Repeated screening of secretions and faeces by RT-PCR and plaque assay revealed no virus shedding, thus also confirming biological safety. In a patient trial involving 29 horses bearing BPV1-induced single or multiple sarcoids, at least one lesion per horse was intratumourally injected and then boosted with iNSA/E6E7equ and/or iNSB/E6E7equ. The treatment induced a systemic antitumour response as reflected by the synchronous regression of injected and non-injected lesions. Irrespective of vaccination schemes, complete tumour regression was achieved in 10/29 horses. In 10/29 horses, regression is still ongoing (May 2021). Intriguingly, scrapings collected from former tumour sites in two patients tested negative by BPV1 PCR. Nine severely affected individuals with a history of unsuccessful therapeutic attempts did not (6/29) or only transiently (3/29) respond to the treatment. INSA/E6E7equ and iNSB/E6E7equ proved safe and effective in significantly reducing the tumour burden even in severe cases.Bovine papillomaviruses types 1 and 2  commonly induce skin tumours termed sarcoids in horses and other equids. Sarcoids seriously compromise the health and welfare of affected individuals due to their propensity to resist treatment and reoccur in a more severe form. We have developed influenza (Flu) A and B virus vectors that harbour a truncated NS1 gene (iNS) assuring interferon induction and co-express shuffled BPV1 E6 and E7 antigens for sarcoid immunotherapy. In a safety trial involving 12 healthy horses, intradermal administration of iNSA/E6E7 Equine sarcoids are non-metastasising, yet locally aggressive cutaneous fibropapillomas induced by bovine delta-papillomaviruses of type 1, 2 and possibly type 13  . SarcoidPapillomaviruses (PVs) are small, non-enveloped viruses consisting of an icosahedral capsid harbouring a short (< 8 kbp) circular dsDNA genome. Whereas most PVs show a pronounced tropism for cutaneous or mucosal keratinocytes and are highly species-specific, δ-PVs do not adhere to this rule. They also or preferentially infect dermal fibroblasts, which may explain their wider host-range, comprising many ungulate species including equids, e.g. horses, donkeys, zebras or mules . GeneticThe widely recognized association of BPV1 and BPV2 with sarcoid development raises the possibility of using therapeutic strategies targeting viral antigens. Whereas naturally acquired sarcoids usually persist and progress without or despite treatment, “pseudo-sarcoids” experimentally induced by wild-type BPV1 or BPV2 virions regress spontaneously due to an effective immune response –28. HencAs early as 1995, it was shown that vaccination of cattle with BPV4 E7 prior to challenge with wild-type (wt) virions retarded the development of papillomas and promoted their early regression. E7-specific T cells were detected at high levels shortly after challenge and three T cell epitopes were identified .+ T helper type 1 cells (Th1) and CD8+ cytotoxic T lymphocytes (CTLs). These observations reflect the potential of E6 and E7 as therapeutic targets and the great importance of T cell responses, particularly CTL responses, for the induction of an immunotherapeutic effect [Considerable efforts have been made to translate this and similar findings obtained in animal PV models into human medicine. Various types of vaccines that mainly target the E6 and E7 oncoproteins are in development for the immunotherapy of high-risk human PV (hrHPV)-induced cancers. They include vector-based viral or bacterial systems as well as peptide-, protein-, DNA- and dendritic cell (DC)-based vaccines , 32, 33.c effect , 32–34.hum, delNSB/E6E7hum) as immunotherapeutic vaccine candidates for prime and boost vaccination. Importantly, intralesional injection of mice with established TC-1 tumours using these viruses induced complete tumour regression in 25% of all animals and significantly reduced tumour growth in 50% of mice [We have previously established human influenza (Flu) viruses lacking the C-terminal part of the non-structural gene NS1 (delNS) . We show of mice –45.equ and iNSB/E6E7equ) as potential sarcoid immunotherapeutics. The specific partial deletion resulted in iNS-based vectors, which induce high levels of IFN while–unlike the fully deleted NS1 deletion mutants–retaining the ability to replicate efficiently in IFN-sensitive tumour cells. In this context, we have previously shown that such partly deleted mutants have better antitumour properties and are less sensitive to the growth inhibitory effects of IFN [To enhance antitumour properties, we then developed influenza A and B viruses harbouring an NS1 gene with a partial deletion (iNS1) and co-expressing shuffled BPV1 E6 and E7 antigens  that originated from A/Puerto Rico/8/34 for PA, PB2, NP, M, and A/Texas/1/77 for PB1. The viral HA and NA genes were derived from H1N1 virus A/New Caledonia/20/99. For FluB, all viral segments were derived from B/Thüringen/02/06 (B/Jiangsu/10/03-like). African green monkey kidney (Vero) cells [2 one day prior to electroporation and then co-transfected with 0.5 μg of plasmid pHW-ANS106-P2A-SP-E6E7shuffled or pHW-BNS106-P2A-SP-E6E7shuffled, expressing both vRNA and mRNA, and 0.5 μg of each bidirectional plasmid encoding the remaining seven segments using Nucleofector™ II technology . INSA/E6E7equ and iNSB/E6E7equ were collected with culture supernatant after cultivation for four to five days.A shuffled BPV1 E6E7 fusion gene was designed and synthesised . The fusion gene was sub-cloned into a pHW2000 plasmid derivative  containio) cells  were seeequ and iNSB/E6E7equ were passaged in Vero cells for a minimum of five rounds. Total viral RNA was isolated using a QIAamp viral RNA mini kit  according to manufacturer’s instructions and subjected to reverse transcription (RT) using uni-12 primer for Flu A (5’-AGCAAAAGCAGG-3’) and Uni-13 primer for Flu B (5’-AGCAGAAGCAGAG-3’)  using a SuperScript® III First-Strand Synthesis System according to manufacturer’s instructions . To amplify the iNSA-and iNSB-E6E7 regions, the following PCR primers were used: FluAf 5´-AGC AAA AGC AGG GTG ACA AAG-3’, FluAr 5´-CTC TTG CTC CAC TTC AAG C-3´ and FluBf 5´- AGC AGA AGC AGA GCA TTT G-3´, FluBr 5´-AGT AGT AAC AAG AGG ATT TTT A-3´ (Eurofins). PCR was carried out with GoTaq® DNA Polymerase following manufacturer’s recommendations . Amplification products (16 μl) were electrophoretically separated in 1.5% TAE-gels and visualised by ethidium bromide staining.To verify transgene stability in the iNS1 reading frame, iNSA/E6E7equ or iNSB/E6E7equ (105 pfu) and then incubated for 12 hours. Cells were harvested, lysed in Laemmli-sample buffer, boiled for 5 minutes, and analysed by SDS-PAGE and Western blot. The antibodies used in these assays comprised mouse anti-influenza A nucleoprotein (NP) blend clone A1/A3  or mouse anti-influenza B NP clone B2 (Millipore); rabbit anti-BPV1 E6 and E7 antibodies  and mouse anti-beta actin clone AC-74 . Proteins were separated on 10–15% gradient Tris-glycine SDS gels (Biorad) using the Tris-glycine SDS buffer system. Western blotting was performed by electrophoretic transfer of proteins to a polyvinyl difluoride (PVDF) membrane (Millipore). Bound antibodies on the blot were detected with HRP-conjugated goat anti-rabbit or goat anti-mouse antibodies  using chemiluminescent substrates. Images were captured and analysed using a FluorChem FC3 system and its image analysis software .For the analysis of transgene expression, equine primary palatal fibroblasts  were grown to sub-confluence for 12 hours, infected with iNSA/E6E7equ or iNSB/E6E7equ in the presence of 0.5 ng/ml amphotericin B (Sigma-Aldrich). After incubation for 20 hours at 37°C (FluA) or 33°C (FluB), cells were harvested, fixed in 4% formalin solution, permeabilized with 0.2% TritonTM X-100 (Sigma-Aldrich) in PBS, stained with anti-influenza A NP or anti-influenza B NP  and anti-mouse IgG Alexa Fluor 488 conjugated . Infected cells were counted on a Cytation Imaging Reader  and virus concentrations were calculated.Vero cells were seeded in 96-well plates at a density of 50,000 cells/well 24 h before the concentration analysis. Cells were then infected with serial dilutions of iNSA/E6E72 one day before infection. For iNSA/E6E7equ, cells were infected at a moi of 0.1 in OptiPro SFM supplemented with 1% 10x TrypLE and 0.25 ng/ml amphotericin B  and incubated at 37°C. The medium was substituted on day 1 after infection and the supernatant collected on day 3 after infection. For iNSB/E6E7equ, cells were infected at a moi of 0.02 in OptiPro SFM supplemented with 1% 10x TrypLE and 0.5 ng/ml amphotericin B, incubated at 33°C and the supernatant was collected on day three after infection. Supernatants were treated with 750U Benzonase Nuclease (Sigma-Aldrich) to reduce cellular DNA and influenza viruses were concentrated by polyethylene glycol (PEG) precipitation. Final viral concentrations ranged between 9.5 and 10.2 log FFU/ml.Vero cells were seeded at a density of 80,000 cells per cm5 infectious virions in 100 μl PBS per injection) as described previously [equ and iNSB/E6E7equ, and in six horses with virus carrier solution  in a blinded approach. Lesions on the right side of the neck were left untouched. The vaccination scheme was the following: iNSA/E6E7equ (500 μl à 9 log FFU) or SNH buffer on days 0, 2 and 4, and iNSB/E6E7equ (500 μl à 9 log FFU) or SNH buffer on days 7, 9 and 11. All horses were closely monitored until all lesions had completely resolved, with special emphasis being put on local and systemic reactions attributable to intratumoural injections. Behavioural parameters , vital signs  and local and systemic effects of injections were recorded twice daily until d15. Since no virus shedding was observed, all horses were subsequently moved from the BSL-2 facility to the Vetfarm providing ideal horse stabling conditions. Pseudo-sarcoid diameters were assessed twice a week until week ten, and then weekly using callipers. After all pseudo-sarcoids had completely resolved, the horses returned to their owner.Twelve horses kindly provided by a cooperating farm were enrolled in the study and kept at the Biosafety Level 2 (BSL-2) horse facility (treatment phase) and then at the Vetfarm of the Vetmeduni Equine University Hospital (post-treatment mentoring phase) . The horeviously , 50. Theequ and iNSB/E6E7equ was assessed by repeated RT/PCR and plaque assays from extracts of nasal swabs and faeces (RT/PCR only) collected on days 1, 3, 5, 8, 10 and 12.Biological safety of iNSA/E6E7TM  according to manufacturer’s recommendations. RT/PCR positive controls consisted of total RNA extracted from nasal swab or faeces aliquots spiked with iNSA/E6E7equ or iNSB/E6E7equ. Plasmids harbouring the iNSA- E6E7 or iNSB-E6E7 sequence served as PCR positive controls. RT/PCR was performed as described above. No-reverse transcriptase  controls were included in all reactions.To extract total RNA from nasal