Maintenance of hematopoietic stem cells by tyrosine-unphosphorylated STAT5 and JAK inhibition.

Adult haematopoietic stem cells (HSCs) are responsible for the lifelong production of blood and immune cells, a process regulated by extracellular cues including cytokines. Many cytokines signal through the conserved JAK/STAT pathway, in which tyrosine-phosphorylated STATs (pSTATs) function as transcription factors. STAT5 is a pivotal downstream mediator of several cytokines known to regulate haematopoiesis but its function in the HSC compartment remains poorly understood. Here, we show that STAT5-deficient HSCs exhibit an unusual phenotype: reduced multi-lineage repopulation and self-renewal, combined with reduced exit from quiescence and increased differentiation. This was driven not only by loss of canonical pSTAT5 signalling, but also by loss of distinct transcriptional functions mediated by STAT5 lacking canonical tyrosine phosphorylation (uSTAT5). Consistent with this concept, expression of an unphosphorylatable STAT5 mutant constrained wild-type HSC differentiation, promoted their maintenance and upregulated transcriptional programs associated with quiescence and stemness. The JAK1/2 inhibitor, ruxolitinib, which increased the uSTAT5:pSTAT5 ratio, had similar effects on murine HSC function: it constrained HSC differentiation and proliferation, promoted HSC maintenance and upregulated transcriptional programs associated with stemness. Ruxolitinib also enhanced serial replating of normal human HSPCs, CALR-mutant murine HSCs and HSPCs obtained from patients with myelofibrosis. Our results therefore reveal a previously unrecognized interplay between pSTAT5 and uSTAT5 in the control of HSC function and highlight JAK inhibition as a potential strategy for enhancing HSC function during ex vivo culture. Increased levels of uSTAT5 may also contribute to the failure of JAK inhibitors to eradicate myeloproliferative neoplasms.

Rapid Assessment of Biomarkers on Single Extracellular Vesicles Using "Catch and Display" on Ultrathin Nanoporous Silicon Nitride Membranes.

Extracellular vesicles (EVs) are particles released from cells that facilitate intercellular communication and have tremendous diagnostic and therapeutic potential. Bulk assays lack the sensitivity to detect rare EV subsets relevant to disease, and while single EV analysis techniques remedy this, they are often undermined by complicated detection schemes and prohibitive instrumentation. To address these issues, a microfluidic technique for EV characterization called "catch and display for liquid biopsy (CAD-LB)" is proposed. In this method, minimally processed samples are pipette-injected and fluorescently labeled EVs are captured in the nanopores of an ultrathin membrane.  This enables the rapid assessment of EV number and biomarker colocalization by light microscopy. Here, nanoparticles are used to define the accuracy and dynamic range for counting and colocalization. The same assessments are then made for purified EVs and for unpurified EVs in plasma. Biomarker detection is validated using CD9 and Western blot analysis to confirm that CAD-LB accurately reports relative protein expression levels. Using unprocessed conditioned media, CAD-LB captures the known increase in EV-associated ICAM-1 following endothelial cell cytokine stimulation. Finally, to demonstrate CAD-LB's clinical potential, EV biomarkers indicative of immunotherapy responsiveness are successfully detected in the plasma of bladder cancer patients treated with immune checkpoint blockade.

Calculating Optic Nerve Planning Organ at Risk Volume Margins for Stereotactic Radiosurgery Using Optic Nerve Motion determined using MRI.

OBJECTIVES: The combination of sharp dose gradients in stereotactic radiosurgery (SRS) and minute optic nerve motion may significantly increase dose to the optic nerves when treating perioptic lesions. The aim of this study was to calculate optic nerve planning organ at risk volume (PRV) margins for CyberKnife SRS treatment planning. METHODS: MRI scans were taken of 10 healthy volunteers looking left, right, up, down, and straight ahead to measure optic nerve motion. The measured optic nerve motion and the uncertainties in the technical accuracy of CyberKnife were used to calculate optic nerve PRV margins. RESULTS: Two optic nerve PRV margins were calculated: a non-isotropic margin of mL/R,PRV=3mm, mSup/Inf,PRV=2mm and mAnt/Post,PRV=1mm which considers the full range of motion measured in a worst case scenario; and an isotropic margin of mPRV=1mm which considers a scenario where patients are asked to look neutrally during imaging and treatment. Applying these PRVs to 8 historical sphenoid wing meningioma CyberKnife plans showed tolerance levels may be exceeded due to optic nerve motion. CONCLUSIONS: Optic nerve PRV margins may be needed in CyberKnife planning to reduce risk to the optic nerves. The use of a mPRV=1mm PRV to account for organ motion, along with instructing patients to hold their gaze neutrally during imaging and treatment, may be a suitable organ sparing strategy. ADVANCES IN KNOWLEDGE: Measured optic nerve motion and the technical accuracy of the CyberKnife system have been used to calculate optic nerve PRV margins.

