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Sensitivity based model agnostic scalable explanations of deep learning.
Pub Date : 2025-03-07 DOI: 10.1101/2025.02.21.639516
Manu Aggarwal, N G Cogan, Vipul Periwal

Deep neural networks (DNNs) are powerful tools for data-driven predictive machine learning, but their complex architecture obscures mechanistic relations that they have learned from data. This information is critical to the scientific method of hypotheses development, experiment design, and model validation, especially when DNNs are used for biological and clinical predictions that affect human health. We design SensX, a model agnostic explainable AI (XAI) framework that outperformed current state-of-the-art XAI in accuracy (up to 52% higher) and computation time (up to 158 times faster), with higher consistency in all cases. It also determines an optimal subset of important input features, reducing dimensionality of further analyses. SensX scaled to explain vision transformer (ViT) models with more than 150, 000 features, which is computationally infeasible for current state-of-the-art XAI. SensX validated that ViT models learned justifiable features as important for different facial attributes of different human faces. SensX revealed biases inherent to the ViT architecture, an observation possible only when importance of each feature is explained. We trained DNNs to annotate biological cell types using single-cell RNA-seq data and SensX determined the sets of genes that the DNNs learned to be important to different cell types.

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引用次数: 0
Functional redundancy between penicillin-binding proteins during asymmetric cell division in Clostridioides difficile. 青霉素结合蛋白在艰难梭菌不对称细胞分裂过程中表现出催化冗余。
Pub Date : 2025-03-07 DOI: 10.1101/2024.09.26.615255
Shailab Shrestha, Jules M Dressler, Gregory A Harrison, Morgan E McNellis, Aimee Shen

Peptidoglycan synthesis is an essential driver of bacterial growth and division. The final steps of this crucial process involve the activity of the SEDS family glycosyltransferases that polymerize glycan strands and the class B penicillin-binding protein (bPBP) transpeptidases that cross-link them. While many bacteria encode multiple bPBPs to perform specialized roles during specific cellular processes, some bPBPs can play redundant roles that are important for resistance against certain cell wall stresses. Our understanding of these compensatory mechanisms, however, remains incomplete. Endospore-forming bacteria typically encode multiple bPBPs that drive morphological changes required for sporulation. The sporulation-specific bPBP, SpoVD, is important for synthesizing the asymmetric division septum and spore cortex peptidoglycan during sporulation in the pathogen Clostridioides difficile. Although SpoVD catalytic activity is essential for cortex synthesis, we show that it is unexpectedly dispensable for SpoVD to mediate asymmetric division. The dispensability of SpoVD's catalytic activity requires the presence of its SEDS partner, SpoVE, and is facilitated by another sporulation-induced bPBP, PBP3. Our data further suggest that PBP3 interacts with components of the asymmetric division machinery, including SpoVD. These findings suggest a possible mechanism by which bPBPs can be functionally redundant in diverse bacteria and facilitate antibiotic resistance.

