Most importantly, pharmacological blockade and gene silence of SLC6A14 both restricted epithelial-mesenchymal transition- (EMT-) driven GC metastasis, in which attenuated activation of the PI3K/AKT/mTORC1 pathway caused by amino acid starvation was involved. In summary, it is conceivable that targeting SLC6A14 has a tremendous promising for the treatment of metastatic GC.Metastasis is the main obstacle for the treatment of gastric cancer (GC), leading to low survival rate and adverse outcomes in CG patients. SLC6A14, a general amino acid transporter, can import all the essential amino acids in a manner dependent on the NaCl-generated osmotic gradients. Herein, we constructed GC cell sublines with high (SGC7901-M and MKN28-M) and low (MKN28-NM and SGC7901-NM) metastatic ability. Putative functional genes advancing GC metastasis were identified using mRNA microarray analysis and High-Content Screening. In particular, most significant change with a dampening trend in the migration potentiality of GC cells emerged after SLC6A14 gene was silenced. SLC6A14 expression was positively correlated with the migrated capability of different GC cell lines, and SLC6A14 was also constitutively expressed in GC patients with venous or lymphatic invasion, lymph node, or distant metastasis and poor prognosis, thus prompting SLC6A14 as a nonnegligible presence in supporting GC migration and invasion. Consistently, SLC6A14 depletion drastically depressed GC metastasis  Gastric cancer (GC) remains one of the most commonly diagnosed cancers of human beings with high incidence and high-grade malignancy . Since iα-SMA characterize the EMT initiation [Epithelial-mesenchymal transition (EMT) refers to a well-established process that epithelial cells lose intercellular “self-discipline” and are provided with mesenchymal features . E-cadheitiation . Transcritiation . Abundanitiation –10. Targ0,+, is special among the SLC family and exhibits distinctive properties which distinguish it from other SLCs. SLC6A14 transports 18 of the 20 proteinogenic amino acids except glutamate and aspartate in an Na+- and Cl−-dependent manner, making the transportation almost unidirectional [As acknowledged, a steady influx of exogenous nutrients is essential for standing up the aggressive biobehaviors of tumor cells . The welectional . Recentlectional , thyroidectional , estrogeectional . HoweverHere, we established GC cell sublines with high (MKN28-M and SGC7901-M) and low (MKN28-NM and SGC7901-NM) metastatic ability. The differentially expressed genes (DEGs) in MKN28-M and SGC7901-M, compared with MKN28-NM and SGC790-NM cells, respectively, were screened using mRNA microarray analysis. Among these DEGs, 10 upregulated transcripts with a fold change ≥ 3 in the mRNA expression in both MKN28-M and SGC7901-M cells have attracted extensive attentions. Of these 10 genes, SLC6A14 was substantiated as a motivation factor in encouraging the migration of GC cells. High expression of SLC6A14 was correlated with improved facility of GC invasion and metastasis and a poor prognosis in GC patients. Pharmacological blockade and gene silence of SLC6A14 enormously wrecked the migrated and invasive ability of GC cells and lessened GC metastasis in tumor-bearing mice. Moreover, SLC6A14 depletion instigated amino acid starvation to weaken the activation of the PI3K/AKT/mTORC1 pathway, thus impeding the EMT development which was considered to be a culprit for GC metastasis. In conclusion, SLC6A14 could be a treated target against metastatic GC.μg/mL). All the cells were cultured at 37°C in a humidified atmosphere with 5% CO2.Human GC cell lines MGC-803, BGC-823, SGC-7901, and MKN-28; human gastric epithelia cell line GES1; human embryonic kidney 293 (HEK-293); and 293T cells were purchased from the American Type Culture Collection (ATCC). These cells were maintained in Dulbecco's modified Eagle's medium (Sigma-Aldrich) that was supplemented with fetal calf serum (Life Technologies), penicillin (100 U/mL) and streptomycin (100 α-methyltryptophan (α-MT) were obtained from Cell Signaling Technology (New England BioLabs Inc.).Recombinant human insulin-like growth factor-1 (IGF-1) and Total RNA from indicated cells was extracted by the TRizol regent (Invitrogen), and cDNAs were synthesized using SuperScript™ III (Thermo Scientific). The expressions of SLC6A14, asparagine synthetase (ASNS), and C/EBP-homologous protein (CHOP) were determined using real-time PCR analysis with the support of TaqMan™ Master Mixes (Thermo Scientific), according to the manufacturer's procedure. GAPDH and HPRT1, the housekeeping genes, were used as the controls for normalization. PCR primers for SLC6A14, GAPDH, HPRT1, ASNS, and CHOP gene were provided as follows: SLC6A14 , GAPDH , HPRT1 , ASNS , and CHOP .SGC-7901 and MKN-28 cells were suspended in a six-well simplified Boyden chamber with an 8-micron polyethylene terephthalate (PET) membrane . Adherent cells were serum-starved, and the bottom chamber was consumed with Tissue Culture Growth Media (1.5 mL) which contains 20% heat-inactivated fetal bovine serum (FBS). After incubation for 24 h at 37°C, the migrated cells on the bottom chamber and the nonmigrated cells on the top chamber were aspirated carefully. These harvested cells were cultured and passaged aseptically. Above steps were repeated until 15-round selection was completed. These cells that failed to migrate through the chamber were characterized as low-metastatic GC sublines (MKN28-NM and SGC7901-NM), and the GC sublines that located into the bottom chamber were named as MKN28-M and SGC7901-M with high metastatic ability.w/v) nonfat dry milk and then cultured with primary antibodies overnight at 4°C, followed by the incubation with Goat Anti-Rabbit IgG H&L (HRP)  for 1 h at room temperature. Immunoreactive proteins were visualized using ChemiDoc™ XRS+System (Bio-Rad) with the help of IMMOBILON WESTERN CHEMILUM HRP SUBSTRATE (Millipore). Relative intensities of indicated proteins towards β-actin were estimated. Detailed information for primary antibodies were shown as follows: rabbit anti-human SLC6A14(ab254786), anti-human ZEB2(ab223688), anti-human Twist1 (ab175430), and anti-human PI3K  mAbs from Abcam; rabbit anti-human β-actin (#4970), anti-human AKT (#4685), anti-human mTOR (#2983), anti-human GSK-3β (#12456), anti-human phospho-AKT (#9018), anti-human phospho-GSK-3β (#5558), anti-human phospho-mTOR (#5536), anti-human N-cadherin (#13116), anti-human Vimentin (#5741), anti-human E-cadherin (#14472), anti-human mLST8 (#3274), anti-human PRAS40 (#2610), anti-human 4EBP1 (#9644), anti-human Phospho-4E-BP1 (Thr37/46) (#2855), anti-human p70S6K (#2708), anti-human Phospho-p70S6K (Thr421/Ser424) (#9204), anti-humanPhospho-Raptor (Ser792) (#2083), anti-human Phospho-PRAS40 (Thr246) (#2997), and anti-human α-SMA (#19245) mAbs from Cell Signaling Technology; and rabbit anti-human Phospho-p85 (Tyr458)/p55 (Tyr199) (PL0304731) from PLLABS (British Columbia).Total proteins from indicated cells or tissue specimens were prepared using RIPA Lysis Buffer . Equal amount of protein samples was separated by 12% SDS-PAGE gels and transferred to the nitrocellulose membranes . The membranes were blocked in Tris-buffered saline with 5% (in vitro transcription (IVT) amplification. CRNA was then converted to biotinylated double-stranded cDNA (ds-cDNA) using GeneAtlas® Hybridization Station (Affymetrix). Arrays were washed and stained using Affymetrix GeneChip Fluidics Station 450 systems, followed by the scanning with GeneChip® Scanner 3000 7G (Affymetrix). Differentially expressed genes (DEGs) (fold change ≥ 1.5 and∗P < 0.05) were generated using R package.Total RNA from high-metastatic (MKN28-M and SGC7901-M) and low-metastatic (MKN28-NM and SGC7901-NM) GC cells was extracted using Cells-to-CT Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. All the RNA was quantified by a UV spectrophotometer , and RNA integrity numbers were inspected by Agilent Bioanalyzer 2000 (Agilent). mRNA expression profiles for individuals were confirmed using human Clariom™ S Assay platform (Affymetrix). RNA samples were employed with the primers containing a T7 promoter and executed by reverse transcription reaction for the generation of single-stranded cDNA (ss-cDNA). 3′Adaptor was added to ss-cDNA, which was further converted to complementary RNA (cRNA) by Total RNA was isolated from MKN28-M cells that were transfected with LV-pTZU-SLC6A14 or LV-pTZU6+1 plasmid for 48 h. DEGs in MKN28-M cells with SLC6A14 silence were screened using mRNA microarray analysis via the Prime View Human GeneChip and GeneChip Hybridization Wash and Stain Kit (Agilent). These DEGs were analyzed through NCBI Gene Expression Omnibus Software based on the GSEA database. