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Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) has shown activity in melanoma, but has not been previously evaluated in metastatic non-small cell lung cancer. We conducted a single-arm open-label phase 1 trial (NCT03215810) of TILs administered with nivolumab in 20 patients with advanced non-small cell lung cancer following initial progression on nivolumab monotherapy. The primary end point was safety and secondary end points included objective response rate, duration of response and T cell persistence. Autologous TILs were expanded ex vivo from minced tumors cultured with interleukin-2. Patients received cyclophosphamide and fludarabine lymphodepletion, TIL infusion and interleukin-2, followed by maintenance nivolumab. The end point of safety was met according to the prespecified criteria of [less than or equal to]17% rate of severe toxicity (95% confidence interval, 3-29%). Of 13 evaluable patients, 3 had confirmed responses and 11 had reduction in tumor burden, with a median best change of 35%. Two patients achieved complete responses that were ongoing 1.5 years later. In exploratory analyses, we found T cells recognizing multiple types of cancer mutations were detected after TIL treatment and were enriched in responding patients. Neoantigen-reactive T cell clonotypes increased and persisted in peripheral blood after treatment. Cell therapy with autologous TILs is generally safe and clinically active and may constitute a new treatment strategy in metastatic lung cancer. Adoptive cell therapy with tumor-infiltrating lymphocytes in metastatic lung cancer patients is safe and elicits antitumor activity, including ongoing complete responses, in association with polyclonal T cell responses against tumor antigens., Author(s): Benjamin C. Creelan [sup.1] , Chao Wang [sup.1] , Jamie K. Teer [sup.2] , Eric M. Toloza [sup.1] , Jiqiang Yao [sup.2] , Sungjune Kim [sup.3] , Ana M. [...]

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations [...]

Cutaneous T cell lymphoma (CTCL), a non-Hodgkin lymphoma, is thought to arise from mature tissue-resident memory T cells. The most common subtypes include Mycosis Fungoides and Sezary Syndrome. The role of skin microbiota remains unclear in the symptom manifestation of MF. Among 39 patients with MF, we analyzed bacteria colonizing MF lesions and non-lesional skin in the contralateral side and characterized regional changes in the skin microbiota related to MF involvement using the difference in relative abundance of each genus between lesional and contralateral non-lesional skin. We investigated the relationship between these skin microbiota alterations and symptom severity. No statistically significant difference was found in bacterial diversity and richness between lesional and non-lesional skin. Different skin microbiota signatures were associated with different symptoms. More pronounced erythema in the lesions was associated with an increase in Staphylococcus. Pain and thick skin in the lesions were associated with a decrease in Propionibacterium. The results of this pilot study suggest that the skin microbiota plays an important role in changing skin phenotypes among patients with MF. Larger skin microbiota studies are needed to confirm these findings and support the use of antibiotic treatment to mitigate CTCL symptoms. [ABSTRACT FROM AUTHOR]

Abstract Background While initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3’ tag digital gene expression (3’DGE) and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h) that could be associated with thermotolerance. Results Using 3’DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5), decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (PHSP90AA1 (PSOD1 or CAT. Conclusions Changes in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene networks involved in embryonic development. It is likely that the increased resistance of morula-stage embryos to heat shock as compared to embryos at earlier stages of development is due in part to developmental acquisition of mechanisms to prevent accumulation of denatured proteins and free radical damage.

