In this study, we investigated the clinical factors, level of serum TNF-α, and polymorphism of c.−308G > A TNF − α gene in inhibitor development in severe PWHA.  The development of factor VIII (FVIII) inhibitor in patients with hemophilia A (PWHA) is a great challenge for hemophilia care. Both genetic and environmental factors led to complications in PWHA. The development of inhibitory antibodies is usually induced by the immune response. Tumor necrosis factor α level were assessed. The genotyping of −380G > A TNF-α gene polymorphism was performed using polymerase chain reaction and Sanger sequencing.  A cross-sectional study was conducted among all PWHA in West Java province. The clinical parameters, FVIII, FVIII inhibitor, and serum TNF-α level and the development of HTI (p = 0.043). The cutoff point of serum TNF-α level, which can be used to differentiate between HTI and LTI, was 11.45 pg/mL. The frequency of FVIII replacement therapy was significant only in HTI of severe PWHA regarding serum TNF-α level (p = 0.028). There is no correlation between polymorphisms of −380G > A TNF-α gene and inhibitor development (p = 0.645).  Among the 258 PWHA, 216 (83.7%) were identified as severe PWHA. The FVIII inhibitor was identified in 90/216 (41.6%) of severe PWHA, consisting of 45 high-titer inhibitors (HTI) and 45 low-titer inhibitors (LTI). There was a significant correlation between serum TNF-α level might be used to differentiate between high and low inhibitor levels in severe hemophilia A, and this might support decision making regarding treatment options for inhibitor in severe hemophilia A. The prevalence of FVIII inhibitor in severe PWHA in West Java, Indonesia, was 41.6%. The frequency of replacement therapy is a risk factor for inhibitor development. Serum TNF- Hemophilia A, the most frequent hereditary bleeding disorder, is an X-linked bleeding disorder due to deficiency in coagulation factor VIII (FVIII) that affects one individual in 5.000–10.000 newborn males . HemophiThe development of an immunogenic response that is characterized by the appearance of alloantibodies called inhibitors against the therapeutic FVIII infused to the patient causes its neutralizing effect of treatment , 4. It iThe prevalence of inhibitor FVIII in severe PWHA varies from 25% to 30%, and it is less often in those with mild/moderate disease and involved multifactorial factors, such as genetic and environmental factors , 5–7. Prα (TNF-α) is considered an important cytokine with potent proinflammatory and immunomodulatory functions associated with autoimmune antibody-mediated diseases included in FVIII inhibitor development. Some studies found polymorphisms of several cytokine genes are risk factors for inhibitor development in PWHA. The TNF-α −308GA polymorphism located in the promoter regions is considered the most extensively studied polymorphism with pathophysiologic effects [Furthermore, individual immune response properties may also affect a patient's reaction to exogenous FVIII, which include certain major histocompatibility complex (MHC) class II system of cytokines and their polymorphisms . Tumor n effects . Environ effects , 10. It  effects . This stThis cross-sectional study included all PWHA (258 patients) who registered at West Java Hemophilia A Registry until 2016 . PatientWritten informed consent for participation in the study was obtained from patients; and for those whose age is <12 years, the consent was obtained from their parents.Ethical approval was obtained from the Ethical Committee of Dr. Hasan Sadikin General Hospital/Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia. This study was conducted in accordance with the principles of the Declaration of Helsinki.α level, also blood sample for polymorphism of −308G > A TNF-α gene test. FVIII and FVIII inhibitor levels were measured using Sysmex CS 2500, and serum TNF-α was measured using the ELISA method . Bethesda assay was used to measure the presence of FVIII inhibitor. FVIII level < 1% was classified as severe hemophilia A, inhibitor level < 5 Bethesda unit (BU) was defined as low titer, and inhibitor level ≥ 5 BU was defined as high titer. All of the measurements were performed in the Clinical Pathology Laboratory of Dr. Hasan Sadikin General Hospital.The clinical parameters of all subjects were assessed to determine age, ethnicity, bleeding episode, age at first therapy, frequency of therapy, and type of FVIII replacement therapy. Data were obtained from patient's medical records and the Indonesian Hemophilia Registry branch of West Java. When this study was conducted, all of the PWHA who classified as severe hemophilia A were in healthy condition, and no signs and symptoms of infection were detected. Peripheral venous blood samples were collected, and a routine hematology test was performed. They were reexamined for the F VIII level to confirm the level regarding the research inclusion at the same time with inhibitor and serum TNF-α gene in subjects, PCR and DNA sequencing were performed in the Molecular Genetics Laboratory of Faculty of Medicine, Universitas Padjadjaran.To investigate the polymorphisms of −308G > A TNF-α level and polymorphism of −308G > A TNF-α gene using independent t-test or Mann–Whitney test. The correlation between serum TNF-α level and inhibitors was stratified by bleeding episode, age of first therapy, and frequency of therapy using the Mann–Whitney test. A p value of < 0.05 was considered statistically significant.Data analyses were performed using SPSS software version 23. Nongenetic risk factor data, including bleeding episode, age at first therapy, frequency of therapy, and product type of VIII replacement therapy, were evaluated using the Kolmogorov–Smirnov test and chi-square test. There was an association between the presence of inhibitor and the serum TNF-The data of 258 PWHA in West Java province were collected from the registry: 216 (83.7%) severe PWHA, 19 (7.4%) moderate PWHA, and 23 (8.9%) mild PWHA. Among the 216 severe PWHA, 90 (41.6%) patients expressed FVIII inhibitor. The mean FVIII inhibitor level was 4.79 (2.00) BU/mL with a range of 0.90-11.04 BU/mL. Forty-five patients with HTI had a mean value of 6.46 (1.24) BU/mL, and its range was 5.04-11.04 BU/mL, whereas the mean of other 45 patients with LTI was 3.13 (0.95) BU/mL with a range of 0.90-4.96 BU/mL. α level that widely varies with a range of 6.5-302.5 pg/mL was 11.34 pg/mL. α levels between two groups of inhibitors, and it is significantly different (p = 0.043). On the basis of Spearman's correlation analysis, there was no significant correlation between serum TNF-α level and inhibitor (p = 0.140).In this study, the median serum TNF-α level used as a predictor of HTI was 11.45 pg/mL  pg/mL versus 12.81 (range 7.73-97.4) pg/mL, respectively. TNF-α level based on age of the start of therapy for <12 months had a higher median in the HTI group than in the LTI group, 14.05 (range 10.03-18.8) pg/mL versus 9.29 (range 8.56-15.9) pg/mL, respectively. Likewise, TNF-α level for frequency ≥ 2 times/month in FVIII frequency replacement therapy had a higher median in the HTI group than in the LTI group, 13.10 (range 7.73–97.4) pg/mL versus 11.25 (range 8–302.5) pg/mL, respectively. On the basis of two clinical variables, bleeding episodes and age of the start of therapy, the correlation between TNF-α level and FVIII inhibitor was not statistically significant. Meanwhile, the correlation between TNF-α levels in the HTI group was influenced by FVIII frequency replacement therapy (p < 0.05)  .α gene was demonstrated in α gene.The assessment of PCR and DNA sequencing polymorphism of −308G > A TNF-p = 0.645). On the basis of an independent t-test, there was no significant difference between polymorphisms of −308G > A TNF-α gene and inhibitor (p = 0.645)  had normal genotype (guanosine–guanosine (GG)), 43 (47.8%) subjects had LTI, and 42 (46.7%) subjects had HTI. However, only 5 subjects had polymorphism genotype (guanosine–adenine (GA)), 2 subjects with LTI, and 3 subjects with HTI (= 0.645) .West Java is the province with the biggest population in Indonesia, including almost 18% of the total population (46 million of 261 million inhabitants). In this study, the prevalence of severe hemophilia A with inhibitor in West Java was 41.6% with a range of inhibitor level being 0.9-11.4 BU/mL. It is reported that the prevalence of inhibitor development  might vary between 3.6% and 52% based on ethnicity , 13. StuThe incidence of inhibitor formation varies on the basis of ethnicity with higher rates found among African-American, Latino, and Hispanic patients than among Caucasian patients , 11, 22.The subject's ABO blood group distribution that developed LTI and HTI groups in this study mostly was A and O (as seen in Hemarthrosis was the most frequent bleeding location developed in both LTI and HTI groups (34.5% versus 36.7%) but not statistically significant. In this study, another frequent bleeding location that was a combination of hemarthrosis- and hematomas-developed inhibitor. A previous study stated that hemarthrosis is a typical bleeding manifestation found in severe hemophilia, especially in patients receiving no maintenance treatment or prophylactic FVIII treatment . Other pβ, TNF-α, and IL-6. Inflammation and proinflammatory cytokines  might be involved in this pathogenesis [α accumulation was found in the hemorrhagic tissues of the injured knee and strong TNF-α gene upregulation observed since day 3 up to 30 days after hemarthroses. Elevated TNF-α level was found in the synovial fluid and plasma which TNF-α level in synovial fluids was 5.2-20 folds higher than in plasma (p < 0.05) in PWHA [In severe hemophilia A, recurrent clinical and subclinical joint bleeding episodes might occur all the time, gradually leading to irreversible changes such as hemophilic arthropathy (HA) , 25. In ogenesis . Both ac in PWHA .p = 0.561). This result is in line with a 25-year case–control study by Maclean et al. in 2011, which showed that there was no correlation between age of therapy and inhibitor development [Most patients reported experiencing inhibitors after ≥2 bleeding episodes. In this study, it did not differ significantly between LTI and HTI groups (38.9% versus 41.1%). Saifudin et al. also reported that bleeding episodes did not affect the inhibitor development in severe PWHA . In thiselopment . Santagoelopment . Howeverelopment .Environmental factors that have been suggested to influence the risk of developing inhibitors include both treatment related  and immune system activating risk factors  . On-demaIn Indonesia, all of the PWHA only received on-demand replacement therapy with plasma-derived FVIII. Fifty-eight subjects who received plasma-derived FVIII infusion developed more inhibitors, especially in HTI groups (32/58). Furthermore, 32 subjects who received mixed therapy (sometimes receiving recombinant or cryoprecipitate) developed lower level inhibitor (19/32) rather than a high-titer inhibitor (13/32). However, these findings were not statistically correlated. Several previous studies concluded different results. The study by Wight and Paisley and the CANAL cohort study demonstrated that some plasma-derived FVIII products may conclude a lower risk of inhibitor development than recombinant FVIII in previously untreated patients (PUPs) with