Fasudil and viscosity of gelatin promote hepatic differentiation by regulating organelles in human umbilical cord matrix-mesenchymal stem cells.

BACKGROUND: Human mesenchymal stem cells originating from umbilical cord matrix are a promising therapeutic resource, and their differentiated cells are spotlighted as a tissue regeneration treatment. However, there are limitations to the medical use of differentiated cells from human umbilical cord matrix-mesenchymal stem cells (hUCM-MSCs), such as efficient differentiation methods. METHODS: To effectively differentiate hUCM-MSCs into hepatocyte-like cells (HLCs), we used the ROCK inhibitor, fasudil, which is known to induce endoderm formation, and gelatin, which provides extracellular matrix to the differentiated cells. To estimate a differentiation efficiency of early stage according to combination of gelatin and fasudil, transcription analysis was conducted. Moreover, to demonstrate that organelle states affect differentiation, we performed transcription, tomographic, and mitochondrial function analysis at each stage of hepatic differentiation. Finally, we evaluated hepatocyte function based on the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4 in mature HLCs. RESULTS: Fasudil induced endoderm-related genes (GATA4, SOX17, and FOXA2) in hUCM-MSCs, and it also induced lipid droplets (LDs) inside the differentiated cells. However, the excessive induction of LDs caused by fasudil inhibited mitochondrial function and prevented differentiation into hepatoblasts. To prevent the excessive LDs formation, we used gelatin as a coating material. When hUCM-MSCs were induced into hepatoblasts with fasudil on high-viscosity (1%) gelatin-coated dishes, hepatoblast-related genes (AFP and HNF4A) showed significant upregulation on high-viscosity gelatin-coated dishes compared to those treated with low-viscosity (0.1%) gelatin. Moreover, other germline cell fates, such as ectoderm and mesoderm, were repressed under these conditions. In addition, LDs abundance was also reduced, whereas mitochondrial function was increased. On the other hand, unlike early stage of the differentiation, low viscosity gelatin was more effective in generating mature HLCs. In this condition, the accumulation of LDs was inhibited in the cells, and mitochondria were activated. Consequently, HLCs originated from hUCM-MSCs were genetically and functionally more matured in low-viscosity gelatin. CONCLUSIONS: This study demonstrated an effective method for differentiating hUCM-MSCs into hepatic cells using fasudil and gelatin of varying viscosities. Moreover, we suggest that efficient hepatic differentiation and the function of hepatic cells differentiated from hUCM-MSCs depend not only on genetic changes but also on the regulation of organelle states.

Ibulocydine Inhibits Migration and Invasion of TNBC Cells via MMP-9 Regulation.

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancer types, indicating a poor survival prognosis with a more aggressive biology of metastasis to the lung and a short response duration to available therapies. Ibulocydine (IB) is a novel (cyclin-dependent kinase) CDK7/9 inhibitor prodrug displaying potent anti-cancer effects against various cancer cell types. We performed in vitro and in vivo experiments to determine whether IB inhibits metastasis and eventually overcomes the poor drug response in TNBC. The result showed that IB inhibited the growth of TNBC cells by inducing caspase-mediated apoptosis and blocking metastasis by reducing MMP-9 expression in vitro. Concurrently, in vivo experiments using the metastasis model showed that IB inhibited metastasis of MDA-MB-231-Luc cells to the lung. Collectively, these results demonstrate that IB inhibited the growth of TNBC cells and blocked metastasis by regulating MMP-9 expression, suggesting a novel therapeutic agent for metastatic TNBC.

Crotamine-based recombinant immunotoxin targeting HER2 for enhanced cancer cell specificity and cytotoxicity.

Crotamine, one of the major toxins present in the venom of the South American rattlesnake Crotalus durissus terrificus, exhibits potent cytotoxic properties and has been suggested for cancer therapy applications. However, its selectivity for cancer cells needs to be improved. This study designed and produced a novel recombinant immunotoxin, HER2(scFv)-CRT, composed of crotamine and single-chain Fv (scFv) derived from trastuzumab targeting human epidermal growth factor receptor 2 (HER2). The recombinant immunotoxin was expressed in Escherichia coli and purified using various chromatographic techniques. The cytotoxicity of HER2(scFv)-CRT was assessed in three breast cancer cell lines, demonstrating enhanced specificity and toxicity in HER2-expressing cells. These findings suggest that the crotamine-based recombinant immunotoxin has the potential to expand the repertoire of recombinant immunotoxin applications in cancer therapy.