Abstract Background Molecular breeding of pepper (Capsicum spp.) can be accelerated by developing DNA markers associated with transcriptomes in breeding germplasm. Before the advent of next generation sequencing (NGS) technologies, the majority of sequencing data were generated by the Sanger sequencing method. By leveraging Sanger EST data, we have generated a wealth of genetic information for pepper including thousands of SNPs and Single Position Polymorphic (SPP) markers. To complement and enhance these resources, we applied NGS to three pepper genotypes: Maor, Early Jalapeño and Criollo de Morelos-334 (CM334) to identify SNPs and SSRs in the assembly of these three genotypes. Results Two pepper transcriptome assemblies were developed with different purposes. The first reference sequence, assembled by CAP3 software, comprises 31,196 contigs from >125,000 Sanger-EST sequences that were mainly derived from a Korean F1-hybrid line, Bukang. Overlapping probes were designed for 30,815 unigenes to construct a pepper Affymetrix GeneChip® microarray for whole genome analyses. In addition, custom Python scripts were used to identify 4,236 SNPs in contigs of the assembly. A total of 2,489 simple sequence repeats (SSRs) were identified from the assembly, and primers were designed for the SSRs. Annotation of contigs using Blast2GO software resulted in information for 60% of the unigenes in the assembly. The second transcriptome assembly was constructed from more than 200 million Illumina Genome Analyzer II reads (80–120 nt) using a combination of Velvet, CLC workbench and CAP3 software packages. BWA, SAMtools and in-house Perl scripts were used to identify SNPs among three pepper genotypes. The SNPs were filtered to be at least 50 bp from any intron-exon junctions as well as flanking SNPs. More than 22,000 high-quality putative SNPs were identified. Using the MISA software, 10,398 SSR markers were also identified within the Illumina transcriptome assembly and primers were designed for the identified markers. The assembly was annotated by Blast2GO and 14,740 (12%) of annotated contigs were associated with functional proteins. Conclusions Before availability of pepper genome sequence, assembling transcriptomes of this economically important crop was required to generate thousands of high-quality molecular markers that could be used in breeding programs. In order to have a better understanding of the assembled sequences and to identify candidate genes underlying QTLs, we annotated the contigs of Sanger-EST and Illumina transcriptome assemblies. These and other information have been curated in a database that we have dedicated for pepper project.

Abstract Background The increasing number of genomic sequences of bacteria makes it possible to select unique SNPs of a particular strain/species at the whole genome level and thus design specific primers based on the SNPs. The high similarity of genomic sequences among phylogenetically-related bacteria requires the identification of the few loci in the genome that can serve as unique markers for strain differentiation. PrimerSNP attempts to identify reliable strain-specific markers, on which specific primers are designed for pathogen detection purpose. Results PrimerSNP is an online tool to design primers based on strain specific SNPs for multiple strains/species of microorganisms at the whole genome level. The allele-specific primers could distinguish query sequences of one strain from other homologous sequences by standard PCR reaction. Additionally, PrimerSNP provides a feature for designing common primers that can amplify all the homologous sequences of multiple strains/species of microorganisms. PrimerSNP is freely available at http://cropdisease.ars.usda.gov/~primer. Conclusion PrimerSNP is a high-throughput specific primer generation tool for the differentiation of phylogenetically-related strains/species. Experimental validation showed that this software had a successful prediction rate of 80.4 – 100% for strain specific primer design.

Abstract Background The family Vitaceae consists of many different grape species that grow in a range of climatic conditions. In the past few years, several studies have generated functional genomic information on different Vitis species and cultivars, including the European grape vine, Vitis vinifera. Our goal is to develop a comprehensive web data source for Vitaceae. Description VitisExpDB is an online MySQL-PHP driven relational database that houses annotated EST and gene expression data for V. vinifera and non-vinifera grape species and varieties. Currently, the database stores ~320,000 EST sequences derived from 8 species/hybrids, their annotation (BLAST top match) details and Gene Ontology based structured vocabulary. Putative homologs for each EST in other species and varieties along with information on their percent nucleotide identities, phylogenetic relationship and common primers can be retrieved. The database also includes information on probe sequence and annotation features of the high density 60-mer gene expression chip consisting of ~20,000 non-redundant set of ESTs. Finally, the database includes 14 processed global microarray expression profile sets. Data from 12 of these expression profile sets have been mapped onto metabolic pathways. A user-friendly web interface with multiple search indices and extensively hyperlinked result features that permit efficient data retrieval has been developed. Several online bioinformatics tools that interact with the database along with other sequence analysis tools have been added. In addition, users can submit their ESTs to the database. Conclusion The developed database provides genomic resource to grape community for functional analysis of genes in the collection and for the grape genome annotation and gene function identification. The VitisExpDB database is available through our website http://cropdisease.ars.usda.gov/vitis_at/main-page.htm.

