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Neurochemical sensing with implantable devices has gained remarkable attention over the last few decades. A promising area of this research is the progress of novel electrodes as electrochemical tools for neurotransmitter detection in the brain. The boron-doped diamond (BDD) electrode is one such candidate that previously has been reported for its excellent electrochemical properties, including a wide working potential, superior chemical inertness and mechanical stability, good biocompatibility and resistance to fouling. Meanwhile, limited research has been conducted on the BDD as a microelectrode for neurochemical detection. Our team has developed a freestanding, all diamond microelectrode consisting of a boron-doped polycrystalline diamond core, encapsulated in an insulating polycrystalline diamond shell, with a cleaved planar tip for electrochemical sensing. This all-diamond electrode is advantageous due to its - (1) batch fabrication using wafer technology that eliminates traditional hand fabrication errors and inconsistencies, (2) absence of metal-based wires, or foundations, to improve biocompatibility and flexibility, and (3) sp 3 carbon surface with resistance to biofouling, i.e. adsorption of proteins or unwanted molecules at the electrode surface in a biological environment that impedes overall electrode performance. Here, we provide findings on further in vitro testing and development of the freestanding boron-doped diamond microelectrode (BDDME) for neurotransmitter detection using fast scan cyclic voltammetry (FSCV). In this report, we elaborate on - 1) an updated fabrication scheme and work flow to generate all diamond BDDMEs, 2) slow scan cyclic voltammetry measurements of reference and target analytes to understand basic electrochemical behavior of the electrode, and 3) FSCV characterization of common neurotransmitters, and overall favorability of serotonin (5-HT) detection. The BDDME showed a 2-fold increased FSCV response for 5-HT in comparison to dopamine (DA), with a limit of detection of 0.16 µM for 5-HT and 0.26 µM for DA. These results are intended to expand on the development of the next generation BDDME and guide future in vivo experiments, adding to the growing body of literature on implantable devices for neurochemical sensing.
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Interactions between acute myeloid leukemia (AML) and the bone marrow microenvironment (BMME) are critical to leukemia progression and chemoresistance. Altered metabolite levels in the tumor microenvironment contribute to immunosuppression in solid tumors, while this has not been studied yet in the leukemic BMME. Metabolomics of AML patient bone marrow serum detected elevated metabolites, including lactate, compared to age- and sex-matched controls. Excess lactate has been implicated in solid tumors for inducing suppressive tumor-associated macrophages (TAMs) and correlates with poor prognosis. We describe the role of lactate in the polarization of leukemia-associated macrophages (LAMs) using a murine model of blast crisis chronic myelogenous leukemia (bcCML) and mice genetically lacking the lactate receptor GPR81. LAMs were CD206hi and suppressive in transcriptomics and cytokine profiling. Yet, LAMs had a largely unique expression profile from other types of TAMs. We demonstrate GPR81 signaling as a mechanism of both LAM polarization and the direct support of leukemia cell growth and self-repopulation. Furthermore, LAMs and elevated lactate diminished the function of hematopoietic progenitors and stromal support, while knockout of GPR81 had modest protective effects on the hematopoietic system. We report microenvironmental lactate as a critical driver of AML-induced immunosuppression and leukemic progression, thus identifying GPR81 signaling as an exciting and novel therapeutic target for treating this devastating disease.
