Helicoverpa armigera, Mythimna separata and Plutella xylostella, encoding predicted proteins of 490 (55.98 kDa), 490 (55.82 kDa) and 491 (56.07 kDa) amino acids, respectively. These sequences shared a high identity with other insect PP5s and contained the TPR (tetratricopeptide repeat) domains at N-terminal regions and highly conserved C-terminal catalytic domains. Tissue- and stage-specific expression pattern analyses revealed these three PP5 genes were constitutively expressed in all stages and in tested tissues with predominant transcription occurring at the egg and adult stages. Activities of Escherichia coli-produced recombinant PP5 proteins could be enhanced by almost 2-fold by a known PP5 activator: arachidonic acid. Kinetic parameters of three recombinant proteins against substrate pNPP were similar both in the absence or presence of arachidonic acid. Protein phosphatases inhibitors, okadaic acid, cantharidin, and endothall strongly impeded the activities of the three recombinant PP5 proteins, as well as exerted an inhibitory effect on crude protein phosphatases extractions from these three insects. In summary, lepidopteran PP5s share similar characteristics and are all sensitive to the protein phosphatases inhibitors. Our results also imply protein phosphatase inhibitors might be used in the management of lepidopteran pests.Protein phosphatase 5 (PP5), a unique member of serine/threonine phosphatases, regulates a variety of biological processes. We obtained full-length PP5 cDNAs from three lepidopteran insects,  Recombinant PP5 proteins were produced in E. coli and purified. Their biochemical properties and sensibility to inhibitors were also investigated.In this study, we identified PP5 genes from three lepidopteran insects, P. xylostella, maintained in the laboratory for >5 yr without exposure to insecticides, was reared on pakchoi cabbage at 25±2°C, 50±5% relative humidity and a photoperiod of 16L:8D. H. armigera larvae were procured from Zhongke Baiyun  and reared on artificial diets at 25±2°C, 50±5% relative humidity and a photoperiod of 14L:10D. M. separata were initially obtained from a culture at the Biorational Pesticides Research and Development Center, Northwest A&F University, Shaanxi, China and reared on corn leaves at 25±2°C, 50±5% relative humidity and a photoperiod of 14L:10D. All moths were supplied with a 5% honey solution as nutrient and permitted to oviposition on moist gauze sterilized with a 1% sodium hypochlorite solution gauze.An insecticide-susceptible strain of Okadaic acid, cantharidin, arachidonic acid, and pNPP were purchased form Sigma . Endothall was purchased from Alfa Aesar . All other chemicals were of research grade or better and were obtained from commercial sources.P. xylostella, H. armigera, and M. separata, respectively, using Trizol plus  according to manual instructions, and treated with DNase using a DNase I kit . Then cDNA was synthesized using a First Strand cDNA Synthesis kit . Degenerate primers were designed based on the PP5 amino acid sequences from Bombyx mori (XP_004923376), Danaus plexippus (EHJ67807), Tribolium castaneum (XP_971407), Dendroctonus ponderosae (AEE62915), Megachile rotundata (XP_003699533), Nasonia vitripennis (XP_001603324), Apis mellifera (XP_624242), Drosophila willistoni (XP_002070260) and Aedes aegypti (XP_001650298), by using two online tools, Blocks WWW Server (http://blocks.fhcrc.org/blocks/) and CODEHOP (http://bioinformatics.weizmann.ac.il/blocks/codehop.html). A pair of primers was used in PCR with first strand cDNA as the template under the following PCR conditions: 94°C for 3 min, 35 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 1.5 min, followed by a final extension at 72°C for 7 min. The purified fragment was inserted into pMD19-T vector  and transformed into E. coli DH5α , then sequenced .Total RNA was extracted from the larvae of the 4th, 5th, and 6th instar larvae of http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Primers used to amplify the ORFs were designed using Primer Premier 5 and were incorporated with a Nde I restriction site at 5′ends of forward ones, while a Hind III restriction site at 5′ends of reverse ones. PCR conditions were as follows: 94°C for 3 min, 35 cycles of 94°C for 30 s, 62°C for 30 s, 72°C for 2 min, followed by a final extension at 72°C for 7 min. PCR products were sequenced as mentioned above. All primers used in this study were listed in Rapid amplification of cDNA ends (RACE) was conducted using the 5′-Full RACE kit  following manual instructions. RACE specific primers were designed from the obtained fragments. PCR conditions were as follows: 94°C for 3 min, 35 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 2 min, and final extension at 72°C for 7 min. PCR products were sequenced as mentioned above. The open reading frame (ORF) was predicted using ORF Finder (http://www.ncbi.nlm.gov/blast/). The phylogenetic analyses were conducted by MEGA 5.0 by the neighbor-joining method with bootstrap test of 1,000 replicates using amino acid sequences of other insect PP5s and the human PP5 (NP_006238) as an outgroup. The ExPASy Compute pI/Mw tool (http://web.expasy.org/compute_pi/) was used to predict the molecular weight and isoelectric points of PP5s. For the analysis of protein domains, the InterProScan 4 from EMBL-EBI (http://www.ebi.ac.uk/Tools/pfa/iprscan/) was used to search the InterPro collection of protein signature databases.The similarity analysis of deduced amino acid sequences was performed using BLAST programs . Primers were designed using an online tool, Primer 3 (http://www.simgene.com/Primer3) (P. xylostella actin gene (accession: JN410820), H. armigera elongation factor-1α (EF-1α) gene (accession: U20129), and M. separata actin gene (accession: GQ856238), were used as references. Using 100-fold diluted cDNAs as templates, the Real-time quantitative PCR (qPCR) reactions were carried out on a thermal cycler  using the Ultra SYBR Mixture  according to the manual instructions. Thermal cycling conditions were 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min, then followed by dissociation analysis to check the homogeneity of the PCR product. The qPCR was repeated three times for each gene. Each replicate was performed with an independent RNA sample preparation and consisted of three technical replicates. Relative transcript levels of PP5 genes were determined by the 2Ct−Δ equation: 2Cttar−Ctref]−[ . The expression data were presented as means ± standard deviation (SD) and were analyzed by One-Way ANOVA.To study stage-specific expression, the eggs, 1st–4th instar larvae, pupae and adults of Primer3) . The P. Nde I site at N-terminal, and Hind III site at C-terminal, were cloned into the corresponding sites of pET28a (+) expression vector , and transformed into E. coli BL21(DE3)plysS cells . The positive transformed cells were amplified in LB medium containing 4 mM Mn2+, 34 µg ml−1 chloramphenicol and 100 µg ml−1 kanamycin. Recombinant PP5 expression was induced with 0.1 mM isopropyl β-D-thiogalactoside (IPTG) when A600 reached 0.4–0.6 and incubated for 48 h at 18°C.The open reading frames (ORF) of PP5 with 2) comprising 0.1% β-mercaptoethanol, 1.0 mg ml−1 iysozyme and 1 mM PMSF. After centrifugation for 20 min at 20,000 g, 4°C, the soluble fraction was loaded onto a Buffer A pre-equilibrated Ni-NTA affinity column  to bind the target protein and the non-target proteins were washed down with Buffer A containing 20 mM imidazole. The target protein was eluted with 5 column volumes of Buffer A containing 150 mM imidazole. Eluted solutions were subjected to 12% SDS-PAGE and stained with Coomassie Blue. The high purity fractions were pooled and dialyzed against Buffer B (Buffer A +50% glycerol) overnight and stored at −20°C. Protein concentration was assessed using the method of Bradford Cells were harvested by centrifugation at 10,000 g for 20 min, 4°C and sonication for 5 min on ice in Buffer A , 0.1% β-mercaptoethanol, 1 mM EDTA, 1 mM benzamidine and 1 mM (PMSF). This was then centrifuged at 12,000 g for 30 min at 4°C. The supernatant extract was desalted using Sephadex G-25 spin columns  to remove the contaminating free phosphate as described in the instruction manual. Each insect extract contained three biological replicates. Protein concentration was estimated using the method of Bradford A410 on an Infinite 200 PRO multimode micro-plate reader . Kinetic parameters of each recombinant PP5 were estimated by using a Michaelis-Menten plot analysis of data obtained under the above assay conditions with pNPP concentrations of 1, 2, 5, 10, 20, 40, and 80 mM.Activity of each recombinant PP5 against the inorganic phosphatase substrate pNPP was determined as described with slight modifications −1 BSA, and pre-warmed to 30°C. Reaction was initiated after adding 100 µM phosphopeptides, RRA(pT)VA to obtain a final volume of 50 µL and incubated at 30°C for 15 min. The reaction was stopped by adding 50 µL of molybdate dye/additive mixture and incubated for an additional 15 min at room temperature for color development. Control samples were determined without enzyme. Sample absorbance was measured at A600 on an Infinite 200 PRO multimode micro-plate reader .Phosphatases activity of crude extract was determined towards phosphopeptides using PPP assay kit  in half-area 96-well plates  following the manual instruction. In brief, 1.0 µg protein was mixed in assay buffer of 50 mM imidazole (pH 7.2), 0.2 mM EGTA, 0.02% β-mercaptoethanol, 0.1 mg mlUsing pNPP as substrate, the optimum reaction temperature of recombinant PP5 protein was assayed at different temperatures and its optimum reaction pH was determined in various pH substation solutions. All assays above were performed with three replications.50 values. The non-enzyme reaction was taken as the background control, while the non-inhibitor reaction was used as the full-activity control.Three candidate inhibitors were dissolved in dimethyl sulphoxide (DMSO) to provide stock solutions of 10 mM for okadaic acid, 100 mM for cantharidin, and 200 mM for endothall, respectively. All solutions were diluted to the desired concentrations with assay buffer before use. Assays were carried out using pNPP as substrate for purified proteins or using RRA(pT)VA as substrate for crude PPP extract. Before initiating the reaction, the enzyme was incubated with the chemicals for 5 min at room temperature. A dose-response assay was used to determine the ICP. xylostella (accession: KF255601) H. armigera and M. separata. Analysis of these three PP5 cDNAs revealed that cDNA of PxPP5 has a 5′ UTR of 102 bp, a 3′ UTR of 620 bp, and an open reading frame (ORF) of 1476 bp that encodes a protein of 491aa, with the theoretical pI of 6.54 and calculated molecular weight of 56073.10 Da; HaPP5 cDNA owns a 5′ UTR of 118 bp, a 3′ UTR of 396 bp, and an ORF of 1473 bp, encoding a protein of 490 aa, with the theoretical pI of 6.20 and calculated molecular weight of 55967.92 Da; while MsPP5 cDNA possesses an 1473 bp ORF which encodes a 490 aa protein, with theoretical pI of 6.20 and calculated molecular weight of 55824.71 Da, and the lengths of 5′ UTR and 3′ UTR are 118 bp and 396 bp, respectively. The full-length cDNAs of HaPP5 and MsPP5 were deposited into the GenBank Database with the accession numbers of KF983016 and KF983017, respectively.Although PP5 genes from several insect species have been identified and are available on NCBI Database, the information of their sequences characterization and their functional roles in biological processes is limited. In our previous work, a full-length PP5 cDNA was cloned from BLAST analysis of deduced amino acid sequences of these three PP5 genes on NCBI revealed their strong similarities with PP5s from other insect species. They also shared >60% identity to human PP5. The constructed phylogenetic tree showed PP5 from lepidopteran insects form a small cluster . PrositeH. armigera, M. separata, and P. xylostella by qPCR reactions. Our results showed that PP5 genes were expressed in all stages of the three insects. Expression levels of the HaPP5 gene were relatively high in the egg and adult stages but significantly lower in the 4th instar, 6th instar, and pupa stages. MsPP5 gene transcripts had the lowest expression at the 6th instar stage and the highest expression in the egg stage. The PxPP5 gene was more abundantly expressed in the egg and adult stages, while it showed the lowest expression level in the pupa stage  was externally added to the culture media E. coli has been reported in mammals Plasmodium falciparumTrypanosoma bruceiE. coli-yielded recombinant PP5 from P. xylostella and the cantharidin-producing beetle, E. chinensisNormally, it is difficult to produce active PPP-type phosphatases in in vitro characterization of recombinant PxPP5, HaPP5, and MsPP5, their ORFs were all subcloned into the pET28a (+) vector. Recombinant proteins were produced and purified under the same conditions. 