Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation

Topics overview: What’s Metafluidics; How to solve the Cpf1 function limitation in BV3L6 and Lachnospiraceae bacterium ND2006; The breakthrough of single-cell genome sequencing to large cell populations; Bacteria can be engineered to function as diagnostics or therapeutics in the mammalian gut; TACCA was applied to produce a high-quality (N50 of 9.76 Mb) de novo chromosome assembly of the wheat line CH Campala Lr22a in only 4 months.

1.  Open-source, community-driven microfluidics with Metafluidics.

Microfluidic devices have the potential to automate and miniaturize biological experiments, but open-source sharing of device designs has lagged behind sharing of other resources such as software. Synthetic biologists have used microfluidics for DNA assembly, cell-free expression, and cell culture, but a combination of expense, device complexity, and reliance on custom set-ups hampers their widespread adoption. David S Kong at Massachusetts Institute of Technology Lincoln Laboratory in Massachusetts, USA and his colleagues present Metafluidics, an open-source, community-driven repository that hosts digital design files, assembly specifications, and open-source software to enable users to build, configure, and operate a microfluidic device. They use Metafluidics to share designs and fabrication instructions for both a microfluidic ring-mixer device and a 32-channel tabletop microfluidic controller. This device and controller are applied to build genetic circuits using standard DNA assembly methods including ligation, Gateway, Gibson, and Golden Gate. Metafluidics is intended to enable a broad community of engineers, DIY enthusiasts, and other nontraditional participants with limited fabrication skills to contribute to microfluidic research.

Read more, please click http://www.nature.com/nbt/journal/v35/n6/full/nbt.3873.html

2. Engineered Cpf1 variants with altered PAM specificities.

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, Linyi Gao at Broad Institute of MIT and Harvard in Massachusetts, USA and his colleagues performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. They engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which they further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ~11 bp.

Read more, please click http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3900.html

3. Single-cell genome sequencing at ultra-high-throughput with microfluidic droplet barcoding.

The application of single-cell genome sequencing to large cell populations has been hindered by technical challenges in isolating single cells during genome preparation. Here Freeman Lan at University of California in California, USA and his colleagues present single-cell genomic sequencing (SiC-seq), which uses droplet microfluidics to isolate, fragment, and barcode the genomes of single cells, followed by Illumina sequencing of pooled DNA. They demonstrate ultra-high-throughput sequencing of >50,000 cells per run in a synthetic community of Gram-negative and Gram-positive bacteria and fungi. The sequenced genomes can be sorted in silico based on characteristic sequences. They use this approach to analyze the distributions of antibiotic-resistance genes, virulence factors, and phage sequences in microbial communities from an environmental sample. The ability to routinely sequence large populations of single cells will enable the de-convolution of genetic heterogeneity in diverse cell populations.

Read more, please click http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3880.html

4. Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation.

Bacteria can be engineered to function as diagnostics or therapeutics in the mammalian gut but commercial translation of technologies to accomplish this has been hindered by the susceptibility of synthetic genetic circuits to mutation and unpredictable function during extended gut colonization. Here, David T Riglar at Harvard Medical School in Massachusetts, USA and his colleagues report stable, engineered bacterial strains that maintain their function for 6 months in the mouse gut. They engineered a commensal murine Escherichia coli strain to detect tetrathionate, which is produced during inflammation. Using their engineered diagnostic strain, which retains memory of exposure in the gut for analysis by fecal testing, they detected tetrathionate in both infection-induced and genetic mouse models of inflammation over 6 months. The synthetic genetic circuits in the engineered strain were genetically stable and functioned as intended over time. The durable performance of these strains confirms the potential of engineered bacteria as living diagnostics.

Read more, please click http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3879.html

5. Rapid cloning of genes in hexaploid wheat using cultivar-specific long-range chromosome assembly.

Cereal crops such as wheat and maize have large repeat-rich genomes that make cloning of individual genes challenging. Moreover, gene order and gene sequences often differ substantially between cultivars of the same crop species. A major bottleneck for gene cloning in cereals is the generation of high-quality sequence information from a cultivar of interest. In order to accelerate gene cloning from any cropping line, Anupriya Kaur Thind at University of Zurich in Zurich, Switzerl and and his colleagues report ‘targeted chromosome-based cloning via long-range assembly’ (TACCA). TACCA combines lossless genome-complexity reduction via chromosome flow sorting with Chicago long-range linkage to assemble complex genomes. They applied TACCA to produce a high-quality (N50 of 9.76 Mb) de novo chromosome assembly of the wheat line CH Campala Lr22a in only 4 months. Using this assembly they cloned the broad-spectrum Lr22a leaf-rust resistance gene, using molecular marker information and ethyl methanesulfonate (EMS) mutants, and found that Lr22a encodes an intracellular immune receptor homologous to the Arabidopsis thaliana RPM1 protein.