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis for these DEGs was implemented using Bioconductor and STRING 9.1 software. The significance levels of gene enrichment in each pathway were analyzed through Fisher's exact test.Supplementary Materials and Methods. The migrated ability of MKN28-M cells was assessed based on the fold change of migration rate (migration rate on day 2 for the group with the treatment of control vector/migration rate on day 2 for the group with the treatment of shRNA vector).MKN28-M cells were seeded into the 48-well plates and transfected with Lentivirus vectors that silenced candidate genes and then prepared for wound healing assay. The 2-day cell migration rate was calculated using the Celigo® Image Cytometer and Cellomics Array Scan System according to the formula described in the α with Amp+LB cover. The clones with correct insertion of target sequence were named as pTZU-SLC6A14 plasmid.siRNA duplexes targeting the open reading frame of SLC6A14 gene with loop ring (sense-loop-antisense) were designed using BLOCK-iTTM RNAi Designer and then synthesized into oligonucleotides (ODNs) with BamHI and EcoRI overhanging ends by GenePhama Biotech Co., Ltd. . Annealed top and bottom chains of single-stranded ODN were mixed, followed by the Polymerase Chain Reaction (PCR) for the generation of shRNA templates. The shRNA templates were cloned into the pTZU6+1 vector using T4 DNA ligase, and the conjugated product was transformed into E. coli DH5Alternatively, the coding sequence of SLC6A14 and Raptor gene was searched in Genbank, and PCR primers targeting SLC6A14 and Raptor gene with EcoRI and XhoI overhangs were designed using Primer 5.0 and DNA Star software. SLC6A14 and Raptor cDNA were acquired from HEK-293 cells using PCR amplification. PCR products were cloned into the pcDNA3.1 plasmid for the construction of pcDNA3.1-SLC6A14 and pcDNA3.1-Raptor plasmid.The recombinant vectors and the packaging plasmids pMD2.G and psPAX2 were transfected into 293 T cells. 48 h later, culture supernatant was collected and filtered through a 0.50 mm filter, followed by the centrifugation  at 4°C for 1 h to capture the viruses containing LV-pTZU-SLC6A14, LV-pcDNA3.1-SLC6A14, and LV-pcDNA3.1-Raptor plasmid.LV-pTZU-SLC6A14, LV-pcDNA3.1-SLC6A14, and LV-pcDNA3.1-Raptor plasmid were transfected into GC cells using Lentivirus Enhancement Reagent Envirus™-LV  according to the manufacturer's instructions.103 GC patients without receiving chemotherapy and radiotherapy but underwent surgical resection were followed up for the evaluation of overall survival (OS) and disease-free survival (DFS) time. Tissue specimens were obtained from these patients, and normal gastric mucosas were taken as the controls. Additionally, 20 paired GC tissues and adjacent normal tissues (ANTs) were kindly provided by the department of pathology in the Affiliated Hospital of Qingdao University. These tissue samples were embedded in paraffin for immunohistochemistry or were stored in liquid nitrogen for Western blot analysis. The Ethics Committee of the Qingdao University approved this study, and full consent for each patient was acquired.μg/mL) administration for the collection of luciferase-labeled MKN28-M cells. These luciferase-labeled MKN28-M cells were then transfected with LV-pTZU-SLC6A14 (MOI = 60) or LV-pcDNA3.1-SLC6A14 (MOI = 60) plasmid and were exposed for two weeks to Puromycin (2.5 μg/mL). BALB/C nude mice (6-8 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd  and were kept under specific pathogen-free (SPF) conditions. The tumor-bearing mice were randomly divided into four groups as follows: (1) control group, which was injected with luciferase-labeled MKN28-M cells via tail vein; (2) α-MT treatment group, for which α-MT (3 mg/mL/kg) was prepared in drinking sucrose water and then continuously administered 3 days prior to intravenous injection of luciferase-labeled MKN28-M cells; (3) SLC6A14 silence group, which was injected intravenously with luciferase-labeled MKN28-M cells after the transfection of LV-pTZU-SLC6A14 plasmid; and (4) SLC6A14 overexpression group, which was injected via tail vein with luciferase-labeled MKN28-M cells after the transfection of LV-pcDNA3.1-SLC6A14 plasmid. The mice in each group were injected intraperitoneally with D-luciferin (Invitrogen) (120 mg/kg) and were observed once a week since cell injection using the Imaging Platform for Small Animals in Vivo IVIS® Lumina III (PerkinElmer). All experimental studies were approved by the Qingdao University Animal Care and Use Committee.MKN28-M cells were transfected with LV-pSin-hyg-GFP/luc2 plasmid  via JetPEI reagent (PolyPlus-transfection FARANCE), followed by the Hygromycin (Sigma-Aldrich) , while culture medium containing 20% FBS was added into the bottom chamber. For the performance of invasion assay, the upper chamber was covered with a film of Matrigel . After incubating for another 48 h, the chamber was take out carefully and washed thoroughly and was fixed in 4% polyformaldehyde for 1 h. Finally, the chamber was stained with 0.1% crystal violet for another 20 min at room temperature and was observed under a microscope.Transwell assay was carried out using the Transwell cell culture chamber units  with an 8-micron polyethylene terephthalate (PET) membrane. Briefly, indicated cells were seeded in the 24-well plates and then starved for 24 h. These cells were reseeded in the top chamber with serum-free medium  at 4°C overnight. After being washed with TBST, tissue slices were incubated with HRP-conjugated secondary antibody (1 : 100 dilution) at room temperature for another 1 h. Finally, these sections were cultured in the diaminobenzidine solution at 37°C for 15 min and were observed under a microscope. Staining scores based on the percentage of positive cells were determined using Image-Pro Plus V6.0 software: Scores 0-1 representing < 10% positive staining ; Scores 1-2 representing 10-30% positive staining ; Scores 2-3, representing 30-50% positive staining ; and Scores 3-5 representing >50% positive staining .Paraffin-embedded tissues were serially sectioned at 4 To find the credible target that got immersed in developing GC metastasis, the differentially expressed genes (DEGs) in high-metastatic (MKN28-M and SGC7901-M) compared with low-metastatic (MKN28-NM and SGC7901-NM) GC cells, were screened using mRNA microarray analysis. Approximately 445 and 487 DEGs exhibiting more than 1.5-fold change were identified in MKN28-M and SGC7901-M compared to MKN28-NM and SGC7901-NM cells, respectively Figures . Among tTo further validate the role of SLC6A14 in touching off GC metastasis, SLC6A14 expression in paired GC tissues and ANTs was assessed. As shown in Figures IHC assay demonstrated that GC patients with muscle invasion and distant metastasis exhibited higher SLC6A14 expression as compared with those in situ (∗P = 0.023), the disposition to distant metastasis (∗∗P = 0.009), venous invasion (∗P = 0.033), lymph node metastasis (∗P = 0.035), and lymphatic invasion (∗∗P = 0.021) in GC patients , p-AKT, and p-mTOR; however, SLC6A14 overexpression considerably increased the protein level of p-PI3K (P85), p-AKT, and p-mTOR in GC cells (Figures To expound the underlining mechanism by which SLC6A14 promoted GC metastasis, the DEGs in SLC6A14-silenced-MKN28-M, compared to MKN28-M cells, were examined using the Prime View Human GeneChip. 28 upregulated and 70 downregulated clusters were presented in MKN28-M cells with the transfection of LV-pTZU-SLC6A14 plasmid . KEGG pa Figures . mTOR bi Figures . Further Figures . SLC6A14 Figures . So we cα-MT and LV-pTZU-SLC6A14 administration both augmented the steady-state mRNA level of ASNS and CHOP in GC cells. Collectively, SLC6A14 depletion animates amino acid starvation to extinguish the activation of the PI3K/AKT/mTORC1 pathway.Strong evidences have authenticated that the delivery of the mTORC1 pathway is essential for nutrients intake by tumor cells , 24, andThen, we explored whether SLC6A14 depletion could mediate the alleviation of the PI3K/AKT/mTORC1 pathway to obstruct the EMT process thus interrupting GC metastasis. As depicted in Figures GC represented the fourth most serious cancer and the third greatest cause of cancer-related death worldwide, especially, ranks second in terms of the cancer-related morbidity and fatality in China . Those pA large number of amino acids were ingested as the nutritional sources for catering to the malignant behavior, and various kinds of amino acid transporters equipped carrier channels for amino acid uptake in tumor cells . SLCs, ein vitro and in vivo. Therefore, our findings are complementary to those of the predecessors, in which the role of SLC6A14 not only in stirring up tumorigenesis but also in facilitating GC metastasis is revealed.Actually, the proliferation-promoting role of SLC6A14 has been stated in certain human cancers –18. SLC6To date, there is no indication about the activated pathways excited by SLC6A14, much less for the precise mechanisms underlined in GC metastasis mediated by SLC6A14. Herein, our findings were firstly enumerated that SLC6A14 depletion distinctly retarded the activation of the PI3K/AKT/mTOR pathway, which was manifested as the decreased expression of the adaptor proteins constituted the mTORC1 complex and the downregulation of signaling proteins manipulated by mTORC1. SLCs have been viewed as the accurate sensors of amino acids for mTORC1 functioning in tumor cells , 37. EmeHere, SLC6A14 depletion provokes amino acid starvation followed by the cripple of the PI3K/AKT/mTORC1 pathway, thus raising the possibility that SLC6A14 can be treated as an amino acid sensor to promote GC metastasis through sensitizing the PI3K/AKT/mTORC1 pathway. However, further efforts should be made to certificate what kind of amino acid sensor can be used for SLC6A14 to activate the PI3K/AKT/mTORC1 pathway.β into cell nucleus, thereby relieving the suppressive effect mediated by GSK-3β on the expression of ZEB2 and Slug in GC cells [κB-α degradation and NF-κB phosphorylation, therefore increasing the protein level of E-cadherin and Vimentin but downregulating the N-cadherin expression [EMT enables many malignant features of GC, in which metastasis has become the most concerned . The actGC cells –46. Phospression –49. HereOur findings remarked the point of view that SLC6A14 initiated GC metastasis, thus leading an unsatisfactory prognosis in GC patients. SLC6A14 depletion held the EMT back to confer reduced GC metastasis for which amino acid starvation was triggered, followed by the mitigated activation of the PI3K/AKT/mTORC1 pathway. Taken together, SLC6A14 might be a druggable target in the treatment of metastatic GC.