severe hemophilia , 34. A mp = 1.000), but in further statistical analysis, there was a correlation between the frequency of therapy ≥ 2 times/month and the HTI level in high serum TNF-α levels. The more the frequent exposure to FVIII replacement therapy, the more the risk of FVIII antibody formation, so it is a higher risk for FVIII inhibitor development as reported by Maclean et al. in 2011 [In this study, there was no significant association between the frequency of replacement therapy and FVIII inhibitor development . The development of inhibitory antibodies against exogenous FVIII is usually considered a Th2 cell-induced immune response, whereas TNF-α is primarily linked to Th1 cells. However, the cytokine profile clearly indicates the formation of inhibitors to be a mixed Th1 and Th2 cell response, which further underscores the fact that the level of TNF-α may also modulate the immune response to the deficient factor in PWHA [in vitro stimuli with FVIII [Some studies suggested that both cytokine patterns and polymorphisms play crucial roles in inhibitor generation . TNF-α i in PWHA . It was  in PWHA , 42. Thes p = 0.0. The devα. Furthermore, the −308A allele has been associated with increased constitutive and inducible transcription levels and with increased production and secretion of TNF-α in patients with autoimmune diseases and healthy controls compared with the −308G allele [α with inhibitor development in PWHA [α gene  in 164 PWHA. Inhibitors were identified in 46.9% of the patients with −308G/A heterozygote gene and implied that −308G > A TNF-α gene polymorphism in Hap2 is a useful marker in PWHA [α was observed with a higher frequency in the inhibitor cohort than in the noninhibitor cohort that was more pronounced for the homozygous A/A genotype [α gene was not statistically associated with inhibitor development (p = 0.645).In some autoimmune diseases, namely, inflammatory bowel diseases and myasthenia gravis, there is an association between polymorphism and increased levels of TNF-genotype . These fα polymorphism and inhibitor level was not statistically significant. It is assumed that the high level of serum TNF-ɑ was not associated with role of the −380G > A TNF-α gene polymorphism, or single polymorphism of cytokine gene was not enough to influence the inhibitor development. Difference characteristic related genetic  background, meanwhile the inhibitor development related to high serum TNF-ɑ level can be induced by other inflammatory substance from damage tissue of the joint. Further studies are required to reinforce these findings. Additionally, there are still many factors that influence the FVIII inhibitor development in severe hemophilia A. It seems that genetic factors predominate in inhibitor development.Our studies showed that serum TNF-ɑ level correlated with HTI. Nevertheless, the correlation between −380G > A TNF-Some of the limitations of the current study could be noted. Because of the cost constraints and limited facilities in this study, not all parameters related to FVIII inhibitor development, such as FVIII gene mutation type, other cytokine levels, and their polymorphisms, were assessed. The levels of all parameters were measured once (these could be the involvement of transient inhibitor), and therefore, there were some factors affecting inhibitor development, which could be missed. The biomarker of inflammation was not measured.α levels in differentiating between high and low inhibitor levels in severe hemophilia A. This finding seems to support decision making on the treatment option of inhibitor in severe hemophilia A. Meanwhile, the frequency of replacement therapy was significantly different between low- and high-titer FVIII inhibitors regarding serum TNF-α levels. It seems that genetic factors predominate in inhibitor development.The prevalence of inhibitors in severe PWHA in West Java, Indonesia, was high. This study provides data on the potential of using serum TNF-


Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death worldwide. Many patients with acute exacerbations need intensive care. There are many cardiovascular risk factors and comorbid conditions linked with COPD. Troponin elevation is used for the diagnosis of myocardial infarction. However, it is commonly elevated in patients with COPD. This systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. PubMed and Scopus were searched for relevant articles. A total of 383 papers were identified. Out of the 383 papers, only 30 papers remained for final synthesis after removing duplicates, screening for relevant articles, and assessing eligibility criteria. After the quality appraisal, 11 papers were chosen for inclusion in this study. COPD is characterized by obstruction and inflammation of the airways. It is caused mainly by smoking, by causing harmful changes in the structure of the airways. It usually presents with dyspnea, cough, and/or production of sputum. Troponins are regulatory proteins found in the myocardium and skeletal muscles. The cause of its elevation in COPD and acute exacerbated chronic obstructive pulmonary disease (AECOPD) remains unclear. However, several reasons and factors have been suggested. The most intriguing fact is that high troponin in COPD, especially in exacerbations, has been linked in many articles to a higher risk of death. Furthermore, it could serve as a tool for better assessment and management of COPD patients. We found that troponin predicts death and poor outcomes in COPD and AECOPD. The exact mechanism of its elevations is not clear. We believe it can be a valuable tool for clinicians in better managing COPD and assessing the severity of the exacerbations. Chronic obstructive pulmonary disease (COPD) is one of the leading causes of mortality and morbidity in the world. It is ranked the fourth cause of mortality worldwide, representing a challenge to public health that can be prevented and treated. Furthermore, the World Health Organization (WHO) predicts that COPD will become the third leading cause of death worldwide by 2030 . Most ofMethodsProtocolThis review article followed the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines .Inclusion/Exclusion CriteriaInclusion criteria were all studies in the English language, all types of studies were included. The studies were collected from inception to March 22, 2021. Studies mentioning a relationship between troponin and COPD patients were only included, and patients were included with no regard to age, gender, sex, or ethnicity. Exclusion criteria were studies that are not in the English language, studies not related to the question of the review, editorials, and letters to the editors.Search Strategy A detailed search of PubMed, PubMed Central, Medical Literature Analysis and Retrieval System Online (MEDLINE), and Scopus was conducted, and the search results were imported to EndNote Web . PubMed was searched using the keywords: (troponin) AND [(COPD) OR (exacerbated COPD)] OR (acute exacerbated COPD) which yielded 175 papers. And using Medical Subject Headings (MeSH) keywords - ("Troponin"[MeSH]) AND "Pulmonary Disease, Chronic Obstructive"[MeSH] - yielded 63 papers. Scopus database search within titles, abstracts, and keywords using the words 'Troponin AND COPD' yielded 145 papers.A total of 383 papers were identified and imported to EndNote Web. Duplicates were removed by EndNote and checked manually by the author, leaving only 238 papers. The results of each search are shown in Table Study SelectionAfter duplicates removal, a total of 238 papers were screened for title and abstracts identifying 54 papers that are relevant to the review question. Of these papers, 24 were excluded based on reasons shown in the PRISMA diagram Figure . After tQuality AssessmentCritical appraisal was performed independently by the first and second authors manually, and three papers were excluded.ResultsA total of 383 papers were identified by searching the databases. After duplicates were removed, 238 papers remained, of which, 184 papers were excluded for being irrelevant. The remaining 54 papers were assessed for eligibility criteria. Twenty-four papers were removed. Thirty papers were included in the final synthesis. Quality assessment of these papers was performed by the first and second authors independently. After the quality appraisal, 27 were of good quality. The papers chosen for this review included five prospective studies, two retrospective studies, two cross-sectional studies, one systematic review, and one comparative study. Included studies are shown in Table Pathophysiology of COPDChronic obstructive pulmonary disease (COPD) is defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) as a disease characterized by limitations of airflow which is not fully reversible . COPD isImportance of measuring troponinTroponin is part of the myocardium and the skeletal muscles . TroponiFactors contributing to troponin elevation in COPDThe mechanism of elevation still is unknown ,10. The Predicting the outcome of COPD using troponinIn a systematic review of 14 studies, troponin was associated with mortality . The morClinical value of measuring troponin during COPD exacerbationsAssessing troponin levels in AECOPD patients helps in identifying those who are at higher risks for poor outcomes ,8. This LimitationsThe main limitation is the relatively small number of patients included in the studies. Also, in some patients, troponin measurements were performed based on the decision of the available clinician at the time of presentation. Also, different time points were studied in the included papers. Hence, further large studies should be conducted for different time points. It is worth mentioning that the studies could have missed acute coronary syndrome in patients with elevated troponin, interfering with the suggested causes of elevation in the studies. How the included studies defined COPD and AECOPD were not the same throughout the papers. This could lead to the false inclusion of patients in the studies. Two of the included studies are cross-sectional in design, limiting the interpretation of the cause and effect relationships in these studies. Another limitation is the relatively narrow patient demographic since most of the included studies were conducted in Norway or New Zealand. Furthermore, we limited our study to papers in the English language and free full-text papers. Measuring troponin in COPD patients was found to predict the outcome of the patients. High troponin predicts worse prognosis and death, ranging from in-hospital death to death after discharge. The cause of elevation of troponin in COPD and AECOPD patients remains unclear. Troponin could be used to assess COPD patients along with other routine investigations to provide a better understanding of the condition of the patients. However, it is not known how troponin levels can be used as a tool for better managing patients with COPD and AECOPD. This paper will help the clinicians to understand the possible factors causing troponin elevation and how different levels of troponin predict different outcomes. Further studies are needed to identify the possible cause(s) for elevated troponin in COPD and AECPD and to study how its measurement could be used in clinical practice to better manage COPD patients, especially during exacerbations.