12% SDS-PAGE analysis showed that three recombinant PP5 were soluble expressed and successfully purified with bands of ∼60 kDa being close to their predicted protein sizes. Relatively high purity recombinant PP5 proteins (∼90%) were eluted by 150 mM imidazole  vector harboring the thioredoxin protein to facilitate the correct-folding of heterologous protein was used to produce the TrxA-tPxPP5 protein. TrxA-tPxPP5 protein showed similar activity with the arachidonic acid activated full-length PxPP5  are significantly higher than those reported for mammalian PP5 (IC50 = 3.5 nM), but are much lower than in E. chinensis PP5 (IC50 = 190.0 nM) 50 values of 0.73–2.38 µM which are consistent with previous reports on other sources of PP5 50 values of endothall were 6.84 µM for PxPP5, 12.64 µM for MsPP5, and 19.33 µM for HaPP5, respectively. According to our results, compared to the other two proteins, MsPP5 was more sensitive to okadaic acid, while cantharidin and endothall blocked PxPP5 activity more potently than they did the other two proteins. These findings indicated that although lepidopteran PP5s possess high sequence similarity and identical key residues, other residues might be involved in the inhibitor binding as well as the conformational differences that may also exist among them.In general, the three proteins showed a similar level of sensitivity to each inhibitor and the inhibiting ability of the three inhibitors against lepidopteran PP5 proteins ranks as: okadaic acid>cantharidin>endothall. The ICArabidopsis thaliana, Lemna paucicostata and E. chinensisMembers of the PPP family are highly conserved at their phosphatase domains. It has been reported that cantharidin and endothall impaired the phosphatase activity of PPP-extract from Specific inhibitors have been proved to be valuable tools to explore the physiological processes of target proteins. Changes in multiple cellular pathways at their transcription levels were observed in plant and in mammals post cantharidin treatment indicating the involvement of PPP members in these pathways E. coli with similar biochemical properties. Three protein phosphatase inhibitors showed a strong inhibitory effect on both recombinant proteins and crude protein phosphatase extracts suggesting their potential utilization value for lepidopteran pest control.Many lepidopteran insects are of great economic importance because many of them are agricultural pests. We identified cDNAs of HaPP5, MsPP5, and PxPP5. Analyses of their expression patterns revealed relatively high transcript abundance in egg and adult stages. Biologically active recombinant proteins were produced in


Members of the Semaphorin 3 family (Sema3s) influence the development of the central nervous system, and some are implicated in regulating aspects of visual system development. However, we lack information about the timing of expression of the Sema3s with respect to different developmental epochs in the mammalian visual system. In this time-course study in the rat, we document for the first time changes in the expression of RNAs for the majority of Class 3 Semaphorins (Sema3s) and their receptor components during the development of the rat retina and superior colliculus (SC).Nrp2, Plxna2, Plxna3, and Plxna4a. In the SC there were also changes in transcript levels for all Sema3s tested, as well as Nrp1, Nrp2, Plxna1, Plxna2, Plxna3, and Plxna4a. These changes correlate with well-established epochs, and our data suggest that the Sema3s could influence retinal ganglion cell (RGC) apoptosis, patterning and connectivity in the maturing retina and SC, and perhaps guidance of RGC and cortical axons in the SC. Functionally we found that SEMA3A, SEMA3C, SEMA3E, and SEMA3F proteins collapsed purified postnatal day 1 RGC growth cones in vitro. Significantly this is a developmental stage when RGCs are growing into and within the SC and are exposed to Sema3 ligands.During retinal development, transcript levels changed for all of the Sema3s examined, as well as These new data describing the overall temporal regulation of Sema3 expression in the rat retina and SC provide a platform for further work characterising the functional impact of these proteins on the development and maturation of mammalian visual pathways. The mammalian visual system is complex, and the developmental events that give rise to this highly organised system are similarly complex and exquisitely ordered. The timing of the major developmental events of the mammalian visual system has been well characterised, and many of the molecular cues controlling these critical events have been elucidated . Ho). Ho7]).Semaphorins (Semas) have been implicated in neural and vascular aspects of visual system development in various species including frog ,9], zeb, zeb9], Sema3a through Sema3g,, bu, buSema3gh Sema3g–44]. Se. SeSema3gh Sema3g,46], fr, frSema3gh Sema3g.in vitro growth cone collapse assay on isolated RGCs from a developmental stage when they would be growing into and within the SC in vivo.In summary, while we understand much about the timing of developmental events in the mammalian visual system, investigation of a potential role for the Sema3s in these events would benefit from knowledge about the timing of expression of Sema3s and their receptors during visual system maturation. We sought to address this gap in our knowledge by building on a previous study from our laboratory , focusinPlxna3 in situ hybridisations at P1 and P7 are presented in Figure Developmental expression profiles for the Sema3s and their receptors are presented in Figures Sema3f and Plxna2; moderate expression of Nrp1 and Plxna1; and relatively low expression of the rest . Plxna4a RNA levels peaked later at P7, but also again in the adult.Relative to other time points, Many of the significant peaks in transcript expression occurred after the main developmental epochs. However, the changes that were quantified in the retina before P21 occurred during periods of RGC apoptosis, and synapse generation and maturation.Plxna1 having the highest; followed by Sema3f, Nrp1, and Nrp2; then Sema3b, Sema3c and Plxna3; and lower amounts for the remainder , peaked at E19, and a decline with increasing postnatal age. The expression profile of Sema3b differed from other transcripts; there was large biological variation at many time points, with CVs greater than 100%, and four apparent peaks in expression, only one of which reached significance (E19). Sema3c transcript expression peaked at E19 and P0, before falling to adult levels by around P14. In contrast to the other Sema3s, Sema3e transcript levels increased steadily from E16 to P14, before a decrease at P21, then again increased in the adult. Sema3f expression was highest at E16 and E19, with RNA levels lower at birth and remaining at those reduced levels during postnatal development. There was relatively high biological variation in expression levels of Nrp1, Nrp2 and all PlexinAs at E16 and E19 (CV > 100%) compared to other time-points. Nrp1 RNA expression peaked at E19 then decreased steadily to P7 at levels that were maintained into the adult. Nrp2 transcript levels peaked at E19 before falling steadily to E16 levels at P7 and beyond. Plxna1 RNA expression also peaked at E19, a pattern that was repeated for the remaining three PlexinA RNAs, all of which had significantly decreased RNA expression by P0 and P7 that remained at low levels into adulthood.The patterns of expression of Sema3s in the developing SC were substantially different from those seen in the retina, with peak expression occurring around or before birth. Temporally, these changes in transcript expression occurred during the period of SC neurogenesis, synapse generation and maturation, and innervation of the SC by extrinsic afferents including those from the retina and visual cortex.Plxna3 RNA expression in the retina occurred during the period of peak RGC apoptosis  immunostaining. This is in agreement with previously published data in the mouse retina . In. InPlxnand Plxna4,77], and and65] aSema3a and Sema3f, have been implicated in apoptosis of neurons including RGCs –[92–[9287]–,59]–[62–[6259]–[is (P14) . The mai the IPL –24]. We. WeSema3 the IPL –70], an, anSema3Sema3f may contribute in helping to guide these axons to their correct retinotopic targets ,,. Pr. PrSema3and L1cam–95], an, anSema3nd Plxna1. While pnd Plxna1, these wnd Plxna1,[71],,,. In. Infree;y values , and they values . BestKeepression .U tests.Normalised data were analysed for changes between developmental groups by Kruskal-Wallis ANOVA . When Kruskal Wallis ANOVA resulted in p < 0.05 pairwise tests of statistical significance between time points were performed by Mann–Whitney Plxna3 riboprobe was kindly provided by Professor Joost Verhaagen . Sense and anti-sense probes were produced by restriction endonuclease  digest followed by in vitro RNA transcription (IVT) with digoxigenin labelled RNA  and RNA polymerase . RE used were: sense, Kpn I; anti-sense, Spe I. Riboprobes were hydrolysed for one hour at 60°C, precipitated with LiCl and 100% ethanol, resuspended in DEPC treated double deionised water (DDW), and stored at −80°C before use.Plasmid for P1 and P7 retinal sections were processed for ISH with the above riboprobe using methodology previously described , with α-After ISH processing, sections were processed for immunofluorescence using standard methods. Antibody diluent was 0.1% (w/v) Triton-X100, 10% (v/v) normal goat serum , in PBS. Primary antibody (α-βIII-tubulin (TUJ1); mouse monoclonal, 1:2,000 dilution; Covance, USA) incubation was 4°C overnight in a humidified chamber, followed by secondary antibody  incubation for 2 hours in a dark humidified chamber at room temperature. Slides were coverslipped using fluorescent mounting media .Cell cultures were stained for βIII-tubulin and F-actin. βIII-tubulin was labelled by IHC according to standard practice, using the same antibody diluent, incubation times, and antibodies as described above. Following immunostaining for βIII-tubulin, cultures were incubated with phalloidin-FITC  diluted 1:1,000 in PBS for 2 hours at room temperature to stain F-actin, then were washed 3 times with PBS, before being coverslipped using DAKO fluorescent mounting media.Sema3a-GFP, Sema3c-FLAG, Sema3e-FLAG, Sema3f-AP) were also a gift from Professor Joost Verhaagen. The Sema3a-GFP and Sema3f-AP plasmids have previously been characterised [Sema3c-FLAG contains mouse Sema3c cDNA, and Sema3e-FLAG contains human Sema3e cDNA. Plasmid constructs were verified by sequencing.Expression plasmids  9:1 fortified with gentamycin , and then grown without the presence of antibiotics overnight. Cells were transfected with plasmids using Lipofectamine 2000 , using Opti-MEM  as per manufacturer’s instructions. Media was changed to original growth media with antibiotics 6 hours after transfection, and conditioned media collected 30 hours later. Control conditioned medium was obtained by the same methods, using sham (no plasmid) transfected cultures.SEMA3A-GFP was purified from conditioned media using magnetic GFP-Trap beads . FLAG tagged proteins (SEMA3C-FLAG and SEMA3E-FLAG), and Myc tagged proteins (SEMA3F-AP) were immunoprecipitated from conditioned media using Dynabeads Protein A/G  and 5 μg α-FLAG antibody , or 5 μg α-Myc antibody . Proteins were eluted in 2X Laemmili Buffer  glycerol, 4% (w/v) SDS, 2% (v/v) β-mercaptoethanol, 0.005% (w/v) bromophenol blue, in DDW pH 6.8), separated by SDS-PAGE  and transferred to nitrocellulose membrane . Recombinant proteins were detected by western blot using 5% (w/v) skim milk powder in Tris buffered saline containing Tween  Tween 20, in DDW pH 7.5) blocking buffer, α-GFP antibody , α-FLAG antibody , α-Myc antibody , and α-mouse-HRP secondary antibody . Proteins were visualised by chemiluminescence  using the Chemi-Doc system .acetyl-cysteine , 6 nM triiodithyronine , 1 mM sodium pyruvate ) fortified with growth factors , 5 μM forskolin , 440 pM rat ciliary neurotrophic factor , 1.85 nM human brain-derived neurotrophic factor  to increase survival as previously described [2.P1 Wistar pups were euthanised by IP Lethabarb overdose and retinas dissected free in PBS. Retinas were dissociated with MiltenyiBiotec Tissue Dissociation Kit – Postnatal Neurons , and RGCs purified using the MACS RGC Isolation Kit  according to manufacturer’s instructions. Purified RGCs were resuspended in growth media , 1X B27 supplement , 1.25 μM transferrin , 0.2 μM progesterone , 100 μM putrescine , 230 nM sodium selenite (Sigma-Aldrich), 1.5 μM bovine