Dephosphorylation of T517 on Hemocyanin Is Required for Antibacterial Activity in Penaeus vannamei.

Posttranslational modifications expand the functions of immune-related proteins, especially during infections. The respiratory glycoprotein, hemocyanin, has been implicated in many other functions, but the role of phosphorylation modification in its functional diversity is not fully understood. In this study, we show that Penaeus vannamei hemocyanin (PvHMC) undergoes phosphorylation modification during bacterial infection. Dephosphorylation of PvHMC mediated by P. vannamei protein phosphatase 2A catalytic increases its in vitro antibacterial activity, whereas phosphorylation by P. vannamei casein kinase 2 catalytic subunit α decreases its oxygen-carrying capacity and attenuates its in vitro antibacterial activity. Mechanistically, we show that Thr517 is a critical phosphorylation modification site on PvHMC to modulate its functions, which when mutated attenuates the action of P. vannamei casein kinase 2 catalytic subunit α and P. vannamei protein phosphatase 2A catalytic, and hence abolishes the antibacterial activity of PvHMC. Our results reveal that phosphorylation of PvHMC modulates its antimicrobial functions in penaeid shrimp.

The effect of human umbilical cord mesenchymal stem cell-derived exosomes on diabetic retinal neurodegeneration in a rat model.

OBJECTIVE: To investigate the effect of human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Exs) on diabetic retinal neurodegeneration (DRN). METHODS: Exosomes were isolated from human umbilical cord mesenchymal stem cells (hucMSC) and identified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blotting (WB). Rats were intraperitoneally injected with Streptozotocin (STZ) to establish a diabetes mellitus model, and blood glucose levels and body weight were assessed. The rats were intravitreally injected with phosphate buffered saline (PBS; diabetic group) or hucMSC-Exs (hucMSC-Exs group). A control group of rats were not treated with STZ and were intravitreally injected with PBS (normal control group). Hematoxylin-eosin (HE) staining was used to observe changes in retinal structure and to count the number of retinal ganglion cells (RGCs) four weeks after intravitreal injection. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL) was used to detect retinal cell apoptosis. The retinal expression of p38 mitogen-activated protein kinase (p38MAPK), phosphorylated p38MAPK (p-p38MAPK), Bcl-2 and Bax was measured using WB to investigate the mechanism by which hucMSC-Exs affects DRN. RESULTS: Using TEM, NTA and WB, hucMSC-Exs were successfully isolated. No significant change was observed after injection in the normal control group. All rats injected with STZ developed hyperglycemia. HE staining revealed that hucMSC-Exs effectively alleviated retinal structure disruption and reduced the apoptosis of RGCs (P < 0.05). Cells positive for TUNEL (TUNEL+) occurred at a higher rate in the diabetic group than in other groups (P < 0.05). Compared with the normal control group, the expression of p-p38MAPK was significantly increased in the diabetic group and decreased in the hucMSC-Exs group (P < 0.01). The expression of Bax was significantly decreased while Bcl-2 expression was significantly increased in hucMSC-Exs group (P < 0.01). CONCLUSION: These findings suggest that intravitreal injection of hucMSC-Exs can reduce DRN and protect retinal structure, and that these effects are mediated through inhibition of the p38MAPK pathway.

The development of epiretinal membrane following rhegmatogenous retinal detachment repair: incidence, risk factors, and outcomes / Desenvolvimento da membrana epirretiniana após reparo do descolamento regmatogênico da retina: incidência, fatores de risco e resultados

