Trypanosoma cruzi, the etiologic agent of Chagas disease, a topoisomerase 3α knockout parasite (TcTopo3α KO) was generated, and the parasite growth, as well as its response to several DNA damage agents, were evaluated. There was no growth alteration caused by the TcTopo3α knockout in epimastigote forms, but a higher dormancy rate was observed. TcTopo3α KO trypomastigote forms displayed reduced invasion rates in LLC-MK2 cells when compared with the wild-type lineage. Amastigote proliferation was also compromised in the TcTopo3α KO, and a higher number of dormant cells was observed. Additionally, TcTopo3α KO epimastigotes were not able to recover cell growth after gamma radiation exposure, suggesting the involvement of topoisomerase 3α in homologous recombination. These parasites were also sensitive to drugs that generate replication stress, such as cisplatin (Cis), hydroxyurea (HU), and methyl methanesulfonate (MMS). In response to HU and Cis treatments, TcTopo3α KO parasites showed a slower cell growth and was not able to efficiently repair the DNA damage induced by these genotoxic agents. The cell growth phenotype observed after MMS treatment was similar to that observed after gamma radiation, although there were fewer dormant cells after MMS exposure. TcTopo3α KO parasites showed a population with sub-G1 DNA content and strong γH2A signal 48 h after MMS treatment. So, it is possible that DNA-damaged cell proliferation due to the absence of TcTopo3α leads to cell death. Whole genome sequencing of MMS-treated parasites showed a significant reduction in the content of the multigene families DFG-1 and RHS, and also a possible erosion of the sub-telomeric region from chromosome 22, relative to non-treated knockout parasites. Southern blot experiments suggest telomere shortening, which could indicate genomic instability in TcTopo3α KO cells owing to MMS treatment. Thus, topoisomerase 3α is important for homologous recombination repair and replication stress in T. cruzi, even though all the pathways in which this enzyme participates during the replication stress response remains elusive.DNA topoisomerases are enzymes that modulate DNA topology. Among them, topoisomerase 3α is engaged in genomic maintenance acting in DNA replication termination, sister chromatid separation, and dissolution of recombination intermediates. To evaluate the role of this enzyme in  Trypanosoma cruzi is the etiological agent of Chagas disease, also known as American trypanosomiasis. This disease, endemic in Latin America, has been spreading to other continents due to migratory flow. Around 8 million people worldwide are infected by this parasite  is a signal transduction pathway responsible for detecting all kinds of DNA damage and replication stress, in which several proteins work in a coordinated way to halt cell cycle progression and activate DNA repair mechanisms . Two pivDNA topoisomerases are conserved enzymes essential to solve topological problems generated during DNA metabolism and gene expression, performing their functions via transesterification reactions . The reaDNA topoisomerases are classified as type I and type II. Type I enzymes cleave DNA single strands, while type II topoisomerases form dimers to cleave DNA double strands, in an ATP-dependent manner. Each topoisomerase type can be subdivided into two subfamilies: IA, IB, IIA, and IIB . Each suEscherichia coli. Prokaryotic proteins are named topoisomerase 1 and topoisomerase 3. Eukaryotes have only topoisomerase 3, and in the model eukaryotes, there are two isoforms of this enzyme, called topoisomerase 3α and topoisomerase 3β , a RecQ DNA helicase, and with RMI1/2 factors, which can dissolute the double Holliday junctions, generating non-crossover products . It is kT. brucei showed that topoisomerase 3α is important for the antigenic variation mediated by homologous recombination , hydroxyurea (HU), and methyl methanesulfonate (MMS) treatments. Indeed, knockout parasites showed a significant reduction in the content of the multigene families DFG-1 and RHS (retrotransposon hot spot), and possible telomere shortening after MMS treatment. Together, these results imply that topoisomerase 3α is necessary to maintain genomic stability in T. cruzi.Genomic analysis in the TriTrypDB has shown that trypanosomatids have a complete set of the various types of DNA topoisomerases in their genome. In these parasites, the enzymes of the subfamily IA are topoisomerase IA, topoisomerase 3α, and topoisomerase 3β. The topoisomerases of this subfamily are phylogenetically well conserved and are compartmentalized in the nucleus and mitochondria of trypanosomatids . Studiesbination . DespiteT. cruzi Dm28c strain were cultivated in liver infusion tryptose (LIT) medium pH 7.4 supplemented with 10% inactivated fetal bovine serum (Gibco) and 1% streptomycin/penicillin (Invitrogen), at 28°C. For the culturing of knockout parasites, 300 μg ml–1 of hygromycin B (Invitrogen) and neomycin (G418 sulfate – Gibco) antibiotics were added to the culture medium. Rhesus monkey kidney monolayers cells (LLC-MK2) , 200 U ml–1 penicillin, and 200 μg ml–1 streptomycin sulfate (2% DMEM). LLC-MK2 cultures were washed daily with PBS+/+ buffer  to remove remaining epimastigotes. Released tissue-culture trypomastigotes (TCTs) were purified as described previously (Epimastigote forms of LLC-MK2)  were maieviously  and usedT. cruzi Dm28c genomic DNA by polymerase chain reaction (PCR). The 5′CDS3α and 3′CDS3α were amplified using the primers described in KpnI and SalI restriction enzymes (New England Biolabs), while 3′CDS3α amplicon was digested with BamHI and XbaI restriction enzymes (New England Biolabs). Both digested amplicons were cloned into pTc2KO-neo or pTc2KO-hygro vectors, which carry neomycin or hygromycin B-resistance gene (T. cruzi epimastigotes using a Gene Pulser II electroporation system (Bio-Rad). The selection occurred in LIT medium with 500 μg ml–1 of G418 until the death of the control parasites. To confirm the insertion of the cassette in the correct loci, a PCR reaction with DNA of transfected parasites was performed using a forward primer located inside the neomycin resistance gene and a reverse primer located 434 bp downstream of the TcTopo3α gene  and 2,391 up to 2,747 (3′CDS3α–356 bp of length) of the coding sequence of the TcTopo3α gene  were amplified from nce gene . The com2HPO4, 3 mM KH2PO4, 55 mM glucose) and mixed with an equal volume of 1% low-melting point agarose. To prepare these agarose blocks containing intact chromosomes, 2 × 107 parasites for each block were used. The blocks were incubated with a lysis solution  at 50°C for 50 h and then stored in this same solution at 4°C. Before the electrophoresis, the blocks were incubated three times with 50 mM EDTA pH 8 for 1 h. The blocks were subjected to PFGE on 1.2 % agarose gel in 0.5× TBE buffer  at 10°C. Chromosomes from yeast Hansenula wingei (1.05 to 3.13 Mbp) were used as a molecular weight marker (CHEF DNA Size Markers-Bio-Rad). The PFGE was carried out in a constant voltage of 100 V for 135 h with five phases of pulses : 90 s for 30 h, 200 s for 30 h, 350 s for 25 h, 500 s for 25 h, and 800 s for 25 h. Following electrophoresis, the gel was stained with ethidium bromide (0.5 μg ml–1) and photographed with L-Pix photodocumentation system (Loccus Biotecnologia). The Southern blot analysis was performed according to standard protocols (32]-dCTP using the Nick Translation Labeling Kit (Invitrogen) following the manufacturer’s recommendations . Exponential growth parasites were recovered from LIT medium and washed with PBS buffer twice. Then the cells were resuspended in PSG buffer  and probed with a 300-bp fragment containing telomeric repeat TTAGGG (TM AlkPhos Direct Labeling and Detection System with AmershamTM CDP-StarTM Detection Reagent (GE Life Sciences).In order to assess the role of TcTopo3α gene in genomic stability maintenance, another Southern blot analysis was performed. To that end, genomic DNA (2 μg) from WT and Topo3α KO cells was treated with CviQI, HpaII, AluI, and HhaI restriction endonucleases at 37°C for 24 h. Digestion products were resolved by gel electrophoresis . DNA was transferred to Hybond-Nt TTAGGG . HybridiT. cruzi, the growth profile of WT parasites was compared with TcTopo3α KO parasites after gamma radiation exposure. The parasites were irradiated with a dose of 1,541 Gy h–1 for 19 min and 28 s using a cobalt (60Co) irradiator located at Laboratório de Irradiação Gama . Additionally, parasites were subjected to replication stress in growth curves performed with 100 μM of cisplatin (Cis) in PBS buffer for 1 h at 28°C; 20 mM of hydroxyurea (HU) in LIT medium at 28°C for 24 h, and 1.5 mM of methyl methanesulfonate (MMS) in PBS buffer for 1 h at 28°C. After each treatment, the cells were washed with PBS to remove the drug. Then the parasites were incubated in a fresh LIT medium at 28°C. For ATR inhibition analysis, parasites were kept in LIT medium containing 5 mM caffeine or 10 μM VE-821 (Sigma). All growth curves were initiated at a density of 1 × 107 cells ml–1 with cells in log phase growth. The cells were counted daily until the control cells reached stationary phase. The number of parasites was determined using a cytometry chamber and vital dye erythrosine. For all treatments tested, it was performed at least three independent experiments in triplicate.To verify whether topoisomerase 3α gene is important during DSB repair in 7 epimastigotes ml–1 were incubated for 20 min at 28°C with 10 mM CFSE in PBS buffer, protected from light. The excess of CFSE was quenched with five volumes of LIT medium for 5 min. After that, cells were centrifuged at 3,000 × g for 10 min and resuspended in fresh LIT medium at a concentration of 1 × 107 cells ml–1. Aliquots from CFSE-stained epimastigote cultures were collected until 144 h for all treatments, fixed overnight with 4% paraformaldehyde (PFA) at 4°C and analyzed by flow cytometry to assess fluorescence intensity. FACSCan or FACSCalibur flow cytometers (Benckton-Dickson) were used for data collection, and 10,000 events for each condition were analyzed using the software FlowJo VX.All treatments were followed by CellTrace CFSE (Thermo Fisher) labeling as previously described . Briefly4 LLC-MK2 cells were suspended in 10% DMEM and added onto 13-mm round glass coverslips inserted into each well of a 24-well plate. Plated cells were then incubated at 37°C with 5% CO2 for 24 h before infection with purified trypomastigotes from WT and TcTopo3α KO parasites, previously labeled with CellTrace CFSE. For trypomastigote CFSE labeling, parasites were incubated for 20 min at 37°C with 10 mM CFSE in PBS buffer. The excess of CFSE was quenched with 2% DMEM for 5 min. After that, cells were centrifuged at 3,000 × g for 10 min and resuspended with five volumes of 2% DMEM. Infection was performed (protected from light) at a multiplicity of infection (MOI) of 50 for 1 h in 2% DMEM. Afterward, cells were washed five times with PBS+/+ and re-incubated in 2% DMEM for additional 24, 48, 72, and 96 h, until overnight fixation with 4% PFA at 4°C temperature in which samples were stored until processed for immunofluorescence. The coverslips with attached cells were washed three times with PBS+/+, incubated for 20 min with PBS containing 2% BSA (PBS/BSA), and processed for an inside/outside immunofluorescence invasion assay as previously described  and 10 μg ml–1 propidium iodide (BD Pharmingen) and incubated in the dark at 37°C for 30 min. The data were collected at FACSCan or FACSCalibur flow cytometer (Benckton-Dickson) from 10,000 events and analyzed with the software FlowJo VX.In order to evaluate the cell cycle alterations due to replication stress, WT and TcTopo3α KO parasites were analyzed by flow cytometry after treatment with Cis (100 μM), HU (20 mM), or MMS (1.5 mM). Cells in log phase growth were treated with the drugs, as described above, and samples were collected each 24 h for cell cycle progression assay. Thus, 1 × 108 cells from each culture were harvested by centrifugation at 2,500 × g for 10 min and was washed twice with PBS buffer. The cells were lysed with 50 μl of sample buffer 2× . Then the samples were sonicated in the Sonic Dismembrator Model 