Age-Dependent Alterations in Meiotic Recombination Cause Chromosome Segregation Errors in Spermatocytes.

Faithful chromosome segregation in meiosis requires crossover (CO) recombination, which is regulated to ensure at least one CO per homolog pair. We investigate the failure to ensure COs in juvenile male mice. By monitoring recombination genome-wide using cytological assays and at hotspots using molecular assays, we show that juvenile mouse spermatocytes have fewer COs relative to adults. Analysis of recombination in the absence of MLH3 provides evidence for greater utilization in juveniles of pathways involving structure-selective nucleases and alternative complexes, which can act upon precursors to generate noncrossovers (NCOs) at the expense of COs. We propose that some designated CO sites fail to mature efficiently in juveniles owing to inappropriate activity of these alternative repair pathways, leading to chromosome mis-segregation. We also find lower MutLγ focus density in juvenile human spermatocytes, suggesting that weaker CO maturation efficiency may explain why younger men have a higher risk of fathering children with Down syndrome.

Platelet mass cytometry reveals dysregulation of prothrombotic pathways in essential thrombocythemia.

Thromboembolic events are common in patients with essential thrombocythemia (ET). However, the pathophysiological mechanisms underlying the increased thrombotic risk remain to be determined. Here, we perform the first phenotypical characterization of platelet expression using single-cell mass cytometry in six ET patients and six age- and sex-matched healthy individuals. A large panel of 18 transmembrane regulators of platelet function and activation were analyzed, at baseline and after ex-vivo stimulation with thrombin receptor-activating peptide (TRAP). We detected a significant overexpression of the activation marker CD62P (p-Selectin) (p = .049) and the collagen receptor GPVI (p = .044) in non-stimulated ET platelets. In contrast, ET platelets had a lower expression of the integrin subunits of the fibrinogen receptor GPIIb/IIIa CD41 (p = .036) and CD61 (p = .044) and of the von Willebrand factor receptor CD42b (p = .044). Using the FlowSOM algorithm, we identified 2 subclusters of ET platelets with a prothrombotic expression profile, one of them (cluster 3) significantly overrepresented in ET (22.13% of the total platelets in ET, 2.94% in controls, p = .035). Platelet counts were significantly increased in ET compared to controls (p = .0123). In ET, MPV inversely correlated with platelet count (r=-0.96). These data highlight the prothrombotic phenotype of ET and postulate GPVI as a potential target to prevent thrombosis in these patients.

Essential thrombocythemia (ET) is a rare disease characterized by an increased number of platelets in the blood. As a complication, many of these patients develop a blood clot, which can be life-threatening. So far, the reason behind the higher risk of blood clots is unclear. In this study, we analyzed platelet surface markers that play a critical role in platelet function and platelet activation using a modern technology called mass cytometry. For this purpose, blood samples from 6 patients with ET and 6 healthy control individuals were analyzed. We found significant differences between ET platelets and healthy platelets. ET platelets had higher expression levels of p-Selectin (CD62P), a key marker of platelet activation, and of the collagen receptor GPVI, which is important for clot formation. These results may be driven by a specific platelet subcluster overrepresented in ET. Other surface markers, such as the fibrinogen receptor GPIIb/IIIa CD41, CD61, and the von Willebrand factor receptor CD42b, were lower expressed in ET platelets. When ET platelets were treated with the clotting factor thrombin (thrombin receptor-activating peptide, TRAP), we found a differential response in platelet activation compared to healthy platelets. In conclusion, our results show an increased activation and clotting potential of ET platelets. The platelet surface protein GPVI may be a potential drug target to prevent abnormal blood clotting in ET patients.

JCAD promotes arterial thrombosis through PI3K/Akt modulation: a translational study.

