Assessment of environmental exposure to betamethasone on the reproductive function of female Japanese medaka (Oryzias latipes).

Betamethasone has been extensively used in medicine in recent years and poses potential hazards to aquatic organisms. This study investigated the reproductive toxic effects of betamethasone exposure in fish, employing female Japanese medaka (Oryzias latipes) as a model. Betamethasone exposure at environmentally relevant concentrations (0, 20, 200, and 2000 ng/L) for a period of 15 weeks resulted in its high accumulation in the ovary, leading to abnormal oogenesis in female Japanese medaka. The production of gonadotropins (LH and FSH) in the pituitary gland was inhibited, and sex steroid biosynthesis in the ovary was significantly influenced at the transcriptional level. The imbalance of androgens and estrogens resulted in a decrease in the E2/T ratio and hepatic VTG synthesis, and the suppression of estrogen receptor signaling was also induced. Furthermore, betamethasone exposure delayed spawning and reduced fertility in the F0 generation, and had detrimental effects on the fertilization rate and hatchability of the F1 generation. Our results showed that environmental betamethasone had the potential to adversely affect female fertility and steroid hormone dynamics in fish.

Regulative Roles of Metabolic Plasticity Caused by Mitochondrial Oxidative Phosphorylation and Glycolysis on the Initiation and Progression of Tumorigenesis.

One important feature of tumour development is the regulatory role of metabolic plasticity in maintaining the balance of mitochondrial oxidative phosphorylation and glycolysis in cancer cells. In recent years, the transition and/or function of metabolic phenotypes between mitochondrial oxidative phosphorylation and glycolysis in tumour cells have been extensively studied. In this review, we aimed to elucidate the characteristics of metabolic plasticity (emphasizing their effects, such as immune escape, angiogenesis migration, invasiveness, heterogeneity, adhesion, and phenotypic properties of cancers, among others) on tumour progression, including the initiation and progression phases. Thus, this article provides an overall understanding of the influence of abnormal metabolic remodeling on malignant proliferation and pathophysiological changes in carcinoma.

Osmoregulatory strategies of estuarine fish Scatophagus argus in response to environmental salinity changes.

BACKGROUND: Scatophagus argus, an estuarine inhabitant, can rapidly adapt to different salinity environments. However, the knowledge of the molecular mechanisms underlying its strong salinity tolerance remains unclear. The gill, as the main osmoregulatory organ, plays a vital role in the salinity adaptation of the fish, and thus relative studies are constructive to reveal unique osmoregulatory mechanisms in S. argus. RESULTS: In the present study, iTRAQ coupled with nanoLC-MS/MS techniques were employed to explore branchial osmoregulatory mechanisms in S. argus acclimated to different salinities. Among 1,604 identified proteins, 796 differentially expressed proteins (DEPs) were detected. To further assess osmoregulatory strategies in the gills under different salinities, DEPs related to osmoregulatory (22), non-directional (18), hypo- (52), and hypersaline (40) stress responses were selected. Functional annotation analysis of these selected DEPs indicated that the cellular ion regulation (e.g. Na+-K+-ATPase [NKA] and Na+-K+-2Cl- cotransporter 1 [NKCC1]) and ATP synthesis were deeply involved in the osmoregulatory process. As an osmoregulatory protein, NKCC1 expression was inhibited under hyposaline stress but showed the opposite trend in hypersaline conditions. The expression levels of NKA α1 and ß1 were only increased under hypersaline challenge. However, hyposaline treatments could enhance branchial NKA activity, which was inhibited under hypersaline environments, and correspondingly, reduced ATP content was observed in gill tissues exposed to hyposaline conditions, while its contents were increased in hypersaline groups. In vitro experiments indicated that Na+, K+, and Cl- ions were pumped out of branchial cells under hypoosmotic stress, whereas they were absorbed into cells under hyperosmotic conditions. Based on our results, we speculated that NKCC1-mediated Na+ influx was inhibited, and proper Na+ efflux was maintained by improving NKA activity under hyposaline stress, promoting the rapid adaptation of branchial cells to the hyposaline condition. Meanwhile, branchial cells prevented excessive loss of ions by increasing NKA internalization and reducing ATP synthesis. In contrast, excess ions in cells exposed to the hyperosmotic medium were excreted with sufficient energy supply, and reduced NKA activity and enhanced NKCC1-mediated Na+ influx were considered a compensatory regulation. CONCLUSIONS: S. argus exhibited divergent osmoregulatory strategies in the gills when encountering hypoosmotic and hyperosmotic stresses, facilitating effective adaptabilities to a wide range of environmental salinity fluctuation.

