The different effects of molybdate on Hg(II) bio-methylation in aerobic and anaerobic bacteria.

In nature, methylmercury (MeHg) is primarily generated through microbial metabolism, and the ability of bacteria to methylate Hg(II) depends on both bacterial properties and environmental factors. It is widely known that, as a metabolic analog, molybdate can inhibit the sulfate reduction process and affect the growth and methylation of sulfate-reducing bacteria (SRB). However, after it enters the cell, molybdate can be involved in various intracellular metabolic pathways as a molybdenum cofactor; whether fluctuations in its concentration affect the growth and methylation of aerobic mercury methylating strains remains unknown. To address this gap, aerobic γ-Proteobacteria strains Raoultella terrigena TGRB3 (B3) and Pseudomonas putida TGRB4 (B4), as well as an obligate anaerobic δ-Proteobacteria strain of the SRB Desulfomicrobium escambiense CGMCC 1.3481 (DE), were used as experimental strains. The growth and methylation ability of each strain were analyzed under conditions of 500 ng·L-1 Hg(II), 0 and 21% of oxygen, and 0, 0.25, 0.50, and 1 mM of MoO4 2-. In addition, in order to explore the metabolic specificity of aerobic strains, transcriptomic data of the facultative mercury-methylated strain B3 were further analyzed in an aerobic mercuric environment. The results indicated that: (a) molybdate significantly inhibited the growth of DE, while B3 and B4 exhibited normal growth. (b) Under anaerobic conditions, in DE, the MeHg content decreased significantly with increasing molybdate concentration, while in B3, MeHg production was unaffected. Furthermore, under aerobic conditions, the MeHg productions of B3 and B4 were not influenced by the molybdate concentration. (c) The transcriptomic analysis showed several genes that were annotated as members of the molybdenum oxidoreductase family of B3 and that exhibited significant differential expression. These findings suggest that the differential expression of molybdenum-binding proteins might be related to their involvement in energy metabolism pathways that utilize nitrate and dimethyl sulfoxide as electron acceptors. Aerobic bacteria, such as B3 and B4, might possess distinct Hg(II) biotransformation pathways from anaerobic SRB, rendering their growth and biomethylation abilities unaffected by molybdate.

Embryo Microinjection for Transgenesis in Drosophila.

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope. Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

CRISPR-Based Modular Assembly for High-Throughput Construction of a UAS-cDNA/ORF Plasmid Library.

Functional genomics screening offers a powerful approach to probe gene function and relies on the construction of genome-wide plasmid libraries. Conventional approaches for plasmid library construction are time-consuming and laborious. Therefore, we recently developed a simple and efficient method, CRISPR-based modular assembly (CRISPRmass), for high-throughput construction of a genome-wide upstream activating sequence-complementary DNA/open reading frame (UAS-cDNA/ORF) plasmid library. Here, we present a protocol for CRISPRmass, taking as an example the construction of a GAL4/UAS-based UAS-cDNA/ORF plasmid library. The protocol includes massively parallel two-step test tube reactions followed by bacterial transformation. The first step is to linearize the existing complementary DNA (cDNA) or open reading frame (ORF) cDNA or ORF library plasmids by cutting the shared upstream vector sequences adjacent to the 5' end of cDNAs or ORFs using CRISPR/Cas9 together with single guide RNA (sgRNA), and the second step is to insert a UAS module into the linearized cDNA or ORF plasmids using a single step reaction. CRISPRmass allows the simple, fast, efficient, and cost-effective construction of various plasmid libraries. The UAS-cDNA/ORF plasmid library can be utilized for gain-of-function screening in cultured cells and for constructing a genome-wide transgenic UAS-cDNA/ORF library in Drosophila.

Video feedback combined with peer role-playing: a method to improve the teaching effect of medical undergraduates.

