Cylindrostoma monotrochum  and Monoophorum striatum  using a phalloidin‐rhodamine technique. As in all rhabdithophoran flatworms, the body‐wall musculature consisted of three muscle layers: on the outer side was a layer of circular muscle fibers and on the inner side was a layer of longitudinal muscle fibers; between them were two different types of diagonally orientated fibers, which is unusual for flatworms. The musculature of the pharynx consisted of a basket‐shaped grid of thin longitudinal and circular fibers. Thick anchoring muscle fibers forming a petal‐like shape connected the proximal parts of the pharynx with the body‐wall musculature. Male genital organs consisted of paired seminal vesicles, a granular vesicle, and an invaginated penis. Peculiar ring‐shaped muscles were only found in M. striatum, predominantly in the anterior body part. In the same species, seminal vesicles and penis only had circular musculature, while in C. monotrochum also longitudinal musculature was found in these organs. Female genital organs were only present in M. striatum, where we characterized a vagina interna, and a bursa seminalis. Transverse, crossover, and dorsoventral muscle fibers were lacking in the middle of the body and greatly varied in number and position in both species.We analyzed the adult musculature of two prolecithophoran species,  It is lacking a vagina and a bursa seminalis, but has paired germaries, one on either side of its conical and remarkably broad pharynx  in March 2016, October 2016, 2017 and in May 2018. The worms were extracted from the algae using a 1:1 7.14% MgCl2 × 6H2O and artificial sea water (ASW) solution, and identified as the two species by histological sections (see below). Animals were maintained in petri dishes with ASW in a climate chamber, at 18°C with 60% humidity and a 14:10 hours day‐night cycle.Specimens of 2.22 × 6H2O and ASW solution for 15–20 min, fixed in 4% formaldehyde  in 1× phosphate buffered saline (PBS) for 1 hr at room temperature (RT) and washed with PBS‐Tx  for 1 hr. Specimens were blocked for 1 hr at RT with BSA‐Tx  and incubated overnight at 4°C in a rabbit anti‐5HT antibody (Sigma‐Aldrich) diluted 1:2,000 in BSA‐Tx (data from antibody staining not shown here). After being washed with PBS‐Tx for 2 days, specimens were incubated in tetramethylrhodamine‐conjugated phalloidin  and the secondary fluorescein isothiocyanate‐conjugated (FITC) swine anti‐rabbit antibody , both diluted 1:250 in BSA‐Tx for 1 hr at room temperature in darkness. Subsequently, specimens were rinsed with PBS‐Tx for 3 days at RT and three nights at 4°C in darkness. Finally, they were mounted in Vectashield (Vector Laboratories). In total, 20 adult M. striatum and five adult C. monotrochum were stained.For whole‐mount stainings, animals were relaxed in a 1:1 7.14% MgCl2.3Three adult animals of each species were relaxed and fixed as described above. Then they were rinsed with distilled water for 5 min and dehydrated in a graded series of ethanol. Subsequently, specimens were infiltrated with a 1:1 mixture of ethanol and Technovit 7100  overnight. Then, they were transferred to 100% Technovit 7100 overnight. Finally, specimens were embedded in Technovit resin overnight at RT and stuck to a wooden block with Technovit 3040.Semi‐thin sections at 3 μm thickness were made with a microtome (Reichert‐Jung Autocut 2040) and stained using a standard hematoxylin‐eosin (H.E.) protocol (Harris hematoxylin and 5% eosin‐Y in ethanol).2.4http://www.gimp.org) and Inkscape v. 0.92 (https://inkscape.org).Live squeeze preparations and H.E. stained sections were observed using a Leica DM 5000 B microscope and photographs were taken with a Leica DFC 490 camera. Confocal stacks were generated using a Leica TCS SP5 II confocal microscope and processed with the open‐source program Fiji v. 1.52j , an inner layer of longitudinal muscle fibers (lm) and a layer of diagonal muscle fibers (dm) in between Figures b,c,f,g. On the dorsal side, sets of two different types of dm dm1 and dm2) extend over the total body length , in C. monotrochum the outer layer is built of approximately 105 evenly spaced cm  are on the dorsal side and about 65 lm (232 lm per mm) on the ventral side , whereas the diameter of the pharynx of C. monotrochum is about 150 ± 6.4 μm (n = 4). The shape of the pharyngeal lumen is also different, cylindrical in M. striatum and conical in C. monotrochum  and lm .In 3.2.5In both species, dorsoventral muscle bundles (dvb), which connect the dorsal and the ventral sides of the body, are present only in the anterior and the posterior parts of the body and in low numbers Figure . Each co3.2.6C. monotrochum than in M. striatum. C. monotrochum has six to eight tmb in the anterior fourth of the body and three dvb in the area posterior to the brain  are possibly associated with their life cycle. In March and in October, we found specimens with a copulatory organ and with testis follicles. In May, specimens had a copulatory organ, but we could not unambiguously identify testis follicles, and the phalloidin staining of these animals did not show any rsm. Specimens from March were only used for histology and not for phalloidin stainings, so we do not know about rsm in animals from March. To clarify the connection of rsm with the life cycle, additional observations about the presence of absence of testis follicles in May, and of rsm in March are needed.The rsm found in half of the phalloidin‐stained specimens of C. monotrochum collected in October, we observed animals with no copulatory organ and no testis follicles, but also animals with copulatory organ, but without testis follicles, and animals with both, copulatory organ and testis follicles. None of the phalloidin‐stained animals showed rsm.In specimens of Ring‐shaped muscles were not previously described in works based on histological sections Böhmig, . A struc4.5Cylindrostoma Ørsted, Cylindrostoma, as well as Pseudostomum, are not monophyletic genera (Norén & Jondelius, Monoophorum Böhmig, Cylindrostoma and Pseudostomum, such as a ciliary groove at the level of the brain, an encapsulated brain with two pairs of eyes, and an orogenital opening on the ventral side at the posterior body half make Monoophorum a likely pseudostomid. In terms of the musculature, M. striatum and C. monotrochum show many shared characters. Both species have the same pattern of body‐wall musculature, namely, showing circular, longitudinal, and two different types of diagonal muscles. Thick muscle bundles, which are extensions of the longitudinal muscles from the muscularis externa and interna of the pharynx, build the anchoring muscles of the pharynx. Still, we find some idiosyncrasies in the musculature differentiating the two observed species. While M. striatum has a creeping sole‐like strengthening of the longitudinal muscles of the body‐wall on the ventral side, C. monotrochum does not. The musculature of the seminal vesicles in M. striatum consists only of circular muscles and in C. monotrochum it has both circular and longitudinal muscles. The musculature of the penis in M. striatum consists only of circular muscles, whereas that in C. monotrochum has both circular and longitudinal muscles. M. striatum has fewer dorsoventral and transverse muscles than C. monotrochum, but M. striatum has special inner crossover muscles at the level of the brain. We could not find any common character for the adiaphanidan clade, which consists of the Prolecithophora, the Fecampiida Rohde, Luton, Baverstock, & Johnson, In molecular phylogenies, the genus The authors declare no potential conflict of interest.A.L.G. and B.E. designed the study. A.L.G. performed stainings, and A.L.G. and P.B. made confocal stacks and prepared the figures. A.L.G. and B.E. wrote the manuscript. All authors agreed on the final manuscript.