Mstn) is a negative regulator of skeletal muscle mass, and its deletion leads to reduced mitochondrial function. However, the exact regulatory mechanism remains unclear. In this study, we used CRISPR/Cas9 to generate myostatin-knockout (Mstn-KO) mice via pronuclear microinjection. The skeletal muscle of Mstn-KO mice significantly increased, and the basal metabolic rate, muscle ATP synthesis, mitochondrial respiratory chain complex activity, tricarboxylic acid cycle (TCA), and thermogenesis decreased. In the muscle tissue of Mstn-KO mice, the expression of SIRT1 and pAMPK decreased, and the acetylation modification of PGC-1α increased. Furthermore, the treatment of isolated muscle cells from Mstn-KO and wild-type mice with AMPK activator (AICAR) and AMPK inhibitor (Compound C) found that Compound C down-regulated the expression of pAMPK and SIRT1 and the activity of citrate synthase (CS), isocitrate dehydrogenase (ICDHm) and α-ketoglutarate acid dehydrogenase (α-KGDH) similar to that of Mstn-KO. However, AICAR partially reversed the inhibitory effect of Mstn-KO on the expression of pAMPK and SIRT1 and activity of three enzymes. Thus, Mstn-KO affects mitochondrial function by inhibiting the AMPK/SIRT1/PGC1α signaling pathway.Myostatin (Mstn) is a major negative regulator of skeletal muscle mass and initiates multiple metabolic changes. The deletion of the Mstn gene in mice leads to reduced mitochondrial functions. However, the underlying regulatory mechanisms remain unclear. In this study, we used CRISPR/Cas9 to generate myostatin-knockout (Mstn-KO) mice via pronuclear microinjection. Mstn-KO mice exhibited significantly larger skeletal muscles. Meanwhile, Mstn knockout regulated the organ weights of mice. Moreover, we found that Mstn knockout reduced the basal metabolic rate, muscle adenosine triphosphate (ATP) synthesis, activities of mitochondrial respiration chain complexes, tricarboxylic acid cycle (TCA) cycle, and thermogenesis. Mechanistically, expressions of silent information regulator 1 (SIRT1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) were down-regulated, while peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) acetylation modification increased in the Mstn-KO mice. Skeletal muscle cells from Mstn-KO and WT were treated with AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR), and the AMPK inhibitor Compound C, respectively. Compared with the wild-type (WT) group, Compound C treatment further down-regulated the expression or activity of pAMPK, SIRT1, citrate synthase (CS), isocitrate dehydrogenase (ICDHm), and α-ketoglutarate acid dehydrogenase (α-KGDH) in Mstn-KO mice, while Mstn knockout inhibited the AICAR activation effect. Therefore, Mstn knockout affects mitochondrial function by inhibiting the AMPK/SIRT1/PGC1α signaling pathway. The present study reveals a new mechanism for Mstn knockout in regulating energy homeostasis.Myostatin ( Mstn) is a negative regulator of skeletal muscle mass [Mstn knockout mice increased muscle fiber number (hyperplasia) and fiber size (hypertrophy) during development, resulting in a significant increase in muscle mass [Mstn gene generate similar muscle hypertrophy phenotypes in many different mammalian species, including cattle, sheep, dogs, and humans [Myostatin , a serine/threonine protein kinase, plays a critical role in intracellular energy homeostasis and is essential for regulating mitochondrial function . SIRT1 , we mated only exon 3 deletion mutant F1 mice. A total of 10 mice (59%) among 17 F2 mice were identified as Mstn mutants, showing nine different genotypes with deletions ranging from 5 to 8 nt . The average body weights of Mstn-KO and WT mice increased continuously from 3 to 10 weeks  of the resting state were examined in the groups. Compared to the WT mice, Mstn-KO mice consumed less O2 (2.40 ± 0.47 ml/min for Mstn-KO and 2.57 ± 0.25 ml/min for WT) and released less CO2 (1.65 ± 0.34 ml/min for Mstn-KO and 1.95 ± 0.26 ml/min for WT)  compared with the WT mice  in  for WT) . RQ was  WT mice a. In a re group. b. HoweveMstn-KO mice , and protects against pThr172 dephosphorylation [Mstn knockout , carbon dioxide production (VCO2), and respiratory quotient (RQ) were measured every hour for 3 min in each cage. Mice were maintained on their normal diet or water throughout the detection process.Mice were individually housed in the metabolic cages (Oxylet) and acclimatized for 24 h before recording. Their 24 h oxygen consumption (VOMstn-KO and WT) were euthanized and tissues were collected for experimental purposes. The organs analyzed were the spleen, brain, lungs, pancreas, heart, liver, kidney, thyroid, testicle, and ovary. Body weight and organ weight were calculated.Healthy mice from each group , mitochondrial electron transport chain complexes I-V assay kit , citrate synthase activity assay kit (CS-1-Y), citrate acid content assay kit (CA-1-W), isocitrate dehydrogenase activity assay kit (ICDHM-1-Y), α-ketoglutarate acid dehydrogenase activity assay kit (KGDH-1-Y), and α-ketoglutarate content assay kit (KGA-4-Q) according to the manufacturer’s protocols from Comin . The optical densities were measured using a microplate reader .Lysates of mice skeletal muscle tissue generated under the addition of proteinase inhibit cocktail Complete Mini  and phosphatase inhibitor cocktail PhosSTOP . The total protein of the lysates was measured by the Pierce BCA Protein Assay Kit . Co-immunoprecipitation (co-IP) was completed using the Thermo Scientific Pierce co-IP kit (#26149) following the manufacturer’s protocol. Ten micrograms of the antibody were incubated with the delivered resin and covalently coupled. The antibody-coupled resin was incubated with 200 µL of the whole mice skeletal muscle protein lysates overnight at 4 °C, respectively. The resin was washed, and the protein complexes bound to the antibody were eluted. Subsequent Western blot analyses were performed as described before.Primary mouse skeletal muscle cell was cultured using a described method before . Cells wp-value of less than 0.05 was accepted as statistical significance.Comparisons between two observations in the same subjects were assessed by Student’s paired t-test. Results were expressed as the mean ± standard deviation (SD). The Mstn knockout decrease basal metabolic rate, body temperature, and mitochondrial activity in skeletal muscle. The probable mechanism is that Mstn knockout suppressed mitochondrial function via inhibiting the AMPK/SIRT1/PGC1α signaling pathway (This study demonstrated that  pathway m.


