Recent advances in acoustic microfluidics and its exemplary applications.

Acoustic-based microfluidics has been widely used in recent years for fundamental research due to its simple device design, biocompatibility, and contactless operation. In this article, the basic theory, typical devices, and technical applications of acoustic microfluidics technology are summarized. First, the theory of acoustic microfluidics is introduced from the classification of acoustic waves, acoustic radiation force, and streaming flow. Then, various applications of acoustic microfluidics including sorting, mixing, atomization, trapping, patterning, and acoustothermal heating are reviewed. Finally, the development trends of acoustic microfluidics in the future were summarized and looked forward to.

Salidroside promoted osteogenic differentiation of adipose-derived stromal cells through Wnt/ß-catenin signaling pathway.

BACKGROUND: Bone disease causes short-term or long-term physical pain and disability. It is necessary to explore new drug for bone-related disease. This study aimed to explore the role and mechanism of Salidroside in promoting osteogenic differentiation of adipose-derived stromal cells (ADSCs). METHODS: ADSCs were isolated and treated with different dose of Salidroside. Cell count kit-8 (CCK-8) assay was performed to assess the cell viability of ADSCs. Then, ALP and ARS staining were conducted to assess the early and late osteogenic capacity of ADSCs, respectively. Then, differentially expressed genes were obtained by R software. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes were further analyzed. The expression of OCN, COL1A1, RUNX2, WNT3A, and ß-catenin were measured by real-time PCR and Western blot analysis. Last, ß-catenin was silenced by small interfering RNA. RESULTS: Salidroside significantly increased the ADSCs viability at a dose-response manner. Moreover, Salidroside enhanced osteogenic capacity of ADSCs, which are identified by enhanced ALP activity and calcium deposition. A total of 543 differentially expressed genes were identified between normal and Salidroside-treated ADSCs. Among these differentially expressed genes, 345 genes were upregulated and 198 genes were downregulated. Differentially expressed genes enriched in the Wnt/ß-catenin signaling pathway. Western blot assay indicated that Salidroside enhanced the WNT3A and ß-catenin expression. Silencing ß-catenin partially reversed the promotion effects of Salidroside. PCR and Western blot results further confirmed these results. CONCLUSION: Salidroside promoted osteogenic differentiation of ADSCs through Wnt/ß-catenin signaling pathway.

Recent Advances in Three-Dimensional Multicellular Spheroid Culture and Future Development.

Three-dimensional multicellular spheroids (MCSs) have received extensive attention in the field of biomedicine due to their ability to simulate the structure and function of tissues in vivo more accurately than traditional in vitro two-dimensional models and to simulate cell-cell and cell extracellular matrix (ECM) interactions. It has become an important in vitro three-dimensional model for tumor research, high-throughput drug screening, tissue engineering, and basic biology research. In the review, we first summarize methods for MCSs generation and their respective advantages and disadvantages and highlight the advances of hydrogel and microfluidic systems in the generation of spheroids. Then, we look at the application of MCSs in cancer research and other aspects. Finally, we discuss the development direction and prospects of MCSs.

[Progress in HIV-1 Env trimer design].

Currently, HIV-1 vaccine development has still been a hot pot in the AIDS research. HIV-1 glycoprotein Env is the sole target in the virion surface that mediates the membrane fusion between the virion and cell in the HIV-1 infection process. Env protein is the significant immunogen for HIV-1 vaccine development. In recent years, there have been breakthroughs in the Env trimer research. For example, the strategies including SOSIP, NFL2P, and UFO had been applied to design and generate HIV-1 Env trimer. The improvement of quantity and stability is beneficial to achieve the HIV-1 native-like Env trimer for elicitation of strong neutralizing antibody responsing in animal immunization. This review focuses on the different strategies for Env trimer design and compares their advantages and disadvantages, combining with our work to give some advice, which might provide relevant information for the future HIV-1 immunogen design.

Characterization of native-like HIV-1 gp140 glycoprotein expressed in insect cells.