Different pediatric physiologically-based pharmacokinetic (PBPK) models have been described incorporating developmental changes that influence plasma drug concentrations. Drug disposition into cerebrospinal fluid (CSF) is also subject to age-related variation and can be further influenced by brain diseases affecting blood-brain barrier integrity, like meningitis. Here, we developed a generic pediatric brain PBPK model to predict CSF concentrations of drugs that undergo passive transfer, including age-appropriate parameters. The model was validated for the analgesics paracetamol, ibuprofen, flurbiprofen and naproxen, and for a pediatric meningitis population by empirical optimization of the blood-brain barrier penetration of the antibiotic meropenem. Plasma and CSF drug concentrations derived from the literature were used to perform visual predictive checks and to calculate ratios between simulated and observed area under the concentration curves (AUCs) in order to evaluate model performance. Model-simulated concentrations were comparable to observed data over a broad age range  for all drugs investigated. The ratios between observed and simulated AUCs (AUCo/AUCp) were within 2-fold difference both in plasma (range 0.92–1.09) and in CSF (range 0.64–1.23) indicating acceptable model performance. The model was also able to describe disease-mediated changes in neonates and young children  related to meningitis and sepsis . Our model provides a new computational tool to predict CSF drug concentrations in children with and without meningitis and can be used as a template model for other compounds that passively enter the CNS. Developmental processes in children affect pharmacokinetics and should ideally be taken into account when establishing drug dosing regimens. One way to incorporate developmental differences is by making use of physiologically-based pharmacokinetic (PBPK) models in which kinetic equations are used to describe drug disposition processes and developmental biology. With these equations the absorption of drugs into the model, the flow of drugs between different compartments (representing major organs/tissues), and excretion from the model are predicted. PBPK models can also be used to describe drug concentrations in different target tissues, which often correlate better with the clinical effects. Here, we developed a generic pediatric PBPK model of drug disposition in the cerebrospinal fluid (CSF), that was able to describe clinically measured drug concentrations of several drugs in neonates and children. The model could be useful in predicting CSF concentrations of other drugs in pediatric populations where clinical data is often sparse or absent and by this means guide first-in-child dose recommendations. Growth and development significantly impact handling of drugs in children across the pediatric age range. Simple linear bodyweight-based extrapolations from adult to pediatric doses have resulted in toxicity or therapy failure . Taking Physiologically-based pharmacokinetic (PBPK) modeling is an important tool to simulate drug exposure and design dosing guidelines. PBPK models are compartmental kinetic models in which physiological and drug-specific parameters are as much as possible separated . PhysiolPBPK models also allow predictions of drug concentrations in target tissues, which often correlate better with the clinical effect. This is especially the case for organs like the brain that are characterized by permeability-limited disposition of various drugs, leading to a significant lag time between their peak plasma and tissue concentrations –7. For det al. developed an adult brain PBPK model consisting of 4 compartments, which was validated using measured paracetamol CSF concentrations . The. The8]. Vbb, Vbm, Vccsf, Vscsf, fubm, fubb, fuccsf, fuscsf, Cbb, Cbm, Cccsf and Cscsf represent volumes, unbound fractions and concentrations in brain blood, brain mass, cranial CSF and spinal CSF, respectively. Qbrain denotes brain blood flow and Cbla denotes concentration in arterial blood. Qsin and Qsout represent CSF shuttle flow between cranial CSF and spinal CSF compartments. Qssink and Qcsink are the flows from CSF compartments to blood. Qbulk represents bulk flow from brain mass to cranial CSF. PSb, PSc and PSe represent permeability surface area products between brain blood and brain mass, brain blood and cranial CSF, and brain mass and cranial CSF, respectively.Where The following assumptions were made: (1) The BBB is a barrier between brain mass and brain blood, and the BCSFB between cranial CSF and brain blood. The barrier separating brain mass and cranial CSF is of high permeability. No barrier exists between cranial CSF and spinal CSF. (2) Compartments are of constant volume and well-stirred. (3) Permeability surface area products of the BCSFB are two-fold smaller than corresponding BBB values, as was previously described , 11, 39.(6) CSF production rate was multiplied by 2 in populations receiving spinal ketamine anesthesia, based on a two-fold increase that was observed in cats . (7) A bin vivo values by using the equation:in vitro permeability is expressed as dm/h, and BBB surface in dm2. A sensitivity analysis was performed to investigate the effect of CSF production rate change on the drug concentration-time curve in the spinal CSF compartment.BBB permeability was estimated for each drug separately from rat carotid artery perfusion data or cell-based passive permeability assays, and in the latter case scaled to To translate the adult model to a pediatric model estimates of height, weight, body surface area, organ volumes, tissue blood flows, hematocrit and albumin concentrations were adjusted using previously reported equations from literature  or the pThe time-based differential equations and assumptions made in the adult brain PBPK model were also applied to the pediatric model. In addition, physiological values for brain parameters were derived from literature leading to the following considerations: (1) Brain volume, brain blood flow, spinal CSF volume and CSF production rate were adjusted as a function of age , 40–43. To validate the physiological parameters included in the pediatric PBPK model, plasma and CSF concentrations were simulated with different drugs not used to build the model, namely ibuprofen, flurbiprofen, and naproxen using both adult and pediatric models. Only drug-specific parameters were adjusted to perform simulations for these drugs, of which brain penetration is like paracetamol known not to be affected by drug transporters.In case of meningitis, blood-brain barrier function is known to be impaired . To haveThe value for blood-brain barrier permeability estimated in the adult model was also used in the pediatric model after correction for the difference in blood-brain barrier surface area. The effect of meningitis on plasma clearance was already incorporated in the clearance parameter derived from the NeoMero study, in which only patients were included suffering from sepsis or meningitis . EffectsGermovsek et al. concentrations were described as ‘time after dose’ at steady state, which was expected to be reached after 24h [Clinical studies describing concentration-time profiles of paracetamol in adults (step 1) and paracetamol (step 2), ibuprofen, flurbiprofen and naproxen (step 3), in children, were used for validation and extracted from original publications with WebPlotDigitizer version 4.1 , 52–56. fter 24h . Becauseth percentile, 95th percentile, minimum, and maximum values were overlaid with clinical observations derived from literature. Also, plasma and CSF AUC0-last were calculated for median simulated data and for observed data using a non-compartmental  