Terminal deoxynucleotidyl transferase (TdT) based template-free signal amplification for the detection of exosomes in MUC1-positive cells.

The Mucin1 (MUC1) protein, involved in cytoprotective and signaling pathways, is abnormally elevated in various cancers, making it a key cancer indicator. Exosomes, which reflect the status of their originating cells, offer potential for cancer diagnosis. Thus, developing a method to detect MUC1-positive exosomes is crucial for the early diagnosis of certain cancers. In this study, we developed a highly sensitive, specific, and simple UV-visible signal amplification method to detect MUC1-positive exosomes using terminal deoxynucleotidyl transferase (TdT). Initially, exosomes were captured on magnetic beads using a CD63 aptamer(apt). The Primer-AuNPs-MUC1 apt complex which we synthesized by low pH loading method was then attached MUC1 proteins on the surface of the exosomes to create a sandwich structure. TdT catalyzed the extension of Biotin-dATP at the 3' end of the primer, introducing multiple biotin sites into the sandwich structure. These sites subsequently bound multiple streptavidin-horseradish peroxidase (streptavidin-HRP), which catalyzed the oxidative color change of the substrate, which can be detected by colorimetric method. This method can detect A549 exosomes in the range of 1.4E+6 to 4.2E+8 particles/mL and shows high specificity for cell lines with different MUC1 expression. Additionally, it successfully distinguished cholangiocarcinoma (CCA) patients (n=11) from healthy individuals (n=7) in clinical serum assays, demonstrating good performance in real sample detection.

Generalization in perceptual learning across stimuli and tasks.

Perceptual learning, known to improve visual perception, demonstrates the plasticity of brain processes underlying vision. Early studies, using the backward-masked texture discrimination task (TDT), focused on the lack of generalizing learning to stimulus features, relating learning specificity to the selectivity of the brain networks involved in the visual task. Learning was found to be highly specific to the stimulus features, as expected from the processing selectivity found in early visual areas as well as to the task employed in training, pointing to top-down effects. More recent studies demonstrate the generalization of learning to untrained features under specifically designed training procedures. Here we suggest that transfer of learning takes place when the trained and untrained stimuli and task activate overlapping brain processes. We tested the effect of TDT learning, under conditions with and without visual adaptation, on the contrast detection (CD) of localized Gabor targets, either alone or backward masked (BM). At the TDT peripheral-target location, we found that the transfer of learning between TDT to CD and BM occurs under the TDT adaptation condition, but not under the no-adaptation condition, whereas at the TDT center-target location we found that transfer occurs for both conditions. Our results suggest that learning generalization across experimental conditions depends on overlapping neural processes within brain networks, here dominated by the inhibitory effects involved in adaptation and in spatiotemporal masking. Importantly, increased adaptation during training, due to increased stimulus consistency, enabled the transfer of learning to other tasks limited by sensory adaptation.

Impact of Empagliflozin on the Outcomes of ß-Thalassemia Major in Patients With Type 2 Diabetes Mellitus: The THALEMPA Observational Study.

OBJECTIVE: Beta-thalassemia major (ß-TM) is a genetic disorder characterized by ineffective erythropoiesis and chronic hemolytic anemia, necessitating lifelong blood transfusions and leading to severe complications. This study, termed THALEMPA by the authors, investigated the effect of empagliflozin (EMPA) on ß-TM outcomes in patients with type 2 diabetes mellitus (T2DM), focusing on disease severity and associated complications of iron overload and hyperuricemia. METHODOLOGY: This study conducted a single-center prospective observational investigation involving adults diagnosed with ß-TM and T2DM. A total of 20 carefully selected patients were stratified into two groups based on their medical condition: the EMPA group, receiving 10 mg of empagliflozin, and a control group, receiving standard care. This focused cohort size was chosen to ensure a detailed, in-depth analysis of the treatment effects within this specific patient population. Over three months, both groups were closely monitored for ß-TM outcomes. The study assessed ß-TM severity parameters such as hemoglobin levels, blood transfusion frequency, aspartate aminotransferase (AST), alanine aminotransferase (ALT), left ventricular ejection fraction percentage, and spleen size. Additionally, ß-TM complications were evaluated through serum ferritin and uric acid levels. RESULTS: Our analysis revealed that EMPA increased hemoglobin levels by up to 0.56 g/dL compared to baseline (P < 0.05). Liver enzyme levels significantly improved with EMPA by the third month. AST and ALT decreased by 36.22% and 33.36%, respectively, from baseline levels (P < 0.05), highlighting EMPA's potential benefits for ß-TM severity. Serum ferritin and uric acid levels decreased by 27.93% and 21.29%, respectively, over three months on EMPA (P < 0.05). However, other parameters did not show significant changes post-EMPA. CONCLUSIONS: This study demonstrates the significant impact of EMPA treatment over three months on ß-TM patients with T2DM, evidenced by notable improvements in hemoglobin levels and reductions in liver enzymes, as well as in complications related to iron overload and hyperuricemia. Future research should confirm these benefits over longer durations and assess broader patient outcomes such as quality of life.

