Trajectory of lung function in diabetic adults: A 16-year follow-up study of community-based prospective cohorts.

BACKGROUND AND OBJECTIVE: To investigate the difference in lung function according to diabetes status in a community-based prospective study. METHODS: Individuals aged 40-69 years from two community-based cohorts were followed prospectively for 16 years. A spirometer was used to evaluate lung function at baseline, and lung function tests were carried out biennially thereafter. Multivariable linear regression analysis was performed for the cross-sectional and longitudinal analyses based on diabetes status. RESULTS: Among the 6483 subjects, 2114 (32.6%) had prediabetes and 671 (10.4%) had diabetes. The prediabetes and diabetes groups had lower baseline % predicted values of forced expiratory volume in 1 s (FEV1) (mean, -1.853; 95% confidence interval [CI] -2.715 to -0.990 for prediabetes and mean, -4.088; 95% CI -5.424 to -2.752 for diabetes) and forced vital capacity (FVC) (mean, -2.087; 95% CI -2.837 to -1.337 for prediabetes and mean, -4.622; 95% CI -5.784 to -3.460 for diabetes) compared to the normoglycemia group after adjusting for relevant covariates. The rate of decline in FEV1% predicted (mean, -0.227; 95% CI -0.366 to -0.089) and FVC % predicted (mean, -0.232; 95% CI -0.347 to -0.117) during follow-up were faster in the diabetes group than in the normoglycemia group. The diabetes group had a lower proportion of normal ventilation (ptrend = 0.048) and higher proportions of restrictive (ptrend = 0.001) and mixed (ptrend = 0.035) ventilatory disorders at the last follow-up. CONCLUSION: Diabetes is associated with a lower baseline lung function and a faster rate of deterioration.

Multiple serum biomarkers for predicting suicidal behaviours in depressive patients receiving pharmacotherapy.

BACKGROUND: Predictive values of multiple serum biomarkers for suicidal behaviours (SBs) have rarely been tested. This study sought to evaluate and develop a panel of multiple serum biomarkers for predicting SBs in outpatients receiving a 12-month pharmacotherapy programme for depressive disorders. METHODS: At baseline, 14 serum biomarkers and socio-demographic/clinical characteristics including previous suicidal attempt and present suicidal severity were evaluated in 1094 patients with depressive disorders without a bipolar diagnosis. Of these, 884 were followed for increased suicidal severity and fatal/non-fatal suicide attempt outcomes over a 12-month treatment period. Individual and combined effects of serum biomarkers on these two prospective SBs were estimated using logistic regression analysis after adjustment for relevant covariates. RESULTS: Increased suicidal severity and fatal/non-fatal suicide attempt during the 12-month pharmacotherapy were present in 155 (17.5%) and 38 (4.3%) participants, respectively. Combined cortisol, total cholesterol, and folate serum biomarkers predicted fatal/non-fatal suicide attempt, and these with interleukin-1 beta and homocysteine additionally predicted increased suicidal severity, with clear gradients robust to adjustment (p values < 0.001). CONCLUSIONS: Application of multiple serum biomarkers could considerably improve the predictability of SBs during the outpatient treatment of depressive disorders, potentially highlighting the need for more frequent monitoring and risk appraisal.

Protecting SOME/IP Communication via Authentication Ticket.

Designed using vehicle requirements, Scalable service-Oriented MiddlewarE over IP (SOME/IP) has been adopted and used as one of the Ethernet communication standard protocols in the AUTomotive Open System Architecture (AUTOSAR). However, SOME/IP was designed without considering security, and its vulnerabilities have been demonstrated through research. In this paper, we propose a SOME/IP communication protection method using an authentication server (AS) and tickets to mitigate the infamous SOME/IP man-in-the-middle (MITM) attack. Reliable communication between the service-providing node and the node using SOME/IP communication is possible through the ticket issued from the authentication server. This method is relatively light in operation at each node, has good scalability for changes such as node addition, guarantees freshness, and provides interoperability with the existing SOME/IP protocol.

The effects of two dietary synbiotics on growth performance, haematological parameters, and non-specific immune responses in Japanese eel (Anguilla japonica).

