Development of a drying method for proteins based on protein-hyaluronic acid precipitation.

This study aims to develop a new method to dry proteins based on protein-hyaluronic acid (HA) precipitation and apply the precipitation-redissolution technique to develop highly concentrated protein formulations. Lysozyme was used as a model protein and HA with various molecular weights (MW) were investigated. Under low ionic strength, low-MW HA (e.g., MW: around 5 K) did not induce lysozyme precipitation. Conversely, high-MW HA (e.g., MW: approximately from 40 K to 1.5 M) precipitated more than 90 % of lysozyme. The dried lysozyme-HA precipitates had moisture levels between 4 % and 5 %. The lysozyme-HA precipitates could be redissolved using PBS (pH 7.4, ionic strength: ∼ 163 mM). The viscosity of the reconstituted solution was dependent on HA MW, e.g., 4 cP for HA40K, and 155 cP for HA1.5 M, suggesting low-MW HA might be a proper excipient for highly concentrated solution formulations for subcutaneous/intraocular injection and high-MW HA may fit for other applications. The tertiary structure of lysozyme after the precipitation-redissolution steps had no significant difference from that of the original lysozyme as confirmed by fluorescence spectroscopy. The denaturation temperature of lysozyme after the precipitation-redissolution steps and that of the original lysozyme were close, indicating they possessed similar thermal stability. The accelerated stability study revealed that lysozyme stored in the dry precipitates was more physically stable than that in the buffer solution. Overall, this new drying technique is suitable for drying proteins and exhibits several benefits such as minimum energy consumption, cost effectiveness, high production yield, and mild processing conditions. In addition, the precipitation-redissolution technique proposed in this study can potentially be used to develop highly concentrated formulations, especially for proteins experiencing poor stability in the liquid state.

Traditional Chinese Medicine Integrated Responsive Microneedles for Systemic Sclerosis Treatment.

Traditional Chinese medicine, such as Tripterygium wilfordii and Paeonia lactiflora, has potential values in treating systemic sclerosis (SSc) and other autoimmune diseases, while their toxic side effect elimination and precise tropical drug delivery are still challenges. Here, we present multiple traditional Chinese medicine integrated photoresponsive black phosphorus (BP) microneedles (MNs) with the desired features for the SSc treatment. By employing a template-assisted layer-by-layer curing method, such MNs with triptolide (TP)/paeoniflorin (Pae) needle tips and BP-hydrogel needle bottoms could be well generated. The combined administration of TP and Pae can not only provide anti-inflammatory, detoxification, and immunomodulatory effects to treat skin lesions in the early stage of SSc but also remarkably reduce the toxicity of single drug delivery. Besides, the additive BPs possess good biocompatibility and near-infrared (NIR) responsiveness, imparting the MN photothermal-controlled drug release capability. Based on these features, we have demonstrated that the traditional Chinese medicine integrated responsive MNs could effectively improve skin fibrosis and telangiectasia, reduce collagen deposition, and reduce epidermal thickness in the SSc mouse models. These results indicated that the proposed Chinese medicine integrated responsive MNs had enormous potential in clinical therapy of SSc and other diseases.

Study on the modification effect and mechanism of tailings powder on coal gangue-based mining cementitious filling material.

Based on the problems of high carbon emission and high cost of traditional mining methods and filling materials, the tailings powder modified Coal Gangue-based Cementitious Backfill Material (CGCBM) was used for paste filling mining. In this study, the samples were prepared with different tailings powder content and different curing ages. The compressive strength test, XRD, SEM test, and NMR test were used to explore the changes of macroscopic strength and microstructure of the material. The results show that adding 50% tailings powder has the most obvious optimization effect on the performance of CGCBM. Tailings powder particles fill the surface holes of the fine aggregate of coal gangue in the early cement hydration process, reduce the water absorption of the aggregate. In addition, the active substances such as Ca2SiO4 play the pozzolanic effect, stimulate the secondary hydration of slurry, make the microstructure closely, and thus improve the macroscopic mechanical strength.