Effects of environmental salinity on the immune response of the coastal fish Scatophagus argus during bacterial infection.

The coastal aquaculture is characterized with environmental salinity fluctuation, and the effects of salinity stress on the immunity of cultured fish are needed to be further explored. Scatophagus argus is an important species in the wild fisheries and aquaculture industry, it would be of great value to reveal the impact of salinity change on the immune response in this species. Understanding the effects of salinity stress on immune response can provide valuable insights into salinity management in the aquacultural process. The head kidney, which is an organ unique for teleost fish, functions not only as a central immune organ but also as a crucial role in the stress response during which the secretion of immunoregulatory molecules i.e. cytokines is facilitated. In the present study, Individuals of S. argus acclimated to 3 different salinities [0‰ (FW), 10‰ (BW), and 25‰ (SW)] were injected intraperitoneally with A. hydrophila, and then monitored throughout one week. The effects of environmental salinity on the immune response in S. argus stimulated by A. hydrophila infection were investigated. mRNA expression profiles of cytokine genes IL-1ß, IL-6, IL-10 and TNF-α in different salinity groups was quite different. mRNA expression of cytokine genes in BW group and SW group rose more quickly and significantly higher than FW group (p < 0.05) at early stages (6-24 hpi) after bacterial injection, and before 96 hpi, the highest value of cytokine expression at each time point was recorded in SW group. Immune parameters such as lysozyme level, complement C3 activity and IgM content in BW and FW groups were lower than SW group at each time point from 24 to 144 hpi after bacterial injection. In addition, leukocyte profiles in the head kidney and blood were also investigated. Although hypoosmotic acclimation could temporarily stimulate monocyte and neutrophil proliferation, it was observed that the number of monocytes, neutrophils and lymphocytes of the head kidney and blood in SW group increased more quickly than BW and FW groups after bacterial infection. Our results indicate that hypoosmotic stress due to the decrease of environmental salinity has suppressive immunoregulatory effects on the immune response of S. argus.

An Efficient Probe-Based Routing for Content-Centric Networking.

With the development of new technologies and applications, such as the Internet of Things, smart cities, 5G, and edge computing, traditional Internet Protocol-based (IP-based) networks have been exposed as having many problems. Information-Centric Networking (ICN), Named Data Networking (NDN), and Content-Centric Networking (CCN) are therefore proposed as an alternative for future networks. However, unlike IP-based networks, CCN routing is non-deterministic and difficult to optimize due to frequent in-network caching replacement. This paper presents a novel probe-based routing algorithm that explores real-time in-network caching to ensure the routing table storing the optimal paths to the nearest content provider is up to date. Effective probe-selections, Pending Interest Table (PIT) probe, and Forwarding Information Base (FIB) probe are discussed and analyzed by simulation with different performance measurements. Compared with the basic CCN, in terms of qualitative analysis, the additional computational overhead of our approach is O(NCS + Nrt + NFIB ∗ NSPT) and O(NFIB) on processing interest packets and data packets, respectively. However, in terms of quantitative analysis, our approach reduces the number of timeout interests by 6% and the average response time by 0.6 s. Furthermore, although basic CCN and our approach belong to the same Quality of Service (QoS) category, our approach outperforms basic CCN in terms of real values. Additionally, our probe-based approach performs better than RECIF+PIF and EEGPR. Owing to speedup FIB updating by probes, our approach provides more reliable interest packet routing when accounting for router failures. In summary, the results demonstrate that compared to basic CCN, our probe-based routing approach raises FIB accuracy and reduces network congestion and response time, resulting in efficient routing.

Inflammatory responses associated with hyposaline stress in gill epithelial cells of the spotted scat Scatophagus argus.

