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To determine if Exendin-4 could be a therapeutic agent for burn-induced hyperglycemia.Male Balb/c mice received a bolus of Exendin-4 intraperitoneally immediately after 15% total body surface area scald injury. Tail glucose levels were recorded and T-cell functions were analyzed at 4 h and 24 h postburn (pb). Pancreatic pathology was observed consecutively. The secretions of cytokines were detected in serum, spleen, and lung. Apoptosis of splenic CD3+ T-cells was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and flow cytometry.Although Exendin-4 could attenuate burn-induced hyperglycemia in mice at 4 h pb, it accelerated their survival dose dependently with progressive depletion of splenocyte number. T-cell function underwent two-phasic changes following Exendin-4 treatment. Compared to placebo mice, T-cell from Exendin-4-treated mice was manifested with increased proliferation, while decreased IL-2 secretion and lower ratio of IL-4/IFN-γ at 4 h pb. However, at 24 h pb, it showed decreased proliferation, while increased IL-2 secretion and higher ratio of IL-4/IFN-γ. Exendin-4 could elicit higher circulating IL-6 and IL-10 levels at 4 h pb, which were pronounced in the lung at 24 h pb. In the meanwhile, severe inflammation could be found in the pancreas. At 24 h pb, the numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling or caspase-3 positive cells and the apoptosis of CD3+ T-cells were significantly increased in the spleens of Exendin-4 mice relative to placebo mice.These data support a pathogenic role of Exendin-4 signaling during thermal injury, warning against its clinical application in acute insults.

Excessive oligodendrocyte precursor cell (OPC) apoptosis occurs during intrauterine infection-induced white matter injury (WMI) in premature infants, preventing excessive apoptosis of OPCs is one of the mechanisms protecting WMI. Micro-RNA-21-5p (miR-21-5p) mediating anti-apoptotic activity was observed in other diseases. Therefore, the aim of this study was to determine whether miR-21-5p protects against WMI by modulating phosphatase and tensin homologue deleted on chromosome 10/phosphatidylinositol-3-kinase/protein kinase B (PTEN/PI3K/Akt) signalling pathway.A lipopolysaccharide (LPS)-induced neonatal Sprague-Dawley (SD) rat model of preterm WMI was established. To explore the effect of miR-21-5p on WMI, we intraventricularly injected miR-21-5p agomir and miR-21-5p antagomir to activate or inhibit endogenous miR-21-5p. Immunofluorescent labelling of myelin basic protein, immunohistochemical labelling of 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), and terminal deoxynucleotidyl transferase dUTP nick end labelling assays were conducted to observe pathological white matter changes. The antibody of anti-oligodendrocyte marker 4 (O4) was used to specifically recognise OPCs. The expressions of miR-21-5p and PTEN mRNA in the brain were detected with quantitative real-time polymerase chain reaction (qRT-PCR). PTEN, Akt, and phosphorylated Akt (p-Akt) protein levels were assayed with western blotting, and apoptotic proteins associated with PI3K/Akt signalling were quantified.Intense white matter dysplasia and excessive OPC apoptosis were observed in the brains of rats with WMI. When the miR-21-5p agonist miR-21-5p agomir was used in the WMI group, apoptosis of OPCs was significantly reduced, and myelin maturation increased. MiR-21-5p agomir relieved WMI. MiR-21-5p agomir inhibited the mRNA and protein expression of PTEN, increased p-Akt phosphorylation, and decreased the expression and activation of related apoptotic proteins.On the other hand, the administration of miR-21-5p specific blocker, miR-21-5p antagomir, reduced the level of p-AKT, increased OPC apoptosis, and worsened WMI.Our findings revealed that miR-21-5p agomir had anti-OPC over-apoptotic effects and enhanced myelin development in WMI by modulating the PTEN/Akt signalling pathway.

Little is known about B-lymphoblastic leukemia (B-ALL) that lacks expression of terminal deoxynucleotidyl transferase (TdT). To address this, we performed the largest study to date of TdT-negative B-ALL using data from St. Jude Total XV and XVI clinical trials. Compared to TdT-positive B-ALL (n = 896), TdT-negative B-ALL (n = 21) was associated with younger age (median, 1.4 versus 6.8 years, P

We describe an isothermal, single-reaction, and one-step method for signal-on quantification of terminal deoxynucleotidyl transferase (TdT) activity based on the periodic elongation and assembly of polythymine embedded activatable molecular beacon (PTA-MB) into DNA nanostructures. PTA-MB is easily designed according to the rule of the conventional molecular beacon (MB) but engineered with a polyT composed loop. Upon exposure to the specific target TdT, the MB is first elongated with an adenine-rich (A-rich) long chain so that it can then act as the anchoring substrate to capture many original PTA-MBs along its strand. Their unfolding contributes to preliminary fluorescence emission. Significantly, the assembled PTA-MBs can also be elongated and hybridized with residual free PTA-MBs for the second round of signal amplification. Accordingly, multiple rounds of elongation, assembly, and activation of initial PTA-MBs can lead to the formation of DNA nanostructures and induce a dramatically enhanced fluorescence signal for qualitative and quantitative evaluation of TdT activity. The final assay indicated a limit of detection (LOD) of 0.042 U mL−1 TdT and showed excellent selectivity for TdT versus other common enzymes. Moreover, the practical applicability was validated by direct/absolute quantification of TdT in real biological specimens and accurate monitoring of the activity of TdT pretreated by low/high temperature and heavy metal ions. These findings demonstrated that this functional PTA-MB and its unique assembly behavior is most likely to promote the study of oligonucleotide probe-based DNA assembly, providing a reliable, convenient, and universal platform for precise and point-of-care monitoring of various biomolecules.

Peptide nucleic acid (PNA) is an amide based structural nucleic acid mimic with potential applications in gene therapeutic drug discovery. in the present study, we evaluated and compared the effects on gene expression, cell viability and apoptosis of two antisense PNA-D-octaarginine conjugates, targeting sequences at the AUG translation start site or the 5 '-UTR of the TdT (terminal deoxynucleotidyl transferase) gene, as well as a sense oligomer corresponding to the 5 '-UTR-antisense, in Molt-4 cells. The protein level of TdT was determined by flow cytometry, and qPCR was used for mRNA expression analysis. Mismatch PNAs were used as control to address the sequence/target spcifity of the biological effects. The results showed that treatment with the AUG- and to slightly lesser extent with the 5'-UTR-antisense PNAs reduced the TdT mRNA as wel as the protein level, whereas only very low effect was observed for the 5 '-UTR-sense PNA. A parallel effect was observed on reduced cell survival and increased rate of apoptosis. Our findings suggest that antisense PNAs can inhibit expression of the TdT gene and induce apoptosis in Molt-4 cells. (C) 2020 Elsevier B.V. All rights reserved., Molecular Medicine Research Center, Tabriz University of Medical Sciences, Tabriz [TBZMED.REC.1394.1104], This work was supported by Molecular Medicine Research Center, Tabriz University of Medical Sciences, Tabriz with the ethical code of TBZMED.REC.1394.1104.

