Reciprocal Increase Research Articles

Immunohistochemical Analysis of GATA2 Expression in Endometrium and its Relationship with Hormone Receptor Expression in Benign and Premalignant Endometrial Disorders.

The GATA gene family encodes highly conserved zinc-finger transcription factors that facilitate the development and function of multiple organ systems including the uterus. In the endometrium, GATA2 functions in a positive autoregulatory loop with the progesterone receptor (PGR) and colocalizes with PGR on chromatin to promote PGR transcriptional programs. GATA2 also has PGR-independent functions that maintain endometrial cell identity, and GATA2 transcripts reportedly are down-regulated in endometrial disorders including endometriosis. This event is accompanied by a reciprocal increase in GATA6. Here, we applied custom anti-GATA2 monoclonal antibodies and performed GATA2 immunohistochemistry (IHC) on patient endometrial tissues corresponding to proliferative, secretory, inactive, and hormone-treated endometrium, as well as endometriosis and endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (EAH/EIN). We also performed IHC for the estrogen receptor, PGR, and GATA6 in relevant groups. The results reveal a tight correlation between GATA2 and PGR expression in the glandular and stromal cells of benign endometrium. GATA2 expression is markedly reduced in stromal but not glandular cells in endometriosis and EAH/EIN. This reduction in GATA2 expression does not lead to a detectable increase in GATA6 expression in endometriosis. Although average glandular GATA2 expression was preserved in endometriosis and EAH/EIN cases, its expression was decoupled from PGR, implying that alternative pathways regulate GATA2 levels in these disorders. Our findings indicate that GATA2 dysregulation is a feature of endometriosis and EAH/EIN, and support a model whereby loss of stromal GATA2 in these disorders contributes to their progesterone insensitivity.

Forest harvest causes rapid changes of maternal investment strategies in ground beetles.

Species recovery following anthropogenic disturbances will depend on adaptations in survivorship and fecundity. Life-history theory predicts increased environmental stress will result in (1) shifts in resource allocation from fecundity to body growth/maintenance and (2) increased provisioning among offspring at the cost of reproductive output. For remnant populations that persist after forest harvesting, selection mediated through anthropogenic disturbances may affect resilience to additional stressors such as climate change. We tested how rapid changes in environmental conditions affected maternal investment strategies in two ground beetle species, Pterostichus pensylvanicus and Pterostichus coracinus, by comparing fecundity and survivorship in populations from recently clear-cut and uncut habitats. Using parents drawn from clear-cut or uncut stands, we reared progeny in both common garden and reciprocal transplant experiments. In P. pensylvanicus, we found that neither lineage nor rearing habitat affected the number of eggs laid per female or survivorship of offspring. However, eggs laid by females from clear-cuts were more likely to hatch and offspring reached maturity more quickly, suggesting increased provisioning per offspring. In P. coracinus, females from clear-cuts laid more eggs, and their eggs hatched more rapidly and had greater hatching success, suggesting increased investment in overall reproductive output and increased offspring provisioning. In the reciprocal transplant, we observed significant habitat by lineage interactions on survival in P. coracinus, with survivorship increasing when progeny were reared in novel habitats. In both species, increased maternal investment among offspring was not associated with a reduction in overall reproductive output, as anticipated. However, maternal investment among offspring declined with increasing female size, implying trade-offs between increased metabolic demand and fecundity. Taken together, our work suggests that females from more stressful, clear-cut habitats increased investment in fecundity, compared to females from uncut habitats, and may compensate for larval mortality. These changes were driven by smaller individuals, suggesting that increased environmental stress can influence the relationship between female size and maternal investment strategy. Additionally, reciprocal increases in offspring survivorship in habitats other than the parents suggest that adjacent areas between unharvested and clear-cut habitat may be useful in maintaining biodiversity under future climate stressors.

LRIG1 engages ligand VISTA and impairs tumor-specific CD8+ T cell responses.

Immune checkpoint blockade is a promising approach to activate antitumor immunity and improve the survival of patients with cancer. V-domain immunoglobulin suppressor of T cell activation (VISTA) is an immune checkpoint target; however, the downstream signaling mechanisms are elusive. Here, we identify leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) as a VISTA binding partner, which acts as an inhibitory receptor by engaging VISTA and suppressing T cell receptor signaling pathways. Mice with T cell-specific LRIG1 deletion developed superior antitumor responses because of expansion of tumor-specific cytotoxic T lymphocytes (CTLs) with increased effector function and survival. Sustained tumor control was associated with a reduction of quiescent CTLs (TCF1+ CD62Lhi PD-1low) and a reciprocal increase in progenitor and memory-like CTLs (TCF1+ PD-1+). In patients with melanoma, elevated LRIG1 expression on tumor-infiltrating CD8+ CTLs correlated with resistance to immunotherapies. These results delineate the role of LRIG1 as an inhibitory immune checkpoint receptor and propose a rationale for targeting the VISTA/LRIG1 axis for cancer immunotherapy.

