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Precursor B cell phenotype Burkitt lymphoma/leukemia with IGH-MYC is a rare subtype of Burkitt lymphoma (BL). BL and B lymphoblastic leukemia/lymphoma (B-ALL/LBL) differ as regards treatment and the distinction between these two entities is crucial. Patients demonstrating a terminal deoxynucleotidyl transferase (TdT)-positive precursor B cell phenotype with IGH-MYC rearrangement have been reported to be molecularly distinct from BL and closer to B-ALL/LBL. We investigated the molecular characteristics of two cases of a rare but distinct TdT-negative precursor B cell phenotype BL. Both patients showed FAB L3 morphology with IGH-MYC translocation, but had precursor B cell immunophenotypes including dim to moderate expression of CD45 and absence of BCL6, CD20, monoclonal kappa, and lambda light chain expression. To characterize the molecular features, we performed exome sequencing and analyzed the breakpoint junction of the IGH-MYC rearrangement. We detected KMT2D mutations in both cases, a rarely acquired chromatin modifying gene mutation in BL. The breakpoint analysis revealed that the IGH-MYC rearrangement occurred due to an aberrant VDJ recombination in one case. The treatment protocols differed, including high-grade lymphoma treatment and standard B-ALL treatment. Complete remission was achieved in the patient who received B-ALL treatment. The degree of resemblance of BL and B-ALL differed between two cases, but the molecular pathogenesis and manifesting features of both TdT-negative precursor B cell phenotype BL case were distinct from classic BL, which indicates the need for a better understanding of this uncommon entity that does not fit in current diagnostic and classification categories.

Repairing of DNA termini is a crucial step in a variety of DNA handling techniques. In this study, we investigated mechanically-sheared DNA 3’-ends (MSD3Es) to establish an efficient repair method. As opposed to the canonical view of DNA terminus generated by sonication, we showed that approximately 47% and 20% of MSD3Es carried a phosphate group and a hydroxyl group, respectively. The others had unidentified abnormal terminal structures. Notably, a fraction of the abnormal 3’ termini (about 20% of the total) was not repaired after the removal of 3’ phosphates and T4 DNA polymerase (T4DP) treatment. To overcome this limitation, we devised a reaction, in which the 3’− > 5’ exonuclease activity of exonuclease III (3’− > 5’ exonuclease, insensitive to the 3’ phosphate group) was counterbalanced by the 5’− > 3’ polymerase activity of T4DP. This combined reaction, termed “SB-repairing” (for scrap-and-build repairing), will serve as a useful tool for the efficient repair of MSD3Es., Ohtsubo et al. show that mechanically sheared DNA 3’ terminus has 47% phosphate group, 20% hydroxyl group with remaining as abnormal termini, half of which is not repaired with T4 DNA polymerase treatment. They devise a method, scrap and build or SB-repairing, to efficiently repair 3’ DNA ends by combining phosphatase and exonuclease (scrap) followed by T4 DNA polymerase (build) reactions.

Salmonella is a widely distributed, extremely harmful bacteria, the presence of which requires confirmation via an on-site visual biosensor. In this study, we constructed a label-free, cascade amplification visualization biosensor for the sensitive and rapid detection of Salmonella enterica subsp. enterica serovar typhimurium based on the RDTG principle (recombinase polymerase amplification (RPA), duplex-specific enzyme (DSN) cleavage, terminal deoxynucleotidyl transferase (TdT) extension and G-quadruplexes output). Following DNA extraction of Salmonella spp., the first step in the construction involved the recognition and amplification of nucleic acids, carried out by RPA, to achieve the first signal amplification within 10 min. This RPA product was then specifically cleaved by DSN to produce a large number of small double-stranded DNA (dsDNA) products with 3′-OH within 15 min to achieve the second signal amplification. Thereafter, TdT was employed to empower these small 3′-OH dsDNA products to extend and produce a large number of long G-rich single-stranded DNAs (ssDNAs) within 20 min, thus realizing the third signal increase. These long G-rich ssDNA products displayed a color change that could be directly observed through the naked eye by adding H2O2/3,3′,5,5′-tetramethylbenzidine (TMB). The RDTG biosensor for the detection of Salmonella spp. has several advantages, including a low limit of 6 cfu/mL. It is an isothermal-free instrument, simple to operate, with a rapid detection time of less than 1.5 h. Furthermore, it can be visually characterized and quantified by a microplate reader to detect Salmonella spp., in food and environmental samples, and it has broad application prospects.

DNA damage seriously threats the genomic stability and is linked to mutagenesis, carcinogenesis, and cell death. DNA damage includes the isolated damage and the clustered damages, but few approaches are available for efficient detection of the clustered damage due to its spatial distribution. Herein, we present a single-molecule counting approach with the capability of detecting both the isolated and the clustered damages in genomic DNAs. We employed the repair enzymes to remove the DNA damage and used the terminal deoxynucleotidyl transferase (TdT) to incorporate biotinylated nucleotides and fluorescent nucleotides into the damage sites in a template-independent manner. The number of total oxidative damaged bases is quantified to be 7328-7406 in a single HeLa cell treated with 150 μM H

Prostate-specific antigen (PSA) is the only biomarker for the diagnosis of prostate cancer. So the PSA screening test is very important due to the high occurrence of prostate cancer in men. In this work, a label-free fluorescent method was developed based on terminal deoxynucleotidyl transferase (TdT) and G-quadruplex-thioflavin T complex for detecting PSA. In the absence of PSA, the PSA aptamer can be used as the primer for TdT extension reactions, resulting in the formation of G-quadruplexes and generation of strong fluorescent signals. After the addition of PSA, the PSA-aptamer complex prevented the TdT extension reaction due to steric hindrance, thus resulting in a poor fluorescent signal. The assay showed a wide linear range (0.1 to 80 pg/mL) and a detection limit of 0.086 pg/mL (S/N = 3). It also has good specificity for PSA determination and gives satisfactory results when applied to biological samples. Conceivably, its merits such as good selectivity and high sensitivity indicate that the proposed method has a promising application potential in the clinical diagnosis and treatment of prostate cancer. Graphical abstract.

DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications. Here, we describe a de novo enzymatic synthesis strategy designed for data storage which harnesses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) in kinetically controlled conditions. Information is stored in transitions between non-identical nucleotides of DNA strands. To produce strands representing user-defined content, nucleotide substrates are added iteratively, yielding short homopolymeric extensions whose lengths are controlled by apyrase-mediated substrate degradation. With this scheme, we synthesize DNA strands carrying 144 bits, including addressing, and demonstrate retrieval with streaming nanopore sequencing. We further devise a digital codec to reduce requirements for synthesis accuracy and sequencing coverage, and experimentally show robust data retrieval from imperfectly synthesized strands. This work provides distributive enzymatic synthesis and information-theoretic approaches to advance digital information storage in DNA., Adoption of DNA as a data storage medium could be accelerated with specialized synthesis processes and codecs. The authors describe TdT-mediated DNA synthesis in which data is stored in transitions between non-identical nucleotides and the use of synchronization markers to provide error tolerance.

As an important base excision repair (BER) enzyme, uracil-DNA glycosylase (UDG) can repair the uracil-induced DNA lesion and maintain the genomic integrity. Herein, we have proposed a sensitive UDG assay based on the terminal deoxynucleotidyl transferase (TdT)-assisted formation of fluorescent copper nanoclusters (CuNCs). In this study, a uracil-containing stem-loop DNA substrate is rationally designed with its 3′-end blocked with 2′, 3′-dideoxycytosine (ddC). UDG can remove the uracil in the DNA substrate to generate an apurinic/apyrimidinic (AP) site, which can then be specifically cleaved by endonuclease IV (Endo IV) to expose a 3′-OH terminus. TdT will initiate the template-free DNA extension along the exposed 3′-OH terminus to produce a quite long poly(T) tail, which will perfectly template the production of fluorescent CuNCs. By recording the fluorescence of the CuNCs, the UDG activity can be faithfully detected. In contrast, if UDG is absent, the 3′-ddC terminus of the DNA substrate cannot be recognized by TdT and thus no TdT-based extension and formation of CuNCs will occur. The use of ddC as a 3′-end blocker can greatly decrease the nonspecific DNA extension and improve the signal-to-noise ratio. Furthermore, TdT is a template-free and sequence-independent DNA polymerase, which can effectively catalyze the tailing process up to a maximum of thousands of thymines, and each tail can form many fluorescent CuNCs. Therefore, an ultrahigh sensitivity is achieved and as low as 0.00005 U/mL of UDG can be clearly detected. Moreover, with an additional AP site-contained poly(A) oligonucleotide during TdT-mediated extension, a branched amplification mechanism is also preliminarily devised, which can further push the detection limit of UDG to an extremely low level of 0.000002 U/mL.

Site-specific modification of synthetic and cellular RNA such as with specific nucleobases, fluorophores and attachment chemistries is important for a variety of basic and applied research applications. However, simple and efficient methods to modify RNA such as at the 3′ terminus with specific nucleobases or nucleotide analogs conjugated to various chemical moieties are lacking. Here, we develop and characterize a one-step enzymatic method to modify RNA 3′ termini using recombinant human polymerase theta (Polθ). We demonstrate that Polθ efficiently adds 30–50 2′-deoxyribonucleotides to the 3′ terminus of RNA molecules of various lengths and sequences, and extends RNA 3′ termini with an assortment of 2′-deoxy and 2′,3′-dideoxy ribonucleotide analogs containing functional chemistries, such as high affinity attachment moieties and fluorophores. In contrast to Polθ, terminal deoxynucleotidyl transferase (TdT) is unable to use RNA as a substrate altogether. Overall, Polθ shows a strong preference for adding deoxyribonucleotides to RNA, but can also add ribonucleotides with relatively high efficiency in particular sequence contexts. We anticipate that this unique activity of Polθ will become invaluable for applications requiring 3′ terminal modification of RNA and potentially enzymatic synthesis of RNA.

We describe a unique flow cytometer (TDI-SFC) for the immunophenotyping of low-abundance cells, particularly when cell counts are sample-limited and operationally difficult for analysis by fluorescence microscopy (>100 cells) or multiparameter flow cytometry (MFC

Terminal deoxynucleotidyl transferase (TdT) is a unique template-free polymerase that randomly adds multiple deoxyribonucleoside triphosphates (dNTPs) to the 3′-OH terminus of ssDNA. This characteristic makes TdT a versatile enzymatic tool in many fields. Moreover, aberrant TdT expression is a well-recognized biomarker of several leukemic diseases and is related to carcinogenesis. In this study, we developed a facile, rapid, label-free, and convenient assay for TdT detection. TdT-generated poly A tails formed a fluorescent enhancement complex in the presence of coralyne. To achieve a better signal-to-noise ratio, we used potassium thiocyanate (KSCN), instead of other halogen anions (KCl, KBr, KI, NaI) as the quenching agent of dissociate coralyne. Our results demonstrate that this assay is extremely facile, rapid, and label-free; at levels as low as 0.025 U/mL, TdT was distinctly detected within 55 min. And the determination of TdT activity in RBL-2H3 and Reh cells lysates exhibited a good sensing performance, demonstrating its potential applications in biochemical research and clinical diagnosis.