Toward a new taxonomy of obstetrical disease: improved performance of maternal blood biomarkers for the great obstetrical syndromes when classified according to placental pathology.

BACKGROUND: The major challenge for obstetrics is the prediction and prevention of the great obstetrical syndromes. We propose that defining obstetrical diseases by the combination of clinical presentation and disease mechanisms as inferred by placental pathology will aid in the discovery of biomarkers and add specificity to those already known. OBJECTIVE: To describe the longitudinal profile of placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and the PlGF/sFlt-1 ratio throughout gestation, and to determine whether the association between abnormal biomarker profiles and obstetrical syndromes is strengthened by information derived from placental examination, eg, the presence or absence of placental lesions of maternal vascular malperfusion. STUDY DESIGN: This retrospective case cohort study was based on a parent cohort of 4006 pregnant women enrolled prospectively. The case cohort of 1499 pregnant women included 1000 randomly selected patients from the parent cohort and all additional patients with obstetrical syndromes from the parent cohort. Pregnant women were classified into six groups: 1) term delivery without pregnancy complications (n=540; control); 2) preterm labor and delivery (n=203); 3) preterm premature rupture of the membranes (n=112); 4) preeclampsia (n=230); 5) small-for-gestational-age neonate (n=334); and 6) other pregnancy complications (n=182). Maternal plasma concentrations of PlGF and sFlt-1 were determined by enzyme-linked immunosorbent assays in 7560 longitudinal samples. Placental pathologists, masked to clinical outcomes, diagnosed the presence or absence of placental lesions of maternal vascular malperfusion. Comparisons between mean biomarker concentrations in cases and controls were performed by utilizing longitudinal generalized additive models. Comparisons were made between controls and each obstetrical syndrome with and without subclassifying cases according to the presence or absence of placental lesions of maternal vascular malperfusion. RESULTS: 1) When obstetrical syndromes are classified based on the presence or absence of placental lesions of maternal vascular malperfusion, significant differences in the mean plasma concentrations of PlGF, sFlt-1, and the PlGF/sFlt-1 ratio between cases and controls emerge earlier in gestation; 2) the strength of association between an abnormal PlGF/sFlt-1 ratio and the occurrence of obstetrical syndromes increases when placental lesions of maternal vascular malperfusion are present (adjusted odds ratio [aOR], 13.6 vs 6.7 for preeclampsia; aOR, 8.1 vs 4.4 for small-for-gestational-age neonates; aOR, 5.5 vs 2.1 for preterm premature rupture of the membranes; and aOR, 3.3 vs 2.1 for preterm labor (all P<0.05); and 3) the PlGF/sFlt-1 ratio at 28 to 32 weeks of gestation is abnormal in patients who subsequently delivered due to preterm labor with intact membranes and in those with preterm premature rupture of the membranes if both groups have placental lesions of maternal vascular malperfusion. Such association is not significant in patients with these obstetrical syndromes who do not have placental lesions. CONCLUSION: Classification of obstetrical syndromes according to the presence or absence of placental lesions of maternal vascular malperfusion allows biomarkers to be informative earlier in gestation and enhances the strength of association between biomarkers and clinical outcomes. We propose that a new taxonomy of obstetrical disorders informed by placental pathology will facilitate the discovery and implementation of biomarkers as well as the prediction and prevention of such disorders.

Antinociceptive and Anti-Inflammatory Effects of Recombinant Crotamine in Mouse Models of Pain.