ISSLS prize in clinical/bioengineering science 2024: How standing and supine positions influence nutrient transport in human lumbar discs?-A serial post-contrast MRI study evaluating interplay between convection and diffusion.

The intervertebral disc being avascular depends on diffusion and load-based convection for essential nutrient supply and waste removal. There are no reliable methods to simultaneously investigate them in humans under natural loads. For the first time, present study aims to investigate this by strategically employing positional MRI and post-contrast studies in three physiological positions: supine, standing and post-standing recovery. A total of 100 healthy intervertebral discs from 20 volunteers were subjected to a serial post-contrast MR study after injecting 0.3mmol/kg gadodiamide and T1-weighted MR images were obtained at 0, 2, 6, 12 and 24h. At each time interval, images were obtained in three positions, i.e. supine, standing and post-standing recovery supine. The signal intensity values at endplate zone and nucleus pulposus were measured. Enhancement percentages were calculated and analysed comparing three positions. During unloaded supine position, there was slow gradual increase in enhancement reaching peak at 6h. When the subjects assumed standing position, there was immediate loss of enhancement at nucleus pulposus which resulted in reciprocal increase in enhancement at endplate zone (washout phenomenon). Interestingly, when subjects assumed the post-standing recovery position, the nucleus pulposus regained the enhancement and endplate zone showed reciprocal loss (pumping-in phenomenon). For the first time, present study documented acute effects of physiological loading and unloading on nutrition of human discs in vivo. While during rest, solutes diffused gradually into disc, the diurnal short loading and unloading redistribute small solutes by convection. Standing caused rapid solute depletion but promptly regained by assuming resting supine position.

Abstract 6944: Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer

Abstract Intratumor heterogeneity (ITH) is a substrate for tumor evolution and confers amenability to stressors and therapies. Sources of ITH include subclonal mutations, microenvironmental factors, and plasticity whereby a cell has the ability to adopt different cell states. Clear cell kidney cancer (ccRCC) has a well-characterized genetic initiating event - VHL inactivation - resulting in activation of HIF-1 and HIF-2 transcription factors. Although VHL inactivation is consistently clonal in nature, ccRCC nonetheless exhibits extensive genetic and morphological ITH. This makes ccRCC an excellent model in which to study ITH. We performed single cell RNA-seq on multiregional samples from kidney tumors and macroscopically normal kidney tissue. We obtained 55 samples from 16 tumors and used the 10x Genomics platform to obtain gene expression data from ~150,000 single cells, including ~80,000 cancer and normal epithelial cells. We identified three cancer cell states, co-existing in all clear cell kidney cancer tumors analyzed and marked by different gene expression programs: 1) differentiated-like cells marked by proximal tubule cell genes characteristic of the cancer cell of origin; 2) epithelial-mesenchymal transition (EMT)-like cells and 3) injured-like cells. Differentiated markers and EMT markers were associated with good and poor prognosis, respectively, indicating that these cell subpopulations are clinically important. Notably, these cell states are also present in the normal proximal tubule although in different proportions to the tumor samples, which contain more cells in the EMT-like and injured-like states. This suggests that the core features of ITH are inherent to the cell of origin of the cancer, but that the cell state trajectories are dysregulated in the tumor. Furthermore, in tumors these states exist as a continuum, whereby transitional cells in between states appear to exist, suggesting that cancer cells are continually and dynamically transitioning between states. Lastly, we used chromosomal copy number variation (CNV) patterns to study genetic ITH. We examined the effect of 14q loss, which is associated with ccRCC metastasis, on gene expression. We found that 14q loss is associated with downregulation of HIF-1 transcriptional activity together with a reciprocal increase in HIF-2 activity, MYC activity and oxidative phosphorylation, highlighting potential mechanisms by which 14q loss may driver ccRCC metastasis. Citation Format: Olivia Lombardi, Ran Li, Lisa Browning, Faiz Jabbar, Hannah Evans, Peter J. Ratcliffe, David R. Mole. Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6944.