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Fast-scan cyclic voltammetry (FSCV) is an electrochemical sensing technique that can be used for neurochemical sensing with high spatiotemporal resolution. Carbon fiber microelectrodes (CFMEs) are traditionally used as FSCV sensors. However, CFMEs are prone to electrochemical fouling caused by oxidative byproducts of repeated serotonin (5-HT) exposure, which makes them less suitable as chronic 5-HT sensors. Our team is developing a boron-doped diamond microelectrode (BDDME) that has previously been shown to be relatively resistant to fouling caused by protein adsorption (biofouling). We sought to determine if this BDDME exhibits resistance to electrochemical fouling, which we explored on electrodes fabricated with either femtosecond laser cutting or physical cleaving. We recorded the oxidation current response after 25 repeated injections of 5-HT in a flow-injection cell and compared the current drop from the first with the last injection. The 5-HT responses were compared with dopamine (DA), a neurochemical that is known to produce minimal fouling oxidative byproducts and has a stable repeated response. Physical cleaving of the BDDME yielded a reduction in fouling due to 5-HT compared with the CFME and the femtosecond laser cut BDDME. However, the femtosecond laser cut BDDME exhibited a large increase in sensitivity over the cleaved BDDME. An extended stability analysis was conducted for all device types following 5-HT fouling tests. This analysis demonstrated an improvement in the long-term stability of boron-doped diamond over CFMEs, as well as a diminishing sensitivity of the laser-cut BDDME over time. This work reports the electrochemical fouling performance of the BDDME when it is repeatedly exposed to DA or 5-HT, which informs the development of a chronic, diamond-based electrochemical sensor for long-term neurotransmitter measurements in vivo.
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Boro , Diamante , Técnicas Electroquímicas , Microelectrodos , Serotonina , Serotonina/análisis , Boro/química , Diamante/química , Técnicas Biosensibles , Dopamina/análisis , Fibra de Carbono , Oxidación-ReducciónRESUMEN
Lead halide perovskite quantum dots (QDs), the latest generation of the colloidal QD family, exhibit outstanding optical properties, which are now exploited as both classical and quantum light sources. Most of their rather exceptional properties are related to the peculiar exciton fine-structure of band-edge states, which can support unique bright triplet excitons. The degeneracy of the bright triplet excitons is lifted with energetic splitting in the order of millielectronvolts, which can be resolved by the photoluminescence (PL) measurements of single QDs at cryogenic temperatures. Each bright exciton fine-structure-state (FSS) exhibits a dominantly linear polarization, in line with several theoretical models based on the sole crystal field, exchange interaction, and shape anisotropy. Here, we show that in addition to a high degree of linear polarization, the individual exciton FSS can exhibit a non-negligible degree of circular polarization even without external magnetic fields by investigating the four Stokes parameters of the exciton fine-structure in individual CsPbBr3 QDs through Stokes polarimetric measurements. We observe a degree of circular polarization up to â¼38%, which could not be detected by using the conventional polarimetric technique. In addition, we found a consistent transition from left- to right-hand circular polarization within the fine-structure triplet manifold, which was observed in magnetic-field-dependent experiments. Our optical investigation provides deeper insights into the nature of the exciton fine structures and thereby drives the yet-incomplete understanding of the unique photophysical properties of this class of QDs for the benefit of future applications in chiral quantum optics.
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Acute myeloid leukemia (AML) is fatal in the majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds, selected 4 hits targeting the IQGAP1-GRD domain, and conducted SAR of the 'fittest hit' to identify UR778Br, a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation, induced apoptosis, resulted in G2/M arrest, and inhibited colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows response to IQGAP1 inhibition, and UR778Br, identified through in-silico studies, selectively targeted AML cells while sparing normal marrow.
