-N antibodies and neutralizing activities throughout the measurement period, indicating the need for careful follow-up. Our data indicate that Elecsys® anti-S can predict the neutralization antibodies in COVID-19.

Inhibition of BCL2A1 by STAT5 inactivation overcomes resistance to targeted therapies of FLT3-ITD/D835 mutant AML.

Tyrosine kinase inhibitors (TKIs) are established drugs in the therapy of FLT3-ITD mutated acute myeloid leukemia (AML). However, acquired mutations, such as D835 in the tyrosine kinase domain (FLT3-ITD/D835), can induce resistance to TKIs. A cap analysis gene expression (CAGE) technology revealed that the gene expression of BCL2A1 transcription start sites was increased in primary AML cells bearing FLT3-ITD/D835 compared to FLT3-ITD. Overexpression of BCL2A1 attenuated the sensitivity to quizartinib, a type II TKI, and venetoclax, a selective BCL2 inhibitor, in AML cell lines. However, a type I TKI, gilteritinib, inhibited the expression of BCL2A1 through inactivation of STAT5 and alleviated TKI resistance of FLT3-ITD/D835. The combination of gilteritinib and venetoclax showed synergistic effects in the FLT3-ITD/D835 positive AML cells. The promoter region of BCL2A1 contains a BRD4 binding site. Thus, the blockade of BRD4 with a BET inhibitor (CPI-0610) downregulated BCL2A1 in FLT3-mutated AML cells and extended profound suppression of FLT3-ITD/D835 mutant cells. Therefore, we propose that BCL2A1 has the potential to be a novel therapeutic target in treating FLT3-ITD/D835 mutated AML.

Activation of SARS-CoV-2 neutralizing antibody is slower than elevation of spike-specific IgG, IgM, and nucleocapsid-specific IgG antibodies.

COVID-19 antibody testing has been developed to investigate humoral immune response in SARS-CoV-2 infection. To assess the serological dynamics and neutralizing potency following SARS-CoV-2 infection, we investigated the neutralizing (NT) antibody, anti-spike, and anti-nucleocapsid antibodies responses using a total of 168 samples obtained from 68 SARS-CoV-2 infected patients. Antibodies were measured using an authentic virus neutralization assay, the high-throughput laboratory measurements of the Abbott Alinity quantitative anti-spike receptor-binding domain IgG (S-IgG), semiquantitative anti-spike IgM (S-IgM), and anti-nucleocapsid IgG (N-IgG) assays. The quantitative measurement of S-IgG antibodies was well correlated with the neutralizing activity detected by the neutralization assay (r = 0.8943, p < 0.0001). However, the kinetics of the SARS-CoV-2 NT antibody in severe cases were slower than that of anti-S and anti-N specific antibodies. These findings indicate a limitation of using the S-IgG antibody titer, detected by the chemiluminescent immunoassay, as a direct quantitative marker of neutralizing activity capacity. Antibody testing should be carefully interpreted when utilized as a marker for serological responses to facilitate diagnostic, therapeutic, and prophylactic interventions.

Vagal stimulation promotes atrial electrical remodeling induced by rapid atrial pacing in dogs: evidence of a noncholinergic effect.

BACKGROUND: Atrial electrical remodeling (AER) is one of the mechanisms by which atrial fibrillation (AF) begets AF. It is known that vagal activity increases the propensity for AF. However, vagal effects on AER have not been fully investigated. METHODS: Adult mongrel dogs were divided in four groups: group I, rapid atria pacing (RAP); group II, RAP plus vagal nerve stimulation (VNS); group III, RAP and VNS with atropine (0.2 mg/kg/h, intravenous), and group IV, group III plus vasoactive intestinal polypeptide (VIP) antagonist ([D-p-Cl-Phe(6), Leu(17)]-VIP, 0.125 µg/kg/h). VNS was performed bilaterally through vagosympathetic trunks to achieve second-degree AV block or sinus rate slowing of >30 beats per minute. Atrial effective refractory periods (AERPs) were determined in the coronary sinus and right atrial appendage every hour at drive cycle lengths (DCLs) 350 ms, 300 ms, and 250 ms. RESULTS: During 5 hours RAP with or without VNS, AERP shortened progressively from baseline at both pacing sites and at all DCLs (P < 0.01). Furthermore, RAP-induced AERP shortening was more pronounced with VNS (P < 0.01). With atropine, the AERP shortening during VNS was blunted (P < 0.01), but was still significantly more pronounced than that in group I (P < 0.05). However, VNS effect on AERP shortening was eliminated completely with the combination of atropine and VIP antagonist (P = 0.15 vs group I). CONCLUSION: Increased vagal activity promotes RAP-induced AER, which could not be totally accounted for by cholinergic effect but could be blocked by the combination of atropine and VIP antagonist. Vagally released VIP may have important role in the vagal promotion of AER.

Phosphorylation of Lamin A/C at serine 22 modulates Nav 1.5 function.

Variants in the LMNA gene, which encodes for Lamin A/C, are associated with cardiac conduction disease (CCD). We previously reported that Lamin A/C variants p.R545H and p.A287Lfs*193, which were identified in CCD patients, decreased peak INa in HEK-293 cells expressing Nav 1.5. Decreased peak INa in the cardiac conduction system could account for patients' atrioventricular block. We found that serine 22 (Ser 22) phosphorylation of Lamin A/C was decreased in the p.R545H variant and hypothesized that lamin phosphorylation modulated Nav 1.5 activity. To test this hypothesis, we assessed Nav 1.5 function in HEK-293 cells co-transfected with LMNA variants or treated with the small molecule LBL1 (lamin-binding ligand 1). LBL1 decreased Ser 22 phosphorylation by 65% but did not affect Nav 1.5 function. To test the complete loss of phosphorylation, we generated a version of LMNA with serine 22 converted to alanine 22 (S22A-LMNA); and a version of mutant R545H-LMNA that mimics phosphorylation via serine 22 to aspartic acid 22 substitution (S22D-R545H-LMNA). We found that S22A-LMNA inhibited Lamin-mediated activation of peak INa by 63% and shifted voltage-dependency of steady-state inactivation of Nav 1.5. Conversely, S22D-R545H-LMNA abolished the effects of mutant R545H-LMNA on voltage-dependency but not peak INa . We conclude that Lamin A/C Ser 22 phosphorylation can modulate Nav 1.5 function and contributes to the mechanism by which R545H-LMNA alters Nav 1.5 function. The differential impact of complete versus partial loss of Ser 22 phosphorylation suggests a threshold of phosphorylation that is required for full Nav 1.5 modulation. This is the first study to link Lamin A/C phosphorylation to Nav 1.5 function.