Abstract 5489: The spatial landscape of early-stage lung adenocarcinoma

Abstract Cellular and molecular basis of ground-glass opacities (GGO), which has been used as a screening indicator for lung cancer, remains a gray area. Our aim was to investigate the tumor-immune microenvironment (TIME) in a cohort of early-stage lung adenocarcinomas (LUAD) through imaging mass cytometry, enabling us to examine the TME spatial composition of nodules at single-cell resolution. We collected 354 imaging mass cytometry (IMC) regions of interest (ROI) from 135 early-stage lung adenocarcinoma patients to investigate the spatial and molecular profiles of early stage LUAD. Specifically, we gathered 27 solid (S), 39 part-solid (PS), 74 pure non-solid (PNS) patients and 89 matched adjacent-normal cases to profile the phenotype of 2.24 million cells across the two IMC panels to characterize the cellular and molecular composition of GGO nodules. Comparing progression from adjacent normal, pure non-solid, part-solid, to solid, we observed consistent reduction in RAGE+ AT1 cells (p*<1.6E-7) and a reciprocal increase in SFTPC+ AT2 cells (p*<1.3E-8) and PanCK+Ki67+ proliferative epithelial cells (p*<2.8E-7). We also report fibrogenesis and an increase in immune cell populations such as regulatory (p*<1.4E-5), CD4 (p*<0.0024), CD8 T (p*<0.017) cells, B (p*<5.5E-9) cells, and macrophages. Spatially, we quantify an upregulation of tumor-tumor and tumor-fibroblast interaction, and a downregulation of tumor-immune interaction. We believe this evidence suggests the role of fibroblasts as liner for tumors, barricading immune infiltration and harvesting uninterrupted tumorigenesis. In summary, we performed a highly multiplexed IMC data to analyze the TIME spatial profiles of GGO nodules in early-stage LUAD at a single-cell level. Spatial analysis of this integrated multidimensional omics dataset provides insight into the pivotal role of fibroblasts in early-stage LUAD in immune suppression, evasion, tumorigenesis, and potentially metastasis. Citation Format: Junbum Kim, Hiranmayi Ravichandran, Liron Yoffe, Bhavneet Binder, Timothy McGraw, Nasser K. Altorki, Vivek Mittel, Olivier Elemento. The spatial landscape of early-stage lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5489.

Clinical implementation of preemptive pharmacogenomics in psychiatry

Pharmacogenomics (PGx) holds promise to revolutionize modern healthcare. Although there are several prospective clinical studies in oncology and cardiology, demonstrating a beneficial effect of PGx-guided treatment in reducing adverse drug reactions, there are very few such studies in psychiatry, none of which spans across all main psychiatric indications, namely schizophrenia, major depressive disorder and bipolar disorder. In this study we aim to investigate the clinical effectiveness of PGx-guided treatment (occurrence of adverse drug reactions, hospitalisations and re-admissions, polypharmacy) and perform a cost analysis of the intervention. We report our findings from a multicenter, large-scale, prospective study of pre-emptive genome-guided treatment named as PREemptive Pharmacogenomic testing for preventing Adverse drug REactions (PREPARE) in a large cohort of psychiatric patients (n=1076) suffering from schizophrenia, major depressive disorder and bipolar disorder. We show that patients with an actionable phenotype belonging to the PGx-guided arm (n=25) present with 34.1% less adverse drug reactions compared to patients belonging to the control arm (n=36), 41.2% less hospitalisations (n=110 in the PGx-guided arm versus n=187 in the control arm) and 40.5% less re-admissions (n=19 in the PGx-guided arm versus n=32 in the control arm), less duration of initial hospitalisations (n=3305 total days of hospitalisation in the PGx-guided arm from 110 patients, versus n=6517 in the control arm from 187 patients) and duration of hospitalisation upon readmission (n=579 total days of hospitalisation upon readmission in the PGx-guided arm, derived from 19 patients, versus n=928 in the control arm, from 32 patients respectively). It was also shown that in the vast majority of the cases, there was less drug dose administrated per drug in the PGx-guided arm compared to the control arm and less polypharmacy (n=124 patients prescribed with at least 4 psychiatric drugs in the PGx-guided arm versus n=143 in the control arm) and smaller average number of co-administered psychiatric drugs (2.19 in the PGx-guided arm versus 2.48 in the control arm. Furthermore, less deaths were reported in the PGx-guided arm (n=1) compared with the control arm (n=9). Most importantly, we observed a 48.5% reduction of treatment costs in the PGx-guided arm with a reciprocal slight increase of the quality of life of patients suffering from major depressive disorder (0.935 versus 0.925 QALYs in the PGx-guided and control arm, respectively). While only a small proportion (∼25%) of the entire study sample had an actionable genotype, PGx-guided treatment can have a beneficial effect in psychiatric patients with a reciprocal reduction of treatment costs. Although some of these findings did not remain significant when all patients were considered, our data indicate that genome-guided psychiatric treatment may be successfully integrated in mainstream healthcare. European Union Horizon 2020.

Heterogeneous treatment effects of Cerebrolysin as an early add-on to reperfusion therapy: post hoc analysis of the CEREHETIS trial.