Metformin's cardioprotective role in isoprenaline-induced myocardial infarction: Unveiling insights into the AMPK, NF-κB, JAK2/STAT3 pathways, and cholinergic regulation.

AIM: Despite advancements in treatment modalities, myocardial infarction (MI) remains a significant global cause of mortality and morbidity. Metformin (MET), a commonly used antidiabetic medication, has demonstrated potential in various cardioprotective mechanisms. This study investigated whether MET could alleviate the histopathological, electrocardiographic, and molecular consequences of MI in rats. MATERIALS AND METHODS: The study hypothesis was tested using an isoprenaline (ISOP)-induced MI model, where male Wistar rats were injected with ISOP (85 mg/kg/day, s.c., for 2 days) and treated with MET at the doses of 500 and 1000 mg/kg/day for 18 days or left untreated. KEY FINDINGS: ISOP-treated rats exhibited several indicators of MI, including significant ST-segment depression and prolonged QT-intervals on ECGs, worsened left ventricular histopathology with increased inflammatory cell infiltration, reduced expression of cardiac CHRM2, a cardioprotective cholinergic receptor, adaptive increases in AMPK and α7nAchR levels, and elevated levels of iNOS, NO, STAT3, JAK2, IL-6, TNF-α, and NF-κB. These effects were attenuated in rats treated with either low or high doses of MET. MET administration restored normal ECG recordings, diminished oxidative stress and inflammatory mediators, and downregulated NF-κB expression. Moreover, MET improved CHRM2 expression and normalized α7nAchR levels. Additionally, MET influenced the expression of key signaling molecules such as Akt, STAT3, and JAK2. SIGNIFICANCE: These findings might suggest that MET exerts cardioprotective effects in ISOP-induced MI in rats by mitigating critical inflammatory signaling pathways and regulating protective cholinergic mechanisms in the heart.

Clonal hematopoiesis and hematological malignancy.

Clonal hematopoiesis (CH), the expansion of hematopoietic stem cells and their progeny driven by somatic mutations in leukemia-associated genes, is a common phenomenon that rises in prevalence with advancing age to affect most people older than 70 years. CH remains subclinical in most carriers, but, in a minority, it progresses to a myeloid neoplasm, such as acute myeloid leukemia, myelodysplastic syndrome, or myeloproliferative neoplasm. Over the last decade, advances in our understanding of CH, its molecular landscape, and the risks associated with different driver gene mutations have culminated in recent developments that allow for a more precise estimation of myeloid neoplasia risk in CH carriers. In turn, this is leading to the development of translational and clinical programs to intercept and prevent CH from developing into myeloid neoplasia. Here, we give an overview of the spectrum of CH driver mutations, what is known about their pathophysiology, and how this informs the risk of incident myeloid malignancy.

The Physiological and Biochemical Response of Ribbed Mussels to Rising Temperatures: Benefits of Salt Marsh Cordgrass.