The molecular processes of immune responses in mucosal tissues such as fish gills under environmental stress are poorly understood. In the present study, pro-inflammatory response under hyposaline stress and its regulation by cortisol/corticosteroid receptors (CRs) in gill epithelial cells of the spotted scat Scatophagus argus were analyzed. The fish were transferred to freshwater for 6 days (144 h) of acclimation. Following freshwater exposure, the cortisol concentration increased transiently before returning to the control level over time. mRNA expression of pro-inflammatory cytokines (TNF-a, IL-1b and IL-6) was stimulated by cortisol through CR signals at early stages of acclimation, but hyposaline stress inhibited their levels by the end of the experimental period. The transcriptional profile of anti-inflammatory cytokine IL-10 was quite different from these pro-inflammatory cytokines, and its value fluctuated within a narrow range during the experimental period. Full-length cDNAs of mineralocorticoid receptor (MR) and glucocorticoid receptor 1 (GR1) (different kinds of CRs) were cloned from the gills. Our results showed that MR and GR displayed mutually antagonistic effects during hyposaline stress. MR responded quickly at early stages, and its expression decreased with the drop of cortisol concentration. By contrast, GR expression was maintained at high levels after the acclimation of freshwater exposure. The tight coordination of GR and MR helps to shape the effects of stress on the immune system, which in turn, regulates the stress response. Our results confirm the interaction between endocrine and cytokine messengers and a clear difference in the sensitivity of GR and MR during the hyposaline challenge in gill epithelial cells of the spotted scat Scatophagus argus.

Modulation of inflammatory response by cortisol in the kidney of spotted scat (Scatophagus argus) in vitro under different osmotic stresses.

Salinity changes on renal osmoregulation have often been investigated while the immune response of the kidney under osmotic stress is poorly understood in teleosts. Acute stress is generally associated with enhancement of circulating cortisol. The effects of osmotic stress on renal immune response and its regulation by cortisol deserve more attention. In the present study, the effects of exogenous cortisol treatment on the lipopolysaccharide (LPS)-induced immune response were analyzed in renal masses of Scatophagus argus under different osmotic stresses in vitro. mRNA expression of pro-inflammatory cytokines (TNF-α, IL1-ß and IL-6) and immune-regulatory related genes (GR and SOCS1) was measured over a short course (15 h). Comprehensive analysis reveals that transcript abundances of pro-inflammatory cytokine genes such as TNF-α, IL-1ß, and IL-6 induced by LPS, alone or in the combination of cortisol, are tightly associated with osmoregulation under acute osmotic stress. Our results showed that osmotic challenge could significantly enhance mRNA expression levels of pro-inflammatory cytokines in renal masses in vitro. Based on our analysis, it can be inferred that cortisol suppresses the magnitude of renal inflammatory response and attenuates LPS-induced immune response through GR signaling in the face of challenging environmental conditions.

Transcriptional analysis of renal dopamine-mediated Na+ homeostasis response to environmental salinity stress in Scatophagus argus.

BACKGROUND: To control the osmotic pressure in the body, physiological adjustments to salinity fluctuations require the fish to regulate body fluid homeostasis in relation to environmental change via osmoregulation. Previous studies related to osmoregulation were focused primarily on the gill; however, little is known about another organ involved in osmoregulation, the kidney. The salinity adaptation of marine fish involves complex physiological traits, metabolic pathways and molecular and gene networks in osmoregulatory organs. To further explore of the salinity adaptation of marine fish with regard to the role of the kidney, the euryhaline fish Scatophagus argus was employed in the present study. Renal expression profiles of S. argus at different salinity levels were characterized using RNA-sequencing, and an integrated approach of combining molecular tools with physiological and biochemical techniques was utilized to reveal renal osmoregulatory mechanisms in vivo and in vitro. RESULTS: S. argus renal transcriptomes from the hyposaline stress (0‰, freshwater [FW]), hypersaline stress (50‰, hypersaline water [HW]) and control groups (25‰) were compared to elucidate potential osmoregulatory mechanisms. In total, 19,012 and 36,253 differentially expressed genes (DEGs) were obtained from the FW and HW groups, respectively. Based on the functional classification of DEGs, the renal dopamine system-induced Na+ transport was demonstrated to play a fundamental role in osmoregulation. In addition, for the first time in fish, many candidate genes associated with the dopamine system were identified. Furthermore, changes in environmental salinity affected renal dopamine release/reuptake by regulating the expression of genes related to dopamine reuptake (dat and nkaα1), vesicular traffic-mediated dopamine release (pink1, lrrk2, ace and apn), DAT phosphorylation (CaMKIIα and pkcß) and internalization (akt1). The associated transcriptional regulation ensured appropriate extracellular dopamine abundance in the S. argus kidney, and fluctuations in extracellular dopamine produced a direct influence on Na+/K+-ATPase (NKA) expression and activity, which is associated with Na+ homeostasis. CONCLUSIONS: These transcriptomic data provided insight into the molecular basis of renal osmoregulation in S. argus. Significantly, the results of this study revealed the mechanism of renal dopamine system-induced Na+ transport is essential in fish osmoregulation.

