Objective. A novel injectable magnesium/calcium sulfate hemihydrate (Mg/CSH) composite with improved properties was reported here. Methods. Composition, setting time, injectability, compressive strength, and bioactivity in simulated body fluid (SBF) of the Mg/CSH composite were evaluated. Furthermore, the cellular responses of canine bone marrow stromal cells (cBMSCs) and bone formation capacity after the implantation of Mg/CSH in tibia defects of canine were investigated. Results. Mg/CSH possessed a prolonged setting time and markedly improved injectability and mechanical property (p < 0.05). Mg/CSH samples showed better degradability than CSH in SBF after 21 days of soaking (p < 0.05). Moreover, the degrees of cell attachment, proliferation, and capability of osteogenic differentiation on the Mg/CSH specimens were higher than those on CSH, without significant cytotoxicity and with the increased proliferation index, ALP activity, and expression levels of integrin β1 and Coll I in cBMSCs (p < 0.05). Mg/CSH enhanced the efficiency of new bone formation at the tibia defect area, including the significantly elevated bone mineral density, bone area fraction, and Coll I expression level (p < 0.05). Conclusions. The results implied that this new injectable bone scaffold exhibited promising prospects for bone repair and had a great potential in bone tissue engineering. Bone defects are usually caused by trauma and nonunion, and autologous bone graft is commonly known as a gold standard in reconstruction of bone defects . In the 4·1/2H2O; CSH) has long been used in clinic as a bone regeneration material, because it is characterized by low curing temperature (about 30°C), rapid setting, excellent biocompatibility without inducing an inflammatory response, and promotion of bone healing [Reportedly, calcium sulfate hemihydrate  and its alloys have been utilized as metallic materials for medical implants because of their unique biocompatibility, nontoxicity, density, and elastic modulus similar to those of human bone and stimulatory effects on new bone formation –18. Howe in vitro bioactivity, and biocompatibility with canine bone marrow stromal cells (cBMSCs) were evaluated. Moreover, in vivo bone formation capacity was also investigated by implanting the Mg/CSH scaffold into a canine tibia defect model. It was expected that the novel Mg/CSH composite cement could be potentially used for the clinical repair of bone defects.In the present study, a composite cement was prepared by adding Mg into CSH; meanwhile the setting time, injectability, mechanical properties,α radiation  in a continuous scan mode. The 2θ range was from 10° to 90° at a scanning speed of 8°/min.Mg powders were primarily prepared by a fluoride coating and micro-arc oxidation treatment . The Mg/CSH powder was prepared by adding Mg powders  into the CSH powder . Then, deionized water was added with a liquid to powder ratio of 0.6 mL/g to moisten these powders. The mixtures were stirred within 60 s to form homogeneous pastes, transferred into Teflon molds (Φ 10 mm × 20 mm) and then stored at 37°C for 24 h; finally the hardened 10% Mg/CSH composite, 20% Mg/CSH composite, and pure CSH cements were obtained. The phase compositions of these samples were characterized by using X-ray diffraction  Technology Co., Ltd., China) based on monochromated CuKØ 5.067 mm) to plunge into the paste no longer leaving a visible print on the surface of the paste, while the final setting time was defined as the time necessary for the heavy needle  to no longer leave a visible print on the surface of the paste. Five replicates were conducted for each group and the average value was calculated.The setting time of CSH and Mg/CSH composite pastes was measured with a Vicat needle  according to ISO9597-1989E. The initial setting time was defined as the time necessary for the light needle , according to a method described previously . Those pFurthermore, the compressive strength of the hardened CSH and Mg/CSH composite samples (10 mm diameter × 20 mm high) was measured at a loading rate of 0.5 mm/min with a universal testing machine  in a way similar to that described in ASTM D695-91. The measurements were performed five times for each group.+, 5.0 mM K+, 1.5 mM Mg2+, 2.5 mM Ca2+, 148.8 mM C1−, 4.2 mM HCO3−, and 1.0 mM HPO42− and was buffered at pH 7.25 with 45 mM hydrochloric acid (HCl) and 50 mM trishydroxymethyl aminoethane ((CH2OH)3(CNH2)). After setting for 24 h, the hardened CSH and Mg/CSH composite specimens were immersed in SBF with a surface-area-to-volume ratio of 0.1 cm2/cm3 at 37°C for 21 days. The temperature of SBF was maintained by using a shaking water bath, and SBF was completely refreshed every day. For each group, five samples were removed from SBF after incubation for 2, 4, 7, 10, 14, and 21 days, respectively. At each time point, the specimens were gently rinsed with deionized water, dried in a 37°C oven for 24 h, and then weighed. To measure in vitro degradation, weight loss percentage was calculated by using the following formula: Degradation ratio = (W0 − Wt)/W0 × 100%, where Wt and W0 represent the dry weights of the degraded specimen and the initial specimen, respectively.According to the method described by Kokubo , SBF was in vitro bioactivity of samples, scanning electron microscopy (SEM) equipped with an energy dispersive X-ray detector  was utilized. Additionally, the pH values of SBF were measured during the test using an electrolyte-type pH meter.Furthermore, in order to evaluate the4/cm2. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. The culture medium was refreshed after 48–72 h and then every 2-3 days. When 80–90% cell confluence was reached, the cells were detached with 0.25% trypsin containing 0.01% EDTA  and then subcultured.After intravenous anesthesia with 5% sodium pentobarbital (0.5 mL/kg), 5 mL bone marrow was harvested from the iliac crests of an adult beagle dog, transferred into a 10 mL preheparinized centrifuge tube, and centrifuged to remove fat and heparin. Mononuclear cBMSCs were separated by percoll  gradient centrifugation  and cultβ-sodium glycerophosphate , and 200 μM ascorbic acid . The medium was replaced every 2 days. All animal care and procedures were done in accordance with the Animal Care Guidelines from Chinese People's Liberation Army General Hospital's Animal ethics committee.For cell osteogenic differentiation, the fourth-generation cBMSCs were cultured in medium containing high glucose-DMEM  with 10% FBS, 100 nM dexamethasone , 10 mM 2 (10 mL extract for each cement). The third-generation cBMSCs after osteogenic differentiation were detached by 0.25% trypsin, and then cell suspension was transferred to 96-well plates . Six wells were set for each cement group. After incubation at 37°C in a humidified atmosphere of 5% CO2 for 24 h, cBMSCs were cultured in the presence of 100 μL extracts, and afterwards the medium was refreshed every 2 days. After 2, 4, 6, and 8 days, 20 μL MTT (5 g/L) was added into each well, and cBMSCs were incubated for further 4 h. Subsequently, the supernatant in each well was carefully removed and 150 μL dimethyl sulfoxide was added. After shaking for 10 min, optical density (OD) values at 490 nm were evaluated by using a microplate reader.The cytotoxicity assay was conducted by culturing cBMSCs in the extracts of the CSH and Mg/CSH composite specimens and using a 3--2,5-diphenyltetrazolium bromide (MTT)  quantitative proliferation assay. According to ISO 10993-1: Biological evaluation of medical devices Part 1: Evaluation and testing within a risk management process, 0.2 g/mL extracts were prepared by soaking cements in cell culture medium for 1 day at 37°C, 5% COμg/mL PI, 0.2% Triton X-100, and 100 μg/mL RNase A for 30 min in dark at 4°C. The numbers of cell at different phases of cell cycle were analyzed by using a flow cytometer at 488 nm excitation band. The proliferation index of cell was calculated as follows: Proliferation index = (S + G2M)/(G0G1 + S + G2M) × 100%.The proliferation of cBMSCs seeded on the pure CSH and Mg/CSH composite cements was evaluated by using cell cycle test based on flow cytometry. After 24, 48, and 72 h of incubations, cBMSCs were collected with trypsinization and centrifugation at 1000 rpm and washed twice with PBS. Then, cBMSCs were resuspended in PBS, fixed in 70% ethanol at 4°C for overnight. Finally, samples were centrifuged and stained with 0.5 mL hypotonic solution containing 50 μL of supernatant was moved to 96-well plates, and 100 μL p-nitrophenyl phosphate  substrate solution was added. After incubation for 30 min at 37°C, the reaction was quenched by adding 50 μL NaOH, and the absorbance at 405 nm was quantified with a plate reader. Each test was conducted five times.For osteogenic differentiation, the biosynthesis of ALP in cBMSCs cultured on the pure CSH and Mg/CSH composite cements was measured. After 7, 14, 21, and 28 days of incubation, culture medium was wiped off, and cBMSCs were washed twice with PBS. Approximately 1 mL of cell lysis buffer containing 0.2% Triton X-100 was added to lyse cBMSCs at room temperature, and cell lysate was obtained. After centrifugation, exactly 100 2. Subsequently, the cell-cement constructs were washed twice with PBS and fixed with 2.5% glutaraldehyde solution for 2 h at 4°C. Then, the fixed constructs were rinsed three times with PBS and dehydrated in graded ethanol . After being CO2-dried overnight in a desiccator, samples were sputtered with gold prior to SEM observation.The cellular morphology and attachment of cBMSCs were examined based on direct visualization under SEM. Firstly, cells were attached to the specimens for 3, 7, and 14 days at 37°C in an atmosphere of 100% humidity and 5% COβ1, and β-actin are shown in −ΔΔCT method [The relative expressions of osteogenesis and attachment-related genes in cBMSCs were examined by using real-time RT-PCR . After being cultured on the hardened constructs for 8, 24, and 48 h, cBMSCs were homogenized in Trizol Reagent. Total RNA was extracted and reversely transcribed into cDNA according to the manufacturer's instructions. The sequences of primers for type I collagen (Coll I), integrin T method .Totally, 24 healthy adult male beagle dogs weighing 16 ± 1 kg were randomly divided into 4 groups (6 dogs for each type of implant). The dogs were anesthetized through 3% pentobarbital  and 1% lidocaine (operative region anaesthesia) and placed in a supine position. Under sterile conditions, a 3 