Oryzias latipes). Specifically, dnd depletion completely ablated host PGCs and fertility, and dnd overexpression remarkably boosted PGCs in donor blastulae. BCT between normal donor and host produced a germline transmission rate of ~4%. This rate was enhanced up to ~30% upon PGC boosting in donors. Most importantly, BCT between PGC-boosted donors and PGC-ablated hosts led to more than 90% fertility restoration and 100% GR. Therefore, BCT features an extremely high efficiency of fertility recovery and GR in medaka. This finding makes medaka an ideal model to analyze genetic and physiological donor-host compatibilities for BCT-mediated surrogate production and propagation of endangered lower vertebrates and biodiversity.Primordial germ cell (PGC) specification early in development establishes the germline for reproduction and reproductive technologies. Germline replacement (GR) is a powerful tool for conservation of valuable or endangered animals. GR is achievable by germ cell transplantation into the PGC migration pathway or gonads. Blastula cell transplantation (BCT) can also lead to the chimeric germline containing PGCs of both donor and host origins. It has remained largely unknown whether BCT is able to achieve GR at a high efficiency. Here we report efficient GR by BCT into blastula embryos in the fish medaka ( Multiple GCT procedures have been developed with respect to germ cell origin, source and treatment, donor-host combination, developmental stage, place and treatment of host. Yoshizaki and his colleagues have pioneered GCT in salmonids by using normal or triploid embryonic gonads as hosts and reported several important scientific advances and innovative procedures for GR in surrogate production28916Oryzias latipes) is an excellent lower vertebrate model to analyze and manipulate embryonic development222527dnd whose RNA forms particles and asymmetrically segregates into 8 blastomeres of 32–64 cleavage embryos. Altering the level of dnd expression can greatly alter the PGC number, ranging from remarkable increase by nearly 3-folds to complete absence of PGC specification2930The laboratory fish medaka  and RFP-labeled liver (Lr for liver red). BGR is 100% phenotypically pigmented and genetically heterozygous (50%) for wild-type melanophores, and 66.6% positive for GFP-labeled PGCs or RFP-labeled liver but 33.3% for Vg or Lr transgenes according to the breeding scheme . Embryo dnd RNA injection at the 1-cell stage did not alter the percentage values of phenotypic traits. Thus, the PGC proportion in donor embryos can greatly be increased by dnd overexpression.In GCT, the majority if not all of donor germ cells are capable of contributing to the host germline. In BCT, only a minority of donor cells have this capability. In medaka, for example, a blastula embryo has approximately 1000 cells including 33 PGCs122i3 as the BCT host. This strain displays albinism and does not contain any transgene. The host was treated in two ways to favor germline chimera formation. Previously we have shown that γ-irradiation can compromise hosts to increase donor cell contribution into the liver and gonaddnd depletion can completely prevent PGC specificationdnd mRNA translationWe used medaka strain dnd-overexpressing donor and normal host, class III between dnd-overexpressing donor and dnd-depleted host, and class IV between dnd-overexpressing donor and γ-irradiated host. In each class, approximately 30 blastula cells were transplanted into the deep cells of a blastula host. It was found that chimeras of classes I-IV were 100% positive for pigmented melanophores of donor origin  significantly increased the chimeras frequency and chimerism degree to 81.2% and 2.04 for Vg, the values become 92.4% and 2.66 upon extrapolation to total PGCs . The majority of classes II-IV chimeras indeed contained 1~3 VgPGCs in the migratory route at earlier stages. Notably, many such chimeras had 2~5 VgPGCs in the gonad, as shown for representatives from class-III chimeras ,a’ and cIn order to evaluate germline transmission, donor fish and BCT-derived chimeras were grown into adulthood and tested for their fertility by breeding as described above for the host. Chimeras of classes I and II from BCT into normal hosts exhibited fertility rates of 100% and 98.4% fully comparable to 98.3 for non-transplanted hosts . Notablyi3, and resultant embryos and fry were observed for pigmentation, liver and gonad. Among 8 donor fish, 8, 6 and 5 were found to be transmitters of pigmentation, Vg and Lr. They produced 494 F1 progeny embryos, of which 48.6%, 33.2% and 33.6% were positive for black pigmentation, Vg and Lr  for donor blastula cell preparation, whereas biopsies may be taken to enable germ cell preparation and propagation into a large for surrogate production of numerous female and male progeny via GCT. Consequently, surrogate progeny from a single super animal may establish a self-mating population to further increase the individual number by normal sexual reproduction. Since the progeny could originate from a single genotype, the self-mating system will minimize genetic variations arising from meiotic segregation and assortment and thus maximize the probability to reconstitute the genotype and phenotype of a super animal.dnd depletion ensures GR-dependent fertility restoration and thus a pure donor origin of progeny from BCT chimeras. As for diversity, donor blastula cells may be collected from hundreds and thousands of PGC-boosted blastulae and thus rich in genetic diversity. BCT may introduce PGCs from different donor embryos into the chimeric germline. Consequently, PGCs in different BCT chimeras and even one and the same chimera may differ in individuality and thus genotype, which makes an apparent sense in terms of effective population size (Ne), a parameter essential for biodiversity conservation and management. In practice, a Ne of 100 is considered as the minimal value necessary for biodiversity restoration and maintenance. If a population has 50 males and 50 females, then Ne is 100 [(4 × 50 × 50)/(50 + 50)] according to the equation Ne = (4NmNf)/(Nm + Nf), where Ne is the effective population size; Nm is the number of males; and Nf is the number of females. In this study we have shown that medaka BCT chimeras often have ~2.5 PGCs of different origins and thus genotypes. Consequently, ~20 male and ~20 female BCT chimeras will approach this minimal Ne value. Blastula cells are easy for dissociation and testing for viability by observation of pseudopodial formation and cell division and do not need to characterize the cell identity as they either contain or produce PGCs in diverse animals1453435BCT is, on the other hand, of choice particularly for biodiversity conservation via restoration and propagation of endangered species. There are three key factors essential for surrogate production of biodiversity, namely donor-host immunocompatibility, purity and diversity of progeny animals. BCT perfectly fulfil these requirements. There is no immune rejection in developing fish embryos as has been well-demonstrated by the production of intra-species chimeras1718213537Several fundamental questions remain to be answered before surrogate production can be used in conservation practice. These include donor-host phylogeny and developmental-physiological compatibility in gametogenesis and fertilization. Our finding that BCT can achieve a high efficiency of fertility restoration and GR makes medaka an ideal model organism for the experimental analysis of these biological parameters towards BCT-mediated surrogate production and propagation of endangered lower vertebrates and biodiversity.i3, HdrR and transgenic lines (see below) were maintained under an artificial photoperiod of 14-h light to 10-h darkness at 26 °C as describedChemicals were purchased from Sigma, enzymes were from New England Biolabs, and PCR reagents were from TaKaRa unless otherwise indicated. Fish work was performed in strict compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Advisory Committee for Laboratory Animal Research in Singapore and approved by this committee (Permit Number: 27/09). Medaka strains HB32C, MOdnd and pCSdnd:chDD were describedMedaka embryos were injected at the 1-cell stage with 20~50 pg of MOdnd or 100 pg of capped dnd:ch mRNA synthesized from pCSdnd:chDD as described122i3 were used as the transplantation host. The transplantation donor was generated in multiple steps as follows. HB32C is a wild-type pigmentation strainet al., 2001 that expresses GFP from the medaka Vasa promoter specifically in germ cellsi3 albino background. F1 fish positive for Vg and Lr were mated to produce F2 generation. F2 embryos positive for Vg and Lr were grown to adults designated as GL fish that were homozygous (1/3) or heterozygous (2/3) for Vg and Lr transgenes and thus 66.6% for Vg and Lr phenotypes. Finally, GL females were crossed with HB32C male, generating BGL embryos that were used as the transplantation donor. BGR is heterozygous (50%) to black melanophores responsible for wild-type pigmentation and 33.3% for Vg and Lr transgenes and phenotypes. In some experiments, donor embryos at the 1-cell stage were injected with 100 pg of dnd:ch RNA to increase the PGC number, and host embryos also at the 1-cell stage were injected with 20–100 ng of MOdnd to ablate PGCs or at the 4- to 8-cell stages were subjected to γ-irradiation at 6 gyPreparation of donor blastula cells, dechorionated host MBEs and cell transplantation were performed essentially as described2122i3 embryos were first maintained on their own for 1 month to observe egg production. They were then maintained for 2 weeks in the presence of fertile HdrR males and another 2 weeks in the presence of fertile HdrR females. Adult chimeras were examined similarly by using fertile HdrR males and fertile HdrR females as the testers. Fertile females were proven by egg-laying. Fertile males were proven by the ability to fertilize eggs in pair-wise mating.Adult fish from MOdnd-injected i3 albino medaka. Progeny were phenotypically analyzed for the donor contribution by pigmentation, GFP and RFP expression.Fertile male and female chimeras were individually test-crossed with The liver, ovary and testis of progeny-tested chimeras were dissected and examined for donor cell derivatives by GFP and RFP expression.Observation and photography on Leica MZFIII stereo microscope, Zeiss Axiovertinvert and Axiovert upright microscopes with a Zeiss AxioCam M5Rc digital camera (Zeiss Corp) were as described previously2128t-test. Genetic data were evaluated by using chi-square test.Statistical analyses were calculated by using Graphad Prism v4.0. Data consolidated were presented as mean ± s.d. and p values were calculated by using non-parametric student’s How to cite this article: Li, M. et al. Germline replacement by blastula cell transplantation in the fish medaka. Sci. Rep.6, 29658; doi: 10.1038/srep29658 (2016).