Efficient protocols were developed for the synthesis of a new compounds - nucleoside 5'-α-iminophosphates using the Staudinger reaction. These substances are alpha-phosphate mimetic nucleotide in which an oxygen atom is replaced by a corresponding imino (=N-R)-group. Various 5'-iminomonophosphates of nucleosides were obtained. A chemical method for the synthesis of triphosphate derivatives based on the iminomonophosphates has been designed. Thymidine 5'-(1,3-dimethylimidazolidin-2-ylidene)-triphosphate (ppp(DMI)T) was synthesized, its hydrolytic stability and substrate properties in relation to some DNA polymerases was firstly studied. It was shown that ppp(DMI)T can serve as substrate for enzyme catalyzed template-independent DNA synthesis by human terminal deoxynucleotidyltransferase TdT.

Bisphenol A (BPA) is a widely used endocrine disrupter that causes male reproductive dysfunction in humans and rodents. Diabetes-induced hyperglycemia alters spermatogenesis and antioxidant status, which negatively impacts male fertility in adults. Zinc (Zn) deficiency is a global health concern maintaining the testicular structure and functions in developing gonads. The present experiment was designed to investigate the role of Zn deficiency on BPA-induced germ cell and male gonadal toxicity in diabetic conditions. Rats were randomly divided into eight different groups - control (normal feed and water), BPA (10 mg/kg/day), ZDD (fed with a Zn-deficient diet), DIA (diabetic), BPA+ZDD, BPA+DIA, ZDD+DIA and BPA+ZDD+DIA for four weeks. Animals' body and organ weight, sperm count, motility and sperm morphology were examined; testes and epididymis histopathology were investigated. Testicular DNA damage and sperm apoptosis were evaluated by halo and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays respectively. Testicular catalase and octamer-binding transcription factor 4 (OCT4) expressions were evaluated by western blot analysis. The present results demonstrated that dietary Zn-deficient condition significantly increased the BPA-induced testicular, epididymal and sperm toxicity in diabetic rats due to hypogonadism, increased sperm abnormalities, epididymis, testicular structure and DNA damages, sperm apoptosis as well as decreased testicular catalase and OCT4 expressions. The present results revealed that dietary Zn-deficient condition exacerbated the BPA-induced testicular and epididymal toxicity as well as perturbed the general male reproductive health in diabetic rats.

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.

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.

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.

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

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.

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.

A novel label-free electrochemical strategy was established based on the unique electro-catalytic activity of graphene oxide (GO)-supported terminal deoxynucleotidyl transferase (TdT)-generated C-rich DNA nanotail-templated silver nanoclusters (DNA-AgNCs). TdT can catalyze the deoxycytidine triphosphate (dCTP) to the 3'-OH terminus of single-stranded DNA (ssDNA) with no template; then, in the presence of Ag(I), TdT-generated C-rich DNA sequence was employed for the synthetic template of AgNCs because of the formed complexes of nitrogen atoms of cytosine based with silver atoms. We proved that in situ grown DNA nanotail-templated AgNCs can be adsorbed on GO-modified electrode and possess high electro-catalytic activity to H2O2 reduction, presenting a good electrochemical indicator for signal readout. Under optimal conditions, the proposed biosensor could be employed for quantitatively monitoring TdT activity and within a dynamic range from 0.4 to 90U/mL and a low limit of detection is 0.08U/mL. With high sensitivity and excellent selectivity, this strategy offers a facile, convenient and specific electrochemical method for TdT activity detection and its relevant inhibitors screening. It holds a promising potential in the practical application of TdT-based biochemical research, disease diagnosis and drug discovery.

Previously reported electrochemical DNA biosensors based on in-situ polymerization approach reveal that terminal deoxynucleoside transferase (TdTase) has good amplifying performance and promising application in the design of electrochemical DNA biosensor. However, this method, in which the background is significantly affected by the amount of TdTase, suffers from being easy to produce false positive result and poor stability. Herein, we firstly present a novel electrochemical DNA biosensor based on grafting-to mode of TdTase-mediated extension, in which DNA targets are polymerized in homogeneous solution and then hybridized with DNA probes on BSA-based DNA carrier platform. It is surprising to find that the background in the grafting-to mode of TdTase-based electrochemical DNA biosensor have little interference from the employed TdTase. Most importantly, the proposed electrochemical DNA biosensor shows greatly improved detection performance over the in-situ polymerization approach-based electrochemical DNA biosensor.

Exosomes are regarded as a promising biomarker in the diagnosis of disease due to their close relationship with the change of physiology and pathology. However, it is still a hard challenge to come up with a highly sensitive method for the exosomes detection. Herein, we propose a spherical nucleic acids (SNAs)-based cascade signal amplification strategy for the exosomes detection with high sensitivity. In this method, SNAs anchoring on exosomes membrane can be extended to form polyT sequence by terminal deoxynucleotidyl transferase (TdT), generating a template strand for the Exo III-catalyzed excision of the designed signal probe (probe A), which may finally induce significant decrease of electrochemical signal due to the consumption of probe A. Benefiting from the SNAs-based cascade signal amplification, this fabricated biosensor achieves a limit of detection for exosomes as low as 44 particles/μL. Moreover, this method shows good performance in the differentiation of healthy and malignancy colorectal cancer patients. Therefore, without complicated nucleic acids sequences design, our approach provides a cascade signal amplification strategy for the highly sensitive detection of exosomes and shows the potential applications in clinical diagnosis.

The assay of detecting DNA methyltransferase activity has been identified as one of the central challenges in cancer diagnostics as DNA methylation is closely related to the diagnosis and treatment of tumors. In this study, a label-free fluorescence probe based on poly-thymine copper nanoclusters engineered by terminal deoxynucleotidyl transferase is proposed for methyltransferase activity assay. Taking advantage of the efficient polymerization extension reaction catalyzed by terminal deoxynucleotidyl transferase and the copper nanoclusters with large Stokes shift instead of labeling fluorescent dyes, the strategy exhibits a broader linear scope from 1 to 300 U mL−1 with a detection limit of 0.176 U mL−1. The economical method is specificity for M.SssI and also provides a convenient and high-throughput platform for screening the DNA methylation inhibitors, which displays a great potential for the practical applications of the drug development and clinical cancer diagnosis in the future.

Specific and sensitive detection of protein biomarkers is of great importance in biomedical and bioanalytical applications. In this work, a dual amplified signal enhancement approach based on the integration of catalytic hairpin assembly (CHA) and terminal deoxynucleotidyl transferase (TdT)-mediated in situ DNA polymerization has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. The presence of the target thrombin leads to the unfolding and capture of a significant number of hairpin signal probes with free 3'-OH termini on the sensor electrode. Subsequently, TdT can catalyze the elongation of the signal probes and formation of many G-quadruplex sequence replicates with the presence of dGTP and dATP at a molar ratio of 6:4. These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a completely label-free fashion. The sensor shows a linear range of 0.5pM-10.0nM and a detection limit of 0.12pM for thrombin. Moreover, the developed sensor can selectively discriminate the target thrombin against other non-target proteins and can be employed to monitor thrombin in human serum samples.