The trimeric HIV-1 envelope glycoprotein (Env) is critical for vaccine development aimed at achieving broadly-neutralizing antibody responses. The use of various recombinant expression systems and construct designs are associated with the resultant nature of produced proteins, especially in terms of glycosylation, antigenicity, and immunogenicity of the glycoprotein. Here, we explored an otherwise baculovirus cassette than classical one designed to express HIV-1 Env protein, including SOSIP mutation and Foldon moiety involvement. This improved design increased the ratio of the Env trimer fraction from ∼40% to ∼60% with respect to that of prototypical design, as indicated by high-performance size-exclusion chromatography and sedimentation velocity analysis. In addition, the design prolonged cell viability and enhanced the final yield (approximately 13-15 mg/L) after affinity purification. gp140 produced from insect cells mimicked the native-like trimer and mainly adopted glycosylation pattern of oligomannose glycans. The native-like Env proteins conferred cross-clade neutralizing antibody production in BALB/c mice. In summary, the expression of Env in insect cells by optimizing the baculovirus vector provides an alternative strategy for HIV-1 immunogen production and may benefit future Env-based HIV vaccine design.

[Expression and self-assembly of HIV-1 CAP2NC protein].

We constructed the CAP2NC prokaryotic expression vector of HIV-1 NL4-3 strain and obtained relatively pure CAP2NC protein by optimizing its purification conditions to explore its in vitro self-assembly conditions. Primers were designed according to the CAP2NC DNA sequence of HIV-1 NL4-3 strain. The target gene was amplified by PCR and cloned into prokaryotic expression vector pTO-T7. Then the recombinant strain was transformed into Escherichia coli BL21 (DE3). IPTG induced protein expression, then the protein was purified by hydrophobic chromatography. SDS-PAGE and Western blotting were performed to analyze the target protein, and the biological activity of the antigen was identified through ELISA. The self-assembly of CAP2NC protein was analyzed by transmission electron microscopy and gel filtration chromatography. The protein had good reaction with the specific antibodies of p24 and formed different structures in various conditions. When 10% yeast RNA was added to the protein complex, the recombinant protein only formed into a tubular structure, which was similar to the self-assembled structure of the HIV-1 virus capsid. The results showed that the HIV-1 CAP2NC protein had in vitro self-assembly activity, and the RNA affected the structure of CAP2NC protein assembly. The protein can be used as a simple and effective molecular model to study its structure, and then it can provide a reference for the study of HIV immature virus particles.

Characterization and epitope mapping of a panel of monoclonal antibodies against HIV-1 matrix protein.

The HIV-1 Gag precursor protein (p55) is the main structural protein comprising the matrix (MA/p17), capsid (CA/p24), and nucleocapsid (NC/p7) proteins, and is uniquely responsible for virion assembly within the virus life cycle. The MA protein plays a critical role in plasma membrane targeting and envelope glycoprotein (Env) uptake during virion assembly. Yet, when viral infection occurs, the MA protein may also be involved in virion uncoating, dissociating from the plasma membrane, and participating in the nuclear importation process. Thus, the MA protein contains a reversibly membrane-binding signal and varied conformation to govern its subcellular localization and biological functions. However, these purported different conformations of the MA protein during assembly are poorly understood, especially in terms of its function as a component of the precursor protein. In this study, we characterized a panel of monoclonal antibodies against MA that showed discrete reactivity to p55, an intermediate (p41), and the final p17 mature form. We suggest that these antibodies could be used to track the different conformations of MA during the HIV-1 life cycle, particularly during HIV-1 assembly and maturation, and contribute to structure determination of MA or MA precursors. These antibodies would also have clinical value, including serving for therapeutic strategy to interfere AIDS progression, reagent in diagnostic kit for the detection of virion-free p17 or p17 derived from virion lysate.

Expression, Purification and Characterization of Hiv-1 Capsid Precursor Protein p41.

Human immunodeficiency virus type 1 (HIV-1) has been a global epidemic since 1983; yet, the virology and immunology related to HIV-1 remain elusive. Furthermore, as there is still no effective chemoprophylaxis or vaccine to treat patients with HIV-1, most research focuses on strategies to prevent HIV-1 infection, such as with antiviral drugs, novel therapeutics, or improved diagnostic kits. The HIV-1 Gag precursor protein (p55)-comprising the matrix (MA/p17), capsid (CA/p24), and nucleocapsid (NC/p7) protein domains-is the main structural HIV-1 protein, and is uniquely responsible for virion assembly within the virus life cycle. Recently, the immature and mature capsid structures were solved; however, the precursor protein structure is still unknown. Here, we expressed two subtypes of HIV-1 MA-CA stretch of the Gag protein, referred to as p41, in a bacterial expression system. We characterized the purified p41 protein, and showed its superior antigenicity over that of p24, highlighting the potential influence of the p17 domain on p24 structure. We further showed that p41 has good immunogenicity to induce an antibody response in mice. These results will aid future investigations into the HIV-1 capsid precursor structure, and potentially contribute to improving the design of diagnostic kits.