Pichia pastoris. The molecular mass of HMG was about 85 kDa and the isoelectric point was 5.3. Circular dichroism spectroscopy suggested that mG-CSF retained nearly all of its native secondary structure, regardless of fusion. The binding capabilities of mG-CSF moiety to G-CSF receptor and HSA moiety to warfarin showed very little change after fusing. The bioactivity of HMG (11.0×106 IU/mg) was more than twice that of rHSA/G-CSF (4.6×106 IU/mg). A mutation was made at the 718th amino acid of HMG, substituting Ala for Thr, to investigate the glycosylation of HMG expressed in P. pastoris. Data indicated that HMG was modified at Thr718, speculatively with the addition of a mannose chain. In conclusion, a novel HSA/G-CSF fusion protein was successfully constructed based on a mutated G-CSF. This protein showed more potent bioactivity than rHSA/G-CSF and thus may be a suitable long-acting G-CSF.The purpose of the present work was to develop a novel, long-acting and potent human serum albumin/granulocyte colony stimulating factor (HSA/G-CSF) therapeutic fusion protein. The novel fusion protein, called HMG, was constructed by genetically fusing mutated human derived G-CSF (mG-CSF) to the C-terminal of HSA and then prepared in  HSA  has a molecular weight of 66.5 kDa and exhibits a long half-life of about 19 days in vivoin vivo. In addition, the albumin molecule masks the protein, which renders it more resistant to proteases and less immunogenic. Albiglutide, a GLP-1/albumin fusion protein, is the first albumin fusion based long-acting drug. It received EMEA and FDA approval in early 2014. The approval of Albiglutide may usher in a promising future for the use of albumin fusion technology as a platform for drug half-life extension. Unfortunately, decreases in the bioactivity of therapeutic proteins and peptides after fusion with HSA are frequent due to steric hindrance. Fleer et al. reported that Neugranin, a recombinant human G-CSF (rhG-CSF) genetically fused to human albumin, only retained 14.3% of its bioactivity For therapeutic proteins and peptides that can be cleared from the body quickly by renal filtration, albumin fusion technology is an attractive means of increasing their circulation time in vitro and in vivoP. pastoris and the structural integrity, conformational stability, ligand binding profile, and bioactivity of HMG and its two components (mG-CSF and HSA) were examined. Additionally, we designed a mutation in the HMG  to explore the glycosylation site and status of HMG.For this reason, a different strategy was designed in current study to generate a novel albumin/G-CSF fusion protein based on a highly potent G-CSF mutant. Thr-1, Leu-3, Gly-4, Pro-5, and Cys-17 in the G-CSF mutant were each substituted with Ala, Thr, Tyr, Arg, and Ser, respectively and this G-CSF mutant showed more potent granulopoietic activity than the intact rhG-CSF both P. pastoris as previously described NdeI and EcoRI and prepared as described by Kuga et al.P. pastoris were stimulated by continuous addition of methanol for about 50 h to induce the expression of proteins. Recombinant proteins were collected from the fermentation broth by centrifugation (5000 rpm). Purification of HMG, rHSA/G-CSF, and mHMG was performed as follows: Cibacron Blue sepharose FF chromatography, phenyl sepharose HP chromatography, Sephadex G25 for buffer exchange, SP sepharose FF chromatography, and a final ultrafiltration/diafiltration (30K MWCO). Purification of mG-CSF was accomplished as follows: DEAE sepharose FF chromatography, phenyl sepharose HP chromatography followed by a final ultrafiltration/diafiltration (10K MWCO). Purified proteins were stored frozen in 5 mg/ml buffer consisting of 20 mM sodium phosphate, pH 7.3.Human G-CSF mutant (mG-CSF) was prepared as previously described The fermentation solution and purified HMG fusion proteins from different purification processes were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with 8% acrylamide gel and 5% condensing gel in the Mini-Protein II electrophoresis unit  and stained with 0.25% Coomassie brilliant blue R-250 .IEF was used to predict the isoelectric point (pI) of HMG. In a separate set of experiments, 2 µg of purified HMG, mG-CSF, HSA and a mixture of HSA, mG-CSF and HMG prepared in 20 mM PB  were loaded and analyzed on a Pharmacia MultiphorII horizontal electrophoresis system  using ampholine, pH 3.5–10 .These samples were also analyzed using size exclusion chromatography on a TSK-GEL G3000SW columns (7.5×300 mm)  at a flow rate of 0.6 ml/min in 20 mM sodium phosphate (pH 7.5) and 0.15 M NaCl. The absorbance was monitored at 280 nm.N-terminal amino acid sequencing was performed by Edman degradation with Shimadzu PPSQ-33A automated protein sequencer. C-terminal amino acid sequencing was performed with Micromass QTOF2 Quadrupole/Time-of-Flight Electrospray ionization tandem mass spectrometry (Q-TOF2 ESI-MS/MS).Far and near-UV CD spectra of equimolar mixtures of HSA and mG-CSF  and HMG fusion protein  were recorded on a JASCO J-715 automatic recording spectropolarimeter  from 190–250 nm and 250–300 nm, respectively.Intrinsic fluorescence emission spectra were used to detect possible conformational changes in mG-CSF after fusion with HSA. mG-CSF, HSA, and HMG were prepared using 67 mM phosphate buffer  as follows: a: mG-CSF (15 µM), b: HSA (15 µM) plus mG-CSF (15 µM), and c: HMG (15 µM). The samples (200 µl/well) were pipetted into a 96-well black plate  and PB was added as a negative control. The plate was placed in SpectraMax M5  to examine the changes in intrinsic fluorescence under 25°C. The excitation wavelength was set to 295 nm and emission wavelength was from 320 to 380 nm.The interaction of warfarin and HSA was examined using fluorescence spectroscopy. Human-plasma-derived HSA , HMG and warfarin sodium  were prepared with 0.01 M phosphate buffered saline (pH 7.4) as follows: a: 5 µM HSA, b: 5 µM HSA plus 50 µM warfarin sodium, c: 5 µM HMG and d: 5 µM HMG plus 50 µM warfarin sodium. The samples were then pipetted into a 96-well black plate  and detected using a microplate reader . The excitation wavelength was set to 320 nm and fluorescence intensity was monitored at 380 nm. The experiment was independently repeated three times.Bio-layer interferometry (BLI) was used to detect the binding of G-CSF to G-CSFR under various conditions using a Streptavidin High Binding Biosensor Kit and a Octet-QK system . Biotinylated rhG-CSFR was desalted with Sephadex G25  and eluted in a final concentration of 15 µg/ml. Sample preparation, hydration of the sensors, and kinetic analysis of macromolecular interactions were performed according to the manufacturer's instructions. Then 200 µl of HMG, rhG-CSF standard , and mG-CSF or buffer were transferred into a 96-well plate  and assayed for rapid assessment of kinetics. Data were analyzed using global fitting to determine the association/dissociation rate constants and affinity constant.Periodic acid–Schiff (PAS) staining is used to detect sugar compounds such as polysaccharides, glycoproteins, and glycolipids. The reaction of periodic acid oxidizes the vicinal diols in glycosyl and creates a pair of aldehydes at the two free tips of each broken monosaccharide ring. These aldehydes then react with the Schiff reagent and form purple-magenta compounds. Purified protein samples (5 µg and 20 µg), a positive control (horseradish peroxidase), and a negative control (soybean trypsin inhibitor) were loaded on SDS-PAGE, subjected to electrophoresis, and then separately stained with periodic acid-Schiff reagent  and Coomassie blue R-250.et al.The carbohydrate content of the three batches of purified HMG or emHmG was further determined by phenol-sulfuric acid method based on Dubois Canavalia ensiformis), can specifically bind to certain structures found in various sugars, glycoproteins, and glycolipids, mainly internal and nonreducing terminal α-D-mannosyl and α-D-glucosyl groups. Because of this, it can be used to separate proteins containing certain sugars from those that do not contain them. In a separate experiment, 30 mg quantities of purified HMG fraction and mHMG were prepared in 0.5 mol/l NaCl and then subjected to affinity chromatography with a ConA Sepharose 4B column  which had been previously equilibrated with 0.5 M NaCl in 20 mM Tris-HCl (pH 7.2). Finally, the proteins were eluted using 0.2 mol/l glucoside.Concanavalin A (ConA) affinity chromatography was conducted to further characterize the glycosylation modification of HMG. ConA, a lectin carbohydrate-binding protein originally extracted from jack-bean (An Agilent 6224 Accurate-Mass Time-of-Flight (TOF) LC/MS system equipped with an Agilent 1260 Infinity LC and electrospray ionization (ESI) was used to investigate the possibility of glycosylated isomers and other posttranslational modifications that might occur in HMG. LC column is packed with polymer-based matrix (polystyrene divinylbenzene). The mobile phase was a: 0.1% formic acid in Milli-Q water and b: 0.1% formic acid in acetonitrile. For ESI-TOF, ion spray voltage and gas temperature were set to 3500 V and 325°C, respectively.8 IU/mg of recombinant hG-CSF standard . The cultures were kept at 37°C in a humidified incubator with 5% CO2 for two days and then 10 µl of 2.5 mg/ml MTT was added. Four hours later, 100 µl of 10% SDS in 0.01 M HCl was added to lyse the cells and dissolve the formazan. The plate was then read at 570 nm with a reference wavelength of 630 nm. The reading at OD570 was plotted against the protein concentrations and the concentration at the inflection point of the sigmoidal curve represented the median effective dose (ED50).Bioactivity of the fusion protein was determined using a cell proliferation assay using murine myeloblastic NFS-60 cells. Briefly, the cells were seeded in a 96-well plate and incubated in 50 µl of assay medium containing serial dilutions of either HMG or 0.7×10Protein expression was induced for 50 h using methanol. Concentrations of HMG and mHMG in broth were then about 0.65 g/l and 0.5 g/l respectively, as detected by SDS-PAGE (data not shown). Blue Sepharose Fast Flow was used to capture protein from broth because the albumins in fusion proteins were able to bind to Cibacron Blue specifically. About 204 mg HMG and 142 mg mHMG were purified from 1 L of cell-free broth, equivalent to total protein recovery rates of 31.3% and 28.4%, respectively.HMG was analyzed after purification using SEC-HPLC and SDS-PAGE. Results indicated purity greater than 97% and molecular mass of about 85 kDa . Only feN-terminal sequencing showed the following results: NH2-DAHKSEV AHRFKDLG, which is identical to the N-terminal sequence of HSA. The three amino acids at the C-terminal were SKE-COOH, also identical to the predicted sequence.CD spectroscopy was used to analyze the changes in the secondary structure that might occur in mG-CSF after fusion with HSA. Near- and far-UV CD spectra of HMG and emHmG are shown in Three aromatic amino acid residues  show contributions to the intrinsic fluorescence of the protein. Intrinsic fluorescence is associated with the tertiary structure of each protein and individual peptides and each one exhibits a characteristic and specific emission profile. There are three tryptophans in the peptide chain of HMG (one in mG-CSF and two in HSA). Possible conformational changes in mG-CSF after fusion with HSA were assessed by measuring the intrinsic fluorescence emission spectra of HMG, emHmG, mG-CSF, and HSA. As illustrated in HSA binds to a large number of endogenous and exogenous ligands , affecting their pharmacokinetics, i.e., rate of distribution and elimination to the tissues The kinetics of the binding interaction of G-CSF and G-CSFR was analyzed by using biolayer interferometry. As illustrated in 6 IU/mg) was greater than that of rhG-CSF (80.2×106 IU/mg) in vitro (6 IU/mg). The activity of HMG was more than twice that of rHSA/G-CSF (4.6×106 IU/mg). These data indicated that the novel fusion protein, in which a mutated mG-CSF was genetically fused to HSA, was more potently bioactive than rHSA/G-CSF and thus may be a suitable therapeutic agent.Regarding bioactivity, a