Feed additives have attracted increased attention in aquaculture due to their ability to modulate fish gut microbiota, resulting in improved fish growth and immunity. This study assessed two synbiotics' effects in Japanese eels: Bacillus subtilis with mannooligosaccharide (MOS) and Enterococcus faecium with fructooligosaccharide (FOS). Six diets, including a control (CON), oxytetracycline (OTC), and four synbiotic diets - B.subtilis at 1 × 106 and 107 CFU/g with 5 g/kg MOS (BS6MO and BS7MO) and E. faecium at 1 × 106 and 107 CFU/g with 5 g/kg FOS (EF6FO and EF7FO) - were fed to triplicate groups of 20 fish averaging 6.00 ± 0.07g for eight weeks. Fish fed the BSMOS diets showed significantly higher weight gain (WG), specific growth rate (SGR), and feed efficiency (FE) compared to the CON and OTC diets (P < 0.05), but not significantly different from those fed the EFFOS diets. Weight gain, SGR of fish fed EFFOS were not significantly different from those fed all other diets (P > 0.05). Fish fed the OTC diet showed a higher mean aspartate aminotransferase (AST) level, though the difference was not statistically significant. The myeloperoxidase (MPO) activity of fish fed the BS7MO diet was significantly higher than in all other diets, and the superoxide dismutase (SOD) activity of fish fed the BS7MO diet was also significantly higher than in the EF7FO diet. Overall, the BSMOS synbiotic diets were significantly more effective than the CON diet in enhancing fish survival against Vibrio anguillarum. Our findings suggest that synbiotics can be a preferable alternative to antibiotics in aquaculture.

Prospective associations of multimodal serum biomarkers with 12-week and 12-month remission in patients with depressive disorders receiving stepwise psychopharmacotherapy.

Prognostic biomarkers for depression treatment outcomes have yet to be elucidated. This study sought to evaluate whether a multi-modal serum biomarker panel was prospectively associated with 12-week and 12-month remission in outpatients with depressive disorders receiving stepwise psychopharmacotherapy. At baseline, 14 serum biomarkers and socio-demographic/clinical characteristics were evaluated in 1094 patients. They received initial antidepressant monotherapy followed, as required by a protocol of successive alternative pharmacological strategies administered in 3-week steps during the acute (3-12 week) phase (N = 1086), and in 3-month steps during the continuation (6-12 month) phase (N = 884). Remission was defined as a Hamilton Depression Rating Scale score of ≤ 7. Remission was achieved in 490 (45.1%) over the 12-week, and in 625 (70.7%) over the 12-month, treatment periods. Combination scores of four serum biomarkers (high-sensitivity C-reactive protein, interleukin-1 beta, interleukin-6, and leptin) were prospectively associated with 12-week remission; and four (high-sensitivity C-reactive protein, tumor necrosis factor-alpha, interleukin-1 beta, and brain-derived neurotrophic factor) were prospectively associated with 12-month remission in a clear gradient manner (P-values < 0.001) and after adjustment for relevant covariates. These associations were evident after the Step 1 treatment monotherapy but weakened with increasing treatment steps, falling below statistical significance after 4 + treatment steps. Application of combined multiple serum biomarkers, particularly on inflammatory markers, could improve predictability of remission at acute and continuation treatment phases for depressive disorders. Patients with unfavourable biomarkers might require alternative treatment regimes for better outcomes.

Modifying Effect of the Interleukin-18 Level on the Association between BDNF Methylation and Long-Term Cardiovascular Outcomes in Patients with Acute Coronary Syndrome.