AIMS: Variants of the junctional cadherin 5 associated (JCAD) locus associate with acute coronary syndromes. JCAD promotes experimental atherosclerosis through the large tumor suppressor kinase 2 (LATS2)/Hippo pathway. This study investigates the role of JCAD in arterial thrombosis. METHODS AND RESULTS: JCAD knockout (Jcad-/-) mice underwent photochemically induced endothelial injury to trigger arterial thrombosis. Primary human aortic endothelial cells (HAECs) treated with JCAD small interfering RNA (siJCAD), LATS2 small interfering RNA (siLATS2) or control siRNA (siSCR) were employed for in vitro assays. Plasma JCAD was measured in patients with chronic coronary syndrome or ST-elevation myocardial infarction (STEMI). Jcad-/- mice displayed reduced thrombogenicity as reflected by delayed time to carotid occlusion. Mechanisms include reduced activation of the coagulation cascade [reduced tissue factor (TF) expression and activity] and increased fibrinolysis [higher thrombus embolization episodes and D-dimer levels, reduced vascular plasminogen activator inhibitor (PAI)-1 expression]. In vitro, JCAD silencing inhibited TF and PAI-1 expression in HAECs. JCAD-silenced HAECs (siJCAD) displayed increased levels of LATS2 kinase. Yet, double JCAD and LATS2 silencing did not restore the control phenotype. si-JCAD HAECs showed increased levels of phosphoinositide 3-kinases (PI3K)/ proteinkinase B (Akt) activation, known to downregulate procoagulant expression. The PI3K/Akt pathway inhibitor-wortmannin-prevented the effect of JCAD silencing on TF and PAI-1, indicating a causative role. Also, co-immunoprecipitation unveiled a direct interaction between JCAD and Akt. Confirming in vitro findings, PI3K/Akt and P-yes-associated protein levels were higher in Jcad-/- animals. Lastly, as compared with chronic coronary syndrome, STEMI patients showed higher plasma JCAD, which notably correlated positively with both TF and PAI-1 levels. CONCLUSIONS: JCAD promotes arterial thrombosis by modulating coagulation and fibrinolysis. Herein, reported translational data suggest JCAD as a potential therapeutic target for atherothrombosis.

Role of Reticulated Platelets in Cardiovascular Disease.

Human platelets differ considerably with regard to their size, RNA content and thrombogenicity. Reticulated platelets (RPs) are young, hyper-reactive platelets that are newly released from the bone marrow. They are larger and contain more RNA compared to older platelets. In comparison to more mature platelets, they exhibit a significantly higher thrombogenicity and are known to be elevated in patients with an increased platelet turnover such as, diabetics and after acute myocardial infarction. Several studies have shown that RPs correlate with an insufficient antiplatelet response to aspirin and specific P2Y12 receptor inhibitors. In addition, RPs are promising novel biomarkers for the prediction of adverse cardiovascular events in cardiovascular disease. However, the reason for RPs intrinsic hyper-reactivity and their association with ischemic events is not completely understood and the biology of RPs is still under investigation. We here present a structured review of preclinical and clinical findings concerning the role of RPs in cardiovascular disease.

Prognostic models for breast cancer: based on logistics regression and Hybrid Bayesian Network.

BACKGROUND: To construct two prognostic models to predict survival in breast cancer patients; to compare the efficacy of the two models in the whole group and the advanced human epidermal growth factor receptor-2-positive (HER2+) subgroup of patients; to conclude whether the Hybrid Bayesian Network (HBN) model outperformed the logistics regression (LR) model. METHODS: In this paper, breast cancer patient data were collected from the SEER database. Data processing and analysis were performed using Rstudio 4.2.0, including data preprocessing, model construction and validation. The L_DVBN algorithm in Julia0.4.7 and bnlearn package in R was used to build and evaluate the HBN model. Data with a diagnosis time of 2018(n = 23,384) were distributed randomly as training and testing sets in the ratio of 7:3 using the leave-out method for model construction and internal validation. External validation of the model was done using the dataset of 2019(n = 8128). Finally, the late HER2 + patients(n = 395) was selected for subgroup analysis. Accuracy, calibration and net benefit of clinical decision making were evaluated for both models. RESULTS: The HBN model showed that seventeen variables were associated with survival outcome, including age, tumor size, site, histologic type, radiotherapy, surgery, chemotherapy, distant metastasis, subtype, clinical stage, ER receptor, PR receptor, clinical grade, race, marital status, tumor laterality, and lymph node. The AUCs for the internal validation of the LR and HBN models were 0.831 and 0.900; The AUCs for the external validation of the LR and HBN models on the whole population were 0.786 and 0.871; the AUCs for the external validation of the two models on the subgroup population were 0.601 and 0.813. CONCLUSION: The accuracy, net clinical benefit, and calibration of the HBN model were better than LR model. The predictive efficacy of both models decreased and the difference was greater in advanced HER2 + patients, which means the HBN model had higher robustness and more stable predictive performance in the subgroup.