500 (Fisher Scientific) apparatus at 30% maximum amplitude for five cycles of 20 s, with an interval of 20 s. After that, the samples were boiled for 10 min and stored at −20°C. Protein concentration was determined using the Bradford method. To separate the proteins according to their size, an SDS-PAGE was performed. Both the 5% stacking gel and the 15% separation gel were prepared following standard protocols (® SD Semi-Dry Transfer Cell (Bio-Rad) for 1 h at 300 mA. The membranes were blocked by incubation in blocking solution, which consists of TBST buffer  with 5% non-fat dry milk for 2 h under agitation. After that, the membranes were washed three times with TBST buffer for 5 min. Then, the membranes were incubated with the primary antibody anti-γH2A  produced in rabbit and kindly provided by Dr. Richard McCulloch’s group. Alternatively, the membranes were incubated with the primary antibody anti-α-tubulin  produced in mouse (Sigma). For both antibodies, the incubation was carried out in blocking solution overnight under agitation. The membranes were washed three times with TBST for 5 min. Then, the membranes were incubated with secondary antibodies  conjugated with peroxidase, anti-rabbit IgG , or anti-mouse IgG (Sigma) for 1 h under agitation. The membranes were washed three times with TBST for 5 min and revealed using ImmobilionTM Western Chemiluminescent HRP Substrate (Millipore) system and ImageQuant LAS 500 (GE) apparatus.To measure the level of DNA damage in WT and Topo3α KO parasites after treatment with Cis (100 μM), HU (20 mM), or MMS (1.5 mM), a Western blot assay was performed. To prepare the protein extracts, 1 × 10rotocols . The prog for 5 min, washed with PBS buffer, and fixed using 4% paraformaldehyde diluted in PBS for 10 min at room temperature. Then parasites were spread out onto slides (previously treated with 0.1% poly-L-lysine), washed with PBS buffer, and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. As a positive control for BrdU incorporation, slides containing samples from WT and Topo3α KO untreated parasites were subjected to DNA denaturation using 2.5 M HCl for 20 min. Then all samples were washed, and accessible BrdU was detected using α-BrdU-rat (Abcam) diluted 1:250 in blocking solution  for 3 h at room temperature, followed by incubation for an additional 3 h with secondary antibody Alexa Fluor 555-conjugated goat anti-rat (Thermo Scientific) diluted 1:1,000 in blocking solution. After that, the slides were washed repeatedly using PBS buffer. VECTASHIELD® Mounting Medium with DAPI (Vector Labs) was used to be the anti-fade mounting solution and to stain DNA content. Images were captured using Olympus BX51 fluorescence microscope coupled with a digital camera  and were analyzed using Olympus-Cell F software. Differences in parasite ssDNA foci were measured using ImageJ software.To verify the involvement of topoisomerase 3α in the replication stress response after HU (20 mM) and MMS (1.5 mM) treatments, a BrdU native detection assay was performed, following the protocol used by Dias and collaborators, with small modifications . Both tr7 cells ml–1 were cultivated in LIT medium supplemented with fetal bovine serum and 1 mM MMS. Every day, for 5 days, cells were centrifuged at 3,000 × g for 5 min at room temperature and resuspended in fresh medium with 0.75 mM MMS. After five generations, cells were counted and washed three times with PBS buffer. DNA extraction was performed using Qiagen Blood and Cell Culture DNA Mini Kit (Cat:13323) following manufacturer protocol for cell culture with an incubation time in proteinase K (kit provided) of 3 h. As control, a non-treated culture was submitted to the same steps of cultivation and DNA extraction, using the same volume of PBS instead of MMS in the medium. The DNA purity and quantity were determined using NanoDropTM 1000 UV–Vis spectrophotometer, and a paired-end library with mean fragment size of 800 bp was constructed using NexteraTM DNA kits (Illumina Inc.). The library was sequenced on Illumina NextSeqTM 500 platform with generation of 75 bp reads.To evaluate the DNA content after MMS treatment a whole genome sequencing (WGS) was performed at Glasgow Polyomics. WT and Topo3α KO cultures containing 1 × 101, and high-quality reads were selected using Trimmomatic.v.0.33 . The absence of a mapping quality filter is important to better estimate read depth in T. cruzi multigene families. To evaluate the deletion of TcTopo3α (TcCLB.508851.170), the read depth of CL Brener Chr18 region that contains this gene was visualized in IGV v2.6.2 . A Dm28c non-TcTopo3α-KO WGS read library SRR7592211 was used as a control for TcCLB.508851.170 gene presence in this analysis.Reads from each WGS library were quality checked using FastQCc.v.0.33 , with a ome v.46  using BWome v.46 . The mapome v.46  for the V v2.6.2 . The reain-house Perl scripts that extract the gene coverages using CDS coordinates in GFF files. Chromosomal copy number variation (CCNV) was evaluated as described by Reis-Cunha and collaborators (p < 0.05), and the median RDC on non-outlier genes in each chromosome was normalized by the genome coverage and assumed as the chromosomal somy. Next, to evaluate segmental duplications/deletions, the coverage of each position of each chromosome was normalized by the genome coverage, and MM1 and MM2 coverages were summed and subtracted from the sum of NT1 and NT2. Hence, values above zero correspond to a higher copy number in MMS-treated isolates, and values below zero correspond to higher copy numbers in MMS-untreated isolates. The image representing these segmental duplications was generated in R (2 R development 2020), using the genoPlotR library3. The gene coordinates were obtained from T. cruzi CL Brener Non-Esmeraldo-like GFF v.46 , with a significance of 0.05. Finally, to evaluate if there was a greater perturbation in DNA content close to replication origins after MMS treatment in TcTopo3α KO isolates, the copy number of DGF-1 genes close to replication origins described by Araujo and collaborators  and untreated (NT1 and NT2) isolates. To estimate alterations in gene and chromosome copies, the read depth of each position in each chromosome was determined with BEDTools genomecov v2.16.2 , and theborators . BrieflyGFF v.46 . The impborators  was compT. cruzi, this gene was deleted in epimastigotes by disrupting both alleles with neomycin and hygromycin selectable resistance markers  or hydroxyurea (HU). Cisplatin interacts with N7 in purine residues generating intrastrand and interstrand crosslinks. These adducts cause DNA double helix distortions and block replication , an almost significant decrease in trans-sialidase (p∼0.14)   and no s(p∼0.14) . The DNA(p∼0.14) .Once the MMS treatment generated sub-G1 cells , with leTo further assess the role of topoisomerase 3α in the replication stress response, a BrdU native detection assay was carried out. In this assay, it is possible to measure the levels of ssDNA foci, which is considered a replication stress marker. In normal culture conditions, both WT and TcTopo3α KO parasites naturally showed very low levels of ssDNA foci either in the nucleus (N) or kinetoplast (K) . After HIt is known that gamma irradiation and its DBS generation activates ATM kinase signaling, while MMS and its intense alkylation on DNA induce formation of ssDNA and ATR kinase signaling. Both pathways act in stalling replication and cellular cycle. Once we observed the discrepancy in CFSE decay between gamma radiation and MMS treatments, but without altering cellular concentration in culture, we checked the response of the TcTopo3α KO and WT parasites to ATM/ATR kinase signaling. To that end, the parasites were treated with caffeine, an unspecific inhibitor of both ATM and ATR kinases, and VE-821, a specific ATR inhibitor, in normal growth conditions. Interestingly, in the presence of caffeine, both cellular types were affected with impaired growth . HoweverT. cruzi amastigotes spontaneously become dormant  and untreated (control) TcTopo3α KO parasites were generated. CCNV analysis showed no differential alteration in chromosomal somies between MMS-treated and untreated parasite populations, suggesting that MMS perturbation was unable to cause chromosomal copy instability in T. cruzi. Even tough ploidy variations are widespread among T. cruzi DTUs  are rare (To further characterize the impact of MMS in uzi DTUs , the ratare rare . As DNA In contrast to what was seen at the chromosomal level, MMS treatment appears to cause short-scale DNA loss in TcTopo3α KO isolates, especially in multigene family’s clusters enriched in DGF-1, RHS, and to a lesser extent, trans-sialidases . There aT. brucei (T. cruzi, Rad51 single knockout parasites were sensitive to treatment with 1.5 mM MMS. Moreover, this treatment was able to increase γH2A levels in TcRad51+/− parasites, suggesting that TcRad51 is necessary to cope with the replication stress generated by this alkylating agent (T. cruzi, however, the duplication was corroborated with the decrease in CFSE labeling intensity after treatment with MMS (T. cruzi. Further studies need to be performed to confirm this hypothesis.Methyl methanesulfonate methylated bases are usually repaired by BER. However, in trypanosomatids, no specific DNA glycosylase capable of removing N-methyl purines has so far been identified . Recentl. brucei . In T. cng agent . Therefong agent . In T. cwith MMS . In factwith MMS . Our hypwith MMS . Thus, iT. cruzi by a pathway other than homologous recombination.Cisplatin treatment also halted WT and TcTopo3α KO growth for 24 h . In accoT. cruzi strains (T. cruzi, there is no difference between TcRad51+/− and WT parasites growth profile after HU treatment. In addition, the level of phosphorylated H2A after treatment with 20 mM HU in these two cells lineages is very similar, suggesting that in T. cruzi, HU-induced lesions are not repaired by homologous recombination (Hydroxyurea treatment for 24 h was able to synchronize both WT and TcTopo3α KO parasites in the G1/S phase of the cell cycle , as stan strains , as well strains . HU remo strains , as well strains . Also, a strains . Upon HU strains . In huma strains . Howeverbination . Once agbination , 5, suggT. cruzi. It is possible to note that not all responses to replication stress are dependent on homologous recombination, which may explain the variation in sensitivity of Topo3α KO parasites to the different genotoxic agents tested. Replication stress induced by MMS can be solved via homologous recombination pathway. However, it is possible that topoisomerase 3α also acts in other pathways to allow the recovery of the replication forks after the replication stress caused by HU and cisplatin. The mechanisms recruited after these treatments need to be elucidated. Furthermore, it was also shown that the absence of TcTopo3α and, therefore, the inability to solve the intermediates of the recombination processes, is an important signal for dormancy or asynchronous replication in epimastigote and amastigote forms. Thus, understanding the role of topoisomerase 3α in T. cruzi may contribute to further elucidate the biology of this trypanosomatid and the mechanisms of its successful infection in the vertebrate host.Taken together, the results obtained here indicate that TcTopo3α is required during homologous recombination repair, as previously described for other organisms . MoreoveSRR13754247, SRR13754250, SRR13754249, and SRR13754248.The data presented in the study are deposited in the NCBI SRA repository, accession numbers: HC-S, BR, LA, JR-C, GF, SF, and CM conceptualized the study. HC-S, BR, AU, WP, MS, SV, AM, ET, LT, ME, LA, JR-C, GF, SF, and CM performed the formal analysis. HC-S, BR, AU, WP, MS, SV, ME, LA, JR-C, GF, SF, and CR performed the investigations. The original draft was written by HC-S, BR, WP, and MS. The study was written by ME, LA, JR-C, GF, SF, and CM. Funding was secured by AM, SP, ET, LT, LA, GF, SF, and CR, while resource sourcing was done by AM, SP, ET, LT, ME, LA, JR-C, GF, SF, and CR. LA, JR-C, GF, SF, and CR did the methodology. All authors contributed to the article and approved the submitted versionThe 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.