secretions, swabs were centrifuged at high speed for 5 minutes. Then RNA was isolated from these nasal secretions by using a Viral RNA Mini Kit (Qiagen) in line with manufacturer’s instructions. RNA isolation from faeces was carried out using a ZR Soil/Fecal RNA microPrep2 in OptiPro 2% Glutamax (Sigma-Aldrich) and incubated for 24 h at 37°C. Then the cells were cultured in infection medium , overlaid with nasal secretions and incubated for 2 h at 37°C. Then the cells were cultured in plaque medium . A 1:1000 dilution of BPV1 virions in medium as well as medium only were used as respective positive and negative controls. Three days post infection, the cytopathic effects were analysed by confocal microscopy.For the plaque assays, Vero cells were seeded at a density of 25,000 cells/cmBased on good general health condition and the informed owner’s written consent, 30 sarcoid-affected horses  were subsequently enrolled in a patient trial. As from March 2019, equine patients consecutively presented at the Vetmeduni Equine University Hospital. Following an owner interview and thorough clinical examination of the horse, its signalment, basic clinical parameters, as well as sarcoid disease specifications were recorded. The latter included the number, type and location of the lesions, maximum tumour diameters as determined with callipers and the disease history  Tables . The intequ or iNSB/E6E7equ in SNH buffer per lesion. In horses bearing more than one sarcoid, at least one tumour was left untreated. The decision as to which tumour(s) should be injected was based on the clinical type , the accessibility of the tumour in the standing horse and the compliance of the animal. In a few cases, animals were sedated by intravenous (i.v.) injection with Detomidine  and Butorphanol  before treatment. Two different time schedules were implemented: Schedule 1 involved the first eight patients and consisted of intratumoural injections of iNSA/E6E7equ on days 0, 2 and 4, and of iNSB/E6E7equ on days 7, 9 and 10. Schedule 2 involved the next 21 patients and consisted of intratumoural injections of iNSA/E6E7equ and/or iNSB/E6E7equ on days 0, 28 and 86 (§) that were unable to present at the Vetmeduni in time due to SARS-CoV2-related travel restrictions  and non-infected EqPALF included as controls tested positive for NP and beta actin, and beta actin only, thus confirming the accuracy of the experiment  within two weeks, or with SNH buffer in an analogous manner (C group). The trial was blinded for co-authors E.K. Hainisch and A. Rümmele carrying out the injections and the monitoring. In both groups, tumours on the right side of the neck were left untouched. Monitoring of horses revealed no alterations regarding behavioural parameters, respiratory rates and heart rates. As shown in equ, which ranged between 38.4°C and 39.5°C  and persisted for a maximum of 24 hours. All horses of the AS group developed oedema at the injected tumour sites, and also at the non-injected tumour sites on the right side of the neck, as exemplarily shown in Twelve horses bearing 2 x 5 experimentally BPV1 virion-induced pseudo-sarcoids on the left and right side of the neck were randomly assigned to an “active substance”  or a control  group. Tumours on the left side of the neck were injected thrice with INSA/E6E7equ or iNSB/E6E7equ were detected from nasal swabs or faeces by RT-PCR, as shown in Biological safety of both viruses was assumed due to the partial NS1 deletion and intratumoural administration. To confirm this assumption, nasal swabs and fresh faeces were collected after each virus administration during the safety study and assessed for virus shedding. No iNSA/E6E7From the 29 sarcoid-bearing horses enrolled in the patient study, 19 presented with severe (S), five with moderate (M), and five with mild (m) disease . Fifteenequ and iNSB/E6E7equ within two weeks and were permanently kept within the BSL-2 facility for this purpose. Two horses with severe disease  initially responded to the therapy as revealed by dramatic tumour regression within the first three months after treatment. However, the sarcoids subsequently resumed growth. Another treatment round in month 6 had a transient therapeutic effect in patient LAW, whereas ROS failed to respond , iNSB/E6E7equ only on days 0, 28, and 86  or iNSB/E6E7equ on days 0 and 28 and iNSA/E6E7equ on day 86 . An overview on current treatment results is given in To explore the possibility of reducing the stabling time at the BSL-2 facility and further improving treatment efficacy, all further patients received intratumoural injections on days 0, 28 and 86. According to this time schedule, the next 10 horses (STF—SAV) were exclusively treated with iNSA/E6E7reatment . In two reatment . Comparaequ on days 0, 28 and 86 (BEG), or iNSB/E6E7equ on days 0, 28 and 86 (ZAC), crusts (BEG) and hair roots (ZAC) collected from previous tumour sites during the last visit were subjected to DNA extraction and subsequent BPV1/2 E5 PCR. Both samples tested negative  for sarcoid immunotherapy. Previous studies have shown that conservation of the first 106 C-terminal amino acids of the NS1 gene (iNS) confers sufficient live attenuation but permits efficient viral replication also in IFN-competent tumour cells. Viral replication is important for the expression of tumour antigens (unpublished). In accordance with these data, we could show that BPV1 E6E7 is stably co-expressed by iNSA/E6E7equ and iNSB/E6E7equ in infected equine primary palatal fibroblasts.Human high-risk PVs (hrHPVs) cause distressing, potentially lethal cancer diseases in humans. In recent years, considerable efforts have been made to develop immunotherapeutic vaccines that induce or enhance a hrHPV-specific immune response and by this, promote the killing of infected tumour cells . In equiequ and then iNSB/E6E7equ/lesion every other day was well tolerated. The immunogenicity of both viruses was clinically reflected by transient fever in four of six, and by oedema at the injection sites in all six treated individuals. Importantly, virus-treated horses also developed oedema at all non-injected tumour sites on the right side of the neck. This finding clearly indicates that iNSA/E6E7equ and iNSB/E6E7equ induced a systemic immune response. This assumption was corroborated in the patient study. Experimental pseudo-sarcoids differ from natural sarcoids in that they regress spontaneously [Previous in vivo studies have shown that (transgene-expressing) NS1-deleted influenza A and B viruses are well tolerated when applied intranasally, subcutaneously or into the muscle , 45, 54.aneously . Virus-,The patient study involved a total of 29 sarcoid-affected horses of different age and breed, and all horses received the active substance. It may be argued that a group of untreated sarcoid patients should have been included as control. We opted against this possibility for the following reasons: First, sarcoids are usually persistent tumours that do not regress spontaneously . Second,equ and/or iNSB/E6E7equ resulted in sarcoid regression in 20/29 patients. Intriguingly, regression was not restricted to injected sarcoids but also observed for non-injected lesions, which is consistent with a systemic and sarcoid-specific immune response. In most cases, both time schedules  led to satisfactory results. Given that the first schedule requires permanent stabling of horses at a BSL-2 facility for more than two weeks, whereas the second schedule only necessitates three stays overnight, the latter has been adopted as standard procedure for welfare reasons.Exclusive or combined intratumoural administration of iNSA/E6E7equ and iNSB/E6E7equ for prime and boost treatment is necessary to achieve a maximum therapeutic effect. Whereas iNSA/E6E7equ and iNSB/E6E7equ contain identical E6 and E7 antigens, the vector backbones stem from antigenically completely different influenza viruses (A and B). The rationale behind the combined approach was that the use of antigenically different vectors may help overcome preexisting immunity to the vector after several treatments, that may reduce the effectiveness of boost treatments. While sarcoids in several horses  started regressing just after the heterologous boost at day 86, three horses treated solely with homologous vector also began to regress after the third treatment . Therefore, we will need to address this issue in larger numbers of patients per group as to be able to provide conclusive data. However, our current findings suggest that exclusive repeated treatment with iNSA/E6E7equ or iNSB/E6E7equ is as efficient as the successive administration of both viruses. In this context, the severity and persistence of the disease as well as a history of previous unsuccessful therapeutic interventions seemed to be more decisive for the therapeutic outcome than injection schedules and schemes. The six non-responders and the three transiently responding horses all presented with severe disease, i.e. multiple sarcoids of all types, including aggressive fibroblastic sarcoids, at different sites of the body. Five of these patients had a history of unsuccessful treatment.We also investigated whether a combination of iNSA/E6E7equ therapy, they are absolutely unique. In cases, where clearance from infection could demonstrably be achieved, it might be possible to reach sustainable freedom from disease.Prior to treatment, all horses of the patient study underwent a clinical examination leading to sarcoid diagnosis. The latter was confirmed by BPV1/2 PCR from DNA of sarcoid scrapings, which also revealed the exclusive presence of BPV1 in the samples. Following successful therapy of severe, periocular sarcoid disease in two horses, BPV1/2 PCR from crusts or hair roots collected from previous tumour sites scored negative. To our knowledge, this is the first report on a sarcoid therapeutic that is not only reported to induce tumour regression, but has also an impact on BPV1 infection levels. This finding is not completely surprising, as we used an immunotherapeutic approach targeting the causative viral agent underlying the disease. Provided these data can be strengthened by in-depth infection screening of further patients with complete sarcoid regression following iNSA and/or B/E6E7equ and iNSB/E6E7equ. Due to ethical considerations, we have addressed this issue in a HPV16 TC-1 mouse model providing consistent evidence that the antitumor effect was mainly due to the expression of the tumour antigens. In this model, we could also demonstrate that tumour regression was highly dependent on virus dosage, and associated with strong infiltration of tumour-specific CD3+ and CD8+ T lymphocytes [equ- and iNSB/E6E7equ-induced tumour regression is ongoing.In this study, we did not include the parenteral influenza virus A and B strains as controls that would allow for assessment of the specific therapeutic effect of iNSA/E6E7phocytes  Click here for additional data file.