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Proliferación Celular , Leucemia Mieloide Aguda , Proteínas Activadoras de ras GTPasa , Humanos , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/antagonistas & inhibidores , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/genética , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Simulación por Computador , Antineoplásicos/farmacología , Dominios Proteicos , Animales , Proteómica/métodosRESUMEN
Post-market surveillance of test performance is a critical function of public health agencies and clinical researchers that ensures tests maintaining diagnostic characteristics following their regulatory approval. Changes in product quality, manufacturing processes over time, or the evolution of new variants may impact product performance. During the COVID-19 pandemic, a plethora of point-of-care tests (POCTs) was released onto the Canadian market. This study evaluated the performance characteristics of several of the most widely distributed POCTs in Canada, including four rapid antigen tests (Abbott Panbio, BTNX Rapid Response, SD Biosensor, and Quidel QuickVue) and two molecular tests (Abbott ID NOW and Lucira Check IT). All tests were challenged with 149 SARS-CoV-2 clinical positives, including multiple variants up to and including Omicron XBB.1.5, as well as 29 clinical negatives. Results were stratified based on whether the isolate was Omicron or pre-Omicron as well as by reverse transcriptase quantitative PCR Ct value. The test performance of each POCT was consistent with the manufacturers' claims and showed no significant decline in clinical performance against any of the variants tested. These findings provide continued confidence in the results of these POCTs as they continue to be used to support decentralized COVID-19 testing. This work demonstrates the essential role of post-market surveillance in ensuring reliability in diagnostic tools.IMPORTANCEPost-market surveillance of diagnostic test performance is critical to ensure their reliability after regulatory approval. This is especially critical in the context of the COVID-19 pandemic as the use of point-of-care tests (POCTs) became widespread. Our study focused on four rapid antigen tests (Abbott Panbio, BTNX Rapid Response, SD Biosensor, and Quidel QuickVue) and two molecular tests (Abbott ID NOW and Lucira Check IT) that were widely distributed across Canada, assessing their performance using many SARS-CoV-2 variants, including up to Omicron subvariant XBB.1.5. Overall, we found no significant difference in performance against any variant, reinforcing confidence in their use. As concerns in test efficacy have been raised by news outlets, particularly regarding the BTNX Rapid Response, this work is even more timely and crucial. Our research offers insights into the performance of widely used COVID-19 POCTs but also highlights the necessity for post-market surveillance.
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COVID-19 , Pruebas en el Punto de Atención , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , COVID-19/diagnóstico , COVID-19/epidemiología , COVID-19/virología , Canadá/epidemiología , Sensibilidad y Especificidad , Prueba de COVID-19/métodos , Vigilancia de Productos ComercializadosRESUMEN
Signals from the microenvironment are known to be critical for development, sustaining adult stem cells, and for oncogenic progression. While candidate niche-driven signals that can promote cancer progression have been identified1-6, concerted efforts to comprehensively map microenvironmental ligands for cancer stem cell specific surface receptors have been lacking. Here, we use temporal single cell RNA-sequencing to identify molecular cues from the bone marrow stromal niche that engage leukemia stem cells (LSC) during oncogenic progression. We integrate these data with our RNA-seq analysis of human LSCs from distinct aggressive myeloid cancer subtypes and our CRISPR based in vivo LSC dependency map7 to develop a temporal receptor-ligand interactome essential for disease progression. These analyses identify the taurine transporter (TauT)-taurine axis as a critical dependency of myeloid malignancies. We show that taurine production is restricted to the osteolineage population during cancer initiation and expansion. Inhibiting taurine synthesis in osteolineage cells impairs LSC growth and survival. Our experiments with the TauT genetic loss of function murine model indicate that its loss significantly impairs the progression of aggressive myeloid leukemias in vivo by downregulating glycolysis. Further, TauT inhibition using a small molecule strongly impairs the growth and survival of patient derived myeloid leukemia cells. Finally, we show that TauT inhibition can synergize with the clinically approved oxidative phosphorylation inhibitor venetoclax8, 9 to block the growth of primary human leukemia cells. Given that aggressive myeloid leukemias continue to be refractory to current therapies and have poor prognosis, our work indicates targeting the taurine transporter may be of therapeutic significance. Collectively, our data establishes a temporal landscape of stromal signals during cancer progression and identifies taurine-taurine transporter signaling as an important new regulator of myeloid malignancies.
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BACKGROUND: The advantages of patent foramen ovale (PFO) closure as protection from a recurrence of stroke remains controversial compared to drug therapy, especially in patients over 60 years. HYPOTHESIS: The aim of the study is to compare recurrence of stroke in patients over 60 years old with PFO closure versus drug therapy alone. METHODS: We included 342 patients over 60 years who suffered a crytopgenic stroke, and were also accepted for a PFO closure. 199 patients refused a PFO closure and were treated with medical therapy alone, whereas 143 patients underwent a PFO closure procedure. RESULTS: The mean follow up time was 5.5 ± 1.5 years. All patients in Group B showed persistent shunt in the follow-up period (n = 199, 100%). In Group A, seven patients were diagnosed with residual shunt during echocardiography examination (5%). A new onset of atrial fibrillation occurred in seven patients in Group A (5%) and six patients in Group B (3%), p = .117. Recurrent stroke occurred in 3 patients in Group A (2%) and 11 patients in Group B (6%), p = .021. One patient died of unknown reason (1%) and two patients were lost due to neurological death (1%) in Group B, whereas no patients in Group A died during the follow-up period. CONCLUSION: Our results show that strict exclusion of patients over 60 years from PFO closure should be reconsidered. As life expectancies are increasing, patients should be considered for same treatment as younger patients, since the outcomes are improved compared to patients treated with medical therapy alone.