C9orf72 expansions. EVs are classified based on their biogenesis and size and can be separated by differential centrifugation. They include exosomes, released by the fusion of multivesicular bodies with the plasma membrane, and ectosomes, also known as microvesicles or microparticles, resulting from budding or pinching of the plasma membrane. In the current study, EVs were obtained from the myotube cell culture medium of ALS patients or healthy controls. EVs of two different sizes, separating at 20,000 or 100,000 g, were then compared in terms of their effects on recipient motor neurons, astrocytes, and myotubes. Compared to untreated cells, the smaller, exosome-like vesicles of ALS patients reduced the survival of motor neurons by 31% and of myotubes by 18%, decreased neurite length and branching, and increased the proportion of stellate astrocytes, whereas neither those of healthy subjects, nor larger EVs of ALS or healthy subjects, had such effects.Extracellular vesicles can mediate communication between tissues, affecting the physiological conditions of recipient cells. They are increasingly investigated in Amyotrophic Lateral Sclerosis, the most common form of Motor Neurone Disease, as transporters of misfolded proteins including SOD1, FUS, TDP43, or other neurotoxic elements, such as the dipeptide repeats resulting from  Cell–cell communication occurs via a variety of mechanisms including secretion of soluble factors, direct contact via tunnelling nanotubes and cytonemes, as well as extracellular vesicles . ExtraceThe skeletal muscle, which represents 40% of total body mass , is knowC9orf72 expansions [Amyotrophic Lateral Sclerosis (ALS) is a disease with an adult onset characterized by the degeneration of upper and lower motor neurons, affecting between 1 and 2.6 per 100,000 people per year . While mpansions ,40,42,43pansions .In this context, we wanted to determine whether MuVs and large muscle vesicles  would be similarly taken up by, and exert similar effects on, motor neurons, astrocytes and muscle cells.n = 7 ALS and n = 7 healthy) [C9orf72 hexanucleotide repeat expansion, ATXN2 repeat length, and the coding regions of SOD1, TARDBP, FUS, UBQLN2 and TBK1. Subject characteristics are given in Primary human myoblasts were obtained from human deltoid muscle biopsies from a previous study  . The prohealthy) , and inc−1 Fetuin, 0.5 ng·mL−1 basic fibroblast growth factor (bFGF), 5 ng/mL epidermal growth factor (EGF), 5 µg·mL−1 insulin and incubated at 5% CO2, 37 °C. Enrichment in myoblasts was confirmed for all cultures, with over 90% of the cells positive for desmin. The healthy myoblasts differentiated into myotubes from subject F aged 50–59 were used to test the uptake and effect of EVs.CD56-sorted myoblasts were expanded in 0.22 µm filtered proliferating medium containing DMEM/M199 (Thermo Scientific) supplemented with 20% FBS, 25 µg·mL−2 as described in [6 myoblasts in 225 cm2 flasks ) for 24 h before washing six times with supplement-free DMEM. The cells were then differentiated in DMEM to form myotubes, and conditioned culture medium was collected at 72 h. All cell cultures were regularly checked for mycoplasma infection.For extraction of extracellular vesicles, myoblasts were plated at a density of 33,400 cells·cmribed in   equipped with fast video capture and particle tracking software. Purified vesicles from differentiated myoblasts were diluted into 1 mL of PBS. Five videos of 1 min duration were recorded for each sample, with a frame rate of 25 frames per second and analyzed using NanoSight software (NTA 3.4 Build 3.4.003). The camera level was set at 11, the temperature was 23.4 °C and the viscosity was 0.920–0.922 cP. The data on the sizes of EVs are expressed as the calculated means ± SD of size distribution.Muscle EVs were lysed by adding 50 μL of RIPA lysis buffer containing 1X Halt™ protease inhibitor cocktail . After 10 min of incubation at 4 °C, the samples were centrifuged at 10,000 g for 10 min at 4 °C. The supernatants were collected, and protein concentration was spectrophotometrically measured at 570 nm using the Pierce™ BCA Protein Assay kit (Thermo Scientific™).−1). Thereafter, 750 µL concentrated sulphuric acid was added to the standard and to the MuV and lmEV samples. After vortexing, standards and samples were incubated at 90 °C for 20 min. The absorbance was measured spectrophotometrically at 540 nm after adding phosphovanillin acid (0.2 mg·mL−1 vanillin in 17% phosphoric acid).The total lipid concentration of EVs in PBS was determined according to the method of . Briefly® Bis-Tris gel  in MOPS-SDS Running buffer  for 60 min at 200 V. The polyacrylamide gel was then incubated in 20% ethanol for 10 min and the separated proteins transferred onto a polyvinylidene fluoride (PVDF) membrane using the iBlot2 Dry Blotting System. The iBind™ Flex Western Device (Life Technologies™) was used for immunoblotting and performed according to manufacturer’s recommendation. The membranes were probed with primary antibodies for CD63 , CD81 , CD82 , Annexin A1 , ARF6 , alpha skeletal-Actin  as well as goat anti-mouse, anti-rabbit or sheep anti-mouse secondary antibodies conjugated with HRP . Subsequently, the membranes were incubated with Amersham ECL Prime Western Blotting Detection Reagent for 2 min at room temperature and images were acquired using the UVP ChemiDoc-It™2 Imager and UVP software.EV protein extracts were prepared under non-reducing or reducing conditions as previously described , heated w/v, 10% w/v, 20% w/v, and 40% w/v iodixanol solutions were prepared by diluting volumes of a stock solution of OptiPrep™ solution (60% w/v in water) with 0.25 M sucrose/10 mM Tris, pH 7.4. The gradient was then formed by adding 3 mL of the 40% iodixanol solution to a polycarbonate tube (Beckman Coulter) using a Pasteur pipette followed by the careful layering of the 20% w/v (3 mL), 10% w/v (3 mL) and 5% w/v (2.5 mL) solutions, respectively. The samples were then centrifuged at 100,000 g for 21 h at 4 °C. Twelve 1 mL fractions were collected from top to bottom and transferred to new tubes. Each fraction was then washed in 9 mL PBS and centrifuged at 100,000 g for 70 min at 4 °C. The resulting pellets were resuspended in 30 µL NuPage, loaded on 4–12% NuPAGE® Bis-Tris gel, electrophoresed and transferred onto PVDF as described above. The membranes were probed for CD63 , Annexin A1  or ARF6 . The iodixanol density of each fraction was determined at 340 nm as previously described [MuVs and lmEVs were resuspended in 500 µL of 0.25 M sucrose in 10 mM Tris, pH 7.4. and carefully loaded on a iodixanol/sucrose gradient as previously described . Brieflyescribed .®) or 96-Well Black/Clear Bottom Plate (Thermo Fischer) were coated with 20 µg·mL−1 poly-L-ornithine overnight. After an initial wash with H2O, followed by PBS, each well was coated with 5µg·mL−1 laminin. After 24 h, human neuron progenitors were then plated and maintained in N2B27 media containing Neurobasal, DMEM/F12-Glutamax, N2 (1×), B27 (1×), Pen/Strep 1%, 0.25 mM 2-mercaptoethanol and 0.1% Glutamax® and supplemented with 100 nM Rock Inhibitor (RI), 100 nM Retinoic Acid (RA), 500 nM SAG, 100 nM DAPT, 10 ng·mL−1 BDNF and 2.5 µg·mL−1 laminin. For the 8-well Ibidi slides for immunostaining, 6000 progenitors/well were plated, for the 96 Well plate for the uptake experiments 10,000 progenitor/well were plated. Two days later, the media was renewed with N2B27 media containing 200 nM RA, 1 µM SAG, 200 nM DAPT, 20 ng·mL−1 BDNF. After 3 days, the culture media was renewed with N2B27 media containing 200 nM RA, 200 nM DAPT, 20 ng·mL−1 BDNF and 20 ng·mL−1 GDNF. Two days later, the media was renewed with N2B27 media containing 20 ngmL−1 BDNF and 20 ng·mL−1 GDNF. At this point, progenitors were fully differentiated into motor neurons and ready for functional studies involving extracellular vesicles.Human iPSC-derived motor neuron progenitors were generated following the protocol as described ,47 and c2., in Gibco Astrocyte Medium comprising Dulbecco’s Modified Eagle’s Medium (DMEM), N-2 Supplement, and One Shot Fetal Bovine Serum (FBS).Healthy mature human astrocytes (Gibco™) derived from human brain tissue were purchased from Thermo Fisher Scientific (N7805200) and cultured according to the manufacturer’s instructions. Tissue culture dishes or slides were coated with Geltrex™ Basement Membrane Matrix™ (prepared in DMEM) and incubated at 37 °C for 1 h. Prior to seeding cells, the dishes or slides were left at room temperature for 1 h followed by a single wash with DPBS containing calcium and magnesium. The cells were cultured at 37 °C, 5% CO2. Rat astrocytes were used for quantification of KI67 and GFAP immunostaining and to assess oxidative stress.Similarly, rat primary cortical astrocytes  were purchased and cultured according to the manufacturer’s instructions and grown in Dulbecco’s Modified Eagle’s Medium  and Fetal Bovine Serum  at 37 °C, 5% COFor immunostaining, 20,000 cells/well were plated on 8-well Ibidi slides. For the uptake experiments, 10,000 cells/well were plated on 96-well plates. For Western blot analysis, 200,000 cells per 10 cm Petri dish were plated.