cm longitudinal skin incision was performed at the anteromedial aspect of left tibia. Skin and subcutaneous tissues were gently dissected down to periosteum, exposing the bone. Then, one tibial bone tunnel (3 mm diameter and 15 mm length) was surgically produced. The defects were filled with 20% Mg/CSH, 10% Mg/CSH, or pure CSH construct, and bone defects of the control group were left unfilled. All wounds were routinely sutured and penicillin  was injected into all animals for 3 days. After surgery, the canines were kept caged freely and given usual regimen of food and water.Animals were sacrificed 4 and 12 weeks after operation and tibia specimens were harvested and fixed in 4% paraformaldehyde. The local bone mineral densities (BMDs) were measured on a dual energy X-ray absorptiometry (DXA) system, and the new bone area fraction (BAF) was quantified 1 day and 4 and 8 weeks after implantation by using the following formula: BAF = AB/AT, where AB is the newly formed bone area and AT is the total material area. For histological analysis after 4, 8, and 12 weeks after surgery, the decalcified and undecalcified bone specimens were, respectively, embedded into paraffin and hard plastic. Tissue sections were stained with hematoxylin and eosin (H&E) and Masson's Trichrome stain, respectively, and then observed under a light microscope . Additionally, the tissue specimens (5 mm × 5 mm × 5 mm) around the implantation materials were removed and immediately immersed into RNAlater solution. Then, the total tissular RNA was extracted and reverse-transcribed into cDNA, and the transcription levels of osteogenesis-related canine osteopontin, bone morphogenetic protein-2 (BMP-2), and Coll I were measured by RT-PCR.p < 0.05.Experimental data were expressed as means ± SD. The one-way and two-way ANOVA with Tukey's post hoc tests were applied to comparison analysis. Differences were considered statistically significant at 4·2H2O  was observed in 20% Mg/CSH composite with the initial and final setting time of 8 ± 0.72 min and 14.5 ± 0.8 min, respectively (p < 0.05). Moreover, the injectability of Mg/CSH composite pastes dramatically increased with the increase of Mg content, and 20% Mg/CSH composite paste exhibited the highest injectability  (p < 0.05) with the increase of Mg content and reached a maximum value of 18.6 ± 2.7 MPa in 20% Mg/CSH composite, while there is no significant difference (p > 0.05) between 10% Mg/CSH and 20% Mg/CSH composites  . After smposites .4 crystals were displayed after 2 and 4 days of immersion  in the degradation ratios among all specimens from 2 to 14 days, while the degradation ratio of 20% Mg/CSH composite was significantly higher (p < 0.05) than that of other constructs after 21 days of soaking (p > 0.05) was observed among the three kinds of construct at selected time points (The degradation ratios of the samples were characterized by the weight loss ratios after soaking in SBF for various time periods. It can be seen that there were no significant differences ( soaking . During e points .p < 0.05) than that of cBMSCs in the extracts of pure CSH after incubating for 8 days  was observed among the proliferation indexes of cBMSCs cultured on different materials for 24 and 48 h. However, the Mg/CSH composites (10% and 20%) could significantly (p < 0.05) increase cell proliferation when compared with the CSH after 72 h of culture (No significant difference ( culture .p > 0.05) among ALP activities of cBMSCs in three groups at a certain time point  in cBMSCs cultured on Mg/CSH composites (10% and 20%) in comparison with CSH construct, while no significant difference (p > 0.05) was observed between 10% and 20% Mg/CSH at 8, 24, and 48 h  BMD values than CSH and control groups at both 4 and 12 weeks. However, there was no marked variation (p > 0.05) between 10% and 20% Mg/CSH grafts (p < 0.05) than that of CSH at both 4 and 8 weeks  after implantation for 4 and 12 weeks, which was similar to BMP-2 expression. Nevertheless, the expression levels of Coll I in both Mg/CSH composite groups were significantly higher (p < 0.05) than that of CSH construct at 4 and 12 weeks  would have a great potential for bone repair in tissue engineering.Because bone is needed for mineral reserve, locomotion, load bearing, and protection of internal organs, bone defect causes disability and represents a medical and socioeconomic challenge. Tissue engineering is playing a critical role in bone regeneration . An ideaThe applicability of a bone cement biomaterial is largely dependent on its self-setting characteristics including injectability and setting time . AdditioBioactivity is defined as the ability of biomaterials to develop an adherent, direct, and strong bonding with the bone tissue . Reporte in vitro cellular responses to biomaterials, including cell attachment, proliferation, and differentiation, are also main components of new bone repair ability of biomaterials [ in vitro and in vivo [β1 in cBMSCs cultured on Mg/CSH composite constructs demonstrated the distinguished ability of cell adhesion and osteogenesis by adding Mg, in comparison with pure CSH. Therefore, the in vitro results indicated that this composite exhibited favorable biocompatibility by improving cell attachment and stimulating cell proliferation and differentiation.It is generally accepted thataterials . The ost in vivo –37. Ther in vivo . Further in vivo . In the  in vivo study, BMDs of Mg/CSH graft were significantly higher than that of pure CSH at 4 and 12 weeks after implantation. Moreover, the increased BAF indicated that more new bone was formed in Mg/CSH composite groups in comparison with CSH 4 and 8 weeks after graft. Histological evaluation also revealed that the new chondrocytes, trabeculae, and mature ossein appeared at the defect area, the boundaries between normal surrounding tissue and the composite were gradually indistinct due to biodegradation, and the residual materials were surrounded by areas of newly formed bone tissue during 8 weeks after implantation of composite cement. On the contrary, no new bone tissue was observed at the interface of CSH implant after 8 weeks, and the boundaries between normal surrounding tissue and CSH were distinct. It may be attributed to the addition of Mg which can accelerate the growth of new bone tissue as previously reported [ in vivo results demonstrated that Mg/CSH composites exhibited not only faster biodegradability but also more effective osteogenesis and osteointegration at bone defect area than pure CSH cement.Unlike autografts which have limited supply and significant potential risk of nerve damage, infecting, disease transmission, and immune response , 39, bonreported , 41. TheA novel injectable Mg/CSH composite was developed by incorporating Mg coated with fluoride into CSH in this study. Mg/CSH composites showed a prolonged setting time with improved injectability. The mechanical strength and biodegradability of the Mg/CSH composite were improved. The Mg/CSH composite could promote the attachment, proliferation, and differentiation of canine cBMSCs and exhibit excellent biocompatibility without cytotoxicity. Additionally, the Mg/CSH composite implant also showed effective osteogenesis and osteointegration. In conclusion, this new kind of injectable biomaterial with improved properties would develop a more promising tissue graft substitute for bone regeneration.


Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system.Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28–34°C is an effective strategy to expand the productive life period of cells and increase their productivity ( Recombinant proteins that require posttranslational modifications, similar to those found in humans, are preferentially produced in mammalian cells . ChineseRecent transcriptomic and proteomic studies have increased our understanding of the genetic and physiological processes in CHO cell cultures ,15. A trNext-generation sequencing (NGS) technologies are more effective for deep sequencing analyses of genomes and transcriptomes compared to Sanger-based sequencing techniques, and are not limited by DNA chips ,20, alloSince high productivity is associated with particular cell responses and different transcriptomic states ,25, we a® medium , supplemented with 6 mM glutamine , 4 mg/mL insulin HUMULIN , 100 U/mL penicillin , 0.01 mg/mL streptomycin , 0.001 g/L Pluronic® F-68 , 200 nM methotrexate , and 4% yeast extract  at pH 7.0. Inocula were prepared in 75-cm2 T-flasks  and incubated at 37°C with humidified air containing 5% CO2. Six spinner flasks were inoculated with 3.5 × 105 cells/mL at 90% viability in a working volume of 100 mL, and were cultured at 90 rpm with 5% CO2 atmosphere in a humidified incubator. After 48 h, three spinners were switched to 30°C (biphasic cultures) and three were kept at 37°C (control cultures). Samples were taken every 24 h until viability dropped to 40%. The cell concentration and cell viability were determined using a cell counter  and a Neubauer chamber following the trypan blue dye exclusion method.The cell line, CHO TF70R, which produces rh-tPA, was obtained from Pharmacia & Upjohn S.A. Sweden and was a kind gift of Torsten Björlig. The cells were cultured in serum-free, chemically defined CD-OptiCHOo-Phenylenediamine dihydrochloride . The reaction color was measured at 450 nm , and the rh-tPA was quantified with a calibration curve using Actilyse® as a standard . The rh-tPA productivity (qtPA) was calculated using The concentration of glucose, glutamine, glutamate, and lactate was measured with a YSI 2900 . The concentration of rh-tPA in the culture supernatants was measured by ELISA using a monoclonal goat anti-human tPA antibody  and an anti-goat IgG (H+L) secondary antibody conjugated to horseradish peroxidase , which was revealed with 7 cells using an RNeasy kit  and quantified on a NanoDrop 1000 spectrophotometer . RNA integrity was verified using an Agilent 2100 Bioanalyzer system . The RNA-seq libraries from mRNA samples were prepared from total RNA using an Illumina® TruSeq RNA Sample Preparation Kit . mRNA sample preparation was performed using an mRNA-Seq Sample preparation kit .Duplicate samples for the transcriptome analysis were taken 24 h and 48 h after TDS. The control RNA was collected at 48 h from cultures grown at 37°C. RNAlater®  was added to preserve RNA in each sample. Total RNA was isolated from 1 × 10The mRNA samples were sequenced using an Illumina GAIIX platform with