Crotamine, a toxin found in the venom of the South American rattlesnake Crotalus durissus terrificus, has been reported to have antinociceptive effects. We purified recombinant crotamine expressed in Escherichia coli and investigated its antinociceptive and anti-inflammatory effects using the hot-plate test, acetic-acid-induced writhing method, and formalin test in mice. Recombinant crotamine was administered intraperitoneally (0.04-1.2 mg kg-1) or intraplantarly (0.9-7.5 µg 10 µL-1) before the tests. The paw volume was measured with a plethysmometer. To evaluate the antagonistic and anti-inflammatory effects of naloxone, subcutaneous naloxone (4 mg kg-1) or intraplantar naloxone (5 µg 10 µL-1) was administered before recombinant crotamine. For tumor necrosis factor (TNF)-α assays, blood was drawn 3 h after formalin injection and measured using enzyme-linked immunosorbent assay. Intraperitoneal and intraplantar recombinant crotamine had antinociceptive and anti-inflammatory effects, neither of which were affected by pre-treatment with naloxone. The mean serum TNF-α levels were significantly lower in the intraperitoneal recombinant crotamine (0.4 and 1.2 mg kg-1) or intraplantar (2.5 and 7.5 µg 10 µL-1) recombinant crotamine groups than in the saline group and were not affected by naloxone pre-treatment. In conclusion, recombinant crotamine possesses significant antinociceptive and anti-inflammatory effects that do not appear to be related to the opioid receptor. The antinociceptive and anti-inflammatory effects of intraperitoneal or intraplantar recombinant crotamine are related to TNF-α.

Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion.

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.

Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor.

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a') tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b'a' Domain.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b'a' domain of protein disulfide isomerase (PDIb'a') greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb'a'-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.

De Novo Development of mtDNA Deletion Due to Decreased POLG and SSBP1 Expression in Humans.

Defects in the mitochondrial genome (mitochondrial DNA (mtDNA)) are associated with both congenital and acquired disorders in humans. Nuclear-encoded DNA polymerase subunit gamma (POLG) plays an important role in mtDNA replication, and proofreading and mutations in POLG have been linked with increased mtDNA deletions. SSBP1 is also a crucial gene for mtDNA replication. Here, we describe a patient diagnosed with Pearson syndrome with large mtDNA deletions that were not detected in the somatic cells of the mother. Exome sequencing was used to evaluate the nuclear factors associated with the patient and his family, which revealed a paternal POLG mutation (c.868C > T) and a maternal SSBP1 mutation (c.320G > A). The patient showed lower POLG and SSBP1 expression than his healthy brothers and the general population of a similar age. Notably, c.868C in the wild-type allele was highly methylated in the patient compared to the same site in both his healthy brothers. These results suggest that the co- deficient expression of POLG and SSBP1 genes could contribute to the development of mtDNA deletion.

Allogeneic epithelial cell sheet transplantation for preventing esophageal stricture after circumferential ESD in a porcine model: preliminary results.

OBJECTIVES: Circumferential endoscopic submucosal dissection (ESD) for large lesions induces severe stricture, requiring subsequent treatment. We aimed to evaluate the efficacy of allogeneic epithelial cell sheet transplantation in preventing esophageal stricture after circumferential ESD in a porcine model. MATERIALS AND METHODS: A total of 15 conventional pigs underwent a 4 cm long circumferential ESD in the mid-esophagus. Out of these animals, 11 were immediately subjected to allogeneic oral mucosal cell sheet transplantation at the resection site, whereas four pigs underwent circumferential ESD only. We performed upper endoscopy 1 and 2 weeks after ESD and assessed the degree of esophageal stricture and histologic characteristics. RESULTS: Dysphagia scores and weight change ratios recorded 1 and 2 weeks after ESD did not differ between the two groups. The stricture rate 2 weeks after ESD was 100% in the control group and 90.9% in the cell sheet group (p = 1.000). The median mucosal constriction rates of the control and cell sheet groups were 73.5% (range 63.0-80.0%) and 53.8% (37.5-73.3%, p = .018), respectively. With regard to microscopic measurements, the length of re-epithelialization was greater in the cell sheet group than in the control group (2,495 µm vs. 369 µm, p = .008). Median fibrosis thickness and degree of muscle damage were not significantly different between groups. CONCLUSIONS: Although allogeneic epithelial cell sheet transplantation showed greater re-epithelialization and less mucosal constriction of post-ESD ulcers, it was not sufficiently effective in preventing post-ESD stricture.