Herein, an aptamer-initiated on-particle template-independent enzymatic polymerization (aptamer-OTEP) strategy for electrochemical aptasensor (E-aptasensor) is developed for analysis of cancer biomarker carcino-embryonic antigen (CEA). A pair of DNA aptamers is employed which can be specifically bond with CEA simultaneously. One of the aptamer is thiolated at 3'-terminal and immobilized onto the gold electrode as a capture probe, while the other one has a thiol group at its 5'-terminal and is modified onto the gold nanoparticles surface to form a nanoprobe. In the present of target, the two aptamers can "sandwich" the target, thus the nanoprobe is attached to the electrode. Then terminal deoxynucleotidyl transferase (TdT) is employed to catalyze the incorporation of biotin labeled dNTPs into the 3'-OH terminals of the DNA aptamer on the nanoprobe. The as-generated long DNA oligo tentacles allow specific binding of numerous avidin modified horseradish peroxidase (Av-HRP), resulting in tens of thousands of HRP catalyzed reduction of hydrogen peroxide and sharply increasing electrochemical signals. Taking advantage of the enzyme based nucleic acid amplification and nanoprobe, this strategy is demonstrated to possess the outstanding amplification efficiency.

DNA double-strand breaks (DSBs) represent a major threat to the genetic integrity of the cell. Knowing both their genome-wide distribution and number is important for a better assessment of genotoxicity at a molecular level. Available methods may have underestimated the extent of DSBs as they are based on markers specific to those undergoing active repair or may not be adapted for the large diversity of naturally occurring DNA ends. We have established conditions for an efficient first step of DNA nick and gap repair (NGR) allowing specific determination of DSBs by end labeling with terminal transferase. We used DNA extracted from HeLa cells harboring an I-SceI cassette to induce a targeted nick or DSB and demonstrated by immunocapture of 3′-OH that a prior step of NGR allows specific determination of loci-specific or genome wide DSBs. This method can be applied to the global determination of DSBs using radioactive end labeling and can find several applications aimed at understanding the distribution and kinetics of DSBs formation and repair.

A simple and highly sensitive biosensing strategy was reported by cascading terminal deoxynucleotidyl transferase (TdT)-catalyzed substrate extension and CRISPR-Cas12a -catalyzed short-stranded DNA probe cleavage. Such a strategy, which is named as TdT-combined CRISPR-Cas12a amplification, gives excellent signal amplification capability due to the synergy of two amplification steps, and thus shows great promise in the design of various biosensors. Based on this strategy, two representative biosensors were developed by simply adjusting the DNA substrate design. High signal amplification efficiency and nearly zero background endowed the biosensors with extraordinary high sensitivity. By utilizing these two biosensors, ultrasensitive detection of uracil-DNA glycosylase (UDG) and T4 polynucleotide kinase (T4 PNK) was achieved with the detection limit as low as 5 × 10−6 U/mL and 1 × 10−4 U/mL, respectively. The proposed UDG-sensing platform was also demonstrated to work well for the UDG activity detection in cancer cells as well as UDG screening and inhibitory capability evaluation, thus showing a great potential in clinical diagnosis and biomedical research.

The terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase expressed in acute myeloid leukemias (AMLs), where it may be involved in the generation of NPM1 and FLT3-ITD mutations. We studied the correlations between TdT expression and FLT3-ITD or NPM1 mutations in primary AML samples, and the impact on patients' survival. TdT expression was analyzed in 143 adult AML patients by flow cytometry as percentage of positivity and mean fluorescence intensity (MFI) on blasts. TdT was positive in 49 samples (34.2%), with a median of 48% TdT-positivity (range 7-98) and a median MFI of 2.70 (range 1.23-30.54). FLT3-ITD and NPM1 mutations were present in 24 (16.7%) and 34 (23.7%) cases, respectively. Median TdT expression on blasts was significantly higher in FLT3-ITD+, as compared with FLT3-ITD- AMLs (median 8% vs 0% respectively, p = 0.035). NPM1 mutational status, FLT3-ITD allelic ratio, karyotype, and ELN risk groups, did not correlate with TdT expression or MFI on blasts. TdT + patients had poorer survival as compared to TdT-, but this result was not confirmed by the multivariable analysis, where ELN risk stratification as well as age and type of treatment remained independent prognostic factors for OS. In summary, our results support the possible implication of TdT enzyme in the generation of FLT3-ITD mutations in AML.

We have developed a new methodology for fluorescence turn-on detection of DNA methyltransferase (MTase) activity based on terminal deoxynucleotidyl transferase (TdT) using a thioflavin T probe. This method is highly selective and sensitive. The fluorescence intensity was direct proportion to Dam MTase concentration in the range from 0.1 to 8.0 U/mL with a detection limit of 0.1 U/mL. And because no labeling with a fluorophore-quencher pair was required, it is simple and low cost. We envision that our novel fluorescent detection method for Dam MTase activity could be applied as a useful tool in biomedical research.

Terminal deoxynucleotidyl transferase (TdT) plays an important role in regulating a wide range of genomic processes. The sensitive and accurate detection of cellular TdT activity, particularly at the single-cell level, is highly significant for leukemia-associated biomedical and biological studies. Nevertheless, owing to the limited sensitivity of the existing TdT assays, the quantification of TdT activity at the single-cell level remains a big challenge. Herein, a simple but ultrasensitive method for assaying TdT activity is proposed based on terminal extension actuated loop-mediated isothermal amplification (TEA-LAMP). By using the TdT-induced extension product as an actuator, TdT activity is amplified twice by terminal extension and LAMP in an exponential manner and finally converted to a remarkably amplified fluorescent signal. In this study, as low as 2 × 10−8 U/μL TdT can be clearly detectable with the elegant TEA-LAMP strategy. Such an ultrahigh sensitivity enables the direct determination of TdT activity in individual single cells. In the meantime, by employing TdT as a co-factor, this strategy can also be applied to detecting other enzymes that can catalyze the DNA terminal hydroxylation. This work not only reports the up-to-now most sensitive TdT detection strategy at a single-cell level but also opens the new gate for versatile enzyme activity detection.

A cascade signal amplification strategy through combining surface-initiated enzymatic polymerization (SIEP) and the subsequent deposition of strepavidin functionalized silver nanoparticles (AgNPs) was proposed. The first step of constructing the electrochemical immunosensor involves covalently immobilizing capture antibody on a chitosan modified glass carbon electrode, which then catalyzes DNA addition of deoxynucleotides (dNTP) at the 3′-OH group by terminal deoxynucleotidyl transferase (TdT), leading to the formation of long single-stranded DNAs labeled with numerous biotins. Following the deposition of numerous strepavidin functionalized AgNPs on those long DNA chains, electrochemical stripping signal of silver was used to monitor the immunoreaction in KCl solution. Using α-fetoprotein as a model analyte, this amplification strategy could detect fetoprotein down to 0.046 pg/mL with a wide linear range from 0.1 pg/mL to 1.0 ng/mL. The achieved high sensitivity and good reproducibility suggest that this cascade signal amplification strategy has great potential for detecting biological samples and possibly clinical application.