ABSTRACT Purpose: To investigate the incidence, risk factors, and visual outcomes of epiretinal membrane development following rhegmatogenous retinal detachment repair. Methods: This was a retrospective study of 309 eyes that underwent initial surgery for primary uncomplicated rhegmatogenous retinal detachment. Examinations were conducted preoperatively and then postoperatively at 1, 3, 6, and 12 months. The study patients were categorized into two groups depending on the presence or absence of the epiretinal membrane. Results: The incidence of postoperative epiretinal membrane was 28.5%; 42.7% of these patients had severe epiretinal membrane development and therefore underwent the epiretinal membrane removal. Logistic regression analyses revealed that giant retinal tears (OR: 2.66; 95% CI: 1.045-6.792, p=0.040) and horseshoe tears (OR: 0.534; 95% CI: 0.295-0.967, p=0.039) were the significant predictors of postoperative epiretinal membrane. Triamcinolone acetonide staining was significantly associated with the prevention of epiretinal membrane (p=0.022). A total of 34 patients showed a better or an equal final best-corrected visual acuity; of which 4 eyes were evaluated at the final follow-up visit and exhibited a reduced best-corrected visual acuity. Conclusion: Our analysis demonstrated that horseshoe tears and giant retinal tears represent the risk factors for the postoperative epiretinal membrane. Triamcinolone acetonide staining had a significant preventive effect on the postoperative epiretinal membrane. Furthermore, a second round of pars plana vitrectomy, including membrane removal, led to a significant improvement in the final best-corrected visual acuity as per the last follow-up examination, albeit the recovery was limited.

RESUMO Objetivos: Investigar a incidência, fatores de risco e desfechos visuais do desenvolvimento da membrana epirretiniana após reparo do descolamento regmatogênico da retina. Métodos: Trata-se de um estudo retrospectivo de 309 olhos submetidos à cirurgia inicial para descolamento regmatogênico da retina primário sem complicações. Os exames foram realizados no pré-operatório aos 1, 3, 6 e 12 meses pós-operatórios. Os pacientes foram divididos em dois grupos, dependendo da presença ou ausência de membrana epirretiniana. Resultados: A incidência de membrana epirretiniana pós-operatória foi de 28,5%; 42,7% desses pacientes apresentaram desenvolvimento grave da membrana epirretiniana e, portanto, foram submetidos à remoção desta membrana. A regressão logística mostrou que as lágrimas retinianas gigantes (RC: 2,66; 95% IC: 1,045 - 6,792, p=0,040) e lágrimas em ferradura (RC: 0,534; 95% IC: 0,295-0,967, p=0,039), foram preditores significativos de membrana epirretiniana pós-operatória. A coloração com acetonida de triancinolona foi significativamente associada à prevenção da membrana epirretiniana (p=0,022). Trinta e quatro pacientes apresentaram acuidade visual melhorada, ou igual, ou acuidade visual final melhor corrigida; 4 olhos foram avaliados na consulta final de acompanhamento e apresentaram redução da acuidade visual melhor corrigida. Conclusão: Nossa análise demonstra que as lágrimas de ferradura e as lágrimas retinianas gigantes representam fatores de risco para a membrana epirretiniana pós-operatória. A coloração com acetonida de triancinolona teve um efeito preventivo significativo na membrana epirretiniana no pós-operatório. Além disso, uma segunda rodada de vitrectomia pars plana, incluindo remoção da membrana, levou a uma melhora significativa da acuidade visual final melhor corrigida na última consulta de acompanhamento, embora a recuperação tenha sido limitada.

Recurrent retinal detachment after pars plana vitrectomy with silicone oil tamponade for rhegmatogenous retinal detachment.

BACKGROUND: The recurrence of retinal detachment following rhegmatogenous retinal detachment (RRD) is a relatively common complication that can lead to reduced visual acuity and requires further surgery. The purpose of this study was to investigate the risk factors and visual outcomes of recurrent RRD following pars plana vitrectomy (PPV) with silicone oil tamponade for primary RRD. METHODS: This was a retrospective follow-up study of 343 eyes that underwent initial PPV surgery with silicone oil tamponade for primary RRD. Patients were divided into a recurrence group and a reattachment group. The main outcome measures included causative factors, visual outcomes related to the recurrence of RRD, and the perioperative factors most affecting the recurrence of RRD. RESULTS: After retinal reattachment, we observed RRD recurrence after PPV for primary RRD in 42 out of 343 eyes (12.2%) during the follow-up period. Most causes of recurrence (69%) occurred within 6 months of surgery. Multivariate logistic regression analysis showed that a PVR ≥ Grade C (odds ratio [OR]: 4.015; 95% confidence interval [CI] 1.721-9.367; P = 0.001) was a significant predictor for the development of recurrent RRD. Compared with the reattachment group, the recurrence group exhibited a significant decline in best-corrected visual acuity (BCVA) at the last follow-up visit (P = 0.000). Eyes with PVR prior to primary surgery, or at the diagnosis of re-detachment, showed a worse final BCVA. CONCLUSIONS: Our analysis shows that the predominant risk factor for the recurrence of RRD is a PVR ≥ Grade C. PVR prior to primary surgery, or at the diagnosis of re-detachment, was also shown to limit the recovery of final visual acuity.