Role of Ischemic Preconditioning in the Cardioprotective Mechanisms of Monomeric C-Reactive Protein-Deposited Myocardium in a Rat Model.

BACKGROUND: The deposition of monomeric C-reactive protein (mCRP) in the myocardium aggravates ischemia-reperfusion injury (IRI) and myocardial infarction. Ischemic preconditioning (IPC) is known to protect the myocardium against IRI. METHODS: We evaluated the effects of IPC on myocardium upon which mCRP had been deposited due to IRI in a rat model. Myocardial IRI was induced via ligation of the coronary artery. Direct IPC was applied prior to IRI using multiple short direct occlusions of the coronary artery. CRP was infused intravenously after IRI. The study included sham (n=3), IRI-only (n=5), IRI+CRP (n=9), and IPC+IRI+CRP (n=6) groups. The infarcted area and the area at risk were assessed using Evans blue and 2,3,5-triphenyltetrazolium staining. Additionally, mCRP immunostaining and interleukin-6 (IL-6) mRNA reverse transcription-polymerase chain reaction were performed. RESULTS: In the IRI+CRP group, the infarcted area and the area of mCRP deposition were greater, and the level of IL-6 mRNA expression was higher, than in the IRI-only group. However, in the IPC+IRI+CRP group relative to the IRI+CRP group, the relative areas of infarction (20% vs. 34%, respectively; p=0.079) and mCRP myocardial deposition (21% vs. 44%, respectively; p=0.026) were lower and IL-6 mRNA expression was higher (fold change: 407 vs. 326, respectively; p=0.376), although the difference in IL-6 mRNA expression was not statistically significant. CONCLUSION: IPC was associated with significantly decreased deposition of mCRP and with increased expression of IL-6 in myocardium damaged by IRI. The net cardioprotective effect of decreased mCRP deposition and increased IL-6 levels should be clarified in a further study.

Clinical chorioamnionitis at term X: microbiology, clinical signs, placental pathology, and neonatal bacteremia - implications for clinical care.

OBJECTIVES: Clinical chorioamnionitis at term is considered the most common infection-related diagnosis in labor and delivery units worldwide. The syndrome affects 5-12% of all term pregnancies and is a leading cause of maternal morbidity and mortality as well as neonatal death and sepsis. The objectives of this study were to determine the (1) amniotic fluid microbiology using cultivation and molecular microbiologic techniques; (2) diagnostic accuracy of the clinical criteria used to identify patients with intra-amniotic infection; (3) relationship between acute inflammatory lesions of the placenta (maternal and fetal inflammatory responses) and amniotic fluid microbiology and inflammatory markers; and (4) frequency of neonatal bacteremia. METHODS: This retrospective cross-sectional study included 43 women with the diagnosis of clinical chorioamnionitis at term. The presence of microorganisms in the amniotic cavity was determined through the analysis of amniotic fluid samples by cultivation for aerobes, anaerobes, and genital mycoplasmas. A broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry was also used to detect bacteria, select viruses, and fungi. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin-6 (IL-6) concentration ≥2.6 ng/mL. RESULTS: (1) Intra-amniotic infection (defined as the combination of microorganisms detected in amniotic fluid and an elevated IL-6 concentration) was present in 63% (27/43) of cases; (2) the most common microorganisms found in the amniotic fluid samples were Ureaplasma species, followed by Gardnerella vaginalis; (3) sterile intra-amniotic inflammation (elevated IL-6 in amniotic fluid but without detectable microorganisms) was present in 5% (2/43) of cases; (4) 26% of patients with the diagnosis of clinical chorioamnionitis had no evidence of intra-amniotic infection or intra-amniotic inflammation; (5) intra-amniotic infection was more common when the membranes were ruptured than when they were intact (78% [21/27] vs. 38% [6/16]; p=0.01); (6) the traditional criteria for the diagnosis of clinical chorioamnionitis had poor diagnostic performance in identifying proven intra-amniotic infection (overall accuracy, 40-58%); (7) neonatal bacteremia was diagnosed in 4.9% (2/41) of cases; and (8) a fetal inflammatory response defined as the presence of severe acute funisitis was observed in 33% (9/27) of cases. CONCLUSIONS: Clinical chorioamnionitis at term, a syndrome that can result from intra-amniotic infection, was diagnosed in approximately 63% of cases and sterile intra-amniotic inflammation in 5% of cases. However, a substantial number of patients had no evidence of intra-amniotic infection or intra-amniotic inflammation. Evidence of the fetal inflammatory response syndrome was frequently present, but microorganisms were detected in only 4.9% of cases based on cultures of aerobic and anaerobic bacteria in neonatal blood.