This study investigated the potential modifying effects of the level of the serum interleukin-18 (IL-18) on the association between BDNF methylation status and long-term cardiovascular outcomes in patients with acute coronary syndrome (ACS). Hospitalized ACS patients were recruited sequentially from 2006 to 2012. At baseline, the IL-18 level and BDNF methylation status were evaluated in 969 patients who were followed for major adverse cardiac events (MACEs) for 5-12 years, until 2017 or death. The time to first composite or individual MACE was compared between individuals with lower and higher average BDNF methylation levels (in the low- and high-IL-18 groups, respectively) using a Cox proportional hazards model. After adjusting for potential covariates, the modifying effects of IL-18 and average BDNF methylation levels on the initial composite and individual MACEs were examined. In the high-IL-18 group, but not in the low-IL-18 group, a higher average BDNF methylation level was associated with increases in composite MACEs (HR (95% CI) = 2.15 (1.42-3.26)), all-cause mortality (HR (95% CI) = 1.89 (1.11-3.22)), myocardial infarction (HR (95% CI) = 1.98 (1.07-3.67)), and percutaneous coronary intervention (HR (95% CI) = 1.81 (1.01-3.23)), independent of confounding variables. The interaction effect between the IL-18 and average BDNF methylation levels on composite MACEs (p = 0.019) and myocardial infarction (p = 0.027) was significant after adjusting for covariates. Analysis of BDNF methylation status and IL-18 levels may help identify ACS patients who are most likely to have adverse clinical outcomes.

Au Hierarchical Nanostructure-Based Surface Modification of Microelectrodes for Improved Neural Signal Recording.

Microelectrodes are widely used for neural signal analysis because they can record high-resolution signals. In general, the smaller the size of the microelectrode for obtaining a high-resolution signal, the higher the impedance and noise value of the electrodes. Therefore, to improve the signal-to-noise ratio (SNR) of neural signals, it is important to develop microelectrodes with low impedance and noise. In this research, an Au hierarchical nanostructure (AHN) was deposited to improve the electrochemical surface area (ECSA) of a microelectrode. Au nanostructures on different scales were deposited on the electrode surface in a hierarchical structure using an electrochemical deposition method. The AHN-modified microelectrode exhibited an average of 80% improvement in impedance compared to a bare microelectrode. Through electrochemical impedance spectroscopy analysis and impedance equivalent circuit modeling, the increase in the ECSA due to the AHN was confirmed. After evaluating the cell cytotoxicity of the AHN-modified microelectrode through an in vitro test, neural signals from rats were obtained in in vivo experiments. The AHN-modified microelectrode exhibited an approximate 9.79 dB improvement in SNR compared to the bare microelectrode. This surface modification technology is a post-treatment strategy used for existing fabricated electrodes, so it can be applied to microelectrode arrays and nerve electrodes made from various structures and materials.

In Vitro Profiling of Laninamivir-Resistant Substitutions in N3 to N9 Avian Influenza Virus Neuraminidase Subtypes and Their Association with In Vivo Susceptibility.

Laninamivir (LAN) is a long-acting neuraminidase (NA) inhibitor (NAI) with a similar binding profile in the influenza NA enzyme active site as those of other NAIs, oseltamivir (OS), zanamivir (ZAN), and peramivir, and may share common resistance markers with these NAIs. We screened viruses with NA substitutions previously found during OS and ZAN selection in avian influenza viruses (AIVs) of the N3 to N9 subtypes for LAN susceptibility. Of the 72 NA substitutions, 19 conferred resistance to LAN, which ranged from 11.2- to 549.8-fold-decreased inhibitory activity over that of their parental viruses. Ten NA substitutions reduced the susceptibility to all four NAIs, whereas the remaining 26 substitutions yielded susceptibility to one or more NAIs. To determine whether the in vitro susceptibility of multi-NAI-resistant AIVs is associated with in vivo susceptibility, we infected BALB/c mice with recombinant AIVs with R292K (ma81K-N3R292K) or Q136K (ma81K-N8Q136K) NA substitutions, which impart in vitro susceptibility only to LAN or OS, respectively. Both ma81K-N3R292K and ma81K-N8Q136K virus-infected mice exhibited reduced weight loss, mortality, and lung viral titers when treated with their susceptible NAIs, confirming the in vitro susceptibility of these substitutions. Together, LAN resistance profiling of AIVs of a range of NA subtypes improves the understanding of NAI resistance mechanisms. Furthermore, the association of in vitro and in vivo NAI susceptibility indicates that our models are useful tools for monitoring NAI susceptibility of AIVs.IMPORTANCE The chemical structures of neuraminidase inhibitors (NAIs) possess similarities, but slight differences can result in variable susceptibility of avian influenza viruses (AIVs) carrying resistance-associated NA substitutions. Therefore, comprehensive susceptibility profiling of these substitutions in AIVs is critical for understanding the mechanism of antiviral resistance. In this study, we profiled resistance to the anti-influenza drug laninamivir in AIVs with substitutions known to impart resistance to other NAIs. We found 10 substitutions that conferred resistance to all four NAIs tested. On the other hand, we found that the remaining 26 NA substitutions were susceptible to at least one or more NAIs and showed for a small selection that in vitro data predicted in vivo behavior. Therefore, our findings highlight the usefulness of screening resistance markers in NA enzyme inhibition assays and animal models of AIV infections.