two different configurations . The first configuration corresponded to 36 bp, with single reads for 48-h (control) and 72-h  samples. The second sequencing configuration corresponded to 72 bp, with paired-end reads for samples collected at 48 h (control) and 96 h . All libraries were prepared and performed with one biological replicate.www.chogenome.org [http://www.sanger.ac.uk/resources/software/smalt/) v7.4. Two libraries were prepared from biological replicates: the first contained samples from 48 h and 72 h (24 h after TDS) of culture, and the second contained samples from 48 h and 96 h (48 h after TDS) of culture. The read counts for each coding DNA sequence (CDS) in each condition were analyzed with the non-parametric and data-adaptive algorithm NOISeq R package [After quality control check and adapter removal, the Illumina reads for each condition were mapped to the CHO-K1 reference genome published on nome.org , using t package , followi package   and 96 h (48 h after TDS) was extracted using an RNeasy Mini Kit . The RevertAid kit  was used to obtain cDNA. For PCR detection, we used SYBR Green PCR Master Mix . Reactions of 12 μL were evaluated, containing 6 μL of SYBR green PCR master mix , 150 nM primers, and 50 ng of template cDNA. The reaction was incubated at 50°C for 2 min, 95°C for 10 min, and for 40 cycles at 95°C for 15 s and 60°C for 1 min. Amplification was carried out in a PikoReal 24 Real-Time PCR System and analyzed with PikoReal software 2.2 . All samples were run in triplicate, and negative controls were included. Data was analyzed using the 2-∆∆Cq method  and normt-test was employed to determine significant differences between the control (37°C) and low temperature treatment (30°C), using a significance level of p < 0.05.Kinetics and stoichiometric parameters in biphasic and control cultures  were calculated from at least three independent experiments and are expressed as mean ± standard deviation. The Student´s -1, which decreased significantly to 0.005 ± 0.001 h-1 (p < 0.05) after TDS. The maximum viable cell concentration was reached in control cultures (37°C) after 4 days (2.70 ± 0.15 × 106 cells/mL), whereas in biphasic cultures (TDS from 37°C to 30°C at 48 h of culture) similar concentration was obtained only after 8 days (2.67 ± 0.10 x 106 cells/mL)  . Cell viells/mL) . SimilarqtPA of 0.82 ± 0.03 pg/cell-day were obtained in biphasic cultures, which represented a 2.2- and 1.6-fold increase, respectively, as compared to control cultures  than control cultures (qlac) in biphasic cultures was 19.6% lower than in the control cultures, while the glutamine consumption rate (qgln) was similar under both conditions (qgln indicated that pyrimidine synthesis and amino acid synthesis were similar in both culture strategies [lac/glc) was about 19% lower after TDS than in 37°C cultures . A totaProtein -coding genes with >1.6 fold change were clustered in eight groups and are presented in gst1, Gapdh, α-tubulin, and ß-actin) were analyzed. Our results indicated that gst1 was the most stably expressed reference gene in CHO TF70R cells under TDS conditions  to validate our transcriptome data, as has been done in previous studies ,40. To dnditions . Genes qTDS (from 37°C to 30°C) after 48 h of culture affected the growth of CHO TF70R cells, but extended their cultivation time and maintained higher cell viability for up to 7 days longer than those cultures that were maintained at 37°C. This resulted in a 2.3-fold and 1.6-fold increase in rh-tPA volumetric and specific productivity, respectively, with a concomitant decrease in glucose consumption and lactate production in TDS cultures as compared to cultures maintained at 37°C. These results are consistent with previous reports that used CHO cell lines that were grown under similar conditions  and thatvim gene, and the overexpression of its coded protein (Vimentin) [vim was upregulated at 96 h , but its function after TDS is still unknown (trpv3), which is activated at warm temperatures [An increase in RBM3 and CIRP transcripts has been associated with a cold response in different cell types –43. CIRPimentin) ,18,45. Vimentin)  and is rimentin) ,48. Here unknown . On the eratures , was maiTranscripts expression of recombinant genes encoding for DHFR and rh-tPA was similar in the biphasic and control cultures, for each gene . Althougmdm2, TP53INP1, waf1/cip1, and Bax [TP53INP2, which codes for the antiproliferative tumor protein p53-inducible nuclear protein 1 homologue [Cdkn1a is consistent with p21 overexpression in CHO-K1 cells after TDS [Cdkn2a, which encodes for two distinct proteins (p16INK4a and p14ARF) [Cdkn1a and Cdkn2a. Moreover, at 24 h after TDS, the gene coding for TSPYL2, a negative cell cycle regulator that could help in arresting cell growth in combination with p21 [Mybl2 inhibition diminishes proliferation of different cells [Api5, Birc3, Hspa8, and Set [Mybl2 and Mybl1, we observed the repression of Hspa8, Set (TAF1), and Birc6  ,65 invol p14ARF) ,67, was with p21 , was uprwith p21 –71, werent cells ,73, whil and Set ,75. In and Birc6 . TDS caund Birc6 . The repnd Birc6 . Interesnd Birc6 . All theRasl11a, which codes for a GTPase associated with positive regulation of the transcriptional activity of an RNA polymerase I [O-linked and N-linked glycosylation, like FUCA1, Man1a1, Ganab, and Neu1, and downregulated Galnt12 and Galnt14, among other genes [FUCA1 and Neu1/2 and downregulation of Galnt1, Galnt7, and Galnt13 , which are probably downstream of c-FOS  [Bcl2l2), which mediate cell survival by suppressing the death-promoting activity of BAX [Bcl2a1, which codes for Bcl-2-related protein A1 and can retard apoptosis and maintain cell survival by interaction with BAK1, BID, BMF, BCL2L11/BIM, and BAX [Grina, which codes for an apoptotic regulator and controls cell death [TDS causes the overexpression of genes coding for cell proliferation promoters such as Cyr61 (upregulated in both times), alarmin IL33, and Fhl2 . In order to avoid apoptosis, genes coding for potent cell death inhibitors such as Bcl-2-like protein 1 (Bcl-X)  and Bcl-y of BAX , were ovy of BAX . Further and BAX ,93, was  and BAX , and BIR and BAX , which cll death , was oveIn contrast, genes coding for pro-apoptotic proteins  were upregulated at both time points analyzed . MoreoveSince anti- and pro-apoptotic genes were upregulated, we suggest a complex interplay in regulatory responses, with the most important signal being the anti-apoptotic response, because the cell culture viability and metabolism were extended.et al. [TDS has been shown to not only increase the productivity of various heterologous proteins, but also to modify their glycosylation patterns ,32,97. Het al. , who obsN-glycosylation. These enzymes include oligosaccharyltransferase, glucosidases I (GIsI) and II (GIsII), and UDP-Glc:glycoprotein glucosyltransferase (UGGT1), which is also a folding sensor in ER quality control (ERQC) [Mogs, which codes for Glucosidase I, was downregulated , was repressed after 48 h of TDS  ,101. Afth of TDS . TRAPG rh of TDS , althougPmm1, which codes for the enzyme localized at ER and is involved in GDP-mannose and dolichol-phosphate-mannose synthesis [Porcn) and dolichol-phosphate mannosyltransferase 3 (Dpm3), both from the ER [Fut1, which codes for the Golgi enzyme galactoside 2-L-fucosyltransferase, was observed  was overexpressed at 48 h after TDS , which codes for a glycosyltransferase that is localized in the Golgi, was downregulated at both timepoints  by endogenous glycoproteins produced after TDS, indicating changes in N-acetylneuraminic acid α did not change, but the genes Neu1 and Neu2  were overexpressed, which could be related to differences in sialic acid content.Hendrick et al.  observedAltogether, the changes observed suggest that TDS caused a positive effect in ribosomal biogenesis and variation in genes involved in posttranslational modification via ER and Golgi . This coLdha/Ldhc, which code for glycolytic enzymes such as the L-lactate dehydrogenase (LDH) A/C chain [Ldha presented similar changes after 48 h  and participates in conversion of lactose into galactose and glucose, was down-regulated , Rbl1 (p107) , and Rb1Ndufb7 and Ndufs7, which code for NADH dehydrogenases and are required to transfer electrons from NADH to the respiratory chain [Cbr2 and Cbr3, which code for NADPH carbonyl reductases  were up-regulated 48 h after TDS , in agrery chain , and Cbrductases .Based on the transcriptomic results, extension of the growth phase in cultures under TDS can be associated with the maintenance of active central metabolism and cellular respiration as well as mitochondrial biogenesis. Altogether, most of the transcriptional changes promote the avoidance of apoptotic cell death, and those changes could be associated with the increase in rh-tPA productivity. Finally, the findings reported in the present study provide an informative transcriptome resource, mainly dealing with genes relevant to extending the culture life combined with the increase of recombinant protein productivity after 24 and 48 h of TDS exposure. In the same sense, we are characterizing the proteomic response and the relationship between glycosylation and secretory machinery in this biological model. The information presented should advance the understanding of the responses involved in cold adaptation, increase the knowledge of recombinant CHO cell biology, and can be useful for establishing improved bioprocesses for optimizing the quality and productivity of biopharmaceutical proteins.S1 FigTranscriptome contigs were aligned with Ensembl, Genome CHO and GenBank using BLAST, an annotation was assigned to each contig. Differential expression was completed using NOISeq algorithm for comparison of samples 48 h (control) and 72 h  single end (q > 0.8) and 48 h (control) and 96 h  paired ends (q > 0.9), and then clustering was conducted using the TopGO algorithm.(DOCX)Click here for additional data file.S1 TableOnly protein-coding genes are shown. Eight main groups are presented. Red color indicates low expression levels, and green color indicates high expression levels. The gene names are shown in the middle of the table and the gene accession numbers are presented. Those differentially expressed genes (>1.6 fold change) were enriched by gene ontology annotation at 24 h (171 genes q ≥ 0.8 and 70 genes 0.6 ≥ q < 0.8) and 48 h  after TDS and classified in sub-ontologies.(DOC)Click here for additional data file.