cell proliferation assay revealed that the activity of mG-CSF  of HMGand mannose residue.LC/MS was used in further analysis of glycosylation isomers and other potential posttranslational modifications of HMG. A series of peaks with MW greater than 85,195.75 Da , were observed in the mass spectrum at 85,357 Da, 85,519 Da, 85,683 Da, and 85,844 Da. Of these, 85,519 Da indicated the greatest abundance . The posThe MWs of a mannose, mannobiose, manninotriose and mannotetraose molecule are 180.2 Da, 342.3 Da, 604.5 Da, and 666.7 Da, respectively. However, one molecule of water is lost during glycosylation, producing a corresponding MW increase of 162.2 Da, 324.3 Da, 586.5 Da, and 648.7 Da, respectively, when a mannose, mannobiose, manninotriose, or mannotetraose is conjugated to an amino acid residue. This indicates that these peaks at 85357 Da, 85519 Da, 85683 Da, and 85844 Da may correspond to 1-, 2-, 3-, and 4-mannose oligosaccharide chains. The areas of the peaks indicate that most of them may be mannobiose. The results of three batches appeared closely consistent .in vitro cell proliferation assay indicated that HMG did not bind to its target as effectively as mG-CSF did. This may have been because of the steric hindrance effects of HSA, but it remained significantly more potent than rHSA/G-CSF.For a fusion protein, retaining individual moiety in the native conformation is essential. For this reason, the structural and functional characteristics of mG-CSF and HSA moieties in HMG were analyzed using various methods. HMG showed a similar CD pattern with emHmG, suggesting that HSA and mG-CSF moieties retained nearly all of their own conformation after fusion. The binding properties of HSA to warfarin were evaluated after fusion and results suggested that this activity of HSA was hardly affected. The G-CSF receptor binding assay revealed that the kinetic binding constants of HMG were similar to those of mG-CSF  althoughHowever, intrinsic fluorescence measurement failed to provide an unambiguous result, proving that the conformation remained unchanged. The fluorescence spectrum of HMG shifted to a shorter wavelength and the intensity of fluorescence became dramatically less pronounced than that of emHmG and HSA. This may be attributed to the mechanism underlying this form of analysis. The fluorescence spectrum of a folded protein is a mixture of the fluorescence of three aromatic amino acid residues , and tryptophan residues contribute most to the emission. mG-CSF alone emits only weak fluorescence, although it contains two Trp in its chain. This may be because they are not sufficiently exposed on the protein's surface. When fused to albumin, mG-CSF may partially block the emission of Trp in albumin, causing the intrinsic fluorescence emissions of HMG to be weaker than those of HSA. Instead, mG-CSF and HSA existed independently in emHmG. They may not interfere with each other, causing emHmG to exhibit a fluorescence pattern similar to that of albumin alone.P. pastoris can be glycosylated on the Asn site of the Asn-Xaa-Thr/Ser consensus sequence or on Ser/Thr hydroxyl groups to provide N-linked or O-linked saccharides, respectively Heterologous proteins expressed by Pichia-derived HMG require study in further depth. Fortunately, HMG does not contain any N-glycosylation site, because it lacks the Asn-X-Ser/Thr sequence. It has been reported that Pichia-derived rhG-CSF is partially glycosylated with mannose, usually at Thr133 et al. reported that the sugar chains of CHO-derived G-CSF were a mixture of galactose and galactosamine The glycosylation situations of There were some peaks in the mass spectrometric pattern that could not be explained theoretically, but they were not considered a result of phosphorylation. This was because the isoelectric point analysis of HMG showed a single band. We speculated that these peaks may be the results of other modifications, such as oxidization or nitration. Of course, further studies are needed to explore the exact modifications.P. pastoris was glycosylated with mannose at Thr718. However, further studies are needed to explore the possible effect of this glycosylation on the efficiency and pharmacokinetics of HMG.In conclusion, a novel HMG fusion protein was here constructed by genetically fusing a mutated human G-CSF to the C-terminus of HSA. The results demonstrated that both mG-CSF and HSA in the fusion protein nearly remained their native structures and the capability of binding to G-CSF receptor and warfarin, respectively. The novel HMG fusion protein exhibited more potent bioactivity than rHSA/G-CSF, which indicated that it may be useful as a therapeutic agent. A glycosylation assay revealed that HMG expressed in


With a view to addressing the moral concerns about the use of donor siblings, the Policy Statement of the American Academy of Pediatrics - Children as Hematopoietic Stem Cell Donors (the Policy) has laid out the criteria upon which tissue harvest from a minor would be permissible.Although tissue harvest serves the best interests of recipient siblings, parents are also obliged to act in the best interests of the donor sibling in the UK. Tissue harvest should proceed if and only if it serves the best interests of both the donor and recipient. Parents should be forbidden, and they are by UK law, to consent to tissue harvest unless there are substantial benefits for an incompetent minor that can outweigh the potential harm. There is no basis to subject a minor to the medical risks of tissue harvest if the recipient sibling can wait without significant risks of complications until the donor becomes Gillick competent. We also argue that the Policy fails to take into account recent advances in haematopoietic transplantation from haploidentical donors or related tissue-matched donors.Unless a recipient sibling will suffer from serious complications or die without the transplantation and no other medically equivalent donors are available, there is no moral or legal basis to violate the donor sibling’s right to bodily integrity. Accordingly, we propose that the Policy should be modified in order to fully satisfy the legal requirements for application in the UK and other commonwealth jurisdictions with similar statute laws protecting minors. In conjunction with preimplantation genetic diagnosis (PGD), assisted reproduction can be used to avoid inheritable diseases. After successful in-vitro fertilization, PGD can identify whether any of the embryos is affected by a genetic condition. Parents now can even request selection of embryos on the basis of tissue type in order to create a child to cure an existing sick child. In response to the moral concerns about the use of paediatric haematopoietic cell donor siblings (donor siblings), the Policy Statement of the American Academy of Pediatrics – Children as Hematopoietic Stem Cell Donors (the Policy) suggests that tissue harvest should only proceed upon satisfaction of all the following five criteria:1) there is no medically equivalent histo-compatible adult relative who is willing and able to donate;2) there is a strong personal and emotionally positive relationship between the donor and recipient;3) there is some likelihood that the recipient will benefit from the transplantation;4) the clinical, emotional, and psychosocial risks to the donor are minimized and are reasonable in relation to the benefits expected to accrue to the donor and to the recipient; and5) parental permission and, where appropriate, child assent have been obtained’.Except that the fourth criterion requires the physician to minimise the risks of medical procedures by, e.g. avoiding central venous catheter insertion and reducing psychological risk by proper communication skills and the fifth requires that child assent should be obtained where appropriate, the remaining criteria seem to merely state the facts common to most cases of tissue harvest rather than regulating the process. We contend that the Policy fails to sufficiently protect the interests of minor donors. In this article we would like to review the prevailing UK law, discuss the ethical relevance, and propose modifications of the Policy for application in the UK and other commonwealth jurisdictions with similar statute laws.One would not dispute that tissue harvest serves the best interests of recipient siblings. But we are also obliged to look at the welfare of the donor sibling. In Birmingham City Council v H (a minor), Balcombe LJ said the question of welfare of both children should be approached 'without giving one priority over the other’. In the The most ethically sound basis to oppose tissue harvest is that it imposes risks of bodily injuries associated with the medical procedures of tissue harvest. The risks depend on the source of cells namely bone marrow, peripheral blood and umbilical cord blood, each with their own benefits and risks. It is known that harvest by umbilical cord blood carries no medical risks except in case of modified delivery. Therefore our analysis below focuses on bone marrow and peripheral blood transplantation only. Minor complications like pain at collection sites, fatigue and pain may occur in a small proportion of donors (6%-20%). Major and life-threatening complications following marrow donation like anesthesia-related events, injuries to bones, sacroiliac joint and sciatic nerve were estimated to occur in 0.1%-0.3% of cases. Major rThe possible benefits for donor siblings are merely psychosocial. In an American case Curran v. Bosze, Calvo J said that 'the psychological benefit is grounded firmly in the fact that the donor and the recipient are known to each other in a family. There may be a psychological benefit to the child from donating bone marrow to a sibling’. We consLong term positive effects for the donor siblings include greater sensitivity to the needs of others, enhanceWhereas the advantages of tissue harvest for donor siblings are long-term and merely psychosocial, the other side of balance sheet consists of short-term psychological burden and life-threatening physical harm. One should bear in mind that the notion of benefits and risks are both relative concepts. To consider the potential gains or losses for donor siblings, we ought to consider what would likely happen to the donors and recipients without tissue harvest. It cannot be said that tissue harvest can offer any significant long-term benefits for the donor sibling if other available source of transplant can offer a similar chance of success or if the sick sibling has a high chance of survival without serious complications even without transplantation. Those who do not agree may argue that a donor sibling may in the future relish and take pride in being a donor and therefore still want to donate even if other donors are available.Parents may legitimately consider this factor when making decisions for incompetent minors. However, the sense of pride may not materialize if the grown-up donor sibling does not accept the filial or social responsibility to save the recipient sibling, is disinterested in altruistic donation, or does not relish in living in the shadows of the recipient sibling. It is too speculative to suggest that an autonomous individual would necessarily prize the honour of 'a donor and saviour’ above all things if there are other suitable donors willing to donate or if the recipient is not in the most dire need of transplantation. As the Policy permits transplantation only if there is no medically equivalent histo-compatible adult relative who is willing and able to donate, it is beyond dispute that a moral agent should not subject a minor to the risks of tissue harvest if there are other equally good alternatives. It implies that the sense of pride alone is not an overriding factor in assessing the benefits and for a minor donor. In short, the only circumstance where tissue harvest would likely serve the best interests of a minor donor is that without the proposed transplantation taking place, the sick sibling will have a high chance of dying or suffering from serious complications.We hold that the above legal approach is in line with the Beauchamp