The impact of transanal drainage tube on the incidence of anastomotic leakage and small bowel obstruction in radical surgery (Dixon) for rectal cancer: a retrospective cohort study.

Background: Anastomotic leakage (AL) and small bowel obstruction (SBO) are common complications after rectal cancer radical surgery (Dixon). Although the commonly used defunctioning stoma (DS) can reduce the incidence and harm of AL, it increases the probability of other adverse consequences, including SBO. Therefore, a safe and effective method for preventing the complications related to the radical surgery of rectal cancer is urgently needed. Previous studies have found that transanal drainage tube (TDT) can have a positive impact on the incidence of these two complications by draining gas and feces from the intestinal lumen, without causing other serious consequences. Therefore, this article further explores the clinical benefits that TDT can bring by analyzing the clinical data of postoperative patients with rectal cancer. Methods: This study included 221 patients who underwent radical surgery (Dixon) for rectal cancer in Hubei Cancer Hospital from September 2020 to February 2023, determine whether it meets the inclusion criteria of this study based on preoperative examination, intraoperative exploration results, and treatment methods. DS was used in 70 patients and TDT in 88 patients during the surgery; meanwhile, no protective anastomotic measures were applied in 63 patients. Seventy patients subjected to DS were categorized as group 1, 88 patients subjected to TDT as group 2, and 63 patients with no protective measures for anastomosis as group 3. Through postoperative clinical manifestations, imaging examinations, and laboratory tests, a total of 18 cases of AL and 30 cases of SBO were identified in the three groups. The effectiveness of TDT and that of other surgical procedures in preventing complications, accelerating postoperative recovery, and reducing surgical costs were compared through univariate and multivariate analyses. Results: The clinical features of the three groups have baseline comparability. No statistically difference was noted in baseline characteristics between three groups (all P>0.05). The incidence of AL and SBO in group 1 are 7.1% and 27.1%, in group 2 are 3.4% and 4.5%, and in group 3 are 15.9% and 11.1%. Compared to patients in no protective anastomotic measures with TDT and DS, TDT has a lower incidence of postoperative AL (P<0.05) and SBO (P>0.05), and faster postoperative recovery (P<0.05). The cost of inpatient surgery is not significantly different (P>0.05). Although DS can reduce the incidence of AL to a certain extent (P>0.05), it significantly increased the incidence of SBO (P<0.05), delayed postoperative defecation time (P<0.05) and caused higher cost (P<0.001). Compared to DS, the incidence of AL in TDT is not significantly different (P>0.05), but the incidence of SBO is noticeably lower (P<0.001), with faster postoperative recovery and less cost (P<0.05). Conclusions: TDT is a safer, more effective, and more economical surgery for preventing postoperative complications.

A Massively Parallel In Vivo Assay of TdT Mutants Yields Variants with Altered Nucleotide Insertion Biases.

Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension of DNA. TdT's de novo DNA synthesis ability has found utility in DNA recording, DNA data storage, oligonucleotide synthesis, and nucleic acid labeling, but TdT's intrinsic nucleotide biases limit its versatility in such applications. Here, we describe a multiplexed assay for profiling and engineering the bias and overall activity of TdT variants with high throughput. In our assay, a library of TdTs is encoded next to a CRISPR-Cas9 target site in HEK293T cells. Upon transfection of Cas9 and sgRNA, the target site is cut, allowing TdT to intercept the double-strand break and add nucleotides. Each resulting insertion is sequenced alongside the identity of the TdT variant that generated it. Using this assay, 25,623 unique TdT variants, constructed by site-saturation mutagenesis at strategic positions, were profiled. This resulted in the isolation of several altered-bias TdTs that expanded the capabilities of our TdT-based DNA recording system, Cell HistorY Recording by Ordered InsertioN (CHYRON), by increasing the information density of recording through an unbiased TdT and achieving dual-channel recording of two distinct inducers (hypoxia and Wnt) through two differently biased TdTs. Select TdT variants were also tested in vitro, revealing concordance between each variant's in vitro bias and the in vivo bias determined from the multiplexed high throughput assay. Overall, our work and the multiplex assay it features should support the continued development of TdT-based DNA recorders, in vitro applications of TdT, and further study of the biology of TdT.

Perylene Diimide-based Fluorescent Aptasensor for Quantitative Analysis of Pb2+ Based on Terminal Deoxynucleotidyl Transferase-assisted Formation of Elongated Aptamer and Gold Nanoparticles.

Due to the exceedingly poisonous properties of Pb2+, it is imperative to conduct a thorough assessment of its quantity in both biological and environmental samples, as this is crucial for safeguarding public health. This study describes an economic turn-off fluorescent aptasensor for the quantitative analysis of Pb2+ employing 3,4,9,10-perylenetetracarboxylic acid diimide (PTCDI) as a cost-effective fluorophore, gold nanoparticles (AuNPs) as separating agent and an elongated aptamer as both targeting agent and PTCDI loading site. The fundamental principle of the suggested fluorescent aptasensor, which is based on PTCDI, relies on detecting variations in the fluorescence intensity of PTCDI when an elongated aptamer (as single-stranded DNA) is present or absent. The advanced aptasensor is advantageous due to the elongation of the lead aptamer sequence length induced by terminal deoxynucleotidyl transferase (TdT), resulting in enhanced sensitivity. The presence of Pb2+ and the centrifugation process causes the separation of the poly A-modified aptamer/Pb2+ conjugate from the poly T sequence. Hence, the interaction of PTCDI with the poly A moiety in the modified aptamer leads to a decrease in its fluorescence emission. The findings showcased that the fluorescent aptasensor exhibited exceptional specificity towards Pb2+ ions, while the biosensing platform accomplished an impressive detection limit of 3.7 pM. Moreover, the suggested aptasensor utilizing PTCDI exhibits a commendable capability in quantitatively analyzing Pb2+ within human serum samples and mineral water.

Computational Modeling Study of the Molecular Basis of dNTP Selectivity in Human Terminal Deoxynucleotidyltransferase.

Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme's selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants-containing either substitution D395N or substitutions D395N+E456N-that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications.

The application of mean number of DNA breakpoints in sperm cryopreservation.

Growing concerns over declining male semen quality and rising infertility have shifted attention to male fertility. Sperm cryopreservation emerges as a crucial tool in preserving male fertility, especially for patients who need proactive preservation, such as cancer patients before undergoing radiation or chemotherapy. Although cryopreservation does not directly address infertility, effective preservation can support future fertility. However, the process may compromise sperm DNA integrity. Despite their impairment, damaged sperm often retain vitality and may still have the potential to fertilize an egg. Nonetheless, if damaged sperm fertilize an egg, excessive DNA damage could impede embryo implantation and development, despite the egg's repair capabilities. Consequently, precise detection of sperm DNA damage is crucial and urgent. To better address the issue of sperm DNA damage detection, we have introduced a novel fluorescence biosensor technology known as the TDT/SD Probe. This technology utilizes terminal deoxynucleotidyl transferase (TdT) and strand displacement probes to accurately detect the number of sperm DNA breakage points during the cryopreservation process. Experimental results reveal that the number of sperm DNA breakpoints significantly increases after both sperm vitrification (8.17 × 105) and conventional slow freezing (10.80 × 105), compared to the DNA breakpoints of fresh semen samples (5.19 × 105). However, sperm vitrification has the least impact on sperm breakage points. This research provides innovative means for further optimizing sperm preservation techniques by offering a novel DNA damage detection method, enabling more precise assessment of sperm DNA damage during the freezing process.