Escherichia coli (aEPEC) is considered to be an emerging enteropathogen that is more prevalent than typical EPEC in developing and developed countries. The major adherence factor, intimin, an outer membrane protein encoded by eae, plays a pivotal role in the pathogenesis of aEPEC. This study investigated the distribution and polymorphisms of intimin subtypes of 143 aEPEC strains from diarrheal patients, healthy carriers, animals, and raw meats in China. These aEPEC strains belonged to more than 71 different serotypes, which comprised 52 O serogroups and 24 H types. Sixty-eight different eae genotypes and 19 intimin subtypes were detected. Eighteen, eight, seven, and five intimin subtypes were identified from 86 diarrheal patients, 14 healthy carriers, 19 animals, and 24 raw meats strains, respectively. Intimin β1 was the most prevalent subtype in strains from diarrheal patients (34.88%) and animals (47.37%). There was a statistically significant difference in the distribution of eae-β1 between diarrheal patients and healthy carriers (P = 0.004). Intimin-θ was more predominant among raw meat strains (50%) than among diarrheal patients strains , healthy carrier strains , or animal strains . The two predominant subtypes (eae-β1 and eae-θ) had considerable polymorphisms with no significant differences among the four sources. PFGE analysis revealed 119 distinct patterns and the strains were clustered into 11 groups with similarity indices ranging from 63% to 100%. These results suggest that in China, aEPEC strains from different sources are highly heterogeneous. Animals and raw meats are important sources of genetically diverse intimin-harboring aEPEC, which might serve as important transmission vehicles of these bacteria.Atypical enteropathogenic  Escherichia coli (EPEC), the first pathotype of diarrheagenic E. coli (DEC) described, represents the main bacterial pathogen that causes severe diarrhea in China [Enteropathogenic in China –3. EPEC in China . Atypicain China , 6. Recein China . Additioin China , 8, 9. Tin China , 8, 9. Iin China . Howeverin China , 8, 9.eae gene localized in the central region of the LEE. Intimin is an adherence factor that plays a pivotal role in intestinal colonization [EPEC has the ability to form attaching and effacing (A/E) lesions on intestinal epithelial cells . The gennization .eae have revealed at least 30 distinct subtypes: α1, α2, α8, β1, β2, β3, γ1, γ2, ε1, ε2, ε3, ε4, ξ, ζ, ζ3, η, η2, θ, τ, ι1, ι2, κ, λ, μ, ν, υ, ο, π, ρ, and σ [eae subtypes and particular E. coli serotypes or pathotypes has been used in the classification of strains from culture collections [eae subtypes in patients and healthy humans remain to be addressed. Moreover, minute variations  within an eae subtype have rarely been described. Currently, the characteristics of aEPEC strains in China have not been well defined. In this study, we investigated the intimin subtypes and polymorphisms of aEPEC isolated from diarrheal patients, healthy carriers, animals, and raw meats in China.The intimin sequences are conserved in the N-terminal region, but highly variable in the last C-terminal region (280 amino acids), where cell binding activity is localized . Analyseρ, and σ . Researcρ, and σ . The asslections , 16; howeae gene by PCR assay [eae-positive enrichment culture was directly streaked onto CHROMagar™ ECC plate  and incubated overnight at 37°C. On each plate, about ten E. coli-like colonies (blue or colorless) were picked randomly to test for the presence of eae gene. Then, the eae-positive colonies were plated onto Luria-Bertani  plates and incubated overnight for further identification. All eae-positive E. coli isolates were confirmed by API 20E biochemical test strips . Isolates with eae+, stx1-/stx2-, and bfpA- were identified as aEPEC [A total of 3401 samples were collected from 2006 to 2014 by local centers for disease control and prevention. Among which, 1418 fecal samples were collected from diarrheal patients in sentinel hospitals, 640 fecal samples were collected from healthy humans during routine physical examination, 897 fecal samples of animals and 446 raw meat samples were collected in routine surveys, in six geographical regions in China . About 200 milligrams of each fecal sample or about one gram of each raw meat sample was enriched in 3 ml or 9 ml of modified Tryptone Soya Broth (mTSB) supplemented with novobiocin (10 μg/μl)  and incubated at 37°C for 16 to 24 h with shaking at 200 rpm. Briefly, 1.5 ml of each enrichment sample was centrifuged and 150 μl of the lysis buffer  was used to suspend the centrifuged enrichment sample, boiled for 10 min and centrifuged. The supernatant was then used as template to detect the presence of et al. [E. coli O antisera  were used to confirm the PCR results. The H type of each isolate was determined by amplifying and sequencing the fliC gene with the primers fliC-F (5′-ATGGCACAAGTCATTAATACCCAAC-3′) and fliC-R (5′-CTAACCCTGCAGCAGAGACA-3′) reported by Fields et al. [https://cge.cbs.dtu.dk/services/SerotypeFinder/) [The O serogroups were screened by O-genotyping PCR created by Iguchi et al. . The coms et al. , and theFinder/) .eae was performed by PCR amplification and sequencing a ~3.2-kb cesT-escD LEE region as reported by Ooka et al. with minor modifications [cesT-F9 (5′-TCAGGGAATAACATTAGAAA-3′)/eae-R3 (5′-TCTTGTGCGCTTTGGCTT-3′) primer pair, and the 3′ half and the downstream region were amplified using the eae-F1 (5′-ACTCCGATTCCTCTGGTGAC-3′)/escD-R1 (5′-GTATCAACATCTCCCGCCCA-3′) primer pair; the primer pairs generated ~1.6 and ~1.9 kb amplicons, respectively. PCR products were analyzed by agarose gel electrophoresis, purified with the QIAquick PCR purification kit , and sequenced using the ABI 3730 Automated DNA Analyzer . An additional primer eae-R3RC (5′-AAGCCAAAGCGCACAAGA-3′) was used for the complete sequencing of the 1.9 kb amplicon.Bacterial genomic DNA was prepared for PCR as previously described . Subtypiications . BrieflycesT-escD region was assembled with SeqMan II . The reference eae sequences downloaded from GenBank [eae sequences obtained in this study were aligned using the ClustalW program, and genetic distances were calculated with MEGA 6 (www.megasoftware.net) [eae sequences acquired in this study were submitted to GenBank with accession numbers KT591191–KT591333.The sequenced ~3.2-kb  GenBank  and the are.net) . A cutofare.net) . A NeighE. coli . The genomic DNA was digested with 45 U of XbaI  at 37°C for 2 h. Macrorestriction fragments were resolved by counter-clamped homogeneous electric field electrophoresis in a CHEF-DRIII apparatus . The run time was 19 h at 6.0 V/cm, with initial and final switch times of 6.76 s and 35.38 s, respectively. The image was captured with a Gel Doc XR+ software . Data analysis was performed and an UPGMA dendrogram was constructed using Bionumerics .PFGE was performed according to the PulseNet protocol developed for non-O157 eae subtypes or polymorphisms were evaluated by Chi-square or Fisher’s exact test. Statistical analyses were performed with Epi Info software [P < 0.05 was considered statistically significant.The associations between isolate sources and software . P < 0.0Fecal samples of humans were acquired with written informed consent of participants and animal fecal samples were collected with the consent of the owners of the lands and animals. Meat samples were purchased from markets. The study was approved by the ethics committee of National Institute for Communicable Disease Control and Prevention, China CDC, according to the medical research regulations of National Health and Family Planning Commission of the People's Republic of China.et al. inferred that ten O serotypes: O26:H[The O-typeable aEPEC strains reported so far belong to >200 different serotypes . Trabulses: O26:H, O55:[H7es: O26:H. In thises: O26:H, 24, 25.E. coli (EHEC) and EPEC typing in routine diagnostics, pathogenesis, and epidemiological, clonal, and immunological studies. Most studies have focused on the allele distribution between diarrhea and control samples, or have characterized the intimin subtypes of EPEC strains from animal and human sources [The differentiation of intimin alleles represents an important tool for enterohemorrhagic  sources , 24–26. eae sequences of the 143 aEPEC strains varied from each other in 0–19.30% of nucleotide sites. Therefore, there was 80.70–100% identity among the sequences of the 143 aEPEC strains. There were 68 different eae sequence types (genotypes) in the 143 aEPEC strains. Except for two major genotypes containing 23 and 12 strains each, 44 genotypes contained only one strain isolated from one of the four sources, while the rest contained two to seven strains  of aEPEC strains from China had not been identified previously.Based on pairwise comparisons, the  strains . NotablyThe 143 aEPEC strains had a wide variety of intimin subtypes. Overall, there were 19 intimin variants: β1 (43 strains), θ (27 strains), ε2 (10 strains), κ (eight strains), η2 (eight strains), ζ3 (eight strains), ι1 (seven strains), ο (five strains), ε1 (five strains), α1 (four strains), λ (three strains), γ1 (three strains), ι2 (two strains), ξ (two strains), μ (two strains), β2 (two strains), ζ (two strains), α2 (one strain), and π (one strain). None of the strains had intimin subtypes α8, β3, ε3, ε4, η, γ2, ν, ρ, τ, υ, or σ .eae subtypes were only detected in diarrheal patients, including intimin subtypes λ, β2, μ, ι2, ξ, ζ, α2, and π. Intimin γ1 was only detected in diarrheal patients and pig. Intimin κ was only identified in diarrheal patients and raw meats, while eae-ε2, η2, and ι1 were only present in humans . There were significant differences in the overall distribution of the intimin subtypes among diarrheal patients, healthy carriers, animals, and raw meats (P = 0.0034). In this study, the distribution of intimin alleles was similar to that reported by other researchers; however, the order of frequency was different [The strains isolated from diarrheal patients had more intimin types than the strains isolated from healthy carriers, animals, or raw meats. Almost all of the 19 intimin subtypes (except for intimin subtype ε1) were detected in 86 diarrheal patients, but only eight, seven, and five intimin subtypes were identified in strains from healthy carriers, animals, and raw meats, respectively. Previously, intimin α2, η, and μ have been mainly detected in human EPEC strains –30, withifferent , 32.eae-β and eae-γ subtypes are considered to be two most frequent variants in animal isolates and clinical isolates associated with human diarrheal diseases [et al. reported that eae-β and eae-γ are present in 34.2 and 31.5%, respectively, of EPEC strains from diarrheal patients in Germany [et al. reported that eae-β and eae-γ are present in 28.6 and 38.6%, respectively, of EHEC isolates from diarrheal patients in Spain [eae-β1 was the most prevalent intimin subtype containing 43 (30.07%) aEPEC strains. Additionally, eae-β1 was the most predominant intimin subtype in strains from diarrheal patients  and animals . However, none of the strains from healthy carriers contained eae-β1. There was a statistically significant difference in the distribution of eae-β1 among the four sources (P = 0.0084). The distribution of eae-β1 in diarrheal patients was significantly higher than that in healthy carriers (P = 0.004), similar to the findings of Wang et al.