A DSN–RNAse–TdT–T7 exo probing system allows the detection of miRNA 21 with very high sensitivity (LOD = 2.57 fM) and selectivity—the result of (i) avoiding the false-positive signal from miRNA reacting with TdT polymerase and (ii) signal amplification occurring through a FRET-breaking mechanism involving T7 exo.

Mature B-cell neoplasms and immature or precursor B-cell neoplasms need to be distinguished because these patients usually require different therapeutic approaches. B-cell neoplasms that express TdT without unequivocal other features of immaturity may therefore present a diagnostic challenge. We describe 13 patients with TdT-positive aggressive B-cell lymphoma. The clinicopathologic features of these patients were highly heterogeneous, but for the purpose of this study we grouped these cases as follows: (1) de novo high-grade B-cell lymphoma with MYC, BCL2, and/or BCL6 rearrangements (double-hit or triple-hit lymphoma) with TdT expression. In this group we included two cases of de novo composite lymphoma in which there were components of diffuse large B-cell lymphoma and TdT-positive blastic B-cell lymphoma; (2) TdT-positive aggressive B-cell lymphoma arising in patients who previously had follicular lymphoma; (3) initial relapse of TdT-negative aggressive B-cell lymphoma in patients who previously had follicular lymphoma, followed by relapses in which the neoplasm acquired TdT expression; and (4) mature B-cell lymphomas that acquired TdT expression at relapse. This group included one case of EBV-positive diffuse large B-cell lymphoma and one case of pleomorphic variant mantle cell lymphoma. All patients in this study had an aggressive clinical course and a dismal outcome despite appropriate therapy. Rather than "squeezing" these cases into current World Health Organization classification categories, we suggest the use of a descriptive term such as high-grade B-cell lymphoma with TdT expression. In these tumors, the cytogenetic findings and poor prognosis of this patient subgroup suggest that these neoplasms need to be distinguished from B-lymphoblastic leukemia/lymphoma. Segregation of these neoplasms also may foster additional research on these neoplasms.

In this study, we develop a novel and reversibleelectrochemical impedance strategy for pH and terminal deoxynucleotide transferase (TdT) analysis based on the TdT-assisted generation of long enough cytosine (C)-rich DNAs. The formation of this special DNA is rationally designed on 5'-thiol DNA modified Au electrode surface, and TdT can catalyze the extension of this 3'-OH end to form a long C-rich DNA in the presence of deoxycytidine triphosphate (dCTP). Here, we discover a reversible process, in which the TdT-generated C-rich DNA maintains an irregular single chain state under neutral conditions and some stable DNA i-motifs (cascade i-motifs) are formed due to the partial protonation of C under acidic conditions. More importantly, the electrochemical impedance spectroscopy (EIS) response varies with the configuration change of the TdT-mediated C-rich DNA under different pH conditions. In view of this, a unique EIS switch ("on-off-on") is constructed faithfully with the configuration change, thus achieving pH analysis well. Additionally, the TdT activity can be also detected well by recording the EIS response, because it can catalyze the DNA tailing process up to hundreds of cytosines; on the contrary, if its inhibitor exists, TdT-based extension and formation of cascade i-motifs will not occur. Using this strategy, the detection of limit for TdT is 0.79 × 10

We developed a rapid and sensitive colorimetric biosensor based on competitive recognition between kanamycin (KAN), magnetic beads-kanamycin (MBs-KAN) and aptamer and terminal deoxynucleotidyl transferase (TdT)-mediated signal amplification strategy. In the absence of KAN, aptamers recognize MBs-KAN. TdT can amplify oligonucleotides to the 3'-OH ends of aptamers, with biotin-dUTP being embedded in the long single stranded DNA (ssDNA). Then the assay produced visual readout due to the horseradish peroxidase (HRP)-catalyzed color change of the substrate after the linkage between biotin and streptavidin (SA)-HRP. In the presence of KAN, however, aptamers tend to bind free KAN rather than MBs-KAN. In this case, aptamers are isolated by magnetic separation, resulting in the failure of signal amplification and catalytic signals. This competitive colorimetric sensor showed excellent selectivity toward KAN with the limit of detection (LOD) as low as 9 pM. And recovery values were between 93.8 and 107.8% when spiked KAN in milk and honey samples.

Objective Regulated cell death is key in the pathogenesis of persistent apical periodontitis. Here, we investigated the mechanisms of regulated cell death in osteoblast-like MG63 cells infected with Enterococcus faecalis OG1RF. Materials and methods MG63 cells were infected with live Enterococcus faecalis OG1RF at the indicated multiplicity of infection for the indicated infection time. We evaluated the cells by flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labelling assay, and lactate dehydrogenase release analysis; measured the activity of caspase-1/-3/-8/-9 and the release of interleukin-1β; and determined the expression of apoptosis-associated proteins and gasdermin D by apoptosis antibody array and western blotting. Results Enterococcus faecalis OG1RF reduced the mitochondrial membrane potential of the infected cells, increased the percentage of apoptotic and terminal deoxynucleotidyl transferase dUTP nick end labelling-positive cells, and enhanced lactate dehydrogenase release. The expression of caspase-3 and survivin and the activity of caspase-3/-8/-9 were upregulated, while the expression of death receptor 6 was downregulated. The activity of caspase-1/gasdermin D and the release of interleukin-1β remained unaltered. Conclusion Enterococcus faecalis OG1RF induced both intrinsic and extrinsic MG63 cell apoptosis via caspase-3/-8/-9 activation but did not activate the pyroptotic pathway regulated by caspase-1/gasdermin D.