Couples' Cognition, Intimacy, and Activity Engagement: An Actor-Partner Interdependence Model Analysis.

OBJECTIVES: This study examines the actor and partner effects of cognition on activity engagement and the potential mediating role of intimate relationship in older couple dyadic context. METHODS: Data for this study were obtained from heterosexual couples who participated in the 2020 wave of the Health and Retirement Study. Constructing a dyadic model of couples' cognitions, activity engagement, and intimacy using structural equation modeling to analyze the relationship between variables and mediating effects. RESULTS: At the actor level, cognition was positively correlated with their activity participation. At the partner level, wives' cognition and husbands' activity engagement were positively correlated. Husband's cognitive score affect wife's subjective feelings about intimacy. The mediation effect of Wife's intimacy on the relationship between husband's cognition and wife's activity engagement was significant. CONCLUSIONS: In older couples, cognition influences activity engagement at a binary level, and this association is influenced by the level of intimacy. Improving intimacy can help increase activity engagement in older couples, which in turn promotes health. Clinical Implications Maintaining cognition helps older people enjoy good marriage and participation in activities. For women, the closer the partnership, the higher the frequency of participating in various activities.

Bioengineered Protein-based Adhesives for Biomedical Applications.

Protein-based adhesives with their robust adhesion performance and excellent biocompatibility have been extensively explored over years. In particular, the unique adhesion behaviours of mussel and sandcastle worm inspired the development of synthetic adhesives. However, the chemical synthesized adhesives often demonstrate weak underwater adhesion performance and poor biocompatibility/biodegradability, limiting their further biomedical applications. In sharp contrast, genetically engineering endows the protein-based adhesives the ability to maintain underwater adhesion property as well as biocompatibility/biodegradability. Herein, we outline recent advances in the design and development of protein-based adhesives by genetic engineering. We summarize the fabrication and adhesion performance of elastin-like polypeptide-based adhesives, followed by mussel foot protein (mfp) based adhesives and other sources protein-based adhesives, such as, spider silk spidroin and suckerin. In addition, the biomedical applications of these bioengineered protein-based adhesives are presented. Finally, we give a brief summary and perspective on the future development of bioengineered protein-based adhesives.

Mass cytometry of platelet-rich plasma: a new approach to analyze platelet surface expression and reactivity.

Mass cytometry (CyTOF) is a new technology that allows the investigation of protein expression at single cell level with high resolution. While several protocols are available to investigate leukocyte expression, platelet staining and analysis with CyTOF have been described only from whole blood. Moreover, available protocols do not allow sample storage but require fresh samples to be obtained, processed, and measured immediately. We provide a structured and reproducible method to stain platelets from platelet-rich plasma to study thrombocyte protein expression and reactivity using mass cytometry. With our method, it is possible to acquire a large number of events allowing deep bioinformatic investigation of platelet expression heterogeneity. Integrated in our protocol is also a previously established freezing protocol that allows the storage of stained samples and to delay their measurement. Finally, we provide a structured workflow using different platelet stimulators and a freely available bioinformatic pipeline to analyze platelet expression. Our protocol unlocks the potential of CyTOF analysis for studying platelet biology in health and disease.

Bioconjugation of Supramolecular Metallacages to Integrin Ligands for Targeted Delivery of Cisplatin.

Cisplatin occupies a crucial role in the treatment of various malignant tumors. However, its efficacy and applicability are heavily restricted by severe systemic toxicities and drug resistance. Our study exploits the active targeting of supramolecular metallacages to enhance the activity of cisplatin in cancer cells while reducing its toxicity. Thus, Pd2L4 cages (L = ligand) have been conjugated to four integrin ligands with different binding affinity and selectivity. Cage formation and encapsulation of cisplatin was proven by NMR spectroscopy. Upon encapsulation, cisplatin showed increased cytotoxicity in vitro, in melanoma A375 cells overexpressing αvß3 integrins. Moreover, ex vivo studies in tissue slices indicated reduced toxicity toward healthy liver and kidney tissues for cage-encapsulated cisplatin. Analysis of metal content by ICP-MS demonstrated that the encapsulated drug is less accumulated in these organs compared to the "free" cisplatin.