Aquaporin-3 Attenuates Oxidative Stress-Induced Nucleus Pulposus Cell Apoptosis Through Regulating the P38 MAPK Pathway.

BACKGROUND/AIMS: Previous studies have shown that oxidative damage is a main contributor to disc nucleus pulposus (NP) cell apoptosis. Aquaporin-3 (AQP-3) facilitates reactive oxygen species (ROS) scavenging and thus alleviates oxidative injury in other cells. This study aims to investigate the role and mechanism of AQP-3 in regulating NP cell apoptosis under oxidative damage. METHODS: Rat NP cells were treated with H2O2 for 48 hours, while control NP cells were free of H2O2. Recombinant AQP-3 lentiviral vectors were used to investigate the effect of enhanced AQP-3 expression levels in NP cells. NP cell apoptosis was assessed by flow cytometry, caspase-3 activity, gene expression of apoptosis-related molecules (Bax, Bcl-2 and caspase-3), and protein expression of cellular apoptosis markers (cleaved PARP and cleaved caspase-3). Additionally, intracellular ROS content and activity of the p38 MAPK pathway were evaluated. RESULTS: Compared with the control NP cells, oxidative damage in the treatment cells significantly increased cell apoptosis ratios and caspase-3 activity, upregulated gene expression of Bax and caspase-3, downregulated gene expression of Bcl-2, and increased protein expression of cleaved PARP and cleaved caspase-3, as well as increased intracellular ROS content and activity of the p38 MAPK pathway. However, AQP-3 overexpression partly alleviated cell apoptosis, decreased intracellular ROS content, and inhibited the p38 MAPK pathway in NP cells under oxidative damage. CONCLUSION: Oxidative damage can significantly downregulate AQP-3 expression. Enhancing AQP-3 expression in NP cells partly attenuates cellular apoptosis through regulating the p38 MAPK pathway under oxidative damage.

Dynamic Compression Promotes the Matrix Synthesis of Nucleus Pulposus Cells Through Up-Regulating N-CDH Expression in a Perfusion Bioreactor Culture.

BACKGROUND/AIMS: An adequate matrix production of nucleus pulposus (NP) cells is an important tissue engineering-based strategy to regenerate degenerative discs. Here, we mainly aimed to investigate the effects and mechanism of mechanical compression (i.e., static compression vs. dynamic compression) on the matrix synthesis of three-dimensional (3D) cultured NP cells in vitro. METHODS: Rat NP cells seeded on small intestinal submucosa (SIS) cryogel scaffolds were cultured in the chambers of a self-developed, mechanically active bioreactor for 10 days. Meanwhile, the NP cells were subjected to compression (static compression or dynamic compression at a 10% scaffold deformation) for 6 hours once per day. Unloaded NP cells were used as controls. The cellular phenotype and matrix biosynthesis of NP cells were investigated by real-time PCR and Western blotting assays. Lentivirus-mediated N-cadherin (N-CDH) knockdown and an inhibitor, LY294002, were used to further investigate the role of N-CDH and the PI3K/Akt pathway in this process. RESULTS: Dynamic compression better maintained the expression of cell-specific markers (keratin-19, FOXF1 and PAX1) and matrix macromolecules (aggrecan and collagen II), as well as N-CDH expression and the activity of the PI3K/Akt pathway, in the 3D-cultured NP cells compared with those expression levels and activity in the cells grown under static compression. Further analysis showed that the N-CDH knockdown significantly down-regulated the expression of NP cell-specific markers and matrix macromolecules and inhibited the activation of the PI3K/Akt pathway under dynamic compression. However, inhibition of the PI3K/Akt pathway had no effects on N-CDH expression but down-regulated the expression of NP cell-specific markers and matrix macromolecules under dynamic compression. CONCLUSION: Dynamic compression increases the matrix synthesis of 3D-cultured NP cells compared with that of the cells under static compression, and the N-CDH-PI3K/Akt pathway is involved in this regulatory process. This study provides a promising strategy to promote the matrix deposition of tissue-engineered NP tissue in vitro prior to clinical transplantation.