Background Mesenchymal stem cells (MSCs) have been shown to enhance neurological recovery after stroke. A rat middle cerebral artery occlusion model was designed to assess neuroprotective effects of stroke pretreated MSCs on cerebral ischemia/reperfusion injury. Methods MSCs were isolated and cultured in medium with 10% fetal bovine serum, normal control serum, or stroke serum (SS). MSCs were then injected into rats (n = 6 in each group) 1 day after middle cerebral artery occlusion, and feeding continued for 28 days. A battery of behavioral tests, 2,3,5-triphenyltetrazolium chloride staining, hematoxylin-eosin staining, enzyme-linked immunosorbent assay, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay were used to assess neural injury. To detect enhancement of neuronal regeneration and angiogenesis, immunofluorescence and Western blotting were performed to assess expression of trophic factors and growth factors. Results After treatment, behavior of rats improved significantly. Infarction area, brain lesion, and apoptosis cells were significantly decreased in the SS-MSCs group compared with the other groups. SS-MSCs also modulated inflammation by attenuating inflammatory cytokines. Furthermore, the number of neurogenesis-positive cells and expression of trophic factors and growth factors were significantly higher in the SS-MSCs group compared with the others. MSCs cultured with fetal bovine serum and normal control serum showed differences in expression of trophic factors and growth factors, but the results were not as good as with SS-MSCs. Conclusions Administration of SS-MCSs after reperfusion led to neuroprotection by inducing the recovery process, including improving pathological changes, behavioral improvement, neurogenesis, suppression of apoptosis and inflammation, and angiogenesis.

ABSTRCT Photocathode-based assay is anti-interference for real sample detection. Photocathode produces low photocurrent signal and gives rise to poor sensitivity. Herein, a novel cathode photoelectrochemical (CPEC) sensing platform based on Ag-AgI-CNTs as photocathode material and K3[Fe(CN)6] as photoelectron acceptor was established. Since [Fe(CN)6]3- effectively accepted photoelectrons from Ag-AgI-CNTs, it greatly enhanced the CPEC response. Combining a surface multisite strand displacement amplification (SMSDA) strategy, the CPEC platform was applied for the activity assay of terminal deoxynucleotidyl transferase (TdT). In this proposal, oligo dT primer tethered on CPEC platform was in-situ extended to generate a polyA tail. Then the polyA tail formed a stable multi-point hybrid structure with the adjacent oligo dT. After launching the SMSDA, the CPEC platform was covered by more elongated polynucleotide chains and network, which acutely hampered the photoelectron transfer (eT) between photocathode and electron acceptor and caused a reduced photocurrent. The CPEC sensor possessed a satisfactory linear response from 6 × 10-5-0.1 U and a low detection limit of 1.1 × 10-5 U. The strategy offered a more specific and sensitive method for TdT activity assay. It was feasible in the field of TdT-based biochemical research, drug screening, and disease diagnosis.

Tumors undergo dynamic immunoediting as part of a process that balances immunologic sensing of emerging neoantigens and evasion from immune responses. Tumor-infiltrating lymphocytes (TIL) comprise heterogeneous subsets of peripheral T cells characterized by diverse functional differentiation states and dependence on T-cell receptor (TCR) specificity gained through recombination events during their development. We hypothesized that within the tumor microenvironment (TME), an antigenic milieu and immunologic interface, tumor-infiltrating peripheral T cells could reexpress key elements of the TCR recombination machinery, namely, Rag1 and Rag2 recombinases and Tdt polymerase, as a potential mechanism involved in the revision of TCR specificity. Using two syngeneic invasive breast cancer transplantable models, 4T1 and TS/A, we observed that Rag1, Rag2, and Dntt in situ mRNA expression characterized rare tumor-infiltrating T cells. In situ expression of the transcripts was increased in coisogenic Mlh1-deficient tumors, characterized by genomic overinstability, and was also modulated by PD-1 immune-checkpoint blockade. Through immunolocalization and mRNA hybridization analyses, we detected the presence of rare TDT+RAG1/2+ cells populating primary tumors and draining lymph nodes in human invasive breast cancer. Analysis of harmonized single-cell RNA-sequencing data sets of human cancers identified a very small fraction of tumor-associated T cells, characterized by the expression of recombination/revision machinery transcripts, which on pseudotemporal ordering corresponded to differentiated effector T cells. We offer thought-provoking evidence of a TIL microniche marked by rare transcripts involved in TCR shaping.

Oxidative stress (OS) plays a significant role in the etiology of male infertility, resulting in the impairment of male reproduction. This condition, characterized by an imbalance in the levels of oxidizing and antioxidant species in the seminal fluid, has a harmful impact on sperm functions and DNA integrity. The present study aimed to evaluate the anti-genotoxic action of ellagic acid, a polyphenolic molecule of natural origin having a powerful antigenotoxic, anti-inflammatory and antiproliferative role. An OS condition was induced in vitro by incubating normozoospermic human semen samples in benzene for 45, 60 and 90 min. DNA integrity was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay, RAPD-PCR was performed to calculate the genome template stability, while the percentage of intracellular reactive oxygen species (ROS) was assessed by the 2ʹ, 7ʹ-dichlorofluorescein assay. Our results showed that ellagic acid has a consistent protective effect on DNA integrity, as well as on sperm vitality and motility, by counteracting generation of intracellular ROS. The results of this study suggest ellagic acid as a suitable molecule to protect sperm DNA from oxidative stress, with a potentially significant translational impact on the management of the male infertility.

