David Baltimore

Bio: David Baltimore is an academic researcher from California Institute of Technology. The author has contributed to research in topics: RNA & Virus. The author has an hindex of 203, co-authored 876 publications receiving 162955 citations. Previous affiliations of David Baltimore include Thomas Jefferson University & Johns Hopkins University.

Dr Baltimore says "sorry".

Abstract: Dr David Baltimore says he had no knowledge of the fabrication of data in a paper in Cell of which he was a co-author, says he will work to develop new guidelines for misconduct and apologizes to Dr Margot O'Toole.

Replicative Intermediate RNAs of Poliovirus Covalent Linkage of a Protein to a Defined Nucleotide Sequence at the 5'-Terminus of Virion and

Abstract: The 5'-terminus of poliovirus polyribosomal RNA is pUp. A candidate for the 5'-terminus of poliovirion RNA was recovered as a compound migrating toward the cathode when 32P-labeled virion RNA was completely digested with ribonucleases TI, T2, and A and analyzed by paper ionophoresis at pH 3.5. Treatment with proteinase K reversed its direction of migration, indicating the presence of rotein. Treatment with venom phosphodiesterase liberated alfof the radioactivity as pUp, suggesting that poliovirion RNA has a protein-pUp 5'-terminus. Treatment of virion RNA with T, ribonuclease alone generated a proteinase K-sensitive oligoribonucleotide. Analysis of the oligoribonucleotide using ribonucleases A and U2 showed its structure to be protein-pU-U-A-A-A-A-CA-C. Digests of replicative intermediate RNA contained sufficient protein-pUp to suggest that this structure is at the 5'-end of most nascent poliovirus RNA molecules. We suggest that a protein-nucleotide structure acts as a primer for initiating synthesis of poliovirus RNA. Although the m7G5'ppp5'N(m)pNp "capping" group has been found on virtually all known mammalian mRNAs (1), poliovirus polyribosomal RNA has pUp at its 5'-end (2-4). Because poliovirion RNA will direct viral protein synthesis in a cell-free system (5), it was thought to be identical to polyribosomal poliovirus RNA. Instead, we found that less than 10% of the virion molecules contained a pUp 5'-end (2). Lee et al. (6) recently presented evidence that a protein might be linked to the 5'-terminus of poliovirion RNA. We also have obtained evidence for a 5'-terminal protein in virion RNA. When total RNase digests of 32P-labeled virion RNA were examined by paper ionophoresis at pH 3.5, some labeled material was found moving toward the cathode, the direction opposite that of pure nucleotides. This material had the properties of a protein-pUp 5'-terminus of the RNA, a structure also suggested by Lee et al. (6). We have further found that cellulose acetate electrophoresis of a RNase T1 digest of virion RNA separates a protein-linked oligonucleotide. We show here that it consists of the structure protein-pU-U-A-A-A-A-C-A-G, which appears to be the 5'-terminus of poliovirion RNA. The protein is also found on replicative intermediate RNA, suggesting that it represents an initiating structure for viral RNA synthesis. MATERIALS AND METHODS Preparation of Poliovirus RNA. Poliovirus-specific RNA labeled with carrier free [32P]orthophosphate (New England Nuclear Corp.) was prepared by infecting actinomycin Dtreated HeLa cells with poliovirus type 1 as described (2). To Abbreviations: NaDodSO4, sodium dodecyl sulfate; P1, Penicillium nuclease. * Present address: Department of Cellular and Developmental Biology, University of Arizona, Tucson, Ariz. 85721. prepare 32P-labeled virions, cells were harvested 6-hr after infection, washed once with Earle's saline, and broken by suspension in cold 1% Nonidet P-40/10mM Tris-HCI (pH 7.5)/10 mM NaCI/1.5 mM MgCI2. The nuclei were immediately removed by centrifugation (5,000 X g, 5 min) and the virions were collected by centrifugation at 45,000 rpm for 2 hr in a type 65 Spinco rotor. The virions were purified by sucrose gradient centrifugation (7), and the RNA was extracted with either 0.5% sodium dodecyl sulfate (NaDodSO4)/O.1 M acetic acid (pH 3.5) (7) or with 0.5% NaDodSO4/phenol/chloroform/isoamyl alcohol (25:24:1) (7). The RNA was purified by sucrose gradient centrifugation and precipitated by ethanol (7). 32P-Labeled replicative intermediate RNA isolated from a cytoplasmic extract of infected cells harvested at 3.5 hr was purified by precipitation with 2 M LiCI, phenol extraction, and chromatography on Sepharose 2B (7). The purified replicative intermediate RNA did not contain any labeled single-stranded 35S RNA when analyzed by electrophoresis on a 1% agarose gel (8). Enzymatic Digestions. Polypropylene tubes and pipettes were used for all enzymatic digestions. Limit digestion of RNA with RNases T1, T2, and A was for 1 hr at 370 in 15 Ail of 0.05 M ammonium acetate (pH 5.0) containing 10 jig of RNA, 40 ,ug of bovine serum albumin carrier protein (electrophoretically pure, Sigma), 5 units/ml of RNase T2 (Calbiochem), 200 units/ml of RNase T, (Calbiochem), and 150 units/ml of pancreatic RNase A (Worthington Biochemicals). Limit digestion of RNA was also performed at 370 for 30 min in 5 ,ul of 6 mM sodium acetate (pH 4.5)/0.6 mM EDTA containing 30 jig/ml of heat-treated takadiastase extract (from Calbiochem; Sanzyme-R) and 0.42 mg/ml of RNase A. The takadiastase extract was prepared as described (9) except 1 mM EDTA was added during the final dialysis step and the extract was used without lyophilization.' Reaction conditions for' RNase U2 (Calbiochem) (0.1 unit/ml) (1O), RNase T, (11), RNase A (11), bacterial alkaline phosphatase (Boehringer Mannheim) (12), and venom phosphodiesterase (Boehringer Mannheim) (12) were as described. Bovine serum albumin (30 Ag) was added when samples were digested with RNase A. Bovine serum albumin was also present in the RNase U2 digests (10). Digestion with proteinase K was for 1 hr at 370 in a 100-,ul solution of 0.5% NaDodSO4/0.1 M NaCI/0.01 M Tris-HCI (pH 7.5)/0.001 M EDTA/proteinase K (200 ,g/ml) (EM Laboratories). The RNA was then extracted with phenol/chloroform/isoamylalcohol (25:24:1) and precipitated with ethanol as described (7). Analytical Separations. lonophoretic separations on cellulose acetate, DEAE-paper, and Whatman 3MM paper at pH 3.5 have been described (10, 12). Compounds labeled with 32p were located by autoradiography with Kodak No-Screen Film. La-