approach. A naïve pooling approach was used for the population data because variability between subjects was expected to cancel out in the analysis. In case of two observations at the same time, the average was used. AUCs were compared between simulated data and observed data according to:Simulations were run for 1000 virtual individuals who were matched with the individuals in the original studies for dosing regimen, age range, and percentage male/female. Simulations of CSF concentration-time profiles were visualized for the spinal compartment as clinical measurements were performed by lumbar puncture. Results were compared with observations using visual predictive checks in which median, 5fference . Only plfference . In contS1 File(R)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Drosophila melanogaster to investigate whether there is a preference between intrachromosomal and interhomolog donor sequences in mitotically dividing cells. Our results demonstrate that, although interhomolog HR is possible and frequent if another donor template is not available, intrachromosomal donor templates are highly preferred. This is true even if the interhomolog donor template is less diverged than the intrachromosomal donor template. Thus, despite the stringent requirements for homology, the chromosomal location of the donor template plays a more significant role in donor template choice.DNA double-strand breaks (DSBs) are especially toxic DNA lesions that, if left unrepaired, can lead to wide-ranging genomic instability. Of the pathways available to repair DSBs, the most accurate is homologous recombination (HR), where a homologous sequence is used as a donor template to restore genetic information at the break site. While much of the biochemical aspects of HR repair have been characterized, how the repair machinery locates and discriminates between potential homologous donor templates throughout the genome remains elusive. We use  They arise from both endogenous (Organisms have evolved multiple mechanisms to repair DSBs. These repair mechanisms have been broadly categorized into two pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is active throughout the cell cycle and has been suggested to play a major role in DSB repair in mammalian cells  was created by a two-step cloning process. First, the iwhiteΔ9 donor template was created by identifying a spontaneous HR repair event from the original DR-white assay . This repair event was used as a template for PCR amplification of iwhiteΔ9 fragment using CloneAmp HiFi PCR Pre-mix (Clontech) with primers 5′ GCTCCACCGCGGTGGCGGCCGCTTGGCCAAGAGGATCAGGAGCTA  and 5′ CTTGATATCGAATTCCTGCAGTTGCAGATCGGCGGCGGAGAAGTTAA . The iwhiteΔ9 PCR fragment with flanking restriction sites was cloned into NotI/PstI–digested pBlueScript.KS−.attB vector  and 5′ GCTCCTGATCCTCTTGGCCAAGCGGCCGCCAACTTTATTATACAAAGTTGTTT  using HiFi PCR Pre-mix. The Sce.white_y+ PCR fragment with flanking restriction sites was cloned into NotI–digested pBSKS−.iwhiteΔ9.attB vector using In-Fusion Cloning to create DR-whiteΔ9. Similarly, for the Sce.white DSB recipient construct , the Sce.white_y+ PCR fragment with flanking NotI restriction sites was cloned into NotI–digested pBSKS−.attB vector using In-Fusion Cloning.All DNA construct development followed manufacturer’s protocol, unless otherwise stated. The DR-iwhite interhomolog donor construct , the y+ transgene was amplified from the original DR-white construct with primers 5′ GAGCTCCACCGCGGTGGCGGCCGCCAACTTTTCTATACAAAGTTG  and 5′ GCTCCTGATCCTCTTGGCCAAGCGGCCGCCAACTTTATTATACAAAGTTGTTT  using HiFi PCR Pre-mix. The y+ PCR fragment with flanking NotI restriction sites was cloned into NotI-digested pBSKS-.iwhite.attB vector using In-Fusion Cloning to create iwhite interhomolog donor construct. The iwhiteΔ9 interhomolog donor construct  was similarly created by inserting the y+ PCR fragment with flanking NotI restriction sites into NotI–digested pBSKS−.iwhiteΔ9.attB vector using In-Fusion Cloning.For Drosophila were maintained on standard NutriFly Bloomington Formulation medium  at 25° with 12-hour light/dark cycles.whiteΔ allele was created using CRISPR/Cas9 tools. Two sequences homologous to the start (5′ GTGTGAAAAATCCCGGCAAT) and stop (5′ ACATATATCCGAAATAACTGCC) codon regions of the white gene were cloned adjacent to guide RNA (gRNA) scaffolds in the pCFD4 Drosophila expression vector as described in (http://www.crisprflydesign.org). Briefly, a pCFD4 fragment including the two described sequences were amplified from pCFD4 (Addgene) with primers 5′ TATATAGGAAAGATATCCGGGTGAACTTCGTGTGAAAAATCCCGGCAATGTTTTAGAGCTAGAAATAGCAAG (forward) and 5′ ATTTTAACTTGCTATTTCTAGCTCTAAAACACATATATCCGAAATAACTGCCGACGTTAAATTGAAAATAGGTC (reverse) using HiFi PCR Pre-mix. The amplified PCR fragment was inserted into BbsI-linearized pCFD4 vector using In-Fusion Cloning. Positive clones, identified by restriction digest, were sequenced (5′ GACACAGCGCGTACGTCCTTCG) to confirm accurate incorporation of gRNA sequences. pCFD4 vector with incorporated gRNAs were injected into NIG-FLY CAS-0001 embryos  (BestGene). G0 males were isolated and crossed into balancer lines to remove the Cas9 transgene. G1 progeny were phenotypically screened for white eyes, crossed out to balancer lines, and their DNA extracted using the previously described DNA Preparation Protocol  and 5′ GTCGTCCAGGCCTCGTTAAT (reverse). One line of each was selected based on fertility and health for downstream applications.For recombination assays, purified DR-I-SceI transgene is located on Chromosome 2 and linked to the dominant marker Sco (Sco–)  and 5′ CGCAGACCCTTAACCAGGTA (reverse).The heat-inducible o (Sco–) . For intSce.white, DR-white or DR-whiteΔ9) were crossed to males containing the heat-inducible I-SceI transgene  and the heat-inducible I-SceI transgene  were crossed to four–five y w tester females in vials. For each experiment/condition, vials containing ≥ 20 F2 progeny from 63-80 individual male germlines were scored. HR repair frequency was determined in F2 progeny containing Sce.white, DR-white, or DR-whiteΔ9.DSB repair assays were performed based on protocol described previously (ransgene . For int2O. PCR reactions were performed on 100 ng of purified genomic DNA using SapphireAmp Fast PCR Master Mix. The target I-SceI recognition site in Sce.white was amplified using primers 5′ GTGGATCAGGTAATCCAGG (forward) and 5′ CTTAAGCCATCGTCAGTTGC (reverse) under the following conditions: three minutes at 94°; 30 s at 94°, 30 s touchdown at 66° (-0.5°/cycle) and 30 s at 72° (16x); 30 s at 94°, 30 s at 58°, 30 s at 72° (20x); five minutes at 72° (1x); and held at 12°. PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega) and sequenced (5′ GAGCCCACCTCCGGACTGGAC). Sequences  were analyzed using the TIDE (Tracking across Indels by DEcomposition) algorithm, a computational protocol previously described . HR products were identified as conversions of the I-SceI sequence to either the wild-type white+ sequence (a 23-bp deletion) or the modified iwhiteΔ9+ sequence (a 32-bp deletion). Other insertions and deletions of up to 35 nucleotides were categorized as NHEJ with processing products. An output of 0 nucleotide indels was classified as the control I-SceI recognition sequence where neither HR nor NHEJ with processing repair events occurred.Genomic DNA was extracted from whole fly samples using a genomic DNA prep protocol adapted from https://doi.org/10.25387/g3.9271007.Strains and plasmids are available upon request. Supplemental Table S1, the R script algorithm , and sequence files  used for TIDE analyses are available on FigShare. white assay effectively measures intrachromosomal HR repair frequency in the Drosophila male