A method is presented that uses photoinduced electron transfer (PET) for the determination of microRNAs (miRNAs) in clinical serum samples and complicated cell samples by using a smartphone. miRNA-21 is adopted as a model analyte. A 3'-phosphorylated DNA probe containing AgNCs is synthesized and hybridized with miRNA-21. Subsequently, the probe is cleaved specifically by duplex-specific nuclease to form 3'-hydroxylated products, then extended by terminal deoxynucleotidyl transferase (TdT) with superlong G for G-quadruplex/hemin units fabrication. In this way, PET occurred between AgNCs and produced G-quadruplex/hemin units, leading to the fluorescence quenching of AgNCs. Notably, the fluorescence images can be captured and translated into digital information by smartphone, resulting in a direct quantitative determination of miRNA. As a result, our strategy for miRNA assay is achieved with a satisfactory detection limit of 1.43 pM. Interestingly, TdT-propelled G-quadruplex/hemin units as multiple electron acceptors promote the sensitivity of miRNA monitoring. Different miRNAs assays are realized by adjusting the complimentary sequences of DNA probe. These qualities not only broaden the practical application of PET-based strategy, but also provide a new insight into the nucleic acid detection. Schematic representation of AgNCs and enzyme-propelled photoinduced electron transfer strategy. It has been successfully applied for detection of miRNA by image analysis software. The method displays portability and accuracy for miRNA determination, meeting the potential for biochemical and clinical applications in resource-limited settings.

New storage technologies are needed to keep up with the global demands of data generation. DNA is an ideal storage medium due to its stability, information density and ease-of-readout with advanced sequencing techniques. However, progress in writing DNA is stifled by the continued reliance on chemical synthesis methods. The enzymatic synthesis of DNA is a promising alternative, but thus far has not been well demonstrated in a parallelized manner. Here, we report a multiplexed enzymatic DNA synthesis method using maskless photolithography. Rapid uncaging of Co2+ ions by patterned UV light activates Terminal deoxynucleotidyl Transferase (TdT) for spatially-selective synthesis on an array surface. Spontaneous quenching of reactions by the diffusion of excess caging molecules confines synthesis to light patterns and controls the extension length. We show that our multiplexed synthesis method can be used to store digital data by encoding 12 unique DNA oligonucleotide sequences with video game music, which is equivalent to 84 trits or 110 bits of data., Writing data in DNA is still a bottleneck due to the reliance on chemical synthesis methods. Here the authors report multiplexed enzymatic DNA synthesis using maskless photolithography.

Chromosomal breakpoints involving the MYC gene locus, frequently referred to as MYC rearrangements (MYC - R+), are a diagnostic hallmark of Burkitt lymphoma and recurrent in many other subtypes of B-cell lymphomas including follicular lymphoma, diffuse large B-cell lymphoma and other high-grade B-cell lymphomas and are associated with an aggressive clinical course. In remarkable contrast, in MCL, only few MYC - R+ cases have yet been described. In the current study, we have retrospectively analysed 16 samples (MYC - R+, n = 15, MYC - R-, n = 1) from 13 patients and describe their morphological, immunophenotypic and (molecular) genetic features and clonal evolution patterns. Thirteen out of fifteen MYC - R+ samples showed a non-classical cytology including pleomorphic (centroblastic, immunoblastic), anaplastic or blastoid. MYC translocation partners were IG-loci in 4/11 and non-IG loci in 7/11 analysed cases. The involved IG-loci included IGH in 3 cases and IGL in one case. PAX5 was the non-IG partner in 2/7 patients. The MYC - R+ MCL reported herein frequently displayed characteristics associated with an aggressive clinical course including high genomic-complexity (6/7 samples), frequent deletions involving the CDKN2A locus (7/10 samples), high Ki-67 proliferation index (12/13 samples) and frequent P53 expression (13/13 samples). Of note, in 4/14 samples, SOX11 was not or only focally expressed and 3/13 samples showed focal or diffuse TdT-positivity presenting a diagnostic challenge as these features could point to a differential diagnosis of diffuse large B-cell lymphoma and/or lymphoblastic lymphoma/leukaemia.

Background High-grade B-cell lymphoma with rearrangements of MYC and BCL2 and/or BCL6 is an aggressive mature B-cell neoplasm, whereas B-lymphoblastic lymphoma is immature cell proliferation, with a frequent positivity for terminal deoxynucleotidyl transferase. The transformation of a low-grade follicular lymphoma into a lymphoblastic neoplasm expressing terminal deoxynucleotidyl transferase is a very rare event. Case presentation A 55-year-old Caucasian man was followed for a grade 1–2 follicular lymphoma carrying a t(14;18) IGH/BCL2+ and was initially treated with R-CHOP. The follicular lymphoma presented two relapses. In the third relapse, the patient had multiple lymphadenopathy and ascites, which motivated a retroperitoneal biopsy and an ascitic tap. These samples were analyzed by histological, cytological, flow cytometric, cytogenetic, and molecular assessments. The patient died of a multiple organ dysfunction syndrome 2 weeks after his third relapse. The biopsy revealed a diffuse proliferation made up of two types of tumor cells: centroblasts (Bcl-6-positive) and immature cells (terminal deoxynucleotidyl transferase-positive). Flow cytometric analysis confirmed the immature phenotype, with an expression of terminal deoxynucleotidyl transferase, combined with a loss of membrane immunoglobulins. The cytogenetic analysis performed on the ascites revealed a clonal evolution characterized by a t(8;22)(q24;q11) MYC+ translocation not previously detected in follicular lymphoma. Fluorescence in situ hybridization confirmed the double rearrangement of the BCL2 and MYC genes. Polymerase chain reactions and sequencing were used to study the clonal relationship between follicular lymphoma and the secondary tumors. The IGVH gene rearrangement revealed a unique clonal rearrangement involving an IGVH4–59 subset in all three specimens. Conclusion These findings suggest a clonal relationship between the two types of lymphoma cells. Furthermore, they support the transformation of an acute follicular lymphoma into a composite lymphoma combining a high-grade B-cell lymphoma and a lymphoblastic neoplasm expressing terminal deoxynucleotidyl transferase. This case report highlights the possible transformation of follicular lymphoma into a highly aggressive and immature proliferation.