Salt marsh ecosystems are heavily reliant on ribbed mussel (Geukensia demissa) populations to aid in rapid recovery from droughts. The focus of this study was thus to document the effects of rising temperatures on ribbed mussel populations in a Georgia salt marsh. Seven lab and eight field experiments were used to assess the effects of current air temperatures on mussels at two high marsh (HM) sites with short and sparse cordgrass and one mid marsh (MM) site with tall and dense cordgrass. Field results in 2018 and 2019 indicate that ribbed mussels were experiencing extremely high temperatures for prolonged periods of time at the landlocked high marsh (LHM) site. In 2018, the highest temperature (54°C) and longest high temperature events, HTEs (58 days), that is, consecutive days with temperatures ≥40°C, were recorded at this site. When laboratory temperatures were increased from 20 to 36°C, mean heart rates increased by an average of 19 bpm for mussels from both high and MM sites respectively. When field temperatures rose from 20°C in April to 40°C in September 2019, mean heart rates increased by an average of 10 bpm for HM mussels and by 26.3 bpm for MM mussels. Under identical laboratory and field conditions, mean heart rates for mussels from the LHM site with the highest temperatures, increased by <1 bpm and 3.7 bpm respectively. Evidence of the potential role of shade on mussel aggregates was provided by examining whether mussels from the edge of mussel aggregates with little to no cordgrass for shade were more stressed than those living at the center of mussel aggregates. In the absence of shade, mean body temperatures for mussels at the edge of mussel aggregates were up to 8°C higher than for those living in the center underneath a dense tuft of cordgrass. Despite high body temperatures, mean heart rates and Hsp70 gene expression were lower for mussels living at the edges. This agrees with the strategy that during prolong exposure to high temperatures, mussels may reduce their heart rate to conserve energy and enhance survival. Alternatively, heat-stressed mussels at the edges of aggregates may not have the resources to express high levels of Hsp70. Increase in the frequency, intensity, and duration of HTEs may stress the physiological and biochemical function of mussel populations to the limit, dictate mussel aggregate size, and threaten the functionality of SE salt marshes.

Les écosystèmes des marais salés dépendent fortement des populations de moules côtelées (Geukensia demissa) pour aider à se remettre rapidement des sécheresses. L'objectif de cette étude était donc de documenter les effets de la hausse des températures sur les populations de moules côtelées dans un marais salant de Géorgie. Sept expériences en laboratoire et huit expériences sur le terrain ont été utilisées pour évaluer les effets des températures actuelles de l'air sur les moules dans deux sites de haut marais avec de la spartine courte et clairsemée et un site de milieu marais avec de la spartine haute et dense. Les résultats obtenus sur le terrain en 2018 et en 2019 indiquent que les moules côtelées ont connu des températures extrêmement élevées pendant de longues périodes sur le site enclavé du haut marais. En 2018, les événements de température la plus élevée (54°C) et les plus longs événements de haute température, les HTE (58 jours), c'est-à-dire des jours consécutifs avec des températures de ≥40°C, ont été enregistrés sur ce site. Lorsque les températures en laboratoire sont passées de 20 à 36°C, la fréquence cardiaque moyenne a augmenté en moyenne de 19 battements/min pour les moules des sites de haut et moyen marais, respectivement. Lorsque les températures sur le terrain sont passées de 20°C en avril à 40°C en septembre 2019, la fréquence cardiaque moyenne a augmenté en moyenne de 10 bpm pour les moules des marais hauts et de 26,3 bpm pour les moules des marais moyens. Dans des conditions identiques en laboratoire et sur le terrain, la fréquence cardiaque moyenne des moules du haut marais enclavé où les températures ont été les plus élevées a augmenté de <1 bpm et de 3,7 bpm respectivement. La preuve du rôle potentiel de l'ombre sur les agrégats de moules a été fournie en examinant si les moules de bordure des agrégats de moules avec peu ou pas de spartine pour l'ombre étaient plus stressées que celles vivant au centre des agrégats de moules. En l'absence d"ombre, la température corporelle moyenne des moules à la lisière des agrégats de moules était jusqu"à 8°C plus élevée que celle des moules vivant au centre, sous une touffe dense de spartine bovine. Malgré des températures corporelles élevées, la fréquence cardiaque moyenne et l'expression du gène Hsp70 étaient plus faibles chez les moules vivant sur les bords. Cela correspond à la stratégie selon laquelle lors d'une exposition prolongée à des températures élevées, les moules peuvent réduire leur fréquence cardiaque pour conserver leur énergie et améliorer leur survie. Par ailleurs, les moules soumises à un stress thermique en bordure des agrégats peuvent ne pas avoir les ressources nécessaires pour exprimer des concentrations élevées de Hsp70. L'augmentation de la fréquence, de l'intensité et de la durée des HTE peut stresser la fonction physiologique et biochimique des populations de moules jusqu'à la limite, dicter la taille des agrégats de moules et menacer la fonctionnalité des marais salés du sud-est.