Association of Triglyceride-Glucose Index with Bone Mineral Density in Non-diabetic Koreans: KNHANES 2008-2011.

The association of insulin resistance (IR), as indicated by the homeostasis model assessment of insulin resistance, with bone metabolism is yet to be clarified. We aimed to investigate the relationship of IR with bone mass by using the triglyceride-glucose (TyG) index as an alternative marker of IR. Data of 4810 non-diabetes individuals (2552 men aged ≥ 50 years and 2258 postmenopausal women) from the Korean National Health and Nutritional Examination Survey IV and V were analyzed. Bone mineral density (BMD) at the lumbar spine, femoral neck, total hip, and whole body were measured using dual-energy X-ray absorptiometry. After adjusting for confounding factors, there were inverse relationships of TyG index with femoral neck, total hip, and whole body BMD in men (ß = - 0.085, P < 0.001 at femoral neck; ß = - 0.046, P = 0.037 at total hip; ß = - 0.098, P < 0.001 at whole body). However, in women, femoral neck and whole body BMD were negatively associated with the TyG index (ß = - 0.071, P = 0.008 at femoral neck and ß = - 0.065, P = 0.005 at whole body). The highest TyG index tertile exhibited reduced femoral neck BMD in both sexes (P = 0.003 in men and P = 0.013 in women) and reduced whole body BMD in men (P < 0.001) after adjusting for confounders. When the study subjects were divided into BMI (body mass index) < 23 kg/m2 and ≥ 23 kg/m2 groups, the TyG index was significantly associated with femoral neck BMD only in the women with BMI < 23 kg/m2 (P = 0.009). We observed a significant association between the highest TyG index tertile and low bone mass at the femoral neck in women with BMI < 23 kg/m2 (P = 0.003) that was not observed in women with BMI ≥ 23 kg/m2 and men. In conclusion, IR evaluated using the TyG index was inversely associated with femoral neck BMD in non-diabetic men aged ≥ 50 years and postmenopausal women. The negative influence of IR on femoral neck BMD was robust in the women with BMI < 23 kg/m2. This indicates a differential effect of IR on BMD according to skeletal site, sex, and BMI.

Interactive effects of systemic inflammation and life stressors on treatment response of depressive disorders.

Inflammation is an important contributor in the pathophysiology of depression and recent evidence suggests that systemic inflammation and life stressors have interactive roles in depression onset. The aim of the present study was to investigate the individual and interactive effects of systemic inflammation and life stressors with short- and long-term treatment responses in outpatients with depressive disorders in a naturalistic one-year prospective design. Serum high-sensitivity C-reactive protein (hsCRP) levels were measured and number of stressful life events (SLEs) during the last 3 months were ascertained from 1094 patients at baseline. These patients received initial antidepressant monotherapy, then, for patients with an insufficient response or uncomfortable side effects, next treatment with alternative strategies were administered at every 3 weeks in the acute treatment phase (3, 6, 9, and 12 weeks) and at every 3 months in the continuation treatment phase (6, 9, and 12 months). 12-week and 12-month remission was estimated, defined as a Hamilton Depression Rating Scale score of ≤ 7. In multivariable logistic regression analyses, individual effects were found only between higher baseline serum hsCRP levels (≥1.0 vs. < 1.0 mg/L) and 12-week non-remission. Significant interactive effects between higher hsCRP levels and higher number of SLEs (≥2 vs. < 2) on both 12-week and 12-month non-remission were observed. Combining serum hsCRP levels and number of SLEs might therefore be a useful predictor for short- and long-term treatment responses in patients with depressive disorders receiving pharmacotherapy.