Abstract Background The Gram-negative, xylem-limited phytopathogenic bacterium Xylella fastidiosa is responsible for causing economically important diseases in grapevine, citrus and many other plant species. Despite its economic impact, relatively little is known about the genomic variations among strains isolated from different hosts and their influence on the population genetics of this pathogen. With the availability of genome sequence information for four strains, it is now possible to perform genome-wide analyses to identify and categorize such DNA variations and to understand their influence on strain functional divergence. Results There are 1,579 genes and 194 non-coding homologous sequences present in the genomes of all four strains, representing a 76. 2% conservation of the sequenced genome. About 60% of the X. fastidiosa unique sequences exist as tandem gene clusters of 6 or more genes. Multiple alignments identified 12,754 SNPs and 14,449 INDELs in the 1528 common genes and 20,779 SNPs and 10,075 INDELs in the 194 non-coding sequences. The average SNP frequency was 1.08 × 10-2 per base pair of DNA and the average INDEL frequency was 2.06 × 10-2 per base pair of DNA. On an average, 60.33% of the SNPs were synonymous type while 39.67% were non-synonymous type. The mutation frequency, primarily in the form of external INDELs was the main type of sequence variation. The relative similarity between the strains was discussed according to the INDEL and SNP differences. The number of genes unique to each strain were 60 (9a5c), 54 (Dixon), 83 (Ann1) and 9 (Temecula-1). A sub-set of the strain specific genes showed significant differences in terms of their codon usage and GC composition from the native genes suggesting their xenologous origin. Tandem repeat analysis of the genomic sequences of the four strains identified associations of repeat sequences with hypothetical and phage related functions. Conclusion INDELs and strain specific genes have been identified as the main source of variations among strains, with individual strains showing different rates of genome evolution. Based on these genome comparisons, it appears that the Pierce's disease strain Temecula-1 genome represents the ancestral genome of the X. fastidiosa. Results of this analysis are publicly available in the form of a web database.

The Arabidopsis thaliana Elongator complex subunit2 (ELP2) genetically interacts with NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), a key transcription coactivator of plant immunity, and regulates the induction kinetics of defense genes. However, the mechanistic relationship between ELP2 and NPR1 and how ELP2 regulates the kinetics of defense gene induction are unclear. Here, we demonstrate that ELP2 is an epigenetic regulator required for pathogen-induced rapid transcriptome reprogramming. We show that ELP2 functions in a transcriptional feed-forward loop regulating both NPR1 and its target genes. An elp2 mutation increases the total methylcytosine number, reduces the average methylation levels of methylcytosines, and alters (increases or decreases) methylation levels of specific methylcytosines. Interestingly, infection of plants with the avirulent bacterial pathogen Pseudomonas syringae pv tomato DC3000/ avrRpt2 induces biphasic changes in DNA methylation levels of NPR1 and PHYTOALEXIN DEFICIENT4 (PAD4), which encodes another key regulator of plant immunity. These dynamic changes are blocked by the elp2 mutation, which is correlated with delayed induction of NPR1 and PAD4. The elp2 mutation also reduces basal histone acetylation levels in the coding regions of several defense genes. Together, our data demonstrate a new role for Elongator in somatic DNA demethylation/methylation and suggest a function for Elongator-mediated chromatin regulation in pathogen-induced transcriptome reprogramming. [ABSTRACT FROM AUTHOR]