Characterization of Postinfusion Phenotypic Differences in Fresh Versus Cryopreserved TCR Engineered Adoptive Cell Therapy Products.

Abstract: Adoptive cell therapy (ACT) consisting of genetically engineered T cells expressing tumor antigen-specific T-cell receptors displays robust initial antitumor activity, followed by loss of T-cell activity/persistence and frequent disease relapse. We characterized baseline and longitudinal T-cell phenotype variations resulting from different manufacturing and administration protocols in patients who received ACT. Patients with melanoma who enrolled in the F5-MART-1 clinical trial (NCT00910650) received infusions of MART-1 T-cell receptors transgenic T cells with MART-1 peptide-pulsed dendritic cell vaccination. Patients were divided into cohorts based on several manufacturing changes in the generation and administration of the transgenic T cells: decreasing ex vivo stimulation/expansion time, increased cell dose, and receiving fresh instead of cryopreserved cells. T-cell phenotypes were analyzed by flow cytometry at baseline and longitudinally in peripheral blood. Transgenic T cells with shorter ex vivo culture/expansion periods displayed significantly increased expression of markers associated with less differentiated naive/memory populations, as well as significantly decreased expression of the inhibitory receptor programmed death 1 (PD1). Patients receiving fresh infusions of transgenic cells demonstrated expansion of central memory T cells and delayed acquisition of PD1 expression compared with patients who received cryopreserved products. Freshly infused transgenic T cells showed persistence and expansion of naive and memory T-cell populations and delayed acquisition of PD1 expression, which correlated with this cohort's superior persistence of transgenic cells and response to dendritic cell vaccines. These results may be useful in designing future ACT protocols.