and Childress’ principled-based approach of ethical analysis. As one In response, one may argue that the requirement that a sick sibling will suffer serious complications or die without the proposed transplantation is too high a bar. However, as we have illustrated in the legal analysis above, unless this requirement is satisfied, the donor sibling is unlikely to benefit from the tissue harvest. Month said 'I do not believe that a day of minor discomfort and an extremely small risk of anesthesia outweigh a lifetime without a healthy older brother or sister and the years of joy it potentially brings’. It is tOne may then question the validity of the claim that transplantation can proceed only if it also serves the best interests of the donor sibling. Beauchamp and Childress gave little guidance regarding the approach of resolving conflicts of interests between two persons. But it is generally accepted that when there are conflicts between two principles, we should first minimise the effects of violation. As the UK court has previously held, pursuant to prevailing family law principles, it was bound to make the least detrimental choice.Transplantation will not benefit the donor sibling, as we contend above, when there is another source of medically equivalent tissue from a mentally competent adult or when the sick sibling will not die or suffer from serious complications without the transplantation. In the former case, to minimize the effect of violation of autonomy, we should accept donation from a competent adult who agrees to donate rather than harvest tissue from a minor who cannot consent. In the latter, as the sick sibling can still survive in an acceptably healthy condition even without transplantation, the least detrimental choice is to wait until the donor can understand the process of transplantation (i.e. Gillick competent) and decide whether he would like to agree to altruistic donation.Lastly, one may argue that there is a special duty to rescue a sick sibling as a result of a family relationship. Dwyer and Vig held that a donation is justifiable even if it does not benefit the donor sibling, so long as there is 'a moral match between the relationship and the risks to the donor relative to the benefits to the recipient’. But putWe submit that it is only fair to impose an obligation to a member of any units or organisations under one of the two following conditions. First, it is considered that the interests of the unit or organisation prevail over the interests of any individual member. Nonetheless, the infringement of a member’s rights as a result of such an imposition of duty must be the least possible infringement without which the primary goal commensurate with the resulting infringement cannot be achieved. In the case of tissue harvest from a donor sibling, this condition will be satisfied only if other sources of medically equivalent tissue from a mentally competent adult are not available. Further, it will not be otherwise proportionate to violate the donor’s bodily integrity unless the recipient sibling has a high chance of dying or suffering from serious complications without transplantation. Second, an individual agrees, either by word or by conduct, to abide by a set of norms or regulations that impose such an obligation while entitling him to certain privileges. In the circumstance, it cannot be legitimate to impose parents’ view of familial obligations on a child who is not yet able to appreciate his rights and obligations in the family. In factIn light of foregoing discussion, to determine whether tissue harvest can be legally and ethically permitted, one should assess first whether the recipient sibling would have a high chance of dying or suffering from serious complications without a transplantation. If the answer is affirmative, then one should see if donation from any mentally competent donors can offer a similar chance of successful transplantation.In the past, tissue-matched related donors were considered to be the best sources of donation. But now, many drawbacks of unrelated or haplo-identical transplants such as graft failure and significant graft-versus-host diseases have been overcome due to the development of new extensive T-cell depletion techniques with mega dose stem cell administration over the last two decades. New approaches such as alloreactive T cell depletion and post-transplant cell therapy have also improved immune reconstitution in tissue-matched unrelated or haplo-identical transplants. Using haplo-identical donors are now considered to be equally safe and effective transplant strategies as tissue-matched donors for advanced leukemia. Leung aIt is already well established that the use of haplo-identical transplant is equally safe and effective for advanced lymphoma or leukemia, whereas studies are still underway to evaluate the results with non-malignant diseases like aplastic anemia, lupus and sickle cell diseases. The above literature search by no means suggests that haplo-identical transplant can replace tissue-matched transplant but, given the success of new regimens of transplantation, doctors should look beyond the availability of tissue-matched related donors for certain types of diseases. Parents and doctors should make sure, if the medical conditions of the sick sibling permit a search, that no other sources of equally or similarly effective transplant are available before subjecting a minor to the physical risks and short-term psychological harm of tissue harvest.In the circumstances, we propose that the following should replace the original first, third, and fifth criteria in the Policy:1. If the medical conditions of the recipient permit a search, there is no medically equivalent haplo-identical/histo-compatible related adult or histo-compatible unrelated adult who is willing and able to donate;3. The recipient sibling will have a high chance of dying or suffering from serious complications without transplantation before the donor sibling becomes Gillick competent;5. Parental permission is obtained if the donor sibling is not Gillick competent.The parental choice to harvest tissues from donor siblings reflects the immense 'emotive desire to do something to help the recipient sibling’. HoweverThe authors declare that they have no competing interests.Corresponding and first author CTK is the main researcher, writer and editor responsible for the legal research and ethical analysis. The co-author TGL, was responsible for identifying ethical issues, overseeing the research and giving expert input into the ethical analysis and review. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6939/14/43/prepub


Consumption levels of prescription opioids (POs) have increased substantially worldwide, particularly the United States. An emerging perspective implicates increasing consumption levels of POs as the primary system level driving factor behind the observed PO-related harms. As such, the present study aimed to assess the correlations between consumption levels of POs and PO-related harms, including non-medical prescription opioid use (NMPOU), PO-related morbidity and PO-related mortality.Pearson’s product-moment correlations were computed using published data from the United States (2001 – 2010). Consumption levels of POs were extracted from the technical reports published by the International Narcotics Control Board, while data for NMPOU was utilized from the National Survey on Drug Use and Health. Additionally, data for PO-related morbidity  and PO-related mortality  were obtained from published studies. Consumption levels of POs were significantly correlated with prevalence of NMPOU in the past month , past year  and lifetime , as well as average number of days per person per year of NMPOU among the general population  and NMPOU users . Similar results were also obtained for PO-related morbidity and PO-related mortality measures.These findings suggest that reducing consumption levels of POs at the population level may be an effective strategy to limit PO-related harms. Prescription opioids (POs) are mainly used as analgesics in the treatment of mild to severe pain, including cancer and chronic non-cancer pain. In the context of expanded pain care, their consumption levels have tripled globally since 1990; however, this expansion has occurred almost exclusively in high-income countries, particularly the United States, which has ranked highest in Coinciding with this expansion in consumption levels of POs in the United States have been several PO-related harms, namely non-medical prescription opioid use (NMPOU), PO-related morbidity and PO-related mortality. For example, prevalence of NMPOU during the past year has risen to almost 5% over the past decade . SimilarGiven previous experiences with other psychoactive substances, one perspective on this area of research, supported by emerging evidence from the United States –8 and CaAnnual consumption levels of POs were derived from technical reports published by the International Narcotics Control Board (INCB), which detail the availability and use of narcotic substances in various countries based on information provided by international governments to the board . Data wiMeasures of NMPOU included annual prevalence of NMPOU during the past month, past year and lifetime, which were drawn from nationally representative surveys of the United States population: National Survey on Drug Use and Health Series . These sThe PO-related morbidity and PO-related mortality measures were extracted from published studies , 13. TheTable The primary analytic strategy involved the computation of Pearson’s product moment correlations. As such, r-values and their respective 95% confidence intervals were estimated for the correlations between consumption level of POs and each of the PO-related harms detailed previously, viz.: NMPOU prevalence , average number of days per year per person of NMPOU , substance abuse treatment admissions for POs per 10,000 people and PO overdose deaths per 100,000 people.Figures This short report documented significant correlations between consumption levels of POs and PO-related harms, including NMPOU, PO-related morbidity and PO-related mortality. These correlations are corroborated by evidence from several other American , 9 and CHowever, the magnitude of three correlation coefficients observed in the present study were noteworthy, as near perfect correlations (r >0.90) were observed with consumption levels of POs: average number of days of NMPOU among general population, substance abuse treatment admissions for POs per 10,000 people and PO overdose deaths per 100,000 people. The strength of these correlations indicate linear increases in PO-related harms with increases in consumption levels of POs. Such near perfect correlations have been demonstrated previously for PO-related morbidity in the United States , but to Based on recent data, there seems to be a convergence of evidence implicating increasing consumption levels of POs as the primary system-level driving factor in the surging opioid epidemic. Interestingly, consumption levels of POs have continued to increase throughout the past decade, despite limitations in evidence regarding the effectiveness of pharmacotherapeutic treatment of cancer and chronic non-cancer pain with POs , 15. ForIrrespectively, the link between consumption levels and public health relevant harms of POs is similar to that observed with other psychoactive substances, including alcohol and tobacco , 17. ThiA key limitation of the present study pertains to the inability to infer causation from the correlations presented. For example, closer surveillance due to increasing public health interest may be responsible for the observed increases in consumption levels of POs or PO-related harms, which would inevitably result in positive correlations. In summary, using data from the United States dating from 2001 – 2010, correlations between consumption levels of POs and PO-related harms were documented. These findings suggest that curbing consumption levels of POs at the population level may be an effective strategy towards limiting PO-related harms.