Reticulated Platelets Predict Cardiovascular Death and Adverse Events in Coronary Artery Disease: A Systematic Review and Meta-analysis.

BACKGROUND: The pro-thrombotic immature or reticulated platelets (RPs) are known to be elevated in high-risk patients and in different pathological settings. It has been shown that RPs correlate with an insufficient antiplatelet response to antiplatelet agents. RPs are emerging novel predictors of adverse cardiovascular events in cardiovascular disease. This study, using the totality of existing evidence, evaluated the prognostic role of RPs in patients with coronary artery disease. METHODS: We performed a systematic review and meta-analysis including trials of acute and chronic coronary syndrome reporting clinical outcomes according to RPs levels in the peripheral blood. We compared patients with elevated RPs (RPshigh) to patients without elevated RPs (RPslow). Odds ratios (ORs) and 95% CIs were used as metric of choice for treatment effects with random-effects models. The primary endpoint was major adverse cardiovascular and cerebrovascular events (MACCE). Secondary endpoints were cardiovascular death, myocardial infarction, ischemic stroke, urgent coronary revascularization and bleedings. RESULTS: A total of 7 studies, including 2213 patients, were included. The risk for MACCE was significantly higher in RPshigh compared to RPslow patients (OR 2.67 [1.87; 3.81], I2 = 43.8%). RPshigh were associated with cardiovascular death (OR 2.09 [1.36; 3.22], I2 = 40.4%). No associations for RPshigh were detected with the other singular components of MACCE: myocardial infarction (OR 1.73 [0.89; 3.38] I2 = 60.5%) and stroke (OR 1.72 [0.59; 4.96] I2 = 21%). The risk of bleeding did not differ between groups(OR 0.58 [0.15; 2.22] I2 = 86.1%). CONCLUSION: Elevated RPs are significantly associated with increased risk of cardiovascular events and cardiovascular death.

Venturi-electrosonic spray ionization cataluminescence sensor array for saccharides detection.

In this article, a Venturi electrosonic spray ionization (V-ESSI) cataluminescence (CTL) sensor array was reported for discriminating saccharides in solution. Integrating electrosonic spray ionization (ESSI), a liquid system of Venturi self-pumping injection for the CTL reaction, was fabricated for enhancing CTL reactivity of aqueous samples. Comparing with simple Venturi injection by air and Venturi easy ambient sonic-spray ionization without electric assistance (V-EASI), the remarkable enhancement of CTL signals resulted from V-ESSI. This system showed higher cross-reactive CTL responses catalyzed by alkaline earth metal-nanomaterials than other catalysts, giving different signals for a given saccharide on different catalysts and different responses for different saccharides on the same catalyst. Then, a 4 × 2 CTL sensor array was used for obtaining "fingerprints" of distinct CTL response patterns. Analyzed by linear discriminant analysis (LDA), this V-ESSI CTL sensor array not only achieved the well discrimination of different saccharides (99.9% of total variation) but also discriminated four groups of urine sugar-level for urine samples from diabetic patients (98.1% of discrimination accuracy). It had good reproducibility and gave a linear range of 22.5-67558 µg/mL (R > 0.99) for xylose with a detection limit of 7.4 µg/mL on MgO. As a new artificial tongue, this system provided a simple, rapid, low cost, low energy consumption, and environmentally friendly pathway for aqueous sample discrimination. It has dramatically expanded applications of the CTL-based senor array and will be applicable to clinical diagnoses, environment monitoring, industrial controls, food industry, and various marine monitoring.

Plasma-assisted cataluminescence sensor array for gaseous hydrocarbons discrimination.