Neuroinflammation plays an important role in the pathogenesis of glaucoma. Tumor necrosis factor-alpha (TNF-α) is a major pro-inflammatory cytokine released from activated retinal glial cells in glaucoma. Here, we investigated how TNF-α induces retinal ganglion cell (RGC) hyperexcitability and injury.+ currents in RGCs. Both intravitreal injection of TNF-α and chronic ocular hypertension (COH) models were used. Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (q-PCR), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) techniques were employed to investigate the molecular mechanisms of TNF-α effects on RGCs.Whole-cell patch-clamp techniques were performed to explore changes in spontaneous firing and evoked action potentials, and Na+ current densities were upregulated. Perfusing TNF-α in RGCs of normal rats mimicked this effect, and the activation curve of Na+ currents shifted toward hyperpolarization direction, which was mediated through p38 MAPK and STAT3 signaling pathways. Further analysis revealed that TNF-α selectively upregulated Nav1.6 subtype of Na+ currents in RGCs. Similar to observations in retinas of rats with COH, intravitreal injection of TNF-α upregulated the expression of Nav1.6 proteins in both total cell and membrane components, which was reversed by the NF-κB inhibitor BAY 11-7082. Inhibition of TNFR1 blocked TNF-α-induced RGC apoptosis.Intravitreal injection of soluble TNF-α significantly increased the spontaneous firing frequencies of RGCs in retinal slices. When the synaptic transmissions were blocked, more than 90% of RGCs still showed spontaneous firing; both the percentage of cells and firing frequency were higher than the controls. Furthermore, the frequency of evoked action potentials was also higher than the controls. Co-injection of the TNF-α receptor 1 (TNFR1) inhibitor R7050 eliminated the TNF-α-induced effects, suggesting that TNF-α may directly act on RGCs to induce cell hyperexcitability through activating TNFR1. In RGCs acutely isolated from TNF-α-injected retinas, Na+ channels, thus contributing to RGC apoptosis in glaucoma.TNF-α/TNFR1 signaling induces RGC hyperexcitability by selectively upregulating Nav1.6 Na Glaucoma, the leading cause of irreversible blindness, is a retinal neurodegenerative disease, which affects over 60 million people around the world –3. Progr2+-permeable GluA2-lacking AMPA receptors in RGCs, thus promoting RGC death quinoxaline , BAY 11-7082 (10 μM), stattic (10 μM), or SB203580 (10 μM)  was intravitreally injected in the same manner. The eyes that received saline injection were used as controls. Details of the operation were described in our previous reports [Recombinant rat TNF-α  (2 μl) was injected into the right vitreous cavity of the anesthetized rat with a micro-injector  under a stereoscopic microscope (Carl Zeiss). Given that the vitreous volume is ∼20 μl , 34, the reports , 35.Rat COH model was produced in accordance with previous studies , 35. In 2 and 5% CO2) artificial cerebral spinal fluid (ACSF) containing the following (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 CaCl2, 1 MgCl2, and glucose 10 (pH 7.4) for 30 min at room temperature (22–24°C) before recording. To isolate RGCs, the retinas of anesthetized rats were removed quickly and incubated in the oxygenated Hank’s solution containing the following (in mM): NaCl 137, NaHCO3 0.5, NaH2PO4 1, KCl 3, CaCl2 2, MgSO4 1, HEPES 20, and glucose 16 (pH 7.4). After digesting in Hank’s solution with 1.6 U/ml papain  and 0.2 mg/ml L-cysteine for 26 min at 34°C, retinal neurons were obtained by mechanical dissociation. The detailed procedures were described in our previous reports [Rat retinal slices (200 μm in thickness) were made on a Leica Vibrotome (VT1000S) and incubated in carbogen saturated (95% O reports .Whole-cell patch-clamp experiments were performed using a patch-clamp amplifier (Axopatch 700B) and Digidata 1440A  at room temperature. Patch pipettes  were pulled by a P-97 Flaming/Brown micropipette puller (Sutter Instrument) with a resistance of 5–8 MΩ after fire-polishing .d-gluconate, 1 EGTA, 10 HEPES, 4 ATP-Mg, 0.3 GTP-Na, 10 phosphocreatine, 0.1 CaCl2, 1 MgCl2, and Alexa Fluor 488 . To record Na+ currents, the bath solution was consisted of (in mM) 130 NaCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 15 tetraethylammonium (TEA)-Cl, 10 4-aminopyridine (4-AP), and 10 glucose . The patch pipette solution contained (in mM) 130 CsCl, 10 NaCl, 5 HEPES, 8 EGTA, 10 TEA-Cl, 2 ATP-Mg, and 1 GTP-Na . Na+ currents were induced by a series of 50 ms depolarizing voltage pulses from a holding potential of −70 to +30 mV in increments of 10 mV. The inactivation curves of voltage-gated sodium channels (VGSCs) were detected by giving RGC a 200 ms pre-pulse from −70 mV  to different voltages and then depolarizing to −10 mV. Both activation and inactivation curves were fitted by Boltzmann function.For spontaneous firing recordings in RGCs, the individual retinal slice was continuously superfused with oxygenated ACSF at a rate of 1–2 ml/min at room temperature. RGCs in retinal slices were identified with the help of infrared-differential interference contrast (IR-DIC) video microscopy  as described , 38. PatWestern blotting analysis was conducted following the procedures previously described . BrieflyThe protocol of immunofluorescence staining was followed as described in previous studies . In shorTo detect neuronal apoptosis, the deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay was performed on whole flat-mounted retinas . The Deauantitative real-time polymerase chain reaction (q-PCR) was carried out using the PrimeScript™ RT reagent Kit with gDNA Eraser , and mRNA was measured using a TB Green® Premix Ex Taq™ II . The qPCR assays were performed on the QuantStudio 3 Real-Time PCR system . The relative mRNA levels were normalized from Ct values according to 2-△△ct calculation method. The sequences of primers used in this study are as follows: Nav1.1, forward 5′-GCG ATT ATG TGA CAA GCA TTT TG-3′, reverse 3′-CGG AGG GAG ATG AGC TTC AG-5′; Nav1.2, forward 5′-TTC ATG GCT TCC AAT CCC TCC-3′, reverse 3′-GGT GTC ACG TCA GTC TTC TCT-5′; Nav1.6, forward 5′-GCA AGC TCA AGA AAC CAC CC-3′, reverse 3′-CCG TAG ATG AAA GGC AAA CTC T-5′; β-actin, forward 5′-AGC CAT GTA CGT AGC CAT CC-3′, reverse 3′-CTC TCA GCT GTG GTG GTG AA-5′.Total RNA was extracted from the whole retina using the TaKaRa MiniBEST Universal RNA Extraction Kit . Qd-(-)-2-amino-5-phosphonopentanoic acid (D-APV), bicuculline, CNQX, 4,9-anhydrotetrodotoxin (AHTTX), and TTX were purchased from Tocris . BAY 11-7082, SB203580 and stattic were from Selleck , and the others were from Sigma-Aldrich .R7050, n” represents cell number in electrophysiological experiments, or animal number in Western blotting, q-PCR, and TUNEL experiments. A Boltzmann function was used to fit the activation and inactivation curves. All experiments and measurements were performed in quadruplicate minimally and analyzed by t test, Brown-Mood test, Mann-Whitney test, or one-way ANOVA with Bonferroni’s post hoc test (multiple comparisons). Before performing t test or one-way ANOVA analysis, the data were analyzed using Shapiro-Wilk test or Brown-Forsythe test to evaluate the normality or the homogeneity of variance. Membrane potential and frequency of spontaneous firing were presented as a median and interquartile range, while the other data were expressed as mean ± SEM. The threshold for statistical significance was P < 0.05 in all tests.Data were analyzed using GraphPad Prism , Clampfit 10.2 , Igor 4.0 , and Origin 2018 . In this work, the “n = 11, P = 0.0018) and 3.40 Hz   in TNF-α3D and TNF-α7D groups, respectively, from the control value of 0.41 Hz  (n = 12) , n = 11, P = 0.0026), as compared to the control group , n = 11)  and 4.22 Hz   in TNF-α3D and TNF-α7D groups, respectively, from the control value of 0.23 Hz  (n = 13) , n = 11, P < 0.001 vs. Ctr; R 7050+TNF-α7D: 0.95 Hz , n = 14, P < 0.001 vs. TNF-α7D alone)  and 143.9 ± 12.4% of control  in TNF-α3D and TNF-α7D groups, respectively. As a positive control, the TNF-α protein levels were increased to 158.5 ± 11.7% of control in COH retinas at week 2 (COH2W)    from the control value of −124.6 ± 4.45 pA/pF (n = 11)  application. TNF-α significantly and voltage-dependently enhanced peak Na+ current densities , and washout pushed the currents almost to control levels  that was significantly different from control value   , a p38 MAPK inhibitor, for 30 min . Similar results were obtained when RGCs were pre-incubated with Stattic (10 μM), a STAT3 inhibitor   and signal transducer and activator of transcription 3 (STAT3) signaling pathways were involved in TNF-α-mediated effects , 44. The+ channels, and Nav1.6 is the predominant one [+ currents. The average current amplitudes were increased to 128.3 ± 4.3% of control   ; however, it was increased to 126.5 ± 12.39% of control  in the TNF-α7D group. As a positive control, the total protein level of Nav1.6 in COH2W was increased to 130.9 ± 8.9% of control , which was reversed by pre-injecting the TNFR1 blocker R7050 (10 μM)   were further examined. Figure n = 4, P < 0.001; TNF-α7D: 143.9 ± 8.2% of control, n = 4, P = 0.0253). In COH retinas, mNav1.6 expression was also increased to 169.6 ± 14.6% of control, n = 4, P < 0.001). Consistently, double immunostaining showed that Nav1.6 was primarily expressed in the ganglion cell layer (GCL) and the inner plexiform layer (IPL), and intravitreal injection of TNF-α increased Nav1.6 expression, especially in the RGC axon-like structures    Fig. a, b. In n = 7, P < 0.001) and 202.0 ± 18.4  in TNF-α3D and TNF-α7D groups, respectively, which could be reversed by co-injection of the TNFR1 blocker R7050    Fig. b. Similaup) Fig. b.Fig. 1It is commonly known that neuroinflammation plays a key role in the pathogenesis of glaucoma although the detailed mechanisms have not been completely elucidated , 48. As In addition, Margolis and Detwiler reported that ON and OFF RGCs showed distinct spiking properties in the mouse retina: resting activity of ON RGCs was dependent on tonic excitatory inputs, whereas OFF RGCs exhibited properties of pacemaker neurons and continued to fire in the absence of synaptic inputs . HoweverIh) and inwardly rectifying potassium channel (Kir) [It should be noted that in dissociated RGCs, most dendrites and part of the axon of RGC may be destroyed during the isolated process. In this study, we usually chose the RGC with axon for recordings. Since dendrites were destroyed in these cells, some ion channels mainly expressed in dendrites, such as hyperpolarization-activated cation channel (el (Kir) , were lo2+ influx through voltage-gated Ca2+ channels, together with activated Ca2+-permeable GluA2-lacking AMPA receptors [2+ overload and triggering cellular death signaling [In the central nervous system (CNS), pleiotropic effects of TNF-α are mainly mediated by two signaling pathways, soluble TNF-α/TNFR1, and transmembrane TNF-α/TNFR2, which may lead to opposing outcomes, deleterious, or beneficial effects for neurons –22, 58. eceptors , resultiignaling .+ channels may change neuronal excitability [+ currents, as evidenced by the following facts. Firstly, intravitreal injection of TNF-α significantly and voltage-dependently increased Na+ current density in RGCs. Secondly, external application of TNF-α reversibly increased Na+ currents. Thirdly, TNF-α shifted the activation curves of Na+ currents toward hyperpolarizing direction, suggesting that TNF-α resulted in Na+ channel activation easier. These results are consistent with observations in dorsal root ganglion (DRG) neurons that TNF-α promoted neuronal excitability by increasing sodium channel currents densities, thus contributing to neuropathic pain hypersensitivity [VGSCs are essential for AP generation and conduction. Modulation of Natability , 62. We sitivity , 63–65.+ channels, such as Nav1.1 and Nav1.2 [+ channels that determine neuronal excitability in the CNS [+ channels was found expressed in mouse RGCs [+ channels, while TTX-resistant Na+ channels seem unlikely expressed in rat RGCs [+ currents, but did not influence the increased Nav1.6 protein expression due to longer TNF-α exposure (7 days), suggesting that these two signaling pathways may directly modulate Na+ channels in a transcriptional-independent manner. Indeed, previous studies reported that p38 MAPK was involved in the upregulation of different subtypes of Na+ currents [+/HCO3- cotransporter 1 [+ channels, such as Nav1.7 and Nav1.3 [Scn8a promoter in DRGs [+ currents by TNF-α in acutely isolated RGCs. In addition, immunohistochemical experiments showed that TNF-α-induced increase in Nav1.6 expression was mainly found in the axon-like structures. It was reported that Nav1.6 expressed in the distal of the AIS of cortical pyramidal neurons promoted action potential initiation [One of the major findings in this study is that TNF-α induced RGC hyperexcitability was mediated by selectively upregulating Nav1.6 currents although RGCs express the other subtypes of Nad Nav1.2 –47. Nav1 the CNS , 66. Eveuse RGCs , 68, ourrat RGCs , 44. It currents , 69–71, porter 1 . Howeverporter 1 . It is pd Nav1.3 , 75. It d Nav1.3 , and TNF in DRGs . Howeveritiation . TherefoNeuroinflammation plays an important role in the pathogenesis of multiple neurodegenerative diseases, including glaucoma, Alzheimer’s disease (AD), and Parkinson’s disease (PD) –81. Nav1Taken together, all these results demonstrated that proinflammatory cytokine TNF-α induced the hyperexcitability of RGCs by enhancing Nav1.6 currents and protein expression through activating TNFR1, thus contributing to RGC apoptosis in glaucoma. Our results revealed a novel mechanism of TNF-α in glaucoma and provided a promising therapeutic target.