Serum IgG anti-nuclear antibodies (ANA) directed to complexes of DNA and histones are a hallmark of systemic lupus erythematosus (SLE) and reflect a failure in lymphocyte self-tolerance. A prior study utilizing spontaneously autoimmune B6.Nba2 mice deficient in terminal deoxynucleotidyl transferase (TdT) and with heterozygous deficiencies in Jh and Igk loci underscored the importance of somatic hypermutation (SHM) as a major generator of SLE-associated ANA. This interpretation had to be qualified because of severely limited opportunities for receptor editing and restricted VHCDR3 diversity. Therefore, we performed the converse study using mice that carried functional Tdt genes and wild type Jh and Igk loci but that could not undergo SHM. Analyses of ANA and ANA-producing hybridomas from B6.Nba2 Aicda(-/-) mice revealed that few animals produced high titers of the prototypical ANA directed to complexes of histones and DNA, that this response was delayed and that those cells that did produce such antibody exhibited limited clonal expansion, unusual Jk use and only infrequent dual receptor expression. This, together with the additional finding of an intrinsic propensity for SHM to generate Arg codons selectively in CDRs, reinforce the view that most IgG autoimmune clones producing prototypical anti-nucleosome antibodies in wild type mice are created by SHM.

In this work, two electrochemical DNA sensors was developed based on the target induced structural switching of stem-loop probe (SLP) and surface initiated enzymatic polymerization (SIEP). Both of the electrochemical DNA sensors employed SLPs with the same sequence. However, one had a thiol label at its 3' terminal (the probe was named 3-SLP and the sensor was named 3-SLP-SENS) and the other at its 5' terminal (the probe was named 5-SLP and the sensor was named 5-SLP-SENS). In the initial state of the sensors, both of the probes adopted the stem-loop structure, which shielded the unlabeled terminals of capture probes from being approached. When the loop regions of the capture probes hybridized with the target DNA the conformation of the SLPs was changed to a rigid double-strand, as a result, the 5-SLP released a 3'-OH terminal for SIEP which could be catalyzed by terminal deoxynudeolidyl transferase (TdT). And the 3-SLP released a 5' phosphate terminal which is not suit for SIEP. Thus a signal probe was employed to hybridize with the 5 terminal of 3-SLP and provide a 3'-OH. Both of the sensors were then submitted to the TdT-mediated SIEP. By using biotinylated 2'-deoxyadenosine 5'-triphosphate (biotin-dATP), biotin labels are incorporated into the SIEP-generated long single-stranded DNA. Then avidin-horseradish peroxidases (Av-HRPs) were employed for specific binding to the biotin labels to produce electrochemical signals. The detection performances of two electrochemical DNA sensors were investigated and compared. It was demonstrated that though the 3-SLP-SENS employed extra signal probes, the background current was lower leading to a better detection limit By taking advantage of SLP and SIEP, this 3-SLP-SENS has been able to detect as low as 0:1 pM DNA targets with excellent differentiation ability for even single mismatches. (c) 2013 Published by Elsevier B.V. All rights reserved.

This study develops a novel electrochemical sensing platform for microcystin-LR (MC-LR) detection. This aptasensor comprises the hybridization of double aptamer to its complementary strand (CS) on the surface of electrode and generation of an Infinity-shaped DNA structure in the absence of target by terminal deoxynucleotidyl transferase (TdT). The formation of Infinity-shaped construction leads to the development of an ultrasensitive aptasensor for MC-LR detection. In the presence of MC-LR, double aptamer is dissociated from its CS because of its high affinity for MC-LR and leaves the surface of electrode. Subsequently, no Infinity-shaped structure is formed following the introduction of TdT and a strong current signal is observed. The proposed method was employed for specific detection of MC-LR in the range from 60 pM to 1000 nM with a detection limit of 15 pM. The credibility of the approach was confirmed by detection of MC-LR in real samples like serum and tap water samples. This study provides a new aptasensor for detection of MC-LR as well as other toxin analysis.

Summary Objective To investigate the hypothesis that strenuous running is a predisposing factor for osteoarthritis. Design Wistar rats were divided into two groups: a control group (CG) and a trained group (TG). The TG underwent a strenuous treadmill running training regimen of controlled intensity, exhibiting progressively improvement of fitness over 12 weeks, running at least 55 km during this period and finally performing an ultra-endurance running exercise to exhaustion. After this period, rats from both groups were euthanized and their knees removed. The articular cartilage was dissected and submitted to histomorphometrical, histomorphological, and immunohistochemical analyses evaluating cell death pathway (caspase-3 and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)) and inflammatory cytokines [interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α)]. In addition, the tissues were analyzed regarding the types and the content of glycosaminoglycans. Results The TG knee joints exhibited increase in the number of chondrocytes and chondrocyte clusters, as well as significantly increased levels of caspase-3, a protein involved in apoptosis, and of inflammatory cytokines IL-1α and TNF-α. In addition, histologically higher grades of osteoarthritis (Osteoarthritis Research Society International – OARSI grading), and significantly decreased levels of chondroitin sulfate and hyaluronic acid. Knee cartilage thickness and TUNEL did not significantly differ between the two groups. Conclusions The articular cartilage of rats subjected to a strenuous running regimen of controlled intensity exhibited molecular and histological characteristics that are present in osteoarthritis.

The diagnostic criteria for acute myeloid leukemia (AML), not otherwise specified, with minimal differentiation (AML-M0, French-American-British classification), have been refined in the 2008 World Health Organization (WHO) classification. Terminal deoxynucleotidyl transferase (TdT) expression in AML-M0 has been proposed by others as a surrogate for RUNX1 (runt-related transcription factor 1) mutations, a mutation associated with distinct gene expression profiles in AML-M0. In this study, we investigated the significance of TdT expression in AML-M0 cases defined using the 2008 WHO classification criteria. Demographic, laboratory and clinical information were obtained from the hospital medical records. Statistical analysis was performed using Student's t-test, log-rank test and Fisher's exact test. The study group included 30 AML-M0 patients (male:female=19:11; median age: 60 years). In all, 10 cases of AML-M0 were positive for TdT(+) and 20 cases were negative for TdT(-). Patients with TdT+ AML-M0 had higher peripheral blood and bone marrow blast counts compared to patients with TdT- AML-M0 (P=0.01). TdT expression in AML-M0 was not associated with a distinct immunophenotype. Monoclonal IgH and TCR gene rearrangements were frequent, but independent of TdT expression in AML-M0. TdT expression in AML-M0 correlated with trisomy 13 and inversely correlated with aberrations of chromosomes 5 and 17. Among six patients with AML-M0 who received a stem cell transplant, overall survival was significantly longer for the three TdT+ patients compared with the three TdT- patients (P=0.03). In the TdT+AML-M0 subgroup, the three patients with stem cell transplant had better overall survival compared with five patients who did not receive stem cell transplant (P=0.01). We conclude that AML-M0, as currently defined in the 2008 WHO classification, can be divided into two groups based on TdT expression. Although there is a need to assess a greater number of patients, our results suggest that TdT positivity in AML-M0 identifies a subset of patients with a better prognosis after stem cell transplant.