The widely cultivated pepper, Capsicum spp., important as a vegetable and spice crop world-wide, is one of the most diverse crops. To enhance breeding programs, a detailed characterization of Capsicum diversity including morphological, geographical and molecular data is required. Currently, molecular data characterizing Capsicum genetic diversity is limited. The development and application of high-throughput genome-wide markers in Capsicum will facilitate more detailed molecular characterization of germplasm collections, genetic relationships, and the generation of ultra-high density maps. We have developed the Pepper GeneChip® array from Affymetrix for polymorphism detection and expression analysis in Capsicum. Probes on the array were designed from 30,815 unigenes assembled from expressed sequence tags (ESTs). Our array design provides a maximum redundancy of 13 probes per base pair position allowing integration of multiple hybridization values per position to detect single position polymorphism (SPP). Hybridization of genomic DNA from 40 diverse C. annuum lines, used in breeding and research programs, and a representative from three additional cultivated species (C. frutescens, C. chinense and C. pubescens) detected 33,401 SPP markers within 13,323 unigenes. Among the C. annuum lines, 6,426 SPPs covering 3,818 unigenes were identified. An estimated three-fold reduction in diversity was detected in non-pungent compared with pungent lines, however, we were able to detect 251 highly informative markers across these C. annuum lines. In addition, an 8.7 cM region without polymorphism was detected around Pun1 in non-pungent C. annuum. An analysis of genetic relatedness and diversity using the software Structure revealed clustering of the germplasm which was confirmed with statistical support by principle components analysis (PCA) and phylogenetic analysis. This research demonstrates the effectiveness of parallel high-throughput discovery and application of genome-wide transcript-based markers to assess genetic and genomic features among Capsicum annuum. [ABSTRACT FROM AUTHOR]

Background: The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system. Results: A total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, NANOG, SOX2, and STAT3) and TE (ELF5, GATA3, and KRT18) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human.Conclusion: Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast. [ABSTRACT FROM AUTHOR]

Simple Summary: Dysregulated fucosylation has been characterized as an underlying cause or a contributor to the pathogenesis of several disease states. However, to date, there is not a clear understanding of how and what proteins, signaling pathways, and cellular processes are impacted by fucosylation. Here, we characterized the proteins recognized by a fucose-binding lectin and unexpectedly discovered that many intracellular proteins are putatively subject to posttranslational fucosylation. We further found that fucosylation on intracellular ribosomal protein S3 responds to stimulus, and that it appears to be independent of the currently characterized fucosylation pathway. This work suggests a to-date-underappreciated role for fucosylation on intracellular proteins and supports the existence of fucosylation capabilities within cells that is not fully known. Alterations in genes encoding for proteins that control fucosylation are known to play causative roles in several developmental disorders, such as Dowling-Degos disease 2 and congenital disorder of glycosylation type IIc (CDGIIc). Recent studies have provided evidence that changes in fucosylation can contribute to the development and progression of several different types of cancers. It is therefore important to gain a detailed understanding of how fucosylation is altered in disease states so that interventions may be developed for therapeutic purposes. In this report, we find that fucosylation occurs on many intracellular proteins. This is an interesting finding, as the fucosylation machinery is restricted to the secretory pathway and is thought to predominately affect cell-membrane-bound and secreted proteins. We find that Ribosomal protein S3 (RPS3) is fucosylated in normal tissues and in cancer cells, and that the extent of its fucosylation appears to respond to stress, including MAPK inhibitors, suggesting a new role in posttranslational protein function. Our data identify a new ribosome-independent species of fucosylated RPS3 that interacts with proteins involved in posttranscriptional regulation of RNA, such as Heterogeneous nuclear ribonucleoprotein U (HNRNPU), as well as with a predominance of non-coding RNAs. These data highlight a novel role for RPS3, which, given previously reported oncogenic roles for RPS3, might represent functions that are perturbed in pathologies such as cancer. Together, our findings suggest a previously unrecognized role for fucosylation in directly influencing intracellular protein functions. [ABSTRACT FROM AUTHOR]