The fetal inflammatory response syndrome: the origins of a concept, pathophysiology, diagnosis, and obstetrical implications.

The fetus can deploy a local or systemic inflammatory response when exposed to microorganisms or, alternatively, to non-infection-related stimuli (e.g., danger signals or alarmins). The term "Fetal Inflammatory Response Syndrome" (FIRS) was coined to describe a condition characterized by evidence of a systemic inflammatory response, frequently a result of the activation of the innate limb of the immune response. FIRS can be diagnosed by an increased concentration of umbilical cord plasma or serum acute phase reactants such as C-reactive protein or cytokines (e.g., interleukin-6). Pathologic evidence of a systemic fetal inflammatory response indicates the presence of funisitis or chorionic vasculitis. FIRS was first described in patients at risk for intraamniotic infection who presented preterm labor with intact membranes or preterm prelabor rupture of the membranes. However, FIRS can also be observed in patients with sterile intra-amniotic inflammation, alloimmunization (e.g., Rh disease), and active autoimmune disorders. Neonates born with FIRS have a higher rate of complications, such as early-onset neonatal sepsis, intraventricular hemorrhage, periventricular leukomalacia, and death, than those born without FIRS. Survivors are at risk for long-term sequelae that may include bronchopulmonary dysplasia, neurodevelopmental disorders, such as cerebral palsy, retinopathy of prematurity, and sensorineuronal hearing loss. Experimental FIRS can be induced by intra-amniotic administration of bacteria, microbial products (such as endotoxin), or inflammatory cytokines (such as interleukin-1), and animal models have provided important insights about the mechanisms responsible for multiple organ involvement and dysfunction. A systemic fetal inflammatory response is thought to be adaptive, but, on occasion, may become dysregulated whereby a fetal cytokine storm ensues and can lead to multiple organ dysfunction and even fetal death if delivery does not occur ("rescued by birth"). Thus, the onset of preterm labor in this context can be considered to have survival value. The evidence so far suggests that FIRS may compound the effects of immaturity and neonatal inflammation, thus increasing the risk of neonatal complications and long-term morbidity. Modulation of a dysregulated fetal inflammatory response by the administration of antimicrobial agents, anti-inflammatory agents, or cell-based therapy holds promise to reduce infant morbidity and mortality.

Alteration of fatty acid oxidation by increased CPT1A on replicative senescence of placenta-derived mesenchymal stem cells.

BACKGROUND: Human placenta-derived mesenchymal stem cells (PD-MSCs) are powerful sources for cell therapy in regenerative medicine. However, a limited lifespan by senescence through mechanisms that are well unknown is the greatest obstacle. In the present study, we first demonstrated the characterization of replicative senescent PD-MSCs and their possible mitochondrial functional alterations. METHODS: Human PD-MSCs were cultured to senescent cells for a long period of time. The cells of before passage number 8 were early cells and after passage number 14 were late cells. Also, immortalized cells of PD-MSCs (overexpressed hTERT gene into PD-MSCs) after passage number 14 were positive control of non-senescent cells. The characterization and mitochondria analysis of PD-MSCs were explored with long-term cultivation. RESULTS: Long-term cultivation of PD-MSCs exhibited increases of senescent markers such as SA-ß-gal and p21 including apoptotic factor, and decreases of proliferation, differentiation potential, and survival factor. Mitochondrial dysfunction was also observed in membrane potential and metabolic flexibility with enlarged mitochondrial mass. Interestingly, we founded that fatty acid oxidation (FAO) is an important metabolism in PD-MSCs, and carnitine palmitoyltransferase1A (CPT1A) overexpressed in senescent PD-MSCs. The inhibition of CPT1A induced a change of energy metabolism and reversed senescence of PD-MSCs. CONCLUSIONS: These findings suggest that alteration of FAO by increased CPT1A plays an important role in mitochondrial dysfunction and senescence of PD-MSCs during long-term cultivation.