Figure 4A as published. The incorrect caption stated NCM460 cell lines are normal gastric epithelial cells, however, NCM460 cell lines are normal colorectal epithelial cells. The correct caption for Figure 4 appears below. Additionally, there was a mistake in Figure 1 as published. Figure 1C is immunofluorescence and Figure 1D is immunocomplex by Co-IP. OE-ControlTrop2 should appear on the top row and OE-Trop2 should appear on the bottom row in Figure 1C. Representative images for these parts contain small issues which have been corrected. The corrected Figure 1 appears below.FIGURE 4. The effects of CORO1C knockdown on CRC growth and metastasis in vitro and in vivo. (A) Levels of CORO1C1 protein expression in CRC cell lines and normal colorectal epithelial cells (NCM460) determined by western blotting. (B) COCA2 and HCT116 cells showed a significant decrease in protein level after shCORO1C transfection. (C) CORO1C downregulation significantly inhibited the proliferation of both cell lines. (D) A significant decrease in cell anchorage-dependent growth was detected after CORO1C knockdown.  Decreased CORO1C expression impaired abilities of migration (E) and invasion (F) of CRC cells . All quantitative data of invitro assays were generated from three replicates (G). The effects of CORO1C downregulation on the tumor growth in the xenograft mouse model (n = 6 mice/per group). *p < 0.05, **p < 0.01, ***p < 0.001.In the original article, there was a mistake in the caption for The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Background: Vitamin D deficiency is prevalent globally and there is lack of evidence as to how 25(OH)D2 contributes to vitamin D status. The aim of this study was to describe vitamin D status and to assess the role of vitamin D2, a dietary vitamin D source, against the vitamin D status of children aged 3–5 years in China. Methods: Data were extracted from the Chinese National Nutrition and Health Surveillance (CNNHS) in 2013. The concentration of serum 25(OH)D2 and 25(OH)D3 was measured by using LC-MS/MS. Results: A total of 1435 subjects were enrolled and serum 25(OH)D were analyzed. The prevalence of total serum 25(OH)D < 30 nmol/L was 8.9%. Serum 25(OH)D2 was detected in 10.9% of the studied children. After adjusting for confounding factors, total 25(OH)D concentration was 8.48 nmol/L lower and odds ratio of vitamin D deficiency was 4.20 times (OR (95%CI): 4.20 ) in children without 25(OH)D2 than those with 25(OH)D2 detected. Conclusions: Vitamin D deficiency was common among children aged 3–5 years in China. Vitamin D2 may play a role in preventing vitamin D deficiency in Chinese children aged 3–5 years. Vitamin D is a critical vitamin that regulates calcium, phosphorus  and boneVitamin D insufficiency is common in China. The prevalence of vitamin D insufficiency (25(OH)D < 50 nmol/L) was reported to be 53.2% for Chinese children and adolescents aged 6–17 years in 2010–2012 . ChildreVitamin D includes both vitamin D2 and vitamin D3. It is commonly considered that human vitamin D is mainly produced by skin through sunlight exposure, with a small amount coming through dietary intake, as it naturally is not present in many foods ,15. SomeData were collected from the Chinese National Nutrition and Health Surveillance in 2013 (CNNHS). The detailed methods of CNNHS 2013 were described previously . BrieflyThe primary caregivers of children aged 3–5 years were interviewed using face-to-face interview by well-trained staff. A standardized questionnaire was used to collect the information of socioeconomic, family, lifestyle, health-related information, food frequency and supplement use information in the past week. A standard stadiometer and electronic weighting scale (Tc-200 K) were used to measure the height and weight respectively, without shoes, hat, heavy coat and untied hair.g for 15 min within 30–60 min. The serum used to test for vitamin D was aliquoted and stored in a brown centrifuge tube at −20~−80 °C in the local laboratory. All samples from the 55 study sites were transported to the National Institution for Nutrition and Health, China CDC by using cold chain. All the samples were preserved at −80 °C refrigerator until analysis. Serum 25(OH)D2 and 25(OH)D3 concentration were measured in qualified laboratory by liquid chromatography–mass spectrometry . The LOD (limit of detection) of 25(OH)D2 and 25(OH)D3 were 2.45 nmol/L and 1.13 nmol/L, respectively. Both low and high concentration of quality control standards  of 25(OH)D2 and 25(OH)D3 were analyzed in every run. Inter-run coefficients of variations (CVs) for serum quality-control pools were 3.47% and 3.10% for low concentration and high concentration for 25(OH)D2 and 3.49% and 3.24% for low concentration and high concentration for 25(OH)D3, respectively.Fasting venous blood (4 mL) was collected into vacutainer tubes (Becton Dickinson) containing potassium ethylene diamine tetraacetic acid (EDTA -K2), and then the samples were centrifuged at 1500× Total serum 25(OH)D concentration was the sum of serum 25(OH)D2 concentration and 25(OH)D3 concentration . Total vp < 0.05.Prevalence of vitamin D deficiency was presented as percentages (%(95% CI)) and concentration of 25(OH)D was described as median . Difference between 25(OH)D concentration was compared by Wilcoxon rank sum test or Kruskal–Wallis H rank sum test according to the classification of two or more groups. Chi-square test was used for categorical variables. Multiple linear regression was used to analyze the association between 25(OH)D concentration and potential related factors. Multiple logistic regression was performed to analyze associated risk factors for vitamin D deficiency. The variables included in the models were found to be significantly different in the concentration of vitamin D and vitamin D deficiency between different groups. The stepwise regression method was used for model selection (significance level for entry (SLE) = 0.20, significance level for stay (SLS) = 0.05). All statistical analyses were performed by SAS 9.4 software . The difference was considered as statistically significance with In total, serum vitamin D was measured in 1481 children aged 3–5 years, 46 samples collected from April to September were excluded because the samples collected during these months were too scattered and the interaction between latitude and survey month could not be analyzed. Finally, 1435 children were included in the current study, 83.6% (1199/1435) of whom were Han ethnic and 2.2% (31/1435) of whom were Muslin ethnic. The percentage of subjects was similar across different latitude groups and almost all of field work was completed in fall/winter (from October to January) .Twenty-five hydroxy-vitamin D3 existed in all children’s blood sample, but 25(OH)D2 was only detected in 10.9% (156/1435) of the samples. The medians of serum 25(OH)D3 and total 25(OH)D for all children were 47.5  nmol/L and 49.3  nmol/L, respectively. The concentration of 25(OH)D2, 25(OH)D3 and total 25(OH)D were 10.1  nmol/L, 41.8  and 55.0 nmol/L in the group with vitamin D2 detected, respectively. The prevalence of children suffered from vitamin D deficiency was 8.9%, which was 3.2% in the group with vitamin D2 detected and 9.5% in the group without vitamin D2 detected .p < 0.001).The associated factors of 25(OH)D concentration were shown in Influencing factors of vitamin D deficiency are shown in The prevalence of vitamin D deficiency (25(OH)D < 30 nmol/L) was 8.9% overall and more than one-fifth of the children had vitamin D deficiency at latitude above 30° N in December. Children with detectable 25(OH)D2 had higher concentration of 25(OH)D and lower odds of vitamin D deficiency.The prevalence of vitamin D deficiency among Chinese preschool children in this study was 8.9%, which was higher than 7.1% of children aged 1–5 years in NHANES 2011–2014  and 7% oVitamin D deficiency was more common at latitude above 30° N in November and December. However, vitamin D deficiency was not severe in October, even in the area of latitude above 35° N. At the areas of latitude below 30° N, vitamin D deficiency was not common from October to January. The prevalence of serum 25(OH)D < 25 nmol/L ranged from approximately <1–5% in the winter/lower latitude subpopulation  and in summer higher latitude subpopulation  of adolescents and adults from NHANES III . It was The prevalence of detectable 25(OH)D2 was 10.9% of children in the current study, which was similar to NHANES 2007–2010 (11% in children aged 1–5 years) . In the Adjusting for the effects of other possible factors, the group of 25(OH)D2 undetected was 4.20 times more likely to have vitamin D deficiency than the detected group. The median total 25(OH)D was significantly higher in the children with detectable vs. undetectable 25(OH)D2 (55.0 nmol/L vs. 48.3 nmol/L). These suggested that vitamin D2 played an important role in maintaining vitamin D status of Chinese children aged 3–5 years.Unlike vitamin D3, vitamin D2 must be derived from dietary sources or nutrient supplements. It was generally considered that vitamin D2 was not commonly found in food. Due to the fact that vitamin D2 data are extremely sparce in food composition tables, it is difficult to calculate vitamin D2 intake and the contribution of vitamin D2 to total vitamin D. In the current study, the concentration of 25(OH)D2 was 10.1  nmol/L and total 25(OH)D was 55.0  nmol/L in the group with detected vitamin D2. In a study about osteoporotic fractures of men ≥65 years old, 25(OH)D2 (18.5 nmol/L) also played an important role to total 25(OH)D (64.5 nmol/L) in the group with detected 25(OH)D2 . VitaminVitamin D2, which is converted by fungal sterol, ergosterol, mainly exists in plant foods. Simon et al. showed that mushrooms are excellent sources of vitamin D2 , and vitOur study has some limitations. The prevalence of vitamin D deficiency may be overestimated, as the survey was conducted from October to January, mainly in the winter. In addition, dietary vitamin D2 information was not available to assess its contribution; however, serum 25OHD2 might provide metabolized vitamin D2 contribution for Chinese children aged 3–5 years. Randomized control trials of dietary vitamin D2 intake study remain warranted to assess the effects.Overall, vitamin D deficiency is relatively common for children aged 3–5 years in China, especially for those living above 30° N latitude in November and December, which could be a focus for vitamin D deficiency interventions. For a proportion of these children, dietary vitamin D2 might contribute to their vitamin D status and dietary vitamin D2 might be considered for improving vitamin D level of children in this age group in China. In the population with risk of vitamin D deficiency , the magnitude of vitamin D2 intake contribution to overall vitamin D status warrants further studies by using longitudinal study designs.