Combining plasma activation and cross-reactivity of sensor array, we have developed a plasma-assisted cataluminescence (PA-CTL) sensor array for fast sensing and discrimination of gaseous hydrocarbons, which can be potentially used for fast diagnosis of lung cancer. Based on dielectric barrier discharge, a low-temperature plasma is generated to activate gaseous hydrocarbons with low cataluminescence (CTL) activities. Extremely increased CTL responses have been obtained, which resulted in a plasma assistance factor of infinity (∞) for some hydrocarbons. On a 4 × 3 PA-CTL sensor array made from alkaline-earth nanomaterials, gaseous hydrocarbons showed robust and unique CTL responses to generate characteristic patterns for fast discrimination. Because of the difference in the component of hydrocarbons in breath, exhaled breath samples from donors with and without lung cancer were tested, and good discrimination has been achieved by this technique. In addition, the feasibility of multidimentional detection based on temperature was confirmed. It had good reproducibility and gave a linear range of 65-6500 ng/mL or 77-7700 ppmv (R > 0.98) for CH(4) with a detection limit of 33 ng/mL (38 ppmv) on MgO. The PA-CTL sensor array is simple, low-cost, thermally stable, nontoxic, and has an abundance of alkaline-earth nanomaterials to act as sensing elements. This has expanded the applications of CTL-based senor arrays and will show great potential in clinical fast diagnosis.

Improved lentiviral vector titers from a multi-gene knockout packaging line.

Lentiviral vectors (LVs) are robust delivery vehicles for gene therapy as they can efficiently integrate transgenes into host cell genomes. However, LVs with lengthy or complex expression cassettes typically are produced at low titers and have reduced gene transfer capacity, creating barriers for clinical and commercial applications. Modifications of the packaging cell line and methods may be able to produce complex vectors at higher titer and infectivity and may improve production of many different LVs. In this study, we identified two host restriction factors in HEK293T packaging cells that impeded LV production, 2'-5'-oligoadenylate synthetase 1 (OAS1) and low-density lipoprotein receptor (LDLR). Knocking out these two genes separately led to ∼2-fold increases in viral titer. We created a monoclonal cell line, CRISPRed HEK293T to Disrupt Antiviral Response (CHEDAR), by successively knocking out OAS1, LDLR, and PKR, a previously identified factor impeding LV titers. Packaging in CHEDAR yielded ∼7-fold increases in physical particles, full-length vector RNA, and vector titers. In addition, overexpressing transcription elongation factors, SPT4 and SPT5, during packaging improved the production of full-length vector RNA, thereby increasing titers by ∼2-fold. Packaging in CHEDAR with over-expression of SPT4 and SPT5 led to ∼11-fold increases of titers. These approaches improved the production of a variety of LVs, especially vectors with low titers or with internal promoters in the reverse orientation, and may be beneficial for multiple gene therapy applications.

ß-Globin Lentiviral Vectors Have Reduced Titers due to Incomplete Vector RNA Genomes and Lowered Virion Production.

Lentiviral vectors (LVs) commonly used for the treatment of hemoglobinopathies often have low titers and sub-optimal gene transfer efficiency for human hematopoietic stem and progenitor cells (HSPCs), hindering clinical translation and commercialization for ex vivo gene therapy. We observed that a high percentage of ß-globin LV viral genomic RNAs were incomplete toward the 3' end in packaging cells and in released vector particles. The incomplete vector genomes impeded reverse transcription in target cells, limiting stable gene transfer to HSPCs. By combining three modifications to vector design and production (shortening the vector length to 5.3 kb; expressing HIV-1 Tat protein during packaging; and packaging in PKR-/- cells) there was a 30-fold increase in vector titer and a 3-fold increase in vector infectivity in HSPCs. These approaches may improve the manufacturing of ß-globin and other complex LVs for enhanced gene delivery and may facilitate clinical applications.

Targeted imaging of integrins in cancer tissues using photocleavable Ru(ii) polypyridine complexes as mass-tags.

Targeted epitope-based mass spectrometry imaging (MSI) utilizes laser cleavable mass-tags bound to targeting moieties for detecting proteins in tissue sections. Our work constitutes the first proof-of-concept of a novel laser desorption ionization (LDI)-MSI strategy using photocleavable Ru(ii) polypyridine complexes as mass-tags for imaging of integrins αvß3 in human cancer tissues.

Self-assembly of highly luminescent heteronuclear coordination cages.

Exo-functionalized Pd2L4 cage compounds with attached Ru(ii) pyridine complexes were prepared via coordination-driven self-assembly. Unlike most of the previously reported palladium(ii) cages, one of these metallocages exhibits an exceptionally high quantum yield of 66%. The presented approach is promising to obtain luminescent coordination complexes for various applications.