OBJECTIVE: The purpose of this study was to investigate the effectiveness of implementation of video feedback combined with peer role-playing (PRP) teaching method in medical undergraduates adopting problem-based learning (PBL) teaching mode. METHODS: The undergraduates of five-year clinical medicine who get enrollment of Wuhan local University from 2016 and 2018 were selected to be the research objects. The same grade level is randomly divided into several groups to carry out PBL, with 6-10 students in each group. Following the principle of voluntary participation, 34 students were enrolled in the study group and 33 students in the control group finally. The research regards group as the unit, and study report in group should be carried out to fulfill the research. In the study group, the students were asked to perform PRP report, and the report videos were used for feedback. At the same time, the control group reported by PPT, and the feedback was carried out according to the PPT. At the end of the study, the "Competency Improvement Satisfaction Questionnaire (CISQ)" was distributed to investigate students' satisfaction with this teaching method to improve their ability, Arizona Clinical Interview Score (ACIR) was administered in Chinese by a trained teacher unrelated using PRP method to assess students' clinical inquiry ability and communication skills, and theory test was performed to assess mastery of theoretical knowledge. RESULTS: The results show that the study group is superior to the control group in improving the interest of learning and the ability of independent learning, interpersonal communication and active problem solving. Although it is in terms of the confidence in becoming a real doctor and the ability of teamwork, language expression, clinical thinking cultivated, active knowledge acquired and understood that study group are better than the control group, the difference was not statistically significant. ACIR shows that the study group is significantly better than the control group in organization, timeline planning, and transition statements, openly questioning, smooth progress, and avoiding repetition, summarizing, understandable language, documentation and total score. There is no significant difference in eye contact and no interruption. The differences between the two groups are not statistically significant in terms of responsing to concerns, positive feedback, and additional questions. The theoretical test scores of the study group are significantly higher than those of the control group. CONCLUSION: Video feedback combined with peer role-playing teaching method implemented in medical undergraduates adopting PBL teaching mode is effective, it could stimulate interest in learning actively, improve interpersonal communication ability, improve learning efficiency and clinical knowledge and skills, and improve the confidence of becoming a real doctor. It is worthy of further research and promotion.

Study on the Gas Release of 3D-Printed Furan Resin Sand Core during the Casting Process.

In sand casting, gas porosity is a common defect that can result in decreased strength, leakage, rough surfaces, or other problems. Although the forming mechanism is very complicated, gas release from sand cores is often a significant contributor to the formation of gas porosity defects. Therefore, studying the gas release behavior of sand cores is crucial to solving this problem. Current research on the gas release behavior of sand cores mainly focuses on parameters such as gas permeability and gas generation properties, through experimental measurement and numerical simulation methods. However, accurately reflecting the gas generation situation in the actual casting process is difficult, and there are certain limitations. To achieve the actual casting condition, a sand core was designed and enclosed inside a casting. The core print was extended to the sand mold surface, with two types of core prints: hollow and dense. Pressure and airflow speed sensors were installed on the exposed surface of the core print to investigate the burn-off of the binder of the 3D-printed furan resin quartz sand cores. The experimental results showed that the gas generation rate was high in the initial stage of the burn-off process. The gas pressure quickly reached its peak in the initial stage and then decreased rapidly. The exhaust speed of the dense type of core print was 1 m/s, lasting for 500 s. The pressure peak of the hollow-type sand core was 1.09 kPa, and the exhaust speed peak was 1.89 m/s. The binder can be sufficiently burned off for the location surrounding the casting and the crack-affected area, so the burnt sand appears white, while the burnt core appears black due to insufficient burning of the binder because of isolation from the air. The gas generated by the burnt resin sand in contact with air was 30.7% less than that generated by the burnt resin sand insulated from the air.

A fluorescent microsphere-based immunochromatographic strip is effective for quantitative fecal blood testing in colorectal cancer screening.

Background: Colorectal cancer (CRC) represents a major health concern that can be screened for by the fecal immunochemical test (FIT), which detects blood in the stool. CRC detection sensitivity for hemoglobin (Hb) combined with transferrin (Tf) is higher than for hemoglobin alone. Methods: We developed a europium fluorescent microsphere-based quantitative lateral flow immunochromatography strip to detect fecal Hb and Tf. Performance was tested using fecal samples from 51 patients with CRC and 122 normal subjects. Test strips were generated using paired mouse anti-human Hb and mouse anti-human Tf monoclonal antibodies and tested using standard Hb and Tf samples. Fluorescence was observed at 365 nm and quantitatively measured using a portable fluorescent strip reader. Results: At cutoff values of 100 ng/mL (10 µg/g feces) and 25 ng/mL (2.5 µg/g feces) for Hb and Tf, respectively, the positive rates for Hb, Tf, and Hb+Tf in normal subjects were 6.56%, 5.74%, and 10.66%, respectively, compared to 88.24%, 64.71%, and 94.12% in patients with CRC. The sensitivity and specificity of the FIT combined detection technique were 87.5% and 89.2%, respectively, and the area under the curve (AUC) was 0.92. The sensitivity, specificity, and AUC for the Tf assay were 63.8%, 68.4%, and 0.759, respectively, and those for Hb testing were 69.7%, 70.2%, and 0.774, respectively. The AUC for Hb+Tf was significantly higher than those for Tf or Hb alone (P < 0.001). Conclusions: Fluorescent microsphere-based immunochromatographic strips sensitively detect fecal Hb and Tf, and sensitivity and specificity are improved for Hb+Tf. This system represents a rapid and portable alternative for on-site early CRC screening.