serum albumin , 872 nM bovine pancreas insulin , 1 mM L-glutamine , 30.6 μM N-escribed . Cells w2. The following day (effectively P2) 30 μL of conditioned media was added to the 300 μL of growth media in each well and incubated at 37°C and 5% pCO2 for 30 minutes. Cultures were then fixed with 4% PFA and stained for βIII-tubulin and F-actin. Epifluorescent photomicrographs were taken of all neurons in all cultures, and encoded such that analysis was completed blind to treatment group. Growth cones were scored as either collapsed or uncollapsed [Purified RGC cultures were grown overnight at 37°C and 5% pCOollapsed : uncollaThe authors declare that they have no competing interests.AS carried out qPCR, in situ hybridisations, immunohistochemistry, data analyses, project coordination, manuscript preparation and revision. CL performed immunopreciptiation and western blotting. GWP participated in experimental design and manuscript revision. ARH conceived experimental design, aided in project management, and contributed heavily to manuscript revision. All authors read and approved the final manuscript.U tests. e: p = 0.05; * p < 0.05; ** p < 0.01.Pairwise statistical comparisons of expression data. Normalised data were analysed for changes between developmental groups by Kruskal-Wallis ANOVA . When Kruskal Wallis ANOVA resulted in p < 0.05 pairwise tests of statistical significance between time points were performed by Mann–Whitney Click here for fileSema3a-GFP, Sema3c-FLAG, Sema3e-FLAG, and Sema3f-AP, as well as a no plasmid control. SEMA3A-GFP expression was visualised directly by epifluorescence (B), SEMA3B-FLAG (C) and SEMA3E-FLAG (D) was detected following fluorescence immunocytochemistry, and SEMA3F-AP was detected by the presence of colour reaction product from NBT/BCIP (F). Scale bar: 50 μM.Recombinant Sema3 protein expression in conditioned media. A: Presented are three different blots, lined up against the same ladder. Controls were from cultures that underwent a sham transfection (no plasmid). Boxes indicated bands of expected molecular weights. Detected bands for SEMA3A-GFP correspond to the approximately 130 kDa and 90 kDa bands reported previously . SEMA3C-Click here for file


HNF1β_shRNA-stable cell lines, we show here that HNF1β drastically alters intracellular metabolism, especially in direction to enhance aerobic glycolysis, so called the “Warburg effect”. The consequence of the metabolic change contributed cell survival under stresses such as hypoxia and chemo-reagent, only when sufficient glucose supply was available. Augmented cell survival was based on the reduced ROS activity derived from metabolic alteration such as shift from oxidative phosphorylation to glycolysis and increased intracellular anti-oxidant, glutathione (GSH). One of the cystine transporters, rBAT is likely to play a major role in this GSH increase. These data suggest that HNF1β, possibly induced by stressful microenvironment in the endometriotic cyst, confers survival advantage to the epithelial cells, which leads to the occurrence of OCCC, a chemo-resistant phenotype of ovarian cancer.HNF1β is expressed exclusively in ovarian clear cell carcinoma (OCCC) and not in other ovarian cancers, regarded as a hallmark of this tumor. This implies its central role in the unique character of OCCC, including resistance to chemotherapy, but its exact role and influence in cancer biology or the molecular bases of its function are largely unknown. Using comprehensive metabolome analysis of  Ovarian clear cell carcinoma (OCCC) is a relatively rare subtype among the ovarian cancers, but is a clinically important entity because it is generally resistant to chemotherapy and is known to have a worse prognosis compared with other ovarian cancers –4. AnothWe have shown previously that the microenvironment within the “chocolate cyst,” which contains high levels of free iron and consequently reactive oxygen species (ROS), is extraordinarily stressful to epithelial cells within the cyst . ROS areWe have also shown that OCCC has a unique gene expression profile consisting of many stress-related genes. Specifically, we suggested that the transcription factor HNF1β (hepatocyte nuclear factor 1 homeobox B), a gene specifically expressed in OCCC, distinguishes this phenotype from other ovarian cancers , 20. HNFIn addition, overexpression of HNF1β has been reported in multiple malignancies including OCCC. So far, reported function of HNF1β includes epithelial morphogenesis and differentiation , regulatWe have previously demonstrated that HNF1β activates glycolysis and increases lactate production in OCCC cell line, RMG2 . RegardiIn this study, we hypothesized that HNF1β, which is expressed exclusively in OCCC confer anti-ROS activity by altering glucose metabolism, thereby contributing to carcinogenesis in high ROS environment of endometriotic cysts and cell survival in OCCC. We attempted to explore the full extent of the metabolic alterations caused by HNF1β in OCCC using a comprehensive metabolic assay. Additionally, we investigated the biological influences of these alterations.http://www.broadinstitute.org/gsea/index.jsp) using the microarray datasets GSE39204, which contain 64 ovarian cancer clinical samples from Kyoto University Hospital, and GSE6008, which contain 99 ovarian cancer clinical samples [To identify characteristics specific for the OCCC subtype, we first performed gene set enrichment analysis   (GSE39204: 01) Fig.  and S1b.In addition, GSEA revealed that 2 transcription motifs of HNF1 (hepatocyte nuclear factor 1) and SF1 (splicing factor 1) were enriched in the OCCC group in both GSE39204 and GSE6008 (Msig DB C3.tft.v4.0) , which sin vitro analysis of intracellular metabolism in HNF1β knockdown RMG2 cells (HNF1β_sh1) and control cells (control) that were established as described [HNF1β_sh1 cells. Of these, 52 metabolites were decreased by at least 0.7-fold and 20 were increased by at least 1.4-fold , and malic acid that was to be converted to citric acid if pyruvic acid was provided to the TCA cycle via acetyl CoA, was decreased, suggesting that oxidative phosphorylation is more active in these cells (or less active in HNF1β-high cells by acetyl CoA supply stagnation)   Fig. . Regardi samples , suggestanalysis . These ranalysis . As for 01) Fig. , which mHK1, G6PD, and LDHA genes, which are assumed to be related to aerobic glycolysis. In HNF1β_sh1 cells, the expression of HK1 and LDHA was significantly decreased (p < 0.0001), but the expression of G6PD was significantly increased (p < 0.0001) . There was no significant difference in the expression of G6PD between the two groups  . In addio groups .HNF1β_sh1, sh2) of both RMG2 and JHOC5 OCCC cells    Fig.  & 3d. Fu02) Fig. .HNF1β_sh1, and HNF1β_sh2 RMG2 cells were cultured in normal growth medium (glucose (+) medium) and in glucose-deprivation medium (glucose (−) medium) in normoxic condition, and cell numbers were measured every 24 hours by WST-8 assay. In glucose (+) medium, HNF1β_sh1, and HNF1β_sh2 RMG2 cells had similar number compared with control cells    Fig.  & 5d. Mo05) Fig.  – 5e. Th05) Fig. .HNF1β_sh1 cells (p < 0.0005) , 097 (p = 0.470), 1.21 (p < 0.001), respectively) , a primary intracellular redox regulator. Metabolome analysis in RMG2 cells showed that intracellular GSH significantly decreased in 05) Fig. . In GSH ly) Fig. . We confly) Fig.  & 6c. ThGCLC expression was significantly decreased in the RMG2 HNF1β_sh1 cells (p < 0.05)   , glutamate-cysteine ligase catalytic subunit (GCLC), and glutathione synthase (GS), as well as GSH transporters of the substrates glutamate, glycine, and cysteine, were analyzed by microarray. Among the synthase-coding genes, only 05) Fig. , whereas05) Fig. . Moreove05) Fig. . In cont01) Fig. . Further01) Fig. .In GSH synthesis, cysteine is known to be the rate-limiting substance because the total levels of cysteine are much lower in cells than the levels of other molecules. Most intracellular cysteine is taken into cells in its oxidized form, cystine. Therefore, we investigated whether the cystine supply altered intracellular GSH. Indeed, decreasing the cystine supply by altering the cystine concentration in the growth medium reduced intracellular GSH Fig. .rBAT , was significantly decreased by HNF1β knockdown (p < 0.001)  Fig.  & 7b. Ex01) Fig. .rBAT_sh1, sh2) of both RMG2 and JHOC5 OCCC cells (p < 0.0001)  Fig.  & 7f. ThIt is well known that cancer cells have unusual metabolic patterns relative to normal cells. A common example of this is the intracellular metabolic shift from oxidative phosphorylation to glycolysis, which is called the Warburg effect –29. AlthComprehensive intracellular metabolites analysis revealed that HNF1β dynamically altered intracellular metabolism. First, we focused on glucose metabolism. As we have previously reported , lactic HK1(Hexokinase-1) that supports glucose supply through phosphating glucose to glucose-6-phosphate, and LDHA that supports conversion pyruvate to lactic acid, [HK1 and LDHA were also expressed at higher levels in the OCCC group than in the non-OCCC group. Expression levels of these genes were correlated significantly to the expression of HNF1β (p < 0.0001)  . CollectSeveral advantages of the Warburg effect have been proposed in cancer cells , 26–28, 2O2. Again, under such oxidative stress, control OCCC cells with high HNF1β expression had lower ROS activity levels compared with their HNF1β knockdown counterparts , was expressed at significantly higher levels in these cells , which is coded by SLC7A9, transports cystine and dibasic amino acids, such as lysine, arginine, and ornithine, and thus is critical for reabsorption of these amino acids in the proximal tubule. With regard to the relation of HNF1β and rBAT, Sauert K. et al. reported that HNF1β mutation decreased rBAT expression [In addition to the suppression of oxidative phosphorylation, our metabolome analysis also showed that HNF1β knockdown is associated with a significant decrease in intracellular GSH, a major regulator of intracellular ROS Fig.  – 6c 4141–43. Inlls Fig. . rBAT ispression . And the-induced aerobic glycolysis increased stress-resistance, hypoxia adaptation, and consequent survival capacity of OCCC, but these effects are highly glucose-dependent. In our assay, actually, the control RMG2 cells with higher HNF1β expression were more sensitive to glucose deprivation than were HNF1β knockdown cells, indicating that glucose uptake and metabolism may be a therapeutic target in OCCC. Secondly, HNF1β inhibition with some type of inhibitor, such as the microRNA mir-802 [In conclusion, we have demonstrated that HNF1β drastically changes glucose and amino acid metabolism and that it promotes cell survival in hypoxic conditions under oxidative stress. These results are compatible with the fact that most OCCCs originate in endometriotic cysts and survive under severely oxidative conditions . It is w mir-802 , may hav mir-802 , 53, or 2 at 37°C.The OCCC cell line RMG2 was kindly provided by Dr. Aoki at Keio University, and the OCCC cell line JHOC5 was purchased from the Riken BioResource Center. Cells were cultured in RPMI1640 medium  supplemented with 10% fetal bovine serum (FBS)  and penicillin–streptomycin  in a humidified atmosphere containing 5% COGIPZ Lentiviral shRNAs and a nonsilencing control RNA  . We used 10 μg /ml puromycin for selection and confirmed that over 95% of cells were successfully transfected with RNA based on GFP fluorescence by fluorescence microscopy. We checked the transfection efficacies of HNF1β and rBAT by RT-PCR and western blotting  primers and probes were designed using Roche Universal ProbeLibrary Assay Design Center software (http://qpcr.probefinder.com/roche3.html) .Total RNA was isolated using the RNeasy Mini Kit , treated with the PrimeScript™ RT reagent Kit , and analyzed using Light Cycler 480 Real-Time PCR system . The ® Deep Red Reagent  with complete medium at 37°C for 30 minutes. The cells were then harvested and washed with PBS and analyzed using a FACSCalibur cytometer . The results were analyzed using FlowJo_V10 software. Differences between groups were assessed based on the mean fluorescence intensity (MFI) of FL4.Each cell culture, both with and without oxidative stress, was washed twice with PBS and then stained with 5 μM CellROXhttp://www.dojindo.com/store/p/824-GSSG-GSH-Quantification-Kit.html).GSH was measured using a GSH/GSSG quantification assay kit , based on the enzymatic recycling method. Cultured cells were harvested, treated, measured by absorption spectrometer, and quantified according to the manufacturer's manual.  