T-Cell Acute Lymphoblastic Leukemia/Lymphoma (T-ALL) With Negative Screening Immaturity Markers and Gamma-Delta Receptor Expression.

T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) is characterized by the combination of T-cell lineage and the presence of immaturity marker(s). Sometimes, the most common immaturity markers for initial flow cytometry screening in T-ALL may be negative, which can be a diagnostic pitfall. When a lack of common first-line immaturity markers is encountered in combination with gamma/delta T-cell receptor expression, a misdiagnosis of mature gamma-delta T-cell leukemia/lymphoma could be rendered. Here, we discuss two T-ALL cases with the absence of common flow cytometry immaturity markers and positive gamma/delta receptor expression.

Magnitude of Bone Disease in Transfusion-Dependent and Non-Transfusion-Dependent ß-Thalassemia Patients.

Introduction ß-Thalassemia is a common inherited disease in the northern part of Iraq. A considerable number of transfusion-dependent (TDT) and non-transfusion-dependent (NTDT) ß-thalassemia patients suffer bone problems. The objective of this study was to evaluate the degree of bone disease in the TDT and NTDT patients using a dual-energy X-ray absorptiometry (DEXA) scan. Patients and methods In this study, 53 TDT and 20 NTDT patients aged ≥10 years were enrolled. Their bone status was assessed using the DEXA scan at the lumbar spine (L1-L4) and femoral neck. The effect of physical, biochemical, and hormonal characteristics on the bone mineral density (BMD) parameters was evaluated. The value of the BMD Z-score was the measure to decide on the magnitude of bone disease. Results and discussion The mean age of the enrolled patients was 24.1 years. The BMD Z-score values were significantly lower among the TDT patients at the lumbar spine and femoral neck (BMD Z-score: -2.05 and -1.51 versus -2.29 and -0.71; p=0.044 and 0.009, respectively). The proportion of osteoporosis at the lumbar spine was significantly higher in the TDT group than in the NTDT group (69.8% versus 40%; p <0.001). The BMD Z-score correlated significantly with patient BMI and parathyroid hormone (PTH) level in both the TDT and NTDT groups. No correlation was found with age, hemoglobin (Hb), and serum levels of calcium, vitamin D, ferritin, phosphorus, and alkaline phosphatase (ALP). Conclusions Impaired bone density was encountered at high proportions in our thalassemia patients. TDT patients suffered more severe bone disease than NTDT patients.

Terminal deoxynucleotidyl transferase expression in different subtypes of childhood B-cell acute lymphoblastic leukemia.

The lack of expression of terminal deoxynucleotidyl transferase (TdT) is frequently associated with KMT2A-rearranged subtype of pediatric acute lymphoblastic leukemia (ALL). However, this association has not been investigated extensively in the Asian population. A retrospective analysis of TdT expression in pediatric B-cell ALL (B-ALL) was performed in patients treated using the Taiwan Pediatric Oncology Group (TPOG) ALL 2002 and 2013 protocols. Among the 331 patients with B-ALL, 12 patients showed TdT negativity at initial diagnosis. Among these, eight patients showed KMT2A rearrangement (66.7%). Other patients showing negative TdT expression had ETV6::RUNX1, MEF2D-rearranged, and other B-ALL subtypes. However, in the context of KMT2A-rearranged B-ALL (n = 20), only eight patients showed TdT negativity. The 5-year event-free survival and overall survival of patients with and without TdT expression were 83.8% versus 46.8% (P <0.001) and 86.3% versus 55.4% (P = 0.004), respectively. Moreover, several aberrant markers, such as CD2, CD56, CD7, and CD117, were rarely expressed in the B-ALL samples, and if expressed, they were enriched in specific genetic subtypes. The results of this study indicate that immunophenotypic features are correlated with specific genetic subtypes of childhood B-ALL.