Background: There has been intensive research into enhancing the effects of reperfusion therapy to mitigate hemorrhagic transformation (HT) in stroke patients. Using neuroprotective agents alongside intravenous thrombolysis (IVT) appears a promising approach. Cerebrolysin is one of the candidates since it consists of neuropeptides mimicking the action of neurotrophic factors on brain protection and repair. Objectives: We looked at treatment effects of Cerebrolysin as an early add-on to IVT in stroke patients with varying HT risk. Methods: It was post hoc analysis of the CEREHETIS trial (ISRCTN87656744). Patients with middle cerebral artery infarction (n = 238) were selected from the intention-to-treat population. To stratify participants according to their HT risk, the DRAGON, SEDAN and HTI scores were computed for each eligible subject using on-admission data. The study endpoints were any and symptomatic HT, and functional outcome measured with the modified Rankin Scale (mRS) on day 90. Favorable functional outcome (FFO) was defined as an mRS ≤2. The performance of each stratification tool was estimated with regression approaches. Heterogeneous treatment effect analysis was conducted using techniques of meta-analysis and the matching-smoothing method. Results: The HTI score outperformed other tools in terms of HT risk stratification. Heterogeneity of Cerebrolysin treatment effects was moderate (I2, 35.8%-56.7%; H2, 1.56-2.31) and mild (I2, 10.9%; H2, 1.12) for symptomatic and any HT, respectively. A significant positive impact of Cerebrolysin on HT and functional outcome was observed in the moderate (HTI = 1) and high (HTI ≥2) HT risk patients, but it was neutral in those with the low (HTI = 0) risk. In particular, there was a steady decline in the rate of symptomatic (HTI = 0 vs. HTI = 4: by 4.3%, p = 0.077 vs. 21.1%, p < 0.001) and any HT (HTI = 0 vs. HTI = 4: by 1.2%, p = 0.737 vs. 32.7%, p < 0.001). Likewise, an mRS score reduction (HTI = 0 vs. HTI = 4: by 1.8%, p = 0.903 vs. 126%, p < 0.001) with a reciprocal increase of the fraction of FFO patients (HTI = 0 vs. HTI = 4: by 1.2% p = 0.757 vs. 35.5%, p < 0.001) was found. Conclusion: Clinically meaningful heterogeneity of Cerebrolysin treatment effects on HT and functional outcome was established in stroke patients. The beneficial effects were significant in those whose estimated on-admission HT risk was either moderate or high.

Clinical decision support to enhance venous thromboembolism pharmacoprophylaxis prescribing for pediatric inpatients with COVID-19.

To design and evaluate a clinical decision support (CDS) module to improve guideline concordant venous thromboembolism (VTE) pharmacoprophylaxis prescribing for pediatric inpatients with COVID-19. The proportion of patients who met our institutional clinical practice guideline's (CPG) criteria for VTE prophylaxis was compared to those who triggered a CDS alert, indicating the patient needed VTE prophylaxis, and to those who were prescribed prophylaxis pre and post the launch of a new VTE CDS module to support VTE pharmacoprophylaxis prescribing. The sensitivity, specificity, positive predictive value (PPV), negative predictive value, F1-score and accuracy of the tool were calculated for the pre- and post-intervention periods using the CPG recommendation as the gold standard. Accuracy was defined as the sum of the true positives and true negatives over the sum of the true positives, false positives, true negatives, and false negatives. Logistic regression was used to identify variables associated with correct thromboprophylaxis prescribing. A significant increase in the proportion of patients triggering a CDS alert occurred in the post-intervention period (44.3% vs. 6.9%, p<.001); however, no reciprocal increase in VTE prophylaxis prescribing was achieved (36.6% vs. 40.9%, p=.53). The updated CDS module had an improved sensitivity (55.0% vs. 13.3%), NPV (44.9% vs. 36.3%), F1-score (66.7% vs. 23.5%), and accuracy (62.5% vs. 42.0%), but an inferior specificity (78.6% vs. 100%) and PPV (84.6% vs. 100%). The updated CDS model had an improved accuracy and overall performance in correctly identifying patients requiring VTE prophylaxis. Despite an increase in correct patient identification by the CDS module, the proportion of patients receiving appropriate pharmacologic prophylaxis did not change. CDS tools to support correct VTE prophylaxis prescribing need ongoing refinement and validation to maximize clinical utility.

Antimicrobial Stewardship and Improved Antibiotic Utilization in the Pediatric Cardiac Intensive Care Unit.

We developed a multidisciplinary antimicrobial stewardship team to optimize antimicrobial use within the Pediatric Cardiac Intensive Care Unit. A quality improvement initiative was conducted to decrease unnecessary broad-spectrum antibiotic use by 20%, with sustained change over 12 months. We conducted this quality improvement initiative within a quaternary care center. PDSA cycles focused on antibiotic overuse, provider education, and practice standardization. The primary outcome measure was days of therapy (DOT)/1000 patient days. Process measures included electronic medical record order-set use. Balancing measures focused on alternative antibiotic use, overall mortality, and sepsis-related mortality. Data were analyzed using statistical process control charts. A significant and sustained decrease in DOT was observed for vancomycin and meropenem. Vancomycin use decreased from a baseline of 198 DOT to 137 DOT, a 31% reduction. Meropenem use decreased from 103 DOT to 34 DOT, a 67% reduction. These changes were sustained over 24 months. The collective use of gram-negative antibiotics, including meropenem, cefepime, and piperacillin-tazobactam, decreased from a baseline of 323 DOT to 239 DOT, a reduction of 26%. There was no reciprocal increase in cefepime or piperacillin-tazobactam use. Key interventions involved electronic medical record changes, including automatic stop times and empiric antibiotic standardization. All-cause mortality remained unchanged. The initiation of a dedicated antimicrobial stewardship initiative resulted in a sustained reduction in meropenem and vancomycin usage. Interventions did not lead to increased utilization of alternative broad-spectrum antimicrobials or increased mortality. Future interventions will target additional broad-spectrum antimicrobials.