The association between serum Retinol Binding Protein 4 (RBP4) and obesity is still controversial. Serum RBP4 levels varies by gender, and estradiol may play a role in the difference. To investigate the participation of sex hormones in the association of RBP4 and obesity in humans, we measured serum RBP4, BMI, and sex hormones in 87 women from the outpatient.Eighty-seven subjects of Chinese women origin from the outpatient (aged 40.22 ± 15.54 years) were enrolled. Subjects with diseases affecting the metabolic state or not suitable to participate in this study were excluded. Anthropometrics and laboratory tests, including lipid profile, luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), estradiol (E2),progesterone (PROG), testosterone (TESTO), and dehydroepiandrosterone (DHEA) were conducted. Serum RBP4 was detected by an enzyme immunoassay kit and validated by quantitative Western blotting.Circulating RBP4 levels were positively associated with BMI, waist circumference, waist-to-hip ratio (WHR), systolic and diastolic (SBP), diastolic blood pressure (DBP), triglycerides (TG), low high-density lipoprotein cholesterol(LDL-c), and testosterone (TESTO) in the total group. While only in obese individuals, serum RBP4 levels were negatively associated with E2. The highest value was in the subjects with both obesity and the low estrogen level. Multiple linear regression analysis revealed that RBP4 correlated independently with TG, TC and insulin in all subjects, TC in non- obese individuals. However, E2 were significantly associated with serum RBP4 only in obese individuals.RBP4 could be a marker of obesity-related factors; estrogen was negatively related to RBP4 and might be one of the influential factors. Retinol-binding protein 4 (RBP4), belongs to the lipocalin family of proteins, which is released primarily by the liver, has also been shown to be released by adipose tissue as the second highest rate of expression , 2. In hSeveral studies show that serum RBP4 levels correlate inversely with the severity of insulin sensitivity, which is positively with obesity and type 2 diabetes, as well as components of the metabolic syndrome, and can be used as a noninvasive serological indexes reflecting the metabolic abnormalities –8. HowevTherefore, the major aim of the present study is to evaluate the association between RBP4 levels, obesity, sex hormones, as well as other metabolic parameters in Chinese women. Additionally, we sought to explore the possible effect of sex hormones on the relation with obesity and RBP4.We consecutively selected 138 subjects from the general population who had undergone medical check-ups at the outpatient department of Endocrinology of Zhongshan hospital, Fudan university from Oct.2013 and Aug.2014. After excluding 51 of the 138 subjects, a total of 87 subjects were enrolled in our study. Exclusion factors were diseases affecting the metabolic state or not suitable to participate in this study. Women presenting endometriosis, uterinefibroid, breast cancer and those with hormone-dependent cancer were excluded. Hyperthyroidism, hypothyroidism, diabetes, mental disease, serious disease with dysfunction of heart, liver, kidney, were excluded as well as those using estrogen replacement therapy. The study was approved by the human research ethics committee of Zhongshan hospital, Fudan University and informed consent was obtained from all subjects.Height (cm) and weight (kg) of participants, wearing light clothing and without shoes, was measured with stadiometers and calibrated balance-beam scales, respectively. BMI was calculated as weight in kilograms divided by the square of height in meters. Waist circumference (cm) was measured with a non-stretchable tape at the end of a normal expiration, at the smallest horizontal circumference between the ribs and iliac crest. Thus, waist–hip ratio (WHR) was calculated as waist circumference divided by hip circumference. Blood pressure was measured in the right arm in the seated position, after at least a 5 min rest, and the average of the three blood pressures was used as the final blood pressure. Blood samples were collected after a fasting period of at least 10 h overnight. Serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) were determined by enzymatic methods with a Hitachi 7600 analyzer . Serum insulin (mU/L) was measured with an insulin radioimmunoassay kit (kit from Beijing North Biotechnology Research Institute). Follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), estradiol (E2), progesterone (PROG), testosterone (TESTO), dehydroepiandrosterone (DHEA) were checked. Methods for measuring blood hormones were electrochemiluminescence immunoassay (ECLIA), and kits were used for ECLIA supplied by Roche Diagnostic Systems, GmbH,Mannheim, Germany. Serum RBP4 (μg/ml) was measured in duplicate by a sandwich ELISA developed in-house, using affinity chromatography purified polyclonal and monoclonal antibodies generated against recombinant human RBP4 protein. The assay system was subsequently cross validated by Western blot analysis . Accordip values <0.05 were considered significant.Mean (standard deviation), median (interquartile range), were tabulated for demographic, and laboratory characteristics by BMI. Differences in the distributions of these variables between non-obese and obese were calculated by the Chi-Square test for categorical variables, ANOVA for normally distributed continuous variables. Following this, Pearson’s correlation was used to examine the association of serum RBP4 and other parameters. Multiple testing was corrected using LSD method  or Games-Howell method . Multiple stepwise regression analysis was used to examine the association of estrogen and serum RBP4 and other parameters. All statistical analyses were performed using SPSS18.0 , The mean age of study participants was 40.22±15.54 years. Demographic and laboratory characteristics stratified by BMI are presented in Table The Pearson correlation coefficients for associations between RBP4 concentrations and other parameters are summarized in Table r = −0.374, p = 0.032), but no longer existed when in non-obese individuals  , compare to those with neither obesity nor low estrogen levels, individuals with no-obese but low estrogen levels, and individuals with obesity but high estrogen levels.Especially, we found that Serum RBP4 level was negatively correlated with estrogen in obese individuals (P <0.001), TC  together with Insulin  were independent variables significantly associated with serum RBP4. In non-obese individuals, TC  was independently associated with serum RBP4, while in obese individuals, Insulin  together with E2  were significantly associated with serum RBP4 (Table A stepwise multiple linear regression analysis was performed using serum RBP4 level as a dependent variable. Independent variables were those parameters, which were significantly associated with RBP4 in Pearson’s correlation analysis. In all subjects, TG (Standardized Coefficients Beta = 0.509, In the current study, we found that serum RBP4 levels were inversely associated with E2 levels in a cohort of Chinese women patients with obesity. To the best of our knowledge, this study is the first showing that circulating RBP4 is associated not only with BMI but also E2 levels in Chinese women patients, which implied that estradiol might be an impact factor on the relation between obesity and circulating RBP4.RBP4 has long-been known to be released by the liver, and it also has been shown that approximately 15% of circulating secretion results from adipose tissue, and down-regulates the glucose transporter GLUT4, which acts as the rate-limiting step in insulin-activated glucose transport across both muscle and adipocyte membranes , 17. ResIn both adults and children, the concentration of serum RBP4 varies by gender, with levels lower in female than in male participants , 24, 25.The close relationship between other metabolic parameters and RBP4 in adult and adolescents has been observed in previous studies, in which RBP4 was positively associated with BP, lipid profiles, and FPG , 26, 40.Some limitations of this study must be taken into consideration. Firstly, the cross-sectional design of the study represents a limitation, implicating that cause-and-effect relationship cannot be discerned. Secondly, our study subjects included only from the outpatient women. And the majority of individuals are middle-aged woman with variation of plasma estrogen, which may influence the levels of RBP4, as it changed during the menstrual cycle. The relation of RBP4 and estrogen is also restricted to only in obese women. Therefore, our results may not be applicable to the whole population. Finally, due to the limitation of data collection on all types of sex hormones, we got small sample size. As well as being small, the trials may have methodological weaknesses that could have resulted in bias.In conclusion, RBP4 could be a marker of obesity-related risk factors, and it is negatively related to estrogen, restricted to obese individuals. The RBP4 levels were higher in the co-presence of obesity and low estrogen, which suggested that their relationship was complicated and might influenced by estrogen and other confounding factors. Further experiments with larger groups of subjects are warranted to clarify the relationship between RBP4 and estrogen.