Involvement of a FAD-Linked Oxidase RSc0454 for Expression of the Type III Secretion System and Pathogenicity in Ralstonia solanacearum.

Ralstonia solanacearum RSc0454 is predicted as a FAD-linked oxidase based on protein homologies, while it contains distinct domains of lactate dehydrogenase and succinate dehydrogenase. A previous study demonstrated that RSc0454 exhibits lactate dehydrogenase activity using pyruvate and NADH as substrates, and is essential for pathogenicity of R. solanacearum. Here, we genetically characterized involvement of RSc0454 on bacterial growth and expression of genes for the type III secretion system (T3SS, a pathogenicity determinant) in R. solanacearum. The RSc0454 mutant grew normally in rich medium but grew faintly in host plants, and failed to grow in minimal medium. Supplementary succinate but not lactate could substantially restore some phenotypes of RSc0454 mutants, including faint growth in host plants, diminished growth in the minimal medium, and lost pathogenicity toward host plants. Expression of T3SS genes is directly controlled by a master regulator, HrpB, and hrpB expression is positively regulated by HrpG and PrhG in parallel ways. Deletion of RSc0454 substantially reduced expression levels of hrpB and T3SS both in vitro and in planta. Moreover, RSc0454 is revealed to be required for the T3SS expression via HrpG and PrhG, although through some novel pathway, and impaired expression of these genes was not due to growth deficiency of RSc0454 mutants. RSc0454 is suggested to be important for redox balance inside cells, and supplementary NADH partially restored diminished growth of the RSc0454 mutant in the minimal medium only in the presence of succinate at some moderate concentrations, indicating that the unbalanced redox in the RSc0454 mutant might be responsible for its diminished growth in the minimal medium. Taken together, these results provide novel insights into the understanding of various biological functions of this FAD-linked oxidase RSc0454 and involvement of the redox balance on expression of the T3SS in R. solanacearum.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

O-GlcNAcylation of TDP-43 suppresses proteinopathies and promotes TDP-43's mRNA splicing activity.

Pathological TDP-43 aggregation is characteristic of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP); however, how TDP-43 aggregation and function are regulated remain poorly understood. Here, we show that O-GlcNAc transferase OGT-mediated O-GlcNAcylation of TDP-43 suppresses ALS-associated proteinopathies and promotes TDP-43's splicing function. Biochemical and cell-based assays indicate that OGT's catalytic activity suppresses TDP-43 aggregation and hyperphosphorylation, whereas abolishment of TDP-43 O-GlcNAcylation impairs its RNA splicing activity. We further show that TDP-43 mutations in the O-GlcNAcylation sites improve locomotion defects of larvae and adult flies and extend adult life spans, following TDP-43 overexpression in Drosophila motor neurons. We finally demonstrate that O-GlcNAcylation of TDP-43 promotes proper splicing of many mRNAs, including STMN2, which is required for normal axonal outgrowth and regeneration. Our findings suggest that O-GlcNAcylation might be a target for the treatment of TDP-43-linked pathogenesis.

Human-Induced Pluripotent Stem Cell Culture Methods Under cGMP Conditions.