® 100 K MWCO column at 15,000× g for 10 min at 4 °C. Labelled MuVs and lmEVs (85 ng of lipids) were then added to human iPSC MNs, astrocytes, or myotubes , and incubated for 6 h at 5% CO2, 37 °C. Thereafter, the wells were gently washed 2 times with PBS, fixed with 4% paraformaldehyde (PAF) and the fluorescence from wells measured with a Biotek microplate reader using 485 and 535 nm as excitation and emission filters, respectively. Representative images were acquired for each cell type and EV type.The uptake of MuVs and lmEVs was measured by PKH26 labelling as previously described . BrieflyMuscle EVs (MuVs or lmEVs) were added to the culture medium of primary human astrocytes. At 72 h after treatment, treated and untreated astrocytes were washed with PBS. Cells were scraped with ice-cold RIPA buffer containing protease cocktail inhibitors, centrifuged at 10,000 g for 10 min at 4 °C. Protein quantification was performed using BCA and 25 µg of protein was separated on a 4–12% polyacrylamide Bis Tris Gel, transferred onto PVDF and blotted as described above. The membrane was probed with GFAP , PCNA , GAPDH  as well as anti-rabbit or anti-goat secondary antibodies conjugated with HRP  overnight. Thereafter, the membrane was incubated with Amersham ECL prime Western blotting detection reagent and images acquired using the UVP ChemiDoc-It™2 Imager and UVP software.All images were acquired using an Olympus IX70 equipped with a Photomatics CoolSNAP™ HQ camera, and images were analyzed using Fiji software.After 72 h of treatment, MN were fixed using 4% formaldehyde, permeabilized, blocked and stained for Tuj1, Islet 1/2 as described . Fifteen−1 DAPI for 1 min. Anti-fading medium fluoromount was then added to each well. To assess the percentage of nuclei positive for Ki67, 15 non-overlapping images were acquired across wells using an Olympus LCPlan FI 401×/0.60 Ph2 objective. To assess GFAP staining, 15 non-overlapping images were acquired across wells using an Olympus UPlan FI 101×/0.30 Ph1 objective.After 72 h of treatment with MuVs or lmEVs, astrocytes were fixed with 4% paraformaldehyde for 15 min. The slides were washed twice with PBS and blocked for 1 h with blocking buffer . Ki-67  or GFAP  primary antibody diluted in blocking buffer was added to the slides and incubated overnight at 4 °C. The slides were washed thrice with PBS and incubated with the secondary antibody  for 90 min at room temperature. The slides were then washed thrice with PBS, and counterstained with 1 μg·mLAfter 72 h of treatment, myotubes were fixed using 4% formaldehyde. The cells were permeabilized, blocked and stained for MF20 as described . To assen = 3, healthy: n = 3, 85 ng of lipids). As a positive control, 100 µM H2O2 was added to the cells; as a negative control, 500 µM NAC (glutathione precursor and ROS scavenger) was added to the cells. Fluorescence was measured with a Biotek Multi-Mode Microplate Reader using 485 nm and 520 nm as excitation and emission filters, respectively.Astrocytes were plated in black 96-well flat bottom plates (Thermo Fisher Scientific Inc.). Post seeding and differentiation/adherence, cells were washed twice with PBS and preincubated with 50 µM 2′,7′-dichlorofluorescin diacetate (DCFH-DA) (Invitrogen™). Astrocytes were treated with MuVs or lmEVs  were loaded into each well, with each well containing the same number of cells , over a 6 h period. Compared to astrocytes or myotubes, motor neurons absorbed a significantly greater amount of MuVs . The myoWe then compared the impact of MuVs and lmEVs on MN survival, neurite length, and neurite branching, over a 72 h period. Similarly to our previous observations , MuVs seSimilarly to our previous observations , ALS MuVCell death effects were not observed on astrocytes at 72 h A. We theEukaryotic cells are reported to release extracellular vesicles under conditions of both normal and pathological physiology, with the function(s) of the released vesicles dependent on the content of the cargo and the donor cell under investigation . EVs par−1, than MuVs, which were found at 1.112 g·mL−1. Ectosomes have previously been reported to have a buoyant density of 1.14–1.20 g·mL−1 [In accordance with our previous studies ,45, MuVs0 g·mL−1 , althoug0 g·mL−1  reported0 g·mL−1 , using SFollowing release from donor cells, EVs are taken up by recipient cells via mechanisms that include plasma or endosomal membrane fusion, endocytosis, phagocytosis and micropinocytosis , allowin2C12 myotubes promoted neuron survival and neurite outgrowth when applied to NSC34 cells [Similarly to our previous study , we conf34 cells . In contOther data support the uptake of CD63-positive EVs by motor neurons. MuVs are enriched in CD63, and when MuVs were coated with anti-CD63, we observed that the uptake by MN was abolished . CD63-poFollowing the release of vesicles into the extracellular space, they can then target other cell types. Notably, skeletal muscle derived exosomes transfer toxic materials to neighboring cells, causing homeostatic alterations  that werThe absorption by astrocytes of exosomes derived from neurons, monocytes, and glial cells, as well as stem cells, has been investigated in some depth . In the In conclusion, since toxicity towards motor neurons was observed only for ALS MuVs and not for Healthy MuVs, ALS lmEVs, or Healthy lmEVs, these findings support a role for muscle EVs of specifically exosomal type in the transmission of neurotoxic elements in ALS pathology. Further study is needed to determine whether this finding is specific to EVs of muscle origin or can be extended to include EVs of any origin. The observation that the absorbance of MuVs is 10 and 2.5 times greater in motor neurons than in myotubes and astrocytes, respectively, may be of interest when considering the cell type specificity of ALS pathology.


Dendrobium officinale have been used as a rare and valuable Chinese tonic medicine, known as “Tiepi Fengdou”, since the Qing dynasty. Because of the increased market demand and continued exploitation of this plant, the reserves of wild D. officinale resources have been depleted, and D. officinale products on the market are being increasingly adulterated. Such changes have strongly affected the sustainable utilization of this valuable medicinal plant resource and the development of related industries. In this study, a species-specific DNA marker was developed for the rapid and accurate authentication of D. officinale. In total, 36 start codon-targeted (SCoT) polymorphism primers were screened in 36 definite Dendrobium species, and a distinct species-specific DNA amplicon (SCoT13-215) for D. officinale was obtained. After the sequence was cloned and sequenced, a sequence-characterized amplified region marker was developed (named SHF/SHR) and validated through PCR amplification of all 38 Dendrobium samples. The marker’s specificity for D. officinale was confirmed through the consistent amplification of a clear 197-bp band. This SCAR marker can be used to rapidly, effectively, and reliably identify D. officinale among various Dendrobium species and may play an important role in ensuring the quality of medicinal preparations and protecting the germplasm of this important medicinal species.The stems of  Dendrobium officinale Kimura et Migo is an important orchid plant endemic to China that has been identified separately from other Dendrobium species in the Pharmacopeia of the People’s Republic of China (2010 Edition). This plant is primarily distributed in Yunnan, Guangxi, Zhejiang, Anhui, Fujian, and Sichuan Provinces of China  are monologs codominant markers that are screened using PCRs with a pair of specific oligonucleotide primers designed from a specific nucleotide sequence generated by RAPD, AFLP, ISSR and inter-retrotransposon amplified polymorphism (IRAP) techniques  markers, developed based on the translation start codon, are PCR-based gene-targeted markers . SCoT maDendrobium species collected from their primary distribution areas in China were used to screen for specific markers by mixing fresh leaves from 3 to 4 individuals 2SO4, 100 mM KCl, 1% Triton X-100, pH 8.8], 2 μL Mg2+ (25 mM), 0.8 μL dNTPs (10 mM), 1 μL primer (10 μM), 0.5 μL Taq DNA polymerase (2 U/μL)  and 50 ng genomic DNA template. Amplification reactions were performed in a thermal cycler  using the following parameters: 5 min at 94°C; 35 cycles of 1 min at 94°C, 1 min at 50–60°C  and 2 min at 72°C; and a final extension at 72°C for 10 min. After purification with an EZ-10 spin column PCR product purification kit , the PCR products were electrophoresed on a 1.5% agarose gel alongside Trans2K DNA markers , followed by staining with GelStain  and photography was performed using a Molecular Imager® Gel DocTM XR + System with Image LabTM Software .For the initial screening, a total of 36 SCoT primers  were synD. officinale was excised and purified from an agarose gel using a SanPrep Column DNA Gel Extraction Kit . The purified product was ligated into a pMDTM 19-T vector  according to the manufacturer’s protocol and transformed into the ultracompetent Escherichia coli strain Trans5α using the heat shock method. The recombinant plasmids were isolated by red/white clone screening and sequenced bidirectionally using M13 universal primers at Shanghai Sunny Biotechnology Co., Ltd. .Any SCoT band present in a particular species and absent in all the other species was considered a species-specific marker. To verify the reproducibility of the results, SCoT-PCR amplifications were performed at least twice, and only repeatable amplicons were selected. A unique band specific to 1 to remove vector sequences. The obtained sequence was identified by performing a BLASTN-based search2 of the nucleotide databases, and it was deposited in GenBank (GenBank accession number: MN746373). Forward and reverse oligonucleotide SCAR primers were designed using Primer Premier 5 (Tm) value was adjusted to 65°C, with a range of 60 to 68°C. SCAR amplification was performed in 20 μL of reaction mixture containing 2 μL 1 × PCR buffer , 2 μL Mg2+ (25 mM), 0.8 μL dNTPs (10 mM), 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 0.5 μL Taq DNA polymerase (2 U/μL) (TaKaRa Bio.), and 50 ng genomic DNA template. PCR analysis was performed in a thermal cycler (MJ Research PTC-100) using the following parameters: 5 min at 94°C; 35 cycles of 1 min at 94°C, 1 min at 65°C and 2 min at 72°C; and a final extension at 72°C for 10 min. The PCR products were run on 1.5% agarose gels and detected by GelStain (TransGen Biotech) staining. The specific amplified fragment was designated a species-specific SCAR marker.The obtained sequence was edited using the online tool VecScreenremier 5  based onremier 5 . The priD. officinale genome DNA template concentration for SHF/SHR, and seven DNA template concentrations, specifically 0, 5, 10, 15, 20, 30, and 50 ng/μL, were set. To further verify the practicality of this marker, four Dendrobium samples of the D. chrysotoxum, D. nobile, D. primulinum, and D. aduncum species were utilized to simulate different adulterations of D. officinale . The nucleotide sequence was 48.84% A + T and 51.16% G + C, as shown in The species-specific band obtained in all three D. officinale were amplified and produced a sharp unique band of 197 bp, but no amplicons were produced for the other Dendrobium species  can be found in the article/supplementary material.JL and SF conceived and designed the experiments, participated in the analysis, and drafted the manuscript. KZ, YC, and YG performed the experiments. JJ performed the statistical analysis. WC and HW collected the plant samples. All authors read and approved the final manuscript.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.


