Functional enhancement of neuronal cell behaviors and differentiation by elastin-mimetic recombinant protein presenting Arg-Gly-Asp peptides

Background: Integrin-mediated interaction of neuronal cells with extracellular matrix (ECM) is important for the control of cell adhesion, morphology, motility, and differentiation in both in vitro and in vivo systems. Arg-Gly-Asp (RGD) sequence is one of the most potent integrin-binding ligand found in many native ECM proteins. An elastin-mimetic recombinant protein, TGPG[VGRGD(VGVPG)6]20WPC, referred to as [RGD-V6]20, contains multiple RGD motifs to bind cell-surface integrins. This study aimed to investigate how surface-adsorbed recombinant protein can be used to modulate the behaviors and differentiation of neuronal cells in vitro. For this purpose, biomimetic ECM surfaces were prepared by isothermal adsorption of [RGD-V6]20 onto the tissue culture polystyrene (TCPS), and the effects of protein-coated surfaces on neuronal cell adhesion, spreading, migration, and differentiation were quantitatively measured using N2a neuroblastoma cells.Results: The [RGD-V6]20 was expressed in E. coli and purified by thermally-induced phase transition. N2a cell attachment to either [RGD-V6]20 or fibronectin followed hyperbolic binding kinetics saturating around 2 μM protein concentration. The apparent maximum cell binding to [RGD-V6]20 was approximately 96% of fibronectin, with half-maximal adhesion on [RGD-V6]20 and fibronectin occurring at a coating concentration of 2.4 × 10-7 and 1.4 × 10-7 M, respectively. The percentage of spreading cells was in the following order of proteins: fibronectin (84.3% ± 6.9%) > [RGD-V6]20 (42.9% ± 6.5%) > [V7]20 (15.5% ± 3.2%) > TCPS (less than 10%). The migration speed of N2a cells on [RGD-V6]20 was similar to that of cells on fibronectin. The expression of neuronal marker proteins Tuj1, MAP2, and GFAP was approximately 1.5-fold up-regulated by [RGD-V6]20 relative to TCPS. Moreover, by the presence of both [RGD-V6]20 and RA, the expression levels of NSE, TuJ1, NF68, MAP2, and GFAP were significantly elevated.Conclusion: We have shown that an elastin-mimetic protein consisting of alternating tropoelastin structural domains and cell-binding RGD motifs is able to stimulate neuronal cell behaviors and differentiation. In particular, adhesion-induced neural differentiation is highly desirable for neural development and nerve repair. In this context, our data emphasize that the combination of biomimetically engineered recombinant protein and isothermal adsorption approach allows for the facile preparation of bioactive matrix or coating for neural tissue regeneration. © 2012 Jeon et al.; licensee BioMed Central Ltd.1

Application of Computed Tomography in the Identification of Hollow Viscus Injuries in Blunt Trauma Patients

Purpose Despite advances in diagnostic and imaging technologies, the diagnosis of traumatic hollow viscus injury (HVI) remains a great challenge in clinical practice. This study aimed to determine the accuracy of computed tomography (CT) in the diagnosis of HVI in emergent blunt trauma patients. Methods The study was conducted on patients with abdominal trauma who were admitted to our center, regional emergency center, Kyung Hee University Medical Center, between January 2008 and December 2018. The clinical data of patients with abdominal trauma who underwent CT and abdominal surgery within 24 hours of hospitalization were analyzed to determine the diagnostic capacity of CT. Results In total, 156 patients were included in the study. There were 88 cases of blunt trauma. Among these patients, 27 were diagnosed with HVI using CT, and 38 patients were diagnosed with HVI in the operating room. The median injury severity score for these patients was 10.0, the revised trauma score was 7.841, and the trauma injury severity score was 0.96. The sensitivity and specificity of CT in predicting HVI in these patients were 65.8%, and 96.0%, respectively. The positive and negative predictive values were 92.6%, and 78.7%, respectively. Conclusion In urgent situations, CT findings alone are insufficient for diagnosing HVI. Further research on the HVI diagnostic capacity of CT is required

Dual-Organ Transcriptomic Analysis of Rainbow Trout Infected With Ichthyophthirius multifiliis Through Co-Expression and Machine Learning

