Characterization of 3D Organotypic Culture of Mouse Adipose-Derived Stem Cells.

Although stem cells are a promising avenue for harnessing the potential of adipose tissue, conventional two-dimensional (2D) culture methods have limitations. This study explored the use of three-dimensional (3D) cultures to preserve the regenerative potential of adipose-derived stem cells (ADSCs) and investigated their cellular properties. Flow cytometric analysis revealed significant variations in surface marker expressions between the two culture conditions. While 2D cultures showed robust surface marker expressions, 3D cultures exhibited reduced levels of CD44, CD90.2, and CD105. Adipogenic differentiation in 3D organotypic ADSCs faced challenges, with decreased organoid size and limited activation of adipogenesis-related genes. Key adipocyte markers, such as lipoprotein lipase (LPL) and adipoQ, were undetectable in 3D-cultured ADSCs, unlike positive controls in 2D-cultured mesenchymal stem cells (MSCs). Surprisingly, 3D-cultured ADSCs underwent mesenchymal-epithelial transition (MET), evidenced by increased E-cadherin and EpCAM expression and decreased mesenchymal markers. This study highlights successful ADSC organoid formation, notable MSC phenotype changes in 3D culture, adipogenic differentiation challenges, and a distinctive shift toward an epithelial-like state. These findings offer insights into the potential applications of 3D-cultured ADSCs in regenerative medicine, emphasizing the need for further exploration of underlying molecular mechanisms.

Differences in gene organization between type I and type II crustins in the morotoge shrimp, Pandalopsis japonica.

Crustins are cysteine-rich cationic antimicrobial peptides (AMPs) found in decapod crustaceans. Six novel crustin genes (Paj-CrusIc, Id, Ie, If, IIb and IIc) were identified in the morotoge shrimp, Pandalopsis japonica. Deduced amino acid sequences of isolated Paj-Crus genes ranged from 99 to 178 amino acid residues (10.6-17.8 kDa). Sequence analysis of nine isolated Paj-Crus genes and 100 different crustins from various decapod crustaceans revealed that a splice site and KXXXCP motif within the WAP domain may be the main criteria for classifying type I and II crustins, suggesting that the two types of crustin genes may have been generated by different processes. We also identified three intron-less crustin I genes (Paj-Crus Id, Ie and If) for the first time, which may have been generated by gene duplication. The tissue distribution profiles showed that Paj-CrusI genes were expressed predominantly in the gill and epidermis, whereas Paj-CrusII genes were expressed ubiquitously, suggesting that the two types of crustins may play different roles in various tissues or under different physiological conditions. Differing from previous results, hemocyte-specific crustin was not isolated from Pandalopsis japonica. This study showed that both types of crustin genes (types I and II) exist in decapod crustaceans and their primary structure and expression profiles differ from each other, suggesting that they may play different biological roles. This will help to extend our knowledge of the crustacean innate immune response, which will provide important basic information of shrimp immunity against various pathogens.

Low-Dose Ionizing Radiation-Crosslinking Immunoprecipitation (LDIR-CLIP) Identified Irradiation-Sensitive RNAs for RNA-Binding Protein HuR-Mediated Decay.

Although ionizing radiation (IR) is widely used for therapeutic and research purposes, studies on low-dose ionizing radiation (LDIR) are limited compared with those on other IR approaches, such as high-dose gamma irradiation and ultraviolet irradiation. High-dose IR affects DNA damage response and nucleotide-protein crosslinking, among other processes; however, the molecular consequences of LDIR have been poorly investigated. Here, we developed a method to profile RNA species crosslinked to an RNA-binding protein, namely, human antigen R (HuR), using LDIR and high-throughput RNA sequencing. The RNA fragments isolated via LDIR-crosslinking and immunoprecipitation sequencing were crosslinked to HuR and protected from RNase-mediated digestion. Upon crosslinking HuR to target mRNAs such as PAX6, ZFP91, NR2F6, and CAND2, the transcripts degraded rapidly in human cell lines. Additionally, PAX6 and NR2F6 downregulation mediated the beneficial effects of LDIR on cell viability. Thus, our approach provides a method for investigating post-transcriptional gene regulation using LDIR.

Molecular cloning and thermal stress-induced expression of a pi-class glutathione S-transferase (GST) in the Antarctic bivalve Laternula elliptica.