Enzymatic Preparation of DNA with an Expanded Genetic Alphabet Using Terminal Deoxynucleotidyl Transferase and Its Applications.

Efficient preparation of DNA oligonucleotides containing unnatural nucleobases (UBs) that can pair with their cognates to form unnatural base pairs (UBPs) is an essential prerequisite for the application of UBPs in vitro and in vivo. Traditional preparation of oligonucleotides containing unnatural nucleobases largely relies on solid-phase synthesis, which needs to use unstable nucleoside phosphoramidites and a DNA synthesizer, and is environmentally unfriendly and limited in product length. To overcome these limitations of solid-phase synthesis, we developed enzymatic methods for daily laboratory preparation of DNA oligonucleotides containing unnatural nucleobase dNaM, dTPT3, or one of the functionalized dTPT3 derivatives, which can be used for orthogonal DNA labeling or the preparation of DNAs containing UBP dNaM-dTPT3, one of the most successful UBPs to date, based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Here, we first provide a detailed procedure for the TdT-based preparation of DNA oligonucleotides containing 3'-nucleotides of dNaM, dTPT3, or one of dTPT3 derivatives. We then present the procedures for enzyme-linked oligonucleotide assay (ELONA) and imaging of bacterial cells using DNA oligonucleotides containing 3'-nucleotides of dTPT3 derivatives with different functional groups. The procedure for enzymatic synthesis of DNAs containing an internal UBP dNaM-dTPT3 is also described. Hopefully, these methods will greatly facilitate the application of UBPs and the construction of semi-synthetic organisms with an expanded genetic alphabet.

Establishment of transgenic fluorescent mice for labeling synapses and screening synaptogenic adhesion molecules.

Synapse is the fundamental structure for neurons to transmit information between cells. The proper synapse formation is crucial for developing neural circuits and cognitive functions of the brain. The aberrant synapse formation has been proved to cause many neurological disorders, including autism spectrum disorders and intellectual disability. Synaptic cell adhesion molecules (CAMs) are thought to play a major role in achieving mechanistic cell-cell recognition and initiating synapse formation via trans-synaptic interactions. Due to the diversity of synapses in different brain areas, circuits and neurons, although many synaptic CAMs, such as Neurexins (NRXNs), Neuroligins (NLGNs), Synaptic cell adhesion molecules (SynCAMs), Leucine-rich-repeat transmembrane neuronal proteins (LRRTMs), and SLIT and NTRK-like protein (SLITRKs) have been identified as synaptogenic molecules, how these molecules determine specific synapse formation and whether other molecules driving synapse formation remain undiscovered are unclear. Here, to provide a tool for synapse labeling and synaptic CAMs screening by artificial synapse formation (ASF) assay, we generated synaptotagmin-1-tdTomato (Syt1-tdTomato) transgenic mice by inserting the tdTomato-fused synaptotagmin-1 coding sequence into the genome of C57BL/6J mice. In the brain of Syt1-tdTomato transgenic mice, the tdTomato-fused synaptotagmin-1 (SYT1-tdTomato) signals were widely observed in different areas and overlapped with synapsin-1, a widely-used synaptic marker. In the olfactory bulb, the SYT1-tdTomato signals are highly enriched in the glomerulus. In the cultured hippocampal neurons, the SYT1-tdTomato signals showed colocalization with several synaptic markers. Compared to the wild-type (WT) mouse neurons, cultured hippocampal neurons from Syt1-tdTomato transgenic mice presented normal synaptic neurotransmission. In ASF assays, neurons from Syt1-tdTomato transgenic mice could form synaptic connections with HEK293T cells expressing NLGN2, LRRTM2, and SLITRK2 without immunostaining. Therefore, our work suggested that the Syt1-tdTomato transgenic mice with the ability to label synapses by tdTomato, and it will be a convenient tool for screening synaptogenic molecules.

How Many Tests Does It Take to Diagnose a Triple-Hit B-Lymphoblastic Lymphoma? (Hint, It's A Lot).