Novel Peripherally Selective Cannabinoid Receptor 1 Neutral Antagonist Improves Metabolic Dysfunction-Associated Steatotic Liver Disease in Mice.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing globally. MASLD is characterized by clinically significant liver steatosis, and a subset of patients progress to more severe metabolic-disorder-associated steatohepatitis (MASH) with liver inflammation and fibrosis. Cannabinoid receptor 1 (CB1) antagonism is a proven therapeutic strategy for the treatment of the phenotypes that underlie MASLD, though work on early centrally penetrant compounds largely ceased following adverse psychiatric indications in humans. We present here preclinical testing of a CB1 neutral antagonist, N-[1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-4-phenylpiperidin-4l]methanesulfonamide (RTI-348), with minimal brain exposure when administered to mice. In a diet-induced model of MASLD-induced MASH, administration of RTI-348 decreased the total body and liver weight gain. Animals treated with RTI-348 showed reduced steatosis. Furthermore, they produced lower plasma alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH), biomarkers associated with liver damage. Mice maintained on the MASH diet had elevated expression of genes associated with profibrogenesis, immune response, and extracellular matrix remodeling, and treatment with RTI-348 mitigated these diet-induced changes in gene expression. Using an intracranial electrical self-stimulation model, we also demonstrated that RTI-348 does not produce an anhedonia response, as seen with the first-generation CB1 inverse agonist rimonabant. Altogether, the results herein point to RTI-348 as a promising neutral antagonist for MASH.

Rocahepevirus ratti: An underrecognised cause of acute hepatitis.

Zoonoses are responsible for many of all emerging infectious diseases as well as for those already established. Rocahepevirus ratti is a rat-originated virus related to the hepatitis E virus (Paslahepevirus balayani) but highly divergent genetically from this, with a high cross-species infection potential and zoonotic transmission. It can infect humans, leading to acute hepatitis, and is primarily transmitted through the consumption of contaminated water. Rocahepevirus ratti was first discovered in Germany in 2010. The first human case was described in 2017 in Hong Kong in an immune-compromised patient. The first case of chronic infection with Rocahepevirus ratti was described in 2023. A meta-analysis based on 38 studies published between 2000 and 2023 identified 21 cases in humans described up to this date and 489 infections in different animals. Raising awareness regarding this virus is essential, as there are probably many cases that remain undiagnosed, and the virus even has the ability to produce chronic infections in selected patients.

SUMOylation of cardiac myosin binding protein-C reduces its phosphorylation and results in impaired relaxation following treatment with isoprenaline.

Systolic and diastolic functions are coordinated in the heart by myofilament proteins that influence force of contraction and calcium sensitivity. Fine control of these processes is afforded by a variety of post-translation modifications that occur on specific proteins at different times during each heartbeat. Cardiac myosin binding protein-C is a sarcomeric accessory protein whose function is to interact transiently with actin, tropomyosin and myosin. Previously many different types of post-translational modification have been shown to influence the action of myosin binding protein-C and we present the first report that the protein can be modified covalently by the small ubiquitin like modifier protein tag. Analysis by mass spectrometry suggests that there are multiple modification sites on myosin binding protein-C for this tag and single point mutations did not serve to abolish the covalent addition of the small ubiquitin like modifier protein. Functionally, our data from both model human embryonic kidney cells and transfected neonatal cardiac myocytes suggests that the modification reduces phosphorylation of the filament protein on serine 282. In cardiac myocytes, the hypo-phosphorylation coincided with a significantly slower relaxation response following isoprenaline induced contraction. We hypothesise that this novel modification of myosin binding protein-C represents a new level of control that acts to alter the relaxation kinetics of cardiac myocytes.

Methodology and development of a machine learning probability calculator: Data heterogeneity limits ability to predict recurrence after arthroscopic Bankart repair.

PURPOSE: The aim of this study was to develop and train a machine learning (ML) algorithm to create a clinical decision support tool (i.e., ML-driven probability calculator) to be used in clinical practice to estimate recurrence rates following an arthroscopic Bankart repair (ABR). METHODS: Data from 14 previously published studies were collected. Inclusion criteria were (1) patients treated with ABR without remplissage for traumatic anterior shoulder instability and (2) a minimum of 2 years follow-up. Risk factors associated with recurrence were identified using bivariate logistic regression analysis. Subsequently, four ML algorithms were developed and internally validated. The predictive performance was assessed using discrimination, calibration and the Brier score. RESULTS: In total, 5591 patients underwent ABR with a recurrence rate of 15.4% (n = 862). Age <35 years, participation in contact and collision sports, bony Bankart lesions and full-thickness rotator cuff tears increased the risk of recurrence (all p < 0.05). A single shoulder dislocation (compared to multiple dislocations) lowered the risk of recurrence (p < 0.05). Due to the unavailability of certain variables in some patients, a portion of the patient data had to be excluded before pooling the data set to create the algorithm. A total of 797 patients were included providing information on risk factors associated with recurrence. The discrimination (area under the receiver operating curve) ranged between 0.54 and 0.57 for prediction of recurrence. CONCLUSION: ML was not able to predict the recurrence following ABR with the current available predictors. Despite a global coordinated effort, the heterogeneity of clinical data limited the predictive capabilities of the algorithm, emphasizing the need for standardized data collection methods in future studies. LEVEL OF EVIDENCE: Level IV, retrospective cohort study.