Protective effects of umbilical cord mesenchymal stem cell exosomes in a diabetic rat model through live retinal imaging.

AIM: To assess the protective effect of human umbilical cord mesenchymal stem cell exosomes (hucMSC-Exs) in a diabetic rat model by using a variety of retinal bioassays. METHODS: hucMSCs were subjected to differential ultracentrifugation for the collection of exosomes, and transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) using a NanoSight analysis system and Western blotting (WB) were used to analyze the expression of surface marker proteins such as CD63, CD9 and Calnexin. Streptozotocin (STZ) was injected into the intraperitoneal cavity to establish a diabetic model. Rats were divided into a normal group, diabetic group and hucMSC-Ex group. Fundus fluorescein angiography (FFA), optical coherence tomography (OCT) and other live imaging methods were used to observe the fundus of the rats. Finally, the eyeballs of rats from each group were collected for hematoxylin-eosin (HE) staining to further analyze the retinal structure. RESULTS: Through TEM, NTA and WB, we successfully isolated hucMSC-Exs. Subsequent FFA and OCT confirmed that hucMSC-Exs effectively prevented early retinal vascular damage and thickening of the retina. Finally, HE staining of rat retinal sections revealed that exosomes effectively alleviated retinal structure disruption caused by diabetes. CONCLUSION: hucMSC-Exs have a protective effect on the retina in diabetic rat through FFA, OCT and HE staining.

The development of epiretinal membrane following rhegmatogenous retinal detachment repair: incidence, risk factors, and outcomes.

PURPOSE: To investigate the incidence, risk factors, and visual outcomes of epiretinal membrane development following rhegmatogenous retinal detachment repair. METHODS: This was a retrospective study of 309 eyes that underwent initial surgery for primary uncomplicated rhegmatogenous retinal detachment. Examinations were conducted preoperatively and then postoperatively at 1, 3, 6, and 12 months. The study patients were categorized into two groups depending on the presence or absence of the epiretinal membrane. RESULTS: The incidence of postoperative epiretinal membrane was 28.5%; 42.7% of these patients had severe epiretinal membrane development and therefore underwent the epiretinal membrane removal. Logistic regression analyses revealed that giant retinal tears (OR: 2.66; 95% CI: 1.045-6.792, p=0.040) and horseshoe tears (OR: 0.534; 95% CI: 0.295-0.967, p=0.039) were the significant predictors of postoperative epiretinal membrane. Triamcinolone acetonide staining was significantly associated with the prevention of epiretinal membrane (p=0.022). A total of 34 patients showed a better or an equal final best-corrected visual acuity; of which 4 eyes were evaluated at the final follow-up visit and exhibited a reduced best-corrected visual acuity. CONCLUSION: Our analysis demonstrated that horseshoe tears and giant retinal tears represent the risk factors for the postoperative epiretinal membrane. Triamcinolone acetonide staining had a significant preventive effect on the postoperative epiretinal membrane. Furthermore, a second round of pars plana vitrectomy, including membrane removal, led to a significant improvement in the final best-corrected visual acuity as per the last follow-up examination, albeit the recovery was limited.

Natural history of persistent subretinal fluid following the successful repair of rhegmatogenous retinal detachment.