B-lymphoblastic leukemia/lymphoma (B-ALL/LBL) is a precursor B-cell neoplasm that often harbors specific cytogenetic/molecular abnormalities with distinctive clinical, phenotypic, and prognostic characteristics. Subcategorization of B-ALL/LBL therefore requires extensive cytogenetic and/or molecular testing to determine the appropriate classification and therapeutic interventions for these patients. Herein, we present a case of a 17-year-old young woman diagnosed with B-LBL harboring not only an IGH::MYC rearrangement but also BCL2 and BCL6 rearrangements (so-called "triple-hit") and somatic biallelic TP53 inactivation. MYC rearrangements are relatively rare in B-ALL/LBL, and the identification of a "triple-hit" elicited an initial diagnostic dilemma. However, a multimodal approach allowed for the classification of this complex case and helped guide selection of an appropriate therapeutic regimen.

Fluorescent enzyme-based biosensor for sensitive analysis of DNA damage in cryopreserved sperm.

The freeze-thaw process can induce irreversible structural and functional changes in human sperm, particularly sperm DNA damage. Selecting a more accurate and sensitive detection method for evaluating sperm DNA integrity is crucial. To accurately assess sperm DNA integrity following the freeze-thaw process and significantly improve the clinical and scientific utilization of cryopreserved sperm. In this study, we utilized a novel fluorescent biosensor, assisted by terminal deoxynucleotidyl transferase (TdT) and Endonuclease IV, to detect DNA breakpoints during sperm cryopreservation. We evaluated the biosensor's performance by comparing it with the conventional DNA fragmentation index (DFI) measured using sperm chromatin structure analysis (SCSA). The cryopreserved group exhibited a significantly higher sperm DFI compared to the fresh group. No significant difference was observed between the antioxidant group and the cryopreserved group. However, the new method revealed a significant reduction in the number of DNA breakpoints in the antioxidant group compared to the cryopreserved group. The novel biosensor demonstrated superior accuracy and effectiveness in assessing sperm DNA integrity during cryopreservation compared to the conventional SCSA method. We believe that the biosensor holds significant potential for widespread use in the field of reproductive medicine.

Insights into Hepatocellular Carcinoma in Patients with Thalassemia: From Pathophysiology to Novel Therapies.

Thalassemia is a heterogeneous congenital hemoglobinopathy common in the Mediterranean region, Middle East, Indian subcontinent, and Southeast Asia with increasing incidence in Northern Europe and North America due to immigration. Iron overloading is one of the major long-term complications in patients with thalassemia and can lead to organ damage and carcinogenesis. Hepatocellular carcinoma (HCC) is one of the most common malignancies in both transfusion-dependent thalassemia (TDT) and non-transfusion-dependent thalassemia (NTDT). The incidence of HCC in patients with thalassemia has increased over time, as better chelation therapy confers a sufficiently long lifespan for the development of HCC. The mechanisms of iron-overloading-associated HCC development include the increased reactive oxygen species (ROS), inflammation cytokines, dysregulated hepcidin, and ferroportin metabolism. The treatment of HCC in patients with thalassemia was basically similar to those in general population. However, due to the younger age of HCC onset in thalassemia, regular surveillance for HCC development is mandatory in TDT and NTDT. Other supplemental therapies and experiences of novel treatments for HCC in the thalassemia population were also reviewed in this article.

Colloidal SERS measurement of enrofloxacin with petaloid nanostructure clusters formed by terminal deoxynucleotidyl transferase catalyzed cytosine-constituted ssDNA.

We developed petal-like plasmonic nanoparticle (PLNP) clusters-based colloidal SERS method for enrofloxacin (EnFX) detection. PLNPs were synthesized by the regulation of single-stranded DNA composed of homo-cytosine deoxynucleotides (hC) catalyzed by terminal deoxynucleotidyl transferase. SERS hot spots were created via the agglomeration process of PLNPs by adding an inorganic salt potassium iodide solution, in which EnFX molecules were attached to the negatively charged PLNPs surface by electrostatic interactions. This approach enabled direct in situ detection of antibiotic residues, achieving a limit of detection (LOD) of 1.15 µg/kg for EnFX. The spiked recoveries of the SERS method were approximately 92.7% to 107.2% and the RSDs ranged from 1.05% to 7.8%, indicating that the method can be applied to actual sample detection. This colloidal SERS measurement platform would be very promising in various applications, especially in real-time and on-site food safety screening owing to its rapidness, simplicity, and sensitivity.