“My Name is Alexa. What’s Your Name?” The Impact of Reciprocal Self-Disclosure on Post-Interaction Trust in Conversational Agents

The use of conversational AI agents (CAs), such as Alexa and Siri, has steadily increased over the past several years. However, the functionality of these agents relies on the personal data obtained from their users. While evidence suggests that user disclosure can be increased through reciprocal self-disclosure (i.e., a process in which a CA discloses information about itself with the expectation that the user would reciprocate by disclosing similar information about themself), it is not clear whether and through which mechanism the process of reciprocal self-disclosure influences users’ post-interaction trust. We theorize that anthropomorphism (i.e., the extent to which a user attributes humanlike attributes to a nonhuman entity) serves as an inductive inference mechanism for understanding reciprocal self-disclosure, enabling users to build conceptually distinct cognitive and affective foundations upon which to form their post-interaction trust. We found strong support for our theory through two randomized experiments that used custom-developed text-based and voicebased CAs. Specifically, we found that reciprocal self-disclosure increases anthropomorphism and anthropomorphism increases cognition-based trustworthiness and affect-based trustworthiness. Our results show that reciprocal self-disclosure has an indirect effect on cognition-based trustworthiness and affect-based trustworthiness and is fully mediated by anthropomorphism. These findings conceptually bridge prior research on motivations of anthropomorphism and research on cognitive and affective bases of trust.

Cerebrolysin as an early add-on to reperfusion therapy: heterogeneous treatment effect analysis in ischemic stroke patients with varying risk of hemorrhagic transformation

The study goal was the assessment of heterogeneous treatment effects of Cerebrolysin as an early add-on to reperfusion therapy in stroke patients with varying risk of hemorrhagic transformation (HT). It was post hoc analysis of the CEREHETIS trial (ISRCTN87656744). Patients with middle cerebral artery infarction (n=238) were stratified by HT risk with the HTI score. The study outcomes were symptomatic and any HT, and functional outcome measured with the modified Rankin Scale (mRS) on day 90. Favorable outcome was defined as an mRS score of ≤2. Heterogeneous treatment effect analysis was performed using techniques of meta-analysis and the matching-smoothing method. Heterogeneity of Cerebrolysin treatment effects was moderate (I2=36.98-69.3%, H2=1.59-3.26) and mild (I2=18.33-32.39%, H2=1.22-1.48) for symptomatic and any HT, respectively. A positive impact of the Cerebrolysin treatment on HT and functional outcome was observed in patients with moderate (HTI=1) and high (HTI≥2) HT risk. However, the effect was neutral in those with low risk (HTI=0). In high HT risk patients, there was a steady decline in the rate of symptomatic (HTI=0 vs. HTI≥2: by 3.8%, p=0.120 vs. 14.3%, p<0.001) and any HT (HTI=0 vs. HTI≥2: by 0.6%, p=0.864 vs. 19.5%, p<0.001). Likewise, Cerebrolysin treatment resulted in an overall decrease in the mRS scores (HTI=0 vs. HTI≥2: by 2.1%, p=0.893 vs. 63%, p<0.001) with a reciprocal increase of the fraction with favorable outcome (HTI=0 vs. HTI≥2: by 2% p=0.634 vs. 19.2%, p<0.001). Clinically meaningful heterogeneity of Cerebrolysin treatment effects on HT and functional outcome was established in stroke patients. The Cerebrolysin positive impact was significant in those whose estimated on-admission HT risk was either moderate or high.

Baseline Large-Scale Network Dynamics Associated with Disease Progression in Huntington's Disease.

Huntington's disease (HD) is a genetically determined disease with motor, cognitive, and neuropsychiatric disorders. However, the links between clinical progression and disruptions to dynamics in motor and cognitive large-scale networks are not well established. To investigate changes in dynamic and static large-scale networks using an established tool of disease progression in Huntington's disease, the composite Unified Huntington's Disease Rating Scale (cUHDRS). Sixty-four mutation carriers were included. Static and dynamic baseline functional connectivity as well as topological features were correlated to 2-year follow-up clinical assessments using the cUHDRS. Decline in cUHDRS scores was associated with higher connectivity between frontal default-mode and motor networks, whereas higher connectivity in posterior, mainly visuospatial regions was associated with a smaller decline in cUHDRS scores. Structural disruptions in HD were evident both in posterior parietal/occipital and frontal motor regions, with reciprocal increases in functional connectivity. However, although higher visuospatial network connectivity was tied to a smaller cUHDRS decline, increased motor and frontal default-mode connections were linked to a larger cUHDRS decreases. Therefore, divergent functional compensation mechanisms might be at play in the clinical evolution of HD.