肽聚糖合成是细菌生长和分裂的重要驱动力。这一关键过程的最后步骤涉及聚合糖链的 SEDS 家族糖基转移酶和交联糖链的 B 类青霉素结合蛋白(bPBP)转肽酶的活性。大多数细菌编码多种 bPBPs,它们在特定细胞过程中发挥专门作用,但有些 bPBPs 可发挥冗余作用,这对抵抗某些细胞壁压力非常重要。然而,我们对这些补偿机制的了解仍不全面。内孢子形成细菌通常编码多种 bPBPs,这些 bPBPs 驱动孢子形成所需的形态变化。病原体难辨梭状芽孢杆菌(Clostridioides difficile)的孢子特异性 bPBP(SpoVD)对于在孢子发生过程中合成不对称分裂隔膜和孢子皮层肽聚糖非常重要。虽然 SpoVD 的催化活性对皮层的合成至关重要,但我们发现 SpoVD 在介导不对称分裂时却意外地不需要这种催化活性。SpoVD 催化活性的可有可无性需要其 SEDS 伙伴 SpoVE 的存在,并受到另一种孢子特异性 bPBP(PBP3)催化活性的促进。我们的数据进一步表明,PBP3 与包括 SpoVD 在内的不对称分裂机制的组分相互作用。这些发现提示了一种可能的机制,即 bPBPs 在不同细菌中可能存在功能冗余,并促进抗生素耐药性的产生。
{"title":"Functional redundancy between penicillin-binding proteins during asymmetric cell division in <i>Clostridioides difficile</i>.","authors":"Shailab Shrestha, Jules M Dressler, Gregory A Harrison, Morgan E McNellis, Aimee Shen","doi":"10.1101/2024.09.26.615255","DOIUrl":"10.1101/2024.09.26.615255","url":null,"abstract":"<p><p>Peptidoglycan synthesis is an essential driver of bacterial growth and division. The final steps of this crucial process involve the activity of the SEDS family glycosyltransferases that polymerize glycan strands and the class B penicillin-binding protein (bPBP) transpeptidases that cross-link them. While many bacteria encode multiple bPBPs to perform specialized roles during specific cellular processes, some bPBPs can play redundant roles that are important for resistance against certain cell wall stresses. Our understanding of these compensatory mechanisms, however, remains incomplete. Endospore-forming bacteria typically encode multiple bPBPs that drive morphological changes required for sporulation. The sporulation-specific bPBP, SpoVD, is important for synthesizing the asymmetric division septum and spore cortex peptidoglycan during sporulation in the pathogen <i>Clostridioides difficile</i>. Although SpoVD catalytic activity is essential for cortex synthesis, we show that it is unexpectedly dispensable for SpoVD to mediate asymmetric division. The dispensability of SpoVD's catalytic activity requires the presence of its SEDS partner, SpoVE, and is facilitated by another sporulation-induced bPBP, PBP3. Our data further suggest that PBP3 interacts with components of the asymmetric division machinery, including SpoVD. These findings suggest a possible mechanism by which bPBPs can be functionally redundant in diverse bacteria and facilitate antibiotic resistance.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11463367/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142396769","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Micromechanics of lung capillaries across mouse lifespan and in positive- vs negative-pressure ventilation.
Pub Date : 2025-03-07 DOI: 10.1101/2025.03.02.641015
Kathryn Regan, Lauren Castle, Robert LeBourdais, Abdulrahman Kobayter, Linzheng Shi, Winita Wangsrikhun, Gabrielle Grifno, Rohin Banerji, Athanasios Batgidis, Bela Suki, Hadi T Nia

The lung undergoes continuous remodeling throughout normal development and aging, including changes to alveolar and capillary structure and function. While histological methods allow static analysis of these age-related changes, characterizing the changes that occur in response to mechanical stimuli remains difficult, particularly over a dynamic, physiologically relevant range in a functioning lung. Alveolar and capillary distension - the change in diameter of alveoli and capillaries, respectively, in response to pressure changes - is one such process, where dynamically controlling and monitoring the diameter of the same capillary or alveolus is essential to infer its mechanical properties. We overcome these limitations by utilizing the recently developed crystal ribcage to image the alveoli and vasculature of a functional mouse lung across the lifespan in postnatal (6-7 days), young adult (12-18 weeks), and aged (20+ months) mice. Using a range of biologically relevant vascular (0-15 cmH2O) and transpulmonary (3-12 cmH2O) pressures, we directly quantify vascular and alveolar distention in the functional lung as we precisely adjust pulmonary pressures. Our results show differences in age-related alveolar and vascular distensibility: when we increase transpulmonary alveolar or vascular pressure, vessels in postnatal lungs expand less and undergo less radial and axial strain, under each respective pressure type, suggesting stiffer capillaries than in older lungs. However, while vessels in young adult and aged lungs respond similarly to variations in vascular pressure, differences in elasticity start to emerge at the alveolar scale in response to transpulmonary alveolar pressure changes. Our results further indicate that differing effects of ventilation mode (i.e., positive vs negative) present themselves at the capillary level, with vessels under positive pressure undergoing more compression than when under negative-pressure conditions. These findings contribute both to the understanding of the functional changes that occur within the lung across the lifespan, as well as to the debate of ventilation effects on lung microphysiology.