STAT3 is a transcription factor which is activated via various signaling transduction pathways or Epstein-Barr virus (EBV) infection and plays an oncogenic role in lymphoid malignancies including Hodgkin lymphoma (HL). The tumor cells of HL are derived from germinal center B-cells and transformed by chromosomal rearrangements, aberrant signal transduction, deregulation of developmental transcription factors, and EBV activity. HL cell lines represent useful models to investigate molecular principles and deduced treatment options of this malignancy. Using cell line L-540, we have recently shown that constitutively activated STAT3 drives aberrant expression of hematopoietic NKL homeobox gene HLX. Here, we analyzed HL cell line AM-HLH which is EBV-positive but, nevertheless, HLX-negative. Consistently, AM-HLH expressed decreased levels of STAT3 proteins which were additionally inactivated and located in the cytoplasm. Combined genomic and expression profiling data revealed several amplified and overexpressed gene candidates involved in opposed regulation of STAT3 and EBV. Corresponding knockdown studies demonstrated that IRF4 and NFATC2 inhibited STAT3 expression. MIR155 (activated by STAT3) and SPIB (repressed by HLX) showed reduced and elevated expression levels in AM-HLH, respectively. However, treatment with IL6 or IL27 activated STAT3, elevated expression of HLX and MIR155, and inhibited IRF4. Taken together, this cell line deals with two conflicting oncogenic drivers, namely, JAK2-STAT3 signaling and EBV infection, but is sensitive to switch after cytokine stimulation. Thus, AM-HLH represents a unique cell line model to study the pathogenic roles of STAT3 and EBV and their therapeutic implications in HL. Hodgkin lymphoma (HL) is a germinal-center-derived B-cell malignancy, although rare cases with T-cell origin have been described . The typHRS and LP cells lack typical B-cell characteristics including expression of B-cell-associated developmental transcription factors (TFs). Even the absence of master TFs PAX5 and EBF1 have been reported; however, their reactivation was insufficient to reconstitute the B-cell phenotype ,15,16. THere, we analyzed the EBV-positive HL cell line AM-HLH . InteresCell lines were obtained from the DSMZ : AM-HLH (ACC 908), HDLM-2 (ACC 17), KM-H2 (ACC 8), L-428 (ACC 197), L-540 (ACC 72), L-1236 ACC 530), SUP-HD1 (ACC 574), U-HO1 (ACC 626). Cell culture conditions, culture media and other relevant information on each cell line are described elsewhere , SUP-HD1. Analyze6 cells by electroporation using the EPI-2500 impulse generator  at 350 V for 10 ms. Transfected cells were harvested after 20 h cultivation. Cell stimulations with 20 ng/mL interleukin (IL)6 or IL27  were performed for 4 h, and for 20 h with 10 µg/mL histone deacetylase inhibitor TSA . All knockdown and stimulation experiments were performed twice generating similar results.Gene activities were suppressed via siRNA-mediated knockdown. Gene-specific siRNA oligonucleotides and AllStars negative control siRNA (siCTR) were purchased from Qiagen . A green fluorescence protein (GFP)-labeled STAT3 expression construct was cloned into vector pCMV6-XL4 and obtained from Origene . SiRNAs (80 pmol) and plasmid DNA (2 µg) were transfected into 1 × 10For functional testing, treated cells were analyzed with the IncuCyte S3 Live-Cell Analysis System . Apoptotic cells were quantified using the IncuCyte Caspase-3/7 Green Apoptosis Assay diluted at 1:2000 . Live-cell imaging experiments were performed twice with four-fold parallel tests.Total RNA was extracted from cell line samples using TRIzol reagent . cDNA was synthesized by random priming from 5 µg RNA using Superscript II . Expression analysis of EBV-encoded genes LMP1, LMP2A, EBNA1, EBNA2, EBNA3A, EBNA3C and of control gene YY1 was performed as described previously . Reverset-test and the derived p-values are indicated by asterisks .Real-time quantitative (RQ)-PCR analysis was performed with the 7500 Real-time System using commercial buffer and primer sets . For normalization of expression levels, we analyzed the transcript of TATA box binding protein (TBP). We used the ddCT-method, and the obtained values are indicated as fold expression in relation to one selected sample which was set to 1. Quantitative PCR analyses were performed in triplicate. Standard deviations are calculated for each experiment and presented in the figures as error bars. Statistical significance was assessed by Western blots were generated using the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma). Proteins were transferred onto nitrocellulose membranes  and blocked with 5% dry milk powder dissolved in phosphate-buffered saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma), STAT3 , phospho-(P)-STAT3 , and IRF4 (Origene). For loading controls, blots were reversibly stained with Poinceau (Sigma), and the detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies  were linked to peroxidase for detection by Western-Lightning-ECL . Documentation was recorded using the digital system ChemoStar Imager .Immuno-cytology was performed as follows: cells were spun onto slides, air-dried and fixed with methanol/acetic acid for 90 s. The STAT3 antibody (see above) was diluted 1:20 in PBS containing 5% BSA and incubated for 30 min. Washing was performed 3 times with PBS. Preparations were incubated with fluorescent secondary antibody (diluted 1:100) for 20 min. After final washing the cells were mounted in Vectashield , containing DAPI for nuclear staining. Images were captured with an Axion A1 microscope using Axiocam 208 color and software ZEN 3.3 blue edition .For genomic profiling by cytogenetic microarray, the genomic DNA of cell line AM-HLH was prepared by the Qiagen Gentra Puregene Kit (Qiagen). The labelling, hybridization and scanning of HD Cytoscan arrays was performed at the Genome Analytics Facility, Helmholtz Centre for Infection Research , using HD arrays according to the manufacturer’s protocols . Data were visualized and interpreted using the Chromosome Analysis Suite, software version 2.0.1.2 (Affymetrix). https://www.ncbi.nlm.nih.gov/gds/, accessed on 1 February 2022) [Gene expression profiling data from cell lines were generated at the Genome Analytics Facility using HG U133 Plus 2.0 gene chips , as reported previously . Expressry 2022) . Transcrry 2022) .In HL, DLBCL and ALCL, STAT3 aberrantly activates NKL homeobox gene HLX which plays an oncogenic role in these lymphoid malignancies ,19,20. AHowever, RQ-PCR analysis of NKL homeobox gene HLX in cell lines derived from HL, DLBCL and ALCL detected only weak expression levels in AM-HLH. This result contrasted to EBV-negative HL cell line L-540, EBV-positive DLBCL cell line DOHH-2 and all examined EBV-negative ALCL cell lines C. HLX isRQ-PCR analysis of STAT3 in eight HL cell lines showed low expression levels in AM-HLH while HDLM-2, L-428 and L-540 expressed the highest levels A. AccordHowever, the treatment of AM-HLH with IL6 for 4 h resulted in STAT3 phosphorylation and translocation from the cytoplasm into the nucleus C, demonsThe treatment of L-540 cells with the deacetylase inhibitor TSA resulted in the translocation of STAT3 from the nucleus into the cytoplasm D. AccordGenomic aberrations play a basic role in the pathogenesis of HL. Accordingly, the tumor cells contain several chromosomal rearrangements and copy number alterations as described in patients and cell lines ,11,12,13We focused on seven amplicons exhibiting at least a 10-fold rise in their copy numbers which were located at 6p25, 9p24, 9p13, 16p13, 16p12, 16p11 and 20q13 . Gene exIRF4 encodes a hematopoietic TF and was amplified at 6p25 and overexpressed in AM-HLH as confirmed by RQ-PCR and Western blot analysis A. IRF4 hBAG1 encodes an activator of survival factor BCL2 . BAG1 waThe amplifications at 16p harbor several overexpressed genes which, however, indicates no connection to STAT3 or EBV C. In conTo validate these findings and to identify additional genes differentially expressed in AM-HLH, we performed genome-wide comparative gene expression profiling analysis, analyzing the top-1000 up/downregulated genes . AccordiOur approach analyzing genomic and gene expression profiling data from AM-HLH and HL control cell lines revealed interesting candidates potentially involved in STAT3 regulation or lymphomagenesis. These genes and their downstream effects were analyzed by the modulation of their activity. SiRNA-mediated knockdown of IRF4 was confirmed by RQ-PCR and Western blot analysis A. The daBoth, IL6- and IL27-signalling activates STAT3 ,22. AccoKMT2A is a direct target of amplified Zinc-finger TF ZFP64 . KMT2A iTaken together, these data showed that overexpressed IRF4 and NFATC act as STAT3 inhibitors. Nevertheless, STAT3 was still ready for reactivation as demonstrated by treatment with IL6 or IL27. Furthermore, we detected downregulation of STAT3-activators NFIB and HIPK2 and of STAT3-target genes HLX, MIR155 and PAX5. CELF2 is a reported inhibitor of MIR155 and was activated by overexpressed NFATC and KMT2A.We identified IRF4 as an amplified and overexpressed STAT3-inhibitor in AM-HLH. To examine its potential oncogenic activity in AM-HLH we performed live-cell-imaging after siRNA-mediated knockdown of IRF4. The results indicated that IRF4 supported proliferation A and inhThe results of this study are summarized in IRF4 played a central role in STAT3 inhibition. Its gene was amplified at 6p25 and consequently overexpressed. IRF4 also inhibited the expression of NFIB which has been shown to activate STAT3 . FunctioJAK2 represents a major STAT3-activator and was downregulated in AM-HLH although located in an amplicon. This observation indicated that this cell line escapes STAT3-activation. As compared to HLX-positive cell line L-540, HLX target gene SPIB was elevated in AM-HLH. SPIB plays an important role for both, B-cell differentiation and EBV activity, thus, indicating a conflict of two pathognomonic mechanisms ,49,50. PAdditional deregulated players identified in AM-HLH included ZFP64, KMT2A, CELF2, and MIR155. ZFP64 was amplified at 20q13 and overexpressed. This TF has been described as potent activator of KMT2A transcription , which wUsing cell line AM-HLH as a model for EBV-positive HL, we recognized downregulated STAT3 activity. Amplified and overexpressed IRF4 played a major role in STAT3 inhibition. Genes located downstream of STAT3 have been described as both oncogenes and regulators of EBV. STAT3 was still ready for reactivation via IL6- and IL27-signalling, indicating a possible switch in EBV activity. Thus, our study revealed conflicting roles of STAT3 in HL and showed that AM-HLH represents a unique cell line model to gauge therapeutic options for EBV-positive HL patients.