Examining the Link Between Physical Activity and Cognitive Function: A Parallel Mediation Model of Health and Wellbeing Among Adolescents.

Background: Adolescents' engagement in daily physical activity brings multiple benefits, including reduction in obesity, improvement of mental health, and enhancement of cognitive function (CF). While prior studies have examined the link between physical activity and cognitive function, little is known regarding the extent to which this relationship is shaped by health and wellbeing factors. This study examines how subjective wellbeing (SWB) and general health (GH) mediate the relationship between adolescents' physical activity and cognitive function. Methods: This study estimates a parallel structural equation model using the Program for International Student Assessment 2018 dataset. Specifically, a total of 63,228 15-year-old subjects in nine countries/economies satisfied the study inclusion criteria, including in Bulgaria, Georgia, Hong Kong, Ireland, Mexico, Panama, Serbia, Spain, and United Arab Emirates. Frequency of moderate physical activity (MPA, ≥3.0 Metabolic Equivalent Task) was reported weekly; SWB and GH were assessed using an internationally validated multi-item standardized questionnaire. SWB was measured by students' self-evaluated satisfaction with their health, life, and schooling. GH was measured by students' physical health and mental health status. Cognitive function (CF) was modeled as a latent function consisting of plausible values derived using item response theory on reading, mathematics, and science achievement tests. Results: Findings indicated that increase in weekly MPA was positively associated with higher levels of SWB (p < .001), GH (p < .001), and CF (p < .001) among the study subjects. Parallel mediation analyses revealed that more frequent weekly MPA had relatively large direct effects (p < .001) on CF, and indirect effects channeling through improvements in SWB and GH were non-trivial (p < .001). Heterogeneity results showed that boosts to CF, associated with MPA, were larger for mathematics and science than for reading (p < .001). Conclusion: This study used a large-scale international dataset to show that the positive relationship observed between MPA and CF among adolescents was robust, and that SWB and GH were two critical mediators through which physical activity positively bolster CF.

Interferon-γ suppresses the proliferation and migration of human placenta-derived mesenchmal stromal cells and enhances their ability to induce the generation of CD4+CXCR5+Foxp3+Treg subset.

We investigate the effects of interferon (IFN)-γ on human placenta-derived mesenchymal stromal cells (hPMSCs), in particular, their adhesion, proliferation and migration and modulatory effects on the CD4+CXCR5+Foxp3+Treg subset. And we compared hPMSCs ability to induce the generation of different Treg subsets in response to treatment with IFN-γ. We found that IFN-γ suppressed the proliferation and migration for hPMSCs. The ability of hPMSCs to induce the generation of CD4+CXCR5+Foxp3+Treg subset was enhanced by IFN-γ. And maximal effectiveness of IFN-γ treated hPMSCs upon inducing the generation of Treg subsets was for CD4+CXCR5+Foxp3+Treg subset as compared with that of CD4+CD25+Foxp3+, CD8+CD25+Foxp3+, CD4+IL-10+ and CD8+IL-10+Treg subsets. These results have important implications for the development and application of hPMSCs in clinical use.

[The effect of human placenta-derived mesenchymal stem cells on cord blood CD8(+);T cells activation, cellcycle and secretion of IL-17].

AIM: To study the effect of human placenta-derived mesenchymal stem cells (hPMSCs) on cord blood CD8(+);T cell activation, cell cycle and secretion of IL-17, and to provide the theoretical basis for it application in the cell-based therapies. METHODS: hPMSCs were isolated from mature placenta by the method of digestion. Then hPMSCs were cultured, expanded in vitro, and were used in test after the third passage. CD8(+);T cells were sorted from cord blood with immunomagetic beads. FCM was used to analyze the expression of early activation phenotype, cell cycle of cord blood CD8(+);T cells and cytokine secretion. RESULTS: CD8(+);T cells stimulated by PHA in the presence of hPMSCs were arrested at G0/G1 phase. The expression of the early activation marker CD25 and CD69 of cord blood CD8(+);T cells was inhibited in the presence of hPMSCs. While, IL-17secretion of cord blood CD8(+);T cells stimulated by PMA was increased. CONCLUSION: hPMSCs can suppress the activation of cord blood CD8(+);T cells by altering T cell cycle; up-regulate the level of IL-17 secreted by cord blood CD8(+);T cells.

