Expression and purification of recombinant human bone morphogenetic protein-7 in Escherichia coli.

Bone morphogenetic protein-7 (BMP-7) is a multifunctional cytokine of the transforming growth factor ß superfamily, which induces bone formation and plays an important role during bone tissue repair and embryonic development. In this study, human BMP-7 (hBMP-7) cDNA was cloned and expressed in Escherichia coli, and its yield was approximately 30% of the total bacterial protein. After the bacteria were lysed by ultrasonication and repeated washing, inclusion bodies were extracted and dissolved using a high-strength denaturant. The monomer of rhBMP-7 was purified by ion-exchange chromatography, and the purity coefficient was approximately 96%. The protein was renatured with refolding buffers at different pH values. The renatured rhBMP-7 dimer protein in this study increased the alkaline phosphatase activity of NIH3T3 cells. This study may be helpful for the in vitro production and biomedical application of rhBMP-7 protein expressed in an E. coli expression system.

Optimisation of reduction for prolapsed silicone tube after lacrimal intubation.

BACKGROUND: A common complication of bicanalicular intubation is dislocation of the silicone tube. METHODS: Eleven patients with prolapsed silicone tubes who had undergone bicanalicular nasal intubation were injected with a 2 per cent lidocaine solution to infiltrate the lacrimal duct mucosa. A memory wire probe was used to pull a 4-0 suture through the lacrimal passage retrogradely, allowing the suture to grab the silicone tube. Paraffin oil was applied to the contact part of the rope and the silicone tube, then the distal end of the silk thread was removed from the nostril until the tube was pulled into place. RESULTS: The prolapsed silicone tubes were restored by surgery in nine patients, with the drainage tube in the correct position in the eye and the lacrimal duct irrigation unobstructed. CONCLUSION: The optimisations made in this study are considered effective adjustments of reduction surgery for a prolapsed silicone tube.

Chip-capillary hybrid device for automated transfer of sample preseparated by capillary isoelectric focusing to parallel capillary gel electrophoresis for two-dimensional protein separation.

In this article, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to reroute the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed.

Enzymatically functionalized RGD-gelatin scaffolds that recruit host mesenchymal stem cells in vivo and promote bone regeneration.

Critical-size bone defects are imposing a substantial biomedical burden. Despite being long regarded as a potential approach to mitigate this burden or an alternative to bone grafts, bone tissue engineering (BTE) has virtually not proceeded to widespread clinical practices. In the BTE field, it is highly required to find a facile method to prepare active scaffolds with tailored biological functions. Here, we immobilized cell adhesive RGD motifs onto gelatin sponge (GS) scaffolds through enzymatic linking. On the basis of the resulting RGD-functionalized GS (RGD/GS) scaffolds, we developed a new and convenient strategy for bone defect repair, in which the scaffolds were first used to recruit mesenchymal stem cells (MSCs) from skeletal muscle, immediately followed by their engraftment into bone defect. We demonstrated significantly enhanced host cells homing into RGD/GS scaffolds as a result of specific RGD-integrin interactions, and the recruited host cells showed a strong osteogenic differentiation potential. After ectopic implantation of cell-laden RGD/GS scaffolds into critical-size mouse bone defects, marked bone tissue regeneration occurred. The presented strategy not only provides an agile route for the preparation of bioactive scaffolds and the construction of osteoinductive bone-graft substitutes, but also avoids or minimizes the complicated and laborious cell isolation, in vitro expansion and cell seeding procedures used in the conventional BTE.

Changes of tear film function after pterygium operation.

OBJECTIVE: To assess changes of tear film function in patients with pterygium after pterygium excision combined with limbal-conjunctival autograft transplantation. METHODS: Sixty eyes of 60 patients were entered in the study. The Schirmer test, tear breakup time (BUT) and mucus fern test (MFT) were evaluated in the patients before and after pterygium excision combined with limbal-conjunctival autograft transplantation. RESULTS: Compared with the opposite healthy eyes, the BUT and MFT in the eyes with pterygium were significantly different before the operation (p < 0.05); however, there was no significant difference in the results of the Schirmer test (p > 0.05). The results of the BUT and MFT in the eyes with pterygium were significantly different before and 4 weeks after the operation (p < 0.05). The BUT was prolonged from 9.89 ± 3.95 to 12.78 ± 4.12 s, and the ratio of normal crystallization in the MFT increased from 46.7 to 80.0%. There was no significant difference in the Schirmer test results before and after the operation (p > 0.05). CONCLUSION: Tear functions were abnormal in the eyes with pterygium. Pterygium excision combined with limbal-conjunctival autograft transplantation can partially restore the tear film function into normal state, and the tear film function was stable 4 weeks after surgery.