[Bipolar femoral head replacement combined with tension band wire fixation for intertrochanteric fracture in elderly osteoporotic patients].

OBJECTIVE: To explore the effectiveness of bipolar femoral head replacement combined with tension band wire fixation for intertrochanteric fracture in elderly osteoporotic patients. METHODS: Bipolar femoral head replacement combined with tension band wire fixation were used for intertrochanteric fracture in 48 elderly osteoporotic patients between January 2004 and December 2010. Of 48 patients, 15 were male and 33 were female, aged 90-99 years (mean, 94.1 years). All fractures were caused by falling, and pathological fracture was excluded. It was 2-7 days (mean, 4.2 days) from fracture to surgery. According to the Tronzo Evans classification, 25 cases were rated as type IV, 20 cases as type III, and 3 cases as type II. And all of the cases were accompanied with severe osteoporosis and accompanied by more than one medical diseases, and 10 cases had spinal compression fracture. RESULTS: All patients underwent the operation successfully. Six cases died of underlying medical illness within 2 years postoperatively. A total of 39 cases were followed up 2-7 years, averaged 3.1 years. After operation, short-term mental disorders occurred in 9 cases, suspected urinary tract infection in 2 cases, sacral rear bedsore in 1 case, hip pain in 1 case, thigh pain in 1 case, and deep vein thrombosis of affected limb in 1 case. All the incisions healed by first intension, and X-ray film showed bone union in all cases; no complications of bone osteolysis, prosthesis loosening, subsidence, rupture, and heterotopic ossification occured postoperatively. No case needed revision. According to the Harris score system, the results were excellent in 5 cases, good in 28 cases, fair in 5 cases, and poor in 1 case, with an excellent and good rate of 84.6%; the score at 2 years was significantly higher than that at 6 weeks (t = -14.79, P = 0.00). The physical health score and mental health score of SF-12 at 2 years postoperatively were significantly higher than those at 6 weeks postoperatively (P < 0.05). The visual analogue scale (VAS) scores at 6 weeks and 2 years postoperatively were significantly lower than those at preoperation (P < 0.05), and the score at 2 years was significantly lower than that at 6 weeks (P < 0.05). CONCLUSION: The bipolar femoral head replacement combined with tension band wire fixation for intertrochanteric fracture in elderly osteoporotic patients has the advantages of firm fixation, early function exercise with load bearing, pain relieving, improving hip function, and avoiding complication in bed.

AKR1B10 induces cell resistance to daunorubicin and idarubicin by reducing C13 ketonic group.

Daunorubicin, idarubicin, doxorubicin and epirubicin are anthracyclines widely used for the treatment of lymphoma, leukemia, and breast, lung, and liver cancers, but tumor resistance limits their clinical success. Aldo-keto reductase family 1 B10 (AKR1B10) is an NADPH-dependent enzyme overexpressed in liver and lung carcinomas. This study was aimed to determine the role of AKR1B10 in tumor resistance to anthracyclines. AKR1B10 activity toward anthracyclines was measured using recombinant protein. Cell resistance to anthracycline was determined by ectopic expression of AKR1B10 or inhibition by epalrestat. Results showed that AKR1B10 reduces C13-ketonic group on side chain of daunorubicin and idarubicin to hydroxyl forms. In vitro, AKR1B10 converted daunorubicin to daunorubicinol at V(max) of 837.42±81.39nmol/mg/min, K(m) of 9.317±2.25mM and k(cat)/K(m) of 3.24. AKR1B10 showed better catalytic efficiency toward idarubicin with V(max) at 460.23±28.12nmol/mg/min, K(m) at 0.461±0.09mM and k(cat)/K(m) at 35.94. AKR1B10 was less active toward doxorubicin and epirubicin with a C14-hydroxyl group. In living cells, AKR1B10 efficiently catalyzed reduction of daunorubicin (50nM) and idarubicin (30nM) to corresponding alcohols. Within 24h, approximately 20±2.7% of daunorubicin (1µM) or 23±2.3% of idarubicin (1µM) was converted to daunorubicinol or idarubicinol in AKR1B10 expression cells compared to 7±0.9% and 5±1.5% in vector control. AKR1B10 expression led to cell resistance to daunorubicin and idarubicin, but inhibitor epalrestat showed a synergistic role with these agents. Together our data suggest that AKR1B10 participates in cellular metabolism of daunorubicin and idarubicin, resulting in drug resistance. These data are informative for the clinical use of idarubicin and daunorubicin.