[Comparison the inhibitory effects of human bone marrow mesenchymal stem cells and human placenta mesenchymal stem cells on T cell proliferation].

AIM: To compare and study the inhibitory effects of human bone marrow mesenchymal stem cells (HBMSCs) and human palacenta mesenchymal stem cells (HPMSCs) on T cell proliferation, and the underlying mechanism. METHODS: The expression of B7H4 on HBMSCs or the expression of PDL1 on HPMSCs were detected by FCM. Blocking experiment was used to analyze the effects of B7H4 or PDL1 on HBMSCs or HPMSCs mediating suppression on T cell proliferation and cell cycle. RESULTS: FCM detection showed that HBMSCs highly expressed B7H4, while HPMSCs highly expressed PDL1, the negative immune molecules. Blockade B7H4 on HBMSCs with B7H4mAb significantly attenuated the inhibitory effects of HBMSCs on T cell proliferation. Likewise, blocking the expression of PDL1 on HPMSCs obviously weakened the suppressive effects of HPMSCs on T cell proliferation activated by PHA. Moreover, Blockade B7H4 on HBMSCs with B7H4mAb or PDL1 on HPMSCs with PDL1mAb significantly weakened the inhibitory effects of HBMSCs or HPMSCs on T cell cycle through down-regulating the cell number in G(0);/G(1); phase and up-regulating the cell number in S phase. CONCLUSION: HBMSCs and HPMSCs could mediate the suppressive effects on T cell proliferation through expressing different negative immune molecules.

Diversity, abundance and distribution of amoA-encoding archaea in deep-sea methane seep sediments of the Okhotsk Sea.

The ecological characteristics of amoA-encoding archaea (AEA) in deep-sea sediments are largely unsolved. This paper aimed to study the diversity, structure, distribution and abundance of the archaeal community and especially its AEA components in the cold seep surface sediments of the Okhotsk Sea, a marginal sea harboring one of the largest methane hydrate reservoirs in the world. Diverse archaeal 16S rRNA gene sequences were identified, with the majority being related to sequences from other cold seep and methane-rich sediment environments. However, the AEA diversity and abundance were quite low as revealed by amoA gene analyses. Correlation analysis indicates that the abundance of the archaeal amoA genes was correlated with the sediment organic matter content. Thus, it is possible that the amoA-carrying archaea here might utilize organic matter for a living. The affiliation of certain archaeal amoA sequences to the GenBank sequences originally obtained from deep-sea hydrothermal vent environments indicated that the related AEA either have a wide range of temperature adaptation or they have a thermophilic evolutionary history in the modern cold deep-sea sediments of the Okhotsk Sea. The dominance of ammonia-oxidizing bacteria over AEA may indicate that bacteria play a significant role in nitrification in the Okhotsk Sea cold seep sediments.

The negative co-signaling molecule b7-h4 is expressed by human bone marrow-derived mesenchymal stem cells and mediates its T-cell modulatory activity.