Muscle-derived stem cells: isolation, characterization, differentiation, and application in cell and gene therapy.

Muscle tissue represents an abundant, accessible, and replenishable source of adult stem cells for cell-based tissue and genetic engineering. A population of cells isolated from muscle exhibits both multipotentiality and self-renewal capabilities. Satellite cells, referred to by many investigators as muscle stem cells, are myogenic precursors that are capable of regenerating muscle and that demonstrate self-renewal properties; however, they are considered to be committed to the myogenic lineage. Muscle-derived stem cells (MDSCs), which may represent a predecessor of the satellite cell, are considered to possess a higher regeneration capacity and to exhibit better cell survival and a broader range of multilineage capabilities. Remarkably, MDSCs are not only able to differentiate into mesodermal cell types including the myogenic, adipogenic, osteogenic, chondrogenic, endothelial, and hematopoietic lineages, but also possess the potential to break germ layer commitment and differentiate into ectodermal lineages including neuron-like cells under certain conditions. This article reviews the current preclinical studies and potential clinical applications of MDSC-mediated gene therapy and tissue-engineering and methods for MDSC isolation, differentiation, and molecular characterization.

Human Bone Marrow Mesenchymal Stem Cells Modified Hybrid Baculovirus-Adeno-Associated Viral Vectors Targeting 131I Therapy of Hypopharyngeal Carcinoma.

Hypopharyngeal carcinoma is one of the most aggressive subtypes of squamous cell carcinoma of the head and neck. Although significant progress has been made in surgical techniques, radiotherapy, and chemotherapy, the prognosis is still poor. Mesenchymal stem cells (MSCs) have attracted substantial attention as tumor-targeted cellular carriers for cancer gene therapy. We have previously shown that recombinant baculovirus-adeno-associated vectors (BV-AAV) possessed high efficiency for multi-gene coexpression in human bone marrow MSCs (BMSCs) and BV-AAV-engineered BMSCs could effectively target hypopharyngeal cancer tissues in vivo. However, it was not clear whether BV-AAV-engineered BMSCs as cellular vehicles, mediating the expression of the sodium iodide symporter (NIS), would be effective in controlling the growth of hypopharyngeal carcinoma by radioiodine therapy. We constructed a hybrid BV-AAV containing the Luc-P2A-eGFP fusion or NIS sequence to modify BMSCs (BMSCs-Bac-Luc-P2A-eGFP or BMSCs-Bac-NIS). The 125I uptake of BMSCs-Bac-NIS was analyzed by an automatic gamma counter in vitro and micro-single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging in vivo. The value of radioiodine therapy for hypopharyngeal carcinoma was evaluated by measuring tumor volume, glucose metabolism (via 2-deoxy-2-[18F] glucose [18F-FDG] positron emission tomography/CT), and proliferation of tumor cells. We demonstrated that 125I uptake of BMSCs-Bac-NIS persists over long-term in vitro (at least 8 h). Radioactive uptake could be detected by SPECT/CT 1 h after 125I injection in the BMSCs-Bac-NIS group, showing that this strategy allows for the tracking of real-time migration and transgene expression of BMSCs. Radioiodine therapy resulted in a significant reduction in tumor growth (386.93 ± 249.23 mm3 vs 816.56 ± 213.87 mm3 in controls), increased survival, and decreased SUVmax of 18F-FDG. The hybrid BV-AAV that can provide a variety of genes and regulatory elements, as a novel gene therapy strategy opens the prospect of NIS-mediated radionuclide therapy of hypopharyngeal carcinoma after MSC-mediated gene delivery.

A LiPO2F2/LiPF6 dual-salt electrolyte enabled stable cycling performance of nickel-rich lithium ion batteries.