N-Cadherin Attenuates High Glucose-Induced Nucleus Pulposus Cell Senescence Through Regulation of the ROS/NF-κB Pathway.

BACKGROUND/AIMS: Diabetes mellitus (DM) is a potential etiology of disc degeneration. N-cadherin (N-CDH) helps maintain the cell viability, cell phenotype and matrix biosynthesis of nucleus pulposus (NP) cells. Here, we mainly aimed to investigate whether N-CDH can attenuate high glucose-induced NP cell senescence and its potential mechanism. METHODS: Rat NP cells were cultured in a base culture medium and base culture medium with a 0.2 M glucose concentration. Recombinant lentiviral vectors were used to enhance N-CDH expression in NP cells. Senescence-associated ß-galactosidase (SA-ß-Gal) activity was measured by SA-ß-Gal staining. NP cell proliferation was evaluated by CCK-8 assay. Telomerase activity and intracellular reactive oxygen species (ROS) content were tested by specific chemical kits according to the manufacturer's instructions. G0/G1 cell cycle arrest was evaluated by flow cytometry. Real-time PCR and Western blotting were used to analyze mRNA and protein expressions of senescence markers (p16 and p53) and matrix macromolecules (aggrecan and collagen II). Additionally, p-NF-κB expression was also analyzed by Western blotting to evaluate NF-κB pathway activity. RESULTS: High glucose significantly decreased N-CDH expression, increased ROS generation and NF-κB pathway activity, and promoted NP cell senescence, which was reflected in the increase in SA-ß-Gal activity and senescence marker (p16 and p53) expression, compared to the control group. High glucose decreased telomerase activity and cell proliferation potency. However, N-CDH overexpression partially attenuated NP cell senescence, decreased ROS content and inhibited the activation of the NF-κB pathway under the high glucose condition. CONCLUSION: High glucose decreases N-CDH expression and promotes NP cell senescence. N-CDH overexpression can attenuate high glucose-induced NP cell senescence through the regulation of the ROS/ NF-κB pathway. This study suggests that N-CDH is a potential therapeutic target to slow DM-mediated disc NP degeneration.

CCL2-CCR2 signaling promotes hepatic ischemia/reperfusion injury.

BACKGROUND: Liver ischemia/reperfusion (I/R) injury is a type of uncontrolled inflammatory cascade in which neutrophils, an early infiltrating immune cell population, elicit significant tissue damage. However, the precise mechanism for neutrophil recruitment and infiltration remains to be fully characterized. METHODS: A hepatic partial I/R model was reproduced in wild-type, CCL2(-/-) and CCR2(-/-) mice. Tissue damage was evaluated by serum enzyme analysis, hematoxylin-eosin staining, and cytokine production measurement. Mobilization of neutrophils from the bone marrow and subsequent infiltration into the liver were measured by flow cytometry. C-C motif chemokine receptor 2 (CCR2) expression on neutrophils and C-C motif chemokine ligand 2 (CCL2) chemotaxis were measured using flow cytometry. The cellular source of CCL2 in the liver was determined by deleting specific cell groups and performing intracellular staining. RESULTS: Liver damage was ameliorated, and neutrophil recruitment and accumulation were decreased in both CCL2(-/-) and CCR2(-/-) mice compared with wild-type mice. Neutrophils displayed upregulated expression of CCR2 during I/R, and these cells were required for CCL2-induced chemotaxis. Depletion of Kupffer cells protected the liver from I/R injury. Furthermore, genetic ablation of CCL2 reduced liver injury, as demonstrated by decreases in the levels of alanine aminotransferase and aspartate aminotransferase and subsequent reductions in neutrophil recruitment and accumulation. CONCLUSIONS: Kupffer cells secrete CCL2 to promote CCR2-expressing neutrophil recruitment from the bone marrow and subsequent infiltration into the liver during I/R. These findings reveal a novel pro-inflammatory role of cell-mediated CCL2-CCR2 interactions during this sterile insult.