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Fibrilación Atrial , Foramen Oval Permeable , Accidente Cerebrovascular , Humanos , Persona de Mediana Edad , Estudios de Seguimiento , Foramen Oval Permeable/complicaciones , Foramen Oval Permeable/diagnóstico por imagen , Foramen Oval Permeable/terapia , Infarto Cerebral , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/prevención & controlRESUMEN
Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are arthropod-borne viruses that are transmitted by biting midges in the genus Culicoides (Diptera: Ceratopogonidae) and can cause hemorrhagic disease in certain ruminants. The objectives of this study were to measure the incidence of BTV and EHDV infections in captive white-tailed deer herd as well as tissues and corresponding presence of Culicoides midges at a location near Clinton, LA. During a 7-yr study with yearly outbreaks of hemorrhagic disease in the deer herd, 15 species of Culicoides were captured using Centers for Disease Control (CDC) black light traps. Reverse transcriptase quantitative polymerase chain reaction (PCR) was performed to screen for BTV and EHDV in pools of midges and tissues of deer. From 2012 to 2018, 1,711 pools of midges representing 24,859 specimens were tested, and specimens from 5 of the 15 collected species (Culicoides debilipalpis, Culicoides stellifer, Culicoides venustus, Culicoides haematopotus, and Culicoides crepuscularis) were found to be PCR positive for BTV and EHDV. Most of the BTV-positive pools of biting midges were from specimens of C. debilipalpis and C. stellifer, and most of the EHDV-positive pools were from specimens of C. venustus and C. stellifer. During the 7-yr period, 112 white-tailed deer that died at the study location were PCR positive for BTV or EHDV: detected BTV serotypes were 10 and 12 and EHDV serotypes were 1, 2, and 6. There was a significant increase in BTV/EHDV antibody prevalence in white-tailed deer during the study; antibody-positive rates increased from 15% to 78% in the deer herd of approximately 100 animals.
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Virus de la Lengua Azul , Lengua Azul , Ceratopogonidae , Ciervos , Virus de la Enfermedad Hemorrágica Epizoótica , Infecciones por Reoviridae , Enfermedades de las Ovejas , Virosis , Animales , Ovinos , Estudios Prospectivos , Incidencia , Insectos Vectores , RumiantesRESUMEN
Developmental science suggests that the consequences of mental health problems for life-course outcomes may depend on the timing of their onset. This study investigated the extent to which mental health predicted educational attainment at ages 17, 20, and 25 and whether gender moderated the links between mental health and educational attainment. It used data from Next Steps, a nationally representative panel survey of individuals born in 1989/1990 in England (N = 15,594, 48% female, 33% ethnic minority). The findings suggest that differences in mental health were more consequential for educational attainment during adolescence than in young adulthood. On average, girls attained higher levels of education than boys, but gender did not moderate the role that mental health played for educational attainment. (PsycInfo Database Record (c) 2024 APA, all rights reserved).
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Éxito Académico , Salud Mental , Masculino , Adolescente , Humanos , Femenino , Adulto , Adulto Joven , Etnicidad , Grupos Minoritarios , EscolaridadRESUMEN
As Western societies become more ethnically and culturally diverse, understanding the acculturation of immigrant youth is essential for fostering social cohesion. How the cultural identity formation of ethnic minority adolescents relates to their academic, social, and psychological adjustment is an important and as yet unresolved research question. This study examined to what extent identifying with the heritage and/or host culture is an individual resource or risk factor for the adjustment of immigrant youth in Germany. A random sample of 15-17-year-olds (N = 1992; Mage_w1 = 15.3 years, SD = 0.64; 44.5% girls; 44.7% students with immigrant background) was assessed twice: at the end of 9th and 10th grade. Academic performance and three dimensions of social/psychological adjustment (school attachment, self-esteem, and life satisfaction) were examined. Results showed that biculturalism was the modal identification pattern. Contrary to expectations, cultural identification did not differ systematically with perceived distance from the majority culture. Multivariate structural equation modeling revealed that both heritage and host identification can be developmental resources, but that their effects are dependent on the dimension of adjustment; biculturalism only proved to be a cumulative resource for school attachment. The domain specificity of the findings challenges the generalization claims of predominant acculturation theories.