Targeting the membrane-proximal C2-set domain of CD33 for improved CAR T cell therapy.

Current CD33-targeted immunotherapies typically recognize the membrane-distal V-set domain of CD33. Here, we show that decreasing the distance between T cell and leukemia cell membrane increases the efficacy of CD33 chimeric antigen receptor (CAR) T cells. We therefore generated and optimized second-generation CAR constructs containing single-chain variable fragments from antibodies raised against the membrane-proximal C2-set domain, which bind CD33 regardless of whether the V-set domain is present (CD33PAN antibodies). CD33PAN CAR T cells resulted in efficient tumor clearance and improved survival of immunodeficient mice bearing human AML cell xenografts and, in an AML model with limited CD33 expression, forced escape of CD33neg leukemia. Compared to CD33V-set CAR T cells, CD33PAN CAR T cells showed greater in vitro and in vivo efficacy against several human AML cell lines with differing levels of CD33 without increased expression of exhaustion markers. CD33PAN moieties were detected at a higher frequency on human leukemic stem cells, and CD33PAN CAR T cells had greater in vitro efficacy against primary human AML cells. Together, our studies demonstrate improved efficacy with CAR T cells binding CD33 close to the cell membrane, providing the rationale to investigate CD33PAN CAR T cells further toward possible clinical application.

PDE4B Missense Variant Increases Susceptibility to Post-Traumatic Stress Disorder-Relevant Phenotypes in Mice.

Large-scale genome-wide association studies (GWAS) have associated intronic variants in PDE4B, encoding cAMP-specific phosphodiesterase-4B (PDE4B), with increased risk for post-traumatic stress disorder (PTSD), as well as schizophrenia and substance use disorders that are often comorbid with it. However, the pathophysiological mechanisms of genetic risk involving PDE4B are poorly understood. To examine the effects of PDE4B variation on phenotypes with translational relevance to psychiatric disorders, we focused on PDE4B missense variant M220T, which is present in the human genome as rare coding variant rs775201287. When expressed in HEK-293 cells, PDE4B1-M220T exhibited an attenuated response to a forskolin-elicited increase in the intracellular cAMP concentration. In behavioral tests, homozygous Pde4b M220T male mice with a C57BL/6JJcl background exhibited increased reactivity to novel environments, startle hyperreactivity, prepulse inhibition deficits, altered cued fear conditioning, and enhanced spatial memory, accompanied by an increase in cAMP signaling pathway-regulated expression of BDNF in the hippocampus. In response to a traumatic event (ten tone-shock pairings), neuronal activity was decreased in the cortex but enhanced in the amygdala and hippocampus of Pde4b M220T mice. At 24 hours post-trauma, Pde4b M220T mice exhibited increased startle hyperreactivity and decreased plasma corticosterone levels, similar to phenotypes exhibited by PTSD patients. Trauma-exposed Pde4b M220T mice also exhibited a slower decay in freezing at 15 days and 30 days post-trauma, demonstrating enhanced persistence of traumatic memories, similar to that exhibited by PTSD patients. These findings provide substantive mouse model evidence linking PDE4B variation to PTSD-relevant phenotypes, and thus highlight how genetic variation of PDE4B may contribute to PTSD risk.Significance Statement Human genetic studies have associated variants in the PDE4B gene, encoding the phosphodiesterase-4B (PDE4B) enzyme, with increased risk for post-traumatic stress disorder (PTSD) and other mental disorders that often occur with it. However, the underlying biological mechanisms of genetic risk involving PDE4B are poorly understood. To examine the effect of PDE4B variation on behaviors relevant to mental disorders, we studied male Pde4b M220T mice that carry a PDE4B variant (M220T), which is also present in humans. Pde4b M220T mice exhibited increased PTSD-like behavior in response to a traumatic event, as well as abnormal neuronal activity in the brain. Our findings provide substantive evidence linking PDE4B variation to PTSD-relevant behaviors, and thus highlight how genetic variation of PDE4B may contribute to PTSD risk.