Domain-swap T cell receptors

Abstract: Disclosed herein are genetically engineered T cell receptors, and methods, vectors, and genetically engineered T cells related to genetically engineered T cell receptors.

I and i

Abstract: There is, I think, something ethereal about i —the square root of minus one. I remember first hearing about it at school. It seemed an odd beast at that time—an intruder hovering on the edge of reality. Usually familiarity dulls this sense of the bizarre, but in the case of i it was the reverse: over the years the sense of its surreal nature intensified. It seemed that it was impossible to write mathematics that described the real world in …

Multiplex Genome Engineering Using CRISPR/Cas Systems

Abstract: Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib

Abstract: BACKGROUND Most patients with non-small-cell lung cancer have no response to the tyrosine kinase inhibitor gefitinib, which targets the epidermal growth factor receptor (EGFR). However, about 10 percent of patients have a rapid and often dramatic clinical response. The molecular mechanisms underlying sensitivity to gefitinib are unknown. METHODS We searched for mutations in the EGFR gene in primary tumors from patients with non-small-cell lung cancer who had a response to gefitinib, those who did not have a response, and those who had not been exposed to gefitinib. The functional consequences of identified mutations were evaluated after the mutant proteins were expressed in cultured cells. RESULTS Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in eight of nine patients with gefitinib-responsive lung cancer, as compared with none of the seven patients with no response (P<0.001). Mutations were either small, in-frame deletions or amino acid substitutions clustered around the ATP-binding pocket of the tyrosine kinase domain. Similar mutations were detected in tumors from 2 of 25 patients with primary non-small-cell lung cancer who had not been exposed to gefitinib (8 percent). All mutations were heterozygous, and identical mutations were observed in multiple patients, suggesting an additive specific gain of function. In vitro, EGFR mutants demonstrated enhanced tyrosine kinase activity in response to epidermal growth factor and increased sensitivity to inhibition by gefitinib. CONCLUSIONS A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib. These mutations lead to increased growth factor signaling and confer susceptibility to the inhibitor. Screening for such mutations in lung cancers may identify patients who will have a response to gefitinib.

Multiplex Genome Engineering Using CRISPR/Cas Systems

Abstract: Genome Editing Clustered regularly interspaced short palindromic repeats (CRISPR) function as part of an adaptive immune system in a range of prokaryotes: Invading phage and plasmid DNA is targeted for cleavage by complementary CRISPR RNAs (crRNAs) bound to a CRISPR-associated endonuclease (see the Perspective by van der Oost). Cong et al. (p. 819, published online 3 January) and Mali et al. (p. 823, published online 3 January) adapted this defense system to function as a genome editing tool in eukaryotic cells. A bacterial genome defense system is adapted to function as a genome-editing tool in mammalian cells. [Also see Perspective by van der Oost] Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

Translating the Histone Code

Abstract: Chromatin, the physiological template of all eukaryotic genetic information, is subject to a diverse array of posttranslational modifications that largely impinge on histone amino termini, thereby regulating access to the underlying DNA. Distinct histone amino-terminal modifications can generate synergistic or antagonistic interaction affinities for chromatin-associated proteins, which in turn dictate dynamic transitions between transcriptionally active or transcriptionally silent chromatin states. The combinatorial nature of histone amino-terminal modifications thus reveals a “histone code” that considerably extends the information potential of the genetic code. We propose that this epigenetic marking system represents a fundamental regulatory mechanism that has an impact on most, if not all, chromatin-templated processes, with far-reaching consequences for cell fate decisions and both normal and pathological development.