Room-temperature cataluminescence from CO oxidation in a non-thermal plasma-assisted catalysis system.

Cataluminescence (CTL) is a kind of chemiluminescence during catalytic reaction on surface of catalysts under a heated condition. Due to the low catalytic reactivity of CO, normally low intensity of CTL is obtained during heterogeneously catalytic oxidation of CO under heated conditions (normally higher than 150°C), even catalyzed by precious-metal-based catalysts. Therefore, seeking enhanced CTL of CO at room temperature and using low-cost catalysts becomes significant. Here, CTL generated from CO oxidation was firstly reported at room temperature, which was carried out in a non-thermal plasma-assisted (NTPA) catalysis system. With air acting as discharge gas, carrier gas as well as oxidant, a Mn/SiO2 nanomaterials-based NTPA catalysis system was fabricated for CO catalytic oxidation at room temperature, whose temperature was much lower than previous CTL methods. Relatively high and selective CTL responses were acquired during CO oxidation on surface of Mn/SiO2 nanomaterials, whereas no significant CTL signal was recorded without plasma assistance or on other metals-doped SiO2 catalysts. Without any excitation light source or heating element, a low cost and simple CO sensor was fabricated by using common and easily synthesized catalysts. The present work has greatly simplified the constructions, and enlarged CTL applications.

Preliminary investigations into surface molecularly imprinted nanoparticles for Helicobacter pylori eradication.

This paper reports investigations into the preparation and characterization of surface molecularly imprinted nanoparticles (SMINs) designed to adhere to Helicobacter pylori (H. pylori). Imprinted nanoparticles were prepared by the inverse microemulsion polymerization method. A fraction of Lpp20, an outer membrane protein of H. pylori known as NQA, was chosen as template and modified with myristic acid to facilitate its localization on the surface of the nanoparticles. The interaction between these SMINs with the template NQA were evaluated using surface plasmon resonance (SPR), change in zeta potential and fluorescence polarization (FP). The results were highly consistent in demonstrating a preferential recognition of the template NQA for SMINs compared with the control nanoparticles. In vitro experiments also indicate that such SMINs are able to adhere to H. pylori and may be useful for H. pylori eradication.

Low temperature CO sensor based on cataluminescence from plasma-assisted catalytic oxidation on Ag doped alkaline-earth nanomaterials.

Based on cataluminescence from plasma-assisted catalytic oxidation, a low temperature CO sensor was fabricated. With Ag doped alkaline-earth catalyst as sensing element, air as discharge gas, carrier gas and oxidant supplier, significant cataluminescence was achieved at low temperature, demonstrating a potential low-consumption and portable sensor of CO.

Lactoferrin conjugated PEG-PLGA nanoparticles for brain delivery: preparation, characterization and efficacy in Parkinson's disease.

A novel biodegradable brain drug delivery system, the lactoferrin (Lf) conjugated polyethylene glycol-polylactide-polyglycolide (PEG-PLGA) nanoparticle (Lf-NP) was constructed in this paper with its in vitro and in vivo delivery properties evaluated by a fluorescent probe coumarin-6. Lf was thiolated and conjugated to the distal maleimide function surrounding on the pegylated nanoparticle to form Lf-NP. TEM observation and ELISA analysis confirmed the existence of active Lf on the surface of Lf-NP. The results of qualitative and quantitative uptake studies of coumarin-6 incorporated Lf-NP showed a more pronounced accumulation of Lf-NP in bEnd.3 cells than that of unconjugated nanoparticle (NP). Further uptake inhibition study indicated that the increased uptake of Lf-NP was via an additional clathrin mediated endocytosis processes. Following intravenous administration, a near 3 fold of coumarin-6 was found in the mice brain carried by Lf-NP compared to that carried by NP. Intravenous injection of urocortin loaded Lf-NP effectively attenuated the striatum lesion caused by 6-hydroxydopamine in rats as indicated by the behavioral test, the immunohistochemistry test and striatal transmitter content detection results. The cell viability test and CD68 immunohistochemistry demonstrated the acceptable toxicity of the system. All these results demonstrated that Lf-NP was a promising brain drug delivery system with reasonable toxicity.