[The diseases , 25. Zha Germany , while Bng et al..eae-θ (18.88%), which was identified in 11 strains from diarrheal patients, 12 strains from raw meats, three strains from animals, and one strain from healthy carriers. Notably, half of the strains from raw meats harbored the eae-θ allele. The overall prevalence of eae-θ in human, animal, and raw meat isolates was significant (P = 0.0003). Intimin θ was more predominant among the raw meat strains than among the diarrheal patient strains , healthy carrier strains , or animal strains . The higher frequency of eae-θ has been reported in children with diarrhea [eae-θ subtype in raw meats. Raw meats represent a possible transmission route of this emerging pathogen.The second most prevalent allele was diarrhea  and animdiarrhea . The reseae-γ subtype represents one of the most frequent variants in animal isolates and clinical isolates associated with human diarrheal diseases and is more frequently isolated in diarrhea cases of longer duration (>7 d) than those of shorter duration [eae-γ1. These three isolates belonged to serotypes more commonly associated with Shiga toxin-producing E. coli (STEC) /enterohemorrhagic E. coli (EHEC) strains [eae-γ in aEPEC could be attributed to the fact that eae-γ is commonly associated with stx-positive EHEC strains such as O157:H7 and O55:H7 that were not examined in this study [The duration . Our res strains . The res strains . The lowis study .eae-κ was present in eight (5.59%) aEPEC strains . It has been reported that eae-κ is more common in animal feces and that it is associated with more severe episodes of diarrhea in humans in comparison with other alleles [eae-κ strains.In this study,  alleles . Raw meaeae-ζ3 strains were obtained in aEPEC isolates from all of four sources with non-significant differences in the prevalence rates. Few studies have reported that aEPEC contained intimin α1, α2, β2, γ1, γ3, λ, ι2, and ε8. eae-α1, eae-ξR/β2, and eae-γ1 strains have been isolated from children with diarrhea [eae-ι2 strains have been isolated from cattle [eae-λ strains have been isolated from children [diarrhea , eae-ι2 m cattle , and eaechildren  in Urugueae-β1 and eae-θ, which were the most prevalent subtypes in this study, were analyzed. Among the 43 eae-β1 strains, 26 polymorphisms were detected based on the eae-β1 reference sequence (GenBank accession number AJ277443) and 12 genotypes were defined  were novel compared to publicly available database (GenBank) by BLAST analysis. In contrast, Marjorie et al. reported that eae-β1 polymorphisms were uncommon or predominantly synonymous [eae-β1 subtype was the predominant intimin variant in clinical isolates, it has been demonstrated that the invasion of differentiated intestinal Caco-2 cell is a sporadic property among aEPEC strains carrying common intimin subtypes [The polymorphisms of  defined . Out of ogeneous . The higogeneous , are resogeneous . Two majnonymous . Howevereae-θ) based on the eae-θ reference sequence (GenBank accession number AF449418); 13 genotypes were defined. Seventeen polymorphisms were non-synonymous, while 13 were synonymous  showed identical PT. Four strains with identical PT showed the same serotype (O51:H7), intimin subtype (β1) and source . It has been found that the pathogenesis of aEPEC seemed to be related to the serotypes [eae-β1 clustered in group III seemed to be specific among diarrheal patients. But the large variety of serotypes, phylogenetic properties and intimin subtypes present in both healthy and diseased human isolates makes it difficult to determine which strains are truly high pathogenic.DNA macrorestriction analysis by PFGE has been considered the ‘gold standard’ for the molecular subtyping of many pathogenic organisms, including EPEC strains . The oveerotypes . aEPEC serotypes , O39:NM erotypes , O55:HNMerotypes , and O76erotypes  have beeeae-β1 and the predominant subtype detected in raw meat-derived strains was eae-θ, isolates from diarrheal patients and animal/raw meats harbored identical serotypes, PFGE patterns or eae-subtypes and even eae genotypes. These results suggested that animals and raw meats are reservoirs of aEPEC possessing these properties in China and represent disease transmission vehicles.In conclusion, aEPEC strains isolated from diarrheal patients, healthy carriers, animal feces, and raw meat samples in China were highly heterogeneous in terms of O:H serotypes, PFGE patterns and intimin subtypes. Even though the main intimin subtype identified in strains isolated from human clinical and animal feces was S1 FigThe dendrogram was generated by Bionumeric software. The strains were clustered into 11 groups generated by the UPGMA algorithm of 67% similarity according to the Dice index.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Pseudomonas chlororaphis EA105, a strain isolated from rice rhizosphere, has shown antagonistic activities against a rice fungal pathogen, and could be important in defense against rice blast. We report the draft genome sequence of EA105, which is an estimated size of 6.6 Mb. Pseudomonas chlororaphis strain EA105 was isolated from rice cultivar M-104 and is capable of inhibiting growth of Magnaporthe oryzae, the fungus responsible for rice blast, as well as reducing disease in the rice plant with pretreatment . The genomic library was prepared using a Nextera XT sequencing library preparation kit (Illumina). Sequencing was carried out at Tufts University Genomics Core using a MiSeq genome sequencer (Illumina), which generated 3,188,442 2 × 250 bp paired end reads. The genome was assembled using CLC v7.5, producing 74 contigs ranging in size from 506 to 829,175 bp. The assembled genome had 121-fold coverage, with an N50 scaffold size of 183,112 bp.Genomic DNA was isolated from P. chlororaphis EA105 consists of 6,595,581 bp, with a G+C content of 59.2%. Annotation was performed using the NCBI PGAP pipeline, which predicted 5,655 protein-coding sequences and 53 tRNAs. The 5S, 16S, and 23S rRNA genes were detected, but because the assembly is based on short reads, the numbers and locations of multiple copies could not be determined. An initial analysis of the genome sequence for genes related to antifungal activity and interactions with other species revealed a cluster of genes putatively involved in synthesis of hydrogen cyanide (HCN) in contig 20 (P. chlororaphis strains (Pseudomonas aeruginosa PAO1’s locus 1-type and locus 4A-type, respectively (Pseudomonas species, including some clustered with the structural components in contigs 2 and 24. This genome sequence will enable further comparative genomic studies among P. chlororaphis strains and facilitate genetic and transcriptomic analysis of the plant protection capability of P. chlororaphis EA105.The draft genome sequence of ontig 20 , but unl strains , 13, we  strains , 15, nor strains . Clusterectively . We alsoJSFK00000000. The version described in this paper is version JSFK01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Resveratrol is a natural polyphenol that has been proposed to improve glycemic control in diabetes, by mechanisms that involve improvement in insulin secretion and activity. In type 1 diabetes (T1D), in which insulin therapy is obligatory, resveratrol treatment has never been investigated. The present study aimed to evaluate resveratrol as an adjunctive agent to insulin therapy in a T1D-like experimental model.Slc2a2), phosphoenolpyruvate carboxykinase (Pck1) and glucose-6-phosphatase catalytic subunit (G6pc) mRNAs (qPCR), glycogen storage and sirtuin 1 (SIRT1) activity were analyzed in liver.Rats were rendered diabetic by streptozotocin (STZ) treatment. Twenty days later, four groups of animals were studied: non-diabetic (ND); diabetic treated with placebo (DP); diabetic treated with insulin (DI) and diabetic treated with insulin plus resveratrol (DIR). After 30 days of treatment, 24-hour urine was collected; then, blood, soleus muscle, proximal small intestine, renal cortex and liver were sampled. Specific glucose transporter proteins were analyzed (Western blotting) in each territory of interest. Solute carrier family 2 member 2 ; besides, it increased  the hepatic glycogen content, and SIRT1 protein.Diabetes induction increased blood glucose, plasma fructosamine concentrations, and glycosuria. Insulin therapy partially recovered the glycemic control; however, resveratrol as adjunctive therapy additionally improved glycemic control and restored plasma fructosamine concentration to values of non-diabetic rats. Resveratrol did not alter the expression of the glucose transporters GLUT2 and SGLT1 in the intestine, GLUT2 and SGLT2 in kidney and GLUT4 in soleus, suggesting that fluxes of glucose in these territories were unaltered. Differently, in liver, resveratrol promoted a reduction in Slc2a2/GLUT2, Pck1, and G6pc expression was observed, favoring reduction of glucose production and efflux. Besides, resveratrol increased SIRT1 nuclear protein content in liver, which may be related to the observed gene expression regulations.Resveratrol is able to improve glycemic control in insulin-treated T1D-like rats. This effect seems not to involve changes in glucose fluxes in the small intestine, renal proximal tubule, and soleus skeletal muscle; but to be related to several changes in the liver, where downregulation of  Diabetes mellitus (DM) is an epidemic metabolic disease growing at exponential rate. Type 2 DM (T2D) accounts for around 90 % of all diabetes subjects, but type 1 DM (T1D) incidence is also increasing worldwide . At pres+ -dependent histone deacetylase sirtuin 1 (SIRT1), the main mechanism related to its effects [Insulin sensitizer agents, which could improve glycemic control, have been extensively investigated for treatment of T2D. However, the recent finding of insulin resistance in T1D patients , 6 has d effects . Numerou effects .db/db mice [Regarding the potential effects of resveratrol in DM treatment, a glycemia-lowering effect was described in normal rats and mice , 15, hig/db mice , and T2D/db mice . In thes/db mice . However/db mice , 18–20, Glycemic homeostasis results from an orchestrated regulation of territorial glucose fluxes, which includes flows into and out of the extracellular/blood compartments , 22. SomMost of these glucose fluxes have been proposed to be altered in DM, and that would involve changes in the expression of specific glucose transporters. On the other hand, regulation of some glucose transporters has been proposed as important targets for the development of preventive and therapeutic approaches for DM , 26, 27.So far, the beneficial effects of resveratrol have been shown in rodent models of T2D, in T2D patients, and in untreated T1D-like rats. This latter condition does not contribute to the investigation of the potential benefits of resveratrol for T1D patients, because it does not reflect their real life situation, since they necessarily require insulin