premeiotic germline .The previously characterized DR-germline . The novel whiteΔ mutant allows experimental control over the donor templates available for repair. Thus, all genetic crosses described herein were carried out in a whiteΔ mutant background.First, to ensure that the endogenous white assay was modified to create the DR-whiteΔ9 assay with similar features: Sce.white, yellow transgene, and an attB targeting sequence  or whiteΔ9+ (orange eyes) indicates interhomolog HR repair, as the only viable donor template was located on the homolog.To confirm that interhomolog recombination is possible and detectable in our system, the interhomolog repair construct ic locus  and 2B. iwhite donor template . A homology-dependent effect on interhomolog recombination frequency was also observed. Interhomolog recombination decreased significantly by about 45% when using the iwhiteΔ9 donor template , similar to the 40% decrease observed in the DR-whiteΔ9 assay . Thus, interhomolog recombination in Drosophila occurs as frequently as intrachromsomal HR repair if no other donor template is available, and the efficiency of interhomolog repair is significantly affected by sequence divergence.When using the template , interhoDrosophila, we investigated the relative contribution of intrachromosomal and interhomolog HR repair when donor templates are simultaneously available on both homologous chromosomes. Females carrying either DR-white or DR-whiteΔ9 were crossed with males carrying the iwhiteΔ9 or iwhite interhomolog donor templates, respectively  compared to intrachromosomal repair using the less diverged iwhite donor sequence. Although total HR frequency declined by about 25% , intrachromosomal recombination still occurred an impressive 85.4 ± 2.3% relative to total HR frequency .The significant preference for intrachromosomal HR repair in the male premeiotic germline prompted us to investigate the HR repair preferences in other ce.white  and 3B. iwhite intrachromosomal donor template, the proportion of intrachromosomal HR repair events relative to total HR frequency was 98.9 ± 0.9% . Expectedly, with an iwhiteΔ9 intrachromosomal donor template, the proportion of intrachromosomal HR repair in all tissues decreased . Furthermore, males and females showed no statistical difference in the relative proportion of intrachromosomal or interhomolog HR repair events . Thus, both somatic and premeiotic germline tissues demonstrate a homology-dependent preference for intrachromosomal HR repair.Of the TIDE-detectable repair events, NHEJ with processing ranged from 15.2 to 93.4% and total HR events were lower on average . Within the HR events, with an Drosophila have shown that interhomolog recombination is possible  or on the sister chromatid. Interestingly, the chromosomal preference holds even when a less diverged donor template is available on the homolog, though total recombination frequencies do decrease. We propose that, in the presence of a diverged intrachromosomal template, repair shifts to true intersister HR , which cannot be distinguished phenotypically or molecularly in our assays from a no DSB or precise NHEJ event. These findings align with previous work in other eukaryotic systems that have established higher frequencies of recombination between sequences on sister chromatids  suggests random sampling of DNA sequences to locate a homologous donor template . Given tDrosophila and ectopic sequences in yeast when no intrachromosomal donor template is available , binds extensively to the resected 3′ ssDNA overhangs to create RecA-ssDNA filaments, which can extend many kilobases (reviewed in E. coli, is limited to a small volume of the cell (cis. Indeed, when the intrachromosomal donor template is less diverged, interhomolog HR occurs <1% of the time.Recent research using single-molecule imaging has revealed a significant amount about the biochemistry of the RecA protein, a necessary component of the prokaryotic HR repair machinery. RecA, and its eukaryotic homolog Rad51 (the cell . These sDrosophila demonstrated that up to 39% of Cas9-mediated DSBs result in recombination between homologous chromosome arms (These results highlight a broader implication in genome editing. As Cas9-mediated genome editing technology becomes more widespread, the exact mechanisms underlying endonuclease-mediated DSB repair will need to be explained to ensure efficient and predictable repair outcomes (reviewed in


The embryonic cerebrospinal fluid (e-CSF) contains various growth factors and morphogens. Recentstudies showed that e-CSF plays significant roles in embryonic brain development. Adipose tissue-derived stemcells (ADSCs) have a mesodermal origin that can be differentiated into mesodermal and ectodermal lineages.This study aimed to evaluate the effects of e-CSF on the proliferation, viability, and neural differentiation ofADSCs in rats.in vitro conditions, ADSCs were cultured in DMEM supplementedwith and without additional 10% e-CSF. These fluids were collected from Wistar rats at the E17, E18, and E19gestational ages. Cellular proliferation and viability were determined using the MTT assay. Immunocytochemistry wasused to study the expression of β-III tubulin in ADSCs. The neurite outgrowth of cultured cells was assessed using theImageJ software. In this experimental study, adipose tissue was dissected out from the inguinal region of adultmale rats. Then, ADSCs were isolated by enzymatic digestion from adipose tissues and mesenchymal cells wereconfirmed using the flow cytometry analysis that measured the cell surface markers including CD90, CD44, CD73,CD105, CD34, CD45, and CD11b. The multi-potential characteristics of ADSCs were assessed by osteogenic andadipogenic potentials of these cells. Under suitable  The results of the present study demonstrated that the viability of ADSCs in cell culture conditioned with E17and E18 e-CSF were significantly increased in comparison with controls. Cultured cells treated with e-CSF from E18and E19 established neuronal-like cells bearing long process, whereas no process was observed in the control groupsor cultured cells treated with E17 e-CSF.This study showed that e-CSF has the ability to induce neuronal differentiation and viability in ADSCs.Our data support a significant role of e-CSF as a therapeutic strategy for the treatment of neurodegenerative diseases. Cerebrospinal fluid (CSF) is a clear and colorlessfluid, secreted mainly (about two-third of its volume)from the epithelial structure in the choroid plexus,and it could also be released from other regionsin the brain such as capillaries surrounded byastrocytes, ependymal epithelium of the ventricles,and subarachnoid plexus . The CSFPrevious studies have shown that embryoniccerebrospinal fluid (e-CSF) is a rich source of proteins,which are involved in the proliferation, differentiation,and migration of neural progenitor cells during braindevelopment. E-CSF affects the neuroepithelial cellsby regulating the proliferation, differentiation, andsurvival of these types of cells. Similar to CSF, e-CSFis a cocktail of various growth and morphogenesisfactors , 5.Adult stem cells are characterized by self-renewal ability,long-time survival, and multipotency . CompareDue to easy accessibility, mesenchymal stem cells(MSCs)-commonly obtained from the bone marrow - area new cell resource for clinical practice and research .HoweverAdipose tissue-derived stem cells (ADSCs) have highproliferation potential that can be differentiated into avariety of mesenchymal cell lineages such as osteoblastsand adipocytes. They also have regenerative propertiesand potency to differentiate into nerve and Schwanncells , 12. As In this experimental study, 22 male and 56 