While the adoption of genomic evaluations in livestock has increased genetic gain rates, its effects on genetic diversity and accumulation of inbreeding have raised concerns in cattle populations. Increased inbreeding may affect fitness and decrease the mean performance for economically important traits, such as fertility and growth in beef cattle, with the age of inbreeding having a possible effect on the magnitude of inbreeding depression. The purpose of this study was to determine changes in genetic diversity as a result of the implementation of genomic selection in Angus cattle and quantify potential inbreeding depression effects of total pedigree and genomic inbreeding, and also to investigate the impact of recent and ancient inbreeding.We found that the yearly rate of inbreeding accumulation remained similar in sires and decreased significantly in dams since the implementation of genomic selection. Other measures such as effective population size and the effective number of chromosome segments show little evidence of a detrimental effect of using genomic selection strategies on the genetic diversity of beef cattle. We also quantified pedigree and genomic inbreeding depression for fertility and growth. While inbreeding did not affect fertility, an increase in pedigree or genomic inbreeding was associated with decreased birth weight, weaning weight, and post-weaning gain in both sexes. We also measured the impact of the age of inbreeding and found that recent inbreeding had a larger depressive effect on growth than ancient inbreeding.In this study, we sought to quantify and understand the possible consequences of genomic selection on the genetic diversity of American Angus cattle. In both sires and dams, we found that, generally, genomic selection resulted in decreased rates of pedigree and genomic inbreeding accumulation and increased or sustained effective population sizes and number of independently segregating chromosome segments. We also found significant depressive effects of inbreeding accumulation on economically important growth traits, particularly with genomic and recent inbreeding.The online version contains supplementary material available at 10.1186/s12711-021-00644-z. It has been more than two decades since the idea of using genomic markers to increase the prediction accuracy of an animal’s genetic value was first laid out . Since tIn the early days of GS, there was speculation about its impact on the accumulation of inbreeding and genetic diversity. Schaeffer  hypothes-Flemish , French -Flemish , and NorIn addition to the loss of genetic diversity associated with high levels of inbreeding , the accThe availability of genomic information has allowed us to capture Mendelian sampling variation and determine the realized inbreeding load of an animal, instead of only the expected load based on pedigree relationships. In addition, it permits the analysis of animals/populations with incomplete or missing pedigree records. Genomic information can be used to estimate inbreeding depression due to marker or segment-based inbreeding. For example, Reverter et al.  found a Not all inbreeding is the same or is expected to have depressive effects on fitness. The ‘age’ of inbreeding is expected to moderate its effects, with older inbreeding having more time for purifying selection to act upon it and purge the population’s detrimental alleles. From this standpoint, pedigree and genomic inbreeding can be split into inbreeding age classes that permit the comparison of inbreeding depression caused by recent and old inbreeding. For example, recent pedigree and genomic inbreeding are more detrimental to milk and milk components yield, heifer and cow reproduction, and health traits than ancient inbreeding in Dutch  and CanaThe aims of our study were to (1) characterize the American Angus population in terms of pedigree and genomic inbreeding levels, (2) determine the magnitude and direction of changes to the rate of inbreeding, generation intervals, effective population size, and the effective number of chromosomal segments since the implementation of genomic evaluations, and (3) quantify the effects of recent and ancient inbreeding that affect reproductive and growth traits in Angus cattle.All data for this study were provided by the American Angus Association (AAA). In total, 569,364 American Angus individuals registered with the AAA born between 1969 and 2019 were used. Pedigree data was obtained from Angus Genetics, Inc.  and contained 1,372,734 animals, including 25,692 founders. Pedigree statistics, including number of complete generations (NCG), complete generation equivalents (CGE), and pedigree completeness index (PCI), were obtained using the optiSel R package . The CGEhttps://genomics.neogen.com/pdf/ag337_ggp_productportfoliobrochure.pdf), HD50K , i50K , and Angus GS v.1, AnGS hereafter, (https://www.neogen.com/neocenter/press-releases/angus-genetics-neogen-introduce-angus-gs-genomic-profile/). Single nucleotide polymorphisms (SNPs) that met one of the following criteria were excluded from the process: (1) SNPs on sex chromosomes; (2) a Mendelian inconsistency higher than 2%; (3) a call rate lower than 90%; and (4) a minor allele frequency lower than 0.1%. Animals with a call rate lower than 90% were removed. Taking advantage of overlapping SNPs between arrays and implementing a multi-step imputation process, all animals were imputed using the FImpute v3.0 program [Animals were genotyped with one of several genotyping platforms used over time at the AAA, including GGP HD, GGP HD150K, GGP LD , runs of homozygosity (ROH), and homozygous-by-descent (HBD) segments.Total pedigree inbreeding  to obtain partial inbreeding coefficients from approximately 2  , 9 on thThe effective number of independently segregating chromosomal segments , which is the binary phenotype of being either pregnant or open at the end of the breeding season. Records were also obtained for three different growth traits: birth weight (BiW), weaning weight (WW), and post-weaning gain (PWG) in males and females. Records for BiW, WW, and PWG were already adjusted for age of calf at time of measurement and records for BiW and WW were also adjusted for the age of dam. Only phenotypic records within three standard deviations of the mean were used. Summary statistics including number of animals, mean, median, standard deviation, and minimum and maximum values for HP, BiW, WW, and PWG, are in Table The effect of a 1% increase of pedigree and genomic inbreeding coefficients relating to recent and ancient inbreeding on the phenotype of heifer pregnancy and growth traits was quantified using a linear mixed model approach. For growth traits, male and female growth was treated as distinct and analyzed with separate regression analysis in order to uncover potential differences between sexes for inbreeding depression. The model used to estimate the regression coefficients of To estimate the effect of a 1% increase in recent and ancient pedigree and genomic inbreeding we arbitrarily grouped inbreeding classes based on whether they represent inbreeding acquired more recently or at a more distant generation, with the threshold for allocating the coefficients into one of the two classes being one of several that could be set. For pedigree inbreeding, we grouped inbreeding acquired 5 or fewer generations ago to create a recent pedigree inbreeding coefficient (Models where HP was the response variable were run using the THRGIBBS1F90 program (v.2.116)  and thosThe mean inbreeding of the population was 5.92% (SD = 2.41%) for  (29.2%) . The hig (29.2%) . Using gFor the partial inbreeding coefficients, the mean inbreeding ranged from 0.10% (SD = 0.03%) for PED with Pearson’s correlation coefficients were calculated for all pairs of inbreeding coefficients and are shown in a heatmap in Fig.  to 0.92 , 24, 27.The correlations of the classical inbreeding coefficient and the partial pedigree-based inbreeding coefficient ranged from low and negative , and birth weight (BiW), weaning weight (WW), and post-weaning gain (PWG) in males and females. The effects of a 1% increase in a cattle , 14. In a cattle , 25. Thea cattle , 36.Tabl, respectively. The effect of a 1% increase in inbreeding on WW ranged from − 0.50 to − 0.61 kg in males and from − 0.47 to − 0.59 kg in females. The effect of a 1% increase in pedigree or genomic inbreeding on PWG was larger in males than females, with male PWG decreasing by − 0.64 kg for When BiW was modeled, a 1% increase in Hereford  and AnguHereford , pedigreHereford , 38, 39.While studies on the effect of genomic inbreeding on growth are scarce, evidence of depression has been reported in Hereford and American Angus cattle. In line 1 Herefords, a 1% increase in ot found . In Amerot found  also fouTo visualize the realized phenotypic depression in the Angus population, we calculated the projected loss in BiW, WW, and PWG, for the animals with low (5th percentile) and high (95th percentile) pedigree and genomic inbreeding, as well as the difference between the lowly and highly inbred animals. These results are in Table http://www.angus.org/nce/genetictrends.aspx) show continuous improvement for these traits. These gains indicate a larger effect of selected regions of the genome for growth genetic gain than the detrimental impact caused by the correlated increase in inbreeding. Therefore, careful monitoring of genetic diversity and inbreeding load in the AAA population is essential to prevent the reversal of this trend.Our results indicate that the current accumulation of pedigree and genomic inbreeding see “” in the In this study, we determined the effect of the accumulation of recent and ancient pedigree and genomic inbreeding. The effects of a 1% increase in recent 1 – BTA29). Number of animals sharing a unique ROH by chromosome position.Additional file 2: Figure S2. Proportion of genome covered by ROH of different lengths. Proportion of the genome covered by ROH of lengths 1–2, 2–4, 4–8, 8–16, and larger than 16 Mb for 100 randomly sampled individuals in the population.Additional file 3: Figure S3. Generation intervals for the four paths of selection (2000–2017). Generation intervals calculated for the sires of sires’, dams of sires’, sires of dams’, and dams of dams’ paths of selection calculated for every year of birth from 2000 to 2017.Additional file 4: Figure S4. Distribution of pedigree inbreeding. Distribution of total pedigree inbreeding and partial pedigree inbreeding.Additional file 5: Figure S5. Distribution of genomic inbreeding. Distribution of genomic inbreeding based on the diagonal of the genomic relationship matrix, based on ROH and partial ROH inbreeding, and model-based HBD segment inbreeding coefficients.Additional file 6: Figure S6. Effective number of independently segregating chromosome segments (2000–2017). Effective number of independently segregating chromosome segments calculated for sires and dams from 2000 to 2017.Additional file 7: Figure S7. Effect of a 1% increase in recent and ancient inbreeding on heifer pregnancy. Estimates for the effect of a 1% increase in recent and ancient pedigree and genomic inbreeding.