In this work, a dual-lithium salt was proposed for constructing an electrolyte for high energy density lithium ion batteries. LiPO2F2 was composed with traditional LiPF6 to enhance the high voltage performance of the electrolyte. The electrochemical performance of the NCM811/Li cells with LiPO2F2/LiPF6 dual-lithium salt at 2.8-4.5 V was investigated. It was found that the dual-lithium salt can inhibit the oxidative decomposition of the electrolyte, suppress the dissolution of the transition metal ions in the electrode material, and reduce the side reaction between the transition metal ions and the electrolyte. We believe that the strategy of a dual-lithium salt electrolyte may provide a new idea to stabilize the electrode/electrolyte interface at high voltage, which is very important for developing high energy density batteries.

A prediction model of outcome of SARS-CoV-2 pneumonia based on laboratory findings.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in thousands of deaths in the world. Information about prediction model of prognosis of SARS-CoV-2 infection is scarce. We used machine learning for processing laboratory findings of 110 patients with SARS-CoV-2 pneumonia (including 51 non-survivors and 59 discharged patients). The maximum relevance minimum redundancy (mRMR) algorithm and the least absolute shrinkage and selection operator logistic regression model were used for selection of laboratory features. Seven laboratory features selected in the model were: prothrombin activity, urea, white blood cell, interleukin-2 receptor, indirect bilirubin, myoglobin, and fibrinogen degradation products. The signature constructed using the seven features had 98% [93%, 100%] sensitivity and 91% [84%, 99%] specificity in predicting outcome of SARS-CoV-2 pneumonia. Thus it is feasible to establish an accurate prediction model of outcome of SARS-CoV-2 pneumonia based on laboratory findings.

Nanocapillaries for open tubular chromatographic separations of proteins in femtoliter to picoliter samples.

We have recently examined the potential of bare nanocapillaries for free solution DNA separations and demonstrated efficiencies exceeding 10(6) theoretical plates/m. In the present work, we demonstrate the use of bare and hydroxypropylcellulose (HPC) coated open tubular nanocapillaries for protein separations. Using 1.5 microm inner diameter (i.d.) capillary columns, hydrodynamically injecting femto- to picoliter volumes of fluorescent or fluorescent dye labeled protein samples, utilizing a pneumatically pressurized chamber containing 1.0 mM sodium tetraborate solution eluent (typically 200 psi) as the pump, and performing on-column detection using a simple laser-induced fluorescence detector, we demonstrate efficiencies of close to a million theoretical plates/m while generating single digit microliter volumes of waste for a complete chromatographic run. We achieve baseline resolution for a protein mixture consisting of transferrin, alpha-lactalbumin, insulin, and alpha-2-macroglobulin.

Electroosmotic pumps for microflow analysis.

With rapid development in microflow analysis, electroosmotic pumps are receiving increasing attention. Compared to other micropumps, electroosmotic pumps have several unique features. For example, they are bi-directional, can generate constant and pulse-free flows with flow rates well suited to microanalytical systems, and can be readily integrated with lab-on-chip devices. The magnitude and the direction of flow of an electroosmotic pump can be changed instantly. In addition, electroosmotic pumps have no moving parts. In this article, we discuss common features, introduce fabrication technologies and highlight applications of electroosmotic pumps.

Cam-based vibration-counter-balanced laser-induced fluorescence scanner for multiplexed capillary detection.

Laser-induced fluorescence (LIF) rotary scanners have been successfully used for multiplexed capillary detection. However, these scanners have a limitation that the capillaries have to be assembled in a circular format, which can be inconvenient for certain applications. A linear LIF scanner works well for flat parallel capillary arrays, but motor accelerations/decelerations (for direction changes) and scanning head vibrations introduce high instrumental noises. The number of capillaries that can be scanned by a linear scanner is limited because of the above constraints. We have constructed a cam-based scanner in an attempt to address these issues. A cam-based scanner eliminates the motor accelerations/decelerations but not the scanning head vibrations. In this work, we attach a second scanning head to the cam on the opposite side of the first scanning head to counter-balance the mechanical vibrations. With this modification, we improve the limit of detection by more than 3 times (from 69 pM to 20 pM fluorescein). We also increase the capillary number capacity by more than 6 times; the total number of capillaries that can be scanned is 426 if 150-µm-o.d. capillaries are used or 320 if 200-µm-o.d. capillaries are used. To demonstrate the utility of this instrument, we assemble a 99-capillary array on one capillary holder and perform capillary electrophoresis of two fluorescent dyes; separations in all capillaries are successfully monitored simultaneously. We also apply it for detecting fluorescently labeled proteins resolved by 24 s-dimension capillaries in a chip-capillary hybrid device; two-dimensional separation results are nicely produced.

