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.

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.

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.

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.

Blood security and management of transfusion-dependent thalassaemia (TDT) patients in Nanning: analysis of " collection-based-supply" blood management mode / 中国输血杂志

【Objective】 To retrospectively analyze the blood use of transfusion-dependent thalassemia (TDT) patients in 9 designated transfusion medical institutions from 2018 to 2023 in Nanning, and to evaluate the effect of " three designated " blood transfusion mode (hereby means TDT patients undergoing blood transfusion in designated transfusion medical institutions regularly) and " collection-based-supply" blood management mode on blood security of TDT patients. 【Methods】 The " three designated" blood transfusion mode was implemented to ensure that TDT patients registered in the local household registration (referred to as the " register" ) obtain the rights and interests of outpatient transfusion and blood security of designated medical institutions. The " collection-based-supply" blood management mode was implemented to assess the blood needs of "register" TDT patients and meet their needs to the maximum extent according to the blood inventory (collection). 【Results】 From 2018 to 2023, the total blood supply of "register" TDT patients was 10.37% of the total red blood supply of all medical institutions (138 509.5 U /1 335 788.0 U), with the highest proportion of type O blood as 46.34% (64 181.0 U/138 509.5 U) and the lowest proportion of type AB blood as 3.85% (5 331.0 U/138 509.5 U). In 2018, 9 transfusion medical institutions were designated for TDT patients.There were a total of 766 TDT patients in the register, with the per capita annual blood transfusion volume increased from 20.28 U (15 531.0 U/766 patients) in 2018 to 36.01 U (27 586.0 U/766 patients) in 2023, maintaining a positive growth every year(30.26%, 4.94%, 11.71%, 8.61%, 4.94% and 7.10%). 【Conclusion】 The " three designated" blood transfusion mode and the " collection-based-supply " blood management mode can effectively guarantee the blood supply of TDT patients.

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.

Genetic variants in genes involved in creatine biosynthesis in patients with severe obesity or anorexia nervosa.

Increased thermogenesis in brown adipose tissue might have an obesity-reducing effect in humans. In transgenic mice, depletion of genes involved in creatine metabolism results in disrupted thermogenic capacity and altered effects of high-fat feeding on body weight. Data analyses of a sex-stratified genome-wide association study (GWAS) for body mass index (BMI) within the genomic regions of genes of this pathway (CKB, CKMT1B, and GATM) revealed one sex-dimorphic BMI-associated SNP in CKB (rs1136165). The effect size was larger in females than in males. A mutation screen of the coding regions of these three candidate genes in a screening group (192 children and adolescents with severe obesity, 192 female patients with anorexia nervosa, and 192 healthy-lean controls) identified five variants in each, CKB and GATM, and nine variants in the coding sequence of CKMT1B. Non-synonymous variants identified in CKB and CKMT1B were genotyped in an independent confirmation study group (781 families with severe obesity (trios), 320 children and adolescents with severe obesity, and 253 healthy-lean controls). In silico tools predicted mainly benign yet protein-destabilizing potentials. A transmission disequilibrium test in trios with severe obesity indicated an obesity-protective effect of the infrequent allele at rs149544188 located in CKMT1B. Subsequent correlation analyses in 1,479 individuals of the Leipzig Obesity BioBank revealed distinct correlations of CKB with the other two genes in omental visceral adipose tissue (VAT) and abdominal subcutaneous adipose tissue (SAT). Furthermore, between-subject comparisons of gene expression levels showed generally higher expressions of all three genes of interest in VAT than in SAT. Future in vitro analyses are needed to assess the functional implications of these findings.

DNTT activation, TdT-aided gene length mutation, and better prognosis in ATG-based regimen allo-HSCT in AML.

This study aimed to investigate the relationship between anomalous DNA nucleotidylexotransferase (DNTT) activation and the mutagenesis of gene length mutations (LMs) in acute myeloid leukemia (AML), and the relevance of their prognosis in antithymocyte globulin (ATG)-based regimen allogeneic hematopoietic stem cell transplantation (allo-HSCT). A cohort of 578 AML cases was enrolled. Next-generation sequencing was performed to screen mutations of 86 leukemia driver genes. RNA-seq was used to analyze gene expression. Prognostic analysis was investigated in 239 AML cases who underwent ATG-based regimen allo-HSCT. We report a refined subtyping algorithm of LMs (type I-IV) based on sequence anatomy considering the TdT-aided mutagenesis mechanism. GC content adjacent to LM junctions, inserted nontemplate nucleotide bases, and DNTT expression analysis supported the DNTT activation and TdT-aided mutagenesis in type II/III LMs in the total AML cohort. Both single-variate and multivariate analyses showed a better overall survival of FLT3 type III compared to type I in a subset of ATG-based regimen allo-HSCT cases. The novel LM subtyping algorithm not only deciphers the etiology of the mutagenesis of LMs but also helps to fine-tune prognosis differentiation in AML. The possible prognostic versatility of this novel LM subtyping algorithm in terms of chemotherapy, targeted therapy, and allo-HSCT merits further investigation.

Effects of elevated temperature on 8-OHdG expression in the American oyster (Crassostrea virginica): Induction of oxidative stress biomarkers, cellular apoptosis, DNA damage and γH2AX signaling pathways.