Though experimental evidence shows that human bone marrow-derived mesenchymal stem cells (hBMSCs) are able to suppress T-cell activation and proliferation, the precise mechanisms are still not completely understood. Here, we investigated the role of the negative costimulatory molecule B7-H4 in the immunosuppressive effect of hBMSCs on T-cell activation. We showed that B7-H4 expresses abundantly on hBMSCs assessed by reverse transcription, immunofluorescence staining, and flow cytometric analysis. Further studies demonstrated that B7-H4 expressed on hBMSCs inhibits T-cell activation and proliferation via induction of cell cycle arrest and inhibition of NF-kappaB nuclear translocation. Blocking B7-H4 would decrease the secretion of transforming growth factor-beta1 (TGF-beta1) in the supernatant of activated T cells co-cultured with hBMSCs. Addition of neutralizing antibodies against B7-H4 significantly attenuated the inhibitory effects of hBMSCs on T-cells. Thus, our study established the novel role of B7-H4 molecule in the suppressive effect of hBMSCs on T-cell activation and proliferation. Taken together, these results highlight the complex role of hBMSCs in regulating the immune response, asserting the possibility of their therapeutic application in transplantation, the treatment of graft-versus-host disease (GVHD), and autoimmune diseases.

[The effects of B7H4 on human bone marrow mesenchymal stem cell inhibiting proliferation of PHA activated T cells].

OBJECTIVE: To investigate the effects of B7H4 on human bone marrow mesenchymal stem cells (HBMSC) mediating immune suppression. METHODS: The expression of the negative immunoregulatory factor B7H4 on HBMSC were analyzed by RT-PCR and flow cytometry (FCM), respectively. The blocking experiment was used to detect the effects of B7H4 on HBMSC mediating suppression on PHA induced T cell activation, proliferation and cell cycle. HBMSC inhibiting T cell proliferation was examined by transwell cell culture system. RESULTS: B7H4 was highly expressed on HBMSC. Blocking the B7H4 expression by B7H4mAb significantly attenuated the inhibitory effects of HBMSC on T cell proliferation. Compared with that of the unblocking group, T cell stimulator index (SI) of the B7H4 blocked group was significantly increased (53 +/- 5 vs 15 +/- 8, P < 0.01) and the inhibitory effects of HBMSC on T cell cycle were weakened significantly through down-regulating the cell number in G(0)/G(1) phase \[(85.6 +/- 9.9)% vs (95.8 +/- 9.9)%\] and up-regulating those in S phase\[(5.8 +/- 3.2)% vs (2.3 +/- 2.2)%, P < 0.05\]. The suppressive effects of HBMSC on T cell proliferation were significantly weakened after separating HBMSC from T cells by transwell cell culture system. Compared with the cell to cell contact group, T cell SI was significantly increased (27 +/- 17 vs 15 +/- 3, P < 0.01). CONCLUSION: HBMSC highly express B7H4, which plays an important role in the suppressive effects of HBMSC on T cell proliferation.

Antheraea pernyi silk fibroin maintains the immunosupressive properties of human bone marrow mesenchymal stem cells.

We reported previously that regenerated Antheraea pernyi silk fibroin (A. pernyi SF) could support the attachment and growth of human bone marrow mesenchymal stem cells (hBMSCs). In this work, the immunosupressive effects of hBMSCs cultured on the A. pernyi SF films on T-cells were investigated in vitro. The production of IL-6, CD80, CD86 and HLA-DR by the hBMSCs was also observed. The study showed that hBMSCs cultured on the regenerated A. pernyi SF films still kept their immunosupression on T-cell proliferation and IL-2 secretion. Moreover, regenerated A. pernyi SF like regenerated Bombyx mori SF and collagen did not elicit T-cell proliferation but it could support the expression of IL-6 and surface antigen of hBMSCs. Regenerated A. pernyi SF can maintain the function of hBMSCs in immunomodulation and cytokines production, which has the potential utility of hBMSCs combined with A. pernyi SF in tissue replacement and repair.

Diversity of both the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China sea.

