A cheap, lightweight smartphone-heated device can test for DNA in blood, urine and other samples in a fraction of the time it takes to test in a lab

Coffee has been linked to changes on our DNA that affect how active certain genes are. The finding may help explain some of coffee's touted health benefits

Gastrointestinal stromal tumor (GIST), the most common sarcoma, is characterized by KIT protein overexpression, and tumors are frequently driven by oncogenic KIT mutations. Targeted inhibition of KIT revolutionized GIST therapy and ushered in the era of precision medicine for the treatment of solid malignancies. Here, we present the first use of a KIT-specific DNA aptamer for targeted labeling of GIST. We found that an anti-KIT DNA aptamer bound cells in a KIT-dependent manner and was highly specific for GIST cell labeling in vitro. Functionally, the KIT aptamer bound extracellular KIT in a manner similar to KIT mAb staining, and was trafficked intracellularly in vitro. The KIT aptamer bound dissociated primary human GIST cells in a mutation agnostic manner such that tumors with KIT and PDGFRA mutations were labeled. In addition, the KIT aptamer specifically labeled intact human GIST tissue ex vivo, as well as peritoneal xenografts in mice with high sensitivity. These results represent the first use of an aptamer-based method for targeted detection of GIST in vitro and in vivo.

Physical and chemical DNA-damaging agents are used widely in the treatment of cancer. Double-strand break (DSB) lesions in DNA are the most deleterious form of damage and, if left unrepaired, can effectively kill cancer cells. DNA-dependent protein kinase (DNA-PK) is a critical component of nonhomologous end joining (NHEJ), one of the two major pathways for DSB repair. Although DNA-PK has been considered an attractive target for cancer therapy, the development of pharmacologic DNA-PK inhibitors for clinical use has been lagging. Here, we report the discovery and characterization of a potent, selective, and orally bioavailable DNA-PK inhibitor, M3814 (peposertib), and provide in vivo proof of principle for DNA-PK inhibition as a novel approach to combination radiotherapy. M3814 potently inhibits DNA-PK catalytic activity and sensitizes multiple cancer cell lines to ionizing radiation (IR) and DSB-inducing agents. Inhibition of DNA-PK autophosphorylation in cancer cells or xenograft tumors led to an increased number of persistent DSBs. Oral administration of M3814 to two xenograft models of human cancer, using a clinically established 6-week fractionated radiation schedule, strongly potentiated the antitumor activity of IR and led to complete tumor regression at nontoxic doses. Our results strongly support DNA-PK inhibition as a novel approach for the combination radiotherapy of cancer. M3814 is currently under investigation in combination with radiotherapy in clinical trials.

ABSTRACT

Merkel cell polyomavirus (MCPyV) is the only polyomavirus known to be associated with tumorigenesis in humans. Similarly to other polyomaviruses, MCPyV expresses a large tumor antigen (LT-Ag) that, together with a small tumor antigen (sT-Ag), contributes to cellular transformation and that is of critical importance for the initiation of the viral DNA replication. Understanding the cellular protein network regulated by MCPyV early proteins will significantly contribute to our understanding of the natural MCPyV life cycle as well as of the mechanisms by which the virus contributes to cellular transformation. We here describe KRAB-associated protein 1 (Kap1), a chromatin remodeling factor involved in cotranscriptional regulation, as a novel protein interaction partner of MCPyV T antigens sT and LT. Kap1 knockout results in a significant increase in the level of viral DNA replication that is highly suggestive of Kap1 being an important host restriction factor during MCPyV infection. Differently from other DNA viruses, MCPyV gene expression is unaffected in the absence of Kap1 and Kap1 does not associate with the viral genome. Instead, we show that in primary normal human dermal fibroblast (nHDF) cells, MCPyV DNA replication, but not T antigen expression alone, induces ataxia telangiectasia mutated (ATM) kinase-dependent Kap1 S824 phosphorylation, a mechanism that typically facilitates repair of double-strand breaks in heterochromatin by arresting the cells in G₂. We show that MCPyV-induced inhibition of cell proliferation is mainly conferred by residues within the origin binding domain and thereby by viral DNA replication. Our data suggest that phosphorylation of Kap1 and subsequent Kap1-dependent G₂ arrest/senescence represent host defense mechanisms against MCPyV replication in nHDF cells.