pregnantfemale Wistar rats were used. The animals were kept inan animal house located in the Department of Biologyat the Kharazmi University. They were kept in large ratboxes with free access to food and water under a 12:12light/dark cycle. All animals were treated according tothe guidelines set by the Kharazmi University based onthe National Institutes of Health (NIH) Guidelines forthe Care and Use of Laboratory Animals (C: 616/919).Individual male and female rats were mated andchecked daily for the vaginal plug presence, designatedas embryonic day 0 (E0). The embryonic age wascalculated from E0. At specific times, pregnant ratswere euthanized with urethane , and the uterus was quickly evacuated intoan icebox. Then, litters were immediately separatedfrom pregnant Wistar rats. Each pregnant rat deliveredseparated from litters in each delivery.To evaluate e-CSF effects on ADSCs cultures, e-CSFwas collected from cisterna magna of rat embryoson day 17 (E17), 18 (E18), and 19 (E19) using a glassmicropipette. Because of large fluid space, the cisternamagna is an ideal site for extracting uncontaminatede-CSF. Besides, the cisterna magna is exposed upondissecting the skin and the removal of the overlyingmuscles. Due to the lack of bone formation in this areaand its flexibility, the risk of blood contamination in CSFwould be decreased. All samples were collected in sterilemicrotubes and centrifuged immediately at 4000 rpmfor 5 minutes to separate the fluid from cellular debris.Afterward, the supernatant was transferred and preservedin a new sterile tube. The samples were stored at -40˚Cuntil the subsequent use. About 10 to 50 μl of e-CSF wascollected from each litter. All steps mentioned above werecarried out on an ice box to avoid denaturation of e-CSFproteins .2. The culture media were renewedevery three days  were anesthetized withurethane (1.5 g/kg i.p.). Under sterile conditions, allparts of white adipose tissue samples were isolatedfrom the abdominal inguinal region and transferred intophosphate-buffered saline . Adiposetissues were minced to pieces in sizes between 1 and2 mm3 using scissors and then washed repeatedly withequal volumes of PBS to remove blood cells. Next,each piece was transferred into a falcon tube containing0.075% collagenase type I enzyme . Tubes were placed on a shaker incubator at 37˚Cfor 30 minutes. After that, samples were centrifugedat 2000 rpm for 5 minutes. Undigested pieces wereremoved, and the remaining suspension containing stemcells were collected. Extracted ADSCs were cultured inDulbecco’s Modified Eagle Medium  containing 10% fetal bovine serum and 1% penicillin/streptomycin. Thecultures were kept in a humidified atmosphere incubatorat 37˚C with 5% COree days . In this4 cells/well), treated with e-CSF  (10% v/v), and β-ME  for six days. The wells without anytreatment were considered as controls. Cell survivaland viability were measured by the MTT assay. MTT supplemented with 10%FBS, 0.1 μM dexamethasone, 10 μM β-glycerophosphate,and 50 μM ascorbate. To assess mineralization, cultureswere stained with 2% Alizarin Red . Adipogenic inducing culture media containedDMEM-LG, supplemented with 10% FBS, 0.5 mMIBMX (3-isobutyl-1-methylxanthine), 10 mg/ml insulin,1 mM dexamethasone, and 100 mM indomethacin. Cellswere stained with Oil Red  for thedetection of adipocytes .After second consecutive passages, to measure thecell surface markers of MSCs and to confirm theirdevelopment, we used the following conjugatedantibodies: PE Mouse Anti-Rat CD44, PE Mouse Anti-Rat CD73, PE Mouse Anti-Rat CD105, FITC MouseAnti-Rat CD90, PE Mouse Anti-Rat CD34, PE MouseAnti-Rat CD45, PE Mouse Anti-Rat CD11b, and isotypecontrol antibody. After trypsinization of the cells with0.25% trypsin/ ethylenediaminetetraacetic acid (EDTA)solution, they were re-suspended in PBS and countedusing hemocytometer. A number of 1×106 cells wereincubated in fluorochrome-conjugated antibody at adilution ratio of 1:10 at room temperature for 20 minutesin the dark place. The stained ADSCs were analyzed usinga BD FACSCaliber™ flow cytometer  and the FlowJo software (version 10.4). A total of200,000 cells were assessed in each sample.In order to evaluate the morphology and growthrate of ADSCs, cells were imaged using an invertedmicroscope  and the obtained imageswere analyzed by the ImageJ software (NIH). Themorphological properties of the cells, as well as thelength of neurite-like processes, were comparedamong cell treatments.In this study, β-III tubulin was considered a neuraldifferentiation marker. After three times washing withPBS, cells were fixed with cold 4% paraformaldehydefor 15 minutes. Then, 0.1% Triton X-100  was employed for the increase of cellpermeability at room temperature for 30 minutes. Thecells were blocked with 1% BSA  in T-PBS (Tween 20 in PBS)  at room temperature for 1 hour. After that, thecells were incubated at 4˚C overnight in the presenceof anti-β III tubulin  as aprimary antibody. The next day, cells were rinsed threetimes with T-PBS and incubated with a Cy3-conjugatedsecondary antibody at room temperature for 1 hour. Finally, cells werewashed with PBS, and nuclear staining was carried outwith 4’,6-Diamidino-2-Phenylindole . Photomicrography was done under afluorescent microscope .Ordinary one-way analysis of variance (one-wayANOVA) was applied for the statistical analysis, followedby Tukey’s post hoc test to compare multiple groups. Dataare expressed as the mean and standard error of the mean(mean ± SEM). The P<0.05 was considered statisticallysignificant. Data were analyzed using the the IBM SPSSStatistics for Windows, Version 24.0. Armonk, NY: IBMCorp. (Released 2016).Cultured cells were adherent and had spindle-shapedmorphology. Besides, cultured ADSCs were pluripotentand differentiated into osteoblasts and adipocytesin specific conditioned media. Alizarin red and OilRed staining were carried out for the confirmation ofADSCs differentiation into osteoblasts and adipocytes.Cell surface markers were utilized on the secondpassage to characterize ADSCs population. The flowcytometry analysis of cell surface markers showed thatthese cells expressed CD90, CD44, CD73, and CD105as MSCs markers, whereas they did not express CD34,CD45, and CD11b markers .After six days of treatment, viability assessment wasimplemented in all cultured groups. As shown in Figure2, the MTT absorbance was increased significantly incultured cells conditioned with CSF (10% v/v) fromE17, E18, and E19. However, the MTT absorbance wasdecreased after the cells were treated with β-ME (positivecontrol) compared with the control group. The highestviability was observed at E17 and E18 in e-CSF treatment.Photomicrographs of the cultured cells are shown in Figure3. Neurite outgrowth and morphological differentiation wereobserved in treatment groups with β-M  and e-CSFor neural induction, ADSCs were incubated whitβ-ME  as the positivecontrol. As shown in Figure 4, the majority of ADSCsin this condition have a high level of β III-tubulinexpression. Moreover, the number of cells werepositive for β III-tubulin were significantly increasedin cultured cells conditioned with CSF at E17, E18,and E19. However, no changes were observed in theexpression of β III-tubulin in ADSCs of the controlgroup .Considering the benefits of the ADSCs in comparisonwith other sources of MSCs, e.g. being less invasivecompared to lipo-aspiration, having less ethical issues, 18, proOur flow cytometry data showed that CD90, CD44,CD73, and CD105 as MSCs-specific markers wereexpressed by ADSCs, whereas CD34, CD45, andCD11b were not, demonstrating the mesenchymal originof ADSCs. ADSCs also share some morphologicalcharacteristics with MSCs, such as being spindle-shapedand possessing