at 37°C in 20% O2 and 5% CO2 were washed by mannitol solution, quenched by methanol and collected. Then, 5 × 106 cells from each condition were diluted with 1000 μl chloroform and 400 μl distilled water, and then centrifuged at 2300 × g for 5 minutes at 4°C. Replicate 400 μl aliquots of each supernatant (for anion/cation) were centrifuged at 9100 × g for 120 minutes at 4°C using a 5 kDa ultrafiltration membrane . The supernatant from each sample was solidified, re-diluted with 50 μl distilled water, and measured by CE-TOFMS (Capillary electrophoresis electrospray ionization time-of-flight mass spectrometry). This measurement was performed by Human Metabolome Technologies (HMT) Inc. . Based on HMT's database of the mass-to-charge ratio (m/z) and the migration time (MT) of peaks detected in CE-TOFMS, 193 peaks were assigned to candidate metabolites. Of these 193 metabolites, 87 were quantified using integrated peak areas obtained from measurements of chemical standards that were analyzed in parallel with experimental samples. Others were compared using their relative peak areas. Data were analyzed using previously reported methods [RMG2_ methods , 55.GSE39204 , which cGSE6008, which was uploaded to publicly available online databases by Wu R et al. and contained 99 individual ovarian tumors ) , was alsHNF1β_sh1 cells and RMG2 control cells, was also analyzed to evaluate the relevance of HNF1β to metabolic function in OCCC.GSE37290 , a microhttp://www.broadinstitute.org/gsea/downloads.jsp).Total RNA expression was analyzed on the HG U133 Plus 2.0 Array (Affymetrix) (GSE39204 and GSE37290) and the HG_U133A array (Affymetrix) (GSE6008). The expression intensities were normalized across all samples using the robust multichip average algorithm . Gene seWhole cell lysates were prepared for western blot analysis. Equal amounts of protein were electrophoresed on SDS–PAGE gels and transferred to polyvinylidene fluoride membranes (Bio-Rad). Then, they were probed with the following antibodies: HNF1β ; rBAT/SLC3A1 ; β-Actin , and visualized by Chemi-Lumi One  and the ChemiDoc™ XRS+ system .Oxidative stress resistance was assessed by MTT assay using WST-8  after treatment with oxidative agents (ferric nitrilotriacetate or hydrogen peroxide) or CDDP. The concentration of oxidative agents required to inhibit cell proliferation by 50% (IC50) was calculated using Prism 5 software .2 for 24 hours and then in either 2% O2 (hypoxia) or 20% O2 (normoxia). Cell proliferation under hypoxia and normoxia was assessed by MTT assay using WST-8 (Cell Count Reagent SF) every 24 hours.Cells were pre-cultured in 20% O2 for 72 hours as previously described and then cultured in either glucose (+) medium , or glucose (−) medium . Both media were supplemented with 10% FBS and contained the same formulation except for the presence or absence of D-glucose. Cell proliferation was assessed by MTT assay using WST-8 (Cell Count Reagent SF) every 24 hours.Cells were pre-cultured in complete medium  at 37°C in 20% O2 and 5% COt-tests using Prism 5 software (GraphPad), and p values of < 0.05 were considered statistically significant. Data are shown as the mean ± standard deviation (SD).Statistical significance was assessed by two-tailed unpaired


Mycobacterium tuberculosis strain MT43, isolated from a pulmonary form of tuberculosis in French Polynesia. Analyzing its 4,145,007-bp, 65.17% G+C chromosome confirmed a fully antibiotic-susceptible Manu2 spoligotype.We announce the draft genome sequence of  Mycobacterium tuberculosis strain MT43 was isolated from a female 56-year-old French Polynesian patient suffering from pulmonary and pleural tuberculosis . Spoligotyping indicated the spoligotype international type SIT1634 (Manu2), which is one “ancestral” lineage of M. tuberculosis comprising only two isolates from the United States, one from Indonesia, and one from Taiwan in the SITVIT database. Recently, at least nine isolates belonging to SIT1634 were described in China, and this spoligotype has been reported to be associated with isoniazid susceptibility testing discrepancies  at 37°C by the cetyl-trimethylammoniumbromide method ; no SNPs conferring resistance were found, confirming the in vitro data that M. tuberculosis strain MT43 is fully susceptible to antibiotics.The genome was shown to encode at least 76 predicted RNAs, including 3 rRNAs, 45 tRNAs, 1 tmRNA, and 27 miscellaneous RNAs. A total of 2,786 genes spanning over 3,722,052 bp were also identified, representing a coding percentage of 89.8%. Among these genes, 274 (6.88%) were assigned as putative proteins and 560 (14.05%) were assigned as hypothetical proteins. Moreover, 2,786 genes matched at least one sequence in the Clusters of Orthologous Groups (COGs) database , 19 withM. tuberculosis strain MT43 has been deposited with its annotations at EMBL under the accession numbers CVMY01000001 to CVMY01000015.The genome sequence of


Neoadjuvant chemotherapy (NAC) is the standard treatment for patients with locally advanced breast cancer, showing improvement in disease-free survival (DFS) and overall survival (OS) rates in patients achieving pathological complete response (pCR). The relationship between immunohistochemistry-based molecular subtyping (IMS), chemo sensitivity and survival is currently a matter of interest. We explore this relationship in a Hispanic cohort of breast cancer patients treated with NAC.A retrospective survival analysis was performed on Colombian females with breast cancer treated at Instituto de Cancerología-Clinica Las Américas between January 2009 and December 2011. Patients were classified according to immunohistochemistry-based subtyping into the following five groups: Luminal A, Luminal B, Luminal B/HER 2+, HER2-enriched, and triple-negative breast cancer. Demographic characteristics, recurrence pattern, and survival rate were reviewed by bivariate and multivariate analysis.p = 0.01) but subtypes had different DFS in Luminal B (p = 0.01) and triple negative (p = 0.02) and also OS in Luminal B (p = 0.01) and triple negative (p = 0.01).A total of 328 patients fulfilled the study’s inclusion parameters and the distribution of subtypes were as follows: Luminal A: 73 (22.3%), Luminal B/HER2−: 110 (33.5%), Luminal B/HER2+: 75 (22.9%), HER2-enriched: 30 (9.1%), and triple-negative: 40 (12.2%). The median follow-up was 41 months (interquartile range: 31–52). Pathological response to NAC was as follows: complete pathological response (pCR) in 28 (8.5%) patients, partial 247 (75.3%); stable disease 47 (14.3%), and progression 6 (1.8%) patients. The presence of pCR had a significant DFS and OS in the entire group (pCR is associated with an improved overall survival and disease-free survival rates in this group of Hispanics patients. Advanced stages, Luminal B subtypes, triple-negative tumours and non-pCR showed lower DFS. Neodjuvant chemotherapy (NAC) has proven either to decrease tumour size in cancer patients not suitable for primary surgery or to increase the possibility of conservative surgery when it had previously been inadvisable. Currently, it must be considered an evaluative tool in designing a therapeutic strategy or biological factors that may help to determine the prognosis and treatment alternatives in a given patients.NAC has shown as significantly improved disease-free survival (DFS) and overall survival (OS) rates as adjuvant therapy in patients with breast cancer ; moreoveIn the last few years, breast cancer intrinsic molecular subtypes have been evaluated by gene expression array data, a technology not widely available worldwide. Immunohistochemistry-based molecular subtype with ER, PR, Ki67, and HER2 Neu has shown good correlation with gene expression assays to identify intrinsic subtypes .Previous reports state that not all patients have an equal response to NAC and that this response may vary depending on biological characteristics of the tumour and their immunohistochemistry-based molecular subtype, In basal-like tumours achieving a complete pathological response (pCR) rate in 45% (95%CI: 24–68), HER2+ in 45% (95%CI: 23–68), and Luminal A in 6% (95%CI:1–21) [Some publications suggest that pCR could be a surrogate marker for OS, particularly in the triple-negative and HER2 subgroups, with HER2+/hormone receptor (HR)-negative presenting a higher OS rate than HER2+/HR+ patients . AlthougAchieving pCR at the time of surgery has been associated with a favourable prognosis , 9. Not This study aims to determine the pathological response and its correlation with breast cancer immunohistochemistry-based molecular subtype as well as its correlation with DFS and OS rates in patients who have undergone NAC at Instituto de Cancerología- Clínica Las Américas (IDC), a comprehensive cancer centre in Colombia.A retrospective survival study was performed in women older than 18 years of age treated with NAC at the IDC between January 2009 and December 2011.The study included patients with histological diagnosis of breast cancer, Stages II and III, who received NAC, breast surgery and had a histopathological assay in our centre . NeitherAll demographic, clinical, and follow-up variables were obtained from the IDC patient’s registry, medical records, and surgical pathology reports. Pathology reports follow the College of American Pathologists (CAP) checklists. The ER, PR, and HER2 Neu were evaluated following the published CAP guideline recommendations , 12. AntPatient’s vital status was traced through phone calls. IDC´s IRB approved the research project.Hormone receptor status (ER and PR) is considered positive if IHC staining is ≥1%. HER2 is positive in tumours showing IHC +++ or IHC ++ by positive FISH (≥ 2.4).Immunohistochemistry-based molecular subtypes definition:‘Luminal A’: ER+ and/or PR+, HER2−, Ki-67 < 14%‘Luminal B’: ER+ and/or PR+, HER2−, Ki-67 ≥ 14%‘Luminal B/HER 2+’: ER+ and/or PR+, HER2+‘HER2-enriched’: ER− and PR−, HER2+‘Triple negative’: ER−, PR−, HER2−The chemotherapy schema utilised were znthracyclines and taxanes, anthracyclines alone, and taxanes only. Patients who had contraindicated the use of anthracyclines was included in the taxane scheme (docetaxel–cyclophosphamide) or treated with cyclophosphamide, methotrexate, and fluorouracil (CMF).in situ cancer in the breast and axillary nodes) [As described in a recent paper, a pCR was defined as ypT0 ypN0 (the absence of invasive cancer and y nodes) .t-test or one-way analysis of variance (ANOVA) was used for numeric variables. Survival rate was calculated up until the date of relapse, death, or last control check. Kaplan–Meier curves were assessed according to pCR, subtypes and clinical variables that were compared with a Breslow test. In the multivariate analysis, all variables with clinical or statistical significance (p < 0.25) in bivariate analysis were included. All variables were categorised for multivariable analysis. Adjusting for potential confounders was performed with Cox regression analysis. The statistical significance for the variables included in the multivariate model was set at a level of statistical significance of p < 0.05. A two-tailed p value was established at > 0.05. SPSS software  was used in the statistical analysis.Bivariate analysis by intrinsic subtypes was performed. The chi-square or Fisher’s exact test were used for the categorical variables, and the Sixty-five (19.8%) patients were Stage II and 263 (80.2%) Stage III; tumour size was ≥ 30 mm in 93.8% of patients. The chemotherapy schema used was anthracyclines and taxanes in 272 (82.9%), anthracycline alone in 46 (14.1%), taxanes only in 6 (1.8%) and CMF in the remaining 4 (1.2%) patients. Out of 105 (32%) patients with HER2-positive cases 87 (82.9%) received trastuzumab in the neoadjuvant setting.After neoadjuvant chemotherapy, 226 (68.9%) patients had mastectomy, 37 (11.3%) of them with early reconstruction, and breast-conserving surgery in 102 (31.1%). According to our institutional protocol at that time, axillar dissection was performed directly in 307 (93.6%) cases, and sentinel lymph node post-NAC was performed in only 28 (8.5%) patients.Involved lymph nodes were found in 187 (57.7%) cases: 88 had 1–3, 60 from 4–9, and in 39, more than nine were involved.Immunohistochemistry-based molecular subtype distribution was as follows: ‘Luminal A’: 73 (22.3%); ‘Luminal B’: 110 (33.5%); ‘Luminal B/HER2+’: 75 (22.9%); ‘HER2-enriched’: 30 (9.1%); and ‘triple negative’: 40 (12.2%). Median time elapsed between histopathological diagnosis and NAC was 56 days (IQR: 34–103), median time of duration of NAC was 175 days (IQR: 144–193). 