Protease-producing bacteria are known to play an important role in degrading sedimentary particular organic nitrogen, and yet, their diversity and extracellular proteases remain largely unknown. In this paper, the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China Sea was investigated. The richness of the cultivable protease-producing bacteria reached 10(6) cells/g in all sediment samples. Analysis of the 16S rRNA gene sequences revealed that the predominant cultivated protease-producing bacteria are Gammaproteobacteria affiliated with the genera Pseudoalteromonas, Alteromonas, Marinobacter, Idiomarina, Halomonas, Vibrio, Shewanella, Pseudomonas, and Rheinheimera, with Alteromonas (34.6%) and Pseudoalteromonas (28.2%) as the predominant groups. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria are serine proteases or metalloproteases. Moreover, these proteases have different hydrolytic ability to different proteins, reflecting they may belong to different kinds of serine proteases or metalloproteases. To our knowledge, this study represents the first report of the diversity of bacterial proteases in deep-sea sediments.

[Effects of MSC on cell cycle, phenotype and cytokine secretion of T cells activated by PHA].

AIM: To study the effect of human bone marrow derived mesenchymal stem cell (MSC) on T cell cycle and activation, and to investigate the inhibitory effect of MSC on T cell proliferation and the underlying mechanism. METHODS: Human bone marrow derived MSC were isolated by gradient centrifugation. then in vitro MSC were cultured, expanded,and were used in test after third passage. FCM analysis and ELISA were used to investigate the effects of MSC on the early activation marker expression of T cells, cell cycle and cytokine secretion. RESULTS: T cells stimulated by PHA in the presence of MSC were arrested at G0/G1 phase. The expression of the early activation marker CD25 and CD69 of T cells was inhibited in the presence of MSC both in CD4(+) and CD8(+) T cell subpopulation. MSC caused a sharp decrease of cytokine secretion in IL-2 and IFN-gamma. CONCLUSION: Human bone marrow derived MSC can suppress the activation and proliferation of T cells by altering T cell cycle.

[Immunoregulatory effects of mesenchymal stem cell and its application].

The immunoregulatory effects of mescenchymal stem cell (MSC) and its application have become a hot research topic in recent years. This article reviews the up-to-dated research advances in the features and mechanisms of immune regulation of MSC and its application.

Attachment and growth of human bone marrow derived mesenchymal stem cells on regenerated antheraea pernyi silk fibroin films.

Silk fibroin of the silkworm Bombyx mori has been studied extensively, while the research on Antheraea pernyi silk fibroin (A. pernyi SF) in biomaterials is only at an early stage. In this study, the attachment, morphology, growth and phenotype of human bone marrow derived mesenchymal stem cells (hBMSCs) cultured on the regenerated A. pernyi SF films were studied in vitro. The results indicated that the attachment of hBMSCs on the regenerated A. pernyi SF films was almost the same as that on the collagen films. MTT and cell counting analyses demonstrated that the growth of hBMSCs on the regenerated A. pernyi SF films was better than that on controls. Moreover, electron scanning microscopy and fluorescence-activated cell sorting assays showed that the regenerated A. pernyi SF supported hBMSCs growth and functional maintenance compared with the controls. These data suggest that the regenerated A. pernyi SF, like Bombyx mori silk fibroin (B. mori SF) and collagen, can support hBMSCs attachment, growth and phenotypic maintenance, and has better biocompatibilities for hBMSCs in vitro culture.

[Experimental study on Rhizoma sparganii and Radices zedoariae treating hepatic fibrosis].

OBJECTIVE: To study the effects of rhizoma sparganii and radices zedoariae on hepatic fibrosis. METHOD: The rat immunohepatic fibrosis model was made by intraperitoneal injection of porcine serum and treated with rhizoma sparganii and radices zedoariae. The ALT, GGT, TP, ALb, A/G, IVC, LN, HA and the pathological change of the liver were observed. RESULT: Rhizoma sparganii and radices zedoariae could increase TP, ALb, A/G, decrease ALT, GGT, IVC, LN, HA and improve the pathological change. CONCLUSION: Rhizoma sparganii and radices zedoariae can protect hepatic cells, alleviate degeneration and necrosis, recover structure and function, and reduce the proliferation of fibrous tissue.