Publications by authors named "Michael R Green"

275 Publications

Cloning Polymerase Chain Reaction (PCR) Products: Blunt-End Cloning.

Cold Spring Harb Protoc 2021 Apr 1;2021(4):pdb.prot101287. Epub 2021 Apr 1.

The following is an elegant and simple protocol for generating and cloning blunt-ended DNA. Incubation of a ligation reaction in the presence of an excess amount of restriction enzyme can dramatically increase the yield of recombinant plasmids. The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are regenerated when plasmid molecules ligate to themselves. The method requires that ligation of the plasmid to a target DNA molecule destroys the restriction site, so preventing the restriction enzyme from digesting recombinants generated during the ligation reaction. The net effect of constant reclamation of unit-length linear vector molecules is to drive the equilibrium of the ligation reaction strongly in favor of recombinants between vector and insert. The method is efficient because regeneration of vector DNA, ligation, and polishing the termini of PCR-generated fragments of DNA all occur simultaneously in the same reaction mixture.
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http://dx.doi.org/10.1101/pdb.prot101287DOI Listing
April 2021

Cloning Polymerase Chain Reaction Products: Addition of Restriction Sites to the Termini of Amplified DNA.

Cold Spring Harb Protoc 2021 Apr 1;2021(4):pdb.prot101279. Epub 2021 Apr 1.

To generate polymerase chain reaction (PCR) products that can be directionally cloned into a vector, different restriction sites are built into the forward and reverse primers that are used in the PCR. After PCR, the amplified product is purified, cleaved with the appropriate restriction enzymes, ligated into a vector with compatible cohesive ends, and used to transform .
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http://dx.doi.org/10.1101/pdb.prot101279DOI Listing
April 2021

Subtype-specific and co-occurring genetic alterations in B-cell non-Hodgkin lymphoma.

Haematologica 2021 Apr 1. Epub 2021 Apr 1.

Department of Lymphoma and Myeloma, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Genomic Medicine, University of Texas MD Anderson Cancer Center, Houston, TX, USA; Center for Cancer Epigenetics, University of Texas MD Anderson Cancer Center, Houston, TX.

B-cell non-Hodgkin's lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL such as diffuse large B-cell lymphoma (DLBCL) have been comprehensively interrogated at the genomic level. But rarer subtypes such as mantle cell lymphoma (MCL) remain sparsely characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth; including DLBCL, MCL, follicular lymphoma (FL), and Burkitt lymphoma (BL). We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumor from each subtype, including the frequent genetic deregulation of the ubiquitin proteasome system (UPS). In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.
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http://dx.doi.org/10.3324/haematol.2020.274258DOI Listing
April 2021

BET proteolysis targeted chimera-based therapy of novel models of Richter Transformation-diffuse large B-cell lymphoma.

Leukemia 2021 Mar 2. Epub 2021 Mar 2.

The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.

Richter Transformation (RT) develops in CLL as an aggressive, therapy-resistant, diffuse large B cell lymphoma (RT-DLBCL), commonly clonally-related (CLR) to the concomitant CLL. Lack of available pre-clinical human models has hampered the development of novel therapies for RT-DLBCL. Here, we report the profiles of genetic alterations, chromatin accessibility and active enhancers, gene-expressions and anti-lymphoma drug-sensitivity of three newly established, patient-derived, xenograft (PDX) models of RT-DLBCLs, including CLR and clonally-unrelated (CLUR) to concomitant CLL. The CLR and CLUR RT-DLBCL cells display active enhancers, higher single-cell RNA-Seq-determined mRNA, and protein expressions of IRF4, TCF4, and BCL2, as well as increased sensitivity to BET protein inhibitors. CRISPR knockout of IRF4 attenuated c-Myc levels and increased sensitivity to a BET protein inhibitor. Co-treatment with BET inhibitor or BET-PROTAC and ibrutinib or venetoclax exerted synergistic in vitro lethality in the RT-DLBCL cells. Finally, as compared to each agent alone, combination therapy with BET-PROTAC and venetoclax significantly reduced lymphoma burden and improved survival of immune-depleted mice engrafted with CLR-RT-DLBCL. These findings highlight a novel, potentially effective therapy for RT-DLBCL.
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http://dx.doi.org/10.1038/s41375-021-01181-wDOI Listing
March 2021

The serine hydroxymethyltransferase-2 (SHMT2) initiates lymphoma development through epigenetic tumor suppressor silencing.