In this work, a novel electrochemical biosensor is developed for facile and highly sensitive detection of lipopolysaccharide (LPS) based on collaboration of dual enzymes for multiple-stages signal amplification. Through ingenious design, the specific recognition of target LPS is transformed to the exonuclease III (Exo III)-assisted interface DNA cycling collaborated with the terminal deoxynucleotidyl transferase (TdT)-catalyzed DNA extension, finally inducing significant electrochemical signal concerned with the concentration of LPS. This paper mainly discusses the detection principle, optimization of key factors, and the analytical performance of the biosensor. With the efficient signal amplification, the biosensor shows high sensitivity with a good linearity and a low limit of detection of 1 pg mL−1 for LPS. Moreover, the developed biosensor can clearly discriminate LPS from interferents and show high specificity for LPS detection. This biosensor has also been successfully employed to measure LPS in real food samples, suggesting potential opportunity for application in food safety detection.

Background. The expression of terminal deoxynucleotidyl transferase (TdT) in myoepithelial cells (MECs) within the breast was recently incidentally observed in our routine practice. This study aimed to elucidate the expression of TdT in MECs. Methods. TdT immunostaining was performed on 180 mammary, 89 cutaneous, and 94 salivary tissues or lesions. Other myoepithelial markers, including P63, calponin, and SMA as well as double staining for TdT and calponin, were also evaluated in some cases. Selected lesions with basal or myoid differentiation were also included in the investigation. Results. MECs were positive for TdT in mammary lesions that contained MECs (132/135) but negative when they did not contain MECs (45/45). MECs in sweat glands (24/30) and their neoplastic counterparts, including those in hidradenoma papilliferum (2/9), spiradenoma (6/6), and cutaneous mixed tumor (9/9), showed weak to moderate TdT positivity. MECs were variably immunolabeled for TdT in salivary or salivary gland–type tumors with myoepithelial differentiation (pleomorphic adenoma, 24/25; basal cell adenoma, 6/7; adenoid cystic carcinoma, 7/7; Warthin tumor, 0/6; mucoepidermoid carcinoma, 0/8; acinic cell carcinoma, 0/4), but MECs in normal salivary gland barely stained for TdT (30/32). Conclusions. Our findings indicate that TdT may be eligible as an additional auxiliary immunohistochemical marker as P63, but not a surrogate, to identify the MECs in the breast with limited cross-reactivity, particularly in lesions with a prominent proportion of MECs. Positivity for TdT, along with other relevant markers, in a subset of sweat gland lesions and salivary tumors may contribute to their diagnosis.

Objectives Merkel cell carcinoma is a rare but very aggressive cutaneous tumor. We evaluated the prognostic potential of B-cell markers (terminal deoxynucleotidyl transferase [TdT], PAX5, CD117), follicular stem cell markers (CK15, CK19), p63, p53, RB, and Merkel cell polyomavirus (MCPyV; CM2B4) in 136 primary cutaneous Merkel cell carcinomas. Methods Clinical, histopathologic, and immunohistochemical analyses were performed. The results were correlated with patient outcomes by Fisher exact test, log-rank tests, and Cox multivariate models. Results By Fisher exact test, although TdT significantly correlated with both lack of progression (P = .0087) and alive status (P = .0056), MCPyV status correlated only with alive status (P = .031). In univariate analyses, TdT, MCPyV, and RB significantly correlated with improved overall survival, whereas p63 and CK15 correlated with worse overall survival. However, in multivariate analyses, only TdT expression remained as an independent predictor of improved overall survival, Merkel cell carcinoma-specific survival, and progression-free survival. By linear regression analyses, significant correlations between MCPyV vs TdT, PAX5, and CD117 were observed. Conclusions TdT expression is a potential marker of better survival in Merkel cell carcinoma. Expression of B-cell markers is associated with MCPyV, suggesting that clonal viral integration might play a role in the expression of these markers.

Exosomes have received increasingly significant attention and have shown great clinical value as biomarkers for a number of diseases. However, there is still a lack of a highly sensitive and visualized method for the detection of exosomes in numerous samples simultaneously. Here, we developed a high-throughput, colorimetric and simple method to detect colorectal cancer (CRC) exosomes based on terminal deoxynucleotidyl transferase (TdT)-aided ultraviolet signal amplification. Anti-A33, a CRC exosomal protein marker, was selected as a capture probe, and a facility-prepared EpCAM (CRC exosomal protein) aptamer-Au-primer complex was used as a signal probe. After the CRC exosomes were captured onto the surface of 96-well plates, the primer was extended to the poly(biotin-adenine) chains with the help of TdT, resulting in an increase in the binding amount of avidin-modified horseradish peroxidase (Av-HRP) for H2O2-mediated oxidation of 3,3′,5,5′-tetramethyl benzidine (TMB) in enzyme-linked aptamer-sorbent assay (ELASA). The results showed that the incorporation of ploy(biotin-A) enabled approximately 10.4-fold signal amplification. This approach achieved a linear range of 9.75 × 103–1.95 × 106 particles/μL for CRC cell-derived exosomes. The feasibility of the developed assay was evaluated using clinical serum samples. CRC patients (n = 16) could be clearly and successfully distinguished from healthy individuals (n = 9). Furthermore, this proposed platform holds considerable potential for the detection of different targets, simply by changing the aptamer and antibody

In this work, a new kind of dendritically amplified fluorescent signal probe on SiO