AIM: To provide a detailed description of the natural history of persistent subretinal fluid (SRF) after successful repair of rhegmatogenous retinal detachment (RRD) and its association with visual outcome. METHODS: This was a prospective long-term follow-up for eyes undergoing scleral buckling (SB) surgery for macula-off RRD. Examinations were carried out preoperatively and postoperatively at 1, 3, 6, 9 and 12mo, until persistent SRF had completely resolved. One month postoperatively, optical coherence tomography (OCT) was used to classify SRF into three patterns: bleb-like loculated (BL), shallow-diffused (SD), and multiple blebs (MB). Serial OCT imaging was used to evaluate morphological changes in SRF until its complete disappearance. Patients were divided into two groups depending on the presence or absence of persistent SRF. RESULTS: A total of 59 patients (59 eyes) were included. There were no statistical differences between two groups at baseline, except for the proportion of patients with high myopia and a younger age. One month after surgery, OCT detected persistent SRF in 49 eyes (83.1%). The 3 morphological patterns of SRF were observed in 27 eyes (55.1%) with BL, 13 eyes (26.5%) with SD, and 9 eyes (18.4%) with MB. The mean time for complete absorption differed significantly across the three SRF patterns (F=8.097, P=0.001), which was 8.8±6.1, 20.1±12.1, and 16.7±10.2mo in BL, SD, and MB, respectively. In 9 of the 13 eyes with SD, the pattern transformed into MB type. In cases involving MB, the size and number of blebs decreased gradually until they had been completely absorbed. Eyes with persistent SRF were more likely to demonstrate disruption of the ellipsoid zone (49.0% vs 10%, P=0.034). The final best-corrected visual acuity of two groups was 0.37±0.11 (with SRF) vs 0.34±0.12 (without SRF) logMAR (P=0.499), respectively. CONCLUSION: High preoperative myopia and younger age are associated with persistent SRF. BL is the most commonly observed pattern with the shortest duration and gradually disappeared. Most cases involving SD SRF transform into MB type during resolution. The size and number of the MBs decrease gradually until they were completely absorbed. The absence of persistent SRF may contribute to slow visual recovery in the short-term but does not influence the final visual outcome.

Labile and recalcitrant components of organic matter of a Mollisol changed with land use and plant litter management: An advanced 13C NMR study.

Soil organic matter (SOM) changes with land use and soil management, yet the controlling factors over the chemical composition of SOM are not fully understood. We applied quantitative 13C nuclear magnetic resonance and spectral editing techniques to measure chemical structures of SOM from different land use types. The land use types included a native grassland (nGL), a crop land with straw burning in the field (bCL), a restored grassland (rGL) and a cropland with straw removed out of the field (rCL) for 28years. The abundances of OCH groups from carbohydrates were higher in the SOMs of the nGL and rGL than in those of the rCL and bCL, while the abundances of OCH3 and aromatic CO groups from lignin were higher in the SOMs of the three-ever cultivated lands (rGL, rCL and bCL) than in that of the nGL. Although aromatic CC groups were most dominant in the Mollisols, they did not consistently decrease after the burnings of straw were ceased in the fields of the rCL and rGL compared to the bCL with continuous burning. In addition, the COO groups were bound with the aromatic CC groups in all the land use types, and the sizes of the aromatic clusters were affected by the land use types. The labile and recalcitrant components were correlated with SOC contents the mineral-associated and particular SOM in a contrasting way. Our results suggested that the chemical composition of SOM in the Mollisol depended on land use types, and that labile and recalcitrant components might be protected through mineral associations and aggregation, respectively. The most abundant aromatics in the Mollisols might not just be pyrogenic and could be oxidized to different extents, depending on field drainage conditions.

C-Terminal Domain of Hemocyanin, a Major Antimicrobial Protein from Litopenaeus vannamei: Structural Homology with Immunoglobulins and Molecular Diversity.