{"title":"Micromechanics of lung capillaries across mouse lifespan and in positive- vs negative-pressure ventilation.","authors":"Kathryn Regan, Lauren Castle, Robert LeBourdais, Abdulrahman Kobayter, Linzheng Shi, Winita Wangsrikhun, Gabrielle Grifno, Rohin Banerji, Athanasios Batgidis, Bela Suki, Hadi T Nia","doi":"10.1101/2025.03.02.641015","DOIUrl":"https://doi.org/10.1101/2025.03.02.641015","url":null,"abstract":"<p><p>The lung undergoes continuous remodeling throughout normal development and aging, including changes to alveolar and capillary structure and function. While histological methods allow static analysis of these age-related changes, characterizing the changes that occur in response to mechanical stimuli remains difficult, particularly over a dynamic, physiologically relevant range in a functioning lung. Alveolar and capillary distension - the change in diameter of alveoli and capillaries, respectively, in response to pressure changes - is one such process, where dynamically controlling and monitoring the diameter of the same capillary or alveolus is essential to infer its mechanical properties. We overcome these limitations by utilizing the recently developed crystal ribcage to image the alveoli and vasculature of a functional mouse lung across the lifespan in postnatal (6-7 days), young adult (12-18 weeks), and aged (20+ months) mice. Using a range of biologically relevant vascular (0-15 cmH2O) and transpulmonary (3-12 cmH2O) pressures, we directly quantify vascular and alveolar distention in the functional lung as we precisely adjust pulmonary pressures. Our results show differences in age-related alveolar and vascular distensibility: when we increase transpulmonary alveolar or vascular pressure, vessels in postnatal lungs expand less and undergo less radial and axial strain, under each respective pressure type, suggesting stiffer capillaries than in older lungs. However, while vessels in young adult and aged lungs respond similarly to variations in vascular pressure, differences in elasticity start to emerge at the alveolar scale in response to transpulmonary alveolar pressure changes. Our results further indicate that differing effects of ventilation mode (i.e., positive vs negative) present themselves at the capillary level, with vessels under positive pressure undergoing more compression than when under negative-pressure conditions. These findings contribute both to the understanding of the functional changes that occur within the lung across the lifespan, as well as to the debate of ventilation effects on lung microphysiology.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11908188/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653122","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
TBCK-deficiency leads to compartment-specific mRNA and lysosomal trafficking defects in patient-derived neurons.
Pub Date : 2025-03-07 DOI: 10.1101/2025.03.02.641041
Marco Flores-Mendez, Jesus A Tintos-Hernández, Leonardo Ramos-Rodriguez, Leann Miles, Tsz Y Lo, Yuanquan Song, Xilma R Ortiz-González

Monogenic pediatric neurodegenerative disorders can reveal fundamental cellular mechanisms that underlie selective neuronal vulnerability. TBCK-Encephaloneuronopathy (TBCKE) is a rare autosomal recessive disorder caused by stop-gain variants in the TBCK gene. Clinically, patients show evidence of profound neurodevelopmental delays, but also symptoms of progressive encephalopathy and motor neuron disease. Yet, the physiological role of TBCK protein remains unclear. We report a human neuronal TBCKE model, derived from iPSCs homozygous for the Boricua variant (p.R126X). Using unbiased proteomic analyses of human neurons, we find TBCK interacts with PPP1R21, C12orf4, and Cryzl1, consistent with TBCK being part of the FERRY mRNA transport complex. Loss of TBCK leads to depletion of C12ORF4 protein levels across multiple cell types, suggesting TBCK may also play a role regulating at least some members of the FERRY complex. We find that TBCK preferentially, but not exclusively, localizes to the surface of endolysosomal vesicles and can colocalize with mRNA in lysosomes. Furthermore, TBCK-deficient neurons have reduced mRNA content in the axonal compartment relative to the soma. TBCK-deficient neurons show reduced levels of the lysosomal dynein/dynactin adapter protein JIP4, which functionally leads to TBCK-deficient neurons exhibiting striking lysosomal axonal retrograde trafficking defects. Hence, our work reveals that TBCK can mediate endolysosomal trafficking of mRNA, particularly along lysosomes in human axonal compartments. TBCK-deficiency leads to compartment-specific mRNA and lysosomal trafficking defects in neurons, which likely contribute to the preferential susceptibility to neurodegeneration.