fibroblast-like properties . MSCs arThe CSF contains various biological factors such asneuropeptides and neurotransmitter, possessing differentconcentrations during various gestational ages. Severalstudies also showed that adult CSF isolated from humanand rat could stimulate the proliferation and viability ofneural stem cells.Additionally, e-CSF comprises of several growthfactors including TGF-β, NGF, BDNF, NT-3, and IGF, 25. In Dual effects of e-CSF on ADSCs proliferation anddifferentiation are probably caused by various growthfactors, their concentration changes in differentembryonic stages, and their interaction in each step.Several reports demonstrated that e-CSF collected fromdifferent embryonic days of the rat brain  has different effects on neuronalprogenitors derived from the embryonic brain of rats.By E16 and E18, these effects have stimulatory roles onthe proliferation and survival of neuronal progenitors.It has been implicated that e-CSF extracted at E20 andE19 has a significant impact on the differentiation ,28, 29.Previous studies indicated that e-CSF isolated at E18and E19 might differentiate cells into neuronal cells ,28. TheThe mechanisms by which the e-CSF contents arealtered in different time points is probably related to thedevelopmental changes in the CSF-brain barrier. It hasbeen reported that the permeability of CSF-brain barrierchanges in an age-dependent manner during embryonicand adult life. The changes in permeability are due to thedifferential distribution of junctional proteins in the CSFbrainbarrier. Therefore, alterations in permeability couldbe a source of e-CSF composition changes in differentgestational periods .In this study, there were limitations on e-CSF extractionfrom the cisterna magna region of rats’ embryo. Due to thelow volume of e-CSF obtained from this area, we suggestthe extraction of e-CSF could be performed on animalmodels since they provide a high amount of e-CSF forthe experiment. Also, we propose the application of othertechniques such as real-time PCR for the precise evaluationof the expression of genes involved in differentiation andproliferation of ADSCs exposed to e-CSF.Previous studies have shown that various factors withdifferent concentrations are presented in embryonic CSFand their level varies on different embryonic days. Theresults of this study indicated the changes in the e-CSFcompositions in a time-dependent manner, which had apositive impact on ADSCs survival and differentiation.Considering specific properties of ADSCs, theirdifferentiation in response to exposure to e-CSF may beregarded as a novel therapeutic strategy for the treatmentof neurodegenerative disorders.


Patients with reduced CSF Aβ42 (< 384.6 pg/mL), increased CSF total tau (> 45.0 pg/mL) and reduced caudate [123I]FP-CIT SPECT uptake (< 1.82) had a 65% risk of developing CI at 36-month follow-up.At the 36-month follow-up, 108/262 (41.2%) PD patients had CI as defined by the MoCA, of which 40/108 (37.0%) had neuropsychological test-defined CI. Baseline CSF Aβ42  that enables identification of early PD patients at risk of developing CI. These findings confirm previous reports of low CSF Aβ42, elevated CSF total tau and reduced dopaminergic integrity being associated with cognitive decline in PD.We report a characteristic profile (reduced CSF Aβ42, increased CSF total tau and reduced caudate [ Cognitive impairment (CI) is an increasingly recognised complication of Parkinson’s disease (PD), with significant impact including increased caregiver burden and mortality . Mild coDiscovering meaningful end points that measure progression is one of the greatest challenges in PD therapeutics. The implementation of suboptimal methods in refining prognosis, diagnosis and tracking disease progression continues to hamper patient management and, particularly, clinical research . Thus, vPrevious reports have demonstrated that vascular and Alzheimer-type (amyloid-β plaques and tau neurofibrillary tangles) pathologies co-exist in PD, and may contribute to the development of CI in PD . In PD, 18F]DOPA uptake and performance in visual and verbal memory [18F]DOPA uptake and impairment in verbal fluency, working memory and attentional functioning FP-CIT single photon emission computed tomography (SPECT) imaging and CSF molecular measures in a group of patients, who at baseline, had early de novo PD.In this study, we hypothesised that molecular markers of PD pathology may have value for predicting cognitive decline and we sought to investigate this by using longitudinal clinical data from the Parkinson’s Progression Markers Initiative (PPMI) and baseline FP-CIT SPECT and CSF data, and a complete 36-month follow-up.Data were acquired from the PPMI database (sampling . Of a to123I]FP-CIT SPECT measures were collected (Table n = 108) or at the complete 36-month follow-up visit if they had not developed CI (n = 154).All PD patients underwent an initial screening visit followed by a baseline visit where demographic, CSF and 23IFP-CIT123I]FP-CIT dopamine transporter imaging.Institutional review boards approved the study and written informed consent was obtained from all participants. All PD patients were recruited between 2010 and 2015, had a disease duration of less than 2 years prior to a screening visit, were not taking dopamine replacement therapy and presented with at least two of the following symptoms: bradykinesia, rigidity, and resting tremor, OR the presence of either an asymmetric resting tremor or asymmetric bradykinesia. PD diagnosis was confirmed by presence of dopamine transporter deficit on the [123I]FP-CIT. [123I]FP-CIT SPECT scans were analysed following the imaging technical operations manual (http://ppmi-info.org/). In brief, SPECT image volumes were spatially normalised to an Ioflupane template. The eight most prominent axial slices containing the striatum were summed and a standardised volume of interest (VOI) template was then applied to this image. VOI analyses were performed on the right and left caudate and putamen, employing the occipital region as the reference tissue. Specific binding ratios were calculated as the ratio of the putamen or caudate VOI count density divided by the occipital cortex count density minus one. This measure approximates the binding potential, BPND, when the radioligand is in equilibrium at the target site and has previously been reported with Ioflupane SPECT.SPECT images were obtained 4 ± 0.5 h after administrating an injection of approximately 185 MBq FP-CIT uptake , which causes a selective dopaminergic lesion, deficits in visuospatial and executive functions were noted [18F]DOPA is associated with verbal and visual memory functioning, verbal fluency and, delayed recall [123I]FP-CIT putamen binding and cognitive impairment, which is in line with findings from [18F]fluorodopa and deficits in neuropsychiatric assessments. This highlights that correct identification of PD patients at risk of CI should include assessment of [123I]FP-CIT uptake in the caudate.We show that AV-1451 [18F]AV-1451 binding in the inferior temporal gyrus and precuneus, which was associated with increased cognitive impairment.The microtubule-associated protein tau gene , cortical/limbic Lewy body pathology and cerebrovascular damage, as well as cortical atrophy and genetic factors. However, PD patient with low caudate FP-CIT SPECT provides valuable information on brain structure and function in the neuropsychiatric aspects of PD. Reduction of [123I]FP-CIT uptake within the putamen is commonly used in clinical practice to assist the diagnosis of PD and here we demonstrated a potential clinical applicability of [123I]FP-CIT uptake within the caudate assisting with disease prognosis, specifically cognitive decline. We show that having low CSF Aβ42, elevated CSF total tau, and low [123I]FP-CIT caudate binding increase the risk of cognitive decline in PD patients by up to 65%. These measures, taken together, may also reflect a characteristic profile of early PD patients most at risk of developing CI over the course of the disease. However, given the restricted number of patients within each subgroup, further studies with larger cohorts of PD patients are required to confirm and validate these exploratory findings.CSF is an accessible source of brain-derived proteins, which reflect molecular changes in the brain and [