The discovery of induced pluripotent stem cells (iPSCs) revolutionized the approach to cell therapy in regenerative medicine. Reprogramming of somatic cells into an embryonic-like pluripotent state provides an invaluable resource of patient-specific cells of any lineage. Implementation of procedures and protocols adapted to current good manufacturing practice (cGMP) requirements is critical to ensure robust and consistent high-quality iPSC manufacturing. The technology developed at Allele Biotechnology for iPSC generation under cGMP conditions is a powerful platform for derivation of pluripotent stem cells through a footprint-free, feeder-free, and xeno-free reprogramming method. The cGMP process established by Allele Biotechnology entails fully cGMP compliant iPSC lines where the entire manufacturing process, from tissue collection, cell reprogramming, cell expansion, cell banking and quality control testing are adopted. Previously, we described in this series of publications how to create iPSCs using mRNA only, and how to do so under cGMP conditions. In this article, we describe in detail how to culture, examine and storage cGMP-iPSCs using reagents, materials and equipment compliant with cGMP standards. © 2020 The Authors. Basic Protocol 1: iPSC Dissociation Support Protocol 1: Stem cell media Support Protocol 2: ROCK inhibitor preparation Support Protocol 3: Vitronectin coating Basic Protocol 2: iPSC Cryopreservation Basic Protocol 3: iPSC Thawing.

Study on the Directional Solidification Process of an Aluminum Alloy Bar in Multishell Mold Being Gradually Immersed in Water.

A multishell mold structure and water-immersion cooling method (MSMWI) is proposed for the directional solidification of castings. A four-layer-shell sand mold was designed for a bar with diameter of 40 mm. As the aluminum melt was poured, the multishell mold was gradually immersed in water, and the water level drove the advancement of the solidification front from bottom to top. The multishell mold was helpful for the heat insulation of its upper part, and its bottom was chilled by the water. Therefore, directional solidification of the bar was vertically realized. The water-cooled solidification process of the bar was 5.8 times faster than that by air natural cooling (MSMNC), and the temperature gradient was increased by 78 times. The secondary dendrite arm spacing (SDAS) and eutectic silicon were significantly refined. Its tensile strength, elongation, and hardness were increased by 56%, 185%, and 62.6%, respectively.

Modeling and Simulation of the Casting Process with Skeletal Sand Mold.

The author-proposed skeletal sand mold, which mainly includes a shell, air cavities and a truss support structure, has been experimentally proven to be very useful in controlling the cooling of casting at local areas and at different periods of the casting process. The modeling and simulation of the casting process using a skeletal sand mold were systemically analyzed. Complicated casting/mold and mold/air boundaries, and the thermal and mechanical behavior of the skeletal sand mold during the casting process were highlighted. A numerical simulation of the casting process of a stress frame specimen using a skeletal sand mold was performed. The temperature, stress and displacement fields of the casting and skeletal sand mold were obtained and compared with those using a traditional sand mold. The simulated results were validated with experiments. Using the skeletal sand mold, the cooling rate of the casting can be greatly improved due to the significant heat release from mold surface to environment. The residual stress and deformation of the casting can be reduced because of the decreased stiffness of this kind of mold. Although the skeletal sand mold is susceptible to cracking, it can be avoided by filleting in the conjunctions and increasing the shell thickness.

Machine Learning and Feature Selection Applied to SEER Data to Reliably Assess Thyroid Cancer Prognosis.

Utilizing historical clinical datasets to guide future treatment choices is beneficial for patients and physicians. Machine learning and feature selection algorithms (namely, Fisher's discriminant ratio, Kruskal-Wallis' analysis, and Relief-F) have been combined in this research to analyse a SEER database containing clinical features from de-identified thyroid cancer patients. The data covered 34 unique clinical variables such as patients' age at diagnosis or information regarding lymph nodes, which were employed to build various novel classifiers to distinguish patients that lived for over 10 years since diagnosis, from those who did not survive at least five years. By properly optimizing supervised neural networks, specifically multilayer perceptrons, using data from large groups of thyroid cancer patients (between 6,756 and 20,344 for different models), we demonstrate that unspecialized and existing medical recording can be reliably turned into power of prediction to help doctors make informed and optimized treatment decisions, as distinguishing patients in terms of prognosis has been achieved with 94.5% accuracy. We also envisage the potential of applying our machine learning strategy to other diseases and purposes such as in designing clinical trials for unmasking the maximum benefits and minimizing risks associated with new drug candidates on given populations.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster.

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

CRISPR-based modular assembly of a UAS-cDNA/ORF plasmid library for more than 5500 Drosophila genes conserved in humans.