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Emigrantes e Inmigrantes , Ajuste Emocional , Femenino , Humanos , Adolescente , Masculino , Identificación Social , Etnicidad/psicología , Grupos Minoritarios , Emigración e Inmigración , Ajuste Social , AculturaciónAsunto(s)
Trastornos de las Plaquetas Sanguíneas , Leucemia Mieloide Aguda , Neoplasias , Humanos , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Eliminación de Gen , Plaquetas , Trastornos de las Plaquetas Sanguíneas/diagnóstico , Trastornos de las Plaquetas Sanguíneas/genética , Susceptibilidad a Enfermedades , Células Germinativas , Leucemia Mieloide Aguda/genética , Predisposición Genética a la EnfermedadRESUMEN
Research has shown that gender role attitudes develop during adolescence; however, the relevant predictors remain a matter of debate. In adolescence, the school environment gains in importance. Thus, the present study investigates how students' and especially teachers' culture and composition predict the development of gender role attitudes in young adolescents. The study addresses this question using a sample of 7360 Flemish students (44.8% girls), who were surveyed three times after entering secondary education between 2012 (Mage = 13.14, SD = 0.56) and 2014. Latent change models reveal that boys' initial gender role attitudes are associated with the students' gender role culture; however, boys with more traditional gender role attitudes do not develop in an even more traditional direction at the beginning of secondary education. In contexts with a more privileged student SES composition, boys develop less traditional attitudes, while a traditional gender role culture among teachers supports the development of more traditional gender role attitudes among boys. Girls with more traditional gender role attitudes find themselves within student contexts with a more traditional culture. However, the development does not vary with the students' gender role culture. Overall, boys seem more susceptible to students' cultural and compositional characteristics.
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Rol de Género , Estudiantes , Masculino , Femenino , Humanos , Adolescente , Actitud , Encuestas y Cuestionarios , MaestrosRESUMEN
Patient-derived xenograft (PDX) tumor models are essential for identifying new biomarkers, signaling pathways and novel targets, to better define key factors of therapy response and resistance mechanisms. Therefore, this study aimed at establishing pancreas carcinoma (PC) PDX models with thorough molecular characterization, and the identification of signatures defining responsiveness toward drug treatment. In total, 45 PC-PDXs were generated from 120 patient tumor specimens and the identity of PDX and corresponding patient tumors was validated. The majority of engrafted PDX models represent ductal adenocarcinomas (PDAC). The PDX growth characteristics were assessed, with great variations in doubling times (4 to 32 days). The mutational analyses revealed an individual mutational profile of the PDXs, predominantly showing alterations in the genes encoding KRAS, TP53, FAT1, KMT2D, MUC4, RNF213, ATR, MUC16, GNAS, RANBP2 and CDKN2A. Sensitivity of PDX toward standard of care (SoC) drugs gemcitabine, 5-fluorouracil, oxaliplatin and abraxane, and combinations thereof, revealed PDX models with sensitivity and resistance toward these treatments. We performed correlation analyses of drug sensitivity of these PDX models and their molecular profile to identify signatures for response and resistance. This study strongly supports the importance and value of PDX models for improvement in therapies of PC.