Read more, please click http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3877.html

Anti-Plant Actin Mouse Monoclonal Antibody (3T3) becomes the latest addition to the Abbkine family

Abbkine Scientific Co. Ltd is known for making quality life science products and tools and it recently announced the official launch of its new antibody – the Anti-Plant Actin Mouse Monoclonal Antibody (3T3).

The antibody otherwise known as AT3G12110 antibody is a Plant Actin Antibody, which is an essential component of cell cytoskeleton. The substance also plays a critical role in the streaming of cytoplasmic, determination of cell shape, cell division and extension growth.

The product is available in a liquid solution and hosted by mouse hence, Plant Actin Mouse mAb. The antibody is also a Recombinant Protein immunogen, with plant reactivity. The antibody like many of its other counterparts is affinity-purified from mouse ascites using specific immunogen by affinity-chromatography.

The Anti-Plant Actin Mouse Monoclonal Antibody (3T3) is made solely for research purpose and not intended for clinical or human use. It can also be stored for as long as one year at -20°C from date of shipment.

Optimal working dilutions for the Anti-Plant Actin Mouse Monoclonal Antibody (3T3) should be determined by the investigator after experiments. However, the suggested starting dilutions are WB 1:2000-5000.

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About Abbkine Scientific

Abbkine Scientific Company Limited is a life science research company headquartered in California. Founded in 2012, the establishment has been able to spread its tentacles across the globe with increasing presence and acceptance from Asia Pacific thanks to its continuous efforts to make the world a better place.

Abbkine combines cutting edge technology with manufacturing engineering and cost advantage to provide innovative, high-quality assay kits and other research and scientific products enhance life science fundamental research and drug discovery amongst others.

TTR Mouse Monoclonal Antibody(1D7) Review

Transthyretin (TTR) is a transport protein in the serum and cerebrospinal fluid that carries the thyroid hormone thyroxine (T4) and retinol-binding protein bound to retinol. The liver secretes transthyretin into the blood, and the choroid plexus secretes TTR into the cerebrospinal fluid. More than 80 different mutations in TTR have been reported; most mutations are related to amyloid deposition, affecting predominantly peripheral nerve and/or the heart, and a small portion of the gene mutations is non-amyloidogenic. The diseases caused by mutations include amyloidotic polyneuropathy, euthyroid hyperthyroxinaemia, amyloidotic vitreous opacities, cardiomyopathy, oculoleptomeningeal amyloidosis, meningocerebrovascular amyloidosis, carpal tunnel syndrome, etc.

Abbkine TTR Mouse Monoclonal Antibody(1D7) was affinity-purified from mouse ascites by affinity-chromatography using TTR recombinant protein antigen. This antibody could be applied to IF, IHC-p, WB, which has been validated. This antibody can react with Human samples. Optimal working dilutions should be determined experimentally by researchers. Abbkine suggest that the starting dilutions are: WB: 1:500-2000, IHC-p: 1:50-2000.

There are a lot of antibody brands in the market, it maybe a little difficult for a beginner to choose an appropriate antibody for his experiment. Hope my experience could help you. I used many antibodies from different firms. Through the comparison, and I think Abbkine is a good selection, which has high quality with lower price, as well as perfect protein specificity and strong technical support. Abbkine provide the primary antibodies that involve many fields. I bought some antibodies to do IHC experiments, including TTR Mouse Monoclonal Antibody(1D7), and they both make good performances. You could save the costs along with satisfactory results.

FH Monoclonal Antibody Review

The FH gene provides instructions for making an enzyme called fumarase (also known as fumarate hydratase). Fumarase participates in an important series of reactions known as the citric acid cycle or Krebs cycle, which allows cells to use oxygen and generate energy. Specifically, fumarase helps convert a molecule called fumarate to a molecule called malate. Mutations in FH can cause fumarase deficiency and lead to progressive encephalopathy.

Abbkine FH Monoclonal Antibody was affinity-purified from mouse ascites by affinity-chromatography using specific immunogen. The antibody has been validated for WB, IF, IHC-P and tested in Human, Mouse and Rat. Abbkine suggested the starting dilutions are as follows: WB: 1:3000, IF, IHC-P: 1:200. The optimal working dilutions should be determined experimentally by users.

Immunofluorescence image of Abbkine FH Monoclonal Antibody (ABM40073) stained human liver cancer tissue sections. The sections were incubated with ABM40073 at 5µg/ml over night at 4°C. The secondary antibody was (red) IFKine red  anti-mouse IgG. DAPI was used to stain the cell nuclei (blue). Target protein signal was strong.