Two hepcidins from spotted scat (Scatophagus argus) possess antibacterial and antiviral functions in vitro.

Hepcidins are small cysteine-rich antimicrobial peptides that play an important role in host immunity against pathogenic organisms. In this study, two hepcidins, SA-hepcidin1 and SA-hepcidin2, were cloned from spotted scat (Scatophagus argus), and the tissue distributions of SA-hepcidins were determined. In addition, mature SA-hepcidin peptides were synthesized to allow evaluation of their antimicrobial and antiviral functions in vitro. SA-hepcidin1 belongs to the HAMP1 class and is widely expressed in all tested tissues from spotted scat, whereas SA-hepcidin2 belongs to the HAMP2 class and present only in the liver. The synthetic SA-hepcidins had similar levels of antibacterial activity against Gram-positive and Gram-negative bacteria; however, the antibacterial activity of SA-hepcidin1 was stronger than that of SA-hepcidin2. The antiviral activities of the synthetic SA-hepcidins were assessed against Siniperca chuatsi rhabdovirus (SCRV) and largemouth bass Micropterus salmoides reovirus (MsReV) in epithelioma papulosum cyprini (EPC) and grass carp fin (GCF) cells. SA-hepcidin2 had antiviral activity, but SA-hepcidin1 did not. The results of this study suggest that SA-hepcidins are important multifunctional proteins in the spotted scat immune system that are involved in resistance to various pathogens.

Comparative renal gene expression in response to abrupt hypoosmotic shock in spotted scat (Scatophagus argus).

Scatophagus argus, a euryhaline fish, is notable for its ability to tolerate a wide range of environmental salinities and especially for its tolerance to a rapid, marked reduction in salinity. Therefore, S. argus is a good model for studying the molecular mechanisms mediating abrupt hyperosmoregulation. The serum osmotic pressure decreased steeply within one hour after transferring S. argus from seawater (SW) to freshwater (FW) and remained at new balance throughout the duration of one week. To explain this phenomenon and understand the molecular responses to an abrupt hypoosmotic shock, hypoosmotic stress responsive genes were identified by constructing two suppression subtractive hybridization (SSH) cDNA libraries from the kidneys of S. argus that had been transferred from SW to FW. After trimming and blasting, 52 ESTs were picked out from the subtractive library. Among them, 11 genes were significantly up-regulated (p < 0.05). The kinetics studies of gene expression levels were conducted for 1 week after the transfer using quantitative real-time PCR. A significant variation in the expression of these genes occurred within 12h after the hypoosmotic shock, except for growth hormone (GH) and polyadenylate binding protein 1 (PBP1), which were significantly up-regulated 2 days post-transfer. Our results suggest different functional roles for these genes in response to hypoosmotic stress during the stress response phase (1 hpt-12 hpt) and stable phase (12 hpt-7 dpt). Furthermore, the plasma growth hormone level was detected to be significantly elevated at 1 hpt and 24 hpt following abrupt hypoosmotic shock. Meanwhile, several hematological parameters, hemoglobin (HGB), red blood cell (RBC) and mean cellular hemoglobin concentration (MCHC), were observed to be significantly increased at 12 hpt and 2 dpt compared with that of control group. Our results provide a solid basis from which to conduct future studies on the osmoregulatory mechanisms in the euryhaline fish.

Single modification at position 14 of siRNA strand abolishes its gene-silencing activity by decreasing both RISC loading and target degradation.