IMPORTANCE We here describe Kap1 as a restriction factor in MCPyV infection. We report a novel, indirect mechanism by which Kap1 affects MCPyV replication. In contrast with from other DNA viruses, Kap1 does not associate with the viral genome in MCPyV infection and has no impact on viral gene expression. In MCPyV-infected nHDF cells, Kap1 phosphorylation (pKap1 S824) accumulates because of genomic stress mainly induced by viral DNA replication. In contrast, ectopic expression of LT or LT MCPyV mutants, previously shown to be important for induction of genotoxic stress, does not result in a similar extent of pKap1 accumulation. We show that cells actively replicating MCPyV accumulate pKap1 (in a manner dependent on the presence of ATM) and display a senescence phenotype reflected by G₂ arrest. These results are supported by transcriptome analyses showing that LT antigen, in a manner dependent on the presence of Kap1, induces expression of secreted factors, which is known as the senescence-associated secretory phenotype (SASP).

ABSTRACT

The initiation of Escherichia coli chromosomal DNA replication starts with the oligomerization of the DnaA protein at repeat sequences within the origin (ori) region. The amount of ori DNA per cell directly correlates with the growth rate. During fast growth, the cell generation time is shorter than the time required for complete DNA replication; therefore, overlapping rounds of chromosome replication are required. Under these circumstances, the ori region DNA abundance exceeds the DNA abundance in the termination (ter) region. Here, high ori/ter ratios are found to persist in (p)ppGpp-deficient [(p)ppGpp⁰] cells over a wide range of balanced exponential growth rates determined by medium composition. Evidently, (p)ppGpp is necessary to maintain the usual correlation of slow DNA replication initiation with a low growth rate. Conversely, ori/ter ratios are lowered when cell growth is slowed by incrementally increasing even low constitutive basal levels of (p)ppGpp without stress, as if (p)ppGpp alone is sufficient for this response. There are several previous reports of (p)ppGpp inhibition of chromosomal DNA synthesis initiation that occurs with very high levels of (p)ppGpp that stop growth, as during the stringent starvation response or during serine hydroxamate treatment. This work suggests that low physiological levels of (p)ppGpp have significant functions in growing cells without stress through a mechanism involving negative supercoiling, which is likely mediated by (p)ppGpp regulation of DNA gyrase.

IMPORTANCE Bacterial cells regulate their own chromosomal DNA synthesis and cell division depending on the growth conditions, producing more DNA when growing in nutritionally rich media than in poor media (i.e., human gut versus water reservoir). The accumulation of the nucleotide analog (p)ppGpp is usually viewed as serving to warn cells of impending peril due to otherwise lethal sources of stress, which stops growth and inhibits DNA, RNA, and protein synthesis. This work importantly finds that small physiological changes in (p)ppGpp basal levels associated with slow balanced exponential growth incrementally inhibit the intricate process of initiation of chromosomal DNA synthesis. Without (p)ppGpp, initiations mimic the high rates present during fast growth. Here, we report that the effect of (p)ppGpp may be due to the regulation of the expression of gyrase, an important enzyme for the replication of DNA that is a current target of several antibiotics.

ABSTRACT

Human adenoviruses (HAdVs) have developed mechanisms to manipulate cellular antiviral measures to ensure proper DNA replication, with detailed processes far from being understood. Host cells repress incoming viral genomes through a network of transcriptional regulators that normally control cellular homeostasis. The nuclear domains involved are promyelocytic leukemia protein nuclear bodies (PML-NBs), interferon-inducible, dot-like nuclear structures and hot spots of SUMO posttranslational modification (PTM). In HAdV-infected cells, such SUMO factories are found in close proximity to newly established viral replication centers (RCs) marked by the adenoviral DNA binding protein (DBP) E2A. Here, we show that E2A is a novel target of host SUMOylation, leading to PTMs supporting E2A function in promoting productive infection. Our data show that SUMOylated E2A interacts with PML. Decreasing SUMO-E2A protein levels by generating HAdV variants mutated in the three main SUMO conjugation motifs (SCMs) led to lower numbers of viral RCs and PML-NBs, and these two structures were no longer next to each other. Our data further indicate that SUMOylated E2A binds the host transcription factor Sp100A, promoting HAdV gene expression, and represents the molecular bridge between PML tracks and adjacent viral RCs. Consequently, E2A SCM mutations repressed late viral gene expression and progeny production. These data highlight a novel mechanism used by the virus to benefit from host antiviral responses by exploiting the cellular SUMO conjugation machinery.