Glutathione S-transferases (GSTs) are multifunctional phase II detoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione. The pi-class GST cDNA (leGSTp) was cloned from the cold-adapted Antarctic bivalve Laternula elliptica. We used degenerated primers designed based on highly conserved regions of known mollusk GSTs to amplify the corresponding L. elliptica mRNA. Full-length cDNA was obtained by rapid amplification of cDNA ends (RACE). The full sequence of the GST cDNA was 1189 bp in length, with a 5' untranslated region (UTR) of 74 bp, a 3' UTR of 485 bp, and an open reading frame of 630 bp encoding 209 amino acid residues with an estimated molecular mass of 23.9 kDa and an estimated isoelectric point of 8.3. Quantitative RT-PCR confirmed basal expression of leGSTp, which was up-regulated upon heat treatment (10 degrees C for different time periods) by a factor of 2.3 (at 24 h) and 2.7 (at 48 h) in the digestive gland and gill tissues, respectively. The recombinant leGSTp expressed in Escherichia coli was purified by affinity chromatography and characterized. The purified leGSTp exhibited high activity towards the substrates ethacrynic acid (ECA) and 1-chloro-2,4-dinitrobenzene (CDNB). The recombinant leGSTp had a maximum activity at approximately pH 8.0, and its optimum temperature was 35 degrees C.

ERdj5 in Innate Immune Cells Is a Crucial Factor for the Mucosal Adjuvanticity of Cholera Toxin.

Cholera toxin (CT) is one of most strong mucosal adjuvants, but it cannot be clinically used owing to its toxicity. The cytosolic A1 subunit of CT (CTA1) is the molecule responsible for its immunostimulatory activity, which increases the concentration of cyclic AMP and causes the induction of pro-inflammatory cytokines in innate immune cells. However, the importance of endoplasmic reticulum (ER) molecules involved in CTA1 retro-translocation to induce immune responses remained to be investigated. ERdj5 is an ER protein which is expected to transfer CTA1 to the Hrd1 complex for the retro-translocation of CTA1. In this study, we investigated the physiological relevance of ERdj5 in immune stimulation by CT. ERdj5-knockout (ERdj5 KO) mice had decreased production of antigen-specific IgG in the serum and IgA in the mucosal secretion after intranasal immunization with Ag and CT. Especially, IgG2c isotypes were specifically reduced in the absence of ERdj5. ERdj5 KO dendritic cells (DCs) failed to full activation with decreased expression of costimulatory molecules, such as MHC class II, CD80, and CD 86. In ERdj5 KO DCs, secretion of pro-inflammatory cytokines, such as IL-1ß, TNF-α, and IL-6, was reduced. The cytokine signatures of several helper T cells were reduced in ERdj5 KO mice following intranasal CT immunization. The absence of ERdj5 affects the immunostimulatory properties of CT but does not affect the response to the CTB pentamer, the response to alum, total antibody production, or cytokine release from DCs exposed to CpG. Interestingly, CT enhanced the expression of ER stress proteins in ERdj5 KO innate immune cells. These results suggested that ERdj5 contributed as a decisive factor to the immunostimulatory capacity of CT via CTA1 retro-translocation.

Stability and efficacy of the 3'-UTR A4G-G5A variant of viral hemorrhagic septicemia virus (VHSV) as a live attenuated immersion VHSV vaccine in olive flounder (Paralichthys olivaceus).

Viral hemorrhagic septicemia virus (VHSV) is the causative agent of viral hemorrhagic septicemia in fish, a disease that affects a number of teleost fish species including olive flounder (Paralichthys olivaceus). In this study, we assessed the safety and efficacy of two recombinant attenuated VHSV strains, termed A4G-G5A and ΔNV, with the purpose to select the most suitable vaccine strain. The virus strains were passaged in two commercially available cell lines, EPC and RTG-2, and the strains were also tested for residual virulence in zebrafish (Danio rerio). The A4G-G5A strain showed an attenuated growth profile in both the EPC and RTG-2 cell lines compared to wild-type (WT) VHSV (JF-09, genotype IVa), whereas the growth profile of ΔNV was comparable to the WT strains in RTG-2 cells in contrast to EPC cells. Moreover, ΔNV had higher residual virulence compared to A4G-G5A and was highly pathogenic to zebrafish. The A4G-G5A strain was chosen as vaccine candidate and tested for efficacy in in vivo fish studies in the target species, olive flounder, using an immersion vaccine scheme. Groups of fish were immunized with 10(2.5), 10(3.5), 10(4.5), and 10(5.5) TCID50/ml of A4G-G5A giving 5-13.3 cumulative percent mortality (CPM) post immunization. Immunization was followed by a challenge experiment using VHSV-WT. The relative percent survival (RPS) in immunized groups ranged from 81.6% to 100%, correlating with vaccination dose. This study demonstrates that while strain A4G-G5A has retained some residual virulence it confers high level of protection in immunized olive flounder.