Randomized phase 2 dose comparison LITESPARK-013 study of belzutifan in patients with advanced clear cell renal cell carcinoma.

BACKGROUND: Belzutifan is a first-in-class HIF-2α inhibitor approved at a dose of 120 mg once daily for certain adults with VHL disease and adults with advanced renal cell carcinoma (RCC) following therapy with a programmed death receptor (or ligand)-1 (PD-[L]1) inhibitor and a vascular endothelial growth factor tyrosine kinase inhibitor. However, whether belzutifan dose could be optimized is unclear. PATIENTS AND METHODS: The phase 2 LITESPARK-013 study (NCT04489771) enrolled patients with advanced clear cell RCC whose disease progressed after 1-3 prior systemic therapies, including an anti-PD-(L)1 regimen. Patients were randomly assigned 1:1 to receive belzutifan 120 mg or 200 mg once daily. The primary endpoint was objective response rate (ORR) per RECIST v1.1. Secondary endpoints included duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. RESULTS: Overall, 154 patients were enrolled (120 mg: n = 76; 200 mg: n = 78). Median follow-up was 20.1 months (range 14.8-28.4). ORR was 23.7% vs 23.1% for the 120 mg and 200 mg groups, respectively (P = 0.5312; -0.5% [95% CI, -14.0 to 12.9]. Median DOR was not reached for the 120 mg arm and was 16.1 months (2.1+ to 23.5+) for the 200 mg arm. No between-group differences were observed for PFS (HR 0.94 [95% CI 0.63-1.40]) or OS (medians not reached; HR 1.11 [95% CI, 0.65-1.90]). Grade 3 or 4 treatment-related adverse events were observed in 35 patients (46.1%) in the 120 mg group and 36 patients (46.2%) in the 200 mg group. CONCLUSION: The efficacy of belzutifan was similar between the 120-mg dose and the 200-mg dose for previously treated clear cell RCC. Safety at both doses was consistent with the known safety profile of belzutifan. These results further support 120 mg once daily as the preferred dose for belzutifan.

Predictors of attrition during acute pharmacotherapy of psychotic depression in a clinical trial.

Little is known about factors that contribute to attrition in clinical trials of the pharmacotherapy of psychotic depression. The purpose of this study was to identify factors associated with attrition during acute pharmacotherapy in the Study of the Pharmacotherapy of Psychotic Depression II (STOP-PD II) clinical trial. Sociodemographic and clinical variables were assessed at baseline in 269 men and women, aged 18-85 years, who were treated with up to 12 weeks of open-label sertraline plus olanzapine. Univariate analyses examined the association of baseline variables with overall non-completion, as well as reasons for non-completion. Logistic regression was used to model the relationship of the significant univariate predictors with non-completion and its reasons. Seventy-four (27.5 %) participants did not complete the acute treatment phase of STOP-PD II. Male gender, younger age, inpatient status, higher Clinical Global Impression (CGI) severity of illness, and higher severity of psychomotor disturbance were associated with non-completion in univariate analyses. In regression models, higher CGI severity of illness score was the only significant independent predictor of non-completion, explained by withdrawal of consent. Our findings have implications for the retention of persons with psychotic depression in clinical trials.

The GPI sidechain of Toxoplasma gondii inhibits parasite pathogenesis.