IMPORTANCE PML nuclear bodies (PML-NBs) are implicated in general antiviral defense based on recruiting host restriction factors; however, it is not understood so far why viruses would establish viral replication centers (RCs) juxtaposed to such "antiviral" compartments. To understand this enigma, we investigate the cross talk between PML-NB components and viral RCs to find the missing link connecting both compartments to promote efficient viral replication and gene expression. Taken together, the current concept is more intricate than originally believed, since viruses apparently take advantage of several specific PML-NB-associated proteins to promote productive infection. Simultaneously, they efficiently inhibit antiviral measures to maintain the viral infectious program. Our data provide evidence that SUMOylation of the viral RC marker protein E2A represents the basis of this virus-host interface and regulates various downstream events to support HAdV productive infection. These results are the basis of our current attempts to generate and screen for specific E2A SUMOylation inhibitors to constitute novel therapeutic approaches to limit and prevent HAdV-mediated diseases and mortality of immunosuppressed patients.

ABSTRACT

Reversible repression of HIV-1 5' long terminal repeat (5'-LTR)-mediated transcription represents the main mechanism for HIV-1 to maintain latency. Identification of host factors that modulate LTR activity and viral latency may help develop new antiretroviral therapies. The heterogeneous nuclear ribonucleoproteins (hnRNPs) are known to regulate gene expression and possess multiple physiological functions. hnRNP family members have recently been identified as the sensors for viral nucleic acids to induce antiviral responses, highlighting the crucial roles of hnRNPs in regulating viral infection. A member of the hnRNP family, X-linked RNA-binding motif protein (RBMX), has been identified in this study as a novel HIV-1 restriction factor that modulates HIV-1 5'-LTR-driven transcription of viral genome in CD4⁺ T cells. Mechanistically, RBMX binds to HIV-1 proviral DNA at the LTR downstream region and maintains the repressive trimethylation of histone H3 lysine 9 (H3K9me3), leading to a blockage of the recruitment of the positive transcription factor phosphorylated RNA polymerase II (RNA pol II) and consequential impediment of transcription elongation. This RBMX-mediated modulation of HIV-1 transcription maintains viral latency by inhibiting viral reactivation from an integrated proviral DNA. Our findings provide a new understanding of how host factors modulate HIV-1 infection and latency and suggest a potential new target for the development of HIV-1 therapies.

IMPORTANCE HIV-1 latency featuring silence of transcription from HIV-1 proviral DNA represents a major obstacle for HIV-1 eradication. Reversible repression of HIV-1 5'-LTR-mediated transcription represents the main mechanism for HIV-1 to maintain latency. The 5'-LTR-driven HIV gene transcription can be modulated by multiple host factors and mechanisms. The hnRNPs are known to regulate gene expression. A member of the hnRNP family, RBMX, has been identified in this study as a novel HIV-1 restriction factor that modulates HIV-1 5'-LTR-driven transcription of viral genome in CD4⁺ T cells and maintains viral latency. These findings provide a new understanding of how host factors modulate HIV-1 infection and latency and suggest a potential new target for the development of HIV-1 therapies.

ABSTRACT

DNA phosphorothioate (PT) modification, in which the nonbridging oxygen in the sugar-phosphate backbone is substituted by sulfur, is catalyzed by DndABCDE or SspABCD in a double-stranded or single-stranded manner, respectively. In Dnd and Ssp systems, mobilization of sulfur in PT formation starts with the activation of the sulfur atom of cysteine catalyzed by the DndA and SspA cysteine desulfurases, respectively. Despite playing the same biochemical role, SspA cannot be functionally replaced by DndA, indicating its unique physiological properties. In this study, we solved the crystal structure of Vibrio cyclitrophicus SspA in complex with its natural substrate, cysteine, and cofactor, pyridoxal phosphate (PLP), at a resolution of 1.80 Å. Our solved structure revealed the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor, suggesting a common binding mode shared by cysteine desulfurases. In addition, although the distance between the catalytic Cys314 and the substrate cysteine is 8.9 Å, which is too far for direct interaction, our structural modeling and biochemical analysis revealed a conformational change in the active site region toward the cysteine substrate to move them close to each other to facilitate the nucleophilic attack. Finally, the pulldown analysis showed that SspA could form a complex with SspD, an ATP pyrophosphatase, suggesting that SspD might potentially accept the activated sulfur atom directly from SspA, providing further insights into the biochemical pathway of Ssp-mediated PT modification.