Nat Cancer 2020 22;1:653-664. Epub 2020 Jun 22.

Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

Cancer cells adapt their metabolic activities to support growth and proliferation. However, increased activity of metabolic enzymes is not usually considered an initiating event in the malignant process. Here, we investigate the possible role of the enzyme serine hydroxymethyltransferase-2 (SHMT2) in lymphoma initiation. localizes to the most frequent region of copy number gains at chromosome 12q14.1 in lymphoma. Elevated expression of cooperates with in lymphoma development; loss or inhibition of impairs lymphoma cell survival. SHMT2 catalyzes the conversion of serine to glycine and produces an activated one-carbon unit that can be used to support -adenosyl methionine synthesis. SHMT2 induces changes in DNA and histone methylation patterns leading to promoter silencing of previously uncharacterized mutational genes, such as and Together, our findings reveal that amplification of in cooperation with is sufficient in the initiation of lymphomagenesis through epigenetic tumor suppressor silencing.
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http://dx.doi.org/10.1038/s43018-020-0080-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872152PMC
June 2020

Restriction Enzymes.

Cold Spring Harb Protoc 2021 Apr 1;2021(4):pdb.top101360. Epub 2021 Apr 1.

Restriction enzymes provided the foundation on which molecular cloning was built, and they remain as essential tools in current recombinant DNA technology. The three classes of restriction enzymes and their features are introduced here.
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http://dx.doi.org/10.1101/pdb.top101360DOI Listing
April 2021

Prognostic Impact of Corticosteroids on Efficacy of Chimeric Antigen Receptor T-cell Therapy in Large B-cell Lymphoma.

Blood 2021 Feb 3. Epub 2021 Feb 3.

The University of Texas MD Anderson Cancer Center, Houston, Texas, United States.

Corticosteroids are commonly used for the management of severe toxicities associated with chimeric antigen receptor (CAR) T-cell therapy. However, it remains unclear whether their dose, duration, and timing may impact clinical efficacy. Here, we determined the impact of corticosteroids on clinical outcomes in patients with relapsed or refractory large B-cell lymphoma treated with standard of care anti-CD19 CAR T-cell therapy. Among 100 patients evaluated, 60 (60%) received corticosteroids for management of CAR T-cell therapy-associated toxicities. The median cumulative dexamethasone-equivalent dose was 186 mg (range, 8-1803 mg) and the median duration of corticosteroid treatment was 9 days (range 1-30). Corticosteroid treatment was started between days 0 and 7 in 45 (75%) patients and beyond day 7 in 15 (25%). After a median follow-up of 10 months (95% CI 8-12 months), use of higher cumulative dose of corticosteroids was associated with significantly shorter progression-free survival. More importantly, higher cumulative dose of corticosteroids, and prolonged and early use after CAR T-cell infusion were associated with significantly shorter overall survival. These results suggest that corticosteroids should be used at the lowest dose and for the shortest duration and their initiation should be delayed whenever clinically feasible, while managing CAR T-cell therapy-associated toxicities.
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http://dx.doi.org/10.1182/blood.2020008865DOI Listing
February 2021

Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension.

Cold Spring Harb Protoc 2021 02 1;2021(2). Epub 2021 Feb 1.

In this procedure, synthesis of cDNA is performed in the presence of saturating concentrations of all four dNTPs and trace amounts of a single radiolabeled dNTP. After subtraction hybridization, the enriched single-stranded cDNA is radiolabeled to high specific activity in a second synthetic reaction by extension of random oligonucleotide primers using the Klenow fragment of DNA Pol I. Because the concentrations of dNTP in the first reaction are nonlimiting, both the amounts and size of cDNA generated are greater than those achieved in standard labeling protocols. The subtractive hybridization step can therefore be performed with higher efficiency. Because the resulting population of cDNA is not vulnerable to radiolytic cleavage, it can be stored indefinitely and radiolabeled to higher specific activity when needed. The protocol works best when the cDNA synthesized in the initial synthetic reaction is full length or close to it. For this reason, synthesis of cDNAs is primed by oligo(dT) rather than random hexanucleotide primers. In contrast, the subsequent radiolabeling reaction is primed by random oligonucleotides, yielding shorter DNA products whose size is ideal for hybridization.
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http://dx.doi.org/10.1101/pdb.prot100644DOI Listing
February 2021

Prostaglandin E2 stimulates cAMP signaling and resensitizes human leukemia cells to glucocorticoid-induced cell death.