{"title":"TBCK-deficiency leads to compartment-specific mRNA and lysosomal trafficking defects in patient-derived neurons.","authors":"Marco Flores-Mendez, Jesus A Tintos-Hernández, Leonardo Ramos-Rodriguez, Leann Miles, Tsz Y Lo, Yuanquan Song, Xilma R Ortiz-González","doi":"10.1101/2025.03.02.641041","DOIUrl":"https://doi.org/10.1101/2025.03.02.641041","url":null,"abstract":"<p><p>Monogenic pediatric neurodegenerative disorders can reveal fundamental cellular mechanisms that underlie selective neuronal vulnerability. TBCK-Encephaloneuronopathy (TBCKE) is a rare autosomal recessive disorder caused by stop-gain variants in the <i>TBCK</i> gene. Clinically, patients show evidence of profound neurodevelopmental delays, but also symptoms of progressive encephalopathy and motor neuron disease. Yet, the physiological role of TBCK protein remains unclear. We report a human neuronal TBCKE model, derived from iPSCs homozygous for the Boricua variant (p.R126X). Using unbiased proteomic analyses of human neurons, we find TBCK interacts with PPP1R21, C12orf4, and Cryzl1, consistent with TBCK being part of the FERRY mRNA transport complex. Loss of TBCK leads to depletion of C12ORF4 protein levels across multiple cell types, suggesting TBCK may also play a role regulating at least some members of the FERRY complex. We find that TBCK preferentially, but not exclusively, localizes to the surface of endolysosomal vesicles and can colocalize with mRNA in lysosomes. Furthermore, TBCK-deficient neurons have reduced mRNA content in the axonal compartment relative to the soma. TBCK-deficient neurons show reduced levels of the lysosomal dynein/dynactin adapter protein JIP4, which functionally leads to TBCK-deficient neurons exhibiting striking lysosomal axonal retrograde trafficking defects. Hence, our work reveals that TBCK can mediate endolysosomal trafficking of mRNA, particularly along lysosomes in human axonal compartments. TBCK-deficiency leads to compartment-specific mRNA and lysosomal trafficking defects in neurons, which likely contribute to the preferential susceptibility to neurodegeneration.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11908138/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653205","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
A patient-derived ovarian cancer organoid platform to study susceptibility to natural killer cells.
Pub Date : 2025-03-07 DOI: 10.1101/2025.03.06.641285
Marisa Mercadante, Armin Scheben, Jacob Estrada, Jan Savas-Carstens, William Sullivan, Nicholas Housel, Tatiana Volpari, Jax Hebner, Maria Sapar, Tom Rusielewicz, Frederick J Monsma, Stefan Semrau, Yinan Wang, Laura A Martin

Intratumoral heterogeneity drives therapy resistance and relapses in advanced stage cancers, such as ovarian cancer. Here, we present a live cell imaging assay using patient-derived ovarian cancer organoids for real time capture and quantification of natural killer cell-mediated apoptotic events in >500 organoids simultaneously. Our assay revealed significant inter- and intratumor response heterogeneity and identified a rare resistant organoid population, opening avenues to test immunomodulatory strategies that overcome resistance.

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引用次数: 0
Relationship between melanoma vemurafenib tolerance thresholds and metabolic pathway choice and Wnt signaling involvement.
Pub Date : 2025-03-07 DOI: 10.1101/2025.03.06.641924
Pratima Nangia-Makker, Madison Ahrens, Neeraja Purandare, Siddhesh Aras, Jing Li, Katherine Gurdziel, Hyejeong Jang, Seongho Kim, Malathy P Shekhar

Vemurafenib constitutes an important therapeutic for BRAFV600 mutant melanomas, but despite high initial response rates, resistance to BRAF and MEK inhibitors quickly develops. Here, we performed an integrative analysis of metabolomic consequences and transcriptome alterations to uncover mechanisms involved in adaptive vemurafenib resistance (VemR) development and their relationship with vemurafenib tolerance thresholds. We developed BRAFV600E isogenic models of VemR utilizing M14 and A2058 lines, and patient-derived melanomas with V600E or normal BRAF to verify vemurafenib selectivity. MEK or PI3K inhibitors only partially inhibited VemR cell proliferation, indicating cross-resistance to these inhibitors. MITF and β-catenin levels were induced and treatment with Wnt/β-catenin inhibitor ICG-001 restored vemurafenib sensitivity with concomitant reductions in β-catenin-regulated gene expressions, phospho-ERK1/2, and VemR-induced mitochondrial mass and respiration. Targeted metabolite, MitoPlate-S1, Mito-stress and transcriptome/metabolomic analysis showed that melanoma cells with elevated vemurafenib tolerance thresholds such as A2058 VemR cells utilize Wnt/β-catenin signaling for mitochondrial metabolism while VemR cells with low tolerance such as M14 VemR cells rely on Wnt/β-catenin signaling for pentose phosphate pathway. Pathways associated with cytokine-cytokine receptor, ECM receptor, and neuroactive ligand receptor interactions were similarly enriched in BRAFV600E patient-derived melanoma as M14 and A2058 cells whereas distinct pathways involving cell cycle, DNA replication, Fanconi anemia and DNA repair pathways are upregulated in wild type BRAF expressing patient derived melanoma. These data show for the first time that the metabolic pathway choices made by VemR BRAF mutant melanomas are controlled by vemurafenib tolerance and endurance thresholds and Wnt/β-catenin signaling plays a central role in coordinating expression of genes controlling VemR and metabolic pathway shifts.