Macaca fascicularis papillomavirus type 3 (MfPV3) antigens delivered via nucleic-acid and adenoviral vectors in outbred mouse models. Ten artificially fused polypeptides comprising early viral regulatory proteins were designed and optionally linked to the T cell adjuvant MHC-II-associated invariant chain. Transfected HEK293 cells and A549 cells transduced with recombinant adenoviruses expressing the same panel of artificial antigens proved proper and comparable expression, respectively. Immunization of outbred CD1 and OF1 mice led to CD8+ and CD4+ T cell responses against MfPV3 antigens after DNA- and adenoviral vector delivery. Moreover, in vivo cytotoxicity of vaccine-induced CD8+ T cells was demonstrated in BALB/c mice by quantifying specific killing of transferred peptide-pulsed syngeneic target cells. The use of the invariant chain as T cell adjuvant enhanced the T cell responses regarding cytotoxicity and in vitro analysis suggested an accelerated turnover of the antigens as causative. Notably, the fusion-polypeptide elicited the same level of T-cell responses as administration of the antigens individually, suggesting no loss of immunogenicity by fusing multiple proteins in one vaccine construct. These data support further development of the vaccine candidates in a follow up efficacy study in persistently infected Macaca fascicularis monkeys to assess their potential to eliminate pre-malignant papillomavirus infections, eventually instructing the design of an analogous therapeutic HPV vaccine.Persistent human papillomavirus (HPV) infections are causative for cervical neoplasia and carcinomas. Despite the availability of prophylactic vaccines, morbidity and mortality induced by HPV are still too high. Thus, an efficient therapy, such as a therapeutic vaccine, is urgently required. Herein, we describe the development and validation of  Despite efficient prophylactic vaccines and the possibility to screen for cervical lesions, infection with human papillomavirus (HPV) was still responsible for more than 340.000 cervical cancer-related deaths worldwide in 2020 . More thWhereas most HPV infections are spontaneously cleared within months, some persist for years . These c+ and CD8+ T cell infiltration  has a close phylogenetic and phenotypic relationship to HPV16 (Macaca fascicularis), making them an ideal non-human primate (NHP) animal model -vectored vaccines encoding ancestral E1 and E2 antigens targeting the most conserved N- and C-terminal domains, we observed strong immune responses in mice and in cynomolgus macaques against these proteins, but cross-reactivity against prevalent MfPV types was only observed in a subset of animals. Nevertheless, 3 out of 3 animals with T-cell responses towards MfPV3-E1/E2 ended up clearing the specific MfPV3 infection . Furthervia DNA vaccination of outbred CD1 mice. Based on this initial characterization, adenoviral vectors from serotype 19a/64 were generated and characterized in vitro as well as in vivo.To develop such a therapeutic vaccine, vectors expressing antigen candidates comprising E1, E2, E6 and E7 of MfPV3 were generated and characterized. The antigens are genetically linked to the intrinsic T cell adjuvant MHC-II-associated invariant chain (Ii) that has been shown to increase viral-vector-induced T cell responses in mice, cynomolgus macaques and humans –27. The Parts of the sequences encoding E1, E2, E6, and E7 of MfPV3 (EF558839.2) were designed and synthesized at Geneart/Thermo Fisher  . Mutatio2 in a non-humidified incubator. HEK293T cells were transfected using the polyethylenimide (PEI) method  and 7.5 µg PEI in DMEM without any supplements. After 6 h incubation, medium was exchanged to DMEM with 10% FCS and 1% Pen/Strep.HEK293T cells and A549 cells were maintained and grown in Dulbecco’s MEM (DMEM) supplemented with 10% Fetal Calf Serum (FCS) and 1% Penicillin/Streptomycin (Pen/Strep). 9E10 mycl hybridoma cells were cultivated in RPMI supplemented with 10% FCS, 1% Pen/Strep and 2 mM glutamine (Pan). All cell lines were maintained at 37°C and 5% CO) method . For PEISubconfluent A549 cells were infected with Ad19a/64 vectors at an MOI of 30 in DMEM without any supplements. 2 h post infection, medium was exchanged to DMEM with 10% FCS and 1% Pen/Strep.via Flp-recombination in E. coli into a BAC vector containing the genome of a replication deficient Ad-based vector deleted in E1/E3 genes. Recombinant viral DNA was released from the purified BAC-DNA by restriction digest with PacI. The obtained linear DNA was transfected into HEK293T cells for virus reconstitution and propagation. Recombinant viruses were released from cells via sodium deoxycholate treatment. Residual free DNA was digested by DNase I. Afterwards, vectors were purified by CsCl gradient ultracentrifugation followed by a buffer exchange to 10 mM Hepes pH 8.0, 2 mM MgCl2 and 4% Sucrose via PD10 columns . Titration was performed using the RapidTiter method by detection of infected HEK293T cells via immunohistochemical staining with anti-hexon antibody ). Insert integrity was confirmed by PCR amplification from the purified vector DNA followed by DNA sequencing.E1/E3 deficient adenoviral vectors of serotype Ad19a/64 (rAd) were generated as previously described . Briefly5 cell per ml in RPMI supplemented with 1% FCS, 1% Pen/Strep and 2 mM glutamine. The supernatant was harvested 5 days after seeding and the antibody was purified via a HiTrap Protein G column . After washing the column with PBS, the antibody was eluted with 0.1 M glycine/HCl (pH 3.2), neutralized with 0.025 volumes of 1 M Tris/HCl (pH 9) and dialyzed against PBS.The antibody against myc (9E10) was obtained from hybridoma cell supernatants. 9E10 mycl hybridoma cells were seeded at 5 × 10Other antibodies used were: mouse anti-p2a peptide , mouse anti-tubulin , mouse anti-ubiquitin-Biotin , goat anti-mouse-HRP , goat anti-rabbit-HRP , Streptavidin-HRP , rat anti-mouse-PE .Western blot analysis was performed as previously described . BrieflyTo analyze ubiquitinylated proteins, 24 h post transfection, cells were treated with 10 µM MG132 proteasome inhibitor for 6 h. Afterwards, cells were harvested in PBS and washed twice. For inactivation of deubiquitination enzymes, 20 mM N-ethylmaleimide from a freshly prepared stock solution were added to the TDLB lysis buffer. Lysates were generated as described above. Before immunoprecipitation, Protein G dynabeads  were loaded with 10 µg of pulldown antibody. Using these beads, target protein was immunoprecipitated out of 500 µg cell lysate over night at 4°C under slow rotation. After washing the beads four times with PBS, SDS-PAGE buffer was added to the beads before heating at 95°C for 10 min. The samples were used for western blot analyses as described above.Intracellular staining of antigens was performed using standard methods . Cells w7 IFU rAd diluted in 50 µL PBS. Mice were anesthetized with isofluorane before rAd injections. One group received a mixture of four rAd, 2×107 IFU each, into one site.BALB/c and CD1 mice were obtained from Envigo  and OF1 mice from Charles River (France). All animals were female, 6-8 weeks old and housed at the Panum Institute, University of Copenhagen. All experiments were initiated after allowing the mice to acclimatize for at least 1 wk. Experiments were approved by the National Animal Experiments Inspectorate  and performed according to national guidelines. DNA immunizations were performed intradermal (i.d.) with 0.5 g DNA coated onto 1.6 µm gold microcarriers  using the Helios Genegun System . The mice received four DNA immunizations at intervals of one week each. One group received a mixture of four plasmids, 0.5 µg each, into one site. Immunizations with adenoviral vectors were performed intramuscular (i.m.) with 2×10Single cell suspensions of splenocytes were obtained by organ harvest in HANKs followed by straining through 70 µm cell strainers. Cells were incubated for 5 hours in 3 µM monensin with or without 1 µg/mL of relevant peptides. The cells were stained against surface markers: APC-Cy7 or BV421 CD8 , PE-Cy7 CD4 , FITC CD44  and PerCP-Cy5.5 B220 . After surface staining, cells were fixed in 1% PFA, permeabilized in 0.5% saponine, and stained intracellularly using APC IFN-γ  and PE TNF-α  antibodies. The peptides used were 16-mers overlapping by 11 amino acids covering the entire Ii-E1E2E6E7 antigen. The peptides were pooled in 5 separate pools containing Ii (45 peptides), E1 (123 peptides), E2  and E7 (21 peptides) peptides respectively. Peptides were obtained from KareBay, Town, China.+ T-cell responses were measured as B220-, CD8+ or CD4+, CD44+, IFN-γ+ cells and are presented in total number of cells per organ. The quality of the IFN-γ+ responses were evaluated by MFI of IFN-γ and fraction of double positive cells (expressing both IFN-γ and TNF-α) in the IFN-γ+ CD8+ or CD4+ populations. The gating strategy is shown in Flow cytometry was performed on the Fortessa 3  flow cytometer and data analysis was performed using FlowJo V10 software. Epitope-specific CD82, 2.5 µg of each peptide/mL, and subsequently stained with combinations of 0.4 or 5 µM CellTrace CFSE and 0.2 and 2.5 µM CellTraceViolet  for 10 minutes at 37°C, 5% CO2. Pulsed and stained splenocytes were mixed at a 1:1:1:1:1 ratio, and a total of 2.5 x107 cells were injected intravenously into rAd-vaccinated recipient BALB/c mice. As the assay requires adoptive transfer of syngeneic target cells, it was necessary to use inbred mice in order to have HLA-matching. 5 hours later, spleens were harvested, and target cells were identified on the Fortessa 3  flow cytometer by CFSE/CTV staining. The percentage of killing was calculated using the following equation:The assay was performed similarly to what was previously described . Briefly+ T cell epitope and a myc-tag and optionally fused to the Ii T cell adjuvant  and MFI of IFN-γ+ was only performed on responders (IFN-γ+ count > avg + 2 × SD of unstimulated samples).In order to aid visual presentation of the results, we applied a threshold for responses based on the average number of B220Statistical analysis was done using GraphPad Prism 8 software . Values with background subtracted were used to compare the individual groups using the Mann-Whitney test with Bonferroni correction for multiple comparisons. Positive control groups were not included in the multiple comparison analyses. Each symbol represents one mouse. Bars indicate median. Significance levels are marked by * (p<0.05), ** (p<0.01), *** (p<0.005), **** (p<0.001).Macaca fascicularis papillomavirus type 3 (MfPV3), which was shown to persist and induce LSIL-like lesions in the cervix of breeding female cynomolgus macaques (Macaca fascicularis)  , 21. Hervia a p2A site , which has been described earlier to act as a T cell adjuvant (Antigens were conceived as (i) read-through polypeptides with all four MfPV3 early antigens encoded in one open reading frame, (ii) alternatively E1-E2 and E6-E7 fusions were linked p2A site  supportiadjuvant . Furtheradjuvant Figure 1Since E6 and E7 are highly potent oncoproteins, only transformation-defective variants should be used in a therapeutic vaccine setting to ensure safety. Based on the homology to HPV16, the oncoprotein E6 was inactivated by introducing a L110Q substitution to prevent binding of E6 to E6BP and subsequent degradation of tumor suppressor p53 . AdditioFor initial biochemical and immunological characterization, the antigen coding sequences Figure 1Following transient transfection of HEK293T cells with the respective DNA vaccine constructs, all antigens were properly expressed yielding proteins chiefly correlating with the calculated molecular weights  or N-terSince proteins of different sizes behave differently during blotting and signal intensities may not reflect the real expression levels, MfPV3 antigens were quantified by flow cytometry after intracellular staining with the anti-myc-antibody   of whether Ii is fused to E1E2E6E7, (ii) of the order of the early antigens in the polyprotein, and (iii) of the use of a p2a site to separate Ii-E1E2 from Ii-E6E7. Only pURVac-Ii-E2, when administered alone, trended to induce slightly higher levels of specific CD4+ and CD8+ positive T cells . Contrasting previous findings, N-terminal fusion of the Ii molecular adjuvant did not enhance antigen specific T cell responses when delivered as DNA  DNA immunization of outbred CD1 mice confirmed that all pURVac DNA vaccines encoding E1 and/or E2 induced E1- and E2-specific IFN-γd as DNA , 49. How+ T cell response in cynomolgus macaques (via a self-separating p2a peptide (Ii-E1E6E7-p2a-Ii-E2) and for control Ii-E1E6E7 lacking E2. In order to confirm proper expression of the encoded antigens, western blot analysis was performed with A549 cells following transduction with the indicated rAds. All vectors readily expressed the antigens with bands resembling the respective fusion proteins at the expected molecular weights  had been shown earlier to be generally suitable to deliver MfPV3 antigens and to efficiently induce CD8macaques . Based o weights 4A, B. A+ and CD4+ T cell responses against E1 and E2 antigens  revealed a p-value of 0.06, which is not strictly significant, but strongly indicates a tendency of Ii improving the magnitude of CD8+ T-cells against E1. Further, the vaccine without Ii was the only one for which the CD4+ and CD8+ T-cell responses against E1 was not significantly different from the negative control. This may confirm earlier observations suggesting that Ii can boost T cell responses in the context of adenoviral vaccine delivery. No difference in the quality of the T cell responses was detected, assessed by the MFI values of IFN-γ , it was remarkable that no responses against E6 or E7 could