Blood 2021 Jan;137(4):500-512

Department of Molecular, Cell, and Cancer Biology.

Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.
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http://dx.doi.org/10.1182/blood.2020005712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845005PMC
January 2021

Northern Hybridization.

Cold Spring Harb Protoc 2021 01 4;2021(1). Epub 2021 Jan 4.

RNA samples that have been transferred and fixed to a membrane may be hybridized with a specific probe to locate the RNA species of interest. Any one of a large number of methods can be used to label and detect probes, at the discretion of the investigator. After treating the membrane with blocking agents that suppress nonspecific absorption of the probe, the membrane is incubated under conditions that favor hybridization of the labeled probe to the immobilized target RNA. The membrane is then washed extensively to remove adventitiously bound probe and finally manipulated to yield an image of the distribution of the tightly bound probe on the membrane. After analysis of the results, the probe may be stripped from the membrane, and the membrane can then be used again in another hybridization experiment.
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http://dx.doi.org/10.1101/pdb.prot101790DOI Listing
January 2021

Transfer and Fixation of Denatured RNA in Polyacrylamide Gels to Membranes by Electrophoretic Transfer.

Cold Spring Harb Protoc 2021 01 4;2021(1). Epub 2021 Jan 4.

Polyacrylamide gels, unlike agarose gels, have a highly cross-linked matrix that does not permit efficient, quantitative, or reproducible transfer by passive diffusion. Polyacrylamide gels should therefore be transferred by electroblotting (also known as "electrophoretic transfer"). Because this approach requires the use of a transfer buffer of low ionic strength, nylon membranes are generally used. The membrane is placed directly on the gel, and the gel together with its membrane is then sandwiched between layers of filter paper and placed in a special cassette that, in turn, is placed in a tank of buffer; the cassette is oriented such that the membrane side of the gel faces the positively charged anode. A voltage gradient is applied perpendicular to the direction of the initial electrophoresis of the gel, forcing the sample to migrate out of the gel and onto the membrane.
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http://dx.doi.org/10.1101/pdb.prot101782DOI Listing
January 2021

Separation of RNA according to Size: Electrophoresis of RNA through Denaturing Urea Polyacrylamide Gels.

Cold Spring Harb Protoc 2021 01 4;2021(1). Epub 2021 Jan 4.

Thin (0.4-1.5 mm) polyacrylamide-urea gels provide high resolution of RNAs up to 1000 nt in size and are capable of resolving single-stranded fragments of RNA that differ in length by as little as 1 nt. The polyacrylamide gel is cast between two glass plates that are separated by two thin Teflon or nylon spacers. A so-called shark's tooth comb or, less frequently, a standard slotted comb forms the sample wells into which the RNA samples are loaded before electrophoresis. In contrast to electrophoresis using agarose gels, which occurs while the gel is horizontal, polyacrylamide gels are run while in the vertical position. Gels are also typically run at 45°C-55°C, which is the melting temperature of RNA, and in the presence of 6-8 m urea. The gel recipe and protocol presented here for 8 m urea/TBE polyacrylamide gels can be used for a variety of applications including mapping RNA with nuclease S1, ribonuclease protection assay, or analysis of RNA by primer extension.
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http://dx.doi.org/10.1101/pdb.prot101766DOI Listing
January 2021

Harnessing lymphoma epigenetics to improve therapies (article not eligible for CME credit).

Hematology Am Soc Hematol Educ Program 2020 Dec;2020(1):95-100

Department of Lymphoma/Myeloma and.