{"title":"Relationship between melanoma vemurafenib tolerance thresholds and metabolic pathway choice and Wnt signaling involvement.","authors":"Pratima Nangia-Makker, Madison Ahrens, Neeraja Purandare, Siddhesh Aras, Jing Li, Katherine Gurdziel, Hyejeong Jang, Seongho Kim, Malathy P Shekhar","doi":"10.1101/2025.03.06.641924","DOIUrl":"https://doi.org/10.1101/2025.03.06.641924","url":null,"abstract":"<p><p>Vemurafenib constitutes an important therapeutic for BRAFV600 mutant melanomas, but despite high initial response rates, resistance to BRAF and MEK inhibitors quickly develops. Here, we performed an integrative analysis of metabolomic consequences and transcriptome alterations to uncover mechanisms involved in adaptive vemurafenib resistance (VemR) development and their relationship with vemurafenib tolerance thresholds. We developed BRAFV600E isogenic models of VemR utilizing M14 and A2058 lines, and patient-derived melanomas with V600E or normal BRAF to verify vemurafenib selectivity. MEK or PI3K inhibitors only partially inhibited VemR cell proliferation, indicating cross-resistance to these inhibitors. MITF and β-catenin levels were induced and treatment with Wnt/β-catenin inhibitor ICG-001 restored vemurafenib sensitivity with concomitant reductions in β-catenin-regulated gene expressions, phospho-ERK1/2, and VemR-induced mitochondrial mass and respiration. Targeted metabolite, MitoPlate-S1, Mito-stress and transcriptome/metabolomic analysis showed that melanoma cells with elevated vemurafenib tolerance thresholds such as A2058 VemR cells utilize Wnt/β-catenin signaling for mitochondrial metabolism while VemR cells with low tolerance such as M14 VemR cells rely on Wnt/β-catenin signaling for pentose phosphate pathway. Pathways associated with cytokine-cytokine receptor, ECM receptor, and neuroactive ligand receptor interactions were similarly enriched in BRAFV600E patient-derived melanoma as M14 and A2058 cells whereas distinct pathways involving cell cycle, DNA replication, Fanconi anemia and DNA repair pathways are upregulated in wild type BRAF expressing patient derived melanoma. These data show for the first time that the metabolic pathway choices made by VemR BRAF mutant melanomas are controlled by vemurafenib tolerance and endurance thresholds and Wnt/β-catenin signaling plays a central role in coordinating expression of genes controlling VemR and metabolic pathway shifts.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11908245/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653000","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
NLRP11 is required for canonical NLRP3 and non-canonical inflammasome activation during human macrophage infection with mycobacteria.
Pub Date : 2025-03-07 DOI: 10.1101/2024.12.11.627830
Mateusz Szczerba, Akshaya Ganesh, Maria Luisa Gil-Marques, Volker Briken, Marcia B Goldberg

The NLRP11 protein is only expressed in primates and participates in the activation of the canonical NLRP3 and non-canonical NLRP3 inflammasome activation after infection with gram-negative bacteria. Here, we generated a series of defined NLRP11 deletion mutants to further analyze the role of NLRP11 in NLRP3 inflammasome activation. Like the complete NLRP11 deletion mutant ( NLRP11 -/- ), the NLRP11 mutant lacking the NACHT and LRR domains ( NLRP11 N_LRR ) showed reduced activation of the canonical NLRP3 inflammasome, whereas a pyrin domain mutant ( NLRP11 PYD ) had no effect on NLRP3 activation. The NLRP11 -/- and NLRP11 N_LRR mutants but not the NLRP11 PYD mutant also displayed reduced activation of caspase-4 during infection with the intracytosolic, gram-negative pathogen Shigella flexneri . We found that the human adapted, acid-fast pathogen Mycobacterium tuberculosis and the opportunistic pathogen M. kansasii both activate the non-canonical NLRP11 inflammasome in a caspase-4/5-dependent pathway. In conclusion, we show that NLRP11 functions in the non-canonical caspase-4/5 inflammasome activation pathway and the canonical NRLP3 inflammasome pathway, and that NLRP11 is required for full recognition of mycobacteria by each of these pathways. Our work extends the spectrum of bacterial pathogen recognition by the non-canonical NLRP11-caspase4/5 pathway beyond gram-negative bacteria.