Integrins are cell-extracellular matrix adhesion molecules considered functionally related to the development of cancer metastasis. Starting from a large dataset of mRNA-seq of papillary thyroid carcinoma (PTC), we investigated the potential role of integrins in the clinical course of PTC patients. Results showed that the PTC “classical” and “tall cell” histology variants display a more similar integrin expression profile with respect to the ‘follicular’ variant. Interestingly, the BRAFV600E mutation was found to be associated with a higher expression of integrins compared to RAS mutations. The integrin subunit ITGA3 was associated with advanced disease stage, lymph node metastasis, extrathyroidal extension, high-risk, and a worst prognosis. In vitro assays with PTC cell lines demonstrated the role of the α3β1 integrin in cell motility and invasion, evidence that supports the role of this adhesion molecule in tumor progression. These results demonstrate the existence of a PTC-specific integrin expression signature that correlates with histopathology, specific driver gene mutations, and aggressiveness of the disease.Integrins are cell-extracellular matrix adhesion molecules whose expression level undergoes quantitative changes upon neoplastic transformation and are considered functionally related to the development of cancer metastasis. We analyzed the largest mRNA-seq dataset available to determine the expression pattern of integrin family subunits in papillary thyroid carcinomas (PTC). ITGA2, 3, 6, V, and ITGB1 integrin subunits were overexpressed in PTC compared to normal thyroid tissue. The PTC histology variants “classical” and “tall cell” displayed a similar integrin expression profile with a higher level of ITGA3, ITGAV, and ITGB1, which differed from that of the “follicular” variant. Interestingly, compared to RAS mutations, BRAFV600E mutation was associated with a significantly higher expression of integrins. Some integrin subunits were associated with advanced disease stage, lymph node metastasis, extrathyroidal extension, and high-risk groups. Among them, ITGA3 expression displayed the highest correlation with advanced disease and was associated with a negative prognosis. In vitro scratch assay and Matrigel invasion assay in two different PTC cell lines confirmed α3β1 role in cell motility and invasion, supporting its involvement during tumor progression. These results demonstrate the existence of a PTC-specific integrin expression signature correlated to histopathology, specific driver gene mutations, and aggressiveness of the disease. Survival of many eukaryotic cell, including epithelial cells, requires appropriate interactions between adhesion molecules and the extracellular matrix (ECM). These interactions are deeply altered in cancer cells, enabling them to invade surrounding tissues, survive, and form secondary tumors .Integrins are the predominant cell surface receptors of ECM proteins such as fibronectin (FN), laminin, vitronectin (VN), and collagen (CO) . These tThus far, integrin expression in normal and pathological thyroid cells has been analyzed mainly by immunodetection and RT-PCR, but their role in thyroid tumorigenesis has been poorly investigated. Alpha3 β1 and αVβ3 are the most abundant integrins in normal thyroid cells, and to a lesser extent α1β1, α2β1, and α6β1 ,6,7,8,9.The availability of gene expression large datasets generated by RNA sequencing provided us the opportunity to extensively investigate the expression of integrin subunits in thyroid cancer samples. Results demonstrate that integrin gene expression hallmarks PTC subtypes and that higher α3β1 expression is associated with a worst outcome of the disease.https://portal.gdc.cancer.gov/ accessed date 21 April 2020). TCGA-THCA dataset comprised raw-counts data with the gene expression profile of 496 PTC tumor samples and 58 solid tissue samples: 358 classical Papillary Thyroid Carcinoma (PTCcl); 37 Tall Cell PTC (PTCtc); 101 Follicular Variant of PTC (PTCfv). Matching clinicopathological data were downloaded from cBioPortal for Cancer Genomics  [https://portal.gdc.cancer.gov/projects/TCGA-THCA accessed date 21 April 2020).The analysis has been performed considering dataset TCGA-THCA, downloaded from The Cancer Genome Atlas (TCGA) (il 2020) . Raw-coup values (FDR ≤ 0.05), computed according to Benjamini–Hochberg [Data analysis of TCGA-THCA data has been performed as described by Salvati et al. . In detaHochberg .2 in DMEN, 10% calf serum (CS).TPC-1 and BCPAP  were papillary thyroid cancer stable cell lines harboring the RET/PTC and BRAFV600E oncogenes, respectively. The cells were cultured at 37 °C, 5% COMonoclonal antibody to α3 was purchased from Santa Cruz Biotechnology ; fluorescein-conjugated anti-mouse antibody was from Sigma-Aldrich . Cells were then analyzed by flow cytometry using a FACScan apparatus . Flow cytometric analysis was performed as follows: cells were harvested by trypsin-PBS, incubated with the primary monoclonal antibody for 1 h at room temperature (RT) in 0.5% BSA-PBS, washed with the same buffer, and incubated again with the secondary fluorescein-conjugated antibody for 30 min at RT. Cells were resuspended in BSA-PBS and analyzed by flow cytometry.The cells were transfected with 3 unique 27mer ITGA3 siRNA duplexes or scrambled negative control siRNA duplex (10 nM) following the manufacturer protocol . Residual α3 expression was assessed 48 h after transfection by flow cytometry with anti-α3 antibody. In BCPAP and TPC-1 cell lines, residual α3 expression was 30.04% +/− 3.1 and 29.5% +/− 3.8, respectively (mean of 4 independent experiments).4 cells. After 1 h at 37 °C, plates were gently washed 3 times with PBS, and the attached cells were fixed with 3% paraformaldehyde for 10 min, followed by 2% methanol for 10 min, and finally stained with 0.5% crystal violet in 20% methanol. After 10 min, the plates were washed with tap water, the stain was eluted with a solution of 0.1 mol/L sodium citrate, pH 4.2, in 50% ethanol, and the absorbance at 540 nm was measured by a spectrophotometer. In the adhesion inhibition assay, the cells were co-incubated with 1 μg/mL anti-α3 monoclonal antibody in plates previously coated with 5 μg/mL FN, CO, or LM. Alternatively, the adhesion assay was performed with cells transfected with ITGA3 siRNA or scrambled negative control siRNA. All experiments were performed in quadruplicate.The assay was performed in 96-well flat-bottomed microtiter plates. The wells were filled with 100 μL of the appropriate dilution in PBS of FN, CO, or LM . After overnight incubation at 4 °C, the plates were washed with PBS, filled with 100 μL 1% heat-denatured BSA, and incubated for 1 h at room temperature. Then, plates were washed and filled with 100 μL/well PBS, 0.9 mmol/L CaCl 2, and 0.5 mmol/L MgCl 2 containing 70 × 10https://imagej.nih.gov accessed date 10 November 2020) after 24 h. After 48 h of transfection, residual α3 expression was assessed by flow cytometry with anti-α3 antibody.Cell plates were coated with ECM with a mix of PBS 100 μg/mL LM, FN, and CO incubated for 24 h at 4 °C. TPC-1 or BCPAP cells were seeded to obtain a confluent monolayer. After 24 h, a scratch was created with a p200 pipet tip, and the anti-α3 integrin subunit monoclonal antibody was added to a fresh medium at a 1 μg/mL concentration. Alternatively, the cells were transfected with ITGA3 siRNA or scrambled negative control siRNA, plated on ECM coated plates and, after 24 h, the scratch was created. The cells were photographed, and scratches were measured by ImageJ software , previously coated with 50 μL of 3 mg/mL Matrigel (Corning) and kept at 37 °C for 24 h to ensure solidification. Cells were placed in 150 μL of serum-free DMEM, 0.1% BSA, whereas the bottom chamber media contained 10% CS. After 48 h, the cells on the top of the membrane were carefully removed by a cotton-tipped applicator. The cells on the bottom of the membrane were fixed with 3% paraformaldehyde for 10 min, followed by 2% methanol for 10 min, and finally stained with 0.5% crystal violet in 20% methanol. After 10 min, the membranes were washed with tap water, the stain was eluted with a solution of 0.1 mol/L sodium citrate, pH 4.2, in 50% ethanol, and the absorbance at 540 nm was measured by a spectrophotometer.2 × 10p-value less than 0.05. Comparisons of integrin expression and clinical features were conducted by one-way analysis of variance (ANOVA), and statistical significance was assumed for p < 0.01. Univariate regression analysis was performed using the Spearman rank correlation test.All data were presented as mean ± standard deviation. We compared groups using the Wilcoxon test, and statistical significance was defined as a n = 130) vs. those with 15–40% (n = 8) (n = 60) vs. those with >30% (n = 53)  . Only th(n = 53) . Only thp < 0.001).Analysis of the TCGA-TCA database confirmed the results previously obtained by immunodetection in normal thyroid tissue. ITGA3, ITGAV, and ITGB1 were the most abundant integrin subunits, followed by ITGA1 and ITGB3 . Very lop < 0.001. Thus, the integrin expression profile highlighted a similarity among PTCcl and PTCtc that distinguishes them from PTCfv.TCGA included a large repository of RNA-seq data relative to PTC, comprising classical (PTCcl), follicular (PTCfv), and Tall Cell variants (PTCtc). The analysis of integrin mRNA expression of this dataset allowed a comparative analysis between PTC variants . PTCtc dp < 0.0001), respectively. Cumulative analysis of all three variants of PTC produced identical results.Of the 321 PTCcl analyzed, the BRAFV600E mutation was detected in 205 and mutated RAS in 43. In general, the expression profile of integrin subunits in BRAF mutant, RAS mutant, and non-BRAF non-RAS mutant PTC was similar, with higher expression levels in BRAF mutant tumors . Variancp < 0.0001) , a single metric parameter designed by the expression level of 16 thyroid metabolism and function genes . ITGA1 p 0.0001) . Advance 0.0001) . The exp 0.0001) , extrath 0.0001) , and hig 0.0001) . In addi 0.0001) C,D. High 0.0001) . Disease = 0.015 F.Two well-characterized papillary thyroid cancer cell lines, TPC-1 and BCPAP, harboring the RET/PTC and BRAFV600E oncogenes, respectively, were used to investigate the role of the integrin α3β1 in adhesion to ECM . Cell atIn vitro scratch assay was used to investigate the role of the integrin α3β1 in cell motility. The cells were plated in ECM coated wells and, after 24 h, a scratch was created and measured immediately . The scrBCPAP and TPC-1 were plated on top of a polycarbonate membrane coated with Matrigel, and cells migrating to the lower face of the membrane were assessed after 48 h. The cells were incubated with or without Ab anti α3 at a final concentration of 1 μg/mL (upper and lower chamber). In separate experiments, the cells were transfected with ITGA3 siRNA or scrambled RNA . In bothReported results of gene expression analysis here confirmed previous immunodetection studies in normal thyroid cells. Considering α and β subunits assembly in the natural heterodimers, only α3β1, αVβ3, and at a minor extent α1β1 and α6β1 receptors are expressed in normal thyroid cells. Thus, integrin repertoire in normal thyroid cells appears restricted but sufficient to enable the cell to bind FN, CO, and LM, besides other non-ECM proteins. These integrins have functional and structural properties, participating in thyroid cell survival and follicle structure. The αVβ3 receptor for FN and VN is a functional component of the cell-ECM interaction and plays a central role in FN-mediated survival in thyroid cells through complex signaling, involving p21 RAS, calcium/CaMKII, and PI3K/AKT ,16,17. TTumor transformation deeply alters the equilibrium between adhesion molecules and basal membrane. Invasive carcinomas generally show aberrant basement architecture and loss of LM around the epithelial structures . Early tAnalysis of the TCGA database highlighted a defined difference between PTC subtypes with higher integrins expression in PTCcl and PTCtc, differing from PTCfv lower integrin expression. Thus, with respect to the integrin profile, PTCcl is closer to PTCtc than to PTCfv. However, when the TCGA database was generated, the histology subtype non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) had not yet been described. Therefore, a number of NIFTPs are present in the PTCfv and this may be responsible for the different integrin profiles of this variant compared to the others.BRAF mutation was associated with higher expression of all integrin subunits and particularly to ITGA2, ITGA3, and ITGAV, contributing to the difference between integrin expression profiles of the three PTC histotypes, as BRAF mutation was the driver mutation in 64% and 82% of PTCcl and PTCtc and only 17% in PTCfv.The α2β1 complex is the one with higher upregulation in thyroid carcinomas (up to 9-fold for ITGA2 vs. 2.5-fold for ITGA3 and ITGAV), although it is not the most abundant integrin expressed. This receptor, whose expression in benign and malignant thyroid tumors was previously detected by immunodetection, binds LM, COL, and heparan sulfate proteoglycan core protein, an integral component of the basement membrane ,33. AlphITGA3 is the most expressed integrin subunit and the most strongly associated with aggressive disease, being found upregulated in stage IV, lymph node metastasis, moderate/advanced extrathyroidal extension, high-risk tumors, and shorter disease-free survival. Recently the role of ITGA3 in thyroid carcinogenesis has been investigated. Knockdown experiments in TPC-1 demonstrated that ZNF367, a member of the zinc finger protein family, regulates cellular adhesion, invasion, and migration through, at least in part, modulation of ITGA3 expression . In TPC-Invasion and metastasis are complex processes that require sequential events: adhesion, proteolysis of ECM, and migration. In principle, well-differentiated epithelial tumors commonly use collective migration mechanisms . CollectIntegrin expression profile is altered in PTC and correlates with histopathology subtype and specific driver gene mutations.Integrin α3β1 is involved in PTC cell motility and invasion and is associated with advanced-stage disease, lymph node metastasis, extrathyroidal extension, high-risk, and a poor prognosis.