therapy. Thus, the present study aimed to investigate if resveratrol could act as an adjunctive agent to insulin therapy in a T1D-like experimental model. For that, insulin-treated STZ-rats were additionally treated with resveratrol; glycemic control and expression of glucose transporters in distinct territories involved on glycemic homeostasis were evaluated. Besides, because the hepatic GLUT2 expression was highly altered, glucose metabolism markers involved in the regulation of glucose fluxes and SIRT1 activity were also investigated in this territory.ad libitum.Forty 60-day old male Wistar rats weighing 250 g were obtained from the Animal Center of the Institute of Biomedical Sciences, University of São Paulo. The animals were housed in a room kept at constant temperature (23 ± 2 °C), in light/dark cycle (12/12 h), receiving standard rat chow  and tap water At 75 days of life, animals were rendered diabetic by intravenous injection of STZ  at a dose of 50 mg/Kg of body weight, solubilized in citrate buffer (pH 4.5); and control rats were injected with citrate buffer. The procedure was performed in halothane  anaesthetized animals.Twenty days later (95-day old rats), a trial was performed to evaluate the efficiency of diabetes induction, and animals with a blood glucose concentration above 300 mg/dL  were included in the study. Immediately, the animals were separated into four groups: non-diabetic control (ND); diabetic treated with 0.9 % NaCl as placebo (DP); diabetic treated with 5 U/day NPH insulin  (DI) and diabetic treated with 5 U/day NPH insulin plus 10 mg/Kg body weight resveratrol  (DIR). Insulin and resveratrol were subcutaneously and intraperitoneally injected, respectively. The treatments were conducted during thirty days, totalizing 50 days of diabetes duration. The experimental protocol was approved by the Ethical Committee for Animal Research of the Institute of Biomedical Sciences, University of São Paulo (#194/2013).At the end of the treatments, 24-hour urine was collected. Then, at 10:00 AM (after 4-hour food deprivation), the animals were anaesthetized with 60 mg/Kg sodium thiopental , and tail blood samples were collected for glucose concentration analysis. After, soleus muscle, liver, kidney and proximal small intestine were sampled and storage at –70 °C for further analysis. Additionally, immediately after liver sampling, blood was collected from the inferior vena cava, for fructosamine concentration analyzes in plasma. Kidneys were removed, and outside cortex slices were excised, as previously described .Plasma fructosamine concentration was measured by a kinetic-colorimetric assay  and blood glucose concentration by a glucometer . The 24-hour urine volume was measured, a sample was centrifuged at 1,000 g (10 min), and the supernatant was used to measure glucose concentrations by an enzymatic-colorimetric assay ; the results were expressed as 24-hour glucose excretion, taking into account the total urinary volume.2SO4 treatment. After hydrolysis, glycogen content was calculated based on the glucose concentrations in the samples, measured by an enzymatic-colorimetric assay .Hepatic glycogen content was measured as previously described . Briefly–ΔΔCt was adopted for analysis. The genes analyzed were: solute carrier family 2 member 4 (Slc2a4), solute carrier family 2 member 2 (Slc2a2), phosphoenolpyruvate carboxykinase 1 (Pck1), glucose-6-phosphatase catalytic subunit (G6pc). The reference gene used was beta-2-microglobulin (B2m), accordingly to RefFinder software analysis. The used primers are depicted in Table Total RNA was isolated from approximately 100 mg of soleus skeletal muscle, and liver. The samples were processed accordingly to TRIzol® Reagent manufacturer specifications . The amount of total RNA in each sample was determined using a spectrophotometer . The integrity of RNA was verified by the presence of 18S and 28S bands and confirmed by a 1.5 % denaturant agarose gel electrophoresis exposed to ultra-violet light . The reverse transcriptase (RT) reaction was performed from 2 μg of total RNA, adding to the reaction: oligo dT (100 μg/mL), 10 mM of each dNTP, 5X First-Strand buffer and 2 μl (200 U/μl) of M-MLV Reverse Transcriptase . The conditions of RT reaction were 65 °C for 10 min, followed by 37 °C for 60 min, and 95 °C for 10 min. The qPCR amplification was performed using Taqman® PCR master mix kit  and carried out with StepOne Plus Instrument . The PCR conditions were 1 cycle of 10 min at 95 °C and 40 cycles of 30 s at 95 °C, 60 s at 60 °C and 60 s at 72 °C. The method of 2 Membrane fractions were prepared as previously described , 30, 31.Total protein content in the samples was determined by Bradford method . Equal amounts of protein  were electrophoresed, transferred to nitrocellulose membrane and immunoblotted, accordingly to the tissue, with anti-GLUT4 , anti-GLUT2 , anti-SGLT1  and anti-SGLT2  antibodies. The appropriate secondary conjugated antibody was used accordingly to manufacturer specifications, followed by enhanced chemiluminescence (ECL) procedure. The optical density of the blots was analyzed using Image J software , and the densities of the respective lanes, stained by Ponceau, were used for normalization. The results were expressed as arbitrary units, related to mean of the controls, which was set as 1.0.Nuclear proteins were extracted from liver samples as previously described , 32. BriP < 0.05.All data were expressed as mean ± standard error of the mean (SEM). The results of four groups were matched by one-way analysis of variance (ANOVA), with Student-Newman-keuls as a post-test. Differences were considered statistically significant at P < 0.001). In both DI and DIR groups, there was a reduction in blood glucose (P < 0.001) to levels similar to those observed in non-diabetic rats. Glycosuria also decreased after insulin treatment , and resveratrol induced an additional reduction , although glycosuria still remained higher in comparison to non-diabetic rats. Fructosamine concentration decreased in the DI group, and a further reduction was observed with resveratrol , achieving mean value similar to that observed in non-diabetic rats.Table Slc2a4 mRNA and its GLUT4 protein were measured in soleus muscle . Insulin treatment restored both mRNA and protein expression, and resveratrol did not alter the effect of insulin.To investigate the skeletal muscle regulation of glucose disposal, and its participation in glycemic regulation, the cle Fig. . Both mRP < 0.05), an effect that was not significantly reversed by insulin with or without resveratrol. Differently, the expression of the luminal SGLT2 in proximal tubule was reduced by DM , which also was not altered by insulin with and without resveratrol. Finally, in liver, GLUT2 protein increased in response to DM , insulin treatment tended to reduced it, but a significant reduction was observed only with the administration of resveratrol to insulin therapy (P < 0.01 vs. DP and P < 0.05 vs. DI).To investigate other territorial fluxes of glucose which could be involved in the resveratrol-induced whole-body improvement of glycemic control, expression of glucose transporters was evaluated in the proximal small intestine Fig.  and b, rSlc2a2 mRNA .RNA Fig.  was moduPck1 mRNA , insulin treatment partially reduced it (P < 0.001 vs. DP), and that was exacerbated with resveratrol , which brought mean expression to the level observed in non-diabetic animals.The expression of RNA Fig.  was simiG6pc mRNA . Importantly, the resveratrol treatment reversed the insulin-induced increase in G6pc mRNA , restoring its expression to the level observed in non-diabetic rats.RNA Fig.  tended tP < 0.001 vs. DP; P < 0.01 vs. DI), becoming higher than in non-diabetic rats (P < 0.01 vs. ND).Finally, the hepatic glycogen content Fig.  was not P < 0.01).Resveratrol effects have been proposed to be mediated by SIRT1, which, once activated in the nucleus, may impair transcriptional activity. The nuclear content of SIRT1 in the liver was unaltered in diabetic rats insulin-treated or not Fig. . HoweverThe present study was carried out to investigate if resveratrol would improve glycemic control in T1D-like rats under insulin therapy. As expected, insulin treatment did not completely restore glycemic control, because it is difficult to mimic endogenous insulin secretion , which iStreptozotocin (STZ) treatment of rats is known to induce a specific pancreatic beta cell necrosis, leading to an insulinopenia syndrome , similarSeveral reports have proposed that resveratrol improves glycemic control in animals and humans with DM , most ofThe present results revealed the impressive effect of resveratrol as adjunct to insulin therapy in T1D-like rats based on fructosamine decay. The serum fructosamine includes all glycated plasmatic proteins produced over the last 2-3 weeks, reflecting recent changes in glycemic control. Since peripheral insulin concentration is unchangeable in this T1D model, the glucose-lowering effect must have been achieved due to changes in one or more peripheral flux of glucose, which invariably is accompanied by changes in the glucose transporters expression.Slc2a4/GLUT4 expression in diabetic rats, and that was completely recovered by insulin treatment, as reported elsewhere [Slc2a4/GLUT4 expression in comparison to insulin alone, showing that, as adjunctive therapy in T1D, this compound does not improve glucose clearance by skeletal muscle.Participation of improved skeletal muscle glucose uptake by resveratrol has already been proposed. Piceatannol, a metabolite of resveratrol, was shown to increase AMPK phosphorylation, GLUT4 translocation, and glucose uptake in L6 myocytes . Besideslsewhere , 36. ResSGLT1- and GLUT2-related increase in intestinal glucose absorption has been proposed in T2D patients  and 2-weSlc5a2 mRNA expression [db/db and Akita mice [It has been extensively reported that inhibition of renal glucose reabsorption by SGLT2 inhibitors contributes to blood glucose reduction, and that is a recent therapeutic approach to DM treatment . STZ-diapression ; howeverita mice , pointinita mice , 31, as Slc2a2 mRNA expression, indicating a transcriptional modulation. Since a reduction in GLUT2 expression has been related to reduced hepatic glucose efflux in several conditions, such as the Fanconi-Bickel syndrome [-/- mice [Relevant results were obtained in the liver. GLUT2 protein increased in diabetic rats and was partially reduced by insulin therapy, in agreement with previous work . Remarkasyndrome  and the -/- mice , this rePck1, which increased in DM, was only restored to non-diabetic levels by treatment with insulin plus resveratrol. G6pc expression tended to increase in DM, and insulin therapy did not reverse this effect, as it was expected, considering the repressor effect of insulin on G6pc gene [G6pc expression to the non-diabetic levels. These findings reveal the resveratrol-induced increase in the local insulin sensitivity, and anticipate a reduction in hepatic glucose production and efflux [To deepen the investigation of liver participation, some markers of glucose metabolism, which have been related to impaired glycemic