271 (82.6%) patients received the whole-planned chemotherapy schema. Of the remaining 57 cases, 15 had drug intolerance, 10 disease progression, 13 poor drug response, six due insurance coverage issues, six because of patients desire, and seven for different comorbidities.n = 28), partial responses in 75.3% (n = 247), the disease remained stable in 14.3% (n = 47), and there was progression in 1.8% (n = 6) patients. The rates of pCR differed (p < 0.01) among subtypes: 27.5% of ‘Triple negative’, 13.3% of ‘HER2-enriched’, 13.5% of ‘Luminal B/HER2+’, 2.7% of ‘Luminal B’, and none in ‘Luminal A’ . despite finding statistical significance. In multivariate analysis, no association was found in DFS and OS for ki67.In 328 patients, we evaluate the Ki-67 ≤ 14. Among the 140 patients with Ki-67 ≤ 14, 20/140 patients (14.3%) had pCR, versus 8/188 patients (8.5%) had pCR with Ki-67 >14 (About 225 of our patients (68.6%) receive hormonotherapy as a adjuvant treatment, tamoxifen (202) was the upfront medication in this group of patients in the majority of the cases 89.9%.Median follow-up was 41 months (IQR: 31–52). Alive without disease evidence 213 (64.9%), alive with active disease 26 (7.9%), 78 (23.8%) had died, and 11 (3.3%) were missing from the follow-up.p < 0.01) (p = 0.03) (p > 0.05).The DFS and OS rates differed according to the intrinsic subtype ( < 0.01) . Differe = 0.03) , but notThe multivariable models are depicted in In the analysis of the results of our study, some important characteristics from the patients treated with NAC in our centre were considered. First, 80.2%, in patients had locally advanced tumours (Stage III), implying a larger tumour load and lower survival expectancy. This could also explain why the most common surgeries for these patients were mastectomy (68.9%).The immunohistochemistry-based molecular subtype distribution of our patients, both in this neoadjuvant group and in our database  show difOur overall frequency of pCR was low, that is 8.5%, and is inferior to the values reported by other groups (13%–15%) , 20, 21,Other papers have reported improved long-term outcomes in patients with pCR , 20–22. et al [The association of pCR with an improved DFS rate is observed in the literature , 23, 25 et al .The immunohistochemistry-based molecular subtype distribution in our series shows important differences with the German series : ‘LuminaIn conclusion, it is evident that for our patients, pCR is associated with an improved DFS and OS rate. In our cohort, achievement of pCR was more frequent in ‘triple negative’ and ‘HER2-enriched’ patients, as has been previously shown in other studies. We believe that the low pCR rate in our trial was due to the advanced cancer stages in the majority of our population (80.2% were Stage III). This needs to be confirmed in prospective studies. We do not have an explanation for the high frequency of HR+ tumours in our population. It could be ethnic and deserves further investigation.None declared.Instituto de Cancerología- IDC, Comprehensive Cancer Center in Colombia.The concept and design of the study was contributed by Rodolfo Gómez, Carlos Andrés Ossa, Héctor García. Rodolfo Gómez, Carlos Andrés Ossa, Fernando Herazo, Carolina Echeverri, Alejo Jiménez, Jorge Madrid, Mónica Gil, Sabrina Herrera María Elvira Montoya, Gonzalo Ángel, Mauricio Borrero, Pedro Reyes, and Eduardo Gutiérrez were involved in material supply and patient referral. Data collection and processing was done by Lina Zuluaga, Johana Ascuntar. Data analysis and interpretation were performed by Rodolfo Gómez, Carlos Andrés Ossa, Héctor García. Rodolfo Gómez, Carlos Andrés Ossa, Fernando Herazo, and Héctor García were involved in the final manuscript approval.


Despite this, the association between LeuRS and lactation in dairy cow mammary epithelial cells (DCMECs) remains unknown. In this study, we found that LeuRS expression in mammary gland tissue was significantly higher during lactation than pregnancy. Moreover, our data demonstrates that LeuRS is localized in the cytoplasm. Treatment with leucine increased DCMECs viability and proliferation, as well as mammalian target of rapamycin (mTOR), p-mTOR, ribosomal protein S6 kinase 1 (S6K1), p-S6K1, β-Casein, sterol regulatory element binding protein 1c (SREBP-1c), glucose transporter 1 (GLUT1), and Cyclin D1 mRNA and protein expression. Secretion of lactose and triglyceride were also increased. siRNA-mediated knockdown of LeuRS led to reduction in all of these processes. Based on these data, LeuRS up-regulates the mTOR pathway to promote proliferation and lactation of DCMECs in response to changes in the intracellular leucine concentration.The role of LeuRS, an aminoacyl-tRNA synthetase, as an intracellular  More specifically, the effect of hormones and amino acids on lactation in the dairy cow has been studied since the last century –3. LeuciThe precise mechanism of how mTORC1 activation is mediated by intracellular leucine sensing is poorly understood. Several studies have identified LeuRS as a key intracellular regulatory factor of the mTORC1 pathway in 293T cells –19. Howe2.2.1.In this study, LeuRS was expressed in both pregnancy and lactating mammary glands in Holstein dairy cows. In pregnancy mammary gland tissue LeuRS showed no difference in the acinar buds  and west2.2.in vitro and cultured for five to ten days to produce a mixed culture of fibroblasts and a small number of mammary epithelial cells , was proposed to be activated by amino acid and involved in the transduction of signals from amino acids to mTORC1 –31. Most signals –34. ThesThe molecular mechanism of leucine-mediated control of lactoprotein synthesis remains poorly understood ,36. Our To address the mechanism of leucine activation of mTORC1, we investigated the effect of LeuRS knockdown on DCMECs proliferation and protein expression. Our data demonstrate that siRNA-mediated silencing of LeuRS decreased cell proliferation and the expression of lactation- and mTOR signaling-related proteins, suggesting that LeuRS is a regulatory factor involved in the amino acid/mTOR signaling pathway in DCMECs. Moreover, LeuRS functions as a GTPase-activating protein (GAP) for Rag GTPase to activate mTORC1, indicating that LeuRS is a key mediator of amino acid signaling to mTORC1 in 293T cells . The amiet al. proposed that leucine stimulates glucose transport in skeletal muscle via the PI3-kinase and PKC pathways independent of mTOR and insulin [et al. reported that to a certain degree, amino acid availability had no effect on lactose secretion [The ability of mammary glands to produce milk is determined by the number of cells and their level of viability . The mai insulin . Howeverecretion ,44. It iecretion –47. Thus3.3.1.n = 3 per group) were approved by the Animal Care Committee of the Northeast Agricultural University.Reagents were purchased from Sigma-Aldrich Canada  unless otherwise stated. Fetal bovine serum (FBS) and Dulbecco modified Eagle medium: F12 (DMEM/F12) base were obtained from GIBCO BRL . Procedures involving animals  to assess whether cells arose from these pieces. The medium was changed once after day 3. For 15–30 days, the cell culture bottle was covered with fibroblasts, myoepithelial cells, mammary epithelial cells, etc. Dairy cow mammary epithelial cells were purified according to previous published reports [Mammary gland parenchymal tissues were individually isolated from three mid-lactation Holstein dairy cows of high genetic merit. The tissue soaked with 75% ethanol for 5 min before removing a portion of the outer periphery of interstitial tissue and fascia, adipose tissue,  reports . Due to 4 cells/cm2 in six-well culture plates, and the medium was replaced with experimental culture medium (based on the basic culture mediun plus 5 μg/mL prolactin and a certain concentration of leucine). DMEM/F12 base media contained leucine (0.45 mmol/L) at a level sufficient for normal cellular processes, but not at a concentration optimal for DCMECs differentiation and lactation. Therefore, it was necessary for the addition of leucine to measure the dosage effects of leucine on lactation in DCMECs.The purified cells were cultured in basic culture medium . Prior to experimental treatments, cells were plated at 3 × 103.2.Cell viability and cell proliferation were determined by using a CASY-TT Analyser System  according to the manufacturer’s instructions. After calibration with live and dead DCMECs, cursor positions were set to 11.75 to 50.00 μm  and 7.63 to 50.00 μm (standardization cursor). DCMECs were trypsinized and then diluted (1:100) with CASY electrolyte solution prior to examination. Three 100 μL aliquots were analyzed in sample .3.3.w/v) paraformaldehyde for 40 min, and then rinsed again with PBS. The lipid droplets in the cells were stained with 5% Oil red O in isopropanol for 15 min and then rinsed again with PBS. Nuclei were stained with Giemsa working solution for 15 min and then examined microscopically. The lipid droplets were stained red while nuclei were stained blue [Cells cultured in six-well culture plates were rinsed three times in phosphate-buffered saline (PBS), fixed in 4% . Analysis of cell cycle distribution was subsequently performed using the Modfit software [The proportion of DCMECs in various different cell cycle phases was measured by flow cytometric analysis. DCMECs were plated at 3 × 10software .3.5.DCMECs were transfected with either LeuRS-specific siRNA  or negative control siRNA , using Lipofectamine™ 2000  according to the manufacturer’s protocol. After about 4–6 h, the medium in the three groups was replaced with experimental culture medium and contained an extra 1.2 mmol/L leucine. The sequences of siRNA were listed below. LeuRS-specific siRNA: sense5′-GCAGGUCCUGUGGAUGAAATT-3′, antisense5′-UUUCAUCCACAGGACCUGCTT-3′; negative control siRNA: sense5′-UUCUUCGAACGUGUCACGUTT-3′, antisense5′-ACGUGACAC GUUCGGAGAATT-3′.3.6.w/v) ice-cold paraformaldehyde for 10 min, and then rinsed in PBS at room temperature for 5 min. Next, fixed tissues were incubated in blocking buffer ) for 1 h at 37 °C, before incubation with anti-LeuRS primary antibody  at 1:100 dilution for 1.5 h at 37 °C. After rinsing in PBS/T, specimens were incubated in the dark with FITC-conjugated donkey anti-rabbit IgG at a 1:200 dilution for 1 h at 37 °C. Then the slides were incubated in blocking buffer again, incubated with anti-Giantin primary antibody  at 1:100 dilution for 1.5 h at 37 °C. Specimens were incubated in the dark with Cy5-conjugated donkey anti-goat IgG at 1:200 dilution for 1 h at 37 °C, and afterwards rinsed three times in PBS/T, then incubated with DAPI for 15 min at 37 °C, and rinsed in PBS/T; the coverslips were visualized using a Leica TCS-SP2 AOBS confocal laser scanning microscope [Slides and coverslips were soaked overnight in chromic acid solution. Then, the slides were rinsed with tap water to remove residual acid, rinsed with distilled water three times, and then rinsed with 75% ethanol. Before use, the frozen sectioning machine  was pre-cooled to −20 °C for 2 h, and then the slices were made into 7–8 μm. The labeled frozen sections were fixed in 4% (croscope .3.7.w/v) ice-cold formaldehyde at 4 °C for 10 min. The slides were rinsed three times with TBS/T for 5 min. To detect endogenous CK18, β-Casein and LeuRS, fixed DCMECs were incubated in blocking buffer  for 1 h at 37 °C, and then incubated with anti-CK18 primary antibody , anti-β-Casein primary antibody  and anti-LeuRS primary antibody at 1:100 dilution for 1.5 h at 37 °C, respectively. After rinsing three times in TBS/T, specimens were incubated in the dark with FITC-conjugated second antibodies at 1:200 dilution for 1 h at 37 °C and incubated with PI or DAPI for 15 min at 37 °C, respectively. Finally, after rinsing three times in TBS/T, the coverslips were visualized using a Leica TCS-SP2 AOBS confocal laser scanning microscope [DCMECs were seeded on glass coverslips to 30%–50% confluency in six-well plates. The cells were rinsed twice with PBS and fixed in 4%  Assay Kit, Megazyme, Ireland, UK) according to the manufacturer’s recommended protocol [The amount of triglyceride and lactose secreted into the culture medium was determined using commercially available assay kits  and were tested for statistical significance (p < 0.05 or p < 0.01) by using SPSS 16.0 . A statistical comparison of the means among the groups was performed using one-way analysis of variance. Differences between the means of individual groups were analyzed by Tukey post hoc tests. Statistical significance was declared at p < 0.05 or p < 0.01.All data were expressed as the mean ± standard deviation (4.Leucine can modulate cell proliferation and lactoprotein, as well as lactose and triglyceride synthesis, by increasing expression of LeuRS in DCMECs. LeuRS is a key mediator for amino acid signaling to mTORC1 that functions in response to changes in the intracellular leucine concentration. LeuRS can mediate cell proliferation and the expression of mTOR, S6K1, SREBP-1c, GLUT1, Cyclin D1, and β-Casein to promote lactation in DCMECs.