Affinity maturation and terminal differentiation of B cells via the germinal center reaction is a complex multistep process controlled by transcription factors that induce or suppress large dynamic transcriptional programs. This occurs via the recruitment of coactivator or corepressor complexes that epigenetically regulate gene expression by post-translationally modifying histones and/or remodeling chromatin structure. B-cell-intrinsic developmental programs both regulate and respond to interactions with other cells in the germinal center that provide survival and differentiation signals, such as T-follicular helper cells and follicular dendritic cells. Epigenetic and transcriptional programs that naturally occur during B-cell development are hijacked in B-cell lymphoma by genetic alterations that directly or indirectly change the function of transcription factors and/or chromatin-modifying genes. These in turn skew differentiation toward the tumor cell of origin and alter interactions between lymphoma B cells and other cells within the microenvironment. Understanding the mechanisms by which genetic alterations perturb epigenetic and transcriptional programs regulating B-cell development and immune interactions may identify opportunities to target these programs using epigenetic-modifying agents. Here, we discuss recently published studies centered on follicular lymphoma and diffuse large B-cell lymphoma within the context of prior knowledge, and we highlight how these insights have informed potential avenues for rational therapeutic interventions.
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http://dx.doi.org/10.1182/hematology.2020006908DOI Listing
December 2020

Harnessing lymphoma epigenetics to improve therapies.

Hematology Am Soc Hematol Educ Program 2020 12;2020(1):95-100

Department of Lymphoma/Myeloma and.

Affinity maturation and terminal differentiation of B cells via the germinal center reaction is a complex multistep process controlled by transcription factors that induce or suppress large dynamic transcriptional programs. This occurs via the recruitment of coactivator or corepressor complexes that epigenetically regulate gene expression by post-translationally modifying histones and/or remodeling chromatin structure. B-cell-intrinsic developmental programs both regulate and respond to interactions with other cells in the germinal center that provide survival and differentiation signals, such as T-follicular helper cells and follicular dendritic cells. Epigenetic and transcriptional programs that naturally occur during B-cell development are hijacked in B-cell lymphoma by genetic alterations that directly or indirectly change the function of transcription factors and/or chromatin-modifying genes. These in turn skew differentiation toward the tumor cell of origin and alter interactions between lymphoma B cells and other cells within the microenvironment. Understanding the mechanisms by which genetic alterations perturb epigenetic and transcriptional programs regulating B-cell development and immune interactions may identify opportunities to target these programs using epigenetic-modifying agents. Here, we discuss recently published studies centered on follicular lymphoma and diffuse large B-cell lymphoma within the context of prior knowledge, and we highlight how these insights have informed potential avenues for rational therapeutic interventions.
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http://dx.doi.org/10.1182/hematology.2020006908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7727577PMC
December 2020

Autoradiography and Phosphorimaging.

Cold Spring Harb Protoc 2020 12 1;2020(12). Epub 2020 Dec 1.

Many of the commonly used techniques in molecular cloning depend on methods to map accurately the distribution of radioactive atoms on two-dimensional (2D) surfaces. Without this ability, methods such as Southern blotting, northern hybridizations, radiolabeled DNA sequencing, and library screening would not have been possible. In the 1970s and 1980s-the pioneering days of molecular cloning-imaging of 2D surfaces was obtained using autoradiography. In this technique, β-particles emitted by radioactive specimens were recorded on X-ray film, producing a latent image that can be converted to a true image by developing and fixing the film. Autoradiography was a lot of fun, but it was also messy. In the impatient excitement of wanting to see how an experiment had turned out, people used to hold the newly developed X-ray films in their metal frames up to the darkroom light. Drips of the final wash would run down their arms, clothes would be stained, and shoes ruined. It is hardly surprising that autoradiography was quickly abandoned when sensitive phosphorimagers came onto the market at the end of the 1990s.
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http://dx.doi.org/10.1101/pdb.top100446DOI Listing
December 2020

Detection of Radioactive DNA in Polyacrylamide Gels.

Cold Spring Harb Protoc 2020 12 1;2020(12). Epub 2020 Dec 1.

Bands of radioactive DNA separated by polyacrylamide gel electrophoresis may be detected by autoradiography or phosphorimaging. Analytical polyacrylamide gels containing radioactive DNA are usually fixed and dried before autoradiography. However, if bands of radioactive DNA are to be recovered from the gel, the gel should generally not be fixed or dried.
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http://dx.doi.org/10.1101/pdb.prot100420DOI Listing
December 2020

Polyacrylamide Gel Electrophoresis.

Cold Spring Harb Protoc 2020 12 1;2020(12). Epub 2020 Dec 1.