{"title":"NLRP11 is required for canonical NLRP3 and non-canonical inflammasome activation during human macrophage infection with mycobacteria.","authors":"Mateusz Szczerba, Akshaya Ganesh, Maria Luisa Gil-Marques, Volker Briken, Marcia B Goldberg","doi":"10.1101/2024.12.11.627830","DOIUrl":"https://doi.org/10.1101/2024.12.11.627830","url":null,"abstract":"<p><p>The NLRP11 protein is only expressed in primates and participates in the activation of the canonical NLRP3 and non-canonical NLRP3 inflammasome activation after infection with gram-negative bacteria. Here, we generated a series of defined NLRP11 deletion mutants to further analyze the role of NLRP11 in NLRP3 inflammasome activation. Like the complete NLRP11 deletion mutant ( <i>NLRP11 <sup>-/-</sup></i> ), the NLRP11 mutant lacking the NACHT and LRR domains ( <i>NLRP11</i> <sup>∆ <i>N_LRR</i></sup> ) showed reduced activation of the canonical NLRP3 inflammasome, whereas a pyrin domain mutant ( <i>NLRP11</i> <sup>∆ <i>PYD</i></sup> ) had no effect on NLRP3 activation. The <i>NLRP11 <sup>-/-</sup></i> and <i>NLRP11</i> <sup>∆ <i>N_LRR</i></sup> mutants but not the <i>NLRP11</i> <sup>∆ <i>PYD</i></sup> mutant also displayed reduced activation of caspase-4 during infection with the intracytosolic, gram-negative pathogen <i>Shigella flexneri</i> . We found that the human adapted, acid-fast pathogen <i>Mycobacterium tuberculosis</i> and the opportunistic pathogen <i>M. kansasii</i> both activate the non-canonical NLRP11 inflammasome in a caspase-4/5-dependent pathway. In conclusion, we show that NLRP11 functions in the non-canonical caspase-4/5 inflammasome activation pathway and the canonical NRLP3 inflammasome pathway, and that NLRP11 is required for full recognition of mycobacteria by each of these pathways. Our work extends the spectrum of bacterial pathogen recognition by the non-canonical NLRP11-caspase4/5 pathway beyond gram-negative bacteria.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11908186/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653134","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
The Th1/Th17 axis regulates chimeric antigen receptor (CAR) T cell therapy toxicities.
Pub Date : 2025-03-07 DOI: 10.1101/2025.03.06.641668
Payal Goala, Yongliang Zhang, Cooper Sailer, Shannon McSain, Muhammad Junaid Tariq, Showkat Hamid, Eduardo Cortes Gomez, Jianmin Wang, Justin C Boucher, Constanza Savid Frontera, Sae Bom Lee, Hiroshi Kotani, Michael Jain, Marco L Davila

CAR-T therapy has led to significant improvements in patient survival. However, a subset of patients experience high-grade toxicities, including cytokine release syndrome (CRS) and immune cell-associated hematologic toxicity (ICAHT). We utilized IL-2Rα knockout mice to model cytokine toxicities with elevated levels of IL6, IFNγ, and TNFα and increased M1-like macrophages. Onset of CRS was accompanied by a reduction in peripheral blood neutrophils due to disruption of bone marrow neutrophil homeostasis characterized by an increase in apoptotic neutrophils and a decrease in proliferative and mature neutrophils. Both non-tumor-bearing and Eμ-ALL tumor-bearing mice recapitulated the co-occurrence of CRS and neutropenia. IFNγ-blockade alleviated CRS and neutropenia without affecting CAR-T efficacy. Mechanistically, a Th1-Th17 imbalance was observed to drive co-occurrence of CRS and neutropenia in an IFNγ-dependent manner leading to decreased IL-17A and G-CSF, neutrophil production, and neutrophil survival. In patients, we observed an increase in the IFNγ-to-IL-17A ratio in the peripheral blood during high-grade CRS and neutropenia. We have uncovered a biological basis for ICAHT and provide support for the use of IFNγ-blockade to reduce CRS and neutropenia.