IMPORTANCE Apart from its roles in Fe-S cluster assembly, tRNA thiolation, and sulfur-containing cofactor biosynthesis, cysteine desulfurase serves as a sulfur donor in the DNA PT modification, in which a sulfur atom substitutes a nonbridging oxygen in the DNA phosphodiester backbone. The initial sulfur mobilization from l-cysteine is catalyzed by the SspA cysteine desulfurase in the SspABCD-mediated DNA PT modification system. By determining the crystal structure of SspA, the study presents the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor. To overcome the long distance (8.9 Å) between the catalytic Cys314 and the cysteine substrate, a conformational change occurs to bring Cys314 to the vicinity of the substrate, allowing for nucleophilic attack.

The selection and firing of DNA replication origins play key roles in ensuring that eukaryotes accurately replicate their genomes. This process is not well documented in plants due in large measure to difficulties in working with plant systems. We developed a new functional assay to label and map very early replicating loci that must, by definition, include at least a subset of replication origins. Arabidopsis (Arabidopsis thaliana) cells were briefly labeled with 5-ethynyl-2'-deoxy-uridine, and nuclei were subjected to two-parameter flow sorting. We identified more than 5500 loci as initiation regions (IRs), the first regions to replicate in very early S phase. These were classified as strong or weak IRs based on the strength of their replication signals. Strong initiation regions were evenly spaced along chromosomal arms and depleted in centromeres, while weak initiation regions were enriched in centromeric regions. IRs are AT-rich sequences flanked by more GC-rich regions and located predominantly in intergenic regions. Nuclease sensitivity assays indicated that IRs are associated with accessible chromatin. Based on these observations, initiation of plant DNA replication shows some similarity to, but is also distinct from, initiation in other well-studied eukaryotic systems.

During meiotic prophase, concurrent transcription, recombination, and chromosome synapsis place substantial topological strain on chromosomal DNA, but the role of topoisomerases in this context remains poorly defined. Here, we analyzed the roles of topoisomerases I and II (Top1 and Top2) during meiotic prophase in Saccharomyces cerevisiae. We show that both topoisomerases accumulate primarily in promoter-containing intergenic regions of actively transcribing genes, including many meiotic double-strand break (DSB) hotspots. Despite the comparable binding patterns, top1 and top2 mutations have different effects on meiotic recombination. TOP1 disruption delays DSB induction and shortens the window of DSB accumulation by an unknown mechanism. By contrast, temperature-sensitive top2-1 mutants exhibit a marked delay in meiotic chromosome remodeling and elevated DSB signals on synapsed chromosomes. The problems in chromosome remodeling were linked to altered Top2 binding patterns rather than a loss of Top2 catalytic activity, and stemmed from a defect in recruiting the chromosome remodeler Pch2/TRIP13 to synapsed chromosomes. No chromosomal defects were observed in the absence of TOP1. Our results imply independent roles for Top1 and Top2 in modulating meiotic chromosome structure and recombination.

Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme...

Histone H2B monoubiquitylation (H2Bub1) has central functions in multiple DNA-templated processes, including gene transcription, DNA repair, and replication. H2Bub1 also is required for the trans-histone regulation of H3K4 and H3K79 methylation. Although previous studies have elucidated the basic mechanisms that establish and remove H2Bub1, we have only an incomplete understanding of how H2Bub1 is regulated. We report here that the histone H4 basic patch regulates H2Bub1. Yeast cells with arginine-to-alanine mutations in the H4 basic patch (H42RA) exhibited a significant loss of global H2Bub1. H42RA mutant yeast strains also displayed chemotoxin sensitivities similar to, but less severe than, strains containing a complete loss of H2Bub1. We found that the H4 basic patch regulates H2Bub1 levels independently of interactions with chromatin remodelers and separately from its regulation of H3K79 methylation. To measure H2B ubiquitylation and deubiquitination kinetics in vivo, we used a rapid and reversible optogenetic tool, the light-inducible nuclear exporter, to control the subcellular location of the H2Bub1 E3 ligase, Bre1. The ability of Bre1 to ubiquitylate H2B was unaffected in the H42RA mutant. In contrast, H2Bub1 deubiquitination by SAGA-associated Ubp8, but not by Ubp10, increased in the H42RA mutant. Consistent with a function for the H4 basic patch in regulating SAGA deubiquitinase activity, we also detected increased SAGA-mediated histone acetylation in H4 basic patch mutants. Our findings uncover that the H4 basic patch has a regulatory function in SAGA-mediated histone modifications.