The drug release profile from the VLP was observed to be gradual, and strictly endosomal pH dependent. We propose that this accessible platform empowers surface functionalization of VLP with numerous ligands containing terminal cysteines, for generating competent delivery vehicles, antigenic display and other biomedical applications.Targeted delivery of hydrophobic chemotherapeutic drugs to tumor cells remains a fundamental problem in cancer therapy. Effective encapsulation of hydrophobic drugs in nano-vehicles can improve their pharmacokinetics, bioavailability and prevent off-target localization. We have devised a method for easy chemical conjugation and multivalent display of a tumor-homing peptide to virus-like particles of a non-mammalian virus, Flock House Virus (FHV), to engineer it into a smart vehicle for targeted delivery of hydrophobic drugs. This conjugation method provides dual functionalization to the VLPs, first, a 2 kDa PEG spacer arm shields VLPs from immune reactivity, and second, attachment of the tumor homing peptide tLyP-1 chauffeurs the encapsulated hydrophobic drugs to target cells. The fortuitous affinity of the FHV capsid towards hydrophobic molecules, and dependence on Ca A variety of nanocarriers have been developed to address this limitation2, however, endosomal escape remains a rate-limiting step for several of these vehicles3. Thus, despite phenomenal advancements in nanoparticle-based delivery system in the recent years, the requirement for smarter packaging and delivery vehicles for hydrophobic drugs persists.Potent therapeutic molecules sometimes fail to make their mark in clinical trials due to their hydrophobic nature, resulting in concomitant difficulty in parenteral administration. Poor aqueous solubility of therapeutic molecules results in lower bioavailability, rapid metabolism, lower retention time in systemic circulation, and adverse clinical effects in patients in consequence. Many effective anticancer drugs are associated with poor water solubility and are therefore difficult to administer4. Virus-like particles (VLPs), which are non-infectious mimics of infectious viruses with a similar quaternary association of capsid proteins, exhibit the same inherent property and can be utilized for deposition of foreign cargo within the cellular cytoplasm5. VLPs fabricated from non-enveloped virus capsids are stable, biocompatible and biodegradable, with a uniform structural organization, which makes them good starting points for developing smart-delivery vehicles7. However, it is essential to address undesirable immune responses elicited by VLPs in order to use them as delivery vehicles9. The hydrodynamic size range of delivery vehicles is an important consideration for conveyance of cargo to the correct site of action without being cleared en route by the immune system. While VLPs in the smaller size range (< 30 nm diameter) are capable of being internalized11, larger VLPs are known to undergo phagocytosis, which may result in inflammation, release of cytokines and rapid systemic clearance12. Cancerous tissues are heavily infiltrated by immune cells, therefore VLPs used in cancer therapy have to be shielded from being hijacked, before delivery of the payload. As VLPs display a similar geometric pattern of capsid proteins as the parental virus, they often induce strong cellular and humoral immune responses10. Previous studies have suggested that coating VLPs with immunologically inert molecules may assist them to bypass immune recognition13. Attachment of inert polymeric molecules, like polyethylene glycol (PEG) on the surface of other drug delivery vehicles have been observed to reduce renal clearance and toxicity, and increase in circulation time and bioavailability of the formulation16. PEGylation further helps in providing an anchor for additional modifications on the surface of VLPs13.Virus particles are programmed to deliver cargos into host cells in an orchestrated fashion. A large majority of viruses with animal hosts are capable of puncturing endosomal membranes to allow the viral genome access to the cytoplasmic milieu18; there are overt safety issues associated with utilizing mammalian viruses, rather than plant or insect viruses, for drug delivery. Modification of non-mammalian viruses with cell specific targeting peptides ensures precise localization since they cannot gain entry into mammalian cells naturally. Targeted drug delivery further reduces systemic toxicity with increased bioavailability and controlled release19.Another requirement for developing VLP-based smart nanotherapeutics is to ensure correct cell specific delivery of the cargo, by genetic or chemical modification of the outer capsid surface with targeting moieties. While Rotavirus and Hepatitis B virus (HBV) have intrinsic tropism toward intestinal cells and hepatocytes respectively, and Canine Parvovirus (CPV) binds to transferrin receptors that are naturally overexpressed on cancer cells20. While native FHV replicates efficiently in cultured Drosophila cells21; expression of the capsid protein of FHV in insect Spodoptera frugiperda (Sf21) cells results in the self-assembly of 180 identical capsid subunits into uniform ~ 30 nm sized VLPs22. The size, uniformity and stability of the particles are conducive to drug delivery. For specific localization of the particles, we chemically conjugated a tumor homing peptide “tLyP-1” on the surface exposed loops of the particle. tLyP-1 (CGNKRTR) is a linear, C-terminally truncated version of Lyp-1 (CGNKRTRGC), a circular peptide capable of specifically recognizing tumor cells, lymphatics of certain tumor types, tumor associated macrophages and macrophages in atherosclerotic plaques24. Binding of Lyp-1 to overexpressed p32/gC1qR on the surface of tumor cells leads to a proteolytic cleavage event, which exposes the C-terminus of the peptide. tLyP-1 subsequently binds to neuropilin receptors NRP-1 and NRP-2, with relatively higher binding affinity for the former25. It has been established that tLyP-1 possesses proapoptotic property and is more effective in specific tumor homing25.In this work, we attempted to design a viral vector that will simultaneously address concerns related to hydrophobic drug encapsulation, specific delivery, safety and immunogenicity. We utilized virus-like particles of Flock House Virus (FHV), a non-enveloped, icosahedral, RNA insect virus, as a base vehicle for development of a smart delivery vehicle26, mass spectrometry and encapsulation reactions with the native virus has clearly established that intact particles are capable of dynamic behavior27, and can allow entry of alkylating agents in the capsid interior, resulting in the inactivation of the viral genome28. An interesting observation was the increased incorporation of hydrophobic alkylating agents compared to charged ones, indicating that the conduit(s) allowing entry of small molecules to the capsid interior are lined with hydrophobic residues28, however, existing structural information does not identify any such channels/pathways for entry of foreign molecules26. This observation clearly indicates the potential for packaging small molecules inside the dynamic capsids. We therefore attempted to encapsulate two hydrophobic chemotherapeutic drugs—doxorubicin and ellipticine—in tLyP-1 conjugated FHV VLPs and monitored the packaging, targeting and delivery of the drugs. Our data suggests that these engineered viral delivery vehicles are capable of successfully encapsulating and specifically delivering hydrophobic chemotherapeutic drugs to breast cancer cells; and can potentially avoid the immune response associated with viral vectors with repetitive subunit structures.Although crystal structures depict FHV as inert, impermeable entities20. The maturation cleavage of all capsid proteins is required for covalent dissociation of γ27, which is a membrane penetrating peptide utilized for endosomal disruption during virus entry29. The maturation cleavage also occurs in FHV VLPs, allowing them to cause endosomal disruption similar to that executed by the native virus30. For optimal attachment of the targeting peptide to the VLP surface, we utilized a modified capsid protein, with a serine to lysine mutation at position 268 (S268K α). K268 is positioned on the outer surface of particles, at the tip of an exposed loop on α, and is ideal for chemical conjugations31. S268K α was expressed in Sf21 cells via recombinant baculovirus infection, and self-assembled particles, which were morphologically similar to wildtype FHV VLPs (data not shown), were purified by standard methods.FHV VLPs are generated from 180 copies of a capsid protein α (43 kDa), which undergoes a post-assembly proteolytic maturation into β (39 kDa) and γ (4 kDa)N-hydroxysuccinimide) ester-primary amine conjugation was carried out between the K268 residues of VLPs and the NHS ester arm of a heterobifunctional PEGylated SMCC crosslinker, SM(PEG)2. The maleimide groups at the free end of the VLP-associated crosslinkers were subsequently allowed to form thioether bonds with the sulfhydryl group of the N-terminal Cysteine residue of tLyP-1 peptides (CGNKRTR). This resulted in the insertion of inert polyethylene glycol (PEG) molecules of 2 kDa between the VLP surface  and ellipticine (denoted EPT_tLyP-1_S268K) were measured at 495 nm and 310 nm respectively.For encapsulating drugs in the interior of the tLyP-1_S268K VLPs, the capsid structure was partially destabilized. It has been shown earlier that the FHV capsid incorporates CaThe amount of doxorubicin and ellipticine present in a particle population was quantified from standard curves of the drugs Fig. A,B. tLyPThe internalization of drugs encapsulated within tLyP-1_S268K VLPs in MDA-MB-231 cells was studied using confocal microscopy  Fig. . In contIn order to assess the cytocompatibility of the base delivery vehicle, different concentrations of S268K FHV VLPs were added to MDA-MB-231 cells for a period of 72 h, followed by a standard MTT assay. The VLPs were found to have significantly high cytocompatibility, as they were non-toxic to MDA-MB-231 cells up to fairly high concentrations. Cell viability was found to be 93% at a concentration of 25 nM of VLPs, which reduced to ~ 85% at double the concentration  was found to be ~ 50% . It is likely that steric clashes result in approximately half of potential conjugation sites remaining unattached. Comparative DLS data and confocal-microscopy based delivery assays clearly show that our particles are surface modified with a substantial number of tLyP-1 moieties which facilitate targeting of encapsulated cargo to specific locations. In addition to the tLyP-1 peptide, we have added a PEG coating on the surface of particles, to protect them from detection and clearance by immune cells. It has been shown before that addition of PEG ameliorates the inherent immunogenicity of virus-based delivery vehicles50. Other reported beneficial effects of PEG conjugation on delivery vehicles includes increase in stability, enhancement of permeability and retention time, prolonged circulation time, better in-vivo pharmacokinetics and bioavailability and reduction in dose dependent toxicity42.The base vehicle for our smart nanocarrier is the insect nodavirus Flock House Virus (FHV), which does not have any natural tropism for mammalian cells. Attachment of the tumor-homing peptide tLyP-1 to FHV VLPs has resulted in the delivery of encapsulated cancer drugs to the breast cancer cell line MDA-MB-231 and triggered cell death by apoptosis. tLyP-1, chosen as a targeting peptide, is an active CendR peptide that binds to neuropilin receptors (NRP1 and NRP2) that are typically overexpressed in the angiogenic vessels of most malignant tumor cells and in the majority of carcinomas20. Any outer surface modification of FHV hinges on two exposed loop regions on the FHV capsid protein, residues 207–208 and 264–268, which are repeated 180 times on the surface of the particle. These loops have been exploited previously for display of a 181 amino acid long sequence of the anthrax toxin receptor by genetic modification of the capsid protein31. Other protein molecules have been chemically attached on to the surface of FHV VLPs by exploiting both covalent and non-covalent interaction chemistry51. Our chemical conjugation method is easy, fast and efficient at mild pH and temperature conditions that do not compromise the stability of the VLPs. This method allows