{"title":"The Th1/Th17 axis regulates chimeric antigen receptor (CAR) T cell therapy toxicities.","authors":"Payal Goala, Yongliang Zhang, Cooper Sailer, Shannon McSain, Muhammad Junaid Tariq, Showkat Hamid, Eduardo Cortes Gomez, Jianmin Wang, Justin C Boucher, Constanza Savid Frontera, Sae Bom Lee, Hiroshi Kotani, Michael Jain, Marco L Davila","doi":"10.1101/2025.03.06.641668","DOIUrl":"https://doi.org/10.1101/2025.03.06.641668","url":null,"abstract":"<p><p>CAR-T therapy has led to significant improvements in patient survival. However, a subset of patients experience high-grade toxicities, including cytokine release syndrome (CRS) and immune cell-associated hematologic toxicity (ICAHT). We utilized IL-2Rα knockout mice to model cytokine toxicities with elevated levels of IL6, IFNγ, and TNFα and increased M1-like macrophages. Onset of CRS was accompanied by a reduction in peripheral blood neutrophils due to disruption of bone marrow neutrophil homeostasis characterized by an increase in apoptotic neutrophils and a decrease in proliferative and mature neutrophils. Both non-tumor-bearing and Eμ-ALL tumor-bearing mice recapitulated the co-occurrence of CRS and neutropenia. IFNγ-blockade alleviated CRS and neutropenia without affecting CAR-T efficacy. Mechanistically, a Th1-Th17 imbalance was observed to drive co-occurrence of CRS and neutropenia in an IFNγ-dependent manner leading to decreased IL-17A and G-CSF, neutrophil production, and neutrophil survival. In patients, we observed an increase in the IFNγ-to-IL-17A ratio in the peripheral blood during high-grade CRS and neutropenia. We have uncovered a biological basis for ICAHT and provide support for the use of IFNγ-blockade to reduce CRS and neutropenia.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11908253/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653248","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Where's my mom? Resilient maternal preference in post-weaning male and female mice within a multi-chamber social behavior task.
Pub Date : 2025-03-07 DOI: 10.1101/2025.03.05.641280
Maggie M Slamin, Indra R Bishnoi, Izabella M Bankowski, Haley A Norris, Evan A Bordt

One of the earliest and most critical social bonds for many mammals is formed with their mother, who provides essential benefits for offspring development and survival. Growing evidence suggests that this social bond is retained even when animals gain independence, such as during the juvenile period immediately post-weaning. Here, we investigated whether juvenile (postnatal day (P)26) mice retain the ability to recognize and prefer their mothers post-weaning. We further investigated the strength of this bond using an acute immune activator. On P26, male and female C57BL/6J mice were intraperitoneally injected with the endotoxin lipopolysaccharide (LPS) or saline control (0.5 mg/kg). Four hours later, mice were subject to a five-chamber social preference task (the AGORA) containing their biological mother, a sex-matched novel mouse, a sex-matched sibling, a novel object, and an empty chamber. Our findings reveal that juvenile mice exhibit a strong maternal preference, significantly greater than chance and higher compared to any other social or non-social stimuli. While LPS exposure reduced the time spent investigating all stimuli, juvenile maternal preference was not significantly altered by LPS exposure. These effects were especially pronounced in females, while subtle shifts towards novel exploration began to emerge in males by P26. These results suggest that juvenile mice have a robust social preference for their mother that is resilient to early-life immune activation. Moreover, the novel multi-chamber task employed in the present study offered a more nuanced understanding of how social bonds evolve and vary across sex.