In conclusion, this study identified potential biomarkers for MHE in HCC. The cytokines investigated may induce neural apoptosis via STAT3 in the pathogenesis of MHE in HCC.Hepatocellular carcinoma (HCC) patients were grouped according to the degree of encephalopathy, with healthy volunteers as controls. We investigated clinical presentation, protein and mRNA expression of 14 cytokines, and activation of six STAT proteins, the downstream signaling mediators. Levels of all 14 cytokines were significantly elevated in HCC patients with clinical hepatic encephalopathy. Statistical analysis showed that levels of IL-1β, IL-6, IFNγ, IL-17α, IFNλ2 and IFNλ3 were correlated with minimal hepatic encephalopathy (MHE). Multivariate regression analysis identified serum IL-6, IFNλ3 and IL-17α as independent risk factors for MHE. Increased mRNA levels of IL-6 and IFNγ were associated with MHE. Among the STAT proteins examined, only STAT3 was elevated in MHE. Treatment with a STAT3 inhibitor protected neurons from cytokine-induced apoptosis  Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, and a major cause of death in areas with a high incidence of viral hepatitis2466Serum cytokines are key mediators for many physiological and pathological modulations including inflammation, cancer development, human nervous system development, bidirectional signal transduction between the central and peripheral nervous systems, and cognitive processes, amongst others911121314111213161718The objective of this study was to analyze the clinical and laboratory data of a group of patients with HCC, and to evaluate the correlations between serum cytokine levels and MHE. This aim of this study was to identify serum markers and potential pathogenetic mechanisms of MHE in patients with HCC in order to facilitate more accurate diagnosis of MHE, and facilitate early intervention that could potentially reduce mortality.Our study population consisted of 121 HCC patients who were treated at our institution from April 2010 to July 2014. Sixty healthy controls were included in this study. The exclusion criteria included history of infectious diseases, autoimmune diseases, allergic response, immune deficiency disorder, diabetes, psychiatric illness, malignancy, severe cognitive impairment, or a systemic or CNS infection two weeks before sample collection. The diagnosis of HCC was determined by liver biopsy with imaging tests such as computed tomography (CT) or magnetic resonance imaging (MRI).Subjects involved in this study were grouped as follows: (1) Controls: healthy volunteers without liver disorders; (2) CHE : HCC patients with a clinical hepatic encephalopathy diagnosis based on the West Haven criteria; (3) HCC patients without CHE were divided into two subgroups: (a) MHE  and (b) No MHE patients: patients were diagnosed by performing the psychometric test .None of the involved patients had undergone a transjugular intrahepatic portosystemic shunt, nor did they have any history of spontaneous bacterial peritonitis. All patients were on normal diet, except for those with ascites who were on hyposodic diet, and for those with CHE who were on protein-restricted diet.Psychometric tests were performed immediately following a standard history and physical examination. Within the same day, blood was drawn for routine laboratory investigations and cytokine level determination.The Beijing You’an Hospital Ethics Committee approved the study, and all involved patients gave written informed consent for their clinical data and samples  to be used in this study. All experiments in this study were performed in accordance with the human experimentation guidelines of the People’s Republic of China.Human neuronal cells were purchased from Procell Inc. . The cells were maintained and grown as described in a previous publicationBlood samples were collected by venipuncture into EDTA-anticoagulation vials. Plasma was harvested from the collected samples within 30 min and stored at −80 °C for subsequent cytokine analysis. Concentrations of IL-2, IL-4, IL-6, IL-8, IL-10, IFNγ, GM-CSF, TNFα , IL-17a , IL-1β , IL1Ra , IFNλ1, IFNλ2, IFNλ3, IFNλ4 , and IL-23  were measured by ELISA according to manufacturers’ instruction16226 cells were briefly trypsinized, washed twice with cold PBS, fixed in 4% paraformaldehyde at 4 °C for 20 min, and washed again with PBS before permeabilization with 0.5 ml 0.5% saponin at 22 °C for 5 min. The cells were washed with PBS, incubated with 80 μl equilibration buffer at 22 °C for 5 min, washed with PBS again, then resuspended in 50 μl Nucleotide Mix and incubated in the dark at 37 °C for 1 h. Cells were washed again with PBS and analyzed by fluorescence microscopy.Neuronal cells were stimulated with IL-1β, IL-6 , IFNλ3 , IFNγ  and IL-17a  for 72 hrs. TUNEL assays to determine DNA fragmentation in apoptotic cells were performed according to the manufacturer’s protocol (Promega). In brief, 3−5 × 10Neuronal cells were first fixed and permeabilized using BD Cytofix/Cytoperm Fixation and Permeability Solution , followed by staining for intracellular proteins, and fixation with 2% formaldehyde. Antibodies against pSTAT1 , pSTAT2 , pSTAT3 , pSTAT4 , pSTAT5a  and pSTAT5b  were purchased from BD Pharmingen . STAT3 inhibitor V (S3I) was obtained from CalBiochem/EMD biosciences . Fluorescence was evaluated with a FACSCalibur flow cytometry and data was analyzed using FlowJo software .MHE was diagnosed by the PHES battery, which has been regarded as the gold standard of MHE diagnosisThis test was used to assess the prognosis of chronic liver diseases, especially cirrhosis. There are five clinical measures involved, which are total bilirubin, serum albumin, prothrombin time, ascites and hepatic encephalopathy. Chronic liver disease prognosis could be classified into Child-Pugh classes A to C.This is a scoring system for assessing the severity of chronic liver disorders, and includes measures such as serum bilirubin, creatinine, and clinical etiology.r2. All analyses were performed by SPSS software (version 12.0). A P value of <0.05 was considered to be statistically significant.Averages of numerical variables were presented as The demographic and clinical data of 121 HCC patients and 60 healthy controls are collected and summarized in To identify potential serum biomarkers to differentiate MHE, we examined the concentration of 14 serum cytokines: IL-1β, IL-1Ra, IL-2, IL-4, IL-6, IL-8, IL-10, IFNγ, IL-17a, IL-23, IFNλ1, IFNλ2, IFNλ3 and IFNλ4. The results of statistical analysis on each cytokine and ammonia are shown in IL-1β and IL-6 levels were dramatically elevated in HCC patients , Table 3Since circulating IL-6 and other cytokines tend to elevate when liver function deteriorates, we analyzed whether the relationship between IL-6 concentration and MHE was independent of liver function (MELD scores). As shown in The mRNA levels of all 14 cytokines were also evaluated, and the results for IL-1β, IL-6, IFNλ3, IFNγ and IL-17a are shown in Since IL-1β, IL-6, IFNλ3, IFNγ and IL-17a all activated the JAK-STAT pathway, we next investigated activation STAT protein family members in circulating PBMCs of patients at different stages of HE. All six STATs were strongly activated in CHE, compared to controls .