HOXB13, a homeodomain transcription factor, is linked to recurrence following radical prostatectomy. While HOXB13 regulates Androgen Receptor (AR) functions in a context dependent manner, its critical effectors in prostate cancer (PC) metastasis remain largely unknown. To identify HOXB13 transcriptional targets in metastatic PCs, we performed integrative bioinformatics analysis of differentially expressed genes (DEGs) in the proximity of the human prostate tumor-specific AR binding sites. Unsupervised Principal Component Analysis (PCA) led to a focused core HOXB13 target gene-set referred to as HOTPAM9 (HOXB13 Targets separating Primary And Metastatic PCs). HOTPAM9 comprised 7 mitotic kinase genes overexpressed in metastatic PCs, TRPM8, and the heat shock protein HSPB8, whose levels were significantly lower in metastatic PCs compared to the primary disease. The expression of a two-gene set, CIT and HSPB8 with an overall balanced accuracy of 98.8% and a threshold value of 0.2347, was sufficient to classify metastasis. HSPB8 mRNA expression was significantly increased following HOXB13 depletion in multiple metastatic CRPC models. Increased expression of HSPB8 by the microtubule inhibitor Colchicine or by exogenous means suppressed migration of mCRPC cells. Collectively, our results indicate that HOXB13 promotes metastasis of PCs by coordinated regulation of mitotic kinases and blockade of a putative tumor suppressor gene. [ABSTRACT FROM AUTHOR]

HOXB13, a homeodomain transcription factor, is linked to recurrence following radical prostatectomy. While HOXB13 regulates Androgen Receptor (AR) functions in a context dependent manner, its critical effectors in prostate cancer (PC) metastasis remain largely unknown. To identify HOXB13 transcriptional targets in metastatic PCs, we performed integrative bioinformatics analysis of differentially expressed genes (DEGs) in the proximity of the human prostate tumor-specific AR binding sites. Unsupervised Principal Component Analysis (PCA) led to a focused core HOXB13 target gene-set referred to as HOTPAM9 (HOXB13 Targets separating Primary And Metastatic PCs). HOTPAM9 comprised 7 mitotic kinase genes overexpressed in metastatic PCs, TRPM8, and the heat shock protein HSPB8, whose levels were significantly lower in metastatic PCs compared to the primary disease. The expression of a two-gene set, CIT and HSPB8 with an overall balanced accuracy of 98.8% and a threshold value of 0.2347, was sufficient to classify metastasis. HSPB8 mRNA expression was significantly increased following HOXB13 depletion in multiple metastatic CRPC models. Increased expression of HSPB8 by the microtubule inhibitor Colchicine or by exogenous means suppressed migration of mCRPC cells. Collectively, our results indicate that HOXB13 promotes metastasis of PCs by coordinated regulation of mitotic kinases and blockade of a putative tumor suppressor gene. [ABSTRACT FROM AUTHOR]

CD19 chimeric antigen receptor (CAR) T cell therapies have been successful in B cell malignancies. Recent reports about CD19 CAR T cell therapy in B-cell acute lymphoblastic leukemia suggest that the median event-free survival of children and young adult patients is longer than that of adult patients. Since the reason is unclear, we compared the functions of CAR-T derived from young or aged mice and also healthy human donors. Young and aged B6 mice spleens (6-12 vs. ≥72 weeks) or young and aged human PBMCs (20-26 vs. 53-60 years) were used for mouse or human CAR-T preparation. 4 types of mouse CD19 CAR and 2 types of human CD19 CAR were evaluated in T cells. Aged mouse CAR-T predominates with CD8+ and effector-like phenotypes at the expense of CD4+ and memory-like phenotypes. Compared to young mouse CAR-T, aged mouse CAR-T exhibited superior cytotoxicity for mouse CD19+ artificial antigen presenting cell (aAPC). Using our immune competent in vivo murine model, aged mouse CAR-T was short-lived and expanded poorly despite superior in vitro cytotoxicity. RNA-Seq suggestes that young mouse CAR-T is advantageous for cell proliferation and regulation of cell differentiation whereas aged mouse CAR-T up-regulates gene expression pathways that regulate responses to stimulus and exocytosis. Furthermore, compared to mouse CAR-T, human CAR-T is complementary with immune phenotypes after human CD19+ aAPC stimulation. Aged donor derived CAR-T exhibited enhanced effector functions but shorter persistence and less memory-like phenotypes. Our results suggest that the difference of clinical outcomes may be due to an age-dependent CAR-T cell phenotype that is reflected by its unique gene expression pattern, secretory profile, and/or transcription factor balance. In our future directions we are identifying potential methods to improve the function of aged donor derived CAR-T. [ABSTRACT FROM AUTHOR]