multivalent attachment of any protein/peptide with a terminal cysteine residue on the surface of S268K FHV VLPs through a heterobifunctional cross linker containing NHS-ester and maleimide groups. Apart from reducing immune reactivity, it is also possible that the PEG spacer arm may reduce steric hindrance towards attachment of multiple copies of proteins on VLP surface.FHV has several properties that make it conducive for biomedical usage. Virus-like particles of FHV can be easily generated in large quantities; the particles are uniform, homogeneous, capable of endosomal membrane penetration and have a stable, adaptable surface which can withstand genetic or chemical modification52. The assembled particles were capable of targeted delivery, in spite of being heterogeneous and non-uniform with a range of sizes. In the current study, we have exploited the dynamicity of fully assembled FHV particles53, the preference of the capsid towards hydrophobic molecules28 and the role of Ca2+ in maintaining capsid integrity26, to trigger the incorporation of two hydrophobic drugs inside the icosahedral capsid by partial destabilization of the shell. Electron-microscopy based visualization clearly shows that particles post-encapsulation are stable and icosahedral, indicating that S268K FHV VLPs are competent for undergoing surface modification and enduring partial-destabilization and stabilization transitions. The particles also show a similar pH sensitivity profile corresponding to native FHV, resulting in controlled release of encapsulated material along the endosomal route.The encapsulation of molecules in the capsid interior is a relatively less clear-cut process. Since recombinant production of FHV capsid protein results in the spontaneous assembly of particles in the expression system, incorporation of molecules of choice during in vitro particle assembly is a challenging task. Our laboratory had earlier devised a procedure involving attachment of a long linker to the N-terminal end of the FHV capsid protein to prevent assembly in the expression system. Proteolytic cleavage of the N-terminal linker from purified, monomeric protein triggered assembly and incorporation of small molecules present in the vicinity, within particles55. Both drugs have significant side effects, with ellipticine showing high toxicity in clinical trials56. Our data shows that while both free and encapsulated drugs can penetrate into the cell nuclei, and trigger cell death by apoptosis; the free drugs are associated with higher cytotoxicity, which is probably due to their relatively smaller size and resultant passive diffusion into cells. A more controlled release of encapsulated drugs within specifically targeted cells is the key to preventing chemotherapy-related side effects, and our study offers a dynamic, smart nano vehicle which can achieve this objective.Both doxorubicin and ellipticine are potent inhibitors of topoisomerase II2 crosslinker, mammalian cell culture media and reagents were from Thermo Fischer Scientific USA, and doxorubicin hydrochloride (DOX.HCl) and ellipticine from Cayman Chemical. TC-100 insect cell media was from Himedia Laboratories, and BacPAK6 viral DNA (Bsu36I digested) was purchased from Clontech. caspase 3 and GAPDH antibodies were from Cell Signalling Technologies, USA. Carbon coated 200-mesh copper grids were from Electron Microscopy Sciences.Peptides were obtained from GenScript Biotech, USA. SM(PEG)Spodoptera frugiperda (IPLB-Sf21 or Sf21) were cultured in TC-100 insect cell media supplemented with 10% FBS and 1% penicillin–streptomycin at 27 °C. MDA-MB-231 cells were cultured in L-15 media supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C.The insect cell line, 6 Sf21 cells in 35-mm tissue culture dishes were co-transfected with 500 ng each of pBacPAK9-S268K FHV capsid and Bsu36I digested linearized BacPAK6 viral DNA, along with 4 μl of Bacfectin transfection reagent in TC-100 serum-free media. After 5 h of incubation, complete growth media was added, followed by a further incubation of 72 h at 27 °C. Transfection supernatant was collected and subjected to plaque purification for isolation of recombinant baculovirus. The initial titre was amplified 3 times in Sf21 cells to generate high titre baculovirus stock for protein expression.The cDNA corresponding to FHV capsid protein, cloned in pBacPAK9 transfer vector, was subjected to site-directed mutagenesis using standard protocol (Stratagene) for conversion of Serine268 to Lysine. 1.5 × 106 Sf21 cells were infected with high-titre recombinant baculovirus and incubated for 4 days at 27 °C. Cells were lysed with 1% IGEPAL at 4 °C for 20 min followed by centrifugation to remove debris. The supernatant was subjected to ultracentrifugation on a 30% sucrose cushion at 27,3620 g at 11 °C. The pellet was resuspended in a buffer containing 50 mM HEPES pH 7.2 and 5 mM CaCl2, and subjected to gradient ultracentrifugation on a 10–40% sucrose gradient at 27,3620 g at 11 °C. The VLP band was dialyzed in 50 mM HEPES pH 7.2, 5 mM CaCl2 buffer for removal of sucrose. The concentration of VLPs was calculated using standard methods from optical density (OD) at 260 nm32.In order to express S268K FHV capsid protein, 8 × 102 F20 Transmission Electron Microscope at 200 kV, and images were captured using a 4 k × 4 k CCD camera (FEI Eagle) using TIA software at a magnification of 50,000×.5 μl of sample (0.5 mg/ml) was adsorbed onto glow discharged carbon coated 200-mesh copper grids, stained with 2% (wt/vol) uranyl acetate and air-dried. Grids were visualized in a FEI Tecnai G2 crosslinker for 2 h at room temperature. After removal of the excess crosslinker by dialysis, SM(PEG)2 conjugated VLPs were incubated with 10-fold molar excess of tLyP-1 peptide overnight at 4 °C. tLyP-1 conjugated VLPs (denoted as tLyP-1_S268K) were again subjected to dialysis for removal of unconjugated peptide.A two-step conjugation reaction was performed to attach tLyP-1 peptides on the surface of S268K FHV VLPs. In the first step, VLPs in 50 mM HEPES, pH 7.2 were incubated with a 50-fold molar excess of SM(PEG)57. For encapsulation of drugs, tLyP-1_S268K was dialysed in Tris–EDTA buffer  at room temperature, followed by incubation with 300 μg of doxorubicin or 200 µg of ellipticine overnight at 4 °C. tLyP-1_S268K encapsulating doxorubicin and ellipticine (denoted as DOX_tLyP-1_S268K and EPT_ tLyP-1_S268K respectively) were further dialysed in Tris CaCl2 buffer  at room temperature, in dark, in order to remove un-encapsulated drug molecules.Dox base (referred to as doxorubicin) was prepared by dissolving 5 mg of Dox.HCl in 2.4 μl of TEA, followed by addition of 1 ml DMSO and overnight incubationThe amount of drugs encapsulated in VLPs was quantified using standard calibration curves. Absorbance of doxorubicin and ellipticine were measured at 495 nm and 310 nm respectively using a UV–Vis spectrophotometer. Number of molecules of encapsulated drug per VLP was calculated using free drug standard calibration curves and densitometric analysis of the capsid protein in VLPs resolved on a 15% SDS-PAGE. Protein concentrations were calculated from a standard calibration curve based on densitometric analysis of predetermined concentrations of lysozyme resolved on a 15% SDS-PAGE. All experiments were repeated three times for confirmation of results.Unconjugated S268K FHV VLPs, and tLyP-1_S268K were subjected to Dynamic Light Scattering (DLS) on a Malvern Zetasizer (Nano ZS90). The average of the mean particle size of three separately generated samples were calculated.4 MDA-MB-231 cells in L-15 media were seeded on glass slides and incubated overnight. Cells were treated with free drugs, free VLPs, or drug encapsulating VLPs for 6 h at 37 °C. After 3 × washes with DPBS, cells were fixed in 2% paraformaldehyde for 15 min, followed by labeling with 300 nM DAPI for 10 min. After washing in DPBS, cells were mounted and dried. For visualization of VLPs with anti-FHV antibody, a rabbit anti-FHV polyclonal antibody was utilized in conjunction with Alexa Fluor™ 555 labeled goat anti-rabbit antibody. Images were collected in an Olympus confocal laser scanning microscope (Olympus FV1200) at a magnification of 60x.For cellular internalization studies, 8 × 10. At predetermined time points, 200 µl of respective dialysis buffers were sampled out accompanied by replenishment with the same volume of respective fresh buffers. Fluorescence intensities of sampled buffers were measured at λex 480 nm and λem 590 nm  and the percentage of drug release were calculated using a standard calibration curve. The experiment was performed in triplicates and average values were reported.In vitro drug release profiles were determined at both pH 7.2 (cytoplasmic pH) and pH 5.5 . 50 µl (0.2 µg/ µl) of Dox_tLyP-1_S268K were dialyzed against PBS (pH 7.2) or sodium acetate buffer (pH 5.5) at 37 °C with gentle stirring3 MDA-MB-231 cells in each well of a 96-well tissue culture plate were treated with different concentrations of S268K FHV VLPs, doxorubicin, ellipticine, Dox_tLyP-1_S268K and EPT_tLyP-1_S268K for 72 h. Media was removed and a 0.5 mg/ml MTT solution was added to the cells, followed by 4 h of incubation in dark at 37 °C. The supernatant was discarded, formazan crystals were dissolved in DMSO and absorbance was measured using a microplate reader  at 570 nm. All experiments were performed in triplicates and repeated three times.For MTT assays, 5 × 106 MDA-MB-231 cells, seeded overnight in a 6 cm dish, were treated with equivalent concentrations of S268K FHV VLPs, doxorubicin and Dox_tLyP-1_FHV S268K respectively for 24, 48 and 72 h. Cells were lysed in SDS-RIPA buffer containing a protease inhibitor cocktail and 1 mM DTT. Samples were separated on a 12% SDS-PAGE, followed by western blotting using a caspase 3 primary antibody and a HRP conjugated anti-rabbit secondary antibody. Control blots were carried out with an anti-GAPDH primary antibody. The full-length western blots are shown in Supplementary Figure S1. Images were acquired using a Typhoon FLA 9000 gel imaging scanner and densitometric analysis of active forms of caspase 3 (p19 and p17) were carried out by normalizing the intensity with an endogenous control (GAPDH) using Image J software.0.8 × 10We have designed a virus-like particle-based nano-vehicle for encapsulation and delivery of hydrophobic drugs to target cells. The vehicles are stable at physiological conditions, and display strict control of targeting and release, suggesting potential inhibition of off-target effects. It is hoped that these particles will prove to be effective in animal models of breast cancer and will show significantly reduced off-target side effects, immunogenicity and toxicity.Supplementary Information


Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine involved in pain processing and hypersensitivity. It regulates not only the expression of a variety of inflammatory mediators but also the functional activity of some ion channels. Acid-sensing ion channels (ASICs), as key sensors for extracellular protons, are expressed in nociceptive sensory neurons and contribute to pain signaling caused by tissue acidosis. It is still unclear whether TNF-α has an effect on functional activity of ASICs. Herein, we reported that a brief exposure of TNF-α acutely sensitized ASICs in rat dorsal root ganglion (DRG) neurons.Electrophysiological experiments on rat DRG neurons were performed in vitro and acetic acid induced nociceptive behavior quantified in vitro.50 value of 0.12 ± 0.01 nM. TNF-α shifted the concentration-response curve of proton upwards with a maximal current response increase of 42.34 ± 7.89%. In current-clamp recording, an acute application of TNF-α also significantly increased acid-evoked firing in rat DRG neurons. The rapid enhancement of ASIC-mediated electrophysiological activity by TNF-α was prevented by p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190, but not by non-selective cyclooxygenase inhibitor indomethacin, suggesting that p38 MAPK is necessary for this enhancement. Behaviorally, TNF-α exacerbated acid-induced nociceptive behaviors in rats via activation of local p38 MAPK pathway.A brief (5min) application of TNF-α rapidly enhanced ASIC-mediated currents in rat DRG neurons. TNF-α (0.1-10 ng/ml) dose-dependently increased the proton-evoked ASIC currents with an ECThese results suggest that TNF-α rapidly enhanced ASIC-mediated functional activity via a p38 MAPK pathway, which revealed a novel peripheral mechanism underlying TNF-α involvement in rapid hyperalgesia by sensitizing ASICs in primary sensory neurons. Data are expressed as mean ± S.E.M. IpH6.0) in DRG neurons. The IpH6.0 could be almost completely blocked by 10 μM of amiloride, a broad-spectrum ASIC channel blocker, and also by 2 μM APETx2, an ASIC3 blocker. In contrast, capsaicin (100 nM) failed to evoke any membrane currents in the presence of AMG9810. Thus, these acid-induced currents were considered to be ASIC currents or ASIC3-mediated currents after TRPV1 activation was blocked by AMG9810.In the present study, AMG9810 (5 μM) was added to external solution to block proton-induced TRPV1 activation. As shown in Fig. IpH6.0. The enhancement of IpH6.0 occurred 5min after the onset of TNF-α application. This enhancement of IpH6.0 was dependent upon the doses of TNF-α treatment. In a representative DRG neuron, the peak amplitude of IpH6.0 progressively increased as doses of pre-treated TNF-α increased from 0.3 to 30 ng/ml  value of 0.12± 0.01 nM (1.96 ± 0.15 ng/ml). The results indicated that TNF-α rapidly enhanced ASIC currents in rat DRG neurons in dose-dependent manner.In some DRG neurons sensitive to acid stimuli, we first evaluated the effects of brief application of TNF-α on the ASIC currents. TNF-α was pre-incubated to DRG neurons for 5min prior to application of pH 6.0 acidic solution. As shown in Fig. /ml Fig. b. FigureIpH6.5, IpH5.5, and IpH4.5 increased after pre-application of 10 ng/ml TNF-α for 5 min. Figure n = 1.30 ± 0.19; TNF-α + pH: n = 1.32 ± 0.21; P > 0.1, post hoc Bonferroni’s test). Third, the pH0.5  values of two curves had also no statistical difference . We therefore concluded that sensitization of ASICs by TNF-α was not due to a change in the apparent affinity of ASICs for protons.We then investigated the effects of TNF-α on concentration-response curve for protons. ASIC currents were measured by applying a range of different pH values in the absence and presence of TNF-α. Figure IpH6.0 increased 44.14 ± 4.26% by TNF-α (10 ng/ml) pre-treatment alone. SB202190, a fast-acting p38 MAPK inhibitor, was applied to DRG neurons for 3 min followed by mixture of SB202190 and TNF-α for additional 5 min. The pretreatment of SB202190 (10 μM) substantially prevented the TNF-α-mediated increase in ASIC currents, and the amplitude of IpH6.0 increased only 2.67 ± 3.71% . These results indicated that p38 MAPK, but not COX, is necessary for TNF-α-induced enhancement of ASIC currents.We further explored the pathway linking TNF-α to its effect on ASIC currents. It has been demonstrated that TNF-α can signal through activation of p38 MAPK in DRG neurons as well as many other cell types , 37, 38.P < 0.01, paired t-test, n = 6, Fig. P > 0. 1, paired t-test; n = 6, Fig. ASICs are non-selective cation channels, once activation by acidification, which leads to membrane potential depolarization and neuronal excitation. We further observed whether TNF-α had effects on acid-evoked action potentials of rat DRG neurons. Although proton-induced TRPV1 activation was blocked in the presence of 5 μM AMG9810, we observed that an acid stimulus of pH 6.0 induced not only a rapid inward current with voltage-clamp recording, but also bursts of action potentials (APs) under current-clamp condition in the same DRG neuron Fig. a and c. p < 0.05 and 0.01, one-way ANOVA followed by post hoc Bonferroni’s test, n = 10; Fig. p < 0.01, one-way ANOVA followed by post hoc Bonferroni’s test, n = 10; Fig. Our above electrophysiological studies showed that TNF-α acutely sensitized ASICs via activation of p38 MAPK in vitro. We further ascertained whether TNF-α had effects on ASIC-mediated nociceptive behaviors through interacting with ASICs in vivo. Our previous studies observe that intraplantar injection of acetic acid into rats elicits ASIC-mediated nociceptive behaviors even if AMG 9810 (10 μM) blocked the activation of TRPV1 , 41. We This study examined the effects of a brief exposure of TNF-α on functional activity of ASICs. We demonstrated that acute application of TNF-α rapidly enhanced ASIC-mediated and acid-evoked currents and action potentials in dissociated rat DRG neurons through a p38 MAPK-dependent mechanism. Behaviorally, TNF-α also exacerbated acid-induced nociceptive responses in rats via activation of local p38 MAPK pathway.After proton-induced TRPV1 activation was block by AMG9810, capsaicin failed to induce any membrane currents. Thus, the low pH-evoked currents may be mediated by only ASICs in the present study. The conclusion was confirmed by the results that these currents were completely blocked by ASIC channel blocker amiloride and ASIC3 blocker APETx2. It has been found that there are seven ASIC subunits in DRG neurons, of which ASIC3 subunit is the most abundant . We ther+ currents, L-type Ca2+ currents, and TRPV1 currents [The present study showed that TNF-α can rapidly sensitized ASICs. A brief (5min) exposure of TNF-α dose-dependently enhanced ASIC currents in rat DRG neurons. TNF-α shifted upwards concentration-response curve for protons with a significant increase in the maximal current response to protons, whereas apparent affinity of ASICs for proton did not change. The rapid pH drop caused an activation of ASICs which promptly depolarized the membrane and initiated a burst of APs. The burst ended quickly because of prolonged depolarization which inactivated the sodium current . The precurrents , 14, 21.+ currents [+ channel currents and hyperpolarization-activated cation currents via a p38 MAPK pathway [in vivo but also in acid-induced nociceptive behaviors in vitro. While acute exposure (5min) of TNF-α enhanced ASIC currents, the effects were likely to result from a posttranslational modification of ASICs by TNF-α, such as phosphorylation. It has been shown that activation of p38 MAPK increases TTX-resistant sodium channel currents by phosphorylation of L1 loop serines of Nav1.8 channels [TNF-α exerts its biological effects by associating with two tumor necrosis factor receptors: TNFR-1 and TNFR-2, which are expressed in DRG neurons –46. TNFRcurrents . TNFR1 acurrents , 38. It  pathway , 17, 18.channels . Future It has been shown that TNF-α leads to COX-2-dependent production of prostaglandin E2 , 49. ProUnder inflammatory and neuropathic pain conditions, various mediators are released, such as ATP, prostanoids, protons, and TNF-α. They contribute to peripheral sensitization in terminals of nociceptor neurons . ClinicaIn summary, our results indicated that acute application of TNF-α rapidly enhanced ASIC-mediated electrophysiological activity and acidosis-evoked pain, which revealed a novel peripheral mechanism underlying rapid sensitization to nociceptive stimuli by peripheral administration of TNF-α. ASICs represent downstream targets of TNF-α and therapy targeting ASICs is likely useful for treating inflammatory and neuropathic pain.


Interdisciplinary collaborations bring lots of benefits to researchers in multiple areas, including precision medicine.This viewpoint aims at studying how cross-institution team science would affect the development of precision medicine.Publications of organizations on the eHealth Catalogue of Activities were collected in 2015 and 2017. The significance of the correlation between coleadership and coauthorship among different organizations was calculated using the Pearson chi-square test of independence. Other nonparametric tests examined whether organizations with coleaders publish more and better papers than organizations without coleaders.P<.001, 2015 and 2017), which received higher citations , compared to those without coleadership. Organizations with coleaders tended to publish papers together .A total of 374 publications from 69 organizations were analyzed in 2015, and 7064 papers from 87 organizations were analyzed in 2017. Organizations with coleadership published more papers (Our findings suggest that organizations in the field of precision medicine could greatly benefit from institutional-level team science. As a result, stronger collaboration is recommended. The concept of a meta-topical brainforest is proposed, to reflect a link between collaborative research and complex ecosystems. Tropical rainforests leverage a diversity of species to capture and convert solar energy into carbon-based life, and research teams can harvest a similar benefit from a diversity of data, tools, and thought paradigms.According to the National Institutes of Health, team science is “a collaborative and often cross-disciplinary approach to scientific inquiry that draws researchers who otherwise work independently or as coinvestigators on smaller-scale projects into collaborative centers and groups” . Thus, tKohane  pointed U test, we validated our hypothesis.This brief viewpoint aims to explore the impact of cross-institution team science on the development of precision medicine. We hypothesized that international organizations with coleaders tend to publish more impactful papers than organizations without coleaders. Using the Pearson chi-square test and the Mann-Whitney Information was collected from the eHealth Catalogue of Activities developed by the nonprofit Global Alliance for Genomics and Health in 2015 . The catIn this paper, coleadership means that a person holds a leadership position in different organizations concurrently. If two papers from separate organizations have at least one author in common, these two organizations are regarded as having a coauthor relationship.U test was used to examine whether the former organizations published papers that received more citations than the latter.Nonparametric tests were performed to verify the hypothesis. We used SPSS  and R to perform two-tailed tests with an α level of .05. The significance of the correlation between the nominal variables coleadership and coauthorship was examined using the Pearson chi-square test of independence and expressed in a contingency table. The Pearson chi-square test of goodness of fit was adopted to evaluate whether organizations with coleaders had a greater number of publications than organizations without coleaders, and the Mann-Whitney We analyzed data from 69 organizations in the catalog and found 16 pairs with coleader relationships in 2015. Among the 374 publications from these organizations at that time, 13 pairs had coauthors. By 2017, the number of institutions in the catalog increased to 87, and there were 37 pairs with coleadership, corresponding to 30 organizations. Information on 7064 papers was collected, showing that 55 organizations had coauthored publications, with 436 papers in total.P<.001, 2015 and 2017).The chi-square goodness of fit test suggests that the number of papers being published is strongly correlated with the category of the organization—organizations in a coleadership network or organizations without coleadership . Papers from the former organizations had a higher mean rank , which means that the organizations with coleaders tended to have a greater number of highly cited papers.The citation number of each paper was obtained from Google Scholar. The results of the Mann-Whitney P<.001, 2015 and 2017).In the chi-square test of independence, the total sample size is the number of lines in a fully connected diagram. The results indicate that in both 2015 and 2017, organizations with coleaders tended to publish papers together, suggesting that coleadership will lead to coauthorship . Moreover, the publications from organizations with coleaders are more frequently cited, indicating a relatively high quality . These results suggest that collaborations among health institutions are becoming stronger, which promotes their working efficiency.We studied how precision medicine can be influenced by institutional-level team science by analyzing coleadership and coauthorship across health organizations. From 2015 to 2017, the number of health organizations grew from 69 to 87, and their publications increased. Concurrent positions held by leaders may incentivize researchers to work for multiple organizations; thus, the researchers will be very likely to have a coauthored paper (These results illustrate the concept of meta-topical brainforests in precision medicine -2 and maOne hopes the analogy persists and the extraordinary natural future-proofing mechanisms in rainforests coincide with similar continued diversification in research networks and widely impactful publications.