A Rare Case of Cord Compression From Extra-Medullary Hematopoiesis (EMH) in Beta Thalassemia.

Cord compression can arise from many different etiologies -- including trauma, degenerative changes, growths, neoplasms, or even abscesses. While some etiologies can cause symptoms such as weakness or motor deficits, others can simply present as pain. A rare cause of cord compression is extramedullary hematopoiesis (EMH), or the growth of blood cells outside the bone marrow. This rare, abnormal growth of cells can result in severe complications such as increased intracranial pressure and motor and sensory impairment. General clinicians should strive for early and prompt diagnosis of cord compression whenever possible, especially in patients who present with acute neurological deficits. We present a case of a 27-year-old female with beta thalassemia major (BTM) and transfusional hemosiderosis, who came in with progressive lower extremity weakness, numbness and urinary retention, and was diagnosed with acute cord compression from EMH.

Progression of follicular lymphoma and related entities: Report from the 2021 SH/EAHP Workshop.

OBJECTIVES: The 2021 Society for Hematopathology and European Association for Haematopathology Workshop addressed the molecular and cytogenetic underpinnings of transformation and transdifferentiation in lymphoid neoplasms. METHODS: Session 4, "Transformations of Follicular Lymphoma," and session 5, "Transformations of Other B-Cell Lymphomas," included 45 cases. Gene alteration analysis and expression profiling were performed on cases with submitted formalin-fixed, paraffin embedded tissue. RESULTS: The findings from session 4 suggest that "diffuse large B-cell lymphoma/high-grade B-cell lymphoma with rearrangements of MYC and BCL2" is a distinct category arising from the constraints of a preexisting BCL2 translocation. TdT expression in aggressive B-cell lymphomas is associated with MYC rearrangements, immunophenotypic immaturity, and a dismal prognosis but must be differentiated from lymphoblastic -lymphoma. Cases in session 5 illustrated unusual morphologic and immunophenotypic patterns of transformation. Additionally, the findings support the role of cytogenetic abnormalities-specifically, MYC and NOTCH1 rearrangements-as well as single gene alterations, including TP53, in transformation. CONCLUSIONS: Together, these unique cases and their accompanying molecular and cytogenetic data suggest potential mechanisms for and unusual patterns of transformation in B-cell lymphomas and indicate numerous opportunities for further study.

A one-pot CRISPR-Cas12a-based toolbox enables determination of terminal deoxynucleotidyl transferase activity for acute leukemia screening.

An isothermal, one-pot toolbox (called OPT-Cas) based on CRISPR-Cas12a collateral cleavage capability is proposed for highly sensitive and selective determination of terminal deoxynucleotidyl transferase (TdT) activity. Oligonucleotide primers with 3'-hydroxyl (OH) terminal were randomly introduced for TdT-induced elongation. In the presence of TdT, dTTP nucleotides polymerized at the 3' terminals of the primers to generate abundant polyT-tails, which function as triggers for the synchronous activation of Cas12a proteins. Finally, the activated Cas12a trans-cleaved FAM and BHQ1 dual-labeled single-stranded DNA (ssDNA-FQ) reporters, producing significantly amplified fluorescence signals. This one-pot assay, that is primer, crRNA, Cas12a protein and ssDNA-FQ reporter are all in one tube, allows simple but high-sensitive quantification of TdT activity with a low detection limit of 6.16 × 10-5 U µL-1 in the concentration scope from 1 × 10-4 U µL-1 to 1 × 10-1 U µL-1, and achieves extraordinary selectivity with other interfering proteins. Furthermore, the OPT-Cas was successfully used to detect TdT in complex matrices and accurate determination of TdT activity in acute lymphoblastic leukemia cells, which might be a reliable technique platform for the diagnosis of TdT-related diseases and biomedical research applications.