A challenging TSH/GH co-secreting pituitary adenoma with concomitant thyroid cancer; a case report and literature review.

BACKGROUND: Thyroid stimulating hormone (TSH) secreting pituitary adenoma (TSHoma) with coexisting thyroid cancer is extremely rare, and proper treatment of both diseases may pose a unique clinical challenge. When TSHoma has plurihormonality, particularly involving the co-secretion of growth hormone (GH), management can be more complicated. Herein, we present a difficult-to-manage case of papillary thyroid cancer with an incurable TSH/GH-secreting pituitary adenoma. CASE PRESENTATION: A 59-year-old man was referred to our hospital due to memory impairment and inappropriate TSH level. Sella magnetic resonance imaging revealed a huge pituitary mass extending to the suprasellar area. Clinical diagnosis of TSH/GH co-secreting pituitary adenoma was made based on elevated free T4, total T3, serum α-subunit, insulin-like growth factor-1 levels and non-suppressible GH levels after oral glucose loading. Rectal cancer and multifocal papillary thyroid microcarcinoma (PTMC) were diagnosed during initial screening for internal malignancy; lower anterior resection was performed and close observation was planned for PTMC. Long-acting octreotide therapy was commenced, which resulted in a dramatic reduction in TSHoma size and facilitated control of hormonal excess. Total thyroidectomy and radioactive iodine (RAI) therapy were needed during follow up due to the growth of PTMC. After the surgery, the pituitary adenoma represented resistance to somatostatin analogue therapy and the tumor size gradually increased despite the addition of dopamine agonist therapy. Furthermore, TSH suppressive therapy with levothyroxine was impossible and an adequate TSH level for RAI therapy was unmountable. Late debulking pituitary surgery was ineffective, and the patient gradually deteriorated and lost to follow up. CONCLUSION: We report the first aggravated case of TSH/GH co-secreting pituitary tumor after total thyroidectomy for concomitant multifocal PTMC. Deferring of thyroid surgery until the TSHoma is well controlled may be the optimal therapeutic strategy in patients with TSHoma and coexistent thyroid cancer; ablative thyroid surgery may result in catastrophic pituitary tumor growth.

Surgery is a safe, effective first-line treatment modality for noninvasive prolactinomas.

PURPOSE: Dopamine agonists (DAs) have long been the recommended first-line treatment for prolactinoma. Given the remarkable developments in surgical techniques, however, surgery is on the rise. We compared the treatment outcomes of patients with noninvasive prolactinomas receiving two different initial treatments (DAs and transsphenoidal surgery). METHODS: We reviewed 745 patients with hyperprolactinemia or pituitary tumors treated from 2004 to 2020 at Chonnam National University Hwasun Hospital and identified 310 with prolactinomas. After selecting patients who had pituitary tumors with Knosp grade 0 to 1 with follow-up period over 1 year, 70 patients (29 who underwent surgery and 41 who received DAs as the initial treatment) were finally included for a comparative study. RESULTS: The surgery group exhibited better outcomes in terms of DA-free remission and the structural response, although the tumor size was significantly larger than in the DA group. The groups exhibited comparable results in terms of symptom control and the biochemical response. Univariate and multivariate analyses indicated that surgery as the initial treatment modality provided significantly better clinical outcomes in terms of DA-free remission. In the surgery group, a postoperative prolactin level < 10 ng/mL was the only significant predictor of DA-free remission. CONCLUSIONS: Transsphenoidal surgery showed comparable clinical outcomes in patients with prolactinomas, and low complication rates. The decision regarding the first-line treatment modality for non-invasive prolactinomas should be made on an individual basis.

Long-term Prognosis of Mild to Moderate Aortic Stenosis and Coronary Artery Disease.