homeostasis in DM , were an6pc gene . Howeverd efflux , 46. Indd efflux , and resd efflux  and G6Pad efflux . HoweverSirt1 mRNA [Increased deacetylase activity of SIRT1 was extensively proposed as the major mechanism by which resveratrol induces beneficial effects in several tissues . Howeverrt1 mRNA , but hadrt1 mRNA ; besidesrt1 mRNA . We are Pck1/G6pc expression in the liver of T1D-like rats receiving resveratrol plus insulin.Interestingly, despite proposals that resveratrol-induced improvement in glycemic homeostasis could involve suppression of gluconeogenic enzymes expression , 48, andSIRT1-mediated decrease of insulin resistance has been proposed, mainly based on resveratrol treatment , 50, altSlc2a2/GLUT2, Pck1 and G6pc expression was observed, favoring reduction of glucose production and efflux. Besides, resveratrol increased SIRT1 nuclear protein content in liver, which may be related to the observed gene expression regulations.In conclusion, resveratrol was able to improve glycemic control in insulin-treated T1D-like rats. This effect seems not to involve changes in glucose fluxes in the small intestine, renal proximal tubule, and soleus skeletal muscle; but to be related to several changes in the liver, where downregulation of B2m, beta-2-microglobulin gene; DI, insulin-treated diabetic rat; DIR, insulin + resveratrol-treated diabetic rat; DM, diabetes mellitus; DP, placebo-treated diabetic rats; G6Pase, glucose-6-phosphatase protein; G6pc, glucose-6-phosphate catalytic subunit gene; GLUT2, glucose transporter 2 protein; GLUT4, glucose transporter 4; ND, non-diabetic rat; Pck1, phosphoenolpyruvate carboxykinase gene; PEPCK, phosphoenolpyruvate carboxykinase protein; qPCR, real-time polymerase chain reaction; RT, reverse transcriptase reaction; SEM, standard error of the mean; SGLT1, sodium-glucose cotransporter 1; SGLT2, sodium-glucose cotransporter 2; SIRT1, sirtuin 1; Slc2a2, solute carrier family 2 member 2 gene; Slc2a4, solute carrier family 2 member 4 gene; STZ, streptozotocin; T1D, type 1 diabetes mellitus; T2D, type 2 diabetes mellitusAMPK, AMP-activated protein kinase; ANOVA, analysis of variance; AU, arbitrary units;


Plumula Nelumbinis is widely used in the world because it has many biological activities, such as anti-inflammation, antioxidant, antihypertension, and butyrylcholinesterase inhibition. However, the action of Plumula Nelumbinis on airway smooth muscle (ASM) relaxation has not been investigated. A chloroform extract of Plumula Nelumbinis (CEPN) was prepared, which completely inhibited precontraction induced by high K+ in a concentration-dependent manner in mouse tracheal rings, but it had no effect on resting tension. CEPN also blocked voltage-dependent L-type Ca2+ channel- (VDCC-) mediated currents. In addition, ACh-induced precontraction was also completely blocked by CEPN and partially inhibited by nifedipine or pyrazole 3. Besides, CEPN partially reduced ACh-activated nonselective cation channel (NSCC) currents. Taken together, our data demonstrate that CEPN blocked VDCC and NSCC to inhibit Ca2+ influx, resulting in relaxation of precontracted ASM. This finding indicates that CEPN would be a candidate of new potent bronchodilators.The traditional herb  Excessive airway obstruction is a cardinal symptom in asthma and COPD –3. Airwadiseases , 8. Howe Plumula Nelumbinis relaxed precontracted ASM induced by high K+ and ACh and the underlying mechanism.Some of traditional herbs have long been used in the treatment of asthma and COPD because they can effectively relax ASM contraction –11. ThisPlumula Nelumbinis is the green germ of a mature lotus (Nelumbo nucifera Gaertn) seed [ Plumula Nelumbinis has many pharmacological and physiological activities, including anti-inflammation [ Plumula Nelumbinis exhibit beneficial biological activities on smooth muscle: neferine markedly inhibits angiotensin II-stimulated proliferation, reduces 45Ca-influx induced by phenylephrine in vascular smooth muscle [ Plumula Nelumbinis extract on ASMC tension have not been studied previously.tn) seed , 13. Thiammation , 15, antammation , antihypammation , and butammation . Severalh muscle , 20, andh muscle , and isoh muscle , 23. How Plumula Nelumbinis (CEPN) on mouse ASM precontraction induced by high K+ and ACh. The results show that CEPN inhibited VDCCs and NSCCs, which then resulted in relaxation of precontracted ASM.The present study investigated the relaxation effects of a chloroform extract ofPlumula Nelumbinis, germs of Nelumbo nucifera Gaertn seeds, were collected in Honghu City, Hubei Province, China, in October 2013, and were identified by Professor Dr. Ding-rong Wan, College of Pharmacy, South-Central University for Nationalities. A voucher specimen (SCUN201310006) is deposited at the Herbarium of the College of Pharmacy, South-Central University for Nationalities, China.Plumula Nelumbinis (1 Kg) were air-dried, milled into powder, and extracted at room temperature with 95% ethanol . Extracts were centrifuged, and the supernatants were collected. The ethanol extract (193 g) was next evaporated to dryness under reduced pressure using a rotary evaporator and immersed in a 2% HCl solution (500 mL). Residues were extracted with petroleum ether  to remove lipids. Ammonia adjusted the pH of the filtrate to 10, and the crude sample was extracted with chloroform . The chloroform extract was evaporated under reduced pressure, and the extraction yield was 0.71% of the raw material dry weight. The dried chloroform extract of Plumula Nelumbinis (CEPN) was dissolved in 3% DMSO for the experiments.Nifedipine, acetylcholine chloride (ACh), niflumic acid (NA), tetraethylammonium chloride (TEA), and pyrazole 3 (Pyr3) were purchased from Sigma Chemical Co. . Other chemicals were purchased from Sinopharm Chemical Reagent Co. .Sexually mature male BALB/c mice were purchased from the Hubei Provincial Center for Disease Control and Prevention . Mice were housed at room temperature (20–25°C) and constant humidity (50–60%) under a 12 h light-dark cycle in an SPF grade laboratory. The animal study was performed according to the guidelines of the Institutional Animal Care and Use Committee of the South-Central University for Nationalities  and approved by the Animal Care and Ethics Committee of the South-Central University for Nationalities .2 1, CaCl2 2, HEPES 10, glucose 10, pH 7.4). Connective tissues were removed, and small rings (~5 mm) were cut from the bottom of tracheae. Each ring was mounted with a preload of 0.5 g in an organ bath with a 10 mL capacity containing PSS bubbled with 95% O2-5% CO2 at 37°C. Tracheal rings were equilibrated for 60 min, precontracted with high K+ (80 mM) or ACh (10−4 M), washed, and rested 3 times. Experiments were performed following an additional 30 min rest.Mouse ASM contraction was measured in tracheal rings , 25. Mic2+ physiological saline solution (LCPSS)  containing 1 mg/mL papain, 0.5 mg/mL dithioerythritol, and 1 mg/mL bovine serum albumin (BSA) at 37°C for 20 min. Tissues were transferred to LCPSS containing 1 mg/mL collagenase H, 1 mg/mL dithiothreitol, and 1 mg/mL BSA and incubated at 37°C for 20 min. Tissues were washed and gently triturated in LCPSS to yield single ASMCs for experiments.Mouse ASMCs were isolated as previous method , 26. Bri2+ as the charge carrier and an EPC-10 patch-clamp amplifier . The pipette solution contained the following (in mM): CsCl 130, EGTA 10, MgCl2 4, Mg-ATP 4, HEPES 10, TEA 10, and pH 7.2 (adjusted with CsOH). The composition of the bath solution was (in mM) NaCl 105, CsCl 6, BaCl2 27.5, glucose 11, HEPES 10, TEA-Cl 10, NA 0.1, and pH 7.4 (adjusted with NaOH). ASMCs were patched and held at −70 mV. Currents were measured following depolarization for 1000 ms from −70 to +30 mV in 10 mV increments every 10 s.VDCC currents were measured using Ba2 1.2, HEPES 10, EGTA 3, CaCl2 1, and pH 7.2 (adjusted with Tris). The free Ca2+ concentration was approximately 70 nM, as calculated using WEBMAXC STANDARD. The bath solution was PSS without K+ containing 10 µM nifedipine, 100 µM NA, and 10 mM TEA to block VDCC, Cl−, and K+ currents, respectively. ACh-induced NSCC currents were recorded with a ramp using a perforated whole-cell configuration with a holding potential of −60 mV. The ramp was performed over 500 ms from −80 to +60 mV.The pipette solution contained the following chemicals for the measurement of NSCC currents (in mM): CsCl 18, cesium acetate 108, MgClt-test using Origin 9.0 software . Statistical significance was defined as P < 0.05. Data are expressed as the means ± SEM.Statistical analysis was performed with Student's +-induced ASM precontraction to investigate whether CEPN relaxed ASM. High K+ (80 mM) induced contraction in a mouse tracheal ring, and CEPN (1 µg–3.16 mg/mL) was cumulatively added to the organ bath. ASM contraction was gradually reduced to baseline (+-induced contraction will restore to 26.7 ± 6.2% (n = 6) after 30 min. The results from 7 experiments yielded a half-maximal inhibition (IC50) of 35.4 ± 1.4 µg/mL and a maximal relaxation of 103.4 ± 1.3% , which completely blocked 80 mM K+-induced contractions ; however, contraction immediately occurred following Ca2+ restoration (2 mM), and CEPN (1 mg/mL) completely inhibited contraction. However, a Ca2+ restoration-induced contraction was not observed in the presence of 1 mg/mL CEPN (+-induced precontraction was completely dependent on the inhibition on VDCC-mediated Ca2+ influx.The above results suggest that the relaxation induced by CEPN might be due to the termination of VDCC-mediated Ca/mL CEPN . This re2+ as the carrier charge) [I-V) curves were constructed based on the results of 5 to 6 experiments (We used patch-clamp techniques to measure VDCC currents (Ba charge)  with voleriments . These dµM ACh-induced contraction . If CEPN was removed, the ACh-induced contraction will recover to 92.2 ± 3.4% (n = 7) within 30 min. Moreover, the precontraction was partially blocked following additions of nifedipine and the resistant component was totally inhibited by 1 mg/mL CEPN  and the remained contraction was completely blocked by CEPN  and Ca2+-free conditions (0 mM Ca2+ and 0.5 mM EGTA)  . Additio2+ influx because ACh activates NSCCs to increase intracellular Ca2+ [n = 8, We measured ACh-activated NSCC currents and observed the effects of CEPN on these currents to further determine the nature of the Calar Ca2+ , 35, 36.+ and ACh through blockade of Ca2+ influx mediated by VDCCs and NSCCs. This finding suggests that CEPN may be a potential bronchodilator.The present study demonstrated that CEPN induced strong relaxation in precontracted mouse ASM induced by high K+ induces membrane depolarization, which opens VDCCs that mediate extracellular Ca2+ influx and induce contractions [+-induced contractions in ASM  ACh by CEPN , and (4) by CEPN . TherefoCEPN induced relaxation of precontracted mouse ASM through the inactivation of VDCCs and NSCCs. This study supports the development of new drugs from CEPN to treat airway hyperresponsiveness in asthmatic and COPD patients. Further investigation will be required to identify the components that are responsible for the relaxation action.