Glycoglycerolipids occur widely in natural products, especially in the marine species. Glycoglycerolipids have been shown to possess a variety of bioactivities. This paper will review the different methodologies and strategies for the synthesis of biological glycoglycerolipids and their analogs for bioactivity assay. In addition, the bioactivities and structure-activity relationship of the glycoglycerolipids are also briefly outlined. Af. Af48 fiO-acetyl-d-glucal 54  to give 58 . To. Toet aliety  to afford the final SQDGs and SQMGs (81).Hanishima et al.  also syn al. [76   group to give 85. The key step was selective α-glycosylation of trichloroacetimidate donor 86 with 26 in diethyl ether (Et2O). Removal of acetonide group of 87 and acylation with different ratios of diverse fatty acids gave 89 and 91. Then deprotection of Bn and Cbz groups and selective acylation of the amino group afforded 90 and 92.The aminoglycoglycerolipids 1,  attracte0 and 92 , which w90 [93 . A better separation of the anomers can be achieved by hydrolysis of the raw isopropylidene-protecting group. Then, the glycerol hydroxyl groups of the α-anomer were acylated with palmitic acid by dicyclohexylcarbodiimide (DCC) method. Reduction of the azido group of 98 using the Staudinger reaction followed by acylation of the amino group afforded 99. Final product 90 was obtained by removal of the protecting groups.Schmidt and co-workers also described the total synthesis of aminoglycoglycerolipid 90 ,90. They90 [93  and the d-gluco-, d-galacto-, d-manno-) moiety were not consistent. Among them, glucosyl trichloroacetimidate donor 107 was used to prepare α-anomeric glucoside at low substrate concentration in Et2O (α:β = 33:1). The 4-O-acetyl protecting group of galactosyl donor 102 was employed as remote neighboring participation group to give the galactoside with high α-anomeric selectivity (α:β = 32:1) in the glycosylation. While utilizing neighboring group participation, mannosyl trichloroacetimidate donor 110 with 2-O-benzoyl protecting group was applied to obtain absolute α-anomeric mannosyl glycoside. Afterwards, hydrolysis of the isopropylidene and reduction of the azido afforded the key intermediates 112. Acylation of the amino and hydroxyl groups processed with different acyl chloride in pyridine followed by deprotection of Bn to give a series 6-deoxy-6-aminoglycoglycerolipids 114.In addition, Li and co-workers completed the synthesis of aminoglycoglycerolipids with different glycosyl and fatty acids for biological activity assay ,92,93. Tsn-3 of the glycerol, have attracted attention as anti-tumor-promoting compounds [O-β-d-glycopyranosyl-sn-glycerol had bioactivities comparable with the corresponding 1-O- and 3-O-isomers [O-β-d-glycoglycerolipid derivatives by chemoenzymatic method and tested their antitumor promoting effect [122 and 123 [O-acylglycoglycerolipids 125 [129 [133 [3CH2OR). Two enzymes, Pseudomonas cepacia (LPS) and Candida antarctica (LCA) lipases, were proved highly regio- and diastereoselectivity, while the former yielded the 2S-l-O-acetyl derivative and the latter yielded its (2R)-diastereoisomer [Although natural glycoglycerolipids, in which the sugar is linked to the ompounds ,22,27,42-isomers . They syg effect ,96,97,98 and 123   showed that SQMG showed potent growth inhibitory effect on the cancer cells while none of the SQDG tested had such an effect, suggesting that the SQDG can not penetrate into the cells [Based on the inhibitory effect of DNA polymerase α and β of the natural sulfoquinovosylacylglycerol, Sakaguchi, Mizushina, and their co-workers, synthesized many derivatives of SQAG to examine the structure-inhibitory function relationship. They demonstrated that not only the chain length of the fatty acid, but also the sulfate moiety of the glycerolipids was important for the inhibitory activity ,108,109.In addition, Li and co-workers have synthesized series of aminoglycoglycerolipids to test their inhibitory activity of Myt 1-kinase ,92. The Various types of glycoglycerolipids isolated from natural products and their wide range of bioactivities have attracted much attention. However, there are challenges of separating the single product and acquiring different type of glycoglycerolipids for evaluating their activity-structure relationship. This review covered the activities of the naturally occurring glycoglycerolipids, total synthesis and their special relationship of structure-activity. The present synthetic methodologies and strategies provided diverse natural and unnatural glycoglycerolipids, including MGDG/DGDG, SQDG/SQMG and aminoglycoglycerolipids for bioactivity evaluation. The biological results of the glycoglycerolipids acting on different models indicate that most bioactivities are closely related with the sugar moiety and acyl chain of the glycoglycerolipids. More structural modification and further mechanism studies are in progress, which will facilitate a great understanding of glycoglycerolipids as candidate drugs.


Cryptorchidism is a condition in which one or both testes have not passed down into the scrotal sac. It is categorized as true undescended testis in which testes are present in the normal path of descent, and as ectopic testis, in which testes are present at abnormal site. Common complications of cryptorchidism are testicular torsion, subfertility, inguinal hernia, and testicular cancer. Here we present a rare case of pantaloon hernia of obstructed indirect component and direct component with cryptorchidism. A 40-year-old male patient presented to the emergency department of RLJ Hospital with history of pain and swelling in the right inguinoscrotal region following lifting heavy metal pipe since 1 day.(i)Abdomen was soft, and diffuse tenderness in the lower abdomen was present.(ii)There was irreducible swelling measuring 6 × 4 cm, which was present in right inguinoscrotal region and was firm in consistency. Skin is erythematous, stretched, and shiny.The following was noticed:right testis was not palpable,left testis is palpable and penis is normal ,in the rest of the examination, nothing was significant. A diagnosis of right irreducible inguinal hernia with undescended testis was made.Regarding the external genitalia,Surgical exploration revealed an indirect hernia of incomplete type  with preAnother direct type sac was noted with omentum as content, which was reduced . In viewPatient tolerated the procedure well and the postoperative period was unremarkable.Histopathology report confirmed the presence of Leydig cells, seminiferous tubule, and Sertoli cells without Testicular Germ Cell Tumors (TGCT) .An undescended testis, sometimes called a cryptorchid testis, can be found in 3% of the term newborns and in 0.5–1.0% of adults . It is cThe complications of unrepaired cryptorchidism are mainly testicular torsion, infertility, inguinal hernia, and testicular cancer. The testicles begin to lose the process of spermatogenesis if they are not in the scrotum because the scrotum is a “cooler location.” This process explains the link between cryptorchidism and infertility. In our case, inspite of old age, histopathology report confirmed the absence of tumor. Because the incidence of testicular cancer generally increases in cryptorchid testes, careful follow-ups are essential , 7.Usually, cryptorchidism resolves without any intervention before the age of 6 months. Surgical repair for cryptorchidism will be carried out if the testicles have not descended. Studies have shown that individuals who had corrective surgery after the age of 13 years had an incidence rate of 5.4%, whereas those who were treated before 13 years had an incidence rate of 2.23% . HistopaIt is difficult to understand the surgical anatomy of inguinal hernias, but once the surgical exploration is performed, surgical repair is simple. It is controversial whether a contralateral orchidopexy is needed or not. Furthermore, the incidence of testicular cancer does not decrease with fixed testes , 10. In This case is particularly notable because of the unusual presentation of cryptorchid testis as an obstructed inguinal hernia at the age of 40 years. The patient remained asymptomatic for 40 years. To our knowledge, the present case represents one of the very few cases of cryptorchid testis revealed at the age of 40 years with an inguinal hernia.“The surgeon must always be alert to the possibility of cryptorchid testis during a surgical exploration of an inguinal hernia.”