P < 0.0001) for those who received mRNA-1273 (GM 601 BAU/mL [GSD 2.05]) versus BNT162b2 (GM 375 BAU/mL [GSD 2.33) vaccines. Results of RBD antibody comparisons (P < 0.0001), and inhibition of ACE-2 binding to the wild-type strain and all tested Delta lineages , were consistent. Adults who received two doses of mRNA-1273 vaccines demonstrated improved wild-type and Delta variant-specific humoral immunity outcomes at 6 months compared with those who received two doses of the BNT162b2 vaccine.While mRNA vaccines are highly efficacious against short-term COVID-19, long-term immunogenicity is less clear. We compared humoral immunogenicity between BNT162b2 and mRNA-1273 vaccines 6 months after the first vaccine dose, examining the wild-type strain and multiple Delta-variant lineages. Using samples from a prospective observational cohort study of adult paramedics, we included COVID-19-negative participants who received two BNT162b2 or mRNA-1273 vaccines, and provided a blood sample 170 to 190 days post first vaccine dose. We compared wild-type spike IgG concentrations using the Mann-Whitney U test. We also compared secondary outcomes of: receptor binding domain (RBD) wild-type antibody concentrations, and inhibition of angiotensin-converting enzyme 2 (ACE-2) binding to spike proteins from the wild-type strain and five Delta-variant lineages. We included 571 adults: 475 BNT162b2 (83%) and 96 mRNA-1273 (17%) vaccinees, with a mean age of 39 (SD = 10) and 43 (SD = 10) years, respectively. Spike IgG antibody concentrations were significantly higher (IMPORTANCE The BNT162b2 and mRNA-1273 mRNA SARS-CoV-2 vaccines have demonstrated high efficacy for preventing short-term COVID-19. However, comparative long-term effectiveness is unclear, especially pertaining to the Delta variant. We tested virus-specific antibody responses 6 months after the first vaccine dose and compared individuals who received the BNT162b2 and mRNA-1273 SARS-CoV-2 vaccines. We found that individuals who received the mRNA-1273 vaccine demonstrated superior serological markers at 6 months in comparison with those who received the BNT162b2 vaccine. Clinical trials have found the BNT162b2 and mRNA-1273 vaccines to be highly efficacious against short-term severe COVID-19 ; 475 (83%) and 96 (17%) received two doses of the BNT162b2 vaccine or two doses of the mRNA-1273 vaccine, respectively. BNT162b2 and mRNA-1273 vaccinees demonstrated similar mean age and sex distribution . The aveP < 0.0001) for mRNA-1273 (GM 601 BAU/mL [GSD 2.05]) versus BNT162b2 (GM 375 BAU/mL [GSD 2.33]) vaccinees.Spike IgG antibody concentrations, measured on the V-PLEX assay , were siP < 0.0001). Receptor binding domain (RBD) antibody concentrations were significantly higher for mRNA-1273, in comparison with BNT162b2, vaccinees .Spike total antibody concentrations were significantly higher for mRNA-1273, in comparison with BNT162b2, vaccinees when measured on the Elecsys assay ; as well as for the AY.1 , AY.2 , B.1.617.2/AY.3/AY.5/AY.6/AY.7/AY.14 , B.1.617.2/AY.4 (GM 14.90 U/mL [GSD 2.39] versus GM 9.71 U/mL ). We reported outcomes as geometric mean (GM) with geometric standard deviation (GSD), and compared using the Mann-Whitney U test. A 2-sided


We found that lipocalin 2 (Lcn2) is a new target of PC-NVs in this process, demonstrating that PC-NVs exert their angiogenic and nerve-regeneration effects by activating MAP kinase and PI3K/Akt and suppressing P53 signaling pathway in an Lcn2-dependent manner. Our findings provide new conclusive evidence that PC-NVs can promote neurovascular regeneration and recovery of erectile function under diabetic conditions via an Lcn2-dependent mechanism. Thus, local administration of PC-NVs may be a promising treatment strategy for the treatment of diabetic ED.Diabetes mellitus is one of the main causes of erectile dysfunction (ED). Men with diabetic ED do not respond well to oral phosphodiesterase-5 inhibitors owing to neurovascular dysfunction. Pericyte-derived extracellular vesicle-mimetic nanovesicles (PC-NVs) are known to promote nerve regeneration in a mouse model of cavernous nerve injury. Here, we report that administration of PC-NVs effectively promoted penile angiogenesis and neural regeneration under diabetic conditions, thereby improving erectile function. Specifically, PC-NVs induced endothelial proliferation and migration and reduced cell apoptosis under diabetic conditions. In addition, PC-NVs induced neural regeneration in STZ-induced diabetic mice in dorsal root ganglion and major pelvic ganglion explants  Erectile dysfunction (ED), defined as incapability to achieve and/or maintain a satisfactory erection during sexual intercourse.Pericytes is a vascular mural cell embedded within the basement membrane of micro-vessels and has the ability to differentiate into completely different cell phenotypes.Extracellular vesicles (EVs) secreted to the extracellular space by cell as micro-vesicles, exosomes, and apoptotic bodies.This verifies erectile function enhancement of exogenic PC-NVs treatment reinstating cavernous mural and endothelial cell content; endothelial NOS (eNOS) phosphorylation, endothelial tight junction integrity; reducing cavernous vessels permeability; and promote nerve regeneration. Furthermore, angiogenic  and neurogenic  effect of PC-NVs under high-glucose conditions. Additionally, RNA sequencing and Signaling Explorer Antibody Arrays analyses revealed that PC-NVs promote penile neurovascular regeneration under diabetic conditions through lipocalin-2 (Lcn2)-dependent stimulating mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/Akt signaling pathways and suppress the tumor protein 53 (Trp53) signaling pathway.Figure 1A), characterized using Zetasizer Nano ZS analysis, transmission electron microscopy (TEM), and Western blotting. Zetasizer Nano ZS analysis  and TEM images  revealed vesicular cup-like structure of PC-NVs with enrichment diameter of 134.5 ± 16.92 nm. Characteristic and similarities of PC-NVs to the natural EV were summarized at  Western blot confirmed positive EV surface markers, TSG101, Alix, and CD81, were present in PC-NVs but not in lysates, whereas the negative EV surface marker GM130 was not presented in purified PC-NVs .PC-NVs were extracted from mouse cavernous pericytes accordinglyFigure S1).To determine the distribution of PC-NVs, DiD a red-dye labeled PC-NVs was injected into the mouse penis and harvested at the indicated timepoints for tracking analysis of PC-NVs by confocal microscopy. Representative images indicate DiD-labeled PC-NVs (red) were present and co-localized with the endothelial cell marker PECAM-1 (green) in corpus cavernosum tissue up to 24 hours . Ratio of maximal and total intracavernous pressure (ICP) to mean systolic blood pressure (MSBP) significantly declined in HBS-treated diabetic mice compared with age-matched control. Marked improvement shown by diabetic mice after two high dose intracavernous injections of PC-NVs (5 µg/20 µL) to 85% of those in controls. Partially improved erection parameters observed in lower doses (0.5 and 1 µg per 20 µL), . No significant MSBP variations were found between experimental and control groups. Immunofluorescence staining for PECAM-1 , α-SMA (smooth muscle cell marker), and NG2 (pericyte marker) in cavernosum tissue demonstrated enhancement of endothelial cell, smooth muscle cell, and pericyte content in diabetic mice after PC-NVs injections . Treatment with PC-NVs  significantly improved levels of p-eNOS , expression of the tight junction proteins claudin-5 and occludin in diabetic mice , suppressed endothelial permeability . Fasting and postprandial blood glucose concentrations significantly higher in diabetic than those in control mice. Regardless of treatment no significant differences between body weight and blood glucose levels of diabetic mice (Table S2) suggesting that PC-NVs restored cavernous endothelial-mural cell content and ameliorate erectile dysfunction in diabetic mice.Pericytes known as regulator of vascular stability and regeneration.Figure 3A). To determine PC-NVs pro-angiogenic activity we performed aortic ring microvessel sprouting assays  and migration assays  under normal-glucose  and HG conditions. Comparably MCECs tube formation assays, aortic ring microvessel-sprouting activity, and endothelial cell migration were significantly reduced under HG conditions (with HBS). HG-related discrepancies were completely rescued by PC-NVs treatment . To understand how PC-NVs induce angiogenesis, we evaluated mouse endothelial cell proliferation  and apoptosis  under HG conditions. Diminished proliferation and augmented apoptosis of MCECs under HG conditions (with HBS); restored to baseline values after addition of PC-NVs . Taken together, these suggest that PC-NVs induce angiogenesis under HG conditions by promoting endothelial cells migration and proliferation also inhibiting cell apoptosis.In diabetes, endothelial dysfunction is a reflection of apoptosis, diminished proliferation and migration.Figure 4A-4C). We sought to determine the effect of PC-NVs on neurite outgrowth in MPG and DRG explants under HG conditions. MPG