be observed in any experiment testing the vaccines in outbred CD1 mice. As the MHC-type of the founders of the CD1 strain is unknown, and has not been controlled for since the establishment of the strain, we wondered whether there were limitations in terms of MHC-restriction in the CD1 mice we had obtained. Limitations of genetic diversity of outbred mouse models has indeed been described previously and almost all outbred mouse strains except OF1 descent from the same 9 founder mice . Hence v+ responses against E1 in BALB/c mice  infects cynomolgus macaques and infection is associated with persistence and cervical intraepithelial neoplasia  was devised in order to separate E2 from the readthrough protein, and Ii-E6E7E1E2 was excluded for further development of rAd-based vaccine prototypes. Antigen delivery with rAds of serotype 19a/64 confirmed that all antigens were immunogenic, with most of them inducing potent CD4+ and/or CD8+ T cell responses  and thus presumably also number of potential epitopes, it was still surprising that none of the 107 outbred CD1 mice in our experiments that were immunized with E6 and/or E7 harboring vaccines were responding. This might be explained by a yet limited number of MHC alleles in CD1 mice. This hypothesis is supported by the fact that this strain descends from only two male and seven female founder mice . In a divia rAds contain the T cell adjuvant Ii (except for E1E2E6E7 which serves as reference), which is known to enhance CD4+ and CD8+ T cell responses when delivered by viral vectors .PH, CT and RW designed the study and acquired funding, PN, DB, BA, TW, CP, SS planned and performed the experiments, PN, DB, BA, PH, CT and RW interpreted the data, PN and DB wrote the draft, PN, DB, BA, PH, RW wrote, reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.This project has received funding from the Eurostars-2 joint programme with co-funding from the European Union Horizon 2020 research and innovation programme (E!12151). The project E!12151 was carried out within the framework of the European funding program “Eurostars” and the German partners were funded by the Federal Ministry of Education and Research. The manuscript reflects only the authors’ view and the European Commission is not responsible for any use that may be made of the information it contains. The funders had no influence on study design, data collection and analysis, decision to publish, or preparation of the manuscript.PH is an inventor on a patent detailing the use of the invariant chain as an adjuvant for virally delivered vaccines owned by the University of Copenhagen. The right to use the patent for treating HPV is licensed to InProTher ApS where author PH is the founder, major shareholder, board member and employee. DB is an employed by the company InProTher ApS. TW, CP, and SS are employed by the company SIRION Biotech GmbH. CT is founder and shareholder of SIRION Biotech GmbH and a board member of InProTher ApS. RW is board member of SIRION Biotech GmbH. PN, BA, RW, DB, PH, CP and CT are inventors on a patent application.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


PS1 mutations  observed in FAD affect mitochondrial functions.Mitochondrial dysfunction and oxidative stress are frequently observed in the early stages of Alzheimer's disease (AD). Studies have shown that presenilin-1 (PS1), the catalytic subunit of γ-secretase whose mutation is linked to familial AD (FAD), localizes to the mitochondrial membrane and regulates its homeostasis. Thus, we investigated how five Methods: We used H4 glioblastoma cell lines genetically engineered to inducibly express either the wild-type PS1 or one of the five PS1 mutants in order to examine mitochondrial morphology, dynamics, membrane potential, ATP production, mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), oxidative stress, and bioenergetics. Furthermore, we used brains of PS1M146V knock-in mice, 3xTg-AD mice, and human AD patients in order to investigate the role of PS1 in regulating MAMs formation.Results: Each PS1 mutant exhibited slightly different mitochondrial dysfunction. Δexon9 mutant induced mitochondrial fragmentation while A431E, E280A, H163R, and M146V mutants increased MAMs formation. A431E, E280A, M146V, and Δexon9 mutants also induced mitochondrial ROS production. A431E mutant impaired both complex I and peroxidase activity while M146V mutant only impaired peroxidase activity. All PS1 mutants compromised mitochondrial membrane potential and cellular ATP levels were reduced by A431E, M146V, and Δexon9 mutants. Through comparative profiling of hippocampal gene expression in PS1M146V knock-in mice, we found that PS1M146V upregulates Atlastin 2 (ATL2) expression level, which increases ER-mitochondria contacts. Down-regulation of ATL2 after PS1 mutant induction rescued abnormally elevated ER-mitochondria interactions back to the normal level. Moreover, ATL2 expression levels were significantly elevated in the brains of 3xTg-AD mice and AD patients.Conclusions: Overall, our findings suggest that each of the five FAD-linked PS1 mutations has a deleterious effect on mitochondrial functions in a variety of ways. The adverse effects of PS1 mutations on mitochondria may contribute to MAMs formation and oxidative stress resulting in an accelerated age of disease onset in people harboring mutant PS1. Alzheimer's disease (AD) is the most common form of dementia for which there is no cure or effective treatment. AD is characterized by pathological phenotypes that include β-amyloid (Aβ) peptide accumulation in the brain, intracellular tau aggregation, chronic neuroinflammation, and mitochondrial dysfunction The most critical risk factor for developing AD is aging, which is a process that results in a gradual and progressive decline in physiological functions, eventually leading to many pathological conditions such as cancer, arthritis, stroke, and neurodegenerative diseases Since aging is the most significant risk factor in developing AD, it is logical to consider that mitochondrial dysfunction contributes to the disease process. In fact, studies have shown that mitochondrial dysfunction and oxidative stress play a significant role in early AD pathogenesis Presenilin-1 (PS1), an aspartyl protease, is the catalytic subunit of γ-secretase that mediates cleavage of type I transmembrane proteins, including amyloid precursor protein (APP) within its transmembrane region PS1 mutations identified in FAD  on mitochondrial functions. We used six H4 glioblastoma cell lines, each genetically engineered to express either PS1 wild-type (WT) or one of the five PS1 mutants in an inducible manner  in the formation of ER-mitochondria contact sites. Moreover, significantly elevated ATL2 expression levels were observed in the brains of both 3xTg-AD mice and AD patients. Overall, our findings suggest a deleterious effect of PS1 mutations identified in FAD on mitochondrial functions.As PS1 is highly enriched in MAM, we investigated the effect of five different Seven-month-old homozygous PS1M146V knock-in mice and their wild-type littermates of the same sex  and genetic background (C57BL/6) were maintained under pathogen-free conditions on a 12 h light-dark cycle with continuous access to food and water. All procedures were reviewed and approved by the NIA Animal Care and Use Committee. Seven and twelve-month-old 3xTg-AD mice  and their wild-type littermates of the same genetic background (C57BL/6) were maintained under pathogen-free conditions on a 12 h light-dark cycle with continuous access to food and water. All experiments and procedures were approved by the Institutional Animal Care and Use Committee of Sungkyunkwan University.Inferior parietal lobule specimens from the brains of three AD patients and three age-matched control subjects that had been enrolled in the University of Kentucky Alzheimer's Disease Center Autopsy Program were used for this study. At autopsy, tissue specimens were rapidly removed, frozen, and stored at -80 °C.http://help.brain-map.org/display/aging/Documentation). RNAseq data from frontal white matter (FWM) of each subject were used for our gene expression level analysis.This database contains RNAseq-derived transcriptome data from 107 subjects obtained from the Adult Change in Thought (ACT) cohort. A detailed description of tissue collection, tissue processing, and quantitative data generation is available in the dataset documentation (Homo sapiens brain neuroglioma H4 cell lines stably expressing wild type (WT) PS1 or EOFAD linked PS1 mutants  under the control of the tetracycline repressor element were a generous gift from Brandon Wustman and Anthony Stevens from Amicus Therapeutics, Inc . Cells were cultured in Dulbecco's modified Eagle medium (DMEM)  supplemented with 10% fetal bovine serum , 50 µg/ml Zeocin , and 2.5 µg/ml Blasticidin . Cultures were maintained at 37 °C with 95% O2 and 5% CO2Tissue sample preparation was performed as described previously TM tissue protein extraction reagent  with protease/phosphatase inhibitor cocktail  and western blot analysis was performed as described previously Cells were collected using T-PERPS1 cell lines were grown in DMEM supplemented with 10% fetal bovine serum, 50 µg/ml Zeocin, and 2.5 µg/ml Blasticidin. For PS1 mutant induction, each cell line was treated with 100 ng/ml of tetracycline  for 5 days TM Red  and 1 µM ER-TrackerTM Green  for 20 min at 37 °C in a 5% CO2 atmosphere, from stocks prepared according to the manufacturer's instructions. For fixation, cells were washed with PBS twice, for 5 min each time, fixed with 4% paraformaldehyde (PFA) for 10 min, and washed in PBS twice (5 min each time). The slides were mounted in Antifade Mounting Medium with DAPI . For live-cell imaging, HBSS was replaced with a fresh probe-free medium after 20 min incubation. Confocal imaging was performed using a LSM700 . 405-nm laser was used for DAPI (Ex/Em = 365/468). 488-nm laser was used for ER-TrackerTM Green (Ex/Em = 504/511) and 555-nm laser was used for MitoTrackerTM Red (Ex/Em = 579/599). Images were acquired, and the fluorescence intensity profile and weighted colocalization coefficient were analyzed using the Zeiss ZEN software. The weighted colocalization coefficients were calculated using the same equation as the Mander's colocalization coefficients, but the value for each pixel was equal to its intensity value.H4PS1 cell line was fixed, processed, and visualized as previously described Each H4TM Red . Cells were incubated with 2 μM MitoSOXTM Red for 30 min at 37 °C in a 5% CO2 atmosphere before washing with PBS. The fluorescence was detected using confocal microscopy (Carl Zeiss LSM700). 