Ichthyophthirius multifiliis is a major pathogen that causes a high mortality rate in trout farms. However, systemic responses to the pathogen and its interactions with multiple organs during the course of infection have not been well described. In this study, dual-organ transcriptomic responses in the liver and head kidney and hemato-serological indexes were profiled under I. multifiliis infection and recovery to investigate systemic immuno-physiological characteristics. Several strategies for massive transcriptomic interpretation, such as differentially expressed genes (DEGs), Poisson linear discriminant (PLDA), and weighted gene co-expression network analysis (WGCNA) models were used to investigate the featured genes/pathways while minimizing the disadvantages of individual methods. During the course of infection, 6,097 and 2,931 DEGs were identified in the head kidney and liver, respectively. Markers of protein processing in the endoplasmic reticulum, oxidative phosphorylation, and the proteasome were highly expressed. Likewise, simultaneous ferroptosis and cellular reconstruction was observed, which is strongly linked to multiple organ dysfunction. In contrast, pathways relevant to cellular replication were up-regulated in only the head kidney, while endocytosis- and phagosome-related pathways were notably expressed in the liver. Moreover, interestingly, most immune-relevant pathways (e.g., leukocyte trans-endothelial migration, Fc gamma R-mediated phagocytosis) were highly activated in the liver, but the same pathways in the head kidney were down-regulated. These conflicting results from different organs suggest that interpretation of co-expression among organs is crucial for profiling of systemic responses during infection. The dual-organ transcriptomics approaches presented in this study will greatly contribute to our understanding of multi-organ interactions under I. multifiliis infection from a broader perspective.publishedVersio

Repair Using Conventional Implant for Ruptured Annulus Fibrosus after Lumbar Discectomy: Surgical Technique and Case Series

Study DesignA retrospective review of annulus fibrosus repair (AR) using a novel technique with a conventional implant.PurposeThe purpose of this study was to present the feasibility and clinico-radiological outcomes of a novel AR technique using a conventional implant to minimize recurrence following a lumbar discectomy (LD).Overview of LiteratureConventional repair techniques to prevent recurrence following LD have several drawbacks. The AR surgical technique has received little attention as an adjunct to LD.MethodsA total of 19 patients who underwent novel AR following LD, and who were available for follow-up for at least three years, were enrolled in this study. Several variables, including the type and size of disc herniation, and the degree of disc degeneration, were evaluated preoperatively. Postoperatively, the presence of clinical and radiological recurrence of disc herniation was evaluated from pain intensity and functional statuses, as well as an enhanced L-spine magnetic resonance imaging at the final follow-up. The presence of a peripheral hollow rim and inserted anchor mobilization were also evaluated during the follow-up.ResultsDuring follow-ups, there were no recurrences of disc herniation or complications, including neurovascular complications. Pain and functional disability improved significantly after surgery, and the improvement was maintained throughout the three-year follow-up period. No mobilization or implant peripheral hollow rim was observed during the follow-up.ConclusionsThis study examined the feasibility of a novel and easily available annulus implant technique following LD. These results suggest performing AR with this technique may be a valuable alternative for optimizing outcomes, if the procedure is performed in proper candidates

Analysis of spike protein variants evolved in a novel in vivo long-term replication model for SARS-CoV-2

IntroductionThe spectrum of SARS-CoV-2 mutations have increased over time, resulting in the emergence of several variants of concern. Persistent infection is assumed to be involved in the evolution of the variants. Calu-3 human lung cancer cells persistently grow without apoptosis and release low virus titers after infection.MethodsWe established a novel in vivo long-term replication model using xenografts of Calu-3 human lung cancer cells in immunodeficient mice. Virus replication in the tumor was monitored for 30 days and occurrence of mutations in the viral genome was determined by whole-genome deep sequencing. Viral isolates with mutations were selected after plaque forming assays and their properties were determined in cells and in K18-hACE2 mice.ResultsAfter infection with parental SARS-CoV-2, viruses were found in the tumor tissues for up to 30 days and acquired various mutations, predominantly in the spike (S) protein, some of which increased while others fluctuated for 30 days. Three viral isolates with different combination of mutations produced higher virus titers than the parental virus in Calu-3 cells without cytopathic effects. In K18-hACE2 mice, the variants were less lethal than the parental virus. Infection with each variant induced production of cross-reactive antibodies to the receptor binding domain of parental SARS-CoV-2 S protein and provided protective immunity against subsequent challenge with parental virus.DiscussionThese results suggest that most of the SARS-CoV-2 variants acquired mutations promoting host adaptation in the Calu-3 xenograft mice. This model can be used in the future to further study SARS-CoV-2 variants upon long-term replication in vivo

Connexin32 inhibits gastric carcinogenesis through cell cycle arrest and altered expression of p21Cip1 and p27Kip1

Gap junctions and their structural proteins, connexins (Cxs), havebeen implicated in carcinogenesis. To explore the involvement ofCx32 in gastric carcinogenesis, immunochemical analysis of Cx32and proliferation marker Ki67 using tissue-microarrayed humangastric cancer and normal tissues was performed. In addition, afterCx32 overexpression in the human gastric cancer cell line AGS,cell proliferation, cell cycle analyses, and p21Cip1 and p27Kip1expression levels were examined by bromodeoxyuridine assay,flow cytometry, real-time RT-PCR, and western blotting.Immunohistochemical study noted a strong inverse correlationbetween Cx32 and Ki67 expression pattern as well as theirlocation. In vitro, overexpression of Cx32 in AGS cells inhibitedcell proliferation significantly. G1 arrest, up-regulation of cellcycle-regulatory proteins p21Cip1 and p27Kip1 was also found atboth mRNA and protein levels. Taken together, Cx32 plays someroles in gastric cancer development by inhibiting gastric cancercell proliferation through cell cycle arrest and cell cycle regulatoryproteins. [BMB Reports 2013; 46(1): 25-30