Invertebrates rely heavily on immune-like molecules with highly diversified variability so as to counteract infections. However, the mechanisms and the relationship between this variability and functionalities are not well understood. Here, we showed that the C-terminal domain of hemocyanin (HMC) from shrimp Litopenaeus vannamei contained an evolutionary conserved domain with highly variable genetic sequence, which is structurally homologous to immunoglobulin (Ig). This domain is responsible for recognizing and binding to bacteria or red blood cells, initiating agglutination and hemolysis. Furthermore, when HMC is separated into three fractions using anti-human IgM, IgG, or IgA, the subpopulation, which reacted with anti-human IgM (HMC-M), showed the most significant antimicrobial activity. The high potency of HMC-M is a consequence of glycosylation, as it contains high abundance of α-d-mannose relative to α-d-glucose and N-acetyl-d-galactosamine. Thus, the removal of these glycans abolished the antimicrobial activity of HMC-M. Our results present a comprehensive investigation of the role of HMC in fighting against infections through genetic variability and epigenetic modification.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) participates in anti-lipopolysaccharide factors (ALFs) gene expression in mud crab.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key cytoplasm signal adaptor that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp TRAF6 (Sp-TRAF6) from Scylla paramamosain contains 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp-TRAf6 has highest identity of 88% with Pt-TRAF6 from Portunus trituberculatus, while the similarity of Sp-TRAF6 with other crustacean sequences was 54-55%. RT-PCR analysis indicated that Sp-TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Moreover, Sp-TRAF6 transcripts were significantly up-regulated after Vibrio parahemolyticus and LPS challenges. RNA interference assay was carried out used by siRNA to investigate the genes expression patterns regulated by Sp-TRAF6. The qRT-PCR results showed that silencing Sp-TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while inhibit SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 expression in hepatopancreas. Taken together, the acute-phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp-TRAF6 plays an important role in host defense against pathogen invasions via regulation of ALF gene expression in S. paramamosain.

Enhancement of the immune response and protection against Vibrio parahaemolyticus by indigenous probiotic Bacillus strains in mud crab (Scylla paramamosain).

In a previous study, bacterial communities of the intestine in three populations of crabs (wild crabs, pond-raised healthy crabs and diseased crabs) were probed by culture-independent methods. In this study, we examined the intestinal communities of the crabs by bacterial cultivation with a variety of media. A total of 135 bacterial strains were isolated from three populations of mud crabs. The strains were screened for antagonistic activity against Vibrio parahaemolyticus using an agar spot assay. Antagonistic strains were then identified by 16S rRNA gene sequence analysis. Three strains (Bacillus subtilis DCU, Bacillus pumilus BP, Bacillus cereus HL7) with the strongest antagonistic activity were further evaluated for their probiotic characteristics. The results showed that two (BP and DCU) of them were able to survive low pH and high bile concentrations, showed good adherence characteristics and a broad spectrum of antibiotic resistance. The probiotic effects were then tested by feeding juvenile mud crabs (Scylla paramamosain) with foods supplemented with 10(5) CFU/g of BP or DCU for 30 days before being subjected to an immersion challenge with V. parahaemolyticus for 48 h. The treated crabs showed significantly higher expression levels of immune related genes (CAT, proPO and SOD) and activities of respiratory burst than that in controlled groups. Crabs treated with BP and DCU supplemented diets exhibited survival rates of 76.67% and 78.33%, respectively, whereas survival rate was 54.88% in crabs not treated with the probiotics. The data showed that indigenous mud-associated microbiota, such as DCU and BP, have potential application in controlling pathogenic Vibriosis in mud crab aquaculture.

HSPC117 is regulated by epigenetic modification and is involved in the migration of JEG-3 cells.