Normally siRNA has to be chemically stabilized in therapeutic applications. It is a challenge to obtain optimal stabilizing effects while maintaining full silencing activity due to a lack of understanding of how different chemical modifications would influence the efficacy of siRNA. In the current study, the effect of single 2'-sugar modifications was profiled across the length of the siRNA guide strand. This led to the surprising finding that a single 2'-OMe modification at position 14 of the siRNA guide strand substantially compromised its gene-silencing activity in a manner that was independent of the nucleotide identity at this site or the sequence context around it. We found that modification at position 14 of the siRNA guide strand reduced its RNA-induced silencing complex (RISC) loading tremendously, whereas the loading of the siRNA sense strand was only marginally affected. When comparing the silencing potency of 14th position-modified siRNA (transfected at 16.7 nM) and native control (transfected at 1 nM) at equivalent Ago2 loading levels, the silencing potency of modified siRNA was much lower, even lower than the level of native siRNA transfected at 0.1 nM. These data indicated that modification at position 14 of the siRNA guide strand abolishes its gene-silencing activity by decreasing both RISC loading and target degradation. Using a computational modeling approach, we demonstrated an intimate interaction between the 14th nucleotide of guide strand and the amino acid Q675 in the AGO protein, which is located in a highly conserved loop of PIWI domain. In addition to gaining insights into siRNA-AGO interactions, this study of structure-activity relationship further established a general principle for siRNA modification in siRNA drug development.

Comparative analysis of the image quality and diagnostic performance of the zooming technique with diffusion-weighted imaging using different b-values for thyroid papillary carcinomas and benign nodules.

Objective: To compare image quality and diagnostic performance using different b-values for the zooming technique with diffusion-weighted imaging (ZOOMit-DWI) in thyroid nodules. Materials and methods: A total of 51 benign thyroid nodules and 50 thyroid papillary carcinomas were included. ZOOMit-DWI was performed with b-values of 0, 500, 1000, 1500 and 2000 s/mm2. The sharpness was evaluated as subjective index. The signal intensity ratio (SIR), signal-to-noise ratio (SNR) and apparent diffusion coefficient (ADC) were measured as objective indices. Pairwise comparisons were performed among the different b-value groups using the Friedman test. A receiver operating characteristic curve of the ADC value was used to evaluate diagnostic performance. The DeLong test was used to compare diagnostic effectiveness among the different b-value groups. Results: In both the papillary carcinoma group (P = 0.670) and the benign nodule group (P = 0.185), the sharpness of nodules was similar between b-values of 1000 s/mm2and 1500 s/mm2. In the papillary carcinoma group, the SIRnodule was statistically higher in DWI images with a b-value of 1500 s/mm2than in DWI images with b-values of 500 s/mm2(P = 0.004), 1000 s/mm2(P = 0.002), and 2000 s/mm2(P = 0.003). When the b-values were 1500 s/mm2(P = 0.008) and 2000 s/mm2(P = 0.009), the SIRnodule significantly differed between the papillary carcinoma group and the benign nodule group. When b = 500 s/mm2, the ADC had an AUC of 0.888. When b = 1000 s/mm2, the ADC had an AUC of 0.881. When b = 1500 s/mm2, the ADC had an AUC of 0.896. When b = 2000 s/mm2, the ADC had an AUC of 0.871. The DeLong test showed comparable diagnostic effectiveness among the different b-value groups except for between b-values of 2000 s/mm2and 1500 s/mm2, with a b-value of 2000 s/mm2showing lower effectiveness. Conclusion: This study suggests that 1500 s/mm2may be a suitable b-value to differentiate benign and malignant thyroid nodules in ZOOMit-DWI images, which yielded better image quality.

Downregulation of VEGFA accelerates AGEs-mediated nucleus pulposus degeneration through inhibiting protective mitophagy in high glucose environments.

Intervertebral disc degeneration (IVDD) contributes largely to low back pain. Recent studies have highlighted the exacerbating role of diabetes mellitus (DM) in IVDD, mainly due to the influence of hyperglycemia (HG) or the accumulation of advanced glycation end products (AGEs). Vascular endothelial growth factor A (VEGFA) newly assumed a distinct impact in nonvascular tissues through mitophagy regulation. However, the combined actions of HG and AGEs on IVDD and the involved role of VEGFA remain unclear. We confirmed the potential relation between VEGFA and DM through bioinformatics and biological specimen detection. Then we observed that AGEs induced nucleus pulposus (NP) cell degeneration by upregulating cellular reactive oxygen species (ROS), and HG further aggravated ROS level through breaking AGEs-induced protective mitophagy. Furthermore, this adverse effect could be strengthened by VEGFA knockdown. Importantly, we identified that the regulation of VEGFA and mitophagy were vital mechanisms in AGEs-HG-induced NP cell degeneration through Parkin/Akt/mTOR and AMPK/mTOR pathway. Additionally, VEGFA overexpression through local injection with lentivirus carrying VEGFA plasmids significantly alleviated NP degeneration and IVDD in STZ-induced diabetes and puncture rat models. In conclusion, the findings first confirmed that VEGFA protects against AGEs-HG-induced IVDD, which may represent a therapeutic strategy for DM-related IVDD.