Construction of a genome-wide transgenic UAS-cDNA/ORF library in Drosophila based on the binary GAL4/UAS system has been severely hampered by technical difficulties, although genome-wide cDNA or ORF resources of Drosophila, human, and mouse have been publicly available for more than a decade. Here, we developed a new method named CRISPR-based modular assembly (CRISPRmass) for the high-throughput construction of a genome-wide UAS-cDNA/ORF library from publicly available cDNA/ORF resources. Through cleavage of shared vector sequences of cDNA/ORF plasmids by CRISPR/Cas9 and subsequent insertion of UAS modules by Gibson assembly, the procedure of construction of such a library by CRISPRmass is standardized as massively parallel two-step test tube reactions before bacterial transformation. Using CRISPRmass, we generated 5551 UAS-cDNA/ORF constructs covering 83% of the Drosophila genes conserved in humans in the Drosophila Genomics Resource Center (DGRC) Gold Collection, and among them, 5518 were generated within 3 mo by three people. Our results show that CRISPRmass allows modulization, simplicity, efficiency, and adaptability in the generation of a genome-wide UAS-cDNA/ORF plasmid library by using publicly available cDNA/ORF resources. CRISPRmass can be applied to editing various genome-wide libraries in general and is an alternative to Gateway technology in high-throughput plasmid library editing. Furthermore, the more than 5500 UAS-cDNA/ORF plasmids of Drosophila genes serve as a powerful resource for gain-of-function (GOF) screening in cultured cells and for generation of a transgenic UAS-cDNA/ORF library in Drosophila.

Long Non-Coding RNAs (lncRNAs) Tumor-Suppressive Role of lncRNA on Chromosome 8p12 (TSLNC8) Inhibits Tumor Metastasis and Promotes Apoptosis by Regulating Interleukin 6 (IL-6)/Signal Transducer and Activator of Transcription 3 (STAT3)/Hypoxia-Inducible Factor 1-alpha (HIF-1α) Signaling Pathway in Non-Small Cell Lung Cancer.

BACKGROUND Long non-coding RNAs (lncRNAs) exert various functions in human cancers. However, the biological functions of lncRNAs in non-small cell lung cancer (NSCLC) are unknown. In the present study we investigated the tumor-suppressive role of lncRNA on chromosome 8p12 (TSLNC8) in the pathogenesis and progression of NSCLC. MATERIAL AND METHODS qRT-PCR was carried out to evaluate the expression of TSLNC8 in lung cancer cell lines. The effects of TSLNC8 on A549 cells proliferation, migration, and invasion were analyzed using CCK-8 assay, wound healing assay, Transwell assay, and Western blot analysis. We used flow cytometry to assess cell apoptosis, and cell autophagy was assessed by Western blot analysis and immunofluorescence staining. Levels of proteins in the IL-6/STAT3/HIF-1alpha pathway were measured by Western blot analysis. RESULTS The results revealed that TSLNC8 was significantly downregulated in lung cancer cells compared to normal bronchial epithelial cells. Further experiments showed that overexpression of TSLNC8 in A549 cells significantly inhibited proliferation in a time-dependent manner and promoted cell apoptosis. We found that TSLNC8 overexpression suppressed cell migration and invasion, and upregulation of TSLNC8 regulated the protein levels of Beclin-1, p62, ATG14, and LC3-II and inhibited the IL-6/STAT3/HIF-1alpha signaling pathway. CONCLUSIONS lncRNA TSLNC8 remarkably inhibited the proliferation and migration and accelerated apoptosis of lung cancer cells by targeting the IL-6/STAT3/HIF-1alpha signaling pathway. TSLNC8 may be a potential therapeutic target for the diagnosis and treatment of NSCLC.

RNF34 modulates the mitochondrial biogenesis and exercise capacity in muscle and lipid metabolism through ubiquitination of PGC-1 in Drosophila.

The transcriptional co-activator PGC-1α is a key regulator of mitochondrial function and muscle fiber specification in the skeletal muscle. The E3 ubiquitin ligase RNF34 ubiquitinates PGC-1α and negatively regulates mammalian brown fat cell metabolism. However, the functional importance of RNF34 in the skeletal muscle and its impact on energy metabolism remain unknown. The Drosophila PGC-1 homolog dPGC-1 and its mammalian counterparts have conserved functions in mitochondria and insulin signaling. Here, we showed that the Drosophila RNF34 (dRNF34) ubiquitinates the Drosophila PGC-1α (dPGC-1) and promotes its degradation in HEK293T cells by immunoprecipitation and western blot analysis. This allows us to use Drosophila as a powerful model system to study the physiological role of RNF34 in mitochondrial function and metabolism. In the in vivo studies, by separately expressing two independent UAS-dRNF34 RNAi transgenes driven by the muscle-specific 24B-Gal4 driver, we found that knockdown of dRNF34 specifically in muscle promotes mitochondrial biogenesis, improves negative geotaxis, extends climbing time to exhaustion in moderate aged flies and counteracts high-fat-diet-induced high triglyceride content. Furthermore, we showed that knockdown of dPGC-1 reversed the effects of the dRNF34 knockdown phenotypes described above. Our results reveal that dRNF34 plays an important role in regulating mitochondrial biogenesis in muscle and lipid metabolism through dPGC-1. Thus, inhibition of RNF34 activity provides a potential novel therapeutic strategy for the treatment of age-related muscle dysfunction.