Site-specific recombinases, such as Cre, are a widely used tool for genetic lineage tracing in the fields of developmental biology, neural science, stem cell biology, and regenerative medicine. However, nonspecific cell labeling by some genetic Cre tools remains a technical limitation of this recombination system, which has resulted in data misinterpretation and led to many controversies in the scientific community. In the past decade, to enhance the specificity and precision of genetic targeting, researchers have used two or more orthogonal recombinases simultaneously for labeling cell lineages. Here, we review the history of cell-tracing strategies and then elaborate on the working principle and application of a recently developed dual genetic lineage-tracing approach for cell fate studies. We place an emphasis on discussing the technical strengths and caveats of different methods, with the goal to develop more specific and efficient tracing technologies for cell fate mapping. Our review also provides several examples for how to use different types of DNA recombinase–mediated lineage-tracing strategies to improve the resolution of the cell fate mapping in order to probe and explore cell fate–related biological phenomena in the life sciences.

Site-specific arrest of RNA polymerases (RNAPs) is fundamental to several technologies that assess RNA structure and function. Current in vitro transcription “roadblocking” approaches inhibit transcription elongation by blocking RNAP with a protein bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires inclusion of a protein factor extrinsic to the minimal in vitro transcription reaction. In this work, we developed a chemical approach for halting transcription by Escherichia coli RNAP. We first established a sequence-independent method for site-specific incorporation of chemical lesions into dsDNA templates by sequential PCR and translesion synthesis. We then show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification or 1,N6-etheno-2'-deoxyadenosine base efficiently and stably halts Escherichia coli RNAP transcription. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables display of nascent RNA molecules from RNAP in a minimal in vitro transcription reaction.

The human immune system relies on highly complex and diverse transcripts and the proteins they encode. These include transcripts encoding human leukocyte antigen (HLA) receptors as well as B cell and T cell receptors (BCR and TCR). Determining which alleles an individual possesses for each HLA gene (high-resolution HLA typing) is essential to establish donor–recipient compatibility in organ and bone marrow transplantations. In turn, the repertoires of millions of unique BCR and TCR transcripts in each individual carry a vast amount of health-relevant information. Both short-read RNA-seq-based HLA typing and BCR/TCR repertoire sequencing (AIRR-seq) currently rely on our incomplete knowledge of the genetic diversity at HLA and BCR/TCR loci. Here, we generated over 10,000,000 full-length cDNA sequences at a median accuracy of 97.9% using our nanopore sequencing-based Rolling Circle Amplification to Concatemeric Consensus (R2C2) protocol. We used this data set to (1) show that deep and accurate full-length cDNA sequencing can be used to provide isoform-level transcriptome analysis for more than 9000 loci, (2) generate accurate sequences of HLA alleles, and (3) extract detailed AIRR data for the analysis of the adaptive immune system. The HLA and AIRR analysis approaches we introduce here are untargeted and therefore do not require prior knowledge of the composition or genetic diversity of HLA and BCR/TCR loci.

Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome (RTT). To identify functional pathways that could inform therapeutic entry points, we carried out a genetic screen for secondary mutations that improved phenotypes in Mecp2/Y mice after mutagenesis with N-ethyl-N-nitrosourea (ENU). Here, we report the isolation of 106 founder animals that show suppression of Mecp2-null traits from screening 3177 Mecp2/Y genomes. Whole-exome sequencing, genetic crosses, and association analysis identified 22 candidate genes. Additional lesions in these candidate genes or pathway components associate variant alleles with phenotypic improvement in 30 lines. A network analysis shows that 63% of the genes cluster into the functional categories of transcriptional repression, chromatin modification, or DNA repair, delineating a pathway relationship with MECP2. Many mutations lie in genes that modulate synaptic signaling or lipid homeostasis. Mutations in genes that function in the DNA damage response (DDR) also improve phenotypes in Mecp2/Y mice. Association analysis was successful in resolving combinatorial effects of multiple loci. One line, which carries a suppressor mutation in a gene required for cholesterol synthesis, Sqle, carries a second mutation in retinoblastoma binding protein 8, endonuclease (Rbbp8, also known as CtIP), which regulates a DDR choice in double-stranded break (DSB) repair. Cells from Mecp2/Y mice have increased DSBs, so this finding suggests that the balance between homology-directed repair and nonhomologous end joining is important for neuronal cells. In this and other lines, two suppressor mutations confer greater improvement than one alone, suggesting that combination therapies could be effective in RTT.