Glycosylphosphatidylinositols (GPIs) are highly conserved anchors for eukaryotic cell surface proteins. The apicomplexan parasite, Toxoplasma gondii, is a widespread intracellular parasite of warm-blooded animals whose plasma membrane is covered with GPI-anchored proteins, and free GPIs called GIPLs. While the glycan portion is conserved, species differ in sidechains added to the triple mannose core. The functional significance of the Glcα1,4GalNAcß1- sidechain reported in Toxoplasma gondii has remained largely unknown without understanding its biosynthesis. Here we identify and disrupt two glycosyltransferase genes and confirm their respective roles by serology and mass spectrometry. Parasites lacking the sidechain on account of deletion of the first glycosyltransferase, PIGJ, exhibit increased virulence during primary and secondary infections, suggesting it is an important pathogenesis factor. Cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants are intact. By contrast, the scavenger receptor CD36 shows enhanced binding to PIGJ mutants, potentially explaining a subtle tropism for macrophages detected early in infection. Galectin-3, which binds GIPLs, exhibits an enhancement of binding to PIGJ mutants, and the protection of galectin-3 knockout mice from lethality suggests that Δpigj parasite virulence in this context is sidechain dependent. Parasite numbers are not affected by Δpigj early in the infection in wild-type mice, suggesting a breakdown of tolerance. However, increased tissue cysts in the brains of mice infected with Δpigj parasites indicate an advantage over wild-type strains. Thus, the GPI sidechain of T. gondii plays a crucial and diverse role in regulating disease outcomes in the infected host.IMPORTANCEThe functional significance of sidechain modifications to the glycosylphosphatidylinositol (GPI) anchor in parasites has yet to be determined because the glycosyltransferases responsible for these modifications have not been identified. Here we present identification and characterization of both Toxoplasmsa gondii GPI sidechain-modifying glycosyltransferases. Removal of the glycosyltransferase that adds the first GalNAc to the sidechain results in parasites without a sidechain on the GPI, and increased host susceptibility to infection. Loss of the second glycosyltransferase results in a sidechain with GalNAc alone, and no glucose added, and has negligible effect on disease outcomes. This indicates GPI sidechains are fundamental to host-parasite interactions.

Dose Estimation Utility in a Population Pharmacokinetic Analysis of Inhaled Δ9-Tetrahydrocannabinol Cannabis Market Products in Occasional and Daily Users.

BACKGROUND: Unusually high variability in blood Δ9-tetrahydrocannabinol (THC) concentrations have been observed in subjects inhaling similar cannabis products over similar time periods when consumption is ad libitum. This makes simple gravimetric dose estimation a poor predictor of THC exposure. Population pharmacokinetic analyses of blood THC concentration versus time data are routinely used to estimate pharmacokinetic parameters. The aim of this study was to estimate the inhaled dose of THC in occasional and daily users of high potency market cannabis. METHODS: Blood THC concentrations were measured for 135 minutes from 29 participants who either smoked high concentration flower or inhaled concentrates ad libitum during a 15-minute session. Frequent blood samples were obtained over the following 135 minutes. RESULTS: The estimated central and rapidly equilibrating volumes of distribution of a 3-compartment model were 19.9 ± 1.2 and 51.6 ± 4.7 L whereas the intercompartmental clearances were 1.65 ± 0.14 and 1.75 ± 0.10 L/min, respectively. Covariate-adjusted analysis revealed that the estimated inhaled THC dose was considerably less among occasional users compared with daily users. CONCLUSIONS: Three-compartment pharmacokinetics of THC did not differ among the 3 user groups, and the early phase (first 135 minutes postinception of inhalation) kinetics were similar to those previously described after smoking low potency cannabis products. Therefore, inhaled THC dose can be estimated from pharmacokinetic data and covariate-driven adjustments can be used to estimate THC doses, based on the participant cannabis usage pattern (occasional versus daily), improving the accuracy of THC exposure estimates compared with those derived from weighed THC content alone.

Quantitative NMR Analysis of Marine Macroalgae for AGE Inhibition by Methylglyoxal Scavenging.

Reactive carbonyl species (RCS) induce a fundamental form of biological stress that has driven the evolution of diverse mechanisms for minimizing its impact on organismal health. The complications that accompany uncontrolled hyperglycemia exemplify the health implications when RCS stress exceeds the body's capacity to prevent the excessive formation of advanced glycation end-products. Presented here is a novel quantitative NMR (qNMR) technique for evaluating scavengers of the prominent sugar-derived carbonyl methylglyoxal (MGO). This tool was employed to screen the chemical diversity of marine macroalgae extracts, with a focus on species that have a history of consumption by the World's healthiest populations and are subject to global scale aquacultural production. Fucus vesiculosus demonstrated the highest capacity for inhibiting glycation and scavenging MGO. Additionally, the Chondrus cripsus, Gracilaria vermiculophyla, and Gracilaria tikvahiae extracts had a high capacity for scavenging MGO, representing the first report of this activity. This new qNMR methodology presented is highly applicable for screening extracts and compounds from diverse sources, and the results highlight the potential of macroalgae extracts to be employed as RCS and AGE targeting therapeutics and food additives.