A seven-color panel was used to detect minimal residual disease (MRD) in T cell acute lymphoblastic leukemia (T-ALL) via flow cytometry (FCM). Its availability and clinical significance were studied in T-ALL patients with newly diagnosed (n = 64), relapsed (n = 48) and morphologically complete remission (n = 103). The following four features were used to identify immature cCD3+ T cells: CD34+, TdT+, but mCD3-/dim+, and CD45dim+. Among these features, either TdT or CD34 expression was the most useful and were found in 93.8% of patients at diagnosis and 86.7% of patients who relapsed. Although some of the immature markers had disappeared in 23 of 59 cases after therapy, only one case presented with a false negative MRD. Of the 74 consecutive patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT), MRD-positive patients showed a higher relapse rate, a higher cumulative incidence of relapse at 4 years and a shorter median relapse-free survival than MRD-negative patients at post-HSCT(72.7% vs 17.3%, P = 0.000; 100% vs 19.9%, P 0.0001; and 16 months vs undefined, P 0.0001). We demonstrated that this panel could be applied to97% of T-ALL patients to detect MRD and predict relapse after allo-HSCT even in the absence of the initial immunophenotype.

Phospholipids are asymmetrically distributed across mammalian plasma membrane with phosphatidylserine (PS) and phosphatidylethanolamine concentrated in the cytoplasmic leaflet of the membrane bilayer. This asymmetric distribution is dependent on a group of P4-ATPases named PS flippases. The proper transport and function of PS flippases require a β-subunit transmembrane protein 30 A (TMEM30A). Disruption of PS flippases led to several human diseases. However, the roles of TMEM30A in the central nervous system remain elusive. To investigate the role of Tmem30a in the cerebellum, we developed a Tmem30a Purkinje cell (PC)-specific knockout (KO) mouse model. The Tmem30a KO mice displayed early-onset ataxia and progressive PC death. Deficiency in Tmem30a led to an increased expression of Glial fibrillary acidic protein and astrogliosis in regions with PC loss. Elevated C/EBP homologous protein and BiP expression levels indicated the presence of endoplasmic reticulum stress in the PCs prior to visible cell loss. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis suggested that apoptotic cell death occurred in the cerebellum. Our data demonstrate that loss of Tmem30a in PCs results in protein folding and transport defects, a substantial decrease in dendritic spine density, increased astrogliosis and PC death. Taken together, our data demonstrate an essential role of Tmem30a in the cerebellum PCs.

Terminal deoxynucleotidyl transferase (TdT) is expressed on precursor lymphoblastic neoplasms and some acute myeloid leukemia (AML) cells. The clinical impact of TdT expression on AML outcomes remains unclear. Here, we conducted a retrospective analysis to identify prognostic implications of TdT expression in AML with an intermediate-risk karyotype. Forty-eight cases of intermediate-risk AML were enrolled. TdT positivity was defined as expression on ≥ 10% of the gated cells. Of 48 cases, 12 (25%) were positive for TdT [median expression rate of TdT 0.9% (range 0–86.9%)]. No significant differences in patient characteristics or complete remission rate were observed between TdT-positive and TdT-negative cases. The probability of overall survival (OS) and event-free survival (EFS) at 1 year was not significantly different between TdT-positive and TdT-negative cases (OS: 58.3% vs. 65.2%, p = 0.32; EFS: 33.3% vs. 57.1%, p = 0.06). Relapse-free survival (RFS) probability at 1 year was significantly lower for TdT-positive than TdT-negative cases (10% vs. 71.3%, p = 0.002). Multivariate analyses revealed that TdT positivity was an independent significant adverse factor for RFS [hazard ratio: 3.309, 95% confidence interval: 1.334–8.209, p = 0.009]. Our results suggest that TdT expression is associated with increased risk of relapse in patients with intermediate-risk AML.

Mitochondrial dysfunction plays a pivotal role in doxorubicin (DOX)-induced cardiomyopathy. Cytochrome c oxidase subunit 5A (COX5A) is a nuclear-encoded subunit of the terminal oxidase involved in mitochondrial electron transport. Although COX5A appears to play a key role in modulating the physiological activity of COX and involve in energy metabolism, the involvement of COX5A in DOX-induced cardiotoxicity remains unclear. In this study, we showed that COX5A was significantly downregulated by DOX treatment of H9c2 cells. Overexpression of COX5A in H9c2 cells effectively attenuated DOX-induced apoptosis. Meanwhile, DOX-induced decrease in mitochondrial membrane potential could be reserved by COX5A overexpression. Furthermore, COX5A overexpression relieved the DOX-induced suppression of mitochondrial respiration, due an increase in basal respiration, maximal respiration, ATP production, and spare respiratory capacity. These findings indicate that up-regulation of COX5A may inhibit the apoptosis and alleviate the mitochondrial dysfunction of DOX-treated H9c2 cells. Thus, COX5A may have potential for clinical use as a therapeutic target in DOX-induced cardiotoxicity.

Tieppo et al. show that the human fetal thymus generates invariant γδ T cells with programmed effector functions. This is due to an intrinsic property of fetal HSPCs caused by high expression of the RNA-binding protein Lin28b., In the mouse thymus, invariant γδ T cells are generated at well-defined times during development and acquire effector functions before exiting the thymus. However, whether such thymic programming and age-dependent generation of invariant γδ T cells occur in humans is not known. Here we found that, unlike postnatal γδ thymocytes, human fetal γδ thymocytes were functionally programmed (e.g., IFNγ, granzymes) and expressed low levels of terminal deoxynucleotidyl transferase (TdT). This low level of TdT resulted in a low number of N nucleotide insertions in the complementarity-determining region-3 (CDR3) of their TCR repertoire, allowing the usage of short homology repeats within the germline-encoded VDJ segments to generate invariant/public cytomegalovirus-reactive CDR3 sequences (TRGV8-TRJP1-CATWDTTGWFKIF, TRDV2-TRDD3-CACDTGGY, and TRDV1-TRDD3-CALGELGD). Furthermore, both the generation of invariant TCRs and the intrathymic acquisition of effector functions were due to an intrinsic property of fetal hematopoietic stem and precursor cells (HSPCs) caused by high expression of the RNA-binding protein Lin28b. In conclusion, our data indicate that the human fetal thymus generates, in an HSPC/Lin28b-dependent manner, invariant γδ T cells with programmed effector functions., Graphical Abstract