and DRG immunofluorescence staining for βIII-tubulin revealed greatly reduced neurite sprouts under HG condition (with HBS); furthermore, PC-NVs exerted a rescue effect, profoundly enhancing neurite outgrowth under HG . To rule whether the effects of PC-NVs on neuronal regeneration are mediated by production of neurotrophic factors, neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF) expression, mouse cavernous tissues were evaluated by Western blotting. Particularly, these neurotrophic factors expression in diabetic mice received PC-NVs were greater compared to those received HBS-injection . These results suggest that PC-NVs promote nerve regeneration under diabetic conditions by increasing the expression of NT-3, BDNF, and NGF.In diabetic related ED, neuropathy is one of contributory factor.Figure 5A and 5B). All seven genes were increased in HG conditions with PC-NVs treatment (HG+PC-NVs) compared with HG+HBS treatment. We assumed that PC-NVs carried and transfer these genes, that directly or indirectly affect neurovascular regeneration under HG conditions. A literature review identified Lcn2 has the closest relationship with neurovascular regeneration. Consistent RNA sequencing result, the expression of Lcn2 protein was significantly increased in MCECs treated with HG+PC-NVs compared with those treated with HG+HBS . Remarkably, Lcn2 protein was also detected in pericyte cell lysates and PC-NVs . To evaluate whether Lcn2 protein is responsible for PC-NV-induced neurovascular regeneration under diabetic conditions, we knockdown Lcn2 expression in mouse cavernous pericytes (MCPs) by infection with lentivirus expressing small hairpin RNA (shRNA) targeting Lcn2 (shLcn2) or scrambled shRNA control (shCon) lentivirus, and isolated Lcn2-KD PC-NVs for in vitro studies . Erectile function was assessed by electrical stimulation of the cavernous nerve after injection of Lcn2-KD PC-NVs as described in Materials and Methods. Maximal and total intracavernous pressure (ICP) relative to MSBP were significantly improved in diabetic mice treated with shCon PC-NVs (5 µg/20 µL). However, treatment with shLcn2-KD PC-NVs had no effects in diabetic mice . No detectable differences in MSBP were found between experimental groups (Table S3). Similar results were observed in MCEC tube-formation assays , aortic ring microvessel sprouting assays , and MPG  and DRG  neurite outgrowth assays. These results suggest that Lcn2 in PC-NVs plays an important role in promoting neurovascular regeneration in diabetic conditions.Extracellular vesicles are known to carry genetic components (mRNAs and miRNAs), lipids, and numerous proteins, which important in physiological and pathological communication between cells.Figure 6A). STRING database were used to analyze the protein-protein interaction (PPI) network, integrates known and predicted PPIs to predict protein functional interactions.Figure 6B). Further validation studies exhibited that the neurovascular regeneration induced by PC-NVs under diabetic conditions was achieved by activating MAPK, PI3K/Akt signaling, and suppressing the Trp53 pathway in an Lcn2-dependent manner . Taken together, these findings suggest that MAPK, Akt, and Trp53 signaling pathways constitute the key pathways for PC-NV/Lcn2-mediated neurovascular regeneration under diabetic conditions.To elucidate the PC-NV/Lcn2-mediated signaling pathway, we profiled mouse cavernosum tissues from age-matched control and diabetic mice received HBS (H), PC-NVs, or Lcn2-KD PC-NVs with Signaling Explorer Antibody Arrays, which employ 1358 unique antibodies covering 20 cell signaling pathways. Out of 1358 protein targets, only 49 contra-regulated proteins showed increases or decreases in DM/Control and Lcn2-KD PC-NVs/shCon PC-NVs expression ratios greater than 1.2  and major pelvic ganglion (MPG) neurite sprouting assays; 50 for RNA sequencing and other signaling pathway studies. All experiments were conducted in blinded manner.Eight-week-old male C57BL/6J mice  were used in this study. All animal experiments were performed in compliance with Institutional Animal Care and Use Committee of Inha University . In this study, total of 150 adult male C57BL/6J mice were used: 30 for mouse cavernous pericytes (MCPs) primary culture and pericyte-derived EV-mimetic NV isolation; 30 for diabetes mouse model preparation and related experiments; 20 for mouse cavernous endothelial cell (MCEC) primary culture and aortic ring sprouting assays; 20 for Primary MCPs were prepared from 8-weeks male C57BL/6J mouse penile Primary MCECs were prepared from mouse penis tissues as previously described Figure 1. Three 100 mm dishes (each dish containing approximately 5 x 106 MCPs ) were washed 3 times with phosphate-buffered saline , detached with 0.25% trypsin-EDTA (Gibco), and re-suspended in 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES)-buffered saline . The cell suspension was sequentially extruded 10 times for each pore-sized polycarbonate membranes   and subjected to ultracentrifugation at 100,000 × g for 2 hours at 4°C using step gradient of 50% iodixanol , overlaid with 10% iodixanol (2 mL), lastly topped with the extruded samples (7 mL). Pellets resuspended in HBS and filtered with 0.45-μm filter before stored at -80°C until further use. PC-NVs were quantified with EXOCET exosome quantitation assay kit , with adjusted concentration 1 µg/µL. Extracted PC-NVs enrichment diameter was measured by dynamic light scattering using a Zetasizer Nano ZS instrument  and the morphology was assessed by transmission electron microscopy .MCP-derived EV-mimetic NVs (PC-NVs) were prepared using a mini extruder system  according to the procedure in our previous studyPC-NVs were labeled with red-fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD)  according to the manufacturer's instructions. 2.5 µL of DiD dye solution was added to 100 µg of PC-NVs with total 500 µL of HBS. Incubated for 10 min at room temperature, diluted with 9.5 mL of cold HBS, and subjected to ultracentrifugation at 100,000 × g for 2 hours at 4°C. The pellets washed with HBS and re-centrifuged at 100,000 × g for 2 hours. DiD-labeled PC-NVs pellets then resuspended in HBS and used for tracking analysis. Penis tissues were harvested at indicated time point after injection and fixed with 4% formaldehyde for 24 hours at 4°C. Tissues were stained with antibodies and mounted with mounting medium containing the nuclear dye, 4',6-diamidino-2-phenylindole . DiD dye tracking was performed using confocal fluorescence microscopy .Eight-week-old male C57BL/6 mice were used in this study. DM was induced by intraperitoneal injections of streptozotocin  in 0.1 M citrate buffer (pH 4.5) at a dose of 50 mg/kg body weight for 5 consecutive days.2O) to mean systolic blood pressure (MSBP).Erectile function was measured according to protocols.in vitro by serum-starving cells for 24 hours then exposed to normal-glucose  or high-glucose  conditions for 3 days (MCECs and MCPs) or 5 days  at 37°C in a humidified 5% CO2 atmosphere.52Diabetes-induced neuro-vasculopathy were mimicked 5 cells/well in 300 µL of M199 or DMEM containing 2% FBS. Tube formation was observed for 18 hours under a phase-contrast microscope, and the amount of master junctions from seven separate experiments were determined in a blinded manner using Image J software .Tube-formation assays were performed based on protocols.http://rsbweb.nih.gov/ij/).Aortic rings were prepared from abdominal and thoracic aorta explants of 8-week-old C57BL/6 mice. Tissue positioned in wells of an 8-well Nunc Lab-Tek Slide System (Sigma-Aldrich). covered with 50 μl Matrigel. The aortas were cultured in complete M199 for 5 days in NG or HG medium with HBS or PC-NVs (1 μg/mL), or as indicated. Images of aortic segments and outgrowth were acquired with a phase-contrast microscope. The sprouting area was analyzed using Image J software  at >95% confluence in 60-mm culture dishes. The blocks were removed 5 hours after seeding, and cells were further incubated with M199 containing 2% FBS for 24 hours. Images were acquired with a phase-contrast microscope. Migrated cells were analyzed by measuring migrated cell proportion along the frame line.MCECs were treated with HBS or PC-NVs (1 µg/mL) under NG or HG conditions for 3 days, then treated with 10 µM BrdU (5'-bromo-2'-deoxyuridine (Sigma-Aldrich) at 37°C for 1 hour. The cells then fixed and stained with an anti-BrdU antibody . The number of BrdU-positive endothelial cells was counted at a screen magnification of 400× and expressed per high-power field.The MCECs cell death after treated with HBS or PC-NVs (1 µg/mL) under NG or HG conditions were evaluated by TUNEL  assay using the ApopTag Fluoresce In Situ Apoptosis Detection Kit  according to the manufacturer's instructions. Numbers of apoptotic cells were determined at a 200× magnification by confocal fluorescence microscope and expressed per high-power field.2 environment. Thereafter, 1 mL of complete Neurobasal medium (Gibco) supplemented with 2% serum-free B-27 (Gibco) and 0.5 nM GlutaMAX-I (Gibco) were added and MPG and DRG tissues were incubated under NG or HG conditions with HBS, PC-NVs (1 μg/mL), or other conditions as described in the text. Five days later, neurite outgrowth was assessed by fixing tissue segments in 4% paraformaldehyde for at least 30 minutes and immunostaining with an anti-βIII tubulin antibody .Mouse MPG and DRG tissues were prepared and maintained as described previously.For RNA-sequencing analysis, MCECs were cultured and treated with HBS or PC-NVs (1 μg/mL) under NG or HG conditions. RNA-sequencing analyses were performed by E-Biogen Inc.  as described previously.