Human Bone Marrow Mesenchymal Stem Cells Functionalized by Hybrid Baculovirus-Adeno-Associated Viral Vectors for Targeting Hypopharyngeal Carcinoma.

Hypopharyngeal carcinoma is a common malignant tumor of the head and neck with a very poor prognosis; the median survival time for curatively treated patients was 17.2 months in India. However, cell-based gene therapy holds promise to improve patient outcomes. In this study, we investigated whether human bone marrow mesenchymal stem cells (BMSCs) possess potential homing capacity for hypopharyngeal carcinoma. To monitor the efficiency of BMSC transplantation therapy through reporter gene imaging, we employed a hybrid baculovirus vector containing the Luc-P2A-eGFP fusion or sodium iodide symporter (NIS) sequence under the control of the cytomegalovirus promoter. To enhance the transfection efficiency, baculovirus vectors (Bac-CMV-Luc-P2A-eGFP-ITR and Bac-CMV-NIS-ITR) were flanked by inverted terminal repeats (ITRs), which are key elements of adeno-associated viruses. The infection efficiency of Bac-CMV-Luc-P2A-eGFP-ITR in BMSCs was as high as 92.84 ± 1.14% with no obvious toxic effects at a multiplicity of infection of 400. Moreover, Bac-CMV-NIS-ITR-infected BMSCs showed highly efficient radioactive iodide (125I) uptake; these high uptake levels were maintained for at least 2 h. Transwell migration assays further demonstrated the chemotaxis of BMSCs to hypopharyngeal carcinoma cells (FaDu cells) in vitro. BMSCs modified by firefly luciferase report gene or NIS were injected into nude mice with hypopharyngeal carcinoma, and changes in the localization of the BMSCs were successfully tracked with bioluminescent imaging and micro-single-photon emission computed tomography imaging. These data indicate the potential utility of BMSCs as a promising targeted-delivery vehicle for hypopharyngeal carcinoma gene therapy. Importantly, BMSCs may represent a promising targeting vector for general tumor radionuclide therapy.

Success rates of in vitro fertilization versus intracytoplasmic sperm injection in men with serum anti-sperm antibodies: a consecutive cohort study.

Antisperm antibodies (ASAs) are assumed to be a possible causative factor for male infertility, with ASAs detected in 5%-15% of infertile men but in only 1%-2% of fertile ones. It remains unclear whether ASAs have an adverse effect on the outcome of in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). This study investigated differences in the rates of fertilization, pregnancy, and live births associated with serum ASA-positive and ASA-negative men following IVF or ICSI. Five hundred and fifty-four consecutive infertile couples undergoing IVF (n = 399) or ICSI (n = 155) were included. The two-sample two-sided t-test and Chi-square or Fisher's exact test was used for statistical analysis. Lower rates of fertilization (41.7% vs 54.8%, P = 0.03), good embryos (18.9% vs 35.2%, P = 0.00), pregnancy (38.5% vs 59.4%, P = 0.00), and live births (25.8% vs 42.5%, P = 0.00) were observed in men of the IVF group with a positive serum ASA than in those with a negative ASA. ASA positivity/negativity correlated with pregnancy rates (P = 0.021, odds ratio [OR]: 0.630, 95% confidence interval [CI]: 0.425-0.932) and live birth rates (P = 0.010, OR: 1.409, 95% CI: 1.084-1.831) after controlling for the female serum follicle-stimulating hormone level and the couple's ages at IVF. Women coupled with ASA-positive men had lower live birth rates with IVF than with ICSI (25.8% and 47.4%, respectively; P = 0.07). Women coupled with ASA-positive men had lower rates of pregnancy and live births following IVF than those coupled with ASA-negative men but had a similar outcome with ICSI.

Bare nanocapillary for DNA separation and genotyping analysis in gel-free solutions without application of external electric field.