Disruption of the Malate-Aspartate Shuttle Leads to Anemia

Erythropoiesis, the process of differentiating multipotent hematopoietic stem cells (HSCs) into mature enucleated red blood cells (RBCs), is a metabolically intensive process, resulting in the generation of over 2 million new RBCs per second. Although anemia due to impaired erythropoiesis is a common condition, therapeutic options are often limited to RBC transfusions. However, the emergence of effective metabolite therapies, such as glutamine supplementation for sickle cell disease, underscores the potential of understanding metabolism for developing additional treatments. Two key enzymes in the malate-aspartate shuttle (MAS), glutamic-oxaloacetic transaminase 1 (GOT1) and 2 (GOT2), play crucial roles in transferring energy in the form of NADH from the cytosol into the mitochondria for oxidative phosphorylation. NADH cannot directly cross the mitochondrial membrane, so its electrons are transported on malate, which enters the mitochondria through a specific transporter. Within this shuttle, mitochondrial GOT2 utilizes glutamine to generate aspartate, which is then transported back into the cytoplasm to be metabolized by GOT1. Previous research has shown that aspartate-dependent nucleotide biosynthesis is essential for HSC regeneration. To investigate the roles of GOT1 and GOT2 in erythropoiesis, we crossed mice expressing tamoxifen-inducible erythroid-specific Cre recombinase ( Gata1-Cre ERT2 BAC transgenic mice) to Got1 or Got2 floxed lines, generating Got1 flox/flox;Gata1-Cre ERT2( Got1 CKO) or Got2 flox/flox;Gata1-Cre ERT2( Got2 CKO) mice, respectively. Upon tamoxifen administration to these mice, conditional Got1 or Got2 deletion occurs in megakaryocytic-erythrocytic progenitor (MEP) cells, which give rise to both RBCs and platelets. Complete blood count analysis revealed decreased hemoglobin levels and RBC counts, along with a reciprocal increase in platelet count in both Got1 and Got2 CKO mice. These results were unexpected, as previous studies showed that hematopoietic Got1 loss led to increased aspartate-driven nucleotide biosynthesis and HSC regeneration, while the opposite effects were observed in mice with hematopoietic Got2 loss. Although these enzymes are expected to have opposite effects on aspartate levels, the deletion of either enzyme disrupts the transfer of reducing equivalents between the cytosol and mitochondria. This results in NADH accumulation and reductive stress, which in turn leads to metabolic dysfunction, affecting glycolysis and oxidative phosphorylation. Therefore, the occurrence of anemia in mice with either Got1 or Got2 deletion in the erythroid compartment suggests that impaired redox balance, rather than aspartate-driven nucleotide biosynthesis, is likely the cause of the anemia. To further understand the basis of anemia in Got1 and Got2 CKO mice, we harvested bone marrow cells and used conventional cell surface markers to quantify MEPs, burst-forming unit erythroid cells (BFU-Es), colony-forming unit erythroid cells (CFU-Es), and terminally differentiated erythroblast populations. Compared to littermate controls with no deletion of Got1 and Got2, the absolute numbers of pre-CFU-Es and CFU-Es were higher in both Got1 and Got2CKO mice, while the absolute numbers of differentiated erythroblasts were lower in both mutant mice. These findings indicate a block in erythroid differentiation, which is currently under further investigation. Collectively, these preliminary data suggest a novel role for MAS in regulating erythropoiesis. Ongoing studies are actively exploring the mechanisms by which dysfunctional MAS leads to anemia.

EPCO-37. DISSECTING GBM EVOLUTION FOLLOWING STANDARD-OF-CARE BY LARGE-SCALE LONGITUDINAL SINGLE NUCLEUS RNA-SEQUENCING

Abstract The heterogeneity within tumors has long been associated with therapy failure and disease progression. Recent advances in single-cell RNA-sequencing technologies have enabled us to dissect the cellular diversity in glioblastoma (GBM). However, how these cellular programs change longitudinally under therapy remains poorly understood. To address this question, we collected and profiled a large-scale longitudinal cohort of 59 matched IDH-wildtype GBM sample pairs provided by 7 centers worldwide by single-nucleus RNA-sequencing (snRNA-seq) and whole-exome/whole-genome sequencing. The majority of patients (51 patients) in this cohort received standard-of-care (temozolomide and radiation) following initial tumor resection. Leveraging this large-scale snRNS-seq dataset of 457,442 cells, we detected novel cellular states and associations between malignant and tumor microenvironment (TME) cells, and then performed longitudinal analyses. The recurrent samples showed significantly lower malignant cell fraction (p=0.002) and reciprocal increase in proportions of glio-neuronal TME cell types (oligodendrocytes, neurons and astrocytes). The TME composition, malignant cell state proportions and baseline expression programs were retained at recurrence more than expected by chance, but overall were not well conserved between primary and recurrent samples. A subset of pairs (12/59 pairs) enriched with glio-neuronal TME at recurrence showed a significant transcriptomic shift and was associated with better clinical course (p=0.02). The tumors that acquired radiation-related small deletion phenotype underwent a transition towards MES/Hypoxia phenotype (0/9 at primary, 6/9 at recurrence, p=0.02). We defined pairs as likely responders or non-responders to treatment based on the MGMT methylation status of the primary tumor sample. This uncovered diverging evolutionary trajectories in cellular programs between the two groups. Strikingly, the changes in malignant state frequency and baseline malignant expression profile were strongly associated with specific changes in the TME composition. Our findings based on high-resolution longitudinal snRNA-seq analyses highlight the diverse evolutionary trajectories in GBM that are shaped by TME changes and treatments.