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) for quality control of raw sequencing data. Adapters, and low-quality reads (<Q20) were removed using FASTX_Trimmer (http://hannonlab.cshl.edu/fastx_toolkit/) and BBMap (https://sourceforge.net/projects/bbmap/), trimmed reads were mapped to reference with TopHat.https://www.r-project.org) Quantile normalization. Data mining and graphic visualized with ExDEGA . RNA-sequencing data have been stored at Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo accession no. GSE183220).Libraries were prepared from total RNA using a SMARTer Stranded RNA-Seq Kit . mRNA was isolated with Poly (A) RNA Selection Kit ; indexed with Illumina indices 1-12; and enriched with PCR. Libraries were assessed for mean fragment size using an Agilent 2100 Bioanalyzer (DNA High Sensitivity Kit), quantified with StepOne Real-Time PCR System . High-throughput sequencing was made as paired-end 100 sequencing with HiSeq 2500 system . FastQC  or scrambled control shRNA (shCon) lentiviruses  were added to culture medium at a concentration of 5 × 10Mouse cavernosum tissues from age-matched control and diabetic mice intracavernous injected with HBS (H), PC-NVs, or Lcn2-knockdown (KD) PC-NVs were used for Signaling Explorer Antibody Array , performed according to the manufacturer's instructions. Assay consists of 1358 unique antibodies covering 20 cell-signaling pathways, was performed as a custom service by E-Biogen (Ebiogen Inc.).https://string-db.org), and scored according to their confidence.38Hierarchical clustering analyses were performed with ExDEGA Graphic plus program  based on Euclidean distance correlation measurement with average linkage.ex vivo samples were incubated at 4°C overnight with indicated primary antibodies against the following proteins: smooth muscle α-actin , neuron-glial antigen 2 , platelet/endothelial adhesion molecule 1 , phosphorylated eNOS , neurofilaments , neuronal NOS , claudin-5 , occludin , βIII tubulin , or oxidized low-density lipoprotein . After several washes with PBS, samples were incubated with species-appropriate tetramethyl rhodamine isothiocyanate (TRITC)- or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies  for 2 hours at room temperature and mounted with nuclear dye DAPI  contained solution. Fluorescence signals were visualized using confocal microscope . Quantitative analyses were performed using Image J software .Explants for immunofluorescence  were fixed in 4% paraformaldehyde for 24 hours at 4°C. Frozen tissue sections (15-µm thick) or http://rsbweb.nih.gov/ij/).Penis tissues were lysed in RIPA buffer (Sigma-Aldrich) with protease inhibitors  and phosphatase inhibitors . Equal amounts of protein (40 µg per lane) electrophoresed on SDS-PAGE gels (8% to 15%), transferred to polyvinylidene difluoride membranes, blocked with 5% nonfat dry milk (BD Biosciences) for 1 hour at room temperature. Membranes were probed with antibodies to eNOS , p-eNOS , neurotrophin-3 , brain-derived neurotrophic factor , nerve growth factor , Lcn2 , MAPK , p-MAPK , PI3K , p-PI3K , AKT , p-AKT , Trp53 , p-Trp53 , or β-actin . The results were quantified by densitometry using Image J software .Data are expressed as means ± SEM from five independent experiments. For parametric data, intergroup assessments made with one-way analysis of variance (ANOVA) followed by Newman-Keuls posthoc tests. Kruskal-Wallis's test was used nonparametric data. Probability value analysis was performed using Graph Pad Prism software. P-values < 0.05 were considered statistically significant; individual P-values are indicated in figure legends (*Supplementary figures and tables.Click here for additional data file.


Influenza-associated hospitalization rates were low during the 2020–21 season. We describe influenza-associated hospitalization rates and prevalence of influenza and SARS-CoV-2 coinfection among patients hospitalized with influenza during 2021–22.We used data from the Influenza Hospitalization Surveillance Network (FluSurv-NET), a population-based surveillance system for laboratory-confirmed influenza-associated hospitalizations active from October—April of each year. We calculated cumulative and weekly hospitalization rates per 100,000 population and compared preliminary rates during 2021–22 with prior season rates (2010–11 through 2020–21). We determined the proportion of influenza-associated hospitalizations with SARS-CoV-2 coinfection during 2021–22.During October 1, 2021—April 23, 2022, 3,262 influenza-associated hospitalizations were reported to FluSurv-NET; the cumulative hospitalization rate of 11.1 was higher than 2011–12 and 2020–21 season rates, but lower than rates observed during all other seasons since 2010–11 . After peaking in the week ending January 1, 2022 (MMWR week 52), weekly hospitalization rates declined until the week ending February 19, 2022 (MMWR week 7) when they began to rise modestly, similar to patterns observed during several prior seasons . Among the 3,262 hospitalizations, 87 (2.7%) had SARS-CoV-2 coinfection; the prevalence by age group was as follows: 0–17 years 3.4%, 18–49 years 2.8%, 50–64 years 3.5%, 65–74 years 2.5%, ≥ 75 years 1.6%. Among the 3,262 influenza-associated hospitalizations, the prevalence of SARS-CoV-2 coinfection by month (October 2021–April 2022), respectively, was 11.4%, 2.5%, 2.6%, 8.9%, 3.4%, 0.8%, and 0.5%.SARS-CoV-2 coinfection was uncommon among patients hospitalized with influenza during 2021–22. Likely due to ongoing COVID-19 mitigation measures, the influenza-associated hospitalization rate during 2021–22 was lower than rates observed in most seasons in the decade preceding the COVID-19 pandemic. A late rise in weekly influenza hospitalization rates in 2021–22 might have been a result of relaxation of COVID-19 mitigation measures and/or a late season peak in influenza activity.Evan J. Anderson, MD, GSK: Advisor/Consultant|GSK: Grant/Research Support|Janssen: Advisor/Consultant|Janssen: Grant/Research Support|Kentucky Bioprocessing, Inc: Data Safety Monitoring Board|MedImmune: Grant/Research Support|Medscape: Advisor/Consultant|Merck: Grant/Research Support|Micron: Grant/Research Support|NIH: Funding from NIH to conduct clinical trials of Moderna and Janssen COVID-19 vaccines|PaxVax: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Regeneron: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant|Sanofi Pasteur: Grant/Research Support|Sanofi Pasteur: Data Adjudication and Data Safety Monitoring Boards|WCG and ACI Clinical: Data Adjudication Board Maya Monroe, MPH, CDC -Emerging Infections Program: Grant/Research Support.


Adjunct immunomodulatory strategies might improve the treatment of this emerging entity, but i90 of a mouse-adapted influenza A/H3N2/Hong Kong/1968 strain, delivered by aerosolization on day (d) 0. On d5 and d8, mice received two intraperitoneal injections of 10 mg cortisone acetate (CA) or mock injections. On d9, mice were intranasally challenged with 50,000 Aspergillus fumigatus (AF) conidia. Infection severity was scored using the viral pneumonia score  and the murine sepsis score . For therapeutic studies, mice received a 30-minute nebulization of the toll-like receptor (TLR) 2/6/9 agonists Pam2+ODN  or PBS (mock treatment) on d8 (single-dose) or d8+12 . Therapeutic studies used a combined morbidity/mortality endpoint, with an event defined as either death or reaching a VPS ≥ 7.8-10-week-old female BALB/c mice were infected with 7.5% LDFigure 1).CA-immunosuppressed mice with IAPA had 64% 16-day mortality and survivors had severe morbidity (median MSS 2.7). In contrast, all non-immunosuppressed mice with IAPA survived and showed consistently low morbidity (median d16 MSS 0.5). All mock-treated CA-immunosuppressed mice with IAPA reached the combined morbidity/mortality endpoint by d11. Single-dose Pam2+ODN treatment led to universal event-free survival until d13 but all mice deteriorated by d16. Dual-dose Pam2+ODN treatment before and after AF infection led to 75% event-free survival until d21, with all survivors fully recovering , AbbVie: Advisor/Consultant|Astellas Pharma: Advisor/Consultant|Astellas Pharma: Grant/Research Support|Astellas Pharma: Honoraria|Cidara Therapeutics: Advisor/Consultant|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|Gilead Sciences: Honoraria|Merck: Advisor/Consultant.


Scientific Reports 10.1038/s41598-022-06498-9, published online 16 February 2022Correction to: In the original version of this Article, the Supplementary Table The Supplementary Information file now accompanies the original Article.Supplementary Table 1.