In this work, we demonstrate DNA separation and genotyping analysis in gel-free solutions using a nanocapillary under pressure-driven conditions without application of an external electric field. The nanocapillary is a approximately 50-cm-long and 500-nm-radius bare fused-silica capillary. After a DNA sample is injected, the analytes are eluted out in a chromatographic separation format. The elution order of DNA molecules follows strictly with their sizes, with the longer DNA being eluted out faster than the shorter ones. High resolutions are obtained for both short (a few bases) and long (tens of thousands of base pairs) DNA fragments. Effects of key experimental parameters, such as eluent composition and elution pressure, on separation efficiency and resolution are investigated. We also apply this technique for DNA separations of real-world genotyping samples to demonstrate its feasibility in biological applications. PCR products (without any purification) amplified from Arabidopsis plant genomic DNA crude preparations are directly injected into the nanocapillary, and PCR-amplified DNA fragments are well resolved, allowing for unambiguous identification of samples from heterozygous and homozygous individuals. Since the capillaries used to conduct the separations are uncoated, column lifetime is virtually unlimited. The only material that is consumed in these assays is the eluent, and hence, the operation cost is low.

Chromatographic separations in a nanocapillary under pressure-driven conditions.

We report a unique property of nanocapillaries for chromatographic separations of ionic species. Due to the electric double layer overlap, ions are unevenly distributed inside a nanochannel, with counterions enriched near the wall and co-ions concentrated in the middle of the channel. As a pressure-driven flow is induced, the co-ions will move faster than the counterions. This differential transport results in a chromatographic separation. In this work, we introduce the fundamental mechanism of this separation technology and demonstrate its application for DNA separations. An outstanding feature of this technique is that each separation consumes less than 1 pL sample and generates less than 0.1 nL waste. We also apply this technique for separations of DNA molecules, and efficiencies of more than 1,00000 plates per meter are obtained.

Efficient transduction of spiral ganglion neurons in vitro by baculovirus vectors.

Degeneration of peripheral auditory neurons constitutes one of the main causes of sensorineural hearing loss. Gene delivery to the inner ear is central to the development of gene therapy for hearing impairment. Thus we investigated the effectiveness of baculovirus-derived vectors to transduce spiral ganglion neurons. We found that baculovirus could efficiently transduce spiral ganglion neurons in vitro and that the highest transduced cell rate could be over 75%. The level of transgene expression exhibited viral dose dependence and was enhanced by the addition of butyrate. Thus, baculovirus is a novel and promising tool for gene transfer into the cochlear nervous system, both in studies of the function of foreign genes and in the development of gene-therapy strategies.

Extracranial head and neck schwannomas: a clinical analysis of 33 patients.

OBJECTIVE: To study the clinical features, diagnosis, and management of the extracranial head and neck schwannomas. STUDY DESIGN: Retrospective study. METHODS: The clinical data of 33 patients with schwannoma of the head and neck from 1996 to 2006 were studied retrospectively. RESULTS: Extracranial head and neck schwannomas usually presented as solitary and well-demarcated lesions with insidious course. Although benign, the lesion can cause secondary symptoms, such as nasal obstruction, dysphasia, and hoarseness, relevant to location of the lesion. Fine needle aspiration cytology, computed tomography scans, and magnetic resonance imaging may provide limited implications in the diagnosis of schwannomas, whereas postoperative pathologic examination establishes the final diagnosis. CONCLUSIONS: Complete surgical excisions with appropriate approaches have proven to be efficient and successful in the treatment of head and neck schwannomas.

Study of recombinant human osteogenic protein-1 expressed in prokaryocyte on the repair of extracted socket in rabbits.

The study investigated the effect of recombinant human osteogenic protein-1 (rhOP-1) expressed in prokaryocyte, to promote the healing of alveolar socket. A model of rabbit extracted socket into which the composites of rhOP-1 and gelatin sponge was immediately implanted was created and the osteoinduction of rhOP-1 was assessed by histological method, quantitative measurement of calcium content and alkaline phosphatase (ALP) activity. The result of histology showed that bone healing in rhOP-1 side is 4-6 weeks earlier than that of the control side. ALP activity and calcium content in rhOP-1 side were significantly high compared with that of the control side. rhOP-1 has a satisfactory osteoinduction ability to promote the healing of extracted socket.