The Cellular Impact, Immediate Targets and Mechanism of Action of NSD2 in t(4;14) Multiple Myeloma

INTRODUCTION: NSD2 is a potential therapeutic target in the 15% of cases of multiple myeloma that carry a t(4;14) translocation. As a consequence of the translocation, strong Ig enhancers are placed in proximity to NSD2 and FGFR3. FGFR3 is deleted at a subsequent time point in 25% of cases, while all cases retain NSD2 expression consistent with it likely being the key oncogenic driver. NSD2 is an H3K36 dimethyl transferase, a histone modification mark associated with active transcription, but the molecular targets and the mechanism by which NSD2 regulates its targets is unknown. The cellular function of NSD2 is also largely unknown, but previous shRNA knockdown studies have suggested it is involved in cell proliferation. The major limitation to understanding the role of NSD2 is the lack of an appropriate experimental system with which to investigate its molecular and cellular function. To address this deficit and to identify the primary direct targets of NSD2 as a means to elucidate its mechanism of action we have generated NSD2 dTAG and NSD2 KO cells together with NSD2 reconstitution systems. METHODS: We have utilized CRISPR gene editing to knock-in a dTAG in NSD2 in the t(4;14) KMS-11 cell line. This system enables the inducible and rapid degradation of NSD2 following the addition of a small-molecule degrader which is otherwise inert. We have also generated clonal NSD2 KO KMS-11 cells that allow us to observe and study the stable phenotypes of NSD2 KO. Because H3K36me2 deposited by NSD2 leads to induction of DNMT3A-mediated DNA methylation and suppression of PRC2-mediated H3K27me3, we have generated DNMT3A KO on top of NSD2dTAG/KO and have used two different PRC2 inhibitors to dissect the dependency of NSD2 function on such secondary modifications. To systematically dissect the dependency on NSD2 isoforms and its methyltransferase activity, we have reconstituted NSD2 in both NSD2 dTAG and NSD2 KO KMS-11 cells, including WT NSD2I WT, NSD2II WT, NSD2 Y1179A (LOF), NSD2 E1099K (GOF). To maximally match the timing of NSD2 perturbation, NSD2 reconstitution was generated in NSD2 dTAG and NSD2 KO cells in a Tet-ON and Tet-OFF expression system, respectively. Further we have generated KMS-11 cells overexpressing Dox-inducible H3.3K36M to investigate the consequences of abrogation of H3K36me2 by this dominant negative mutant of H3.3. We have done SLAM-seq to assess the transcriptional targets of NSD2 and will extend this with ATAC-seq and CUT&amp;Tag to identify the molecular targets of NSD2. RESULTS: We show that NSD2 is rapidly degraded within 1 h when a degrader is added and subsequently a significant reduction of H3K36me2 is seen by 24 hours although it takes more than 3 days to see its deep depletion. We observed a reciprocal increase of H3K27me3 as previously noted. Depletion of H3K36me2 and increase in H3K27me3 was also seen in the NSD2 KO KMS-11 cells. Paradoxically we didn't see an induction of apoptosis in response to the degradation of NSD2 or its KO. In sharp contrast to the previously published shRNA results only a mild inhibition of cell proliferation was observed in NSD2 KO cells and over an extended period of 3 weeks in the dTAG system. We didn't observe an effect of H3.3 K36M overexpression on cell proliferation although it abolished H3K36me2 very efficiently. These experiments demonstrate that NSD2 is not essential for viability of KMS-11 cells but is required for optimal cell growth, which is likely independent of its H3K36me2 methyltransferase activity. SLAMseq results show that NSD2 degradation in the dTAG system causes transcriptional changes of a specific set of newly synthesized mRNAs. ATAC-seq and CUT&amp;Tag experiments are being run and will be superimposed on the SLAM-seq data to identify immediate targets of NSD2. Subsequently, use of PRC2 inhibitors and DNMT3A KO cells in the dTAG cells and the NSD2 reconstitution experiments will uncover the mechanisms of how NSD2 controls the transcription of its targets. CONCLUSION: We show that NSD2 is not essential for viability of KMS-11 cells but is required for optimal cell growth and that NSD2 controls the transcription of a specific set of genes and does not exert a global impact on the transcriptome. We will further define the immediate targets of NSD2 by leveraging our SLAM-seq, ATAC-seq, and CUT&amp;Tag data in NSD2 dTAG/KO and NSD2-reconstituted cells and by blocking the crosstalk with other epigenetic marks (i.e. H3K27me3 and DNAme).