The mammalian target of rapamycin (mTOR) pathway is crucial in energy metabolism and cell proliferation. Previously, we reported transcriptome-wide 3′-untranslated region (UTR) shortening by alternative polyadenylation upon mTOR activation and its impact on the proteome. Here, we further interrogated the mTOR-activated transcriptome and found that hyperactivation of mTOR promotes transcriptome-wide exon skipping/exclusion, producing short isoform transcripts from genes. This widespread exon skipping confers multifarious regulations in the mTOR-controlled functional proteomics: AS in coding regions widely affects the protein length and functional domains. They also alter the half-life of proteins and affect the regulatory post-translational modifications. Among the RNA processing factors differentially regulated by mTOR signaling, we found that SRSF3 mechanistically facilitates exon skipping in the mTOR-activated transcriptome. This study reveals a role of mTOR in AS regulation and demonstrates that widespread AS is a multifaceted modulator of the mTOR-regulated functional proteome. AlternaMammalian target of rapamycin (mTOR) is a serine/threonine kinase and is the major component of the mTOR Complex 1 (mTORC1) and 2 (mTORC2) ,10,11. TIn this study, we investigated mTOR-driven transcriptome changes in alternative splicing and their impact on the resulting proteome using various cellular models. We found that widespread exon skipping by mTOR activation diversifies the proteome by changing functional domains, post-translational modifications, and protein stabilities.Tsc1-/- mouse embryonic fibroblasts (MEFs), human breast cancer cell line MCF7, and MDA-MB361 treated with DMSO (mock) or Torin 1, a potent inhibitor of mTOR. From our initial interrogation focused on the RNA processing pathway, we found numerous RNA-binding proteins (RBPs) involved in alternative splicing (AS) and alternative polyadenylation to be differentially expressed (Tsc1-/- MEFs (Tsc1-/- MEF transcriptome). This discrepancy may come from the degree of mTOR activation in these cell lines examined: Tsc1-/- MEFs are genetically programmed to hyperactivate mTOR signaling while MCF7 and MDA-MB361 cells have a reduced level of mTOR hyperactivation  transcriptome, it showed fewer exon inclusion-biased AS events compared to that of the Tsc1-/- transcriptome with Torin 1 treatment. We further delved into the nucleotide contexts surrounding the 3′-splice site and found that mTOR activation promotes the skipping of the cassette exon that contains a strong, longer stretch of the poly-pyrimidine tract (−3 to −20 position) and a weak poly-pyrimidine tract with biased C at the −1 position in the alternative 3′-splice site in both Tsc1-/- MEFs and human cancer cells (Tsc1-/- MEFs mock vs. Torin 1 treatment)  D. These Tsc1-/- MEFs, and human breast cancer cell lines MCF7 and MDA-MB361 showed an overall differential intron retention upon mTOR inhibition.  overlap in exon skipping cases and a 19% (4/21) overlap in exon-inclusion cases  while 33% of CDS AS events were linked to changes in the Pfam  Dulbecco’s Modified Eagle Media (DMEM) , in supplements with 10% FBS (Gibco), 100 ug/mL streptomycin, and 100 U/mL penicillin. All cell lines were cultured at 37 °C with 5% CO2. WT and iversity . The folCycloheximide (No. 14126) and Torin 1 (No. 10997) were purchased from Cayman Chemical, Ann Arbor, MI, USA (No. 14126) and reconstituted in DMSO.Sirt2 long-form (NM_022432), Sirt2 short-form (NM_001122765), mouse mdm2 long-form (NM_010786.4), mdm2 short-form (NM_001288586.2), mouse Adnp long-form (NM_009628.3), and Adnp short-form (NM_001310088.1) were cloned into the p3xFlag-PGK plasmid.cDNAs of mouse Total RNAs were isolated using the Trizol method recommended by the manufacturer’s protocol. cDNAs were created using reverse transcription using Oligo-d(T) and superscript III . cDNA templates were used to determine alternative splicing isoform expression in a gel-based RT-PCR analysis. Isoform expression levels were determined using densitometry.Ranbp3 RT-PCR forward: 5′-AAGCCTGCCGTCGCACCGTCTGTCT; mouse Ranbp3 RT-PCR reverse: 5′-CTTCTCCGGCTTCGGGACTGGAGC-3′; mouse Mdm4 RT-PCR forward: 5′-GCTAAGAAAGAATCTTGTTACATCAGC-3′; mouse Mdm4 RT-PCR reverse: 5′-ATGTCGTGAGGTAGGCAGTGTGTGA-3′; mouse Syce2 RT-PCR forward: 5′-AGCATCGGCAGAGTGAGAAC-3′; mouse Syce2 RT-PCR reverse: 5′-CCGTTTCCACAGTTTGGCAG-3′; mouse Prrc2b RT-PCR forward: 5′-GTTGAAAGGCTTCCACTTTGCCGA-3′; mouse Prrc2b RT-PCR reverse: 5′-CAGGCACCTTCAGGCTTTGCTTC-3′; mouse Asph RT-PCR forward: 5′-CGCAGAACCATCCAAATGACA-3′; mouse Asph RT-PCR reverse: 5′-GACCCCTTCATGCTCTGAGG-3′; mouse Map2k7 RT-PCR forward: 5′-CCGCAGGAGGATCGACCTCAACTT-3′; mouse Map2k7 RT-PCR reverse: 5′-GGAGCTCTCTGAGGATGGTGAGCG-3′; mouse Pcdh19 RT-PCR forward: 5′-TACCTGTCCCCAGCTCTTGATG-3′; mouse Pcdh19 RT-PCR reverse: 5′-AAGGGAGGAGCAACTGACAACAT-3′; mouse Rpl12 RT-PCR forward: 5′-TCAAGGAGATCCTGGGTACTGC-3′; mouse Rpl12 RT-PCR reverse: 5′-CCCCAAAAAGCAGCCAAGCC-3′; mouse Wbp1 RT-PCR forward: 5′-GCCCTGTTCCAACCGGTTCACTGCTTG-3′; mouse Wbp1 RT-PCR reverse: 5′-GGGGTATGTGAACTGGGCTCAATCCTG-3′; mouse 2310033P09Rik RT-PCR forward: 5′-GACAGCTGCTCCTCATCTCC-3′; mouse 2310033P09Rik RT-PCR reverse: 5′-GGTGTCCTTTGGTGTAGCCA-3′; mouse Anks1 RT-PCR forward: 5′- GCTGACTCGAAAGGCTGCTACC-3′; mouse Anks1 RT-PCR reverse: 5′- TCCAGGGGCGTTTCAAACTTGTTG-3′; mouse Ect2 RT-PCR forward: 5′- GAAATGCCGCAGGTTGAAGCAAG-3′; mouse Ect2 RT-PCR reverse: 5′- ACTGGTGGCCCAACAATCCTACA-3′; mouse Adgrl2 RT-PCR forward: 5′- TCCTCTGTGAGGCTGATGGAAC-3′; mouse Adgrl2 RT-PCR reverse 5′- CAACAACATTGTGGCTGTGTGCG-3′.Mouse Srsf3: 5′-CGUGAUAUCAAGAAUUGU-3′. The knockdown efficiency of Srsf3 was determined by qPCR with forward primer 5′-GCTGCCGTGTAAGAGTGGAA-3′ and reverse primer 5′-AGGACTCCTCCTGCGGTAAT-3′. The antibodies used for Western blot analysis include anti-Flag , anti-Actin , anti-4EBP1 , anti-phospho-S6 , anti-S6 , anti-Tubulin , Anti-Secondary antibody against mouse: goat-anti-mouse IgG-HRP .Cells were transfected with siRNAs synthesized by Integrated DNA Technologies (IDT) using RNAiMax Transfection reagents (Thermo Fisher Scientific) recommended by the manufacturer’s protocol. siRNA targeting mouse Cells were treated with 30 ug/mL cycloheximide to inhibit protein synthesis and were harvested at different time points. Cells were then lysed and 15–30 ug of lysates (depending on the expression level of the Flag-tagged protein) were run on SDS PAGE gels for Western blot analysis. Tsc1-/- MEFs, MCF7, and MDA-MB361 cells. Paired-end reads were aligned to the mouse mm10 reference genome or the human hg19 reference genome using TopHat2 [MEFs were treated with either DMSO (control) or Torin 1 at 50 nM for 24 h. Human cancer cell lines were treated with either DMSO or 100 nM Torin 1 for 24 h. To evaluate the transcriptome features under mTOR-hyperactivation at single-nucleotide resolution, we performed RNA-Seq analyses of poly(A+) RNAs isolated from WT,  TopHat2  with up  TopHat2  was appl TopHat2 .n) from these alternatively spliced exons were measured and compared to reads (N) coverage of the rest of the transcripts. A 2 × 2 Chi-Square test was performed to determine the statistical significance for each alternative splicing event using the ratio of n/N. Events with a p-value < 0.1 and a ratio difference >0.1 were considered real alternative splicing exons.Alternative splicing analysis was performed using the AS-Quant pipeline . BrieflyThe input samples were aligned with the reference genome to be converted into BAM files and the read coverage files were generated for each chromosome. For the detection and analysis of both the annotated and novel intron retention events, our pipeline generated a predefined list of independent introns, which do not overlap with any exons on any isoforms of that gene. Using the RefSeq annotation, the independent introns were generated by merging all the annotated exons of a specific gene and subtracting the merged region from the total genomic region. In general, the read coverages in the retained introns are distributed flatly. Based on this speculation, we integrated several measures to characterize the intronic region as the retained one. For each pre-generated independent intron, we considered five different regions , which span over the intron and its surrounding exons. A, B, and C are the average read coverage of the 100 bp-long regions, where A and C are from the upstream and downstream boundary regions of the targeted intron, and B is from the middle position of the range, respectively. D and E denote the average read coverage of the upstream and downstream exons surrounding the target intron. To make the computation consistent, intron lengths smaller than 100 were excluded from the analysis. To characterize the ‘flatness’ of the introns and to filter out the false positives, several filtering criteria were introduced in the analysis: (a) the minimum reads spanning over the 3 regions  >20, (b) the change in the read coverage of the 3 regions  does not exceed 30% of the highest read coverage, and (c) the average read coverage of the regions D and E is at least 5 times larger than the average read coverage of the sections A, B, and C.−5 e-value cutoff [Pfam-Scan was used to search Pfam databases for the matched Pfam domains on each transcript with a 1 × 10e cutoff . Only the cutoff  providede cutoff  providedt test unless stated otherwise.Signal intensity from gel electrophoresis or Western blots was quantified using the Ver 5.2 of the ImageStudio software . Statistical analysis was performed using the two-tailed Student’s