Modified Nanoantibodies Increase Sensitivity in Avidin-Biotin Immunohistochemistry.

Revealing the spatial arrangement of molecules within a tissue through immunohistochemistry (IHC) is an invaluable tool in biomedical research and clinical diagnostics. Choosing both the appropriate antibody and amplification system is paramount to the pathologic interpretation of the tissue at hand. The use of single domain VHH nanoantibodies (nAbs) promise more robust and consistent results in IHC, but are rarely used as an alternative to conventional immunoglobulin G (IgG) antibodies. nAbs are originally obtained from llamas and are the smallest antigen-binding fragments available. To determine whether the unique biophysical properties of nAbs give them an advantage in IHC, we first compared a basic fibroblast growth factor nAb to polyclonal IgG antibodies using tissue isolated from pancreatic adenocarcinoma. The nAb was extremely effective in antigen signal detection and allowed for a more streamlined and reproducible protocol. Furthermore, because nAbs are expressed in Escherichia coli from a single gene, they are quite amenable to genetic engineering. As such, we then covalently bound a highly biotinylated amplifier protein to basic fibroblast growth factor and p16 nAbs (termed nAb Plus), resulting in improved IHC sensitivity. The use of a biotinylated nAb Plus not only achieved local, covalent signal amplification, but also eliminated the need for a secondary antibody and subsequent amplification steps. These results highlight nAbs as valuable alternatives to conventional IgG antibodies, decreasing overall processing time and costs of reagents while increasing sensitivity and reproducibility across individual IHC assays.

cGMP Generation of Human Induced Pluripotent Stem Cells with Messenger RNA.

Reprogramming somatic cells to generate induced pluripotent stem cells (iPSCs) has presented the biomedical community with a powerful platform to develop new models for human disease. To fully realize the promise of this technology in cell therapy and regenerative medicine, creating iPSCs under current Good Manufacture Practice (cGMP) conditions is paramount. Some reports have described efforts in this regard, resulting in iPSC lines that are cGMP compliant. The technology developed at Allele Biotechnology for footprint-free, feeder-free, and xeno-free reprogramming using only mRNA is very suitable for creating iPSC lines through an established cGMP process. This technology has resulted in a licensing agreement between Allele Biotechnology and Ocata (formerly ACT, now a wholly owned division of Astellas) for clinical applications. All reagents and vessels are certified as cGMP-produced, all equipment and software are certifiable, and all procedures are carried out in Industry ISO 7 or Class 10,000-grade cleanrooms. In this revised version of the unit, we describe the core improvements to implement steps toward cGMP-compliant generation of iPSCs. Recreating a process close to cGMP production in academic research will make these findings more applicable to translational research. © 2016 by John Wiley & Sons, Inc.

The effects of land use change and precipitation change on direct runoff in Wei River watershed, China.

The principles and degrees to which land use change and climate change affect direct runoff generation are distinctive. In this paper, based on the MODIS data of land use in 1992 and 2003, the impacts of land use and climate change are explored using the Soil Conservation Service Curve Number (SCS-CN) method under two defined scenarios. In the first scenario, the precipitation is assumed to be constant, and thus the consequence of land use change could be evaluated. In the second scenario, the condition of land use is assumed to be constant, so the influence only induced by climate change could be assessed. Combining the conclusions of two scenarios, the effects of land use and climate change on direct runoff volume can be separated. At last, it is concluded: for the study basin, the land use types which have the greatest effect on direct runoff generation are agricultural land and water body. For the big sub basins, the effect of land use change is generally larger than that of climate change; for middle and small sub basins, most of them suffer more from land use change than from climate change.

Scalable generation of universal platelets from human induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid "surge" capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the ß2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.