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Introduction Acute otitis media (AOM) is a common condition of children encountered in general practice. A proportion of children develop otitis media with effusion (OME), which may require tympanostomy and ventilation tube insertion. Aim The aim of the study was to compare the incidence of AOM in Maori and New Zealand (NZ) European children in general practice and the referral practices to secondary care for tympanostomy and ventilation tube insertion. Methods The study was conducted in two parts: (1) an analysis of the incidence of AOM and OME in a rural Waikato general practice (Otorohanga) with a high Maori population over a 2-year period; and (2) an analysis of all referrals to the otorhinolaryngology (ORL) department at Waikato District Health Board and tympanostomy and ventilation tube insertion by this service over the same period. Results The incidence of AOM was similar in Maori compared with NZ European children. The incidence declined significantly between 2019 and 2020 and 50% of children with AOM were treated with antibiotics. Referral rates to the ORL department were greater for Maori compared with NZ European children as were tympanostomy and ventilation tube insertion rates. Discussion Although AOM is common, OME was rarely diagnosed. The clinical guidelines regarding antibiotic use for common conditions are not being readily adopted and further research is needed into this matter. The COVID-19 pandemic had a substantial effect on demand both in general practice and in the hospital sector. This may have been due to a reduction in the incidence of AOM or due to system changes caused by the pandemic.
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COVID-19 , Otitis Media , Niño , Humanos , Pandemias , COVID-19/epidemiología , Pueblo Maorí , Otitis Media/epidemiología , Medicina Familiar y Comunitaria , Antibacterianos/uso terapéuticoRESUMEN
BACKGROUND: Echocardiographic assessment of right ventricular (RV) measurements may be challenging. The aim of this study was to develop a formula for calculation of RV volumes and function based on measurements of linear dimensions by 2-dimensional (2D) transthoracic echocardiography (TTE) in comparison to cardiovascular magnetic resonance (CMR). METHODS: 129 consecutive patients with standard TTE and RV analysis by CMR were included. A formula based on the geometric assumptions of a truncated cone minus a truncated rhomboid pyramid was developed for calculations of RV end-diastolic volume (EDV) and RV end-systolic volume (ESV) by using the basal diameter of the RV (Dd and Ds) and the baso-apical length (Ld and Ls) in apical 4-chamber TTE views: RV EDV = 1.21 * Dd2 * Ld, and RV ESV = 1.21 * Ds2 * Ls. RESULTS: Calculations of RV EDV (ΔRV EDV = 10.2±26.4 ml to CMR, r = 0.889), RV ESV (ΔRV ESV = 4.5±18.4 ml to CMR, r = 0.921) and RV EF (ΔRV EF = 0.5±4.0% to CMR, r = 0.905) with the cone-pyramid formula (CPF) highly agreed with CMR. Impaired RV function on CMR (n = 52) was identified with a trend to higher accuracy by CPF than by conventional echocardiographic parameters (tricuspid annular plane systolic excursion (TAPSE) and fractional area change (FAC)). CONCLUSION: Calculations of RV volumes and RV function by 2D TTE with the newly developed CPF were in high concordance to measurements by CMR. Accuracy for detection of patients with reduced RV function were higher by the proposed 2D TTE CPF method than by conventional echocardiographic parameters of TAPSE and RV FAC.
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Músculos de la Espalda , Insuficiencia Cardíaca , Humanos , Ecocardiografía , Ventrículos Cardíacos/diagnóstico por imagen , Tractos PiramidalesRESUMEN
Wastewater surveillance (WS) helps to improve the understanding of the spread of communicable diseases in communities. WS can assist public health decision-makers in the design and implementation of timely mitigation measures. There is an increased need to use reliable, cost-effective, simple, and rapid WS systems, given traditional analytical (or 'gold-standard') programs are instrument/time-intensive, and dependent on highly skilled personnel. This study investigated the application of the portable GeneXpert platform for WS of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), influenza B virus (IBV), and respiratory syncytial virus (RSV). The GeneXpert system with the Xpert Xpress-SARS-CoV-2/Flu/RSV test kit uses reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to analyze wastewater samples. From September 2022 through January 2023, wastewater samples were collected from the influents of municipal wastewater treatment plants (MWTPs) of Saskatoon, Prince Albert, and North Battleford in the province of Saskatchewan, Canada. Both raw and concentrated wastewater samples were subjected to the GeneXpert analysis. Results showed that the Saskatoon wastewater viral loads were significantly correlated to Saskatchewan's influenza and COVID-19 clinical cases, with a lead time of 10 days for IAV and a lag time of 4 days for SARS-CoV-2. Additionally, the GeneXpert analysis of the three cities' wastewater samples showed that the raw WS could capture the dynamics of SARS-CoV-2 and IAV due to their correlation with concentrated WS. Interestingly, IBV loads were not detected in any wastewater samples, while the Saskatoon and Prince Albert wastewater samples collected following the 2023 holiday season (end of December and beginning of January) were positive for RSV. This study indicates that the GeneXpert has excellent potential for use in the development of an early warning system for transmissible disease in municipalities and limited-resource communities while simultaneously providing stakeholders with an efficient WS methodology.