We examined cell isolation, viability, and growth in adipose-derived stem cells harvested from whole adipose tissue subject to different cryopreservation lengths (2–1159 days) from patients of varying ages (26–62 years). Subcutaneous abdominal adipose tissue was excised during abdominoplasties and was cryopreserved. The viability and number of adipose-derived stem cells isolated were measured after initial isolation and after 9, 18, and 28 days of growth. Data were analyzed with respect to cryopreservation duration and patient age. Significantly more viable cells were initially isolated from tissue cryopreserved <1 year than from tissue cryopreserved >2 years, irrespective of patient age. However, this difference did not persist with continued growth and there were no significant differences in cell viability or growth at subsequent time points with respect to cryopreservation duration or patient age. Mesenchymal stem cell markers were maintained in all cohorts tested throughout the duration of the study. Consequently, longer cryopreservation negatively impacts initial live adipose-derived stem cell isolation; however, this effect is neutralized with continued cell growth. Patient age does not significantly impact stem cell isolation, viability, or growth. Cryopreservation of adipose tissue is an effective long-term banking method for isolation of adipose-derived stem cells in patients of varying ages. Adipose-derived stem cells (ASCs) are adult mesenchymal stem cells that have garnered significant attention since their description in humans in 2001 by Zuk et al. subsequent to their initial identification in animal models  and becaBecause ASCs can differentiate into any cell type of mesenchymal origin, including muscle, fat, bone, and cartilage, they have been hypothesized to have broad clinical applications in regenerative medicine including cellular repair after myocardial infarction, breast reconstruction, bone and cartilage regeneration after trauma, cancer, and autoimmune disorders . Recent Several animal models have previously examined the effect of cryopreservation on ASCs. It has been shown that human lipoaspirate frozen for seven days and injected into mice displayed similar fat graft growth and resorption rates compared to freshly injected lipoaspirate . LikewisA limited number of studies in humans have examined the effects of cryopreservation on lipoaspirate and isolated ASCs. Lipoaspirate frozen for less than a month demonstrated no change in phenotypic markers, proliferative capacity, or differentiation potential . Further2 and did not differ significantly between the young (27 ± 3 kg/m2) and advanced age groups (29 ± 6 kg/m2). The majority of patients were of Caucasian descent (87%), while the remaining patients were of African American descent (13%), without significant differences between the young and advanced age groups. No patients had been diagnosed as prediabetic or diabetic prior to adipose isolation.Subcutaneous abdominal fat was excised during abdominoplasties between November 2010 and January 2015 from patients of different ages. Tissue was obtained from 32  patients, chosen at random. All procedures were conducted using informed consent under the University of Pennsylvania IRB approval (Protocol number 812150). On the day of the procedure, tissue was excised, maintained on ice, transported to the lab, aliquoted into 50 mL conical tubes, and stored at −70°C. No cryopreservation or other agents were used in the freezing of the whole adipose tissue specimens. Tissue from 32 patients with an age range of 26–62 years (average 43.2 ± 9.7 years) and cryopreservation time (−70°C) of 2–1159 days (average 596.4 ± 369.9 days) was analyzed. Average patient BMI was 28 ± 5 kg/mμm cell strainer . An additional 2 mL of Stem Cell Media was rinsed through the strainer to obtain any residual cells from the filter. The plate was incubated at 37°C in 5% CO2 and the Stem Cell Media was changed 48 hours after plating to remove debris and nonadherent cells.Standard methods for isolating and purifying ASCs, separating them from the stromal vascular fraction containing fibroblasts, pericytes, preadipocytes, monocytes, and macrophages, as well as smooth muscle, endothelial progenitor, and red blood cells of the SVF, have been well established and were employed here , 26, 27.n = 28, p0-1) were replated to a density of 1 × 105 cells/well on a 12-well plate. Cells were subsequently measured again at 9, 18, and 28 days after the initial analysis. Data were analyzed with respect to the following independent variables: cryopreservation time: <1 year (N = 10), 1-2 years (N = 5), and >2 years (N = 17), and patient age: <40 years (N = 12) versus ≥40 years (N = 20) and <50 years (N = 23) versus ≥50 years (N = 9).Cells from the SVF pellet were grown in Stem Cell Media (as described above), that was changed twice weekly. In accordance with accepted ASC isolation protocols, 48 hours after SVF pellet plating, viable and adherent cells were considered to represent adipose-derived stem cells , 26. 17 5 cells were resuspended in 100 μL FACS buffer containing 0.1% BSA  in PBS. For FACS analysis of surface markers, each sample was incubated with 5 μL of anti-human PE-conjugated, PE-Cy7-conjugated, or APC-conjugated antibodies: CD34 , CD45, and CD105 , and then diluted in FACS buffer for 30 minutes at 4°C. Unstained cells and single-color-stained cell controls were prepared for compensation. After incubation, the excess and nonspecifically bound antibodies were removed by multiple washes in FACS buffer. Cells were pelleted and resuspended in 500 μL of FACS buffer and transferred to polypropylene tubes. Propidium Iodide  staining was used to determine cell viability. The surface expression of live cells (approximately 104 cells per patient) was characterized by FACS  using the BD FACSCalibur flow cytometer . Results were analyzed using FlowJo software version 10.0.6 .ASCs were grown until confluent, trypsinized, and pelleted by centrifugation at 500 g for five minutes. For fluorescence-activated cell sorting (FACS) analysis, approximately 2.5 × 103 cells/well and grown in adipogenic media comprised of DMEM/F12 Medium supplemented with 10% FBS, 1 μmol/L dexamethasone, 10 μg/mL insulin, and 0.5 mmol/L isobutylmethylxanthine . ASCs were grown for 14 days, with media changes every 3 days, after which ASCs were stained with Oil Red O  as per manufacturer's protocol.ASCs were seeded in a 24-well plate at a density of 5 × 103 cells/well and grown in osteogenic media comprised of DMEM/F12 Medium supplemented with 10% FBS, 0.1 μmol/L dexamethasone, 10 mmol/L β-glycerol phosphate, and 50 μmol/L ascorbate. Cells were grown for 21 days, with media changes every 3-4 days, after which ASCs were stained with alkaline phosphatase  as per manufacturer's protocol.ASCs were seeded in a 24-well plate at a density of 5 × 10n = 11) and the human lung fibroblast IMR90 cell line (n = 3) using standard TRIzol-chloroform extraction  and the RNeasy Mini Kit . cDNA was produced using a High Capacity RNA-to-cDNA Kit  as per manufacturer instructions. Forward and reverse primers were designed for GAPDH , Nanog , Oct4 , and Sox2 . qPCR was prepared as a 10 μL reaction using a SYBR Green PCR Master Mix Kit , run on a 7900HT Fast Real-Time PCR machine , and analyzed using SDS 2.4 software. Transcript levels were normalized against expression levels of the housekeeper GAPDH using the 2−ΔΔCT method. Three technical replicates were performed for each assay and the results were averaged.Total mRNA was extracted from individual ASC lines . Unpaired, two-tailed T-tests were utilized to generate P values comparing all involved cohorts.Student 4 cells/g adipose tissue harvested, averaging 2.95 × 104 ± 2.5 × 104 cells/g tissue. ASC isolation from frozen tissue of cohorts <1 year, 1-2 years, and >2 years had average live cell count values of 4.06 × 104 ± 1.36 × 104 cells/g tissue, 2.29 × 104 ± 2.24 × 104 cells/g tissue, and 1.87 × 104 ± 1.12 × 104 cells/g tissue, respectively. We observed an inverse relationship between the number of live ASCs isolated in relation to the number of days the tissue was frozen . No significant differences were found between other groups (P > 0.05). In contrast, cryopreservation length was independently associated with initial number of cells isolated irrespective of patient age , 18 days , or 28 days in culture , <50 years versus ≥50 years , and <40 years versus ≥50 years . No significant differences in live ASC isolation were observed between any of the age cohorts, P > 0.05  <40 years versus ≥40 year cohorts after 9 days of growth , 18 days of growth , or 28 days of growth  in culture; (2) <50 years versus ≥50 year cohorts after 9 days of growth , 18 days of growth , or 28 days of growth  in culture; (3) <40 years versus ≥50 cohorts after 9 days of growth , 18 days of growth , or 28 days of growth  comprising a range of patient ages (26–62 years) and cryopreservation times (2–1159 days) as compared to IMR90 (n = 3) controls  significantly reduces the number of live ASCs isolated relative to short-term cryostorage <1 year, . FurtherConsequently, our data support whole adipose tissue cryopreservation as a valuable method for long-term ASC preservation. The observation that patient age had little impact on initial cell number, viability, or long-term cell growth further supports the suitability of this biobanking technique Figures –6 and thThis study provides evidence that whole adipose tissue cryopreservation can serve as the gold standard for long-term ASC biobanking. Because of the incremental decrease in subcutaneous adipose tissue mass with advancing patient age, it behooves us, when feasible, to bank this valuable tissue prior to its diminution, ideally by middle age. Additional studies are required to further elucidate putative effects of long-term cryopreservation on precise ASC functions and differentiation potentials in patients of different ages. This is particularly important in light of recent literature questioning the ability of directly cryopreserved ASCs to undergo osteogenic differentiation , 34. FutAdipose-derived stem cells are multipotent, abundant, and easily isolated cells that represent a powerful tool for regenerative medicine. Unfortunately, much of the research regarding these cells is performed on fresh lipoaspirates that may not be readily available in therapeutic clinical scenarios. Though cryopreservation technologies continue to advance, the effects of long-term adipose tissue cryopreservation on ASC isolation have not been adequately characterized and remain a limiting factor in advancing ASC-based regenerative medicine applications. Herein we demonstrate that long-term adipose tissue cryopreservation (>2 years at −70°C) does significantly reduce the number of ASCs initially isolated per gram of tissue. However, this primary attenuation in ASC isolation is neutralized upon extended culturing as demonstrated by comparable ASC proliferative capacity, irrespective of cryopreservation duration or patient age. Together, our data suggest that ASCs are capable of enduring long-term cryopreservation while maintaining their stemness, further reinforcing their important contribution to regenerative medicine applications. We anticipate that data such as those reported here will be critical for the development of safe and effective long-term human adipose tissue biobanking technologies, optimization of ASC isolation protocols using minimal tissue/cell processing, and validation of specific clinical therapeutic applications for ASCs. In conclusion, we advocate for whole adipose tissue cryopreservation in preparation for future ASC-based regenerative medicine therapies.