Global temperature is increasing due to anthropogenic activities and the effects of elevated temperature on DNA lesions are not well documented in marine organisms. The American oyster (Crassostrea virginica, an edible and commercially important marine mollusk) is an ideal shellfish species to study oxidative DNA lesions during heat stress. In this study, we examined the effects of elevated temperatures (24, 28, and 32 °C for one-week exposure) on heat shock protein-70 (HSP70, a biomarker of heat stress), 8­hydroxy-2'-deoxyguanosine (8-OHdG, a biomarker of pro-mutagenic DNA lesion), double-stranded DNA (dsDNA), γ-histone family member X (γH2AX, a molecular biomarker of DNA damage), caspase-3 (CAS-3, a key enzyme of apoptotic pathway) and Bcl-2-associated X (BAX, an apoptosis regulator) protein and/or mRNA expressions in the gills of American oysters. Immunohistochemical and qRT-PCR results showed that HSP70, 8-OHdG, dsDNA, and γH2AX expressions in gills were significantly increased at high temperatures (28 and 32 °C) compared with control (24°C). In situ TUNEL analysis showed that the apoptotic cells in gill tissues were increased in heat-exposed oysters. Interestingly, the enhanced apoptotic cells were associated with increased CAS-3 and BAX mRNA and/or protein expressions, along with 8-OHdG levels in gills after heat exposure. Moreover, the extrapallial (EP) fluid (i.e., extracellular body fluid) protein concentrations were lower; however, the EP glucose levels were higher in heat-exposed oysters. Taken together, these results suggest that heat shock-driven oxidative stress alters extracellular body fluid conditions and induces cellular apoptosis and DNA damage, which may lead to increased 8-OHdG levels in cells/tissues in oysters.

Effects of Au@Ag core-shell nanostructure with alginate coating on male reproductive system in mice.

Despite the widespread use of silver nanoparticles (NPs), these NPs can accumulate and have toxic effects on various organs. However, the effects of silver nanostructures (Ag-NS) with alginate coating on the male reproductive system have not been studied. Therefore, this study aimed to investigate the impacts of this NS on sperm function and testicular structure. After the synthesis and characterization of Ag-NS, the animals were divided into five groups (n = 8), including one control group, two sham groups (received 1.5 mg/kg/day alginate solution for 14 and 35 days), and two treatment groups (received Ag-NS at the same dose and time). Following injections, sperm parameters, apoptosis, and autophagy were analyzed by the TUNEL assay and measurement of the mRNA expression of Bax, Bcl-2, caspase-3, LC3, and Beclin-1. Fertilization rate was assessed by in vitro fertilization (IVF), and testicular structure was analyzed using the TUNEL assay and hematoxylin and eosin (H&E) staining. The results showed that the NS was rod-shaped, had a size of about 60 nm, and could reduce sperm function and fertility. Gene expression results demonstrated an increase in the apoptotic markers and a decrease in autophagy markers, indicating apoptotic cell death. Moreover, Ag-NS invaded testicular tissues, especially in the chronic phase (35 days), resulting in tissue alteration and epithelium disintegration. The results suggest that sperm parameters and fertility were affected. In addition, NS has negative influences on testicular tissues, causing infertility in men exposed to these NS.

Apoptotic biliary epithelial cells and gut dysbiosis in the induction of murine primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a female-predominant liver autoimmune disease characterized by the specific immune-mediated destruction of the intrahepatic small bile duct. Although apoptosis of biliary epithelial cells (BECs) and alterations in gut microbiota are observed in patients with PBC, it is still unclear whether these events happen in the early stage and cause the breakdown of tolerance in PBC. In this study, we examined the early events in the loss of tolerance in our well-defined 2-OA-OVA-induced murine autoimmune cholangitis (AIC) model. We report herein that apoptosis of BECs was notable in the early stage of murine AIC. An altered gut microbiota, in particular, an increased percentage of gram-positive Firmicutes in AIC mice was also observed. BECs in AIC mice expressed adhesion molecule ICAM-1, cytokines/chemokines TNF-α, CCL2, CXCL9, CXCL10, and toll-like receptor (TLR) 2. Moreover, BECs treated with TLR2 ligand had elevated apoptosis and CXCL10 production. These data collectively suggest a new mechanism of tolerance breakdown in AIC. Altered gut microbiota induces apoptosis of BECs through TLR2 signaling. BECs secrete chemokines to recruit CD8 T cells to damage BECs further.

Primer-Engineered Transferase Enzyme for One-Pot and Amplified Detection of Cobalt Pollution and Peptide Remover Screening.

Extensive consumption of cobalt in the chemical field such as for battery materials, alloy, pigments, and dyes has aggravated the pollution of cobalt both in food and the environment, and assays for its on-site monitoring are urgently demanded. Herein, we utilized enzyme dependence on metal cofactors to develop terminal transferase (TdT) as a recognition element, achieving a one-pot sensitive and specific assay for detecting cobalt pollution. We engineered a 3'-OH terminus primer to improve the discrimination capacity of TdT for Co2+ from other bivalent cations. The TdT extension reaction amplified the recognition of Co2+ and yielded a limit of detection of 0.99 µM for Co2+ detection. Then, the TdT-based assay was designed to precisely detect cobalt in food and agricultural soil samples. By end-measurement of fluorescence using a microplate reader, the multiplexing assay enabled the rapid screening of the peptide remover for cobalt pollution. The TdT-based assay can be a promising tool for cobalt pollution monitoring and control.