Polymer Micelle Formulation for the Proteasome Inhibitor Drug Carfilzomib: Anticancer Efficacy and Pharmacokinetic Studies in Mice

Carfilzomib (CFZ) is a peptide epoxyketone proteasome inhibitor approved for the treatment of multiple myeloma (MM). Despite the remarkable efficacy of CFZ against MM, the clinical trials in patients with solid cancers yielded rather disappointing results with minimal clinical benefits. Rapid degradation of CFZ in vivo and its poor penetration to tumor sites are considered to be major factors limiting its efficacy against solid cancers. We previously reported that polymer micelles (PMs) composed of biodegradable block copolymers poly(ethylene glycol) (PEG) and poly(caprolactone) (PCL) can improve the metabolic stability of CFZ in vitro. Here, we prepared the CFZ-loaded PM, PEG-PCL-deoxycholic acid (CFZ-PM) and assessed its in vivo anticancer efficacy and pharmacokinetic profiles. Despite in vitro metabolic protection of CFZ, CFZ-PM did not display in vivo anticancer efficacy in mice bearing human lung cancer xenograft (H460) superior to that of the clinically used cyclodextrin-based CFZ (CFZ-CD) formulation. The plasma pharmacokinetic profiles of CFZ-PM were also comparable to those of CFZ-CD and the residual tumors that persisted in xenograft mice receiving CFZ-PM displayed an incomplete proteasome inhibition. In summary, our results showed that despite its favorable in vitroperformances, the current CFZ-PM formulation did not improve in vivo anticancer efficacy and accessibility of active CFZ to solid cancer tissues over CFZ-CD. Careful consideration of the current results and potential confounding factors may provide valuable insights into the future efforts to validate the potential of CFZ-based therapy for solid cancer and to develop effective CFZ delivery strategies that can be used to treat solid cancers

Expression of aldo-keto reductase family 1 member C1 (AKR1C1) gene in porcine ovary and uterine endometrium during the estrous cycle and pregnancy

<p>Abstract</p> <p>Background</p> <p>The aldo-keto reductase family 1 member C1 (AKR1C1) belongs to a superfamily of NADPH-dependent reductases that convert a wide range of substrates, including carbohydrates, steroid hormones, and endogenous prostaglandins. The 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) is a member of AKR family. The aims of this study were to determine its expression in the ovary and uterus endometrium during the estrous cycle and pregnancy.</p> <p>Methods</p> <p>Rapid amplification of cDNA ends (RACE) experiments were performed to obtain the 5' and 3' ends of the porcine <it>20alpha-HSD </it>cDNA. Reverse-transcriptase-PCR (RT-PCR), real-time PCR, northern blot analysis, and western blot analysis were performed to examine the expression of porcine 20alpha-HSD. Immunohistochemical analysis was also performed to determine the localization in the ovary.</p> <p>Results</p> <p>The porcine 20alpha-HSD cDNA is 957 bp in length and encodes a protein of 319 amino acids. The cloned cDNA was virtually the same as the porcine <it>AKR1C1 </it>gene (337 amino acids) reported recently, and only differed in the C-terminal region (the <it>AKR1C1 </it>gene has a longer C-terminal region than our sequence). The <it>20alpha-HSD </it>gene (from now on referred to as <it>AKR1C1</it>) cloned in this paper encodes a deletion of 4 amino acids, compared with the C-terminal region of <it>AKR1C1 </it>genes from other animals. Porcine AKR1C1 mRNA was expressed on day 5, 10, 12, 15 of the cycle and 0-60 of pregnancy in the ovary. The mRNA was also specifically detected in the uterine endometrium on day 30 of pregnancy. Western blot analysis indicated that the pattern of AKR1C1 protein in the ovary during the estrous cycle and uterus during early pregnancy was similar to that of <it>AKR1C1 </it>mRNA expression. The recombinant protein produced in CHO cells was detected at approximately 37 kDa. Immunohistochemical analysis also revealed that pig AKR1C1 protein was localized in the large luteal cells in the early stages of the estrous cycle and before parturition.</p> <p>Conclusions</p> <p>Our study demonstrated that AKR1C1 mRNA and protein are coordinately expressed in the luteal cell of ovary throughout the estrous cycle and in the uterus on day 30 of pregnancy. Thus, the porcine AKR1C1 gene might control important mechanisms during the estrous cycle.</p