A new haplotype variability in complement C6 is marginally associated with resistance to Aeromonas hydrophila in grass carp.

Aeromonas hydrophila, a widespread bacterium in the aquatic environment, causes haemorrhagic septicemia in fish. In the last decade, the disease has caused mass mortality and tremendous economic loss in cultured grass carp in the mainland China. The complement component C6 is a constituent of a biochemical cascade that serves as a major effector of the human innate and adaptor immunity, and eliminates infected cells. The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the C6 gene and to assess their association with A. hydrophila resistance in grass carp. A resource population consisting of 186 susceptible and 191 resistant grass carp was constructed. The gcC6 genomic sequence is composed of 9292 bp, containing 18 exons and 17 introns. The promoter sequence of gcC6 gene contained several consensus sequences for hepatic-specific transcription factors. We sequenced a total of 9744 bp of the C6 gene from a diverse population of grass carp and identified 8 SNPs that were genotyped in the resource population. Statistical analysis revealed a lack of association between any individual SNPs and resistance to A. hydrophila in grass carp. The SNPs 1214G>A, 1380G>C, 2095A>C and 2167T>C were linked together (r(2) > 0.8). The haplotype GCCC generated with these four SNPs was associated marginally with resistance to A. hydrophila in grass carp. These findings suggest a lack of strong association of the C6 polymorphisms with the A. hydrophila resistance in grass carp.

Reproductive endocrine disruption and gonadal intersex induction in male Japanese medaka chronically exposed to betamethasone at environmentally relevant levels.

The broad utilization of betamethasone in medical treatments may pose a significant ecotoxicological risk to aquatic organisms, yet its potential reproductive toxicity remains unclear. The present study examined the impacts of environmental exposure on male reproduction using Japanese medaka (Oryzias latipes). After 110 days of betamethasone exposure at environmentally relevant concentrations (0, 20 and 200 ng/L), LH/FSH synthesis and release in the pituitary was inhibited, and the production of sex hormones and their signaling pathways in the gonads of male medaka were greatly influenced. This synthetic glucocorticoid restrained testosterone (T) synthesis and gave rise to a significant increase in E2/T and E2/11-KT ratios. Furthermore, chronic betamethasone exposure (20 and 200 ng/L) led to the suppression of androgen receptor (AR) signaling and enhancement of estrogen receptors (ERs) signaling. An increase in hepatic vitellogenin contents was also detected, and testicular oocytes were observed in both 20 and 200 ng/L betamethasone-treated groups. It showed that 20 and 200 ng/L betamethasone could induce male feminization and even intersex, triggering abnormal spermatogenesis in medaka males. With its adverse effects on male fertility, betamethasone could potentially influence the fishery productivity and population dynamics in aquatic ecosystems.

Ameliorating Osteoarthritis in Mice Using Silver Nanoparticles.

Knee osteoarthritis (KOA) is one of the most commonly encountered degenerative diseases of the joints in people over 45 years of age. Currently, there are not any effective therapeutics for KOA,and the only end-point strategy is total knee arthroplasty (TKA); therefore, KOA is associated with economic burdens and societal costs. The immune inflammatory response is involved in the occurrence and development of KOA. We previously established a mouse model of KOA using type II collagen. Hyperplasia of the synovial tissue was present in the model, alongside a large number of infiltrated inflammatory cells. Silver nanoparticles have substantial anti-inflammatory effects and have been widely used in tumor therapy and surgical drug delivery. Therefore, we evaluated the therapeutic effects of silver nanoparticles in a collagenase II-induced KOA model. The experimental results showed that silver nanoparticles significantly reduced synovial hyperplasia and the infiltration of neutrophils in the synovial tissue. Hence, this work demonstrates the identification of a novel strategy for OA and provides a theoretical basis for preventing the progress of KOA.