Prokaryotic soluble overexpression and purification of oncostatin M using a fusion approach and genetically engineered E. coli strains.

Human Oncostatin M (OSM), initially discovered as a tumour inhibitory factor secreted from U-937 cells, is a gp130 (IL-6/LIF) cytokine family member that exhibits pleiotropic effects in inflammation, haematopoiesis, skeletal tissue alteration, liver regeneration, cardiovascular and metabolic diseases. Cytoplasmic expression of OSM in Escherichia coli results in inclusion bodies, and complex solubilisation, refolding and purification is required to prepare bioactive protein. Herein, eight N-terminal fusion variants of OSM with hexahistidine (His6) tag and seven solubility-enhancing tags, including thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (Nusa), human protein disulphide isomerase (PDI) and the b'a' domain of PDI (PDIb'a'), were tested for soluble OSM expression in E. coli. The His6-OSM plasmid was also introduced into genetically engineered Origami 2 and SHuffle strains to test expression of the protein. At 18 °C, MBP-tagged OSM was highly expressed and solubility was dramatically enhanced. In addition, His6-OSM was more highly expressed and soluble in Origami 2 and SHuffle strains than in BL21(DE3). MBP-OSM and His6-OSM were purified more than 95% with yields of 11.02 mg and 3.27 mg from a 500 mL culture. Protein identity was confirmed by mass spectroscopy, and bioactivity was demonstrated by in vitro inhibition of Th17 cell differentiation.

An oncogenic activity of PDGF-C and its splice variant in human breast cancer.

Despite strong evidence for the involvement of PDGF signaling in breast cancer, little is known about the PDGF ligand responsible for PDGFR activation during breast cancer progression. Here, we found PDGF-C to be highly expressed in breast carcinoma cell lines. Immunohistochemical analysis of invasive breast cancer revealed an association between increased PDGF-C expression and lymph node metastases, Ki-67 proliferation index, and poor disease-free survival. We also identified a PDGF-C splice variant encoding truncated PDGF-C (t-PDGF-C) isoform lacking the signal peptide and the N-terminal CUB domain. While t-PDGF C homodimer is retained intracellularly, it can be secreted as a heterodimer with full-length PDGF-C (FL-PDGF-C). PDGF-C downregulation reduced anchorage-independent growth and matrigel invasion of MDA-MB-231 cells. Conversely, ectopic expression of t-PDGF-C enhanced phenotypic transformation and invasion in BT-549 cells expressing endogenous FL-PDGF-C. The present study provides new insights into the functional significance of PDGF-C and its splice variant in human breast cancer.

Umbilical cord-derived mesenchymal stem cell extracts ameliorate atopic dermatitis in mice by reducing the T cell responses.

Mesenchymal stem cells derived from Wharton's jelly of the umbilical cord (UC-MSCs) have immunomodulatory properties. The aim of this study was to explore whether extracts of MSCs (MSC-Ex) could augment the low therapeutic efficacy of the whole cells in an Aspergillus fumigatus (Af)-induced atopic dermatitis (AD) model. LPS- or TNF-α/IFN-γ-stimulated keratinocytes (HaCaT cells) were treated with MSC-Ex, and the Af-induced AD model was established in BALB/c mice. In HaCaT cells, MSC-Ex treatment significantly reduced the inflammatory cytokine (IL-6, IL-1ß, IL-4, IL-5 and TNF-α), iNOS and NF-κB levels, and upregulated the anti-inflammatory cytokines (IL-10 and TGF-ß1). In the AD mice, the MSC-Ex group showed greatly reduced dermatitis, and lower clinical symptom scores and IgE levels. The histological dermatitis scores were also markedly lower in the MSC-Ex-treated animals compared with the MSC-treated group. Decreased levels of IFN-γ (Th1) and IL-17 (Th17), IL-4 and IL-13 (Th2) were detected in T cells and the skin tissue from the MSC-Ex treated AD mice. The therapeutic capacity of MSC-Ex was preserved after lyophilization and reconstitution. MSC-Ex treatment reproducibly suppresses dermatitis and inhibits the induction of inflammatory cytokines in the skin of AD mice. MSC-Ex is therefore a potential new treatment agent for AD.