We have established the pig, a large natural host animal for influenza, with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies (mAbs). In this study we demonstrated that prophylactic intravenous administration of 15 mg/kg of porcine mAb pb18, against the K160–163 site of the hemagglutinin, significantly reduced lung pathology and nasal virus shedding and eliminated virus from the lung of pigs following H1N1pdm09 challenge. When given at 1 mg/kg, pb18 significantly reduced lung pathology and lung and BAL virus loads, but not nasal shedding. Similarly, when pb18 was given in combination with pb27, which recognized the K130 site, at 1 mg/kg each, lung virus load and pathology were reduced, although without an apparent additive or synergistic effect. No evidence for mAb driven virus evolution was detected. These data indicate that intravenous administration of high doses was required to reduce nasal virus shedding, although this was inconsistent and seldom complete. In contrast, the effect on lung pathology and lung virus load is consistent and is also seen at a one log lower dose, strongly indicating that a lower dose might be sufficient to reduce severity of disease, but for prevention of transmission other measures would be needed. Influenza virus infection remains a global health threat to humans and animal influenza A viruses are the CDC’s top zoonotic pathogen. Although influenza vaccines are available, most inactivated and live-attenuated influenza vaccines are strain specific, requiring constant updating, and influenza is still a cause of serious morbidity and mortality in high-risk groups, such as young children, the elderly and immunocompromised patients . Monocloin vitro studies or efficacy in small animals can be further tested. Pigs can provide such a model. Pig anatomy and physiology closely resembles that of humans. They have a similar distribution of sialic acid receptors in their respiratory tract and are infected with similar influenza A viruses, making them a powerful natural host large animal model to study immunity to influenza  virus load and greatly reduced lung pathology after an intravenous administration at 10 mg/kg in vivo, although nasal shedding was not eliminated  according to the UK animal (Scientific Procedures) Act 1986 under project license P47CE0F2. Both Institutes conform to the ARRIVE guidelines.The generation of porcine H1N1pdm09-specific mAbs was described previously . For ani6 PFUs of pandemic swine H1N1 isolate, A/swine/England/1353/2009 (H1N1pdm09) in 2 ml (1 ml per nostril) using a mucosal atomization device . Clinical signs  were observed and recorded. Clinical signs were mild and no animal developed a moderate or severe disease.Twenty 5-week old, Landrace × Hampshire cross female pigs were obtained from a commercial high-health status herd and were screened for antibody-free status against four swine influenza virus antigens: H1N1pdm09, H1N2, H3N2, and avian-like H1N1. Pigs weighed between 8 and 11 kg (average 9.5 kg). Pigs were randomized into four groups of five animals: the first group received 15 mg/kg of pb18; the second group received 1 mg/kg of pb18; the third received a combination of 1 mg/kg of pb27 and 1 mg/kg pb18 and; the fourth control group was left untreated. The mAbs were administered to the ear vein of the animals sedated with stresnil . Twenty-four hours after mAb administration, all animals were challenged intranasally with 1 × 10postmortem, the lungs were removed and digital photographs were taken of the dorsal and ventral aspects. Gross pathology was scored by quantitating the lesion areas as previously described by Halbur  pieces of the lung were homogenized in 0.1% BSA using the gentle MACS Octo dissociator, the homogenate was clarified by centrifugation and the supernatant was used for virus titration.Two nasal swabs (one per nostril) were taken each day following the challenge with H1N1pdm09. The swabs were placed into 2 ml of virus transport medium (VTM) comprising tissue culture medium 199 (Sigma-Aldrich) supplemented with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.035% sodium bicarbonate, 0.5% bovine serum albumin, penicillin 100  IU/ml, streptomycin 100 µg/ml, and nystatin 0.25 µg/ml, vortexed, centrifuged to remove debris, aliquoted and stored at −80°C for subsequent virus titration. Serum samples were collected at the start of the study  and at 0, 1, 3, and 4 DPI of the challenge. Broncho-alveolar lavage fluid (BAL) was collected from the entire left lung with 100 ml of 0.1% BSA+PBS. BAL samples were centrifuged at 300×Virus titers in nasal swabs, BAL fluid, and accessory lobe were determined by plaque assay on MDCK cells. The samples were 10-fold serially diluted in Dulbecco’s Modified Eagle’s Medium (DMEM) and 200 µl overlaid on confluent MDCK cells in 12-well tissue culture plates. After 1 h, the wells were washed and overlaid with 1 ml of 0.6% agarose in culture medium with 2 µg/ml of TPCK  trypsin. Plates were incubated for 48 h at 37°C. The overlay was removed, and plaques were visualized by staining the monolayer with 0.1% (v/v) crystal violet and enumerated.ahead RT-PCR kits (#220213) to amplify HA-gene products for Sanger sequencing and whole genome products for Illumina MiSeq sequencing (primer sequences available on request). Amplification products were then purified, sequenced, curated, and analyzed as described recently using the Staden package for Sanger sequencing and an in-house pipeline developed to analyze MiSeq outputs  was transferred to a 2 ml Eppendorf tube and 1.5 vol (375 µl) 100% ethanol added, mixed by inversion, then centrifuged briefly. The liquid was transferred to a vRNA capture column  and extracted following the manufacturer’s instructions before eluting in 50 µl of supplied AVE-buffer. RT-PCRs were performed using QIAGEN OneStep  outputs .Quantification of administered mAbs, pb18, and pb27, in serum, BAL and nasal swabs were determined by ELISA. Ninety-six well microtiter plates  were coated with 1 µg/ml of HA in PBS overnight at 4°C. Plates were blocked with 200 µl of blocking solution composed of 4% milk powder in PBS, supplemented with 0.05% Tween-20 (PBS-T) for 2 h at room temperature. A standard curve for pb18 mAb was prepared as 1:2 serial dilutions starting at 500 ng/ml in dilution buffer and added in duplicate to the assay plate. Samples and standard were incubated for 1 h at room temperature. The plates were washed four times with PBS-T and incubated with detecting antibodies; polyclonal goat-anti pig IgG HRP  (Bio-Rad). Binding of Abs was detected by developing with 50 µl/well 3,3’,5,5’-tetramethylbenzidine (TMB) high sensitivity substrate solution  and stopping with 50 µl 1 M sulfuric acid. The plates were read at 450 and 630 nm with the Cytation3 Imaging Reader (Biotek).4 MDCK-SIAT1 cells after overnight infection in 96-well flat-bottomed plates. Sera were heat inactivated at 56°C for 30 min. Sera were diluted in 50 µl of VGM to 1:4 and serially double diluted. Approximately 50 µl of diluted virus was added to the serum and incubated for 1 h at 37°C. In each well 100 µl of 3 × 104 MDCK-SIAT1 cells were added and incubated overnight at 37°C. The monolayer was fixed with 4% paraformaldehyde and permeabilized with Triton-X100 and stained with mouse anti-NP IgG1  followed by goat anti-mouse HRP (DAKO) antibody. TMB substrate was added and incubated for 2–5 min and the reaction was stopped with 50 µl of 1 M sulfuric acid and absorbance was measured at 450 and 630 nm (reference wavelength) on the Cytation3 Imaging Reader (Biotek). The 50% inhibition titer was defined as the final dilution of serum that caused ≥50% reduction in NP expression.Neutralizing antibody titers were determined in serum using a microneutralization (MN) assay. Briefly, viruses were diluted in virus growth medium  and titrated to give plateau expression of NP in 3 × 10Statistical analyses were performed using Graphpad Prism 8.3 (GraphPad software). Normally-distributed data sets were subjected to a one-way ANOVA with Bonferroni’s multiple comparisons test Figure 2in vivo efficacy of porcine mAbs, pb18 was administered at 15 mg/kg, 1 mg/kg or in combination with pb27 at 1 mg/kg each (pb18 + pb27). Control animals were untreated. Twenty-four hours after mAb administration, all the animals were challenged with H1N1pdm09 and culled at 4 days post infection (DPI) . The AUC for the animals treated with combined pb18 + pb27 at 1 mg/kg was also reduced (p = 0.056). However, 1 mg/kg of pb18 alone was insufficient to reduce nasal virus shedding (p = 0.126). We also assessed virus load in BAL and lung at 4 DPI .ET, BP, AT, and JH conceived, designed and coordinated the study. BP, AM, and BC, performed animal experiments, processed samples and analyzed the data. AN carried out postmortem and pathological analyses. AT and PR designed experiments, provided reagents and developed assays for assessment of antibody function. RD and JM performed sequencing analysis. ET, BP, PR and RD wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Bill & Melinda Gates Foundation grants OPP1201470 and OPP1215550 (Pirbright Livestock Antibody Hub); the UKRI Biotechnology and Biological Sciences Research Council (BBSRC) grants BBS/E/I/00007031, BBS/E/I/00007038, and BBS/E/I/00007039. AT and PR were funded by the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (CIFMS), China Grant 2018-I2M-2-002, the Townsend-Jeantet Prize Charitable Trust (charity number 1011770) and the Medical Research Council (MRC) Grant MR/P021336/1. The Worldwide Influenza Centre is supported by the Francis Crick Institute receiving core funding from the Cancer Research UK (FC001030), the Medical Research Council (FC001030) and the Wellcome Trust (FC001030).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.