Annelies Wouters, Jan-Pieter Ploem, Sabine A. S. Langie, Tom Artois, Aziz Aboobaker, and Karen Smeets

Pluripotent stem cells hold great potential for regenerative medicine. Increased replication and division, such is the case during regeneration, concomitantly increases the risk of adverse outcomes through the acquisition of mutations. Seeking for driving mechanisms of such outcomes, we challenged a pluripotent stem cell system during the tightly controlled regeneration process in the planarian Schmidtea mediterranea. Exposure to the genotoxic compound methyl methanesulfonate (MMS) revealed that despite a similar DNA-damaging effect along the anteroposterior axis of intact animals, responses differed between anterior and posterior fragments after amputation. Stem cell proliferation and differentiation proceeded successfully in the amputated heads, leading to regeneration of missing tissues. Stem cells in the amputated tails showed decreased proliferation and differentiation capacity. As a result, tails could not regenerate. Interference with the body-axis-associated component β-catenin-1 increased regenerative success in tail fragments by stimulating proliferation at an early time point. Our results suggest that differences in the Wnt signalling gradient along the body axis modulate stem cell responses to MMS.

Molecular radiotherapy using ¹⁷⁷Lu-DOTATATE is a most effective treatment for somatostatin receptor–expressing neuroendocrine tumors. Despite its frequent and successful use in the clinic, little or no radiobiologic considerations are made at the time of treatment planning or delivery. On positive uptake on octreotide-based PET/SPECT imaging, treatment is usually administered as a standard dose and number of cycles without adjustment for peptide uptake, dosimetry, or radiobiologic and DNA damage effects in the tumor. Here, we visualized and quantified the extent of DNA damage response after ¹⁷⁷Lu-DOTATATE therapy using SPECT imaging with ¹¹¹In-anti-H2AX-TAT. This work was a proof-of-principle study of this in vivo noninvasive biodosimeter with β-emitting therapeutic radiopharmaceuticals. Methods: Six cell lines were exposed to external-beam radiotherapy (EBRT) or ¹⁷⁷Lu-DOTATATE, after which the number of H2AX foci and the clonogenic survival were measured. Mice bearing CA20948 somatostatin receptor–positive tumor xenografts were treated with ¹⁷⁷Lu-DOTATATE or sham-treated and coinjected with ¹¹¹In-anti-H2AX-TAT, ¹¹¹In-IgG-TAT control, or vehicle. Results: Clonogenic survival after external-beam radiotherapy was cell-line–specific, indicating varying levels of intrinsic radiosensitivity. Regarding in vitro cell lines treated with ¹⁷⁷Lu-DOTATATE, clonogenic survival decreased and H2AX foci increased for cells expressing high levels of somatostatin receptor subtype 2. Ex vivo measurements revealed a partial correlation between ¹⁷⁷Lu-DOTATATE uptake and H2AX focus induction between different regions of CA20948 xenograft tumors, suggesting that different parts of the tumor may react differentially to ¹⁷⁷Lu-DOTATATE irradiation. Conclusion: ¹¹¹In-anti-H2AX-TAT allows monitoring of DNA damage after ¹⁷⁷Lu-DOTATATE therapy and reveals heterogeneous damage responses.

Prostate-specific membrane antigen (PSMA)–targeting α-radiation therapy (TAT) is an emerging treatment modality for metastatic castration-resistant prostate cancer. There is a subgroup of patients with poor response despite sufficient expression of PSMA in their tumors. The aim of this work was to characterize PSMA-TAT–nonresponding lesions by targeted next-generation sequencing. Methods: Of 60 patients treated with ²²⁵Ac-PSMA-617, we identified 10 patients who presented with a poor response despite sufficient tumor uptake in PSMA PET/CT. We were able to perform CT-guided biopsies with histologic validation of the nonresponding lesions in 7 of these nonresponding patients. Specimens were analyzed by targeted next-generation sequencing interrogating 37 DNA damage-repair–associated genes. Results: In the 7 tumor samples analyzed, we found a total of 15 whole-gene deletions, deleterious or presumably deleterious mutations affecting TP53 (n = 3), CHEK2 (n = 2), ATM (n = 2), and BRCA1, BRCA2, PALB2, MSH2, MSH6, NBN, FANCB, and PMS1 (n = 1 each). The average number of deleterious or presumably deleterious mutations was 2.2 (range, 0–6) per patient. In addition, several variants of unknown significance in ATM, BRCA1, MSH2, SLX4, ERCC, and various FANC genes were detected. Conclusion: Patients with resistance to PSMA-TAT despite PSMA positivity frequently harbor mutations in DNA damage-repair and checkpoint genes. Although the causal role of these alterations in the patient outcome remains to be determined, our findings encourage future studies combining PSMA-TAT and DNA damage-repair–targeting agents such as poly(ADP-ribose)-polymerase inhibitors.