BACKGROUND: There is an incomplete understanding of the natural course of mild to moderate aortic stenosis (AS). We aimed to evaluate the natural course of patients with mild to moderate AS and its association with coronary artery disease (CAD). METHODS: We retrospectively analyzed 787 patients diagnosed with mild to moderate AS using echocardiography between 2004 and 2010. Cardiac death and aortic valve replacement (AVR) for AS were assessed. RESULTS: A median follow-up period was 92 months. Compared to the general population, patients with mild to moderate AS had a higher risk of cardiac death (hazard ratio [HR], 17.16; 95% confidence interval [CI], 13.65-21.59; P < 0.001). Established CAD was detected in 22.4% and associated with a significantly higher risk of cardiac mortality (adjusted HR, 1.62; 95% CI, 1.04-2.53; P = 0.033). The risk of cardiac death was lower when patients were taking statin (adjusted HR, 0.64; 95% CI, 0.41-0.98; P = 0.041), which was clear only after 7 years. Both patients with CAD and on statin tended to undergo more AVR, but the difference was not statistically significant (the presence of established CAD; adjusted HR, 1.63; 95% CI, 0.51-3.51; P = 0.214 and the use of statin; adjusted HR, 1.86; 95% CI, 0.76-4.58; P = 0.177). CONCLUSION: Mild to moderate AS does not have a benign course. The presence of CAD and statin use may affect the long-term prognosis of patients with mild to moderate AS.

In Vitro and In Vivo Characterization of Novel Neuraminidase Substitutions in Influenza A(H1N1)pdm09 Virus Identified Using Laninamivir-Mediated In Vitro Selection.

Neuraminidase (NA) inhibitors (NAIs) are widely used antiviral drugs for the treatment of humans with influenza virus infections. There have been widespread reports of NAI resistance among seasonal A(H1N1) viruses, and most have been identified in oseltamivir-exposed patients or those treated with other NAIs. Thus, monitoring and identifying NA markers conferring resistance to NAIs-particularly newly introduced treatments-are critical to the management of viral infections. Therefore, we screened and identified substitutions conferring resistance to laninamivir by enriching random mutations in the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] virus followed by deep sequencing of the laninamivir-selected variants. After the generation of single mutants possessing each identified mutation, two A(H1N1)pdm09 recombinants possessing novel NA gene substitutions (i.e., D199E and P458T) were shown to exhibit resistance to more than one NAI. Of note, mutants possessing P458T-which is located outside of the catalytic or framework residue of the NA active site-exhibited highly reduced inhibition by all four approved NAIs. Using MDCK cells, we observed that the in vitro viral replication of the two recombinants was lower than that of the wild type (WT). Additionally, in infected mice, decreased mortality and/or mean lung viral titers were observed in mutants compared with the WT. Reverse mutations to the WT were observed in lung homogenate samples from D199E-infected mice after 3 serial passages. Overall, the novel NA substitutions identified could possibly emerge in influenza A(H1N1)pdm09 viruses during laninamivir therapy and the viruses could have altered NAI susceptibility, but the compromised in vitro/in vivo viral fitness may limit viral spreading.IMPORTANCE With the widespread emergence of NAI-resistant influenza virus strains, continuous monitoring of mutations that confer antiviral resistance is needed. Laninamivir is the most recently approved NAI in several countries; few data exist related to the in vitro selection of viral mutations conferring resistance to laninamivir. Thus, we screened and identified substitutions conferring resistance to laninamivir by random mutagenesis of the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] virus strain followed by deep sequencing of the laninamivir-selected variants. We found several novel substitutions in NA (D199E and P458T) in an A(H1N1)pdm09 background which conferred resistance to NAIs and which had an impact on viral fitness. Our study highlights the importance of continued surveillance for potential antiviral-resistant variants and the development of alternative therapeutics.

Comparison of Continuous ECG Monitoring by Wearable Patch Device and Conventional Telemonitoring Device.