Non-esterified fatty acids (NEFAs) are one of the main lipid components of follicular fluid at concentrations that depend on circulating levels. Elevated levels of NEFAs impair oocyte quality, development potential, and may subsequently influence the metabolism and reproductive fitness of offspring. Granulosa cells (GCs) are the follicular cells that are closely communicating with the oocyte. However, the responses of GCs exposed to high levels of NEFAs when cocultured with cumulus-oocyte complexes (COCs), and how they attenuate the negative effects of NEFAs on oocytes, are unclear.in vitro maturation (IVM) in the presence of elevated levels of NEFAs. Genomic expression analysis was conducted to explore the responses of the GCs to the elevated levels of NEFAs. After limma algorithm analysis, 1,013 genes were differentially expressed between GCs cultured with and without elevated NEFAs. Among them, 438 genes were upregulated and 575 were downregulated. The differentially expressed genes were enriched in pathways related to metabolism, inflammation, and epithelial-mesenchymal transition.To better understand this protective effect, monolayers of porcine GCs were cocultured with COCs during The pathways and upstream regulators suggested that the cocultured GCs responded to the elevated NEFAs with (1) inhibition of the transition from granulosa to luteal cell, (2) interactions of metabolism change, anti-inflammation, mitochondrial function, and cell transition, (3) intercommunication with cocultured COCs of anti-inflammatory factors.The online version contains supplementary material available at 10.1186/s13048-021-00904-y. In vitro studies showed that increased NEFA levels can affect follicular growth through impaired viability and steroidogenesis of granulosa cells (GCs) [In mammals, especially in bovine and humans, the contexts of negative energy balance and obesity result in elevated levels of circulating non-esterified fatty acids (NEFAs) and may affect fertility , 2. The ls (GCs) , 5. Morels (GCs) . High lels (GCs) . In our ls (GCs) .The coculture of follicle shell-like GCs encapsulated in an agarose matrix with cumulus-oocyte complexes (COCs) indicated that GCs could improve oocyte developmental competence and blastocyst formation, reduce the intra-oocyte ROS content, and reduce apoptosis . The stuIn vitro studies on human follicles and embryos are limited because of ethical concerns and limitations. The pig is considered one of the optimal experimental models for human biomedical and reproductive research due to physiological, digestive, and reproductive similarities including the duration of oocyte in vitro maturation, the kinetics of cell division, and the timing of the embryo genome activation [in vitro fertilization.tivation . In thisFollowing loess and quantile normalization and the limma algorithm with Flexaray, 1,381 out of the 43,794 probes were differentially expressed in cocultured GCs with NEFAs relative to the control (P<0.05). Of these, 1,295 probes were differentially expressed with the cut off criterion of 1.2-fold change , Toll Like Receptor 6 (TLR6), Interleukin 33 (IL33), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) had similar trends, and TLR6 and GAPDH were significantly expressed by both methods (P<0.05), confirming the reliability of the microarray analysis , and Cytochrome P450 Family 19 Subfamily A Member 3 (CYP19A3) in response to exposure to excessive NEFAs, and only the abundance of Cytochrome P450 Family 11 Subfamily A Member 1 (CYP11A1) was increased  were “Clathrin-mediated endocytosis signaling”, “phospholipase C signaling”, “glucocorticoid receptor signaling”, “semaphoring signaling in neurons”, “estrogen-dependent breast cancer signaling”, “HOTAIR regulatory pathway”, “regulation of eIF4 and p70S6K signaling”, “EGF signaling”, “Huntington’s disease signaling”, and “Insulin secretion signaling pathway”  may promote the EMT process in bovine GCs [Granulosa cells are one of the epithelial cell types that display polarity, cellular adhesion, and cell-cell junctions. Following the LH surge, GCs lose their epithelial nature and undergo an epithelial-mesenchymal transition (EMT) known as “luteinization” –20. Lutein vitro . In thisin vitro . Moreovein vitro –25. Predvine GCs . Oleic avine GCs , 27, 28.vine GCs , 30. Besvine GCs . In humavine GCs , 33. Morvine GCs .HSD3B1, which is involved in the conversion from pregnenolone to progesterone, was not affected by the elevated level of NEFAs, suggesting that progesterone production was not increased. The protein STAR is responsible for the transportation of cholesterol, the substrate for steroidogenesis; and CYP11A1 encodes P450scc, which catalyzes the formation of pregnenolone, the precursor for both progesterone and estradiol [Moreover, elevated NEFA levels were also reported to decrease progesterone production in bovine thecal cells, and inhibit testosterone production in mouse Leydig cells , 36. Thestradiol . In the stradiol , 39. TheIVM resulted in blastocysts with increased inflammation, apoptosis, and mitochondrial dysfunction associated with ROS production [2+ level in oocytes [When cells are in the presence of an excess of NEFAs, these NEFAs are stored as lipid droplets, or they enter mitochondria to be β-oxidated to produce ATP. However, elevated NEFAs remaining in the cytosol may undergo lipid peroxidation, generating ROS and depleting protective glutathione levels . The regoduction . In lineoduction , 41, and oocytes . The mos oocytes . This stThe mitochondrion is the principal organelle that produces ATP, and its well-organized functioning guarantees the efficiency of cellular processes. The increased ROS content due to the β-oxidation of excess fatty acids may cause mitochondrial dysfunction. A balance of pyruvate and fatty acid oxidation is required in healthy mammalian oocyte mitochondria to maintain a low level of ROS production . BesidesThere is a possible protection mechanism in follicular cells that can desaturate the potentially toxic saturated fatty acids into mono-unsaturated fatty acids . Insteadin vivo  or in vitro, the different ovarian cell types respond differently. Commonly, apoptosis is induced by elevated levels of NEFAs in pig, human, bovine, and mouse granulosa cells [When simultaneously exposed to elevated levels of NEFAs either sa cells , 49, 50,sa cells , 7, 51. sa cells , 51, 52.sa cells , 53, 54,sa cells , 55, 56.sa cells , 58.The model for the mechanisms of the effects of high levels of NEFAs on GCs and how the GCs protect COCs is shown in Fig. All supplements and reagents used in this study were purchased from sigma-Aldrich  unless otherwise specified.2 in air for 24 h for equilibration before use.PA, SA, and OA were dissolved in absolute ethanol at 468 mM, 194mM, and 534mM, respectively to make the stock solution as described previously . The NEFPorcine ovaries were collected from a local slaughterhouse and transported within 2 h to the laboratory in physiological saline supplemented with 100 IU/L of penicillin G and 100 mg/mL of streptomycin sulfate at approximately 35 °C. The ovaries were washed three times with saline warmed to 37 °C and COCs, GCs, and follicular fluid were aspirated from follicles 3-6 mm in diameter with an 18-gauge needle attached to a 10-ml syringe, avoiding blood contamination. The cell mixture was washed twice in warmed Tyrode’s lactate-buffered HEPES supplemented with 0.1 % polyvinyl alcohol (PVA-TL-HEPES).5 per mL in DMEM/F12  supplemented with 10 % fetal calf serum , penicillin-streptomycin (100 U per mL) and 50 µg/mL gentamycin and cultured in 4-well plates  at 38.5 °C under a humidified atmosphere (5 % CO2). At the 24-h point, cells were washed with PBS twice and re-supplemented with fresh Dulbecco’s minimal essential medium/Ham’s F12 (DMEM/F12) with 10 % fetal calf serum for 6 more days of culture during which the medium (70 %) was changed every 48 h.After the COCs had fallen to the bottom of the container due to their larger size, the granulosa cell-containing superstratum of follicular fluid was passed through a Falcon 40-µm mesh cell strainer , centrifuged at 800 × g for 8 min and washed three times with warm phosphate-buffered saline  containing penicillin-streptomycin  and 50 µg/mL gentamycin. The cells were re-suspended at 5 × 10After 7 days of culture, the DMEM/F12 culture medium was replaced with NEFA-containing NCSU-23 (or NCSU-23 without NEFA but with same amount of BSA and ethanol as control) supplemented with 10 ng/mL epidermal growth factor (EGF), 0.5 g/mL follicle-stimulating hormone (FSH), 0.5 g/mL luteinizing hormone (LH), 1 mM dibutyryl-cyclic adenosine monophosphate, 1 % (v/v) purified porcine follicular fluid, and 50 µg/mL gentamycin. COCs were collected from the follicular fluid of a different batch of ovaries then washed with PVA-TL-HEPES. Those with cytoplasmic uniformity and at least three layers of cumulus cells fully surrounding the oocyte  were selTM RNA Miniprep Plus  was used to extract the RNA of GCs in Trizol. Cells from four wells of one 4-well plate were pooled as one replicate, and eight biological replicates were collected on different days  validation). An equal volume of ethanol was added to cells lysed in Trizol and mixed thoroughly. The mixture was passed through a Zymo-SpinTM IIIGG column with a DNase I treatment, followed by the washing buffer. The quality and concentration of RNA were analyzed with a Bioanalyzer . All the extracted RNA samples were of good quality, with integrity numbers > 8.5. Samples were stored at −80 °C.The Direct-zolFive ng of RNA from four replicates from two conditions were amplified linearly using a RiboAmp® HS PLUS RNA Kit (Applied Biosystems). Antisense RNA (aRNA) was produced using T7 RNA polymerase and labeled with Cy3 and Cy5 using a ULS Fluorescent Labeling Kit . Two corresponding treatments (900 ng aRNA per replicate) were mixed in a two-color dye-swap design and hybridized on an Agilent-manufactured EmbryoGENE 4 × 44 K porcine transcriptome microarray slide  for 17 h at 65 °C. The slide was washed for 1 min in Expression Wash Buffer 1 (RT), 3 min in Expression Wash Buffer 2 (42 °C), 10 s in 100 % acetonitrile (RT), and 30 s in Stabilization and Drying Solution. Microarray slide was then scanned with the PowerScanner  and features were extracted with Array-pro 6.3 . Intensity files were normalized and analyzed with FlexArray. The background was subtracted from raw fluorescence intensity prior to normalization within Cy-3/Cy-5 and between each array with Loess and quantile, respectively.Sus scrofa 11.1) in NCBI [Sus scrofa background [www.ingenuity.com/) with human, mouse, and rat backgrounds. IPA analysis considers the general tendency in gene expression variations, including the direction in which each tends to change and the concordance between the variations of the significantly affected genes and the signaling pathways. It then determines the most plausible upstream regulators (explaining the observed gene expression changes) and tox list  by taking into consideration the results and the usual context in which the same genes are affected. The extremely high similarity between the porcine and human genomes and the availability of more annotated functions in human studies made the IPA analysis a necessary complement.Treatment effects were analyzed with the limma algorithm, which attributed to each probe a probability of differing significantly from the control. The differentially expressed genes (DEGs) were then annotated by the latest version (Revision 6) of the Affymetrix porcine annotation database combined with the current annotation data of pig . Real-time PCR reactions were conducted on a LightCycler 480 System  with LightCycler 480® SYBR Green I Master. The efficiency of the primers and the standard curve for each gene were deduced from serial dilutions (1 pg to 0.1 fg) of the corresponding cDNA fragment used as template. The geometric mean of two housekeeping genes, succinate dehydrogenase complex flavoprotein subunit A (SDHA) and peptidylprolyl isomerase A (PPIA), [Total RNA extracted from the other four biological replicates was reverse transcribed using oligo-dT and random primers with the qScript (PPIA), , was useAdditional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.