Manoella Gemaque Cavalcante, Cleusa Yoshiko Nagamachi, Julio Cesar Pieczarka, and Renata Coelho Rodrigues Noronha

Eukaryotic genomes exhibit substantial accumulation of repetitive DNA sequences. These sequences can participate in chromosomal reorganization events and undergo molecular cooption to interfere with the function and evolution of genomes. In turtles, repetitive DNA sequences appear to be accumulated at probable break points and may participate in events such as non-homologous recombination and chromosomal rearrangements. In this study, repeated sequences of 5S rDNA, U2 snRNA and Tc1/Mariner transposons were amplified from the genomes of the turtles, Podocnemis expansa and Podocnemis unifilis, and mapped by fluorescence in situ hybridization. Our data confirm the 2n=28 chromosomes for these species (the second lowest 2n in the order Testudines). We observe high conservation of the co-located 5S rDNA and U2 snRNA genes on a small chromosome pair (pair 13), and surmise that this represents the ancestral condition. Our analysis reveals a wide distribution of the Tc1/Mariner transposons and we discuss how the mobility of these transposons can act on karyotypic reorganization events (contributing to the 2n decrease of those species). Our data add new information for the order Testudines and provide important insights into the dynamics and organization of these sequences in the chelonian genomes.

Recent advances in the study and clinical applications of circulating tumor DNA (ctDNA) are limited by practical considerations of sample collection. Whole-genome sequencing (WGS) is increasingly used for analysis of ctDNA, identifying copy-number alterations and fragmentation patterns. We hypothesized that low-depth/shallow WGS (sWGS) data may be generated from minute amounts of cell-free DNA, and that fragment-size selection may remove contaminating genomic DNA from small blood volumes. Dried blood spots have practical advantages for sample collection, may facilitate serial sampling, and could support novel study designs in humans and animal models.We developed a protocol for the isolation and analysis of cell-free DNA from dried blood spots using filter paper cards and bead-based size selection. DNA extracted and size-selected from dried spots was analyzed using sWGS and polymerase chain reaction (PCR).Analyzing a 50 μL dried blood spot from frozen whole blood of a patient with melanoma, we identified ctDNA based on the presence of tumor-specific somatic copy-number alterations, and found a fragment-size profile similar to that observed in plasma DNA. We found alterations in different chromosomes in blood spots from 2 patients with high-grade serous ovarian carcinoma. Extending this approach to serial dried blood spots from mouse xenograft models, we detect tumor-derived cell-free DNA and identified ctDNA from the originally grafted ascites.Our data suggest that ctDNA can be detected and monitored in dried blood spots from archived and fresh blood samples, enabling new approaches for sample collection and novel study/trial designs for both patients and in vivo models.

Plasma DNA fragmentomics is an emerging area of research covering plasma DNA sizes, end points, and nucleosome footprints. In the present study, we found a significant increase in the diversity of plasma DNA end motifs in patients with hepatocellular carcinoma (HCC). Compared with patients without HCC, patients with HCC showed a preferential pattern of 4-mer end motifs. In particular, the abundance of plasma DNA motif CCCA was much lower in patients with HCC than in subjects without HCC. The aberrant end motifs were also observed in patients with other cancer types, including colorectal cancer, lung cancer, nasopharyngeal carcinoma, and head and neck squamous cell carcinoma. We further observed that the profile of plasma DNA end motifs originating from the same organ, such as the liver, placenta, and hematopoietic cells, generally clustered together. The profile of end motifs may therefore serve as a class of biomarkers for liquid biopsy in oncology, noninvasive prenatal testing, and transplantation monitoring.

Significance:

Plasma DNA molecules originating from the liver, HCC and other cancers, placenta, and hematopoietic cells each harbor a set of characteristic plasma DNA end motifs. Such markers carry tissue-of-origin information and represent a new class of biomarkers in the nascent field of fragmentomics.

This article is highlighted in the In This Issue feature, p. 627

In the plasmaMATCH trial, researchers performed circulating tumor DNA testing on patients with advanced breast cancer and matched those with ESR1, HER2, or AKT1 alterations to targeted therapies. Patients with HER2 and AKT1 mutations experienced response rates greater than 22% with durable benefit.