Echinochrome A (Ech A) is a natural pigment from sea urchins that has been reported to have antioxidant properties and a cardio protective effect against ischemia reperfusion injury. In this study, we ascertained whether Ech A enhances the mitochondrial biogenesis and oxidative phosphorylation in rat cardio myoblast H9c2 cells. To study the effects of Ech A on mitochondrial biogenesis, we measured mitochondrial mass, level of oxidative phosphorylation, and mitochondrial biogenesis regulatory gene expression. Ech A treatment did not induce cytotoxicity. However, Ech A treatment enhanced oxygen consumption rate and mitochondrial ATP level. Likewise, Ech A treatment increased mitochondrial contents in H9c2 cells. Furthermore, Ech A treatment up-regulated biogenesis of regulatory transcription genes, including proliferator-activated receptor gamma co-activator (PGC)-1α, estrogen-related receptor (ERR)-α, peroxisome proliferator-activator receptor (PPAR)-γ, and nuclear respiratory factor (NRF)-1 and such mitochondrial transcription regulatory genes as mitochondrial transcriptional factor A (TFAM), mitochondrial transcription factor B2 (TFB2M), mitochondrial DNA direct polymerase (POLMRT), single strand binding protein (SSBP) and Tu translation elongation factor (TUFM). In conclusion, these data suggest that Ech A is a potentiated marine drug which enhances mitochondrial biogenesis. Of thin drugs ,11,12.® drug is produced as an isotonic solution of di- and trisodium salts of Ech A at a concentration of 0.2 mg/mL. Histochrome® normalizes metabolic processes and eliminates inflammation in the retina, vascular membrane, and cornea of the eye. It improves trophic functions, reduces edema, and accelerates epithelialization, when administered into patients [Besides antioxidant properties, Ech A has a cardioprotective effect against reperfusion injury . The Hispatients . However2+ levels during ischemic reperfusion [Mitochondria are essential and pivotal regulators of cellular bioenergetics . Mitochoerfusion ,21. As aerfusion . HoweverMitochondrial biogenesis is regulated by a network of signaling factors. The peroxisome proliferated receptor gamma co-activator (PCG) family of transcription co-activators (PCG-1α/β) is a major group of regulators involved in mitochondrial biogenesis an important regulator of oxidative metabolism in the heart . PCG-1α In this study, we focus on the natural compound Ech A, which has a beneficial effect on cardiac mitochondrial biogenesis and OXPHOS function without cytotoxicity. We investigated how Ech A affects cardiac mitochondrial function in cardiomyocytes via the PGC-1α signal pathway by measuring changes in downstream gene expression after treatment with Ech A.First, we investigated whether Ech A influences cell proliferation and cell viability using rat cardio myoblast H9c2 cells. The cells were cultured with 0, 2.5, 5, 7.5, 10, 50, and 100 μM of Ech A for 24 h. As Next, we investigated whether Ech A influences oxidative phosphorylation and energy production. We measured cellular oxygen consumption rate (OCR) 24 h after Ech A treatment. Interestingly, Ech A treatment significantly increased cellular oxygen consumption rate A. Cells Next, we investigated whether the observed increase in mitochondrial function was due to increased mitochondrial contents. We measured mitochondrial contents using 10-nonylacridine orange (NAO) staining and confocal microscopy. We found that mitochondrial mass as measured by NAO intensity, was increased by Ech A treatment B. This rTo further evaluate mitochondrial biogenesis, we examined mitochondrial biogenesis-regulated gene expression levels by quantitative real-time PCR. As describe in previous studies, mitochondrial biogenesis is regulated by various transcription factors, such as PGC-1α and NRF-1 ,36,37,38It seems plausible that the effects of Ech A, such as increasing of oxygen consumption rate , are conOne of the important activators, CREB (cAMP response element-binding protein), increases expression of PGC-1α ,42. TherScaphechinus mirabilis (Agassiz) by a previously described method [®), produced by Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences as a stock solution [Ech A was isolated from the sea urchin d method . The purd method ); and sid method . We usedsolution .Rat cardio myoblast H9c2 cells  were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% of heat-inactivated fetal bovine serum, 50 U/mL penicillin and 50 μg/mL streptomycin .4 cells/well in 96-wells tissue culture plate. After 16 h, the cells were treated with 0, 5, or 10 μM of Ech A for 24 h. Cell viability was measured by quantitative colorimetric assay with MTT -2,5-diphenyltetrazolium bromide; Sigma-Aldrich, St. Louis, MO, USA), which measures the mitochondrial activity of living cells. The extent of reduction of MTT to formazan within the cells was quantified by measuring the optical density at 570 nm using a microplate reader .H9c2 cells were plated 2 × 104 cells/well in black and clear bottom 96-wells tissue culture plate. After 16 h, the cells were treated with 0, 2.5, 5, 7.5, 10, 50, and 100 μM of Ech A for 24 h. Cytotoxicity was assessed by quantitative fluorescence assay with CellTox Green cytotoxicity assay . This cytotoxicity assay measures changes in membrane integrity that occur as results of cell death. For a positive control , lysis buffer (as 0.02% digitonin) was added in non-treated cells. Cells were quantified by measuring fluorescence (excitation/emission = 485 nm/530 nm) using a microplate reader .H9c2 cells were plated 2 × 106 cells in 60 mm cell culture plate. After 16 h, the cells were treated with 0, 5, or 10 μM of Ech A for 24 h. Then the cells were stained with 200 nM TMRE for 30 min at 37 °C. Cells were washed twice with PBS, and then the relative signal intensity of TMRE in cells was analyzed using a FACsCalibur flow cytometer .To evaluate the effect of Ech A in mitochondria, mitochondrial inner membrane potential (ΔΨm) was compared in cells treated with 0, 5, or 10 μM of Ech A using the fluorescent dye tetramethylrhodamine, ethyl ester  which is sequestered by active mitochondria. H9c2 cells were plated 2 × 102DCF-DA , a general ROS indicator. H9c2 cells were plated 2 × 106 cells in 60 mm cell culture plate. After 16 h, the cells were treated with 0, 5, or 10 μM of Ech A for 24 h. The cells were stained with 10 μM CM-H2DCF-DA for 30 min at 37 °C. After washing twice with PBS, relative signal intensity of CM-H2DCF-DA in cells was analyzed using a FACsCalibur flow cytometer .To evaluate the effect of Ech A in mitochondria, ROS was measured in cells treated with Ech A  using the fluorescent dye CM-H6 cells/well in 60 mm tissue culture plate. After 16 h, the cells were treated with 0, 5, or 10 μM of Ech A for 24 h. Treated cells were harvested and resuspended by pipetting until the cells were evenly dispersed. Resuspended H9c2 cells were plated 2 × 104 cells/well in white and clear bottom 96-well culture plate. Plates were centrifuged at 200× g for 10 min to remove medium and after it 50 μL of fresh medium lacking glucose and supplemented with 10 mM galactose. The plate incubated at 37 °C in a humidified and CO2-supplemented incubator for 90 min. Assay solution (100 μL) was added to the plate, and then the plate was incubated at room temperature for 30 min. Luminescence was measured using a luminometer .Mitochondrial ATP level was measured by Mitochondrial ToxGlo™ assay  according to the manufacture’s protocol. Briefly, H9c2 cells were plated 2 × 104 cells/well in XF24 cell culture plate . After 16 h, the cells were treated with various doses of Ech A. After 24 h, the medium was exchanged for 500 μL of XF Assay Medium-modified DMEM  and then incubated at 37 °C without CO2 for 1 h. OCR was measured by XF24 analyzer and XF24 software. After measuring the OCR, the XF24 assay results were normalized to cell number. Cell number for each well was counted using a Luna™ automated cell counter .OCR was measured using a XF24 analyzer  as previously described . BrieflyTo measure the mitochondrial mass, cells treated with 0, 5, or 10 μM of Ech A were stained with acridine orange 10-nonyl bromide at a final concentration of 2.5 μM in phosphate buffered saline (PBS)  This dye binds with mitochondrial membrane cardiolipin. Importantly, binding is independent of ΔΨm over the physiologically relevant range. The cells were incubated in dark at 37 °C for 30 min then washed twice with PBS. NAO fluorescence for the each group was measured in laser confocal microscope . The cells were excited using 488 nm and emission of NAO was measured beyond 585 nm. The mean intensity of the region of interest (ROI) was measured in each cell and analyzed by ZEN2009 software . Nucleoli of cells were co-stained with Hoechst 33342 as a counter stain.6 cells in 60 mm cell culture plate. After 16 h, the cells were treated with 0, 5, or 10 μM of Ech A for 24 h. The cells were stained with 2.5 μM NAO for 30 min at 37 °C. After washing twice with PBS, relative signal intensity of NAO in cells was analyzed using a FACsCalibur flow cytometer .To confirm the mitochondrial mass, H9c2 cells were plated 2 × 10Total RNA of H9c2 cells was extracted using Trizol reagent . One microgram of total RNA was reverse transcribed using Revet Aid First Strand cDNA Synthesis kit  according to the manufacturer’s instructions. The forward and reverse primers are shown in Total DNA, including chromosomal and mitochondrial DNA, was extracted from H9c2 cells treated with 0, 5, or 10 μM Ech A using a Gentra Puregene kit  following the manufacturer’s instructions. The forward and reverse primers are shown in Cell lysates were centrifuged at 14,000 rpm for 15 min at 4 °C. Protein concentrations were determined by Bradford protein assay , and 30 μg of protein was loaded per lane onto 10% SDS polyacrylamide gels. Gels were transferred onto nitrocellulose membranes  and incubated with specific antibodies , and beta-tubulin; Cell Signaling, Danvers, MA, USA, PGC-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Western blot analysis was performed using these antibodies and a western blotting detection kit, Ab signal™ . Blots were visualized with an LAS-3000 Plus imager .p ≤ 0.05 was considered significant.Unless stated otherwise, all experiments were performed in triplicate. Data are presented as means ± standard error of the mean (SEM). One-way ANOVA was used to compare values between groups, and ® drug used to treat acute myocardial ischemia in patients [ex vivo and animal models.To the best of our knowledge, this is the first study that investigates the net effect of Ech A on mitochondrial biogenesis and OXPHOS function. Our results indicate that Ech A increased mitochondrial mass and OXPHOS function significantly, which enhanced mitochondrial energy efficiency by modulating major mitochondria biogenesis regulatory genes, including PGC-1α and NRF-1. These results provide some evidence that Ech A has the potential to enhance mitochondrial energy metabolism, which may be clinically beneficial for the treatment of various mitochondrial dysfunctions implicated in metabolic diseases. These results can explain the ATP-saving effect of the Histochromepatients . In futu