BACKGROUND: Detection of arrhythmias is crucial for the treatment of cardiovascular diseases. However, conventional devices do not provide sufficient diagnostic accuracy while patients should suffer from bothersome diagnostic process. We sought to evaluate diagnostic capability and safety of the new adhesive electrocardiogram (ECG) monitoring device in patients who need ECG monitoring during admission. METHODS: We enrolled 10 patients who admitted to Seoul National University Bundang Hospital and required continuous ECG monitoring between October 31, 2019 and December 18, 2019. New adhesive ECG monitoring device and conventional ECG monitoring device were simultaneously applied to the patients and maintained for 48 hours. From each patient, 48 pairs of ECG signal were collected and analyzed by two cardiologists independently. Discrepancy of diagnosis and frequency of noise or signal loss were compared between the two devices. RESULTS: From analyzable ECG data, discrepancy of arrhythmia diagnosis was not observed between the two devices. Noise rate was higher in conventional ECG monitoring device (2.5% vs. 17.3%, P < 0.001) and signal loss was not observed in new adhesive device while there was 9.4% of signal losses in conventional Holter recorder group. The new device was well-tolerated among 48 hours of monitoring period and no adverse event was observed. CONCLUSION: A newer adhesive ECG monitoring device demonstrated similar diagnostic accuracy compared to conventional ECG monitoring device.

Screening for Neuraminidase Inhibitor Resistance Markers among Avian Influenza Viruses of the N4, N5, N6, and N8 Neuraminidase Subtypes.

Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs.IMPORTANCE The frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs), as the only available class of drugs for AIVs in humans, have been constantly used for treatment, leading to the inevitable emergence of drug-resistant variants. To screen for substitutions conferring NAI resistance in AIVs of N4, N5, N6, and N8 NA subtypes, random mutations within the target gene were generated, and resistant viruses were selected from mutant libraries in the presence of individual drugs. We identified 16 NA substitutions conferring NAI resistance in the tested AIV subtypes; some are novel and subtype specific, and others have been previously reported in other subtypes. Our findings will contribute to an increased and more comprehensive understanding of the mechanisms of NAI-induced inhibition of influenza virus and help lead to the development of drugs that bind to alternative interaction motifs.

Comparison of Minimally Invasive Direct Coronary Artery Bypass and Percutaneous Coronary Intervention Using Second-Generation Drug-Eluting Stents for Coronary Artery Disease　- Propensity Score-Matched Analysis.

BACKGROUND: Some studies comparing minimally invasive direct coronary artery bypass (MIDCAB) and percutaneous coronary intervention (PCI) have reported MIDCAB's superiority, but they did not investigate contemporary PCI with newer generation drug-eluting stents (DES). We compared clinical outcomes after MIDCAB with previously reported outcomes after PCI with second-generation DES.Methods and Results:We retrospectively reviewed the records of patients treated with MIDCAB. Baseline characteristics and clinical outcomes after MIDCAB were compared with those for left anterior descending artery disease treated via PCI. The primary outcomes were major adverse cardiovascular and cerebrovascular events (MACCE), a composite of cardiovascular death, non-fatal myocardial infarction, ischemic stroke, and target vessel revascularization (TVR). A propensity score-matching (PSM) analysis was conducted to adjust for between-group differences in baseline characteristics. We analyzed 77 patients treated with MIDCAB and 2,206 treated with PCI. The MIDCAB group was older and had more severe coronary disease and a higher incidence of left ventricular dysfunction. Over a 3-year follow-up, the PCI group had favorable MACCE outcomes. After PSM, there were no between-group differences in MACCE (MIDCAB, 15.6% vs. PCI, 23.4%; hazard ratio [HR], 0.80; 95% CI: 0.38-1.68, P=0.548) or TVR (MIDCAB, 2.6% vs. PCI, 5.2%; HR, 0.51; 95% CI: 0.10-3.09, P=0.509). CONCLUSIONS: Clinical outcomes were similar between MIDCAB and PCI using second-generation DES over 3 years of follow-up.

Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform.

BACKGROUND: In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans. METHODS: We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses). RESULTS: We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%). CONCLUSIONS: Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.

Supine lateral radiographs at 90° of knee flexion have a similar diagnostic accuracy for chronic posterior cruciate ligament injuries as stress radiographs.