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Small-molecule inhibitors of PD-L1 are postulated to control immune evasion in tumors similar to antibodies that target the PD-L1/PD-1 immune checkpoint axis. However, the identity of targetable PD-L1 inducers is required to develop small-molecule PD-L1 inhibitors. In this study, using chromatin immunoprecipitation (ChIP) assay and siRNA, we demonstrate that vitamin D/VDR regulates PD-L1 expression in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells. We have examined whether a VDR antagonist, MeTC7, can inhibit PD-L1. To ensure that MeTC7 inhibits VDR/PD-L1 without off-target effects, we examined competitive inhibition of VDR by MeTC7, utilizing ligand-dependent dimerization of VDR-RXR, RXR-RXR, and VDR-coactivators in a mammalian 2-hybrid (M2H) assay. MeTC7 inhibits VDR selectively, suppresses PD-L1 expression sparing PD-L2, and inhibits the cell viability, clonogenicity, and xenograft growth of AML cells. MeTC7 blocks AML/mesenchymal stem cells (MSCs) adhesion and increases the efferocytotic efficiency of THP-1 AML cells. Additionally, utilizing a syngeneic colorectal cancer model in which VDR/PD-L1 co-upregulation occurs in vivo under radiation therapy (RT), MeTC7 inhibits PD-L1 and enhances intra-tumoral CD8+T cells expressing lymphoid activation antigen-CD69. Taken together, MeTC7 is a promising small-molecule inhibitor of PD-L1 with clinical potential.
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The efficient clearance of dead and dying cells, efferocytosis, is critical to maintain tissue homeostasis. In the bone marrow microenvironment (BMME), this role is primarily fulfilled by professional bone marrow macrophages, but recent work has shown that mesenchymal stromal cells (MSCs) act as a non-professional phagocyte within the BMME. However, little is known about the mechanism and impact of efferocytosis on MSCs and on their function. To investigate, we performed flow cytometric analysis of neutrophil uptake by ST2 cells, a murine bone marrow-derived stromal cell line, and in murine primary bone marrow-derived stromal cells. Transcriptional analysis showed that MSCs possess the necessary receptors and internal processing machinery to conduct efferocytosis, with Axl and Tyro3 serving as the main receptors, while MerTK was not expressed. Moreover, the expression of these receptors was modulated by efferocytic behavior, regardless of apoptotic target. MSCs derived from human bone marrow also demonstrated efferocytic behavior, showing that MSC efferocytosis is conserved. In all MSCs, efferocytosis impaired osteoblastic differentiation. Transcriptional analysis and functional assays identified downregulation in MSC mitochondrial function upon efferocytosis. Experimentally, efferocytosis induced mitochondrial fission in MSCs. Pharmacologic inhibition of mitochondrial fission in MSCs not only decreased efferocytic activity but also rescued osteoblastic differentiation, demonstrating that efferocytosis-mediated mitochondrial remodeling plays a critical role in regulating MSC differentiation. This work describes a novel function of MSCs as non-professional phagocytes within the BMME and demonstrates that efferocytosis by MSCs plays a key role in directing mitochondrial remodeling and MSC differentiation. Efferocytosis by MSCs may therefore be a novel mechanism of dysfunction and senescence. Since our data in human MSCs show that MSC efferocytosis is conserved, the consequences of MSC efferocytosis may impact the behavior of these cells in the human skeleton, including bone marrow remodeling and bone loss in the setting of aging, cancer and other diseases.