405-nm laser was used for Hoechst (Ex/Em = 361/497) and 555-nm laser was used for MitoSOXTM Red (Ex/Em = 510/580).Mitochondrial superoxide was detected using the fluorescent MitoSOXPS1 cell line was determined using the Complex I Enzyme Activity Microplate Assay Kit  according to the kit instructions. Briefly, 200 μg of protein was incubated in 'incubation buffer' for 3 h at room temperature and washed three times with '1× buffer'. Assay buffer was added, and samples were analyzed on a Synergy HTX Multi-Mode Microplate Reader  .Mitochondrial complex I activity of each H4PS1 cell line was determined using the EZ-Hydrogen Peroxide/Peroxidase assay kit  according to the manufacturer's instructions. Briefly, 50 µl of sample and HRP standard solution was incubated with 50 ul of Oxi-Probe/H2O2 working solution for 30 min at room temperature in the dark. Each well was analyzed on a Synergy HTX Multi-Mode Microplate Reader (Abs: 560 nm).Peroxidase activity of each H4PS1 cell line was seeded in a XF24 cell culture plate. One day prior to the experiment, 1 ml of XF calibrator was added to each well of the XF cartridge and incubated overnight at 37 °C in humidified atmosphere and 0% CO2. Thirty minutes prior to the experiment, cells were washed with PBS, and 625 μl of respective XF assay medium was added per well before incubating for 30 min at 37°C in a humidified atmosphere and 0% CO2. For the XF Cell Mito Stress analysis, XF assay medium was supplemented with 5 mM glucose and 2 mM glutamine. After a 15 min equilibration time, OCR was measured every 8.30 min , 4 times after the addition of respective compounds. The different compounds were added to the injection ports of the XF cartridge in 10x of final concentration, and they were diluted in XF assay medium before the experiment.The Seahorse analyzer XF24  was used according to the kit instructions to monitor the oxygen consumption rate (OCR) in real-time. Briefly, two days before the experiment, each H4P-dCTP . Radiolabeled cDNA was allowed to hybridize overnight at 43 °C to the mouse NIA 17K cDNA filters Mice were sacrificed by cervical dislocation and hippocampus was removed and flashes frozen. The tissue was processed with a Bead Beater  and RNA was purified using RNEasy Mini Kits . Purified RNA was evaluated for quality and quantity with a Bioanalyzer . Probe preparation and hybridization were done as previously described P value < 0.05 were selected as DEGs. The z-ratio is a measure of fold change between comparisons P values between the selected samples were drawn by in-house R scripts. The GO terms enriched in the DEGs were determined using g:Profiler (ver 0.6.7).All data were processed by z-score transformation ATL2 sense, 5'-UAG AGU UUG UUA CAG ACU G-3';ATL2 antisense, 5'-CAG UCU GUA ACA AAC UCU A-3'.Cells were seeded in 6-well dishes at 70% confluence and transfected with siRNA against ATL2  using OptiMEM and INTERFERin (Polyplus-transfection), according to the manufacturer's instructions. Transfected cells were maintained for 24 h prior to further experiment. The sequences of siRNA are as follow:PS1 cell lines and reverse transcribed into cDNA using PrimeScript RT Master Mix (TaKaRa). qPCR was performed to detect mRNA expression of ATL1, ATL2, ATL3 by TB Green PCR Kit (TaKaRa), and GAPDH was used as a reference gene. The qPCR primer sequences used are as follow:ATL1 forward, 5'-CCC TGT GCA CTT GGG CAT AT-3';ATL1 reverse, 5'-TTG TAC AAA GCC TGG TCC CAC-3';ATL2 forward, 5'-TTG CCA CAT CCT GGT CTT AAA-3';ATL2 reverse, 5'-GCA GCA ATG GAA CCA GAT TT-3';ATL3 forward, 5'-ACA AGC CCT GAC TTT GAT GG-3';ATL3 reverse, 5'-TGC AGC TGC TAA GTT GTT GG-3';GAPDH forward, 5'-AGC CAC ATC GCT CAG ACA C-3';GAPDH reverse, 5'-GCC CAA TAC GAC CAA ATC C-3'.Total RNA was extracted by RNAiso plus  from H42DCFDA) , which is converted to the highly fluorescent 2',7'-dichlorofluorescein (DCF) in the presence of an oxidant. Briefly, each H4PS1 cell line was plated in 96-well plates. Twenty-four hours later, cells were incubated with CM-H2DCFDA (5 μM) in serum-free medium for 30 min. The fluorescence intensity was detected using a Victor X3 plate reader  according to the manufacturer's protocol (Ex/Em = 492/527).Intracellular ROS levels were measured using a fluorescent dye, chloromethyl 2',7'-dichlorofluorescein diacetate   or TMRM . The fluorescence intensity for aggregates and monomeric forms of JC-1 was detected using a TECAN Infinite 200 multifunctional microplate reader  according to the manufacturer's protocol . The fluorescence intensity for TMRM was detected using Synergy NEO multi-mode microplate reader  according to the manufacturer's protocol (Ex/Em = 548/574). Total cellular ATP levels were detected using an ATP bioluminescence detection kit  according to the manufacturer's protocol.Mitochondrial membrane potential was measured in H4t-test (two-tailed) was used to compare each H4PS1 cell line before and after PS1 induction. A P-value < 0.05 was considered statistically significant.All statistical analyses were performed using GraphPad Prism 8.0 software . All data are presented as mean ± standard error (SEM). Depending upon the variables, one-way or two-way analysis of variance (ANOVA) with Bonferroni post-hoc test was used to compare group differences. Student PS1Δexon9 cell line, 44 kDa full-length PS1 was accumulated upon tetracycline treatment. This is because deletion of exon 9, which contains endoproteolysis site, prevents the cleavage of full-length PS1. Furthermore, the endogenous PS1 was diminished in H4PS1Δexon9 cell line upon tetracycline treatment . Each PS1 mutant was successfully induced by 100 ng/ml of tetracycline treatment and the degree of overexpression was observed to be 2.2 to 3.4-fold  is the proximal mitochondrial ROS, we measured the production of O2•- in each H4PS1 cell line. After induction, there was a significant increase in mitochondrial O2•- in H4PS1A431E, H4PS1E280A, H4PS1M146V, and H4PS1Δexon9 cell lines based on MitoSOX fluorescence  and Complex III (ubiquinol cytochrome c oxidoreductase). Complex III is responsible for most O2•- production in the heart and lung, while complex I is the main source of O2•- in the brain + decreases, which leads to an enhanced superoxide production 2•- level was due to abnormal complex I activity. The H4PS1A431E cell line exhibited significantly reduced complex I activity after induction  and molecular oxygen (O2) 2O2 can have a deleterious effect on cells because it can be converted to highly reactive hydroxyl radical (•OH) via the Fenton reaction in the presence of Fe2+2O2 to H2O and O2. Unfortunately, catalase is not present in the mitochondria. In mitochondria, Glutathione Peroxidase (GPx), an enzyme with peroxidase activity, reduces H2O2 to H2O and lipid hydroperoxides to their corresponding alcohols to protect the organism from oxidative damage PS1 cell line was perturbed by PS1 mutant expression. After induction, H4PS1A431E and H4PS1M146V cell lines showed significantly impaired peroxidase activity  catalytically converts Oactivity A-D. FurtPS1 mutation affects mitochondrial respiration. We used a Seahorse XF24 extracellular flux analyzer to measure OCR during sequential treatment with compounds that modulate mitochondrial activity and examine the functional bioenergetic capacity of mitochondria in each H4PS1 cell line. There was no significant difference in basal OCR before and after the induction in most H4PS1 cell lines except for H4PS1H163R cell line , the raw reads were preprocessed and analyzed with DIANE 1.0. Eventually, a total of 409 DEGs were identified, of which 203 (49.63%) were upregulated, and 206 50.37%) were down-regulated . Hierarc0.37% wer-in mice B.Atl2, which was the most upregulated ER-related genes in PS1M146V knock-in mice. Atl2 encodes an ER-resident membrane-bound GTPase Atlastin 2 (ATL2), which mediates ER membrane fusion and tethering. We first examined whether PS1 mutant induction increases ATL2 expression in each H4PS1 cell line. While there was no significant difference in ATL1 and ATL3 mRNA expression, ATL2 mRNA expression was significantly increased in H4PS1A431E and H4PS1M146V cell lines after induction . The study contained clinical data from 107 subjects, of which 57 were free from dementia, and 50 were clinically diagnosed with dementia. Among 57 non-dementia subjects, we excluded any individuals with one of the following conditions: (1) Braak stage 6, (2) NIA-Reagan stage 3, (3) three or more traumatic brain injuries (TBIs). This resulted in 36 subjects (Non-AD). Among 50 subjects that were clinically diagnosed with dementia, 43 met NINDS-ARDA Alzheimer's criteria for either 'Probable AD' or 'Possible AD'. Of these 43 AD subjects, we excluded any individuals with one of the following conditions: (1) Braak stage 0, (2) NIA-Reagan stage 0, (3) three or more TBIs. This resulted in 33 subjects (AD). To determine whether ATL2 gene expression is elevated in AD subjects, we analyzed RNA-seq datasets. Within frontal white matter (FWM), significantly increased expression levels were observed in AD subjects for ATL2 but not for ATL1 or ATL3  knock-in mouse compared to that of wild-type mouse. We identified a total of 409 DEGs of which 21 were related to mitochondria and 26 were related to ER. Since PS1 is mostly localized to ER and Golgi, the DEGs were significantly enriched in biological processes, such as 'translation', 'protein transport', 'intracellular transport', and 'organic substance transport'. Additionally, the DEGs were also significantly enriched in cellular components, such as 'organelle', 'intracellular organelle', 'membrane-bounded organelle', and 'membrane-enclosed lumen'. This suggests that PS1 might have a significant role in the endomembrane system.Atl2, a gene that encodes ER-shaping protein ATL2, was the most upregulated gene. ATL2 regulates ER structure and plays a critical role in ER membrane fusion and tethering, which are crucial for the formation of ER-mitochondria contact sites. In fact, down-regulation of ATL2 after PS1 mutant induction rescued abnormally elevated ER-mitochondria colocalization back to a normal level suggesting a crucial role of ATL2 in the formation of ER-mitochondria contact sites. Moreover, significantly elevated ATL2 expression levels were observed in the brains of both 3xTg-AD mice and AD patients.Among ER-related DEGs, Although it is unclear how PS1 mutant regulates ATL2 expression level, PS1 might play an important role in maintaining and regulating MAMs structure formation through ATL2, which is significantly related to AD pathogenesis. Therefore, discovering the precise role of PS1 and ATL2 in MAMs formation might open a new therapeutic window for AD treatment.PS1 mutations have detrimental effect on different mitochondrial functions. Most importantly, most of PS1 mutations increased ER-mitochondria contacts and mitochondrial ROS production while reducing mitochondrial membrane potential. We also suggest that PS1 might play a critical role in maintaining and regulating ER-mitochondria interactions through ATL2, an ER-shaping protein whose expression level is significantly elevated in brains of both FAD mouse model and AD patients. We believe that our discovery of the effects of PS1 mutations on mitochondria function and ER-mitochondria interactions will have significant clinical and biological implications in pathology of AD. However, further studies are needed to elucidate how PS1 mutant regulates ATL2 expression level.Overall, our findings suggest that each of five Supplementary figures and tables.Click here for additional data file.Supplementary table S1.Click here for additional data file.Supplementary table S2.Click here for additional data file.


Pathological growth of cardiomyocytes during hypertrophy is characterized by excess protein synthesis; however, the regulatory mechanism remains largely unknown. Using a neonatal rat ventricular myocytes (NRVMs) model, here we find that the expression of nucleosome assembly protein 1 like 5 (Nap1l5) is upregulated in phenylephrine (PE)-induced hypertrophy. Knockdown of Nap1l5 expression by siRNA significantly blocks cell size enlargement and pathological gene induction after PE treatment. In contrast, Adenovirus-mediated Nap1l5 overexpression significantly aggravates the pro-hypertrophic effects of PE on NRVMs. RNA-seq analysis reveals that Nap1l5 knockdown reverses the pro-hypertrophic transcriptome reprogramming after PE treatment. Whereas, immune response is dominantly enriched in the upregulated genes, oxidative phosphorylation, cardiac muscle contraction and ribosome-related pathways are remarkably enriched in the down-regulated genes. Although Nap1l5-mediated gene regulation is correlated with PRC2 and PRC1, Nap1l5 does not directly alter the levels of global histone methylations at K4, K9, K27 or K36. However, puromycin incorporation assay shows that Nap1l5 is both necessary and sufficient to promote protein synthesis in cardiomyocyte hypertrophy. This is attributable to a direct regulation of nucleolus hypertrophy and subsequent ribosome assembly. Our findings demonstrate a previously unrecognized role of Nap1l5 in translation control during cardiac hypertrophy. The heart develops ventricular hypertrophy to compensate for increased hemodynamic workload under various stresses, and progressively leading to heart failure , 2. CardPrimary regulation of translation occurs in nucleolus, where ribosomal DNA is transcribed and ribosome subunits are assembled. Cardiac hypertrophy is one of the first diseases identified to be associated with ribosomal DNA transcriptional disorders , 9. AcceThe mechanistic target of rapamycin (mTOR) plays a central role in protein synthesis by phosphorylating 70 kD ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein-1 (4E-BP1), which subsequently initiate a series of signal transduction to promote the operation of ribosomes , 14. ThoNucleosome assembly protein 1 like (NAP1L) protein family has been identified as evolutionarily conserved histone chaperones