As one of the key initiators of the base excision repair process, uracil-DNA glycosylase (UDG) plays an important role in maintaining genomic integrity. It has been found that aberrant expression of UDG is associated with a variety of diseases. Thus, accurate and sensitive detection of UDG activity is of critical significance for biomedical research and early clinical diagnosis. Here, we developed a novel fluorescent sensing platform for UDG activity detection based on a terminal deoxynucleotidyl transferase (TdT) and T7 exonuclease (T7 Exo)-aided recycling amplification strategy. In this strategy, only two DNA oligonucleotides (DNA substrate containing one uracil base and Poly dT probe labeled with a fluorophore/quencher pair) are used. UDG catalyzes the removal of uracil base from the enclosed dumbbell-shape DNA substrate to give an apyrimidinic site, at which the substrate oligonucleotide is cleaved by endonuclease IV. The released 3'-end can be elongated by TdT to form a long deoxyadenine-rich (Poly dA) tail, which may be used as a recyclable template to initiate T7 Exo-mediated hybridization-digestion cycles of the Poly dT probe, giving a significantly enhanced fluorescence output. The proposed UDG-sensing strategy showed excellent selectivity and high sensitivity with a detection limit of 1.5 × 10-4 U/mL. The sensing platform was also demonstrated to work well for UDG inhibitor screening and inhibitory activity evaluation, thus holding great potential in UDG-related disease diagnosis and drug discovery. The proposed strategy can be easily used for the detection of other DNA repair-related enzymes by simply changing the recognition site in DNA substrate and might also be extended to the analysis of some DNA/RNA-processing enzymes, including restriction endonuclease, DNA methyltransferase, polynucleotide kinase, and so on.

A versatile flow cytometric strategy is developed for the sensitive detection of plant microRNA (miRNA) by coupling the target-templated click nucleic acid ligation (CNAL) with on-bead terminal enzymatic DNA polymerization (TEP). Unlike ligase-catalyzed ligation reaction, the plant miRNA-templated enzyme-free CNAL between two single-stranded DNA (ssDNA) probes, respectively modified with Aza-dibenzocyclooctyne (Aza-DBCO) and N3, can not only simplify the operation, but also achieve a much higher ligation efficiency. More importantly, the undesirable nonspecific ligation between the Aza-DBCO- and N3-modified ssDNA, can be effectively eliminated by adding Tween-20, which allows the use of cycling CNAL (CCNAL) in a background-free manner. So each plant miRNA can template many rounds of CNAL reaction to produce numerous ligation products, forming efficient signal amplification. The ligated ssDNA can be anchored on the magnetic beads (MBs) with the 3′-OH termini exposed outside. Then terminal deoxynucleotidyl ...

There is a growing demand for sustainable methods in research and development, where instead of hazardous chemicals, an aqueous medium is chosen to perform biological reactions. In this Perspective, we examine the history and current methodology of using enzymes to generate artificial single-stranded DNA. By using traditional solid-phase phosphoramidite chemistry as a metric, we also explore criteria for the method of template-independent enzymatic oligonucleotide synthesis (TiEOS). As its key component, we delve into the biology of one of the most enigmatic enzymes, terminal deoxynucleotidyl transferase (TdT). As TdT is found to exponentially increase antigen receptor diversity in the vertebrate immune system by adding nucleotides in a template-free manner, researchers have exploited this function as an alternative to the phosphoramidite synthesis method. Though TdT is currently the preferred enzyme for TiEOS, its random nucleotide incorporation presents a barrier in synthesis automation. Taking a closer look at the TiEOS cycle, particularly the coupling step, we find it is comprised of additions > n+1 and deletions. By tapping into the physical and biochemical properties of TdT, we strive to further elucidate its mercurial behavior and offer ways to better optimize TiEOS for production-grade oligonucleotide synthesis.

Herein, we proposed a terminal deoxynucleotidyl transferase (TdT), a potential biomarker of lymphoid tumors, responsive fluorescence polarization (FP)- sensing protocol based on framework nucleic acid (FNA)-wrapped protein-inorganic hybrid nanoflowers. To achieve this goal, a pair of poly-A-composed extension primers (EPa and EPb) was designed, and protein-inorganic hybrid nanoflowers were synthesized by a biomineralization reaction. EPa was labeled with carboxyfluorescein (FAM) fluorophore to create the preliminary FP signal. EPb was labeled with biotin to conjugate with hybrid nanoflowers. Upon introduction of TdT into the dTTP pool, both EPa and EPb can be catalyzed by TdT to incorporate numerous T bases, thereby facilitating intermolecular hybridization between ‘A’ and ‘T’ bases. The final assembled FNA-wrapped hybrid nanoflowers with greatly enhanced molecular volume and weight restrict the free rotation of attached FAMs, causing a great FP enhancement from a designated three-stage FP amplification. Under optimized conditions, the TdT can be detected with a detection limit of 0.023 U/mL and a linear detection from 0.1 U/mL to 100 U/mL within 20 min. As a proof-of-concept study, the first exploitation of FNA and protein-inorganic nanoflowers to improve the FP signal and the merit of FP without sample separation and washing opens a new avenue for biochemical study and disease diagnosis.