Immune checkpoint inhibitors have emerged as a potent new class of anticancer therapy

Topics overview: Immune checkpoint inhibitors have emerged as a potent new class of anticancer therapy, learned carry-over effects generalize over time and across routes of drug administration—independent of conscious expectations, Infection with a mutant parasite with impaired hemoglobin digestion that produces little hemozoin, a major Plasmodium by-product, did not cause bone loss, proteasome variations influence T cell repertoire selection and may contribute to the difference in individual susceptibility to autoimmunity,  pre-DC compartment contains distinct lineage-committed subpopulations.

1. Combined immune checkpoint blockade as a therapeutic strategy for BRCA1-mutated breast cancer.

Immune checkpoint inhibitors have emerged as a potent new class of anticancer therapy. They have changed the treatment landscape for a range of tumors, particularly those with a high mutational load. To date, however, modest results have been observed in breast cancer, where tumors are rarely hypermutated. Because BRCA1-associated tumors frequently exhibit a triple-negative phenotype with extensive lymphocyte infiltration, Emma Nolan at Walter and Eliza Hall Institute of Medical Research in Victoria, Australia and her colleagues explored their mutational load, immune profile, and response to checkpoint inhibition in a Brca1-deficient tumor model. BRCA1-mutated triple-negative breast cancers (TNBCs) exhibited an increased somatic mutational load and greater numbers of tumor-infiltrating lymphocytes, with increased expression of immunomodulatory genes including PDCD1 (PD-1) and CTLA4, when compared to TNBCs from BRCA1–wild-type patients. Cisplatin treatment combined with dual anti–programmed death-1 and anti–cytotoxic T lymphocyte–associated antigen 4 therapy substantially augmented antitumor immunity in Brca1-deficient mice, resulting in an avid systemic and intratumoral immune response. This response involved enhanced dendritic cell activation, reduced suppressive FOXP3+ regulatory T cells, and concomitant increase in the activation of tumor-infiltrating cytotoxic CD8+ and CD4+ T cells, characterized by the induction of polyfunctional cytokine-producing T cells. Dual (but not single) checkpoint blockade together with cisplatin profoundly attenuated the growth of Brca1-deficient tumors in vivo and improved survival. These findings provide a rationale for clinical studies of combined immune checkpoint blockade in BRCA1-associated TNBC.

Read more, please click http://stm.sciencemag.org/content/9/393/eaal4922

2. The effects of treatment failure generalize across different routes of drug administration.

 Failure of medical treatments can hamper responses to subsequent treatments. It has been suggested that changing the route of drug administration could reduce such negative carry-over effects, but direct evidence for this approach is lacking. Matthias Zunhammer at University Hospital Essen in Essen, Germany and his colleagues therefore investigated in 211 healthy volunteers whether changes in drug administration route reduce such carry-over effects. A positive or negative treatment history with topical analgesic treatments was induced experimentally in a mock clinical trial setting. Subsequently, a different inert drug was introduced via the same (topical) or another (oral) route of administration and its analgesic efficacy was tested. Changing the route of drug administration induced expectations of positive treatment effects in the subjects but did not actually counteract the negative carry-over effects on treatment efficacy. These findings indicate that learned carry-over effects generalize over time and across routes of drug administration—independent of conscious expectations. Other strategies are needed to prevent negative carry-over effects of treatment failure from influencing the results of subsequent treatment attempts.

Read more, please click http://stm.sciencemag.org/content/9/393/eaal2999

3. Plasmodium products persist in the bone marrow and promote chronic bone loss.

Although malaria is a life-threatening disease with severe complications, most people develop partial immunity and suffer from mild symptoms. However, incomplete recovery from infection causes chronic illness, and little is known of the potential outcomes of this chronicity. Michelle S. J. Lee at Immunology Frontier Research Center (IFReC), Osaka University in Osaka, Japan and his colleagues found that malaria causes bone loss and growth retardation as a result of chronic bone inflammation induced by Plasmodium products. Acute malaria infection severely suppresses bone homeostasis, but sustained accumulation of Plasmodium products in the bone marrow niche induces MyD88-dependent inflammatory responses in osteoclast and osteoblast precursors, leading to increased RANKL expression and overstimulation of osteoclastogenesis, favoring bone resorption. Infection with a mutant parasite with impaired hemoglobin digestion that produces little hemozoin, a major Plasmodium by-product, did not cause bone loss. Supplementation of alfacalcidol, a vitamin D3 analog, could prevent the bone loss. These results highlight the risk of bone loss in malaria-infected patients and the potential benefits of coupling bone therapy with antimalarial treatment.