assisting the assembly of nucleosomes with different histone variants –20. It cHere we find that NAP1L5 expression is significantly upregulated in phenylephrine (PE)-induced cardiomyocyte hypertrophy. siRNA-mediated knockdown and adenovirus-mediated overexpression experiments suggest that NAP1L5 is required for the development of cardiomyocyte hypertrophy. Transcriptome analysis and puromycin incorporation assay reveal a crucial regulation of nucleolus hypertrophy, ribosome assembly and protein synthesis rate by NAP1L5. Our findings demonstrate a novel role of NAP1L5 in translation control during the pathological growth of cardiomyocytes, and provide potential molecular targets to treat cardiac hypertrophy.We previously performed an RNA-seq analysis in PE-induced cardiomyocyte hypertrophy . After rWe then constructed an Adenovirus carrying rat Nap1l5 gene (Ad-Nap1l5) to overexpress it in NRVMs. Both qRT-PCR and Western blot analyses confirmed the overexpression of NAP1L5 48h after infection . NAP1L5 vice versa , after siNap1l5 transfection . Principce versa , suggestce versa . These dGene ontology (GO) analysis showed that the upregulated genes after NAP1L5 knockdown mainly related to immune responses, whereas the down-regulated genes covered oxidative phosphorylation, cardiac muscle contraction, and ribosome . Gene seInterestingly, the genes altered by siNap1l5 were negatively correlated with the regulations by polycomb repressive complex II PRC2;  and compConsistently with the GO analysis results, GSEA also revealed a down-regulation of ribosomal genes after NAP1L5 knockdown , coverinWe next analyzed the interactome of NAP1L5 from the String database, and found that NAP1L5 linked to core ribosomal proteins through binding to ribosome assembly or transport factors, including SDAD1, RRP7A, and NOL6 . InteresTo further explore the molecular mechanism, we tried to validate the interaction between NAP1L5 and SDAD1 or NOL6. Unfortunately, we did not observe a direct interaction between NAP1L5 and SDAD1, nor the full-length or spliced NOL6 isoforms . These dThe pathogenesis of cardiac hypertrophy is a programmed process mediated by regulations at multiple layers such as transcription, translation, and metabolism , 47–49. Ribosome biogenesis is a highly energy-consuming process. After translation, ribosomal proteins need to be transported into nucleus and precisely folded with mature rRNA inside nucleolus . CardiacDuring evolution, higher ordered plants and mammals have acquired several paralogues of NAP1, named NAP1 like family, and five different NAP1L proteins (NAP1L1-5) have been identified. The overall structure of NAP1L proteins is highly conserved and the protein sequences of human NAP1L homologs to hNAP1L1 show identities ranging between 31% for hNAP1L5 and 64% for hNAP1L4 . These NSDAD1 (SDA1 domain containing 1) is a shared hub protein interacting with both NAP1L1 and NAP1L5 . It has Regulation of cell growth is a fundamental process in development and disease that integrates a vast array of extra- and intra-cellular information. A central player in this process is PolI, which transcribes ribosomal RNA (rRNA) genes in the nucleolus. Rapidly growing cancer cells are characterized by increased Pol I-mediated transcription, and consequently nucleolar hypertrophy. An aberrant increase in nucleolar size in cancer cells was documented more than a century ago. Nucleolar hypertrophy is a common feature in cancer and that nucleolar size can be used as a histopathological marker to grade the malignancy of tumors , 71. DesmTOR is at the core of the translational regulation by converging signaling transduction from a variety of nutrients and growth factors , 72–76. in vitro cellular model induced by an α1-receptor agonist, PE. Whether this mechanism also applies in vivo and in human systems need to be further investigated.One limitation of this study is that all observations are from an Taken together, we demonstrate that NAP1L5 is upregulated in PE-induced cardiomyocyte hypertrophy, which functions to promote protein synthesis through facilitating ribosome assembly. Our findings provide novel insights into the translation control during cardiac hypertrophy, and provide potential molecular targets to treat cardiac hypertrophy and heart failure.Neonatal rat ventricular myocytes (NRVMs) were isolated and cultured as described previously . NeonataGCCACCatggccgaccccgagaag−3′) and rNap1l5 CZR (5′- TACGTCGTATGGGTATCTAGATcttctcggcagagtcgggacc−3′) was used to amplify a cDNA fragment encoding the mature rNap1l5 peptide. Massive plasmid replication was performed by transforming the pcDNA3.1-rNap1l5-Flag plasmid into E. coli DH-5α. In addition, in order to overexpress Nap1l5 in NRVM, the pcDNA3.1-rNap1l5-Flag plasmid was sent to Shanghai Hanheng Biotechnology Co., Ltd. for adenovirus packaging.The full-length cDNA sequence of rat Nap1l5 (rNap1l5) were obtained by the National Center for Biotechnology Information (NCBI). The pcDNA3.1-rNap1l5-Flag recombinant plasmid were constructed by cloning the entire coding region of rNap1l5 into the NheI and XhoI sites of the pcDNA3.1-HA plasmid. The primer pair rNap1l5 CZF (5′- GGGAGACCCAAGCTGGCTAGCACTATAGGGAGACCCAAGCTGGCTAGCGCCACCatgtccgggaggaacaac−3′) and SDAD1 CZR (5′- ATCTGGTACGTCGTATGGGTATCTAGActtcattcttttcctctttttcag−3′) was used to amplify a cDNA fragment encoding the mature SDAD1 peptide. Again, to get the mature Nol6 peptide, the primer pair Nol6 CZF (5′- ACTCACTATAGGGAGACCCAAGCTGGCTAGCGCCACCatgggaccagcccccgccgga−3′) and Nol6 CZR (5′- ATCTGGTACGTCGTATGGGTATCTAGAcacagtccacctctcacttcggg−3′) was used to amplify a cDNA fragment encoding the mature Nol6 peptide. However, the sequencing results showed that the deletion of exon 18 of this Nol6 resulted in frameshift mutation after exon 18, so we named this spliced NOL6 isoforms as Nol6Δ. To get the full-length Nol6, we used genomic DNA of Nol6 as a template and designed primers NOL6 gBF (tggtttgtcacctggagggcagcgg) and NOL6 gBR (cttgaggacgtcagtgtgtgtggcag) amplified in the upstream and downstream of exon 18 to obtain the missing fragment, and then the-full length Nol6 was obtained by bridging and recombination.The full-length cDNA sequence of rat Nol6 and SDAD1 were obtained by the National Center for Biotechnology Information (NCBI). The pcDNA3.1-SDAD1-HA recombinant plasmid were constructed by cloning the entire coding region of SDAD1 into the NheI and XhoI sites of the pcDNA3.1-Flag plasmid. The primer pair SDAD1 CZF (5′- Cell size was measured using wheat germ agglutinin (WGA) staining as previously described. Briefly, cardiomyocytes were stimulated with 50 μM of PE for 48 h after transfection with different siRNA or infection with different adenoviruses for 24 h, the culture medium was discarded, washed with PBS, and fixed with 4% paraformaldehyde for 15 min at room temperature. Then 5.0 μg/mL WGA (Invitrigen) was applied to incubate with cardiomyocytes for 10 min at 37°C. When labeling was complete, removed the labeling solution, and washed cells three times with PBS. Then dyed with DAPI for 10 min at room temperature, and washed 5–7 times with PBS for 5 min each time. Finally, Images collected under a fluorescence microscope. Image Pro Plus 6.0 software was used to measure cardiomyocyte surface area. Each field of view measured more than 50 to calculate the surface area of cardiomyocytes.The method of immunofluorescence staining of cardiomyocytes was as described above. The cardiomyocytes used for immunofluorescence staining were cultured in a six well cell culture plate containing sterile glass slides. Cardiomyocytes were stimulated with 50 μM of PE for 48 h after transfection with different siRNA or infection with different adenoviruses for 24 h, The cells were then fixed with 4% formaldehyde in PBS for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 10 min, goat serum was blocked at room temperature for 30 min, and then incubated with α-actinin antibody  or Fibrillarin antibody  at 4°C overnight. The next day, incubated the cell sample with the corresponding secondary antibody for 1 h at room temperature. Then dyed with DAPI for 10 min at room temperature. Then washed 5–7 times with PBS for 5 min each time. Finally, removed the slides from the 6-well plate and mounted the slides. Then, the fluorescence staining pictures of cardiomyocytes were collected under a fluorescence microscope. Image Pro Plus 6.0 software was used observe and take pictures.Cultured HEK293T cells co-transfected with the appropriate plasmids were collected and lysed in an IP buffer (50mM Tris-HCl (pH 8.0), 150mM NaCl, 2mM EDTA, 1% NP-40 and 5% Glycerol) supplemented with a protease inhibitor cocktail (Roche) and PMSF. After incubating for 30 min at 4°C, followed by centrifuging 13,000 g for 15 min, the cell lysates were precleared with normal mouse or rabbit immunoglobulin G and protein A/G-agarose beads (Roche) for 1h at 4°C. The precleared lysates (500 μl) were then incubated with 1 μg of antibody and 10 μl of protein A/G-agarose beads on a rocking platform at 4°C overnight. The immunocomplex was collected, washed 5–6 times with cold IP wash buffer (50mM Tris-HCl (pH 8.0), 150mM NaCl, 2mM EDTA, 0.1% NP-40) and blotted using the indicated primary antibodies.Total proteins were extracted from NRVM cells. NRVM cells lysed in Radio Immunoprecipitation Assay (RIPA) lysis buffers . Cell lysates were centrifuged at 12,000 × g for 15 min. The proteins concentration was detected by the BCA method, and equal quantities of protein extracts were loaded on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred onto polyvinylidene fluoride (PVDF) membranes . The membranes were blocked with 5% (w/v) non-fat milk for 1 h at room temperature, the membranes were incubated with specific primary antibodies overnight at 4°C. After washing 3 times with TBST, the membranes were incubated with the HRP-linked secondary antibody at room temperature for 1 h, and then washed 3 times with TBST again. Finally, the protein bands on the membranes were detected by chemiluminescent reagents . Chemiluminescence signals were quantified using an ECL imager, and analyzed using Quantity One software . The specific primary antibodies were: anti-GAPDH (Proteintech), anti-Histone3 (Proteintech), anti-H3K4me3 (CST), anti-H3K9me3 (CST), anti-H3K27me3 (CST), anti-H3K36me3 (CST), anti-Flag (Sigma), and anti-Puromycin (Santa Cruz).Different treatments of NRVM cells, 30 min before harvesting, add 1μM puromycin to each well for 30 min, then harvest the cells and extract the protein, and detect the binding level of puromycin by Western blot with anti-Puromycin antibody.2, 0.5% Nonidet P-40) containing 100 μg/ml CHX, 1x protease inhibitors and 1:1,000 dilution of RiboLock RNase inhibitor for 15 min on ice. Following centrifugation, lysates were ultracentrifuged on 10–50% sucrose gradients at 190,000g for 1.5 h at 4°C. Following ultracentrifugation, fractions were collected from each sample using a BioComp Piston Gradient Fractionator instrument fitted with a TRIAX flow cell to measure absorbance.Ribosome profiling was performed as described. Cardiomyocytes were transfection with different siRNA or infection with different adenoviruses for 48 h, and were treated with with CHX (100 μg/mL) for 10–30 min before harvested. Cells were harvested using trypsin and then lysed in polysome extraction buffer  Enrichment analysis of DEGs using Metascape online database with default parameters. (iii) Gene Set Enrichment Analysis (GSEA) is used to screen significantly enriched signaling pathways and transcription factors with default parameters, was performed as described previously.NRVMs were transfected with siNap1l5 or siNeg using Lipofectamine RNAiMAX. After transfected 24 h, 50 μM phenylephrine (PE) were added in the media to induce cardiomyocyte hypertrophy, was performed as described previously . 