Background/Purpose: Viral infections and innate immunity signaling, especially Toll-like receptor 7 (TLR7) have been implicated in the pathogenesis of biliary atresia (BA). Administration of rhesus rotavirus-type A to newborn Balb/c mice produces inflammatory obstruction of bile ducts, which resembles human BA. However, whether activation of TLR7 signaling plays a role in neonatal hepatobiliary injury remains to be investigated. Methods: TLR7 agonist, imiquimod (R837), was intraperitoneally administered to Balb/c mice within 24 hours of birth and then every other day. Morphological and histological injuries of liver and gallbladder were examined at 2 weeks. Hepatic messenger RNA expression of TLR7 signaling was studied. Terminal deoxynucleotidyl transferase 2′-deoxyuridine 5′-triphosphate nick end labeling staining was used to delineate hepatobiliary apoptosis upon TLR7 stimulation. Results: TLR7 agonist, imiquimod, induced hypoplasia of the biliary system of neonatal Balb/c mice both in atrophic gallbladder and in paucity of intrahepatic bile ducts. There was significantly higher hepatic expression of TLR7 and downstream innate immunity-mediated interferon regulatory factor 7, interferon-α, and tumor necrosis factor-α. In addition, terminal deoxynucleotidyl transferase 2′-deoxyuridine 5′-triphosphate nick end labeling-positive cells in the liver were increased after injections of TLR7 agonist. Conclusion: The results demonstrate that TLR7 activation may trigger innate immunity pathways and induce apoptosis and hypoplasia of neonatal biliary trees in Balb/c mice. The novel findings give an implication of pathogenesis of infantile cholestasis, such as BA. Keywords: animal model, Balb/c mice, biliary atresia, infantile cholestasis, innate immunity, toll-like receptor 7

Nearly every DNA polymerase characterized to date exclusively catalyzes the incorporation of mononucleotides into a growing primer using a DNA or RNA template as a guide to direct each incorporation event. There is, however, one unique DNA polymerase designated terminal deoxynucleotidyl transferase that performs DNA synthesis using only single-stranded DNA as the nucleic acid substrate. In this chapter, we review the biological role of this enigmatic DNA polymerase and the biochemical mechanism for its ability to perform DNA synthesis in the absence of a templating strand. We compare and contrast the molecular events for template-independent DNA synthesis catalyzed by terminal deoxynucleotidyl transferase with other well-characterized DNA polymerases that perform template-dependent synthesis. This includes a quantitative inspection of how terminal deoxynucleotidyl transferase binds DNA and dNTP substrates, the possible involvement of a conformational change that precedes phosphoryl transfer, and kinetic steps that are associated with the release of products. These enzymatic steps are discussed within the context of the available structures of terminal deoxynucleotidyl transferase in the presence of DNA or nucleotide substrate. In addition, we discuss the ability of proteins involved in replication and recombination to regulate the activity of the terminal deoxynucleotidyl transferase. Finally, the biomedical role of this specialized DNA polymerase is discussed focusing on its involvement in cancer development and its use in biomedical applications such as labeling DNA for detecting apoptosis.

Perfluoroalkyl acid (PFAA)-induced apoptosis has been reported in many cell types. However, minimal information on its mode of action is available. This study explored the possible involvement of apoptotic signaling pathways in a nine-carbon-chain length PFAA-perfluorononanoic acid (PFNA)-induced splenocyte apoptosis. After a 14-day exposure to PFNA, rat spleens showed dose-dependent levels of apoptosis. The production of pro-inflammatory and anti-inflammatory cytokines was significantly increased and decreased, respectively. However, protein levels of tumor necrosis factor receptor 1 (TNFR1), fas-associated protein with death domain (FADD), caspase 8 and caspase 3, which are involved in inflammation-related and caspase-dependent apoptosis, were discordant. Peroxisome proliferator-activated receptors alpha (PPARalpha) and PPARgamma genes expression was up-regulated in rats treated with 3 or 5 mg/kg/day of PFNA, and the level of hydrogen peroxide (H2O2) increased concurrently in rats treated with the highest dose. Moreover, superoxide dismutase (SOD) activity and Bcl-2 protein levels were dramatically decreased in spleens after treatment with 3 and 5 mg/kg/day of PFNA. However, protein levels of Bax were unchanged. Apoptosis-inducing factor (AIF), an initiator of caspase-independent apoptosis, was significantly increased in all PFNA-dosed rats. Thus, oxidative stress and the activation of a caspase-independent apoptotic signaling pathway contributed to PFNA-induced apoptosis in rat splenocytes.

Terminal deoxynucleotidyl transferase (TdT) expression is controlled at the transcriptional level, however, the TdT core promoter combining D, D′, an initiator (Inr) and downstream basal elements (DBE) does not recapitulate the whole complex regulation of TdT expression. We hypothesized that important cis-regulatory elements of the gene are located outside of the TdT promoter. In an attempt to identify these elements, we performed DNase I hypersensitivity assays over 24 kb including a 10 kb region located upstream of the transcription start site (+1) and a 14 kb region spanning exons and introns I to VI. Hypersensitive sites (HS) HS1 and HS2 were localized 8.5 and 8 kb upstream of the transcription start site, respectively, and were exclusively detected in TdT + cell types. HS3, HS4 and HS5 were mapped at positions −7, −3.4 and −3 kb, respectively, and detected in both TdT negative and positive cells. HS6, HS7 and HS8 were detected immediately upstream of the TdT promoter. HS10 and HS11 were localized in the first and third intron of the gene. Luciferase reporter assays revealed that HS1, HS2 and HS3 synergize with the TdT promoter to activate transcription in a TdT + pre-T cell line but not in a TdT + pro-B cell line. In summary novel cis-regulatory elements have been identified in the 5′ region of the TdT locus that synergize with the promoter to activate gene expression and our results suggest these elements may be more active in T cells.

We have used selected rabbit anti-human polyclonal antibodies as an example of useful and easily available tools for studies on immune system structure and development in important veterinary species, many of which also represent animal models in biomedicine. The cocktail of anti-human Igkappa-FITC/anti-Iglambda-RPE F(ab')(2) fragments was used for two-colour and, in combination with the cross-reactive anti-CD79alpha monoclonal antibody HM-57, for three-colour flow cytometry of canine, feline, bovine and porcine peripheral B-cells. A possible application of such immunoreagents in studies on primary B-cell differentiation has been suggested in pigs; the same approach can be used in other species of interest. Rabbit anti-human lactoferrin-FITC F(ab')(2) fragment was used for visualizing neutrophils in dogs, pigs and cattle and an application for two-colour immunophenotyping of canine granulocyte subsets has been designed. Affinity isolated rabbit anti-human CD3 and anti-human TdT have been shown to represent a ready-to-use tool for in situ studies on primary T-lymphopoiesis in pigs with possible extensions both to the B-lineage development in pigs and other animal models. Altogether, our study show that carefully selected polyclonal antibodies available on the market may possess broad cross-reactivity with important applications in veterinary research.