The human hematopoietic stem/progenitor cell 117 (HSPC117) protein is an essential component of protein complexes and has been identified to be involved in many important functions. However, how this gene expression is regulated and whether the HSPC117 gene affects cell migration is still unknown. The aim of this study was to identify whether HSPC117 mRNA expression is regulated by epigenetic modification and whether HSPC117 expression level affects the expression of matrix metalloproteinase 2 (MMP 2), matrix metalloproteinase 14 (MMP 14), and tissue inhibitor of metalloproteinases 2 (TIMP 2), and further affects human placenta choriocarcinoma cell (JEG-3) migration speed. In our epigenetic modification experiment, JEG-3 cells were cultured in medium with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), the histone deacetylase (HDAC) inhibitor trichostatin A (TSA), or both inhibitors. Then, the HSPC117 mRNA and protein expressions were assessed using real-time quantitative PCR (qPCR) and Western blot assay. The results showed that, compared to the control, HSPC117 mRNA expression was increased by TSA or 5-aza-dC. The highest HSPC117 expression level was found after treatment with both 5-aza-dC and TSA. Further, in order to investigate the effect of HSPC117 on MMP 2, MMP 14, and TIMP 2 mRNA expressions, pEGFP-C1-HSPC117 plasmids were transfected into JEG-3 cells to improve the expression of HSPC117 in the JEG-3 cells. Then, the mRNA expression levels of MMP 2, MMP 14, TIMP 2, and the speed of cell migration were assessed using the scratch wound assay. The results showed that over-expression of HSPC117 mRNA reduced MMP 2 and MMP 14 mRNA expression, while TIMP 2 mRNA expression was up-regulated. The scratch wound assay showed that the migration speed of JEG-3 cells was slower than the non-transfected group and the C1-transfected group. All of these results indicate that HSPC117 mRNA expression is regulated by epigenetic modification; over-expression of HSPC117 decreases MMP 2 and MMP 14 transcription, reduces cell migration speed, and increases TIMP 2 transcription.

Dodecamer is required for agglutination of Litopenaeus vannamei hemocyanin with bacterial cells and red blood cells.

Hemocyanins are multi-functional proteins, although they are well known to be respiratory proteins of invertebrate to date. In the present study, the agglutination ability of two oligomers of hemocyanin, hexamer and dodecamer, with pathogenic bacteria and red blood cells (RBCs) is investigated in pacific white shrimp, Litopenaeus vannamei. Hexameric hemocyanin exhibits an extremely high stability even in the absence of Ca(2+) and in alkaline pH. Dodecamer (di-hexamer) is easily dissociated into hexamers in unphysiological conditions. Hexamer and dodecamer are interchanged reciprocally with environmental conditions. Both oligomers can bind to bacteria and RBCs, but agglutination is observed only using dodecamer but not using hexamer in agglutination assay. However, the agglutination is detected when hexamer is utilized in the presence of antiserum against hemocyanin. These results indicate that dodecamer of hemocyanin is required for agglutination with bacteria and RBCs. It can be logically inferred that there is only one carbohydrate-binding site to bacterial cells and RBCs in the hexamer, while at least two sites in the dodecamer. Our finding has provided new insights into structural-functional relationship of hemocyanin.

[Effects of ginkgolides injection on experimental cerebral ischemia in mice and rats].

OBJECTIVE: To investigate the effects of ginkgolides injection on experimental cerebral ischemia and its related mechanism of action. METHOD: The middle cerebral artery occlusion (MACO) model was induced by the FeCl3-occluding method to explore the protective effects of ginkgolides injection on the score of neurological deficits, the rate of cerebral infarction and the histomorphology of cerbral ischemia in rats. Thrombosis formation in vivo was induced by adrenaline-collagen in mice to explore the antithrombotic effect. Platelet aggregation was induced by ADP and hemorrheological parameters with hyper-viscosity by dextran T-500 were used to explore the effects of antiplatelet aggregation and decreasing viscosity of blood. RESULT: Ginkgolides injection could markedly decrease the infarct size and behavior deficits score, inhibit the thrombus formation in mice, decrease blood viscosity and ameliorate hemorrheological parameters in rat. CONCLUSION: Ginkgolides injection has the protective effects on focal cerebral ischemia, and its mechanism may be relative to its inhibition of platelet-dependent thrombosis and amelioration of hemarheological partments.