There is increasing international interest in clinical practice and research related to mental health in the international sports society. The athlete-specific psychological distress assessment tool that addresses potential mental health needs can help promote early detection and recovery of mental illness, as well as physical illnesses. Currently, little is known about the applicability of the useful assessment tool for Japanese elite athletes. The Athlete Psychological Strain Questionnaire (APSQ) is a brief, effective and reliable screening tool to identify early signs of athlete-specific distress and potential mental health symptoms. We examined the applicability and reliability of a Japanese version of the APSQ (APSQ-J) in a Japanese elite athlete context. Further, we examined the construct validity of the APSQ-J.We collected web-based anonymous self-report data from 219 currently competing Japanese professional male rugby players. A two-stage process was conducted to validate the factor structure of the APSQ-J using exploratory factor analysis (EFA) in a randomly partitioned calibration sample and confirmatory factor analysis (CFA) in a separate validation sample. Cronbach’s alpha is used to assess internal consistency. Pearson product-moment correlation coefficients were calculated to determine if the APSQ-J was significantly associated with measures of psychological distress and well-being using Kessler-6 (K6) and the WHO-5 Well-Being Index, respectively.β = 0.41–0.83 (p < 0.001). A Cronbach’s alpha of 0.84 was obtained for the APSQ-J. The APSQ-J demonstrated significant correlations with the K-6  and WHO-5 .We identified a one-factor structure for the APSQ-J. Confirmatory factor analysis supports this one-factor model, revealing good model fit indices. The standardized path coefficients for each of the items were The APSQ-J can be an appropriate and psychometrically robust measure for identifying athlete-specific distress in elite athletes in Japan. Widely disseminating and utilizing this scale in Japanese sports society may support athletes' mental health via early detection of symptoms of psychological distress.The online version contains supplementary material available at 10.1186/s40798-021-00385-9. Athlete Psychological Strain Questionnaire (APSQ) is an athlete-specific psychological distress assessment tool that addresses potential mental health needs.Current findings from the sample of Japan Rugby Top League players supported the factor structure, validity, and reliability of the Japanese version of the APSQ (APSQ-J) scale, with a one-factor structure, indicating the higher score, the worse the psychological distress and strain.APSQ-J is a brief and effective evaluation tool that facilitates the development and social implementation of mental health support for elite athletes in Japan.Recent research and clinical practice for mental health in elite athletes has increased, similar to the trajectory for physical health and injury care. Most of the findings have come from the USA , 2, EuroThe Athlete Psychological Strain Questionnaire (APSQ) is a mental health assessment tool that addresses these potential needs specifically within an elite sports context , 23. TheThe APSQ is included as the triage measure in the Sports Mental Health Assessment Tool-1 (SMHAT-1), a mental health screening procedure developed by the International Olympic Committee (IOC) to better support the mental health of athletes and stimulate more international research . At presThe current study employed a web-based cross-sectional design. We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines for observational cross-sectional studies  registered with the Japan Rugby Players Association. The participants were all aged 18 years and over and belonged to the Japan Rugby Top League. The current survey was available in both Japanese and English. No exclusion criteria were applied. Overall, 227 of the 612 players agreed to complete the survey (response rate: 37.1%). The response rate of this survey was consistent with other mental health surveys in Japan . We analM = 23.67, SD = 4.16). The exploratory and confirmatory factor analysis and tests of differential item functioning were conducted with the samples randomly partitioned into calibration (n = 497) and validation (n = 510) samples. Exploratory factor analysis, with parallel analysis, conducted on the calibration sample supported a second-order with three-factor model. The subscales included Self-Regulation, Performance, and External Coping domains, accounting for 50.44% of total scale variance. In the second-order model, the path coefficient from the upper factor “Athlete psychological strain” to each factor was 0.8 or more. In the confirmatory factor analysis, excellent model goodness-of-fit indicators were provided. The mean score for the 10 items among the Australian male athletes was 14.67 (SD = 5.47).The APSQ is a brief, self-report questionnaire specific to the elite sporting context, comprised of 10 items  scored on a 5-point scale (from ‘none of the time’ (1) to ‘all of the time’ (5)). A total score ranging from 10 to 50, with higher scores representing more psychological distress, is calculated by summing the answers on the 10 items . A more For this study, the original APSQ was translated into Japanese by the first author (Y.O.). The professional athletes (S.K. and K.H.) in this research team modified the terms and sentences to improve the readability for athletes. A bilingual English speaker then produced a back-translation. Finally, the back-translated version of the APSQ was confirmed and approved by the researchers who had originally developed the APSQ (S.M.R and R.P).We used the Kessler-6 (K6) and the WHO-5 Well-Being Index, widely used measures of psychological distress and well-being to assess convergent validity with the APSQ-J. Previous studies have shown that the performance of the K6 in screening mood and anxiety disorders, as assessed by the areas under the receiver operating characteristic curves (AUCs), was excellent , with vaThe background information and demographic survey items included age, country of birth, educational attainments, marital status, the number (if any) of dependent children, residential status, the national team's experience, and playing status for the last season.Descriptive statistics were used to characterize the sample. We conducted a two-stage process to validate the factor structure of the APSQ-J using exploratory factor analysis (EFA) in a randomly partitioned calibration sample and confirmatory factor analysis (CFA) in a separate validation sample. EFA was undertaken to determine the underlying factor structure of the APSQ-J. To determine which items belonged to each factor, we extracted items if they loaded ≥ 0.3. In addition, we examined the number of factors based on scree plots. We adopted the oblique rotation (promax) with principal factor method. In CFA, we evaluated the fit of the model with the data using the chi-squared statistic (CMIN), root mean square error of approximation (RMSEA), and comparative fit index (CFI). According to conventional criteria, an excellent fit would be indicated by CMIN/df < 2, RMSEA < 0.05, CFI > 0.97, while CMIN/df < 3, RMSEA < 0.08, CFI > 0.95 demonstrates an acceptable fit . After cWe analysed 219 players born in Japan. The demographic characteristics of the participants are shown in Table In EFA, considering the results of the scree plots Fig. , we empldf = 2.48 (p < 0.001), RMSEA = 0.117, CFI = 0.871. Along with modification indices which suggest a high parameter change, covariances were added between some of the error variables of items. After the modification, we obtained a final model with excellent fit indices: CMIN/df = 1.024 (p < 0.001), RMSEA = 0.015; CFI = 0.998. The standardized path coefficients for each item were β = 0.41–0.83 (p < 0.001).The results of CFA are presented in Fig. r = 0.80, p < 0.001) and a significant moderate negative correlation with WHO-5 . The total score for the 10 items was significantly higher than that for Australians (mean = 19.16 (SD = 6.30) vs. mean = 14.67 (SD = 5.47), t = 10.70; df = 1224; p < 0.001) in Additional file The Item-test, Item-rest correlation for the 10-items APSQ-J is shown in Table This study evaluated the psychometric properties of the Japanese version of APSQ in order to contribute to improving mental health promotion and support for Japanese elite athletes. We confirm the factor structure, validity, and reliability of the scale in Japanese male professional rugby players. The APSQ-J demonstrated a one-factor structure which is different from the three-factor of the original version of APSQ from the factor analysis. The APSQ-J could be considered a useful assessment tool for athlete-specific psychological distress in Japanese elite athletes, with relatively high Cronbach’s alphas, as well as convergent validity with the K-6 and WHO-5.The Japanese version of APSQ did not match the factor structure of the original version of APSQ. The original version had three factors: Self-regulation, Performance, and External Coping , 23, whiOur findings suggest that the APSQ-J confirmed a good convergent and divergent validity with the K-6 and WHO-5, respectively. The advantage of using the APSQ-J in elite athlete populations is the ability to detect athlete-specific distress, which can then enable mental health support for affected athletes. The APSQ is a 10-item, sufficiently brief tool highly relevant to the competitive sport context, by its inclusion of items related to injury, training, and selection, as well as mental health-related externalization issues such as alcohol and substance abuse. While K6 focuses on internalized symptoms, the APSQ includes several externalized symptoms in addition to such internalized symptoms. This feature was included as elite athletes conforming to traditional masculinity norms may be more likely to experience and manifest externalized, rather than internalized symptoms, as a sign of poor mental health , 39. FocThe current study showed that the items of APSQ-J are summed as factor to generate a total score, suggesting that the higher the score, the worse the psychological distress and strain. We also demonstrated that the total score in the sample of Japanese male rugby players was higher than that of Australian male elite athletes. Considering the difference of the total score in each country, the next step in developing the APSQ-J will be to further explore the validity of APSQ-J cut-off values, including relative to the original version . FurtherThe major strengths of this study were developing a Japanese version of the APSQ and confirming its psychometric properties. Our efforts are expected to advance mental health research among elite athletes in Japan, which has been delayed in comparison with the USA, European countries and Australia. In translating the scale development process, we coordinated athlete-friendly terminology with active elite athletes. This co-production with elite athletes may have improved the accuracy of answering APSQ-J questions. However, we also recognize several limitations that may impact the findings. The current samples were all male professional rugby players and the response rate was less than 40%, although this is consistent with other mental health surveys in Japan. Future research should aim to examine the psychometric properties of the APSQ-J in a wider, more representative cohort, including female athletes and those from other sports. Furthermore, because we employed a cross-sectional survey design, the test–retest for the stability of the APSQ-J was not evaluated. Finally, the effectiveness of scale convergence and divergence could be better understood by consideration of critical stages of risk for athlete mental health, such as injury duration and severity and the transition from competitive sports.We have developed the APSQ-J, which enables screening for mental health status in Japanese elite athletes. Utilizing this scale may support athletes’ mental health via early detection of symptoms of psychological distress. There is increasing international interest in clinical practice and research related to mental health in the international sports society. APSQ-J, a brief and effective evaluation tool, is expected to be widely used in the Japanese sports society. In addition, early detection of psychological distress by this tool may help promote mental health support for elite athletes.Additional file 1. Comparison of Means and SDs of participants’ responses to APSQ between Japanese and Australian elite athletes.