Cross-linked chains of polyacrylamide can be used as electrically neutral gels to separate double-stranded DNA fragments according to size and single-stranded DNAs according to size and conformation. Polyacrylamide gels have the following three major advantages over agarose gels: (1) Their resolving power is so great that they can separate molecules of DNA whose lengths differ by as little as 0.1% (i.e., 1 bp in 1000 bp). (2) They can accommodate much larger quantities of DNA than agarose gels. Up to 10 µg of DNA can be applied to a single slot (1 cm × 1 mm) of a typical polyacrylamide gel without significant loss of resolution. (3) DNA recovered from polyacrylamide gels is extremely pure and can be used for the most demanding purposes (e.g., microinjection of mouse embryos). However, polyacrylamide gels have the disadvantage of being more difficult to prepare and handle than agarose gels. Methods are presented here for preparing and running nondenaturing polyacrylamide gels and for detection of DNA in these gels by staining.
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http://dx.doi.org/10.1101/pdb.prot100412DOI Listing
December 2020

Harnessing lymphoma epigenetics to improve therapies.

Blood 2020 Nov 18. Epub 2020 Nov 18.

University of Texas MD Anderson Cancer Center, Houston, Texas, United States.

Affinity maturation and terminal differentiation of B-cells via the germinal center reaction is a complex multi-step process controlled by transcription factors that induce or suppress large dynamic transcriptional programs. This occurs via the recruitment of co-activator or co-repressor complexes that epigenetically regulate gene expression by post-translationally modifying histones and/or remodeling chromatin structure. B-cell-intrinsic developmental programs both regulate and respond to interactions with other cells in the germinal center that provide survival and differentiation signals, such as T follicular helper cells and follicular dendritic cells. Epigenetic and transcriptional programs that naturally occur during B-cell development are hijacked in B-cell lymphoma by genetic alterations that directly or indirectly change the function of transcription factors and/or chromatin modifying genes. These in turn skew differentiation towards the tumor cell-of-origin and alter interactions between lymphoma B-cells and other cells within the microenvironment. Understanding the mechanisms by which genetic alterations perturb epigenetic and transcriptional programs regulating B-cell development and immune interactions may identify opportunities to target these programs using epigenetic modifying agents. Here, we discuss recently published studies centered on follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) within the context of prior knowledge, and highlight how these insights have informed potential avenues for rational therapeutic interventions.
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http://dx.doi.org/10.1182/blood.2020006908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685211PMC
November 2020

dagLogo: An R/Bioconductor package for identifying and visualizing differential amino acid group usage in proteomics data.

PLoS One 2020 6;15(11):e0242030. Epub 2020 Nov 6.

Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

Sequence logos have been widely used as graphical representations of conserved nucleic acid and protein motifs. Due to the complexity of the amino acid (AA) alphabet, rich post-translational modification, and diverse subcellular localization of proteins, few versatile tools are available for effective identification and visualization of protein motifs. In addition, various reduced AA alphabets based on physicochemical, structural, or functional properties have been valuable in the study of protein alignment, folding, structure prediction, and evolution. However, there is lack of tools for applying reduced AA alphabets to the identification and visualization of statistically significant motifs. To fill this gap, we developed an R/Bioconductor package dagLogo, which has several advantages over existing tools. First, dagLogo allows various formats for input sets and provides comprehensive options to build optimal background models. It implements different reduced AA alphabets to group AAs of similar properties. Furthermore, dagLogo provides statistical and visual solutions for differential AA (or AA group) usage analysis of both large and small data sets. Case studies showed that dagLogo can better identify and visualize conserved protein sequence patterns from different types of inputs and can potentially reveal the biological patterns that could be missed by other logo generators.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0242030PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7647101PMC
December 2020

Dephosphorylation of Plasmid DNA.

Cold Spring Harb Protoc 2020 11 2;2020(11). Epub 2020 Nov 2.

This protocol presents a method to remove 5'-phosphate residues from protruding or blunt termini of linearized plasmids. This suppresses the recircularization of plasmid DNA during subsequent ligation procedures.
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http://dx.doi.org/10.1101/pdb.prot101253DOI Listing
November 2020

Cloning in Plasmid Vectors: Blunt-End Cloning.

Cold Spring Harb Protoc 2020 11 2;2020(11). Epub 2020 Nov 2.