Motor function in school-aged children with attention-deficit/hyperactivity disorder in Korea.

OBJECTIVE: Motor function critically influences daily activities and academic performance. We compared motor function in school-aged children with Attention-Deficit/Hyperactivity Disorder (ADHD) to that of normal children. METHODS: Participants were 58 children with ADHD [51 males, 7 females; mean age 9 years 6 months±2 years 0 months (SD)] and 70 normal controls [56 males, 14 females; mean age 9 years 2 months±1 years 7 months (SD)]. We assessed motor function with the Bruininks-Oseretsky Test of Motor Proficiency, Second Edition. RESULTS: The ADHD group had a significantly lower total motor composite score (t=-9.32, p<0.001) than that of the control group. Standard scores of four motor-area composites such as fine manual control (t=-3.76, p<0.001), manual coordination (t=-6.87, p<0.001), body coordination (t=-7.14, p<0.001), and strength and agility (t=-8.54, p<0.1) were significantly lower in the ADHD group than those in the control group. Among the subtests, scores on fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, running speed and agility, and strength were significantly lower in the ADHD group than those in the controls, whereas upper-limb coordination was not significantly different between the groups. CONCLUSION: School-aged children with ADHD in Korea had significantly lower motor function compared to that of controls. Thus, it is suggested that appropriate target intervention for motor function is important in children with motor impairment in addition to pharmacotherapy or psychosocial therapy for improving the core symptoms.

Molecular characterization of three crustin genes in the morotoge shrimp, Pandalopsis japonica.

Crustins are among the most important antimicrobial peptides (AMPs) found in decapod crustaceans. They are small cationic AMPs (5-7 kDa) characterized by a proline-rich amino-terminal domain and a cysteine-rich carboxyl-terminal domain. Here, the first 3 crustin-like cDNAs (Pj-crus Ia, Ib, and II) were identified from the morotoge shrimp, Pandalopsis japonica. The full-length cDNAs of Pj-crus Ia, Ib, and II consisted of 1135, 580, and 700 nucleotides and encoded putative proteins containing 109, 119, and 186 amino acids residues, respectively. All 3 identified Pj-crus sequences exhibited the conserved domain organization for crustins, including a signal sequence, a cysteine-containing region, a glycine-rich region, and a whey-acidic protein (WAP) domain. Amino acid sequence comparisons and phylogenetic analysis revealed that the Pj-crus Ia and Ib belong to type I crustins (e.g., carcinin), which have been mostly identified from Brachyura and Astacidea, whereas Pj-crus II was classified as belonging to the type II crustins, which are mainly found in Dendrobranchiata. An analysis of the organization of these 3 Pj-crus genes revealed that the splicing site within the WAP domain may be an important key for classifying types I and II crustin family members. The tissue distribution profile results showed that the Pj-crus I genes were expressed in a tissue-specific manner but that the Pj-crus II gene was expressed ubiquitously, suggesting that these crustins may play different roles in various tissues or under different physiological conditions. The bacterial challenge results suggested that the Pj-crus genes may be transcriptionally influenced by different bacterial types. This comparative study of various crustin family members will help extend the knowledge on the crustacean innate immune response, which will provide important basic information for controlling shrimp immunity against various pathogens.

Five hepatopancreatic and one epidermal chitinases from a pandalid shrimp (Pandalopsis japonica): cloning and effects of eyestalk ablation on gene expression.