RBP4 in cystic follicles. However, the role of RBP4 in mammalian follicular granulosa cells (GCs) remains largely unknown. To illustrate the molecular pathways associated with the effects of RBP4 on GCs, we used high-throughput sequencing to detect differential gene expression in GCs overexpressing RBP4. A total of 113 differentially expressed genes (DEGs) were identified in RBP4-overexpressing GCs, and they included 71 upregulated and 42 downregulated genes. The differential expressions of the top 10 DEGs were further confirmed by real-time quantitative polymerase chain reaction. Pathway analysis indicated that the DEGs are mostly involved in oxidative phosphorylation, Parkinson’s disease, non-alcoholic fatty liver disease, Huntington’s disease, cardiac muscle contraction, Alzheimer’s disease, fatty acid biosynthesis, AMP-activated protein kinase signaling pathway, and insulin signaling pathway. Genes in these pathways should be useful for future studies on GCs. Altogether, the results of our study establish a framework for understanding the potential functions of RBP4 in porcine GCs.Retinol binding protein 4 (RBP4), mainly secreted by the liver and adipocytes, is a transporter of vitamin A. RBP4 has been shown to be involved in several pathophysiological processes, such as obesity, insulin resistance, and cardiovascular risk. Reports have indicated the high expression levels of  RBP4 expression in adipose tissues is regulated by 17-β-estradiol. RBP4 levels in serum are elevated in polycystic ovary syndrome (PCOS) women with obesity [Retinols  mediate various reproductive processes, such as follicle development, oocyte maturation, early embryogenesis, and steroidogenesis ,2,3,4. R obesity . RBP4 ca obesity .RBP4 in reproduction have been supported by accumulating studies. A recent study identified a new single nucleotide polymorphism in RBP4 affecting the production of large litter size of sows [RBP4 expresses in bovine ovarian cells [RBP4 is expressed during the period of pig blastocyst [RBP4 plays important roles in ovarian, uterine, and conceptus physiology.The roles of  of sows . Studiesan cells , early van cells , and ovaan cells . In addiastocyst , in the astocyst . RBP4 haastocyst . Researcastocyst . These eRBP4 and retinols in the fluids of large follicles are higher than those in the fluids of medium or small follicles [RBP4 can also be detected in ovarian GCs of various species, including pigs [RBP4 is highly expressed in follicular fluids of large porcine follicles. RBP4 can also increase the expressions of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in GCs [RBP4 might be involved in normal ovarian function. However, current available information on the function and regulatory mechanism of RBP4 in GCs is limited. This study aimed to analyze the function of the porcine RBP4 gene using gene overexpression and high-throughput sequencing.Ovarian granulosa cells (GCs) are important somatic cells and play a central role in steroidogenesis, which is critical for female reproduction. The growth and development of GCs are regulated by a complex cell signaling system, which significantly influences follicular development and oocyte maturation. Factors regulating functions of GCs may therefore play important roles in follicular health and female reproduction . The levollicles . The exping pigs , mice [2ing pigs , and cowing pigs . Furthering pigs . A recen) in GCs . These dAll experiments in the present study were performed in accordance with the guidelines of the Animal Care and Use Committee of Jilin University Changchun, China .2 at 37 °CUnless otherwise specified, all the chemicals and reagents were purchased from Takara . The 293T cell line , which were used to generate lentivirus, were cultured in Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM/ F12)  supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin–streptomycin under humidified atmosphere containing 5% COg for 5 min and washed thrice with phosphate buffer saline (PBS). The viability of cells before cell culture was assessed by trypan blue exclusion assay. The GCs were seeded at an initial density of 1 × 106 cells/mL in six-well plates. The culture medium consisted of DMEM/F12 with 10% FBS and 1% penicillin–streptomycin. The cells were incubated under humidified atmosphere containing 5% CO2 at 37 °C for 24 h and then washed with PBS to remove any unattached cells.The current study followed the culture procedure of a previous method . LandracRBP4 amplification containing XhoI and NotI restriction enzyme sites in the forward and reverse primers included 5′-TTC CTC GAG ACT ATG GAA TGG GTT TGG GCG CTC GTG CT-3′ and 5′-CCG CGG CCG CTC CTA CAA AAT GTT TCT TTC CGA TTT GC-3′. Full-length porcine RBP4 complementary DNA  was amplified and inserted into the XhoI/NotI sites of the pLVX-IRES-ZsGreen lentiviral vector. RBP4 polymerase chain reaction (PCR) products and pLVX-IRES-ZsGreen plasmid were excised by digesting with XhoI and NotI respectively. Porcine RBP4 gene was inserted into the pLVX-IRES-ZsGreen lentiviral vector to generate the pLVX-RBP4-IRES-ZsGreen construct. Approximately 24 h before transduction, 293T cells were seeded into 100 mm plates at 4 × 106 cells/plate and 10 mL growth medium and then incubated at 37 °C, 5% CO2 overnight. Then, the cells were further incubated until 80–90% confluency. Four packaged systems of plasmids  included pLVX-IRES-ZsGreen vector, structure plasmid PLP1, PLP2, and envelope plasmid VSVG. FuGENE HD reagent , following the manufacturer’s instructions, was used to transfect the 293T cells by mixing vector plasmid pLVX-RBP4-IRES-ZsGreen, structure plasmid PLP1, PLP2, and envelope plasmid VSVG at a mass ratio of 4:2:1:1. The cells with blank pLVX-IRES-ZsGreen (vector) transfection were used as negative controls. After 48 and 72 h, viral particles from the culture supernatants were collected by ultracentrifugation at 4 °C, 72,000× g for 2.5 h. A limiting dilution assay established that the titer of the virus detected by the green fluorescent protein (GFP)-labeled method was 1.2 × 107  transduction units (TU)/mL according to previously reported protocol [RBP4-IRES-ZsGreen (pLVX-RBP4) and pLVX-IRES-ZsGreen (CTRL) groups. Each group was performed in biological triplicate. The lentiviral particles were added to the GCs at a multiplicity of infection of 50. The GCs were infected with lentiviral particles for 72 h, and the medium with lentiviral particles was replaced every 24 h. After 72 h of incubation, enhanced GFP (EGFP) was observed under a fluorescence microscope. RBP4 expression was quantified by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting analysis.The primer sequences used for protocol . GCs werRBP4 and CTRL groups with TRIZOL reagent  was extracted according to manufacturer’s protocol. RNA concentration and purification were assessed using a NanoDrop 2000 spectrophotometer ; the number of OD260/OD280 must be higher than 1.8. RNA integrity was detected by agarose gel electrophoresis. Total RNA was reverse-transcribed into cDNA by using PrimeScript 