Memory B cells and the antibodies they encode are important for protective immunity against infectious pathogens. Characterization of naïve and memory B cell antibody repertoires will elucidate the molecular basis for the generation of antibody diversity in human B cells and the optimization of antibody structures that bind microbial antigens. In this study we aimed to investigate the influence of antigenic selection on the antibody genes of the two CD27+ memory B cell subsets, comparing them with the naïve repertoire in CD27- cells. We analyzed and compared the Ig heavy chain gene transcripts in three recently defined circulating naïve and memory B cell subsets (CD19+IgD+CD27- [naïve], CD19+IgD+CD27+ [un-class-switched memory] or CD19+IgD- CD27+ [class-switched memory]) at the single cell level. We found similar biased patterns of variable, diversity and joining heavy chain gene usages in all three groups of cells. CD19+IgD+CD27+ memory B cells harbored as diverse an antibody gene repertoire as CD19+IgD-CD27+ memory B cells. Interestingly, CD19+IgD+CD27+ memory B cells possessed a lower frequency of somatic mutations, a higher incidence of exonuclease activity at the 3' end of D regions, and a lower frequency of N and P nucleotide additions at both VH-D and D-JH junctions of CDR3 regions compared to CD19+IgD-CD27+ memory B cells. These data suggest distinct functional mechanisms underlying selection of this unique subset of un-class-switched memory B cells.

The interleukin 7 (IL-7) signaling pathway is critical for early lymphoid differentiation. We found dramatic perturbations in fetal liver B cell development and confirmed a complete absence of developing B cells in the adult bone marrow in mice lacking the IL-7 receptor alpha (IL-7Ralpha) gene. We show that peripheral B-2 and B-1 cell populations are deficient in IL-7Ralpha-/- mice. B-2 follicular cell and peritoneal B-1 cell percentages are reduced, while B-2 marginal zone cell percentages are increased. A comparison of bone marrow and splenic populations at different ages revealed that the splenic B cell populations seen in adult IL-7Ralpha-/- mice first appear during neonatal development. We have measured N-nucleotide addition at the joints of V(D)J rearrangements in splenic B cells and have used it as a somatic marker to define and separate bone marrow-derived B cells from fetal liver-derived B cells. B cells isolated from the bone marrow and spleen of adult and neonatal IL-7Ralpha-deficient mice harbor high levels of N-nucleotide additions similar to those found in equivalent wild-type B cell populations. We conclude that the majority of splenic B cells in IL-7Ralpha-deficient mice originate from the bone marrow and not the fetal liver.

Cadmium causes cellular damage but the exact mechanism of apoptosis in cadmium induced acute lung injury is not clear. We investigated the sequential expression of apoptotic nuclei and detected related molecules in tissue of cadmium-induced acute lung injury. Forty Sprague–Dawley rats were sacrificed at days 1, 3, 7 and 10 after intra-tracheal cadmium injection (2.5 mg/kg). Light microscopic, ultrastructural terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (TUNEL), and Western blot analysis for detection of FasL, Bid, cytochrome c, caspase 3 and PARP were carried out. Apoptosis occurred at day 1, and markedly decreased at days 3, 7 and 10 (11.8, 2.8, 0.9, 0.5%, respectively) determined by light microscopy and TUNEL assay. Ultrastructural TUNEL revealed two patterns of nuclear morphology according to the apoptotic stage. One pattern showed chromatin fragmentation and apoptotic nuclear body formation. The other pattern had bleb formation in the chromatin, budding with projection out to the nuclear membranes, fragmentation, segregation of chromatin clumps and apoptotic body formation. Western blot analysis showed prominent expression of FasL at days 1 and 3. Expression of Bid, cytochrome c and caspase 3 were prominent at day 1 compared to days 3, 7 and 10. PARP cleavage was prominent at day 1. In conclusion, intra-tracheal cadmium injection showed active alveolar cell apoptosis at day 1. Ultrastructural TUNEL showed various expressions according to the apoptotic nuclear stage. These studies suggest that cadmium-induced alveolar cell apoptosis is mediated by FasL and caspase-dependent mitochondrial apoptosis pathways.

CD4+CD56+ hematodermic neoplasms are rare, aggressive hematopoietic malignancies usually presenting with cutaneous masses followed by a leukemic phase. The blastic morphology, CD56 expression and lack of definitive myeloid or T-cell markers initially resulted in assignment of this tumor to the NK-cell lineage. Accumulating evidence now suggests that these neoplasms represent malignant counterparts to the plasmacytoid dendritic cell. BDCA-2 is a cell surface protein whose expression is restricted to human plasmacytoid dendritic cells, in a differentiation stage-specific manner. In the current study, we assessed expression of BDCA-2 in CD4+CD56+ hematodermic neoplasms using a new antibody reagent we developed for use in fixed tissue sections. In 10 of 19 cases of CD4+CD56+ hematodermic neoplasm, BDCA-2 immunoreactivity was detected, whereas no expression was observed in NK-lineage tumors (0 of six). Interestingly, expression of terminal deoxynucleotidyl transferase, a marker of immaturity/blast stage, was significantly and negatively correlated with BDCA-2 in CD4+CD56+ hematodermic neoplasms whereas a positive correlation was observed between BDCA-2 and CD7. These findings demonstrate that BDCA-2 is expressed predominantly in the CD7+ subset of hematodermic neoplasms, and similar to non-neoplastic plasmacytoid dendritic cells, expression indicates a relatively more mature differentiation state. Clinical follow-up data confirm the aggressiveness of these tumors and suggests that BDCA-2 immunoreactivity, as identified here, may herald a significant reduction in survival.

Recent studies concerning the pathophysiology of myelodysplastic syndromes (MDS) have shown evidences for the existence of complex interactions between hematopoietic stem cells and the bone marrow (BM) microenvironment. We analyzed the B-lymphocyte maturation in BM of patients with MDS. For this purpose, 41 newly-diagnosed patients were analyzed. Enumeration and characterization of CD34+ and CD34- B-cell precursors and mature B-lymphocytes was performed using multiparameter flow cytometry. BM from eight transplant donors and six orthopedic surgery patients were used as controls. CD34+/CD45(lo) B-cells were found in 17/22 patients with RA/RARS and in 5/13 with RAEB. In patients with RAEB-t and CMML no CD34+ B-cell precursors could be detected. A positive correlation was found between CD34+ and CD34- B-cell precursors (r=0.52). CD34+ B-cell precursors presented an inverse correlation with BM percentage of blasts and peripheral leukocytes and a positive one with hemoglobin. Asynchronous antigen expression (CD19+/CD79a- cells) was found in 7/11 cases of RA/RARS and 6/18 cases of RAEB in which this phenotype was examined. Abnormal patterns of expression for at least one antigen was found in 91% of RA/RARS cases and in 74% of RAEB. Underexpression of TdT and CD79a were the most frequent abnormalities. Our results present evidences of an abnormal B-cell maturation in MDS. This may be an evidence that B-lymphocytes are derived of the abnormal clone. But it may also be the consequence of influences of abnormalities of BM microenvironment leading to an impaired commitment and maturation of the B-cell line in MDS. Studies performed with purified well-characterized B-cells may further elucidate these abnormalities.