Escherichia coli DNA Pol I can carry out three enzymatic reactions: It possesses 5' -> 3' DNA polymerase activity and 3' -> 5' and 5' -> 3' exonuclease activity. Pol I can be cleaved by mild treatment with subtilisin into two fragments; the larger fragment is known as the Klenow fragment. The Klenow fragment retains the polymerizing activity and the 3' -> 5' exonuclease of the holo-enzyme but lacks its powerful 5' -> 3' exonuclease activity. These enzymes and their applications in molecular cloning are introduced here.

The Klenow fragment, which retains the template-dependent deoxynucleotide polymerizing activity and the 3' -> 5' exonuclease of the holo-enzyme but lacks its powerful 5' -> 3' exonuclease activity, is used to fill recessed 3' termini of dsDNA. In this protocol, fragments suitable as templates for the end-filling reaction are produced by digestion of DNA with an appropriate restriction enzyme. The Klenow enzyme is then used to catalyze the attachment of dNTPs to the recessed 3'-hydroxyl groups.

A cheap, lightweight smartphone-heated device can test for DNA in blood, urine and other samples in a fraction of the time it takes to test in a lab

Coffee has been linked to changes on our DNA that affect how active certain genes are. The finding may help explain some of coffee's touted health benefits

A north London teenager, 16, has been found guilty of kidnapping a stranger in the street and forcing him to withdraw money from a cash machine.

There are decisions being made right now that could have an effect on global populations for generations to come. As part of this project, we commissioned an artist to investigate some of the themes raised in the podcasts. This work of fiction imagines a future where gene editing has become mainstream and discusses the moral, ethical and political divides that this might create

Continue reading...

Scientists have created glowing flowers after editing the DNA of a plant.

A St. John’s genetics specialist has found DNA connections that link the long-vanished Beothuk people to contemporary people, almost two centuries after the last known Beothuk died. 

All the clues you need in one picture

An international team of researchers including the University of Adelaide, has completed the first large-scale study of DNA belonging to ancient humans of the central Andes in South America and found early genetic differences between groups of nearby regions, and surprising genetic continuity over thousands of years.

Agents with the FBI, Immigration and Customs Enforcement and the Wyoming Department of Criminal Justice arrested Wyoming Highway Patrol Trooper Franklin Joseph Ryle late yesterday in Douglas, Wyo., on criminal civil rights charges.

The U.S. Court of Appeals for the Fifth Circuit today rejected a challenge to the conviction of James Ford Seale, a former member of the White Knights of the Ku Klux Klan of Mississippi. Seale was convicted by a federal jury in Mississippi in 2007 and sentenced to three life terms in prison.

Additional charges and defendants have been added to a federal case arising from the abduction of a boy from his Las Vegas home in October 2008.

Franklin Joseph Ryle Jr., a former Wyoming Highway Patrol trooper, pleaded guilty on July 20, 2009, in federal court in Wyoming to depriving a man of his constitutional right to be free from unreasonable seizures by kidnapping him. Ryle also pleaded guilty to one count of using his firearm in relation to the crime.

“This officer abandoned his law enforcement role and engaged in a bizarre scheme to target innocent victims for his own personal gain. Officers who abuse their power in this way will be prosecuted to the fullest extent the law allows,” said Assistant Attorney General Thomas E. Perez of the Civil Rights Division.

Vagan Adzhemyan, 41, of Costa Mesa, Calif.; Suren Garibyan, 32, of North Hollywood, Calif.; and Galvin Shaun Gibson, 30, of Mira Loma, Calif., were charged in August 2009 with conspiracy to commit kidnapping and kidnapping.

“DNA evidence is one of the most powerful tools available to the criminal justice system, and these new steps will ensure the department can use DNA to the greatest extent possible to solve crimes and ensure the guilty are convicted,” Attorney General Holder said.

One hundred and two members and associates of transnational organized criminal groups operating in the United States have been charged in indictments unsealed today in Los Angeles; Santa Ana, Calif.; Miami and Denver.

Sheena Flores, 34, of Manassas, Va., pleaded guilty before U.S. District Judge Gerald Bruce Lee to one count of transmitting in foreign commerce, with intent to extort money, a communication containing a threat to injure another person.

Larry Lujan, 33, will receive a life prison sentence for a kidnapping that resulted in the death of a 16-year-old, after a federal jury today announced it could not reach a unanimous decision on whether to impose the death penalty.

Sheena Flores, 34, of Manassas, Va., was sentenced by U.S. District Judge Gerald Bruce Lee in the Eastern District of Virginia.

Larry Lujan, 33, was sentenced today to life in prison for a kidnapping that resulted in the death of a 16-year-old