Tuberculous meningitis continues to be a serious problem for physicians because it is difficult to make an early diagnosis and the consequences of delaying treatment are severe. The objective of this study is to provide data for the optimization of diagnostic and timely treatment of tuberculous meningitis. Of the 401 human immunodeficiency virus (HIV)-negative tuberculous meningitis patients in our study, 332 were found to have an impaired blood brain barrier (82.8%). Nearly 17.0% of patients failed to be timely diagnosed. Headache (53.6%) and fever (48.6%) were the most common features, and Computed Tomography/Magnetic Resonance Imaging (CT/MRI) detected 96 patients (23.9%) with abnormal meningeal imaging. Cerebrospinal fluid real-time polymerase chain reaction was positive in 73.8% of the tuberculous meningitis patients, whereas, smears and cultures detected only 6.7% and 5.2%, respectively. Further analysis identified striking differences between drug-resistant and drug-susceptible tuberculous meningitis. Patients with drug resistance correlated with grave prognosis. Tuberculous meningitis diagnosis should overall embody clinical symptoms, laboratory and cerebral imaging findings, and more sensitive diagnostic approaches are still warranted. Our data suggest cerebrospinal fluid polymerase chain reaction for mycobacterial DNA and molecular drug susceptibility testing as routine assays for suspected tuberculous meningitis patients, and observation of the blood brain barrier function could be performed for individual management. Mycobacterium tuberculosis (MTB)256789101113Tuberculous meningitis (TBM) is the most severe form of tuberculosis (TB) and causes death or severe neurological defects in more than half of affected patients, despite advancements in available anti-tuberculosis treatments1A total of 218 men and 183 women who were HIV-negative TBM patients were recruited. The age of the patients ranged from 11 to 84 years old, with a median age of 39 years old. Of these, 105 (26.2%) patients had a history of tuberculosis infection. In a clinical examination, 53.6% of the patients complained mainly of headache, whereas 48.6% of the patients complained mainly of fever. Physical examinations revealed that 24.9% of the patients exhibited signs of meningeal irritation, 26.2% had altered mentation, and 25.9% displayed confusion. The prevalence of all other symptoms and signs was less than 20%. However, these symptoms and signs of TBM patients were atypical and difficult to differentiate TBM from other neurological diseases.Only 6.7% and 5.2% of the 401 patients had acid-fast positive and culture positive results in CSF samples, respectively. This rate was lower than rates reported in other studies715et al.et al.According to the methods of Thwaites et al.According to the guidelines proposed by Marais Thirty-five patients (19 men and 16 women with a mean age of 42 years old) were randomly sampled from 401 TBM patients for MTBDRplus and MTBDRsl assays, and 17 (48.6%) of these patients had experienced a prior TB infection. As shown in According to the CSF analyses, an increased number of leukocytes, with a lymphocytic predominance of more than 50%, a decreased glucose concentration and a markedly increased protein concentration was detected in the CSF of most of the patients . We did 2O, and 14 (40.0%) had an increased CSF opening pressure (>200 cmH2O). The median CSF total leukocyte count was 140 × 106/L, and 31 (88.6%) of the cases had an increased leukocyte count. The median CSF glucose level in these 35 cases was 2.1 mmol/L, and 26 (74.3%) of the cases had decreased glucose levels (<2.5 mmol/L). The median CSF protein concentration was 1.4 mg/dl, and 32 (91.4%) of the cases had an increased protein concentration (>0.45 mg/dl). The median Cl− concentration in 35 of the cases was 111.4 mmol/l, and 28 (80.0%) of the cases had a decreased Cl- concentration (<120 mmol/L) (In the 35 cases that underwent drug susceptibility testing (DST), the median CSF opening pressure was 170 cmH mmol/L) .In all, 23 of the 35 cases had a CSF IgG index and a CSF IgG synthesis rate analysis. Of these, 4 (17.4%) cases had a CSF IgG index that was increased, and 18 (78.3%) cases had a CSF IgG synthesis rate that was increased. Using Protis software, we found that 22 (62.9%) of the 35 cases exhibited BBB damage, 4 (11.4%) cases exhibited increased CSF IgG synthesis rates and BBB damage, and 1 case had only increased CSF IgG synthesis rates. The phenomenon of increased CSF IgG synthesis rates calculated by using Protis software is different from the routine CSF laboratory analysis results in that there was BBB damage in TBM. Results calculated by using Protis software may be concerned that IgG entering the CSF as a result of brain barrier damage can cause false positives in these cases.As shown in Further analysis of the results of genotypic assays indicated that among the analyzed mutations , mutatioAs shown in Eleven of the 14 drug-resistant TBM patients (78.6%) exhibited high opening pressure, whereas only three of the 21 drug-sensitive patients (14.3%) exhibited high opening pressure (p < 0.01). No significant difference was observed in other comparisons with respect to the laboratory results of CSF.As an anti-drug therapy, all patients were administered Isoniazid + Rifampicin + Pyrazinamide + Ethambutol + Levofloxacin (HRZEO) chemotherapy regimens as an initial treatment. Two cases exhibited a poor response to treatment and had their therapeutic regimen changed. Both of these cases exhibited resistance to both RFP and INH. Drug-sensitive TBM patients did not have their therapeutic regimen changed. Only two patients among the drug-resistant TBM patients had their therapeutic regimen changed.et al.et al.et al.We compared our results to those of Marais et al.From our statistical analysis of the clinical data as well as previous studies, we recognize that the diagnosis of TBM in children and adults is a major difficulty and may be obscured by days to weeks of non-specific meningitis symptoms in the early stage of the disease20Previous evidences have indicated that among anti-tuberculosis drugs, only lipophilic drugs such as isoniazid and pyrazinamide can penetrate the blood-brain barrier (BBB) and reach CSF concentrations that are active against MTBDrug-resistance in TBM is the primary factor that influences tuberculosis treatment. In our previous studyet al.et al.1419The study of Marais There are some limitations to this study. First, our study was based in southwest China and was focused on HIV-negative TBM, and our findings may not be applicable in other regions. Second, although 401 patients were included in this study, only 35 patients were involved in the genotype DST assay, and some significant differences between groups may not have been detected during the statistical analysis. It is necessary to accumulate more phenotypic and genotypic data for drug resistance in patients with TBM from different regions to obtain a better picture of drug resistance patterns in China. Third, data related to drug concentrations needs to be collected to more fully to describe the relationships between the effects of anti-TB treatments and the permeability of the BBB.In conclusion, the present study was performed using samples from TBM patients without HIV infection. Our data suggest that TBM diagnosis should overall embody clinical symptoms, laboratory investigations and cerebral imaging findings. Among laboratory tests, molecular diagnostics achieves a rapid diagnosis and relatively higher sensitivity when compared to conventional smear and culture in CSF. Most TBM patients are accompanied with impaired BBB status. Patients with drug-resistance and HIV co-infection may easily evolve into unfavorable prognosis. We recommend that a molecular DST assay should be immediately requested if a positive CSF-PCR result is obtained and that the function of the BBB should be better analyzed for individual therapy requirement.All details of the study design and all procedures involved were in accordance with the Declaration of Helsinki. Informed written consent was obtained from all enrolled patients. The protocols and procedures for the protection of human subjects were approved by the Ethics Committee of West China Hospital, Sichuan University (reference No. 198 (2014)). Furthermore, all of the methods were performed in accordance with the approved guidelines.From January 2010 to September 2015, a total of 8164 cases of suspected meningitis in West China Hospital of Sichuan University underwent lumbar puncture (LP) and CSF analysis. Of these, 975 cases were suspected to have meningitis based on CSF abnormalities. A total of 401 cases  were finally diagnosed with TBM. Thirty-five patients  with CSF TB-DNA positive results were randomly sampled and submitted to drug susceptibility testing (DST).All patients we enrolled underwent a clinical assessment and had blood drawn for hematological and biochemical tests. A lumbar puncture (LP) was performed, and CSF cell counts and protein and glucose concentrations were determined using routine CSF laboratory analysis techniques . CSF was centrifuged, and a portion of the deposit was examined using Gram, India ink, and Ziehl-Neelsen stains. The remaining portion of the sample was cultured on blood and chocolate agar for pyogenic bacteria and on Löwenstein–Jensen medium in liquid 7H9 media  for mycobacteria. Patients also underwent chest radiography and, in cases that were clinically indicated, a cranial computer tomography or magnetic resonance imaging scan was performed.A definite diagnosis was made by neurological physicians if the patients exhibited typical TBM clinical manifestations and/or cerebrospinal fluid pathological changes, including a positive result in the CSF in any one of the following three examinations: a direct acid fast bacilli (AFB) smear examination, culture by liquid or solid medium, or a nucleic acid amplification, such as PCR.The cellular and biochemical analyses were performed using routine CSF laboratory analysis protocols .CSF/IgGSerum)/(AlbCSF/AlbSerum) and applied to reflect the production rate of intrathecal immunoglobulin G (IgG), usually increases significantly in TBMProtis software , which could combine protein analytic results with Reibergrams histogram28The presence of HIV antibodies in serum samples was determined using a Modular Analytics E170 automated immunoassay analyzer .For CSF that tested positive for TB DNA, the DNA was extracted using a NucliSens EasyMag  and was then directly input into a Genotype MTBDRplus and MTBDRsl assay .Clinical and laboratory features were compared. We applied a diagnostic scoring system that required the presence of symptoms or signs indicative of meningitis plus symptoms derived from clinical, CSF, or imaging criteria to calculate a patient’s score for each feature. Patients then moved up or down the diagnostic pyramid shown in Ethical approval: Ethical approval was given by the Clinical Trials and Biomedical Ethics Committee of West China Hospital, Sichuan University (reference number: 198 (2014)).How to cite this article: Zhang, J. et al. Clinical features, Outcomes and Molecular Profiles of Drug Resistance in Tuberculous Meningitis in non-HIV Patients. Sci. Rep.6, 19072; doi: 10.1038/srep19072 (2016).


Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) modulate progression of certain solid tumors. The G-CSF- or GM-CSF-secreting cancers, albeit not very common are, however, among the most rapidly advancing ones due to a cytokine-mediated immune suppression and angiogenesis. Similarly, de novo angiogenesis and vasculogenesis may complicate adjuvant use of recombinant G-CSF or GM-CSF thus possibly contributing to a cancer relapse. Rapid diagnostic tools to differentiate G-CSF- or GM-CSF-secreting cancers are not well developed therefore hindering efforts to individualize treatments for these patients. Given an increasing utilization of adjuvant G-/GM-CSF in cancer therapy, we aimed to summarize recent studies exploring their roles in pathophysiology of solid tumors and to provide insights into some complexities of their therapeutic applications. Granulocyte and granulocyte-macrophage colony-stimulating factors  regulate maturation of progenitor cells in the bone marrow into differentiated granulocytes, macrophages, and the T cells. In clinical oncology, recombinant G- or GM-CSFs are routinely used to correct neutropenia subsequent to chemotherapy and radiation. However, adjuvant G-/GM-CSF treatments have been suggested to occasionally enable tumor growth. Such adverse treatment outcomes are thought to occur due to certain solid tumors being addicted to G-/GM-CSF-dependent signaling by expressing endogenous cytokines and/or their cognate receptors (G-/GM-CSFR). Clinical case reports reveal that newly diagnosed patients with G-/GM-CSF(R)-positive tumors present with an advanced often metastatic disease suggesting an accelerated progression of such cancers. The putative mechanisms of progression bear similarities to those observed with an adjuvant use of recombinant cytokines, that is, induction of immune tolerance and angiogenesis. It is therefore an imperative to explore roles of G-CSF and GM-CSF in cancer in order to improve treatment outcomes and to better define eligible patients' cohorts. In this review, we aimed to present recent advances in studies addressing putative mechanisms and therapeutic uses of G-CSF and GM-CSF in several cancers of a nonmyeloid origin.α-subunits and the two nonselective βc subunits; each α-/βc-subunits dimer is thought to bind one ligand molecule  Primary and metastatic lung cancers are by far the most frequently occurring type of malignancy driven by the ectopically secreted G-/GM-CSF Various cell types have been proposed as putative sources of G-/GM-CSF in lung cancers. Specifically, tumor-associated endothelial cells may secrete cytokines and thus promote angiogenesis and metastasis via increases in expression of cell adhesion molecules Glioma is the most common type of a primary malignant brain tumor in adults with universally poor prognosis. Gliomas more often than other G-/GM-CSF-secreting cancers also express intratumoral cognate receptors; augmented G-/GM-CSF(R) levels have been found to correlate with higher tumor grade α-subunits of GM-CSFR in bladder cancers compared to normal tissues A G-CSF was initially purified from the human bladder carcinoma cell line 5637 The elevated GM-CSF plasma levels have been found in certain patients with colorectal cancers thus implying this cytokine contributions to a disease progression α augments transcription of a VEGF receptor gene and enhances the antiangiogenic actions of GM-CSF in this model Melanoma is a rapidly progressing incurable skin cancer with a propensity to metastasize early due to immunosuppression and growth induction in part occurring via G-/GM-CSF. Clinical case studies report existence of are albeit severe G-CSF-secreting melanomas A role for G- or GM-CSF in cancers of prostate is less defined. Reports of G-/GM-CSF-secreting prostate tumors in English scientific literature are uncommon thus implying lesser significance of these cytokines for a prostate cancer progression. However, in vitro and animal models of this disease implicate G-/GM-CSF in promoting dissemination and bone metastasis. Namely, costimulation with G-CSF and a stem cell factor enhances cancer stem cell phenotype via upregulation of Oct3/4 transcription factor, NANOGP8 pseudogene and ABCG2 transporter Solid tumors of every origin present with many dissimilarities in their cellular composition and the molecular mechanisms of progression. Tumors addicted to G-/GM-CSF(R) signaling represents a very distinct molecular subset among other malignancies. For example, they utilize signaling pathways of normal hematopoiesis and the cytokine-mediated auto-/paracrine growth augmentation in addition to immune suppression. The aforementioned signaling modalities albeit not common in solid neoplasms contribute to some of the most advanced cases. Table


Due to the effort of several research teams across the world, today we have a solid base of knowledge on the liquid contained in the brain cavities, its composition, and biological roles. Although the cerebrospinal fluid (CSF) is among the most relevant parts of the central nervous system from the physiological point of view, it seems that it is not a permanent and stable entity because its composition and biological properties evolve across life. So, we can talk about different CSFs during the vertebrate life span. In this review, we focus on the CSF in an interesting period, early in vertebrate development before the formation of the choroid plexus. This specific entity is called “embryonic CSF.” Based on the structure of the compartment, CSF composition, origin and circulation, and its interaction with neuroepithelial precursor cells (the target cells) we can conclude that embryonic CSF is different from the CSF in later developmental stages and from the adult CSF. This article presents arguments that support the singularity of the embryonic CSF, mainly focusing on its influence on neural precursor behavior during development and in adult life. We will first discuss what makes embryonic CSF different from fetal and adult CSF. According to the classic concept, which applies to the fetal and adult periods, CSF fills the cavities of the ventricular system and subarachnoid space. It is in direct contact with different cellular types such as the ventricular layer in the ventricular system and the pial layer in the subarachnoid space, as well as with specific cellular populations such as the choroid plexus cells and the subventricular organs. In this period, CSF is enclosed in a non-distensible cavity system with permanent production, circulation, and reabsorption .At the earliest stages of brain development, embryonic CSF fills a brain restricted cavity, which is closed and undergoes a quick change in volume and morphology. Embryonic CSF is located in a cavity in the anterior part of the neural tube, the brain anlagen where we can clearly differentiate three major parts of the brain , which evolve quickly . This caRegarding embryonic CSF composition, many studies in different species found proteins to be the most important components of CSF during embryonic and fetal development . For exaIncrease in the accuracy and sensitivity of the proteomic techniques allowed the analysis of the complete molecular composition of the CSF in different species and at different stages of development. Such studies are necessary to assess the CSF usefulness in therapeutic strategies.Another specificity of the embryonic CSF is that it is confined in a restricted space. In fetal and adult stages, the CSF is located in the brain cavities or ventricles, medullar cavity, and subarachnoid space. However, at the earliest developmental stages, there is no subarachnoid space in the mesenchyme and no functional communication between the brain cavity and the space outside. Consequently, embryonic CSF is restricted to the cavity of the brain vesicles. A specificity of the neural tube in these stages is that the cavity that encloses the CSF is completely surrounded and sealed by the apical end of the homogeneous population of neuroepithelial precursors, leading us to two conclusions: 1) the origin of the CSF must necessarily be mediated by neuroepithelial precursors and 2) neuroepithelial precursors must be the target cells.Regarding the first point, the content of the sealed cavity must come from the cavity wall, which consists of the brain neuroepithelium. Two different mechanisms of the CSF origin have been proposed : the firOn the basis of these data, we can conclude that embryonic CSF can be a means of inner communication between neuroepithelial precursor cells, in which some populations are involved in the creation of a complex signaling fluid, while other answer these signals.Here we will try to review the specific roles attributed to the embryonic CSF during early brain development. As we have stated before, one of the main characteristics of embryonic CSF is the permanent interaction with the apical end of the neuroepithelial precursors, which can be considered brain stem cells during development . Today wThe first is that embryonic CSF creates an expansive force inside the brain cavity, involved in the generation and regulation of the brain anlagen growth and morphogenesis. As we have already said, in the earliest stages of development, the brain cavity becomes a sealed system filled by a fluid that exerts positive pressure against the neuroepithelial wall. This positive pressure is necessary for the brain’s expansive growth both in the cavity and neuroepithelial volume and also for the normal morphogenesis ,25. ThisRegulation of brain growth and morphogenesis by embryonic CSF has been partially explained in the sense that it is a physical mechanism that creates a positive hydrostatic pressure inside the sealed cavity against the neuroepithelial wall, which has local growth differences. This mechanism is created and developmentally regulated by neuroepithelial precursor cells synthesizing and apically secreting into the brain cavity osmotically active molecules such as proteoglycans, together with a simultaneous and specific ionic transport across the neuroepithelium. Proteoglycans are responsible for trapping of water inside the cavity, which generates the hydrostatic pressure ,20,23,26The second mechanism is the regulation of basic cellular behavior of brain neuroepithelial precursors by embryonic CSF. When Desmond and Jacobson  showed aAfter these studies, we focused on the identification of the CSF molecules responsible for these trophic properties. To date, several individual factors have been found to be involved in each mechanism, ie, survival, replication, and differentiation. Many studies have focused on the identification of mitogenic factors in CSF, such as FGF2, IGF1, NGF, and EGF -31, whicOn the other hand, it was overlooked that embryonic CSF had a role as an activator of neurogenesis in the adult brain. Surprisingly, neural precursors have been shown to have “astrocytic” nature in the subventricular zone (SVZ) and in the dentate gyrus (DG) of the hippocampus in adult mammals . DespiteThese data lead us to the key question: Why does the same cellular lineage result in a quite different behavior at different life stages? In fact, the main difference between the embryonic and the adult stages is the intensity of activity rate, which is at its maximum in the embryonic stages and shows a permanent decrease later on, leading to a restricted regenerative ability.In the last decades, many research groups have reported that at any life stage the degree of neural precursor activity is determined by the “cellular niche,” which is defined as the surroundings of the neural precursor cells and includes the mature cells (neurons and glia), immature cells generated by the precursors, extracellular matrix and all types of intercellular signals, including those from the microvessels, and the fluid content in the ventricular system -40. The Finally, literature data support the fact that the CSF is a part of the niche. In this way, neural precursors are in permanent and direct contact with the CSF content in the brain cavities, which directly influences the precursor behavior and is consequently considered a source of instructive signals that play a key role in the niche activity ,41,42. IIn conclusion, embryonic CSF plays a key role in brain growth by creating a hydrostatic pressure inside the brain anlagen cavity. Furthermore, it is a powerful mitogenic and neurogenic inductor in neural progenitors at the earliest stages of development, when the activity of neural precursors is at its maximum. Neural progenitors in adult mammal brain remain responsive to the embryonic factors present in embryonic CSF, which increases the niche activity and enables the use of embryonic CSF-specific factors as a tool to induce brain neuroregeneration.