Studies of mammalian terminal deoxyribonucleotidyltransferase (TdT) are facilitated by use of inhibitors that selectively knock down the activity of the enzyme. We have screened for selective inhibitors of TdT and identified a natural compound with this property in the Japanese vegetable, Arctium lappa. The compound has little effect on the activities of mammalian DNA polymerases, such as alpha, beta, delta or lambda polymerase, and prokaryotic DNA polymerases, such as Taq DNA polymerase, T4 DNA polymerase and Klenow fragment. H1- and C13-NMR spectroscopic analyses showed the compound to be baicalin, a compound previously reported as an anti-inflammatory or antipyretic agent. The IC50 value of baicalin to TdT was 18.6 microM. We also found that genistin, a baicalin derivative known to be antimutagenic, more selectively inhibited TdT activity than baicalin, although its IC50 value was weaker (28.7 microM). Genistin and baicalin also inhibited the activity of truncated TdT (the so-called pol beta core domain) in which the BRCT motif was deleted in its N-terminal region. In kinetic analyses, inhibition by either genistin or baicalin was competitive with the primer and non-competitive with the dNTP substrate. The compounds may, therefore, bind directly to the primer-binding site of TdT and simultaneously disturb dNTP substrate incorporation into the primer. Genistin and baicalin should prove to be useful agents for studying TdT.

Three Pol X family members have been linked to nonhomologous end joining (NHEJ) in mammals. Template-independent TdT promotes diversity during NHEJ-dependent repair of V(D)J recombination intermediates, but the roles of the template-dependent polymerases mu and lambda in NHEJ remain unclear. We show here that pol mu and pol lambda are similarly recruited by NHEJ factors to fill gaps when ends have partially complementary overhangs, suggesting equivalent roles promoting accuracy in NHEJ. However, only pol mu promotes accuracy during immunoglobulin kappa recombination. This distinctive in vivo role correlates with the TdT-like ability of pol mu, but not pol lambda, to act when primer termini lack complementary bases in the template strand. However, unlike TdT, synthesis by pol mu in this context is primarily instructed by a template from another DNA molecule. This apparent gradient of template dependence is largely attributable to a small structural element that is present but different in all three polymerases.

We investigated the prognostic relevance of immunophenotype and other clinical pathological features in 379 adult patients with de novo (acute myeloid leukemia) AML diagnosed and treated at our institution during an 8-year period. Acute promyelocytic leukemia (APL) cases were excluded because they received different treatment. The overall complete remission (CR) rate post-induction therapy with Ara-C and daunorubicin (DNR) was 60% with a median disease free survival (DFS) of 72 weeks, and a median overall survival (OS) of 54 weeks. At diagnosis, CD34, deoxynucleotidyl transferase (TdT), CD7, CD56, HLADR and CD19 were expressed in 65, 19, 32, 15, 87 and 5%, respectively, of 379 evaluable cases. CD34 positive patients had a significantly lower CR rate (P=0.0003) than CD34 negative patients and there was a trend to a lower remission rate in HLADR positive patients (P=0.067). In multi-variate analysis, co-expression of CD34 and HLADR was an independent adverse factor for achieving CR (P=0.0364). CD56 expression was associated with a significantly shorter overall survival (P=0.0262), but did not affect remission rate or disease free survival. Neither TdT nor CD7 expression was associated with treatment outcome. Age (60 years or older) and cytogenetic features (classified by favorable, intermediate and unfavorable groups) were associated with a lower CR rate, shorter disease free survival and shorter OS. Patients with higher white cell counts (WBC) also had a significantly lower remission rate (P=0.0064) and OS (P=0.0127). We propose a prognostic score for achieving CR in AML patients based on age, WBC, cytogenetics and CD34/HLADR status as four independent factors. Defined by number of factors, this score system may help to stratify AML patients to alternative treatment for better outcome.

DNA polymerase lambda is a novel enzyme of the family X of DNA polymerases. The recent demonstration of an intrinsic 5'-deoxyribose-5'-phosphate lyase activity, a template/primer dependent polymerase activity, a distributive manner of DNA synthesis and sequence similarity to DNA polymerase beta suggested a novel beta-like enzyme. All these properties support a role of DNA polymerase lambda in base excision repair. On the other hand, the biochemical properties of the polymerisation activity of DNA polymerase lambda are still largely unknown. Here we give evidence that human DNA polymerase lambda has an intrinsic terminal deoxyribonucleotidyl transferase activity that preferentially adds pyrimidines onto 3'OH ends of DNA oligonucleotides. Furthermore, human DNA polymerase lambda efficiently elongates an RNA primer hybridized to a DNA template. These two novel properties of human DNA polymerase lambda might suggest additional roles for this enzyme in DNA replication and repair processes.

To understand the extent and specificity of astrocyte pathology in sporadic frontotemporal dementia (FTD), we examined several FTD cases for molecular and morphologic characteristics of astrocyte degeneration. We quantified reactive and degenerating astrocytes in sections of frontal, temporal, parietal, and occipital cortex identified using glial fibrillary acidic protein (GFAP) immunoreactivity, terminal deoxynucleotidyl transferase (TdT) labeling, and morphological characteristics and compared them with nondemented, age-matched control brains. Conventional and confocal microscopy revealed that a subpopulation of GFAP(+) astrocytes exhibited positive TdT labeling and beading of their processes in the frontal, temporal, and parietal cortices in 5 of 7 FTD cases that also exhibited gliosis. This morphology was reproduced in cultured astrocytes using ischemic insults. Degenerating astrocytes in FTD correlated inversely with cerebral blood flow as measured by single photon emission computed tomography (SPECT) analysis of (133)Xe inhalation (r = 0.55, p < 0.05). Furthermore, areas of significant astrogliosis corresponded to areas of SPECT hypoperfusion, suggesting that astrocytes may be affected by or perhaps have a causal role in the disturbances of cerebral perfusion in FTD.