{"title":"Where's my mom? Resilient maternal preference in post-weaning male and female mice within a multi-chamber social behavior task.","authors":"Maggie M Slamin, Indra R Bishnoi, Izabella M Bankowski, Haley A Norris, Evan A Bordt","doi":"10.1101/2025.03.05.641280","DOIUrl":"https://doi.org/10.1101/2025.03.05.641280","url":null,"abstract":"<p><p>One of the earliest and most critical social bonds for many mammals is formed with their mother, who provides essential benefits for offspring development and survival. Growing evidence suggests that this social bond is retained even when animals gain independence, such as during the juvenile period immediately post-weaning. Here, we investigated whether juvenile (postnatal day (P)26) mice retain the ability to recognize and prefer their mothers post-weaning. We further investigated the strength of this bond using an acute immune activator. On P26, male and female C57BL/6J mice were intraperitoneally injected with the endotoxin lipopolysaccharide (LPS) or saline control (0.5 mg/kg). Four hours later, mice were subject to a five-chamber social preference task (the AGORA) containing their biological mother, a sex-matched novel mouse, a sex-matched sibling, a novel object, and an empty chamber. Our findings reveal that juvenile mice exhibit a strong maternal preference, significantly greater than chance and higher compared to any other social or non-social stimuli. While LPS exposure reduced the time spent investigating all stimuli, juvenile maternal preference was not significantly altered by LPS exposure. These effects were especially pronounced in females, while subtle shifts towards novel exploration began to emerge in males by P26. These results suggest that juvenile mice have a robust social preference for their mother that is resilient to early-life immune activation. Moreover, the novel multi-chamber task employed in the present study offered a more nuanced understanding of how social bonds evolve and vary across sex.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11908228/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143652955","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Pathogenicity and transmissibility of bovine-derived HPAI H5N1 B3.13 virus in pigs.
Pub Date : 2025-03-07 DOI: 10.1101/2025.03.04.641414
Taeyong Kwon, Jessie D Trujillo, Mariano Carossino, Heather M Machkovech, Konner Cool, Eu Lim Lyoo, Gagandeep Singh, Sujan Kafle, Shanmugasundaram Elango, Govindsamy Vediyappan, Wanting Wei, Nicholas Minor, Franco S Matias-Ferreyra, Igor Morozov, Natasha N Gaudreault, Udeni B R Balasuriya, Lisa Hensley, Diego G Diel, Wenjun Ma, Thomas C Friedrich, Juergen A Richt

Since the first emergence of highly pathogenic avian influenza (HPAI) H5N1 viruses in dairy cattle, the virus has continued to spread, reaching at least 17 states and at least 970 dairy herds in the United States. Subsequently, spillovers of the virus from dairy cattle to humans have been reported. Pigs are an important reservoir in influenza ecology because they serve as a mixing vessel in which novel reassortant viruses with pandemic potential can be generated. Here, we show that oro-respiratory infection of pigs resulted in productive replication of a bovine-derived HPAI H5N1 B3.13 virus. Infectious virus was mainly identified in the lower respiratory tract of principal infected pigs, and sero-conversion was observed in most of the principal pigs at later time points. In one animal, we detected the emergence of a mutation in hemagglutinin (HA) previously associated with increased affinity for "mammalian-type" α2,6-linked sialic acid receptors, but this mutation did not reach consensus levels. Sentinel contact pigs remained sero-negative throughout the study, indicating lack of transmission. The results support that pigs are susceptible to a bovine-derived HPAI H5N1 B3.13 virus, but this virus did not replicate as robustly in pigs as mink-derived HPAI H5N1 and swine-adapted influenza viruses.

{"title":"Pathogenicity and transmissibility of bovine-derived HPAI H5N1 B3.13 virus in pigs.","authors":"Taeyong Kwon, Jessie D Trujillo, Mariano Carossino, Heather M Machkovech, Konner Cool, Eu Lim Lyoo, Gagandeep Singh, Sujan Kafle, Shanmugasundaram Elango, Govindsamy Vediyappan, Wanting Wei, Nicholas Minor, Franco S Matias-Ferreyra, Igor Morozov, Natasha N Gaudreault, Udeni B R Balasuriya, Lisa Hensley, Diego G Diel, Wenjun Ma, Thomas C Friedrich, Juergen A Richt","doi":"10.1101/2025.03.04.641414","DOIUrl":"https://doi.org/10.1101/2025.03.04.641414","url":null,"abstract":"<p><p>Since the first emergence of highly pathogenic avian influenza (HPAI) H5N1 viruses in dairy cattle, the virus has continued to spread, reaching at least 17 states and at least 970 dairy herds in the United States. Subsequently, spillovers of the virus from dairy cattle to humans have been reported. Pigs are an important reservoir in influenza ecology because they serve as a mixing vessel in which novel reassortant viruses with pandemic potential can be generated. Here, we show that oro-respiratory infection of pigs resulted in productive replication of a bovine-derived HPAI H5N1 B3.13 virus. Infectious virus was mainly identified in the lower respiratory tract of principal infected pigs, and sero-conversion was observed in most of the principal pigs at later time points. In one animal, we detected the emergence of a mutation in hemagglutinin (HA) previously associated with increased affinity for \"mammalian-type\" α2,6-linked sialic acid receptors, but this mutation did not reach consensus levels. Sentinel contact pigs remained sero-negative throughout the study, indicating lack of transmission. The results support that pigs are susceptible to a bovine-derived HPAI H5N1 B3.13 virus, but this virus did not replicate as robustly in pigs as mink-derived HPAI H5N1 and swine-adapted influenza viruses.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11908241/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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