Read more, please click http://immunology.sciencemag.org/content/2/12/eaam8093

4. Human thymoproteasome variations influence CD8 T cell selection.

The proteasome is a multi-subunit protease complex essential for housekeeping protein degradation and the production of the major histocompatibility complex (MHC) class I-bound antigen peptides that are essential for recognition by CD8 T cells. MHC variations dramatically contribute to T cell selection and autoimmunity, but genetic variations of peptide processing machinery including proteasome genes have been poorly explored in this context. In the computational analysis of human proteasome gene variation, Takeshi Nitta at University of Tokyo in Tokyo, Japan and his colleagues documented that PSMB11 was highly enriched for nucleotide changes that interfere with protein function. This gene encodes β5t, a thymus-specific catalytic subunit that regulates positive selection of CD8 T cells by producing a distinct set of MHC class I-bound peptides. The introduction of PSMB11 variations into the mouse genome by genome-editing revealed that these variations impaired the development of CD8 T cells in vivo. One of the PSMB11 polymorphisms altered the CD8 T cell repertoire in mice and was associated with a higher risk of an autoimmune disease in humans. Their findings suggest that, in addition to the MHC haplotype, proteasome variations influence T cell repertoire selection and may contribute to the difference in individual susceptibility to autoimmunity.

Read more, please click http://immunology.sciencemag.org/content/2/12/eaan5165

5. Mapping the human DC lineage through the integration of high-dimensional techniques.

Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises two main functionally specialized lineages, whose origins and differentiation pathways remain incompletely defined. Here, Peter See at Singapore Immunology Network (SIgN) in Singapore and his colleagues combine two high-dimensional technologies—single-cell messenger RNA sequencing (scmRNAseq) and cytometry by time-of-flight (CyTOF)—to identify human blood CD123+CD33+CD45RA+ DC precursors (pre-DC). Pre-DC share surface markers with plasmacytoid DC (pDC) but have distinct functional properties that were previously attributed to pDC. Tracing the differentiation of DC from the bone marrow to the peripheral blood revealed that the pre-DC compartment contains distinct lineage-committed subpopulations, including one early uncommitted CD123 high pre-DC subset and two CD45RA+CD123low lineage-committed subsets exhibiting functional differences. The discovery of multiple committed pre-DC populations opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting.

Read more, please click http://science.sciencemag.org/content/356/6342/eaag3009

 

Abbkine Scientific adds PurKine™ His-Tag Ni-Super Resin to the its product list

Wuhan, China. 430074, 15^th June 2017. Abbkine Scientific has added the PurKine™ His-Tag Ni-Super Resin to its long list of high-quality scientific research product. Also known as the His tag resin, the product is made using the latest synthetic process of resin production, combined with cutting-edge techniques for the resin development and accessories optimization.

The product is created to simplify and enhance the process of purification, based on innovative high-capacity IMAC matrix, ensuring single-step purification of His-tag proteins from total lysates. The Ni-Super resin as it is also known is effective for purifying his-tag proteins from samples that can cause Ni stripping from the medium, such as secreted proteins in liquids containing chelators.

The resin comes with two distinct benefits compared to Ni-NTA and Ni-IDA, which are high tolerance and easy cleaning. Unlike other resins, the product is more tolerable to various chemicals including reducing, chelating agents, with easy cleaning using NaOH.

The resin comes in a liquid solution and is available in multiple formats including bulk resin, spin columns and complete kits, allowing for flexibility. In addition to its easy cleaning feature, the product is also versatile and cost-effective, showing no decrease in performance after about five repeated uses of the same batch of resin.

The Ni-Super resin is for research use only and is not intended for use in human or clinical diagnosis.

About Abbkine Scientific Co. Ltd

Abbkine Scientific Co. Ltd is headquartered in California. The life science research company was founded in 2012 and has subsequently reached the rest of the world, with a wide range of high quality science products and services.

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Abbkine Scientific combines cutting edge technology with manufacturing engineering and cost advantage, providing high-quality and innovative assay kits and other research and scientific products designed to enhance life science fundamental research and drug discovery amongst others.

CD10 Monoclonal Antibody review

CD10 is a cell surface enzyme with neutral metalloendopeptidase activity. CD10 is also known as CALLA (common acute lymphocytic leukemia antigen). It serves as a marker for the common form of ALL (acute lymphocytic leukemia) as well as for Burkitt lymphoma and follicular germinal center lymphoma. CD10/CALLA is normally present on the surface of early lymphoid cells as well as on a number of other types of normal cells, such as especially cells in the kidney.

Abbkine CD10 Monoclonal Antibody was affinity-purified from mouse ascites by affinity-chromatography using specific peptide to detect endogenous CD10 proteins. The antibody has been validated for IHC-P, IF and tested in Human, Mouse and Rat. Supplied in liquid solution, the concentration of the antibody is 1mg/ml. The antibody is for research use only.

To validate the staining in rat kidney tissue sections, we chose Abbkine CD10 Monoclonal Antibody as the primary antibody. A goat polyclonal to mouse IgG1 was used as the secondary antibody. A positive result was found and no non-specific background. Negative control was used by secondary antibody only. I was happy with the result. I found it to be worth the cost and plan to use them again.