Acute lymphocytic leukemia (ALL) is an aggressive neoplasm that has been defined by the presence of more than 30% lymphoblasts in the bone marrow or peripheral blood in the French-American-British (FAB) Cooperative Group classification system. 7 In the more recently proposed World Health Organization (WHO) Classification of Neoplastic Diseases of Hematopoietic and Lymphoid Tissues or Lymphomas, a blast count above 20% is sufficient for a diagnosis of acute leukemia, 29 although most ALLs have a markedly hypercellular marrow composed predominantly of lymphoblasts. Acute lymphocytic leukemia occurs most frequently in children and is the most common type of leukemia of childhood. 50 Roughly 80% of the cases of acute leukemia in children but only 20% of cases of acute leukemia in adults are ALL. Clinical manifestations are often related to the extensive replacement of the bone marrow with blasts. Examination of the peripheral blood may give the first indication of this illness. Although the diagnosis of ALL may be readily apparent in some cases with high peripheral blood blast counts, other cases may show less specific findings, such as neutropenia, thrombocytopenia, and normochromic or normocytic anemia with reticulocytopenia. An alternative presentation is a leukoerythroblastic picture with or without eosinophilia. 40, 76 The relationship between ALL and lymphoblastic lymphomas has been extensively studied. Approximately 80% of the lymphoblastic lymphomas are of precursor T-cell immunophenotype, whereas 85% of cases of ALL have a precursor B-cell immunophenotype. Rare cases of lymphoblastic lymphoma of precursor B-cell type have been reported. They most frequently involve the skin, bone, and soft tissues and are associated with a better prognosis than ALL of the precursor B-cell type. 44 In contrast with T-cell ALL, lymphoblastic lymphomas of the precursor T-cell lineage usually have no or minimal peripheral blood or bone marrow involvement and have normal or minimally decreased levels of hemoglobin, white blood cells, and platelets. 51, 52 Arbitrary criteria such as the presence of more than 25% blasts in the bone marrow also have been used to distinguish ALL from lymphoblastic lymphoma. 9 Nevertheless, this distinction remains unclear in some cases, and the discrimination between a leukemic conversion of lymphoblastic lymphoma and de novo ALL cannot always be made. 51 Recently, because lymphoblastic lymphomas and ALL have more similarities than differences in pathology, immunophenotype, and genotypes, they have been regarded as processes falling within the spectrum of a single disease entity. Thus, ALL and lymphoblastic lymphomas are grouped under the category labeled lymphoblastic leukemia/lymphoma in the Revised European and American Classification of Lymphoid Neoplasms (REAL) and in the WHO Classification Scheme. 28, 29 The pattern of disease involvement (i.e., bone marrow versus extramedullary involvement) is an important factor for clinical staging, however. Despite the advent of modern ancillary techniques for ALL diagnostic evaluation, morphologic examination and cytochemical staining of well-prepared air-dried bone marrow and peripheral blood smears are critical in the pathologic diagnosis and classification of acute leukemias. 5, 7 Careful morphologic examination allows certain differential diagnostic possibilities to be considered at the very beginning of the diagnostic process so that bone marrow aspirate specimens can be sent for appropriate laboratory studies. This article gives an overview of the diagnostic workup for suspected cases of ALL. A summary of the typical morphologic, cytochemical, and immunophenotypic features of ALL and a few unusual ALL variants is presented. Emphasis is given to the discussion of a list of entities that ALL mimics which the clinician and pathologist must consider in the differential diagnosis of ALL.

The BRCA1 COOH terminus (BRCT) motif is present in many nuclear proteins that contribute to cell cycle regulation or DNA repair. Polymerase chain reaction-based screening with degenerate primers targeted to the BRCT motif resulted in the isolation of a human cDNA for a previously unidentified DNA polymerase (designated DNA polymerase beta2) that is closely related to DNA polymerase beta (Pol beta). The predicted Pol beta2 protein contains a BRCT motif in its NH(2)-terminal region; its COOH-terminal region exhibits 33% sequence identity to a corresponding region of human Pol beta. The Pol beta2 gene is expressed in a tissue-specific manner, with transcripts being most abundant in testis. A fusion construct comprising Pol beta2 and green fluorescent protein exhibited a predominantly nuclear localization in transfected HeLa cells. Recombinant human Pol beta2 from insect cells exhibited substantial DNA polymerase activity, but it did not possess terminal deoxyribonucleotidyl transferase activity. A truncated Pol beta2 mutant lacking the BRCT motif retained substantial DNA polymerase activity, whereas a mutant Pol beta2 with two alanine point mutations within the DNA polymerase active site did not. These results indicate that Pol beta2 is a Pol beta-related DNA polymerase with a BRCT motif that is dispensable for its polymerase activity.

A major obstacle in neural transplantation is a severe loss of neurons in grafts soon after implantation. In the present study, we have investigated whether the systemic administration of synthetic fibronectin peptide V can increase the survival of neural grafts. Synthetic fibronectin peptide V is derived from the 33,000 mol. wt carboxyl-terminal heparin-binding domain of fibronectin. Previous studies have shown that these polypeptides possess anti-inflammatory properties. However, it is currently unknown whether this peptide has anti-apoptotic properties. Dissociated neural grafts were prepared from the ventral mesencephalon of pregnant Sprague-Dawley rats and were stereotaxically injected as a cell suspension into the striatum of adult Sprague-Dawley rats. A group of recipient rats received i.v. injections of peptide V (5mg/kg, dissolved in saline) at 24 and 4h prior to transplantation, at the time of transplantation, and 24, 48 and 72h post-transplantation. Saline-treated rats served as controls. The rats were killed at two, four and 42 days post-grafting and the brain tissue was immunologically processed for tyrosine-hydroxylase, major histocompatibility complex class I and class II antigens, complement receptor type 3 and leukocyte common antigen immunocytochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. We found a significant increase (approximately twofold) in the number of dopamine neurons in the grafts for the peptide-treated group at four and 42 days compared with the controls. In contrast, there was no significant difference in the patterns of inflammation using different immunocytochemical markers in the two different groups. The levels of expression for these markers, however, were reduced over time. Interestingly, the number of apoptotic cells in the graft areas was significantly smaller in the peptide-treated group than in the control group two days after grafting. The results demonstrate that the systemic administration of synthetic fibronectin peptide V can dramatically increase the survival of nigral grafts in the brain and substantially reduce the number of apoptotic cells in the graft site, suggesting that this peptide may exert a beneficial effect on survival of nigral grafts through an anti-apoptotic mechanism.

The phosphorylation and phosphonylation of the 3'-hydroxyl of oligodeoxynucleotide 3'-termini (oligodeoxynucleotidyl kinase activity) catalyzed by calf thymus terminal deoxynucleotidyl transferase (TDT) are discussed. Palpha and Palpha, Pgamma-substituted modified triphosphates serve as low-molecular weight substrates in this reaction to give oligodeoxynucleotides with a 3'-phosphorylated or phosphonylated hydroxyl. The reaction is specific for TDT, and it is not catalyzed by avian myeloblastosis virus reverse transcriptase. The phosphate or phosphonate donor activities of modified triphosphates depend on their structure and increase with hydrophobicity. Several modified triphosphates demonstrated very high substrate activity, in some cases, up to one order of magnitude higher than that for dTTP. It has also been shown that TDT catalyzes primer extension with dinucleoside 5',5'-tetraphosphates as substrates.