Evaluation of Mechanical Stress Distribution Across the Calcaneus following Calcaneal Autograft: A Finite Element Analysis

Category: Other Introduction/Purpose: The purpose of this study was to utilize finite element analysis to evaluate progressive bone removal from the calcaneus at various angles and its effect on the mechanical stress distribution at the calcaneus, and potential increase in fracture risk. Methods: A calcaneus CT scan was imported into Autodesk Fusion360 (Autodesk, v.2.0.14569). After rendering the initial mesh body using tetrahedral elements, a copy of the calcaneus was made. A simulated force equal to the average weight of person (876N) was applied to the middle and posterior talus facets while the calcaneus was oriented in the heel strike position during gait, with constraints applied around the heel. The initial harvest site was modelled as a 6mm diameter 30mm deep cylinder. From this center point, the diameter of the harvest site was increased to 8, and 10mm, with the depth being adjusted such that there was no risk of breaching the cortex on the opposite side. For each iteration, the safety factor, stress (MPa), and strain were recorded. This was repeated at an angle of 90°, 45° and a combination of 45° and 90°. Results: The 90° model demonstrated that as the diameter of the harvest site increased, there was a reciprocal increase in stress across the calcaneus. The number of cycles to failure (fracture) for all diameters was 10^6-10^7. The 45° model demonstrated that as the diameter of the harvest site increased, there was a reciprocal increase in stress applied across the calcaneus with a corresponding decrease in stress at the harvest site. The number of cycles to failure increased from 10^3 at 6mm to 10^4-10^5 at 10mm. The combination of the 45°-90° model demonstrated that as the diameter of the harvest site increased, there was a reciprocal increase in stress applied across the calcaneus. The cycles to failures decreased as the diameter of the harvest site increased. Conclusion: This finite element analysis demonstrated that an increase in the diameter of the harvest site led to an increase in stress along the calcaneus. There was variation in the correlation between size of the diameter and the number of cycles to failure depending on the angle of the harvest. The greatest amount of stress applied at the calcaneus was with the 45°-90° model. It was determined that harvesting at an angle of 45° at 6mm produced the lowest number of cycles to failure.

Selenium-dependent glutathione peroxidase 1 regulates transcription of elongase 3 in murine tissues

We have previously shown dysregulated lipid metabolism in tissues of glutathione peroxidase 1 (GPX1) overexpressing (OE) or deficient (KO) mice. This study explored underlying mechanisms of GPX1 in regulating tissue fatty acid (FA) biosynthesis. GPX1 OE, KO, and wild-type (WT) mice (n = 5, male, 3–6 months old) were fed a Se-adequate diet (0.3 mg/kg) and assayed for liver and adipose tissue FA profiles and mRNA levels of key enzymes of FA biosynthesis and redox-responsive transcriptional factors (TFs). These three genotypes of mice (n = 5) were injected intraperitoneally with diquat, ebselen, and N-acetylcysteine (NAC) at 10, 50, and 50 mg/kg of body weight, respectively, and killed at 0 and 12 h after the injections to detect mRNA levels of FA elongases and desaturases and the TFs in the liver and adipose tissue. A luciferase reporter assay with targeted deletions of mouse Elovl3 promoter was performed to determine transcriptional regulations of the gene by GPX1 mimic ebselen in HEK293T cells. Compared with WT, GPX1 OE and KO mice had 9–42% lower (p < 0.05) and 36–161% higher (p < 0.05) concentrations of C20:0, C22:0, and C24:0 in these two tissues, respectively, along with reciprocal increases and decreases (p < 0.05) of Elovl3 transcripts. Ebselen and NAC decreased (p < 0.05), whereas diquat decreased (p < 0.05), Elovl3 transcripts in the two tissues. Overexpression and knockout of GPX1 decreased (p < 0.05) and increased (p < 0.05) ELOVL3 levels in the two tissues, respectively. Three TFs (GABP, SP1, and DBP) were identified to bind the Elovl3 promoter (−1164/+33 base pairs). Deletion of DBP (−98/−86 base pairs) binding domain in the promoter attenuated (13%, p < 0.05) inhibition of ebselen on Elovl3 promoter activation. In summary, GPX1 overexpression down-regulated very long-chain FA biosynthesis via transcriptional inhibition of the Elovl3 promoter activation.

It’s me again… Ask avoidance and the dynamics of charitable giving

Abstract Charities typically ask potential donors repeatedly for a donation. These repeated requests might trigger avoidance behaviour. Considering that, this article analyses the impact of offering the option to opt-out of receiving future fundraising asks on charitable giving. In a proposed utility framework, any opt-out option decreases the social pressure to donate. At the same time, an unconditional opt-out option induces feelings of gratitude toward the fundraiser, which may lead to a reciprocal increase in donations. The results of a lab experiment designed to disentangle the two channels show no negative impact of the option to avoid repeated asking for donations. Instead, the full model indicates a positive impact of the reciprocity channel. This finding suggests that it might be beneficial for charities to introduce an unconditional opt-out option during high-frequency fundraising campaigns.

Renal function in patients with chronic kidney disease and hypertension degree 1–2 against the background of SARS-CoV-2

Renal function in patients with chronic kidney disease and hypertension degree 1–2 against the background of SARS-CoV-2