Six cDNAs encoding chitinase proteins in Pandalopsis japonica were isolated by using polymerase chain reaction (PCR) cloning methods and bioinformatic analysis of expressed sequence tags (ESTs). The cDNAs, designated Pj-Cht1, 2, 3A, 3B, 3C, and 4, encoded proteins ranging from 388 to 607 amino acid residues in length (43.61-67.62kDa) and displayed a common structural organization: an N-terminal catalytic domain, a Thr/Pro-rich linker region, and either 0 (Pj-Cht2, 3A), 1 (Pj-Cht1, 3B, and 3C), or 2 (Pj-Cht4) C-terminal chitin-binding domain(s) (CBD). Pj-Cht1 and 2 lacked the 5' end of the open reading frame (ORF); the other Pj-Chts contained the complete ORF. All known decapod crustacean chitinases were segregated into at least four groups based on phylogenetic analysis and domain organization. Group 1 chitinases, represented by Pj-Cht1, were most closely related to insect group I chitinases and may function in the digestion of the peritrophic membrane. Group 2 chitinases including Pj-Cht2 show different domain organizations and pI value from other chitinases and appear to function in degradation of the old exoskeleton during the premolt period. Group 3 chitinases, represented by Pj-Cht3A, 3B, and 3C, may function in digestion of chitin-containing food and defense against pathogens. Group 4 chitinases, represented by Pj-Cht4, have two CBDs and their functions are unknown. Five Pj-Chts (Pj-Cht1, 3A, 3B, 3C, and 4) are expressed in the hepatopancreas and intestine, whereas Pj-Cht2 is expressed in epidermis and SG/XO complex suggesting crustacean chitinases can be classified into two groups (hepatopancreatic and epidermal) based on the expression profile. Eyestalk ablation (ESA) down-regulated the hepatopancreatic chitinase expression (Pj-Cht1, 3A, and 3C); Pj-Cht3B expression was not significantly affected by ESA. By contrast, mRNA levels of Pj-Cht2 were significantly upregulated in 7days post-ESA. Pj-Cht4 mRNA levels were too low for measurement with quantitative polymerase chain reaction. ESA had no significant effect on chitinase expression in the intestine. These data indicate that Pj-Cht1, 3A, 3B, 3C, and 4 are hepatopancreatic chitinases that may function in the digestion of ingested chitin and the modification of peritrophic membrane in the intestine. By contrast, epidermal chitinase, Pj-Cht2 may play a role in chitin metabolism during molt cycle as shown in other crustacean group 2 chitinases.

Molecular characterization and induction of heat shock protein 90 in the Antarctic bivalve Laternula elliptica.

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that plays a key role in protein synthesis, folding, denaturation prevention, and signal transduction. We cloned the complete complementary DNA (cDNA) sequence of the Laternula elliptica HSP90. The full-length cDNA was 2,823 bp in size and contained an open reading frame of 2,190 bp that was translated into 729 amino acids with a calculated molecular weight of 83.4 kDa. The deduced amino acid sequence of HSP90 showed the highest homology to Haliotis tuberculata HSP90 (83%). Reverse-transcriptase polymerase chain reaction analysis revealed the presence of HSP90 transcripts in all of the tissues examined. We also studied the transcriptional expression pattern of HSP90 exposed to thermal stress with real-time polymerase chain reaction. The relative expression level of HSP90 messenger RNA was upregulated and peaked at 12 h in the digestive gland and at 24 h in the gills, then dropped progressively.

Analysis of ESTs and expression of two peroxiredoxins in the thermally stressed Antarctic bivalve Laternula elliptica.

A total of 2592 expressed sequenced tags (ESTs) were generated from a cDNA library of the Antarctic bivalve Laternula elliptica, which is an important species in Antarctic coastal ecosystems. A cluster of 1789 unique sequences was revealed, which consisted of 273 contigs and 1076 singletons. Based on BLAST searches, we identified 508 genes, 56 of which were functionally related to immunity and stress responses, including the peroxiredoxins (Prxs). Prxs comprise an antioxidant protein family with conserved catalytic redox-active cysteine residues. We characterised two full-length cDNAs of the genes that encode peroxiredoxin V (lePrxV) and peroxiredoxin VI (lePrxVI) in L. elliptica. The lePrxV cDNA contains a 480-bp open reading frame (ORF), which encodes 159 amino acids, including two conserved cysteine residues that are characteristic of the atypical 2-Cys subgroup of the Prx family. LePrxVI contains a 702-bp ORF, which encodes 233 amino acids, including one conserved cysteine residue that is characteristic of the 1-Cys subgroup of the Prx family. RT-PCR analysis revealed the presence of lePrxV and lePrxVI transcripts in all the examined tissues. In addition, real-time PCR analysis indicated significant increases in the levels of lePrxV and lePrxVI transcripts were induced by thermal stress. These results suggest that lePrxV and lePrxVI play protective roles against oxidative stress caused by thermal exposure.