PURPOSE: This study aimed to evaluate whether supine lateral radiographs (SLRs) could replace stress radiographs for diagnosing chronic posterior cruciate ligament (PCL) injuries and identifying combined PCL injuries (defined as PCL injury with medial collateral ligament or posterolateral ligament complex injury). METHODS: In this retrospective study, both SLRs at 30° and 90° of knee flexion (30/90 SLRs) and Telos stress radiographs of patients with chronic PCL injuries (n = 38) and only 30/90 SLRs of healthy controls (n = 84) were taken. Injured-to-normal differences on 30/90 SLRs and stress radiographs were assessed. Correlation analysis was performed to evaluate injured-to-normal differences on 30/90 SLRs and stress radiographs in patients with chronic PCL injury. Subgroup analysis was performed to compare injured-to-normal differences on 30/90 SLRs and stress radiographs between the isolated and combined PCL injury groups. Receiver operating characteristic curves based on 30/90 SLRs were calculated to determine the cut-off value for diagnosing chronic PCL injury and identifying combined PCL injury. RESULTS: Injured-to-normal differences on both 30 SLRs (3.1 ± 3.6 vs 1.6 ± 1.2, P = 0.019) and 90 SLRs (7.5 ± 3.5 vs 1.2 ± 1.0, P < 0.001) were significantly greater in patients with chronic PCL injuries than in healthy controls. Further, 90 SLRs had a highly accurate diagnostic value for chronic PCL injuries (area under the curve 0.958). The cut-off value for diagnosing chronic PCL injuries based on 90 SLRs was 3.0 mm (sensitivity, 94.7%; specificity, 92.9%). Injured-to-normal differences on 30/90 SLRs were significantly correlated with those on stress radiographs. The correlation coefficients were 0.397 (P = 0.014) for 30 SLRs and 0.605 (P < 0.001) for 90 SLRs. The cut-off value for diagnosing combined PCL injuries based on 90 SLRs was 9.6 mm (area under the curve 0.72). CONCLUSIONS: The diagnostic accuracy of 90 SLRs for chronic PCL injuries was similar to that of stress radiographs. Therefore, the 90 SLRs are reliable alternative method to assess the posterior knee laxity when the stress radiographs are not available. LEVEL OF EVIDENCE: Level IV, case series.

An I436N substitution confers resistance of influenza A(H1N1)pdm09 viruses to multiple neuraminidase inhibitors without affecting viral fitness.

The resistance of influenza viruses to neuraminidase (NA) inhibitors (NAIs; i.e. oseltamivir, zanamivir, peramivir and laninamivir) can be associated with several NA substitutions, with differing effects on viral fitness. To identify novel molecular markers conferring multi-NAI resistance, the NA gene of oseltamivir-resistant (H275Y, N1 numbering) 2009 pandemic influenza [A(H1N1)pdm09] virus was enriched with random mutations. This randomly mutated viral library was propagated in Madin-Darby canine kidney (MDCK) cells under zanamivir pressure and gave rise to additional changes within NA, including an I436N substitution located outside the NA enzyme active site. We generated four recombinant A(H1N1)pdm09 viruses containing either wild-type NA or NA with single (I436N or H275Y) or double (H275Y-I436N) substitutions. The double H275Y-I436N mutation significantly reduced inhibition by oseltamivir and peramivir and reduced inhibition by zanamivir and laninamivir. I436N alone reduced inhibition by all NAIs, suggesting that it is a multi-NAI resistance marker. I436N did not affect viral fitness in vitro or in a murine model; however, H275Y and I436N together had a negative impact on viral fitness. Further, I436N alone did not have an appreciable impact on viral replication in the upper respiratory tract or transmissibility in ferrets. However, the rg-H275Y-I436N double mutant transmitted less efficiently than either single mutant via the direct contact and respiratory droplet routes in ferrets. Overall, these results highlight the usefulness of a random mutagenesis approach for identifying potential molecular markers of resistance and the importance of I436N NA substitution in A(H1N1)pdm09 virus as a marker for multi-NAI resistance.