Moreover, matrix remodeling and production were decreased under sµG, which is in line with the impaired fibroblast differentiation. We further investigated changes on a transcriptomic level using RNA sequencing. The results demonstrated that sµG has less effect on fibroblast transcriptomes, while sµG triggers changes in the transcriptome of myofibroblasts. Several genes and biological pathways found through transcriptome analysis have previously been reported to impair fibroblast differentiation. Overall, our data indicated that fibroblast differentiation, as well as matrix production and remodeling, are impaired in 3D culture under sµG conditions.Exposure to microgravity affects astronauts’ health in adverse ways. However, less is known about the extent to which fibroblast differentiation during the wound healing process is affected by the lack of gravity. One of the key steps of this process is the differentiation of fibroblasts into myofibroblasts, which contribute functionally through extracellular matrix production and remodeling. In this work, we utilized collagen-based three-dimensional (3D) matrices to mimic interstitial tissue and studied fibroblast differentiation under simulated microgravity (sµG). Our results demonstrated that alpha-smooth muscle actin (αSMA) expression and translocation of Smad2/3 into the cell nucleus were reduced upon exposure to sµG compared to the 1 The current interest in spaceflight and long-term stays in space is increasing. However, little is known about how the lack of gravity will affect the health of space travelers. It has been shown that spaceflight and microgravity conditions can cause adverse effects on the immune system, neurological function, and bone density ,2,3. SevWound healing is a dynamic process involving many cell types , each with distinct roles in the regulation of different wound healing phases, namely homeostasis, inflammation, proliferation and remodeling . ThroughFibroblasts are the key players in wound healing during the tissue repair process ,13. DuriCip1/Waf1 [Under real microgravity conditions, human fibroblast samples cultured during space flight reveal increased levels of collagen synthesis compared to control samples , while aip1/Waf1 . In addiip1/Waf1 . Similarip1/Waf1 . These dg . To ensure the physiological relevance of our model, we utilized a three-dimensional (3D) cell culture system based on a collagen matrix as a biomimetic tissue model. Three-dimensional collagen matrices allow us a more in depth understanding of mechanisms due to their structural complexity, and are widely used due to their ability to better mimic interstitial tissue compared to traditional 2D cell culture surfaces [In this work, studies whether sμG conditions affects fibroblast differentiation into myofibroblasts when compared to 1surfaces ,36. Currg). As mentioned, the essential step in the tissue repair process is the differentiation of fibroblasts into myofibroblasts. To induce myofibroblast differentiation, cell culture medium was supplemented with 10 ng/mL of TGF-β1. After 3 days of cultivation, cells were analyzed in terms of differentiation state by means of αSMA expression, nuclear translocation of Smad2/3, transcriptome analysis using RNA sequencing (RNA-seq), matrix remodeling using a custom-made image analysis toolbox and cytokine secretion profile using multiplex bead-based ELISA. A schematic illustration of the experimental setup is depicted in Fibroblast differentiation is a critical step during the tissue repair process ,13, and αSMA gene expression, a prominent marker of myofibroblasts [g and sμG conditions, but a significant reduction in αSMA expression was observed under sμG conditions when compared to 1g conditions, whether sμG samples were TGF-β1 stimulated or not. To verify the gene expression results, cells were stained with αSMA antibodies and visualized using epi-fluorescence microscopy , LAMA1 (laminin), VTN (vitronectin), as well as HAS1-3 and HYAL1-3 , as shown in MMP1, MMP2 and MMP9, to be less expressed in fibroblasts when compared to myofibroblasts in both sµG and 1g conditions  using RNA-Seq data. Upon differentiation, fibroblasts produce high amounts of matrix components, especially collagen, fibronectin and hyaluronic acid ,40,42. Anditions E. Again,g. These results support the impairment of fibroblast differentiation under sµG, as demonstrated in the previous sections. This emphasizes the effects of sµG conditions on fibroblast differentiation and ECM remodeling in our biomimetic cell culture model. Overall, we found that matrix remodeling through the expression of collagen and other ECM components, as well as matrix metalloproteinases (MMPs), were reduced in myofibroblasts under sµG conditions when compared to cell culture at 1g and under sµG, in an attempt to explain the impairment in fibroblast differentiation under sµG at a transcriptomic level.As demonstrated above, fibroblast differentiation and function are impaired in sµG. Using RNA-Seq we analyzed the transcriptome of fibroblasts and myofibroblasts, both under 1g and sµG conditions in both fibroblasts and myofibroblasts were found. By analyzing differentially expressed genes (DEGs) in fibroblasts between both cell culture conditions, we found only 16 DEGs with a fold change (FC) higher or equal to two, and a false discovery rate (FDR) smaller or equal to 0.05  and therefore this could affect the poor performance of the DEGs analysis. However, our finding is consistent with another report demonstrating that few genes  were differentially expressed in WI-38 human fibroblasts cell line after 5 days of spaceflight [As shown by the heat map in  to 0.05 B. Only tceflight  taking iceflight .g and sµG respectively, while cells in both conditions shared 1224 upregulated genes fibroblast survival in chronic fibrotic disorders [g condition. IGF signaling is known for selective suppression of Smad3 activation [KLF2  and kept in PBS prior to use.Three-dimensional collagen matrices for cell culture of fibroblasts were made by mixing rat tail type I collagen , 500 mM phosphate buffer  and 0.1% acetic acid  as a collagen solution at a concentration of 2 mg/mL, as previously published . The pre2 and 95% humidity (standard cell culture conditions). Cell culture medium and supplements were all purchased from Gibco, Invitrogen, Thermo Fisher Scientific Inc, Dreieich, Germany.Primary human dermal fibroblasts were maintained in Dulbecco’s Modified Eagle Medium (DMEM) cell culture medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C, 5% CO5 fibroblast cells were seeded onto 3D collagen matrices and kept at standard cell culture conditions for 2 h to allow for cell attachment. Afterwards, the cell culture medium was removed and biocompatible microvessels were placed onto the well plate, as previously described [g or sµG conditions in an incubator at standard cell culture conditions.For all experiments, the 3D collagen matrices were placed into four well-plates . 1 × 10escribed . Subsequescribed . Cells wAll experiments using sµG were performed on desktop Random Positioning Machine (RPM) . The RPM was operated in 3D random mode, using random motion and random direction, maintaining an average velocity of 60 deg/s. Four-well cell culture well plates were placed at the center of the rotation, as previously described . All expFor cell staining, microvessels were removed from the wells and cells were fixed with 4% paraformaldehyde  for 10 min and subsequently permeabilized with 0.1% Triton X100  for 10 min. Cells were washed three times with PBS after each step. Afterwards, cells were stained with Phalloidin conjugated with Alexa Fluor 594 , and Hoechst-33342 . For additional staining of αSMA, cells were blocked with 1% bovine serum albumin for 1 h at room temperature, incubated with mouse anti-human αSMA  overnight at 4 °C, and incubated with goat anti-mouse IgG conjugated with Alexa Fluor 488  for 2 h.For additional staining of Smad2/3, cells were blocked with 1% bovine serum albumin for 1 h at room temperature, incubated with mouse anti-human Smad2/3 conjugated with Alexa Fluor 488  overnight at 4 °C.Cell imaging was performed using a Lionheart FX automated microscope  using a 10× objective. Stacked images were gathered at a z-interval of 5 µm with overall z-layer of 500 µm. The representative images are of cells at the z-layer approximately 10–20 μm below the collagen surface, where many cells are located. Quantitative image analysis was done using an automated custom-built 3D cell analysis software . First, https://git.sc.uni-leipzig.de/pe695hoje/topology-analysis (accessed on: 15 September 2021)) [Collagen matrices were decellularized by osmotic shock through incubation with distilled water for 1 h, as previously published . Afterwar 2021)) . Experim−ΔΔCT method. Experiments were performed with at least four independent replicates.Gene expression analysis was performed using an established protocol, as published . BrieflyRNA for sequencing was isolated as mentioned in the RNA isolation and gene expression analysis, followed by a purification step using the RNeasy mini kit  as described by the manufacturer’s protocol. RNA quantity and quality were quantified using Nanodrop  and Qi RNA kit . Samples were prepared with NEB Ultra II RNA kit  as per protocol instructions using NEBNext Poly(A) mRNA Magnetic Isolation module , and uniquely dual indexed. The resulting libraries concentration, size distribution, and quality were assessed on a Qubit 4 fluorometer  with a dsDNA high sensitivity kit  and on a 4200 TapeStation using a High Sensitivity D5000 kit . Based on these results, libraries were normalized according to their molarity and pooled, then quantified with a library quantification kit for Illumina platforms  on a StepOnePlus qPCR machine . Finally, pooled libraries were loaded at 350pM with 1% PhiX on S2 FlowCell, and paired end sequenced (2 × 150 bp) on a NovaSeq 6000 next generation sequencer . RNA-Seq was performed in triplicate.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on: 15 September 2021)) [ILLUMINACLIP: trimmomatic_adapter.fa:2:30:10 TRAILING:3 LEADING:3 SLIDINGWINDOW:4:15 MINLEN:36). The surviving trimmed read pairs were then processed with Fastp [–dta flag. The resulting SAM alignments were then converted to BAM format and coordinate sorted using SAMtools v1.3.1 [-s no -t exon -I gene_id) for raw count generation. Concurrently, the sorted alignments were processed through Stringtie v1.3.0 [Raw FASTQ sequenced reads were first assessed for quality using FastQC v0.11.5 (available online at r 2021)) . The rear 2021))  for qualth Fastp  in orderth Fastp  with thes v1.3.1 . The sors v1.3.1  using the v1.3.0  for trane v1.3.0  was usedhttp://nasqar.abudhabi.nyu.edu/ (accessed on: 15 September 2021)) [http://bioinformatics.sdstate.edu/idep93/ (accessed on: 15 September 2021); publicly accessible by South Dakota State University) [http://pantherdb.org (accessed on: 15 September 2021)) [RNA-Seq data were merged using the NASQAR toolbox (publicly accessible at r 2021))  and the versity) . For theversity) . The DEGr 2021)) .p ≤ 0.05. Unless otherwise stated, all experiments were performed with at least three replicates and data are represented as mean ± standard deviation (SD).Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc test using Prism 9  and the level of significance was set to