48 h afTotal RNA was extracted from NRVM cells using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), following the manufacturer's instructions. RNA was quantified using NanoDrop (Thermo Fisher Scientific). The cDNAs were synthesized from 1μg RNA using a RevertAid First Strand cDNA Synthesis Kit . Real-time PCR was performed using the specific primers and Ultra SYBR Mixture  on CFX96M Touch Real-Time PCR Detection System . The PCR primer sequences used in this study include rNap1l5-forward: GAGCACAGCAGCTGACAGAC; rNap1l5-reverse: ATGACGTCGTTCTTGGGTTC; rNppa-forward: ATACAGTGCGGTGTCCAACA; rNppa-reverse: AGCCCTCAGTTTGCTTTTCA; rNppb-forward: CAGCTCTCAAAGGACCAAGG; rNppb-reverse: GCAGCTTGAACTATGTGCCA; rGAPDH-forward: ACAGCAACAGGGTGGTGGAC; rGAPDH-reverse: TTTGAGGGTGCAGCGAACTT. Relative expression of genes was determined with GAPDH as an endogenous control.post hoc analysis. Student's t-test was used for comparisons between two groups. P < 0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism 8 Software. All experimental data are presented as mean ± SEM of at least three independent experiments unless denoted elsewhere. Statistical significance for multiple comparisons was determined by one-way ANOVA or two-way ANOVA followed by Tukey's test. Bonferroni adjustment was used for GSE173737).All relevant data supporting the findings of this study are available within the article and its supplementary information or from the authors upon reasonable request. RNA-seq data have been deposited in NCBI's Gene Expression Omnibus (GEO) repository , National Health Commission of China (No. 2017ZX1030440 2001-008), State Key Laboratory of Cardiovascular Disease (Nos. GZ2021015 and 2021-YJR01), and start-up funds from Fuwai Hospital Chinese Academy of Medical Sciences, Shenzhen (GSP-RC-2020002).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The approach operates under mild conditions and is selectivetoward the semihydrogenated olefins with good to very good Z isomer stereoselectivity. (Spectro)electrochemistry supportsthat the electrocatalytic cycle is initiated in an atypical mannerwith a nickelacyclopropene complex, which upon further protonationis converted into a putative cationic Ni(II)–vinyl intermediatethat produces the olefin after electron–proton uptake. Thiswork establishes a proof of concept for homogeneous electrocatalysisapplied to alkyne semihydrogenation, with opportunities to improvethe yields and stereoselectivity.Electrifying theproduction of base and fine chemicals calls forthe development of electrocatalytic methodologies for these transformations.We show here that the semihydrogenation of alkynes, an important transformationin organic synthesis, is electrocatalyzed at room temperature by asimple complex of earth-abundant nickel, [Ni(bpy) The semihydrogenationof alkynes to form alkenes  were recently disclosed. The electrochemical counterpart, electrocatalyticalkyne semihydrogenation, has to date been explored only with heterogeneouscatalytic systems,32 often based on noble metals.32The increasing electrificationof the energy sector is expected to significantly impact process technologiesin other sectors, including the chemical industry. Electrocatalysisis therein instrumental to facilitate the conversion of chemicalsusing renewable electricity as the energy income. alkenes 1a is a p3](BF4)2 (Ni(BF4)2)33 -olefin. In addition, mechanistic investigations conductedby (spectro)electrochemical methods indicate the involvement of annickelacyclopropene complex as a central intermediate, which is protonatedto form a putative cationic Ni(II)–vinyl species.Here we report the firstexample of electrocatalytic alkyne semihydrogenationusing a homogeneous molecular catalyst, the readily accessible complex2+ into [Ni(bpy)3]0, followed by release of abipyridine ligand to give [Ni(bpy)2]0 .35 The following reversiblewaves at E1/2 = /2 = −2.39 and −2.65 VFc  are assigned to the [Ni(bpy)2]0/– couple and the bpy0/– coupleof released free bipyridine, respectively.34Aninitial investigation of the electrochemical behavior of ) in DMF 2a and S11) (10 equiv vs Ni) produces a positive shift of the [Ni(bpy)3]2+ reduction wave and the [Ni(bpy)2]0/– couple , plausibly showingcoordination of the alkyne to reduced Ni.33 Further addition of benzoic acid (BA) (50 equiv vs Ni) as a proton source results in anenhanced cathodic peak current for the reduction of [Ni(bpy)3]2+  and the appearance of an irreversible reduction wave fromca. −1.80 VFc , discarding electrocatalytic protonreduction at that wave. We thus hypothesized that the larger cathodiccurrents building upon concomitant alkyne and H+ additiontestify to electrocatalytic reduction of the alkyne.The addition of 4-octyne (1/1H2)redox couple is ca. E°(1/1H2) = −0.82 VFc in our conditions.Alkyne semihydrogenation is thus thermodynamicallyfeasible in the potential range −1.5 to −2.0 VFc considered here and even favored compared with the hydrogen evolutionreaction (HER) (cf. Supporting Information (SI) section 2 for details).The standard potentialof the alkyne/alkene .37 Under our standard conditions , we observed that 1 is successfullyfully converted within 40 min to the respective (Z)-olefin in 69% yield with 53% Faradaic efficiency (F.E.), correspondingto a turnover number (TON) of 6.8 (Table S2). The same conditions exempt of Ni or BA showed only traces of activity .Substituting Ni(BF4)2 with theNi(II) complex [Ni(MeCN)6](BF4)2 resultsin only 6% conversion . Inaddition, nonrinsed postactivity electrodes39 are barely active in the absence of Ni, despite thepresence of residual adsorbed Ni (see SI section 3.2.3.6 for details). These points all strongly support thatelectrocatalysis occurs at well-defined molecular species of the type[Ni(bpy)n]q.The actual electrocatalytic activityof BA] (Table S2). Applying largeror smaller overpotentials  still afford full conversion of 1 but lower the F.E.and, for the more anodic conditions, also the yield (46%). Balancingthe activity and excess acid equivalents, we thus selected the standardconditions summarized in Screening for optimized reaction conditions evidenced thatincreasingalkyne concentration results in lower conversions, yields, F.E., andTONs, whereas the opposite is observed for increasing acid concentration,reaching up to 83% yield, 58% F.E., and a TON of 8.3 at 500 mM [BA] S2. ApplyS , which produce thecorresponding olefins in good to high yields (68–93%). Thereaction is highly stereoselective toward cis-olefins except inthe case of diphenylacetylene, which evolves a 61:39 mixture of (Z)- and (E)-stilbene40 .Terminal alkynes (5 and 6) are also fullyconverted, although in limited olefin yields ,likely because of secondary reactions to give oligomeric species (videinfra).The electrocatalytictransformation of 10 terminal and internalalkynes 1 to 10; 1 was inv7) predominantly produces the corresponding enynes. Chlorosubstitution(8) slows the conversion but still affords high olefinyield, whereas bromo substitution (9) quenches the reactivity.Good tolerance is observed for the propargylic alcohol of substrate 10, which evolves the corresponding (Z)-olefinin high yield and F.E. Importantly, full alkyne conversion in generalproceeds with overhydrogenation below traces, featuring intrinsicsemihydrogenation selectivity of the system (see SI section 3.2.3.2). The F.E. values in olefin at full conversionare usually moderate, which result from the occurrence of side reductions,the most plausible being hydrogen evolution over Ni species.The intermediate conversion of a conjugated diyne (Ni through CV and in situ ultraviolet–visible(UV) and infrared (IR) spectroelectrochemistry (SEC). Upon additionof excess 1 (≤5 equiv), the CV wave for two-electronreduction of Ni to [Ni(bpy)2]0  and triggers a reoxidationevent at Ep,a = −1.40 VFc in the backward scan . Inconjunction, the [Ni(bpy)2]0/– coupleat E1/2 = −2.36 VFc evolvesinto a reversible wave at E1/2 = −2.30VFc. A stoichiometricamount of 1 (Ni:1 = 1:1) sufficesto provoke these changes  andto induce the release of a second bipyridine per Ni upontwo-electron reduction . We thus conclude that the complex evolvedfrom the doubly reduced Ni and alkyne 1 isformulated as [Ni(bpy)1]0 (Ni-I) (33 Thisintermediate is best described as a Ni(II) nickelacyclopropene complex.42 The in situ UV-SEC electrolysis of Ni and 1 at an applied potential of −1.93 VFc  evolves charge-transfer bands (at438 and 695 nm) in spectral regions reported for the formation ofnickelacyclopropene [Ni(bpy)] fragments.41We then investigated possible mechanisms for alkyne semihydrogenationelectrocatalyzed by and refs −35) becoard scan S2. Inco2 (to avoid overlay with the solvent signature) produces aband at 1924 cm–1 . We tentatively attribute this band to the C–C stretchingof the nickelacycle in [Ni(bpy)2] by comparison withthe literature (proposed at 1770 cm–1 in [Ni(bpy)4];42 see SI section 3.3 for details). These results further strengthenour assignment of the structure of the electrochemically generated Ni-I. The CV wave at E1/2 = −2.30VFc is then attributed to a [Ni(bpy)1]0/– couple and the reoxidation wave at Ep,a = −1.40 VFc to [Ni(bpy)1]0 oxidation .Moreover, the corresponding IR-SEC experiment using alkyne BA is added to the reaction mixture, thetwo-electron-reduction wave of Ni is only marginallyaffected with regard to peak potential and current density . We thus discard the formation ofa catalytically competent nickel hydride species at the potentialof this wave,43 from which it follows thatthe electrocatalytic cycle is initiated via nickelacyclopropene Ni-I .When only Ni and alkyne 1, raising the concentrationof BA (Ni:1:BA = 1:100:0–50) was found to magnifythe first reduction wave , indicatingthat protonation of Ni-I is related to the buildup ofelectrocatalytic current. At the potential of this wave, we can ruleout a stepwise electron transfer (ET) to Ni-I precedingprotonation, as the reduction of [Ni(bpy)1] requiresa more negative potential (E1/2([Ni(bpy)1]0/–) = −2.30 VFc; videsupra). Rather, a plausible transformation involves opening of the Ni-I nickelacycle via proton transfer (PT) to form a cationicNi(II)–vinyl complex Ni-II (S42).44Ni-II can then bifurcate betweena second PT (protonation-first pathway) to give the dicationic Ni(II)olefin complex [Ni(bpy)]2+ (Ni-III) (0 (Ni-IV) (At fixed concentrationsof ex Ni-II 3 and S42(Ni-III) 3 or anE (Ni-IV) 3.4] results in release of the respective olefin 4H2.45 Furthermore, the absenceof electrocatalytic activity for olefin hydrogenation  points to rapid and irreversibleolefin release from the Ni center (presumably via exchange with solventor bpy). Once the olefin is displaced, the complex can then re-engagein reduction and alkyne activation to form Ni-I.The occurrenceof a protonation-first pathway is supported by thereported observation that twofold protonation of Ni(II) nickelacyclopropene in the presenceof large excess of 2  gradually restores the inhibitedelectrocatalytic waves. These results indicate that excess alkyne(s)coordination occurs at stages preceding protonation, likely Ni–vinylspecies Ni-II and Ni-IV . Additional alkyne binding to Ni–vinylintermediates is also supported by the GC-MS analysis of the electrolyticmixture of phenylacetylene (6), which shows the formationof unsaturated dimeric coupling products . Such di/oligomerization processes are known with similarNi species50 and here constitute side reactionscompeting with the desired hydrogenation to olefins.Finally, we observed thatthe electrocatalytic waves at −1.65and from −1.80 V3]2+ at room temperature using a simple organic proton donor.The system generally discards overhydrogenation and predominantlyproduces the semihydrogenated olefins with good to very good Z stereoselectivity. (Spectro)electrochemical experimentssupport that the electrocatalytic cycle is entered via a nickelacyclopropeneintermediate, which is a rather atypical initiation route in homogeneousalkyne hydrogenation. This intermediate opens upon protonation togive a putative cationic Ni(II)–vinyl intermediate, from whicheither a second protonation or a reduction–protonation sequenceevolves the olefin. Additional alkyne insertion competes with protonationsteps at on-cycle intermediates and leads to oligomeric byproductsdetrimental to the olefin yield. This proof of concept for homogeneouselectrocatalytic alkyne semihydrogenation offers the potential tooperate under mild conditions using readily accessible (Ni) complexeswithout precautions for the handling of sensitive catalysts or theuse of hydrogen. Studies are ongoing to suppress undesired oligomerizationsand to control the stereoselectivity by structural optimization ofthe molecular catalyst.In summary,we have shown here that the semihydrogenation of internaland terminal alkynes is electrocatalyzed by [Ni(bpy)