Gloriosa superba and Catharanthus roseus are useful in traditional medicine for treatment of various skin diseases and cancer. However, their molecular effect on psoriasis has not been investigated. In this study, the effect of ethanol extracts derived from G. superba leaves and C. roseus stems on the expression of psoriatic marker, keratin 17 (K17), was investigated in human keratinocytes using biochemical and molecular experimental approaches. Both extracts could reduce the expression of K17 in a dose-dependent manner through JAK/STAT pathway as demonstrated by an observation of reduced phosphorylation of STAT3 (p-STAT3). The inhibitory activity of G. superba extract was more potent than that of C. roseus. The Pearson's correlation between K17 and cell viability was shown positive. Taken together, the extracts of G. superba and C. roseus may be developed as alternative therapies for psoriasis. Psoriasis is a chronic inflammatory skin disease with hyperproliferation and abnormal differentiation of keratinocytes . This diK17 is classified as a type of epithelial keratins functioning in providing the epidermis integrity , and it Gloriosa superba and Catharanthus roseus are herbs used in traditional medicine for centuries. G. superba has been used to treat intestinal worms, bruises, infertility, joint pain, cancer, Kapha and Vata conditions, and skin problems [ C. roseus consists of many alkaloids that are useful for treatment of menorrhagia, diabetes, hypertension, and cancer [problems , 15. C. d cancer . G. superba and C. roseus on K17 expression was investigated for use as an alternative medicine for psoriasis treatment.In this study, the molecular effect of ethanol extracts ofμg/mL of streptomycin) were purchased from HyClone . Recombinant human IFN-γ was purchased from PeproTech . 3--2,5-diphenyltetrazolium bromide (MTT) was purchased from Calbiochem .All reagents used in this study were of analytical grade. Dimethyl sulfoxide (DMSO) and ethanol were purchased from Merck . 1,4-Dithiothreitol (DTT) was purchased from Bio Basic Inc. . Phenylmethyl sulphonyl fluoride (PMSF) was purchased from United States Biochemicals . Bovine pancreatic ribonuclease A (RNAse A) was purchased from US Biological . Triton X-100 was purchased from SPI Supplies . Propidium iodide (PI) and trypan blue solution were purchased from Invitrogen . 4′,6-Diamidino-2-phenylindole (DAPI), piceatannol, PD98059, and Kodak processing chemicals for autoradiography films were purchased from Sigma Aldrich Co. . Dulbecco's modified Eagle medium (DMEM)/high glucose, fetal bovine serum (FBS), and penicillin-streptomycin solution  was purchased from Biorad . Ammonium persulfate (APS) was purchased from EMD Millipore . Amersham ECL Select Western blotting detection reagent and Hyperfilm ECL were purchased from GE Healthcare . The monoclonal rabbit anti-keratin 17 , polyclonal rabbit anti-phospho (p)-STAT3 , HRP-linked anti-rabbit IgG (cat#7074S), and anti-rabbit IgG conjugated Alexa Fluor 488 (cat#4412S) antibodies were purchased from Cell Signaling Technology . The polyclonal rabbit anti-GAPDH  antibody was purchased from Santa Cruz Biotechnology .μg/mL streptomycin) at 37°C in a humidified atmosphere at 5% CO2.HaCaT cells, an immortalized human epidermal keratinocyte cell line, were purchased from cell line service . They were cultured in DMEM/high glucose containing 10% FBS and antibiotics  and authenticated by Professor Dr. Thaweesakdi Boonkerd . The voucher specimens deposited at Prof. Dr. Kasin Suvatabhandhu Herbarium  were 013412 (BCU) and 013415 (BCU) for C. roseus and G. suberba, respectively. Parts used for this study were leaves and stems of G. superba and C. roseus, respectively. G. superba and C. roseus were extracted using ethanol (ratio1 : 10) at room temperature for 48 hours. The resulting products were subsequently filtered, and residues were extracted twice. Ethanol was removed by evaporation at 45°C. Final concentration of crude extracts was 100 mg/mL dissolved in DMSO as stock solutions and stored at −20°C until use. For experiments, working concentrations were prepared from the original stocks.Bothγ at the final tested concentration of 1 ng/mL for 24 hours. After stimulation, media were changed. Cells were treated with G. superba extract (working concentrations of 0 to 100 μg/mL to result in tested concentrations of 0 to 10 μg/mL) or C. roseus extract  or 100% DMSO as a positive control or media alone as a negative control for 48 hours. MTT stock solution (5 mg/mL) was added to each well (20 µL/well) and incubated for 4 hours. In this step, formazan product was formed. Media were removed carefully and formazan product was dissolved with 150 μL of 100% DMSO. Supernatant was collected by centrifugation, transferred to a new 96-well plate, and measured the absorbance of the reaction with a spectrophotometer at 550 nm. The percentage of cell viability was calculated using the formulaCells were seeded at 10,000 cells/well in 96-well plates for MTT and 100,000 cells/well in 12-well plates for trypan blue and incubated at 37°C for 24 hours. Having been incubated, cells were stimulated by IFN-50) of each plant extract was calculated from the graph plotting inhibition percentage against extract concentration. For trypan blue exclusion, having been incubated with each extract for 48 hours, cells were harvested and resuspended in media. The mixture of cell suspension and 0.4% trypan blue (ratio 1 : 10) was incubated 5 min at room temperature. In the following step, cell viability was counted with a hemocytometer.It should be noted that an inhibitory concentration  or C. roseus  for 48 hours. In the following day, cells were harvested and washed by centrifugation in cold PBS at 200 g, 5 min, and 4°C. Cell suspension was added to 9 mL of cold 70% ethanol and stored at −20°C to −40°C for at least 2 hours. Cells were washed in cold PBS at 200 g, 10 min, and 4°C. Cells were resuspended in 500 μL of PI/Triton X-100 staining solution: 10 mL of 0.1% (v/v) Triton X-100 in cold PBS and 2 mg of DNAse-free RNAse A in 1 mL water and 0.40 mL of 500 μg/mL of PI. If RNAse was not DNAse-free, boiling of 2 mg/mL RNAse A solution for 5 min was required. Cell suspension was incubated at 37°C for 15 min, transferred tubes to ice, stored at 4°C, and protected from light. Data were acquired on flow cytometer within 48 hours.HaCaT cells were seeded at 500,000 cells/well in 6-well plates and incubated at 37°C for 24 hours. After that, cells were stimulated by IFN-γ at the final concentration of 1 ng/mL to induce K17 expression. After cytokine stimulation for 24 hours, they were treated with G. superba extract  or C. roseus  or inhibitors (either piceatannol or PD98059) for 48 hours. Both inhibitors were prepared in the working concentrations of 10 and 100 μM, thus resulting in the tested concentrations of 1 and 10 μM. In the following day, protein extraction was carried out using 1 mM of DTT and 1 mM of PMSF in NP-40 lysis buffer. Total protein (20 μg) was mixed with Laemmli buffer (ratio 1 : 1) and boiled for 5 min. Protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk either for 1 hour at room temperature or overnight at 4°C. Membranes were incubated with K17, p-STAT3, and GAPDH primary antibodies for 1 hour at room temperature or overnight at 4°C. After incubation, membranes were washed by 1X TBS-Tween 20 (TBST) for 5 min 3 times, incubated with secondary antibodies  for 45 min at room temperature, and washed by TBST for 5 min 3 times. Protein bands were visualized by adding the enhanced chemiluminescence detection reagent, visualized by using Amersham Hyperfilm ECL and Kodak processing chemicals for autoradiography films . Each band was normalized against GAPDH as an internal control.For Western blot analysis, HaCaT cells were stimulated with IFN-γ for 24 hours. Media were changed, and cells were treated with either G. superba or C. roseus extract for 48 hours. Cells were washed with PBS 3 times and fixed with 4% paraformaldehyde for 20 min. Cells were permeabilized with 0.5% Triton X-100 for 30 min, and blocking with 1% FBS in PBS for 1 hour was performed. After that, cells were incubated in K17 primary antibody (1 : 200) at 4°C overnight and anti-rabbit IgG Alexa Fluor 488 (1 : 200) for 1 hour and stained with DAPI (1 : 2000 from stock solution of 5 mg/mL) for 10 min.For immunofluorescence, cells were seeded in 12-well plate at 100,000 cells/well, until cell confluence was 70%, and then stimulated with IFN- post hoc Dunnett's test  using cytokine-treated cells as a control group.Data were presented as the mean ± standard deviation (SD). Means were from at least three independent experiments. Data were analyzed by one-way analysis of variance (ANOVA) followed byγ. Significantly increased K17 expression was found when HaCaT cells were treated with IFN-γ at the final concentration of 1 ng/mL for 24 hours. To confirm that IFN-γ could upregulate the K17 expression by activating the transcription factor, STAT3, we employed 2 inhibitors, piceatannol and PD98059. According to their mechanisms of action, piceatannol can inhibit STAT3 in JAK/STAT pathway, which controls the cellular response to extracellular cytokines, whereas PD98059 can inhibit MAP kinase kinase (MEK). We found that IFN-γ could induce the K17 expression, and the K17 protein level was significantly decreased when HaCaT cells were treated with piceatannol. Therefore, IFN-γ could induce the K17 expression through JAK/STAT pathway  and C. roseus extract  were prepared. These concentrations tested were based on our obtained IC50 values, 6.09 μg/mL and 74.28 μg/mL for G. superba and C. roseus, respectively. Experimentally, IFN-γ-treated HaCaT cells were therefore treated with the final concentrations of G. superba extract  and C. roseus extract . We found that both plant extracts could decrease the cell viability of IFN-γ-treated HaCaT cells in a dose-dependent manner after treatment for 48 hours. The effect on cell viability was detected by MTT assay and trypan blue exclusion as revealed in Figures  G. superba and Figures  C. roseus.Using the working concentrations of both plant extracts,γ was lower than in untreated group. IFN-γ induced cells from resting phase (G0/G1) into cell cycle. The marked induction was observed in extract-treated groups. The percentage of cells in G0/G1 phase after treatment with such working concentrations as 0.1, 1, 10, and 100 μg/mL of G. superba and 100, 250, 500, and 1000 μg/mL of C. roseus extracts was found to be significantly decreased as compared with the pretreated group (P < 0.05) as depicted in Figures  G. superba extract at all concentrations applied in this study significantly increased the percentage of cells in G2/M phase. In particular, G. superba extract at the highest concentration applied to HaCaT cells in our experiment was the most potent one for inducing the cell cycle arrest at G2/M phase, while the effect of C. roseus extract on cell cycle with respect to all three higher concentrations was found but was not different among them. Nevertheless, the effect of G. superba extract on cell cycle arrest was more obvious when comparing to that of C. roseus.The percentage of cells in G0/G1 phase when treated with IFN- G. superba extract  when cells were treated with G. superba extract at the working concentrations of 0.1, 1, 10, and 100 μg/mL, and a tendency of decreased p-STAT3 was observed in response to the treatment with C. roseus extract at the working concentrations of 100, 250, 500, and 1000 μg/mL. Based on the decreased expression of p-STAT3, our results suggested that both extracts decreased the K17 expression through JAK/STAT pathway.STAT3 is an important transcription factor that plays a role in K17 expression. To confirm that both plant extracts could decrease the K17 expression through JAK/STAT pathway, p-STAT3 expression was detected using Western blot. As revealed in  in vitro studies showed that downregulated K17 expression could inhibit cell proliferation in HaCaT. In this study, we found that G. superba extract  and C. roseus extract  could lessen cell viability  and could also diminish the level of K17 expression in a dose-dependent manner .K17 is known as a hyperproliferative marker, and many G. superba and C. roseus extracts, the level of K17 expression was positively correlated with the percentage of viable cells  as depicted in Figures Therefore, we investigated the correlation between K17 expression and cell proliferation by Pearson's coefficient. For both G. superba and C. roseus on cell division was an interesting result. Chemicals may disrupt cell division by inhibiting the polymerization of tubulin, a monomer of mitotic spindle causing cells arrest at metaphase and cell division disruption [γ-treated HaCaT cells.The inhibitory effect ofsruption . Our resγ is a cytokine that plays a critical role in innate and adaptive immunity for viral and intracellular bacterial infections. It is secreted by T helper 1 (Th 1), CD8+ cytotoxic lymphocytes, natural killer cells (NK), and antigen presenting cells (APCs) such as monocytes, macrophages, and dendritic cells (DCs) [γ secretion. IFN-γ alters keratinization in activated keratinocytes through JAK/STAT pathway [IFN-ls (DCs) , 19. In  pathway . It causγ-treated HaCaT cells was alleviated by treating with piceatannol for 48 hours, indicating that IFN-γ mediated the K17 expression via JAK/STAT pathway. In this work, the authors explored the effect of plant extracts on K17 expression using not only Western blot technique but also immunofluorescence as a quantitative and qualitative approach, respectively. Although the modulation of K17 mRNA transcripts by both studied extracts was not assessed, we believed in our results according to the validity, accuracy, and potential of anti-K17 antibody utilized in this work. Our promising finding was based on the K17 protein level since the protein is the biomarker actually acting in the cells. Using the anti-K17 antibody for Western blot analysis, previous study conducted by Tang and collogues discovered the effect of the active fraction of Flos Daturae on IFN-γ-induced apoptosis in HaCaT cells [Induced K17 expression in IFN-aT cells . cis-element IFN-stimulated response element (ISRE), thereby originating transcription of numerous IFN-inducible genes. This transcriptional activation by STAT3 proteins requires the recruitment of coactivators such as CREB-binding protein (CBP)/p300. STAT3 proteins recognize a conserved element in the gene promoters and increase the mRNA expression of the cell cycle regulatory genes, thus modifying cell growth or transformation [In addition, we confirmed the molecular involvement of JAK/STAT signaling using Western blot to examine the level of p-STAT3, a critical transcription factor playing a role in K17 expression, and we found that both plant extracts could suppress the p-STAT3 expression. It should be worth addressing that STAT3 proteins activate gene in response to cytokine receptor stimulation. Upon tyrosine phosphorylation, STAT3 proteins dimerize, translocate to the nucleus, and activate certain target genes specifically such as theormation , 23. Intormation , thus ma G. superba extract exerted its effect not only in a dose-dependent fashion but also in a stronger effectiveness. The correlation between K17 and cell viability was shown positive, indicating that K17 expression was directly proportional to cell viability in IFN-γ-treated HaCaT cells.In this investigation, all the concentrations of both extracts could decrease cell viability and also decrease K17 expression. Particularly