A chemical conjugate between HER2-targeting antibody fragment and Pseudomonas exotoxin A fragment demonstrates cytotoxic effects on HER2-expressing breast cancer cells.

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins. [BMB Reports 2019; 52(8): 496-501].

Adrenal Cortical Neoplasm with Uncertain Malignant Potential Arising in the Heterotopic Adrenal Cortex in the Liver of a Patient with Beckwith-Wiedemann Syndrome.

Patients with Beckwith-Wiedemann syndrome (BWS) are predisposed to developing embryonal tumors, with hepatoblastoma being the most common type. Our patient showed hemihypertrophy, macroglossia, and paternal uniparental disomy in chromosome 11 and was diagnosed with BWS. When the patient was 9 months old, a 2.5×1.5 cm oval hypoechoic exophytic mass was detected in the inferior tip of his right liver. Preoperative imaging identified it as hepatoblastoma; however, histologic, immunohistochemistry, and electron microscopic findings were compatible with adrenal cortical neoplasm with uncertain malignant potential. The origin of the adrenal tissue seemed to be heterotopic. Here, we describe for the first time an adrenal cortical neoplasm with uncertain malignant potential arising in the heterotopic adrenal cortex located in the liver of a patient with BWS.

Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia.

Preeclampsia is a disease of the mother, fetus, and placenta, and the gaps in our understanding of the complex interactions among their respective disease pathways preclude successful treatment and prevention. The placenta has a key role in the pathogenesis of the terminal pathway characterized by exaggerated maternal systemic inflammation, generalized endothelial damage, hypertension, and proteinuria. This sine qua non of preeclampsia may be triggered by distinct underlying mechanisms that occur at early stages of pregnancy and induce different phenotypes. To gain insights into these molecular pathways, we employed a systems biology approach and integrated different "omics," clinical, placental, and functional data from patients with distinct phenotypes of preeclampsia. First trimester maternal blood proteomics uncovered an altered abundance of proteins of the renin-angiotensin and immune systems, complement, and coagulation cascades in patients with term or preterm preeclampsia. Moreover, first trimester maternal blood from preterm preeclamptic patients in vitro dysregulated trophoblastic gene expression. Placental transcriptomics of women with preterm preeclampsia identified distinct gene modules associated with maternal or fetal disease. Placental "virtual" liquid biopsy showed that the dysregulation of these disease gene modules originates during the first trimester. In vitro experiments on hub transcription factors of these gene modules demonstrated that DNA hypermethylation in the regulatory region of ZNF554 leads to gene down-regulation and impaired trophoblast invasion, while BCL6 and ARNT2 up-regulation sensitizes the trophoblast to ischemia, hallmarks of preterm preeclampsia. In summary, our data suggest that there are distinct maternal and placental disease pathways, and their interaction influences the clinical presentation of preeclampsia. The activation of maternal disease pathways can be detected in all phenotypes of preeclampsia earlier and upstream of placental dysfunction, not only downstream as described before, and distinct placental disease pathways are superimposed on these maternal pathways. This is a paradigm shift, which, in agreement with epidemiological studies, warrants for the central pathologic role of preexisting maternal diseases or perturbed maternal-fetal-placental immune interactions in preeclampsia. The description of these novel pathways in the "molecular phase" of preeclampsia and the identification of their hub molecules may enable timely molecular characterization of patients with distinct preeclampsia phenotypes.