1st strand cDNA Synthesis Kit (Takara). The total volume of 20 µL RT-PCR reaction mixture contained 10 µL SYBR® Green Real-time PCR Master Mix (Takara), 2 µL cDNA, 7 µL ddH2O, and 0.5 µM each of forward and reverse primers. The program used for all genes consisted of a denaturing cycle of 120 s at 95 °C, 40 cycles of PCR , a melting cycle consisting of 95 °C for 30 s, 72 °C for 5 min, and a step cycle starting at 65 °C with a 0.2 °C/s transition rate to 95 °C. RT-qPCR analysis was conducted on an Mx3000P system  with SYBR-Green detection according to manufacturer’s instructions. The expression level of GAPDH was used as endogenous control. Relative expression was calculated using the 2−ΔΔCt method. The primer sequences are listed in Total RNA of GCs from three independent biological repetitions of the pLVX-Extraction of proteins from GCs and subsequent quantification were performed as previously described . Equal aRBP4 group and three RNA-seq libraries for CTRL group) were generated using an NEBNext Ultra RNA Library Prep Kit for Illumina  according to the manufacturer’s recommendations. A total amount of 1 µg RNA per sample was submitted to HiSeqTM 2500  for cDNA library preparation. The library was sequenced by pair-end sequencing, and 125 bp paired end reads were generated. Primary sequencing data produced using the Illumina HiSeqTM 2500 system were considered raw reads. Raw sequencing data generally featured clean reads, reads with a sequencing primer, tags containing N, and adapter sequences. Raw reads were filtered into clean reads by FAST-QC [Six RNA-sequencing (RNA-seq) libraries (three RNA-seq libraries for pLVX- FAST-QC . http://biit.cs.ut.ee/gprofiler/). Gene expression levels were calculated by fragments per kilo bases per million mapped reads (FPKM). The screening of DEGs used Cuffdiff command of Cufflinks Software. The criteria filtering DEGs were as follows: (1) p-value < 0.05 and q-value  < 0.05; (2) |fold change| ≥ 2 or ≤1 in both groups. The criteria for screening the top 10 genes are based on fold change values.TopHat2 software was used for aligning the clean reads to the current pig reference genome (Sscrofa10.2) . The unihttp://www.genome.jp/kegg) was used to identify the metabolic pathways or signal transduction pathways that were significantly enriched with DEGs by comparing them to the entire genome background . The accession record of GEO is GSE132962.Go term enrichment analysis was conducted for upregulated and downregulated genes using the R package clusterProfiler . In GO tckground . Fisher’enriched . Proteinenriched . All RNAt-test was performed to analyze statistical differences between means. All values were expressed as mean ± SEM (standard error of the mean). p < 0.05 was considered statistically significant. SPSS version 17.0  was used for statistical analysis. The Student’s RBP4 messenger RNA (mRNA) abundance in the pLVX-RBP4 group  groups of GCs were sequenced. RBP4 group. After quality control, 30.104–35.282 Mb of clean reads were obtained. Furthermore, Q20, Q30, and total mapped ratio were calculated  by high-throughput sequencing, and this finding was confirmed by RT-qPCR. RT-qPCR also confirmed the downregulation of nine genes revealed by high-throughput sequencing. RNA sequencing showed that CCDC33 and CD248 were differentially expressed compared with the CTRL group, but RT-qPCR failed to confirm the data. The results demonstrated that differential expression was validated for 18 of 20 genes , cellular components (CC), and molecular functions (MF). GO terms with To better illustrate their biological functions, we divided the DEGs into two groups: upregulated and downregulated. In the BP analysis, the enriched GO terms for upregulated DEGs converged on protein folding, regulation of interleukin-1 production, and interleukin-1 production. On the other hand, the downregulated DEGs were focused on exocytosis and cell migration . In the All the DEGs were mapped to the KEGG database to further investigate their functions. A total of 28 DEGs were categorized into 16 pathways . The DEGAn integral PPI network a and thrRBP4 affected proliferation, differentiation, and mineralization of MC3T3-E1 cell line in vitro [Granulosa cells are important somatic cells that surround oocytes. These cells are involved in ovarian follicular development in pigs. RBP4 is an approximately 21 KD peptide that acts as a carrier of vitamin A in the plasma. This protein is one of the identified adipokines that are associated with IR, obesity, and type 2 diabetes . RBP4 isin vitro . RBP4 on GCs by transcriptomic analysis following RBP4 overexpression. RNA-based overexpression of RBP4 changed the expression levels of 113 DEGs. In addition, our data revealed the effects of RBP4 of DEGs that are important to pathways in GCs, including oxidative phosphorylation, NAFLD, Alzheimer’s disease, fatty acid biosynthesis, AMPK signaling pathway, and insulin signaling pathway.In the present study, we identified the effects of Oxidative phosphorylation is involved in GC apoptosis and follicular atresia . AbnormaRBP4 in ovarian follicles increased with follicular sizes. GCs treated with increasing FSH or LH promoted the expression of RBP4 [RBP4 and type 2 diabetes mellitus, PCOS, and ovarian cancer have been originated from population-based prospective studies. However, in present study, some attractive DEGs related to reproduction and cell cycle were detected, such as MMP1, IGF signaling related genes , HSPB1, and cell-cycle gene (CDK1).The significant role of RBP4 is the transport of retinol from the liver to peripheral tissues. A recent study found that RBP4 was located in the granulosa and inner theca cell layers and the expression level of  of RBP4 . HoweverRBP4 knockdown decreased cell invasion, whereas RBP4 overexpression increased the invasion of HTR8/SVneo cells. Moreover, the production of MMPs increased with the increase in RBP4 expression [RBP4-stimulated expression of MMP1 in the present study supports the above notion. Furthermore, our results confirmed for the first time that IGFBP5, IGFBP7, and IGFALS were detected as DEGs in GCs overexpressing RBP4. Among these DEGs, IGFBP5 and IGFALS were downregulated, and IGFBP7 was upregulated. Interestingly, IGFBP5 and IGFALS were downregulated simultaneously, as IGF1, IGFBP5, and IGFALS function in a form of ternary complex for IGF signaling, which has been shown to play important roles in a variety of physiological processes, including protein chaperoning, steroidogenesis, and protection against apoptosis [HSPB1 by RBP4 may have significant consequences for the production of HSPB1 in GCs. HSPB1 is a protein that appears to antagonize a molecular pathway for stress [CDK1) is a key regulator of the cell cycle and RNA transcription [CDK1 are upregulated in bovine GCs in luteal phase and small healthy follicles [CDK1, which also acts as a hub of PPI, was upregulated in GCs challenged with RBP4. In addition, previous studies have shown that RBP4 could promote cell proliferation. These results suggest that overexpression of RBP4 promoted cell cycle in porcine GCs. Our data indicates that RBP4 plays a potential role in in ovarian folliculogenesis and pathogenesis.Li et al. observed that pression . The RBPpoptosis ,48,49,50r stress , and HSPr stress . Cyclin-cription . The hubollicles . MeanwhiRBP4 by using high-throughput sequencing. In addition, our transcriptomic data support that several genes are involved in important biological processes associated with folliculogenesis and pathogenesis. As such, these findings provide novel insights on the role of RBP4 in porcine GCs.In this study, we compared the differential expression of genes of porcine GCs overexpressing