This protocol describes procedures for cloning blunt-ended DNA fragments into linearized plasmid vectors. To obtain the maximum number of "correct" ligation products when cloning blunt-ended target fragments, the two components of DNA in the ligation reaction must be present at an appropriate ratio. If the molar ratio of plasmid vector to target DNA is too high, then the ligation reaction may generate an undesirable number of circular empty plasmids, both monomeric and polymeric; if too low, the ligation reaction may generate an excess of linear and circular homopolymers and heteropolymers of varying sizes, orientations, and compositions. For this reason, the orientation of the foreign DNA and the number of inserts in each recombinant clone must always be validated by restriction endonuclease mapping or some other means.
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http://dx.doi.org/10.1101/pdb.prot101246DOI Listing
November 2020

Cloning in Plasmid Vectors: Directional Cloning.

Cold Spring Harb Protoc 2020 11 2;2020(11). Epub 2020 Nov 2.

This protocol describes the standard, old-fashioned but reliable procedure for cloning linear DNA fragments whose ends are incompatible with each other but are compatible with those of the linearized vector.
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http://dx.doi.org/10.1101/pdb.prot101238DOI Listing
November 2020

OneStopRNAseq: A Web Application for Comprehensive and Efficient Analyses of RNA-Seq Data.

Genes (Basel) 2020 Oct 2;11(10). Epub 2020 Oct 2.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA.

Over the past decade, a large amount of RNA sequencing (RNA-seq) data were deposited in public repositories, and more are being produced at an unprecedented rate. However, there are few open source tools with point-and-click interfaces that are versatile and offer streamlined comprehensive analysis of RNA-seq datasets. To maximize the capitalization of these vast public resources and facilitate the analysis of RNA-seq data by biologists, we developed a web application called OneStopRNAseq for the one-stop analysis of RNA-seq data. OneStopRNAseq has user-friendly interfaces and offers workflows for common types of RNA-seq data analyses, such as comprehensive data-quality control, differential analysis of gene expression, exon usage, alternative splicing, transposable element expression, allele-specific gene expression quantification, and gene set enrichment analysis. Users only need to select the desired analyses and genome build, and provide a Gene Expression Omnibus (GEO) accession number or Dropbox links to sequence files, alignment files, gene-expression-count tables, or rank files with the corresponding metadata. Our pipeline facilitates the comprehensive and efficient analysis of private and public RNA-seq data.
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http://dx.doi.org/10.3390/genes11101165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650687PMC
October 2020

Characteristics of anti-CD19 CAR T cell infusion products associated with efficacy and toxicity in patients with large B cell lymphomas.

Nat Med 2020 12 5;26(12):1878-1887. Epub 2020 Oct 5.

Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Autologous chimeric antigen receptor (CAR) T cell therapies targeting CD19 have high efficacy in large B cell lymphomas (LBCLs), but long-term remissions are observed in less than half of patients, and treatment-associated adverse events, such as immune effector cell-associated neurotoxicity syndrome (ICANS), are a clinical challenge. We performed single-cell RNA sequencing with capture-based cell identification on autologous axicabtagene ciloleucel (axi-cel) anti-CD19 CAR T cell infusion products to identify transcriptomic features associated with efficacy and toxicity in 24 patients with LBCL. Patients who achieved a complete response by positron emission tomography/computed tomography at their 3-month follow-up had three-fold higher frequencies of CD8 T cells expressing memory signatures than patients with partial response or progressive disease. Molecular response measured by cell-free DNA sequencing at day 7 after infusion was significantly associated with clinical response (P = 0.008), and a signature of CD8 T cell exhaustion was associated (q = 2.8 × 10) with a poor molecular response. Furthermore, a rare cell population with monocyte-like transcriptional features was associated (P = 0.0002) with high-grade ICANS. Our results suggest that heterogeneity in the cellular and molecular features of CAR T cell infusion products contributes to variation in efficacy and toxicity after axi-cel therapy in LBCL, and that day 7 molecular response might serve as an early predictor of CAR T cell efficacy.
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http://dx.doi.org/10.1038/s41591-020-1061-7DOI Listing
December 2020

Isolation of Total RNA Using Monophasic Lysis Reagents.

Cold Spring Harb Protoc 2020 09 1;2020(9):101642. Epub 2020 Sep 1.

A typical mammalian cell contains ∼10 µg of RNA, 80%-85% of which is ribosomal RNA (rRNA; chiefly the 28S, 18S, 5.8S, and 5S species). Most of the remaining 15%-20% consists of a variety of low-molecular-weight species (e.g., transfer RNAs [tRNAs] and small nuclear RNAs). These abundant RNAs are of defined size and sequence. In contrast, messenger RNA (mRNA), which makes up between 1% and 5% of the total cellular RNA, is heterogeneous in both size-from a few hundred bases to many kilobases in length-and sequence. In this introduction, the development and use of monophasic lysis reagents for the isolation of total RNA from eukaryotic cells is discussed.
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http://dx.doi.org/10.1101/pdb.top101642DOI Listing
September 2020

Total RNA Extraction from .

Cold Spring Harb Protoc 2020 09 1;2020(9):101683. Epub 2020 Sep 1.

This protocol is for isolating total RNA from either larvae (L1-L4) or adult worms using a monophasic lysis reagent. It is preferable when multiple samples have to be processed.
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http://dx.doi.org/10.1101/pdb.prot101683DOI Listing
September 2020

Total RNA Isolation from .

Cold Spring Harb Protoc 2020 09 1;2020(9):101675. Epub 2020 Sep 1.

Of the methods available for extracting total RNA from , monophasic lysis reagents (such as TRIzol) yield consistent and reliable results and are considerably cheaper than other kit-based techniques for RNA isolation. In this protocol, up to 50 mg of tissue is homogenized in a microcentrifuge tube using a plastic pestle in the presence of the monophasic lysis reagent. For larger amounts of starting material, homogenization can be performed using a mortar and pestle or a Dounce homogenizer, and the rest of the protocol can be readily scaled up.
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http://dx.doi.org/10.1101/pdb.prot101675DOI Listing
September 2020

Prostaglandin E2 stimulates cAMP signaling and re-sensitizes human leukemia cells to glucocorticoid-induced cell death.

Blood 2020 Aug 5. Epub 2020 Aug 5.

University of Massachusetts Medical School, Worcester, Massachusetts, United States.

Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia (ALL) where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, shRNA screen in murine T cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cAMP signaling and are under-expressed in GC resistant or relapsed ALL patients. Silencing of the cAMP activating guanine nucleotide binding protein, alpha stimulating Gnas gene, interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that Prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC re-sensitization in relapsed pediatric T-ALL.
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http://dx.doi.org/10.1182/blood.2020005712DOI Listing
August 2020

Alkaline Phosphatase.

Cold Spring Harb Protoc 2020 08 3;2020(8):100768. Epub 2020 Aug 3.

Several types of alkaline phosphatases (or alkaline phosphomonoesterase) are commonly used in molecular cloning, including bacterial alkaline phosphatase (BAP) and calf intestinal alkaline phosphatase (CIP, CIAP, or CAP). Similar enzymes isolated from more esoteric cold-blooded organisms (e.g., SAP from shrimp) have become available in recent years and have the advantage of being easier to inactivate than BAP or CIP at the end of dephosphorylation reactions. The uses and properties of these enzymes are introduced here.
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http://dx.doi.org/10.1101/pdb.top100768DOI Listing
August 2020

Dephosphorylation of DNA Fragments with Alkaline Phosphatase.

Cold Spring Harb Protoc 2020 08 3;2020(8):100669. Epub 2020 Aug 3.

The removal of 5' phosphates from nucleic acids is used to enhance subsequent labeling with [γ-P]-ATP, reduce the circularization of plasmid vectors in ligation reactions, and render DNA susceptible or resistant to other enzymes that act on nucleic acids (e.g., λ exonuclease). Essentially, any nucleotide phosphatase (e.g., bacterial alkaline phosphatase, calf intestinal alkaline phosphatase [CIP], placental alkaline phosphatase, shrimp alkaline phosphatase [SAP], or several acid phosphatases such as sweet potato and prostate acid phosphatase) will catalyze the removal of 5' phosphates from nucleic acid templates. In fact, these enzymes prefer small substrates such as -nitrophenyl phosphate (PNPP) and the exposed 5' phosphates of nucleic acids to bulky globular protein substrates.
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http://dx.doi.org/10.1101/pdb.prot100669DOI Listing
August 2020