Redundant Roles Research Articles

Redundancy protects processing speed in healthy individuals with accelerated brain aging.

Recent advancements in computational learning techniques have enabled the estimation of brain age (BA) from neuroimaging data. The difference between chronological age (CA) and BA, known as the BA gap, can potentially serve as a biomarker of brain health. Studies, however, have documented low correlations between BA gap and cognition in healthy aging. This suggests that protective mechanisms in the brain may help counter the effect of accelerated brain aging. Here, we investigated whether redundancy in brain networks may protect cognitive function in individuals with accelerated brain aging. First, we employed deep learning to estimate individual brain ages from structural magnetic resonance imaging (MRI). Next, we associated CA, BA, and BA gap, with cognitive measures and network topology derived from diffusion MRI and tractography. We found that CA and BA were both similarly related to cognitive measures and network topology, while BA gap did not show strong relationships in either domain. Despite observing no strong relationships between brain-age gap (BA gap) and demographic variables, cognitive measures, or topological features in healthy aging, individuals with accelerated aging (BA gap+) exhibited lower average degree and redundancy within the dorsal attention network compared to those with delayed aging (BA gap-). Furthermore, redundancy in the dorsal attention network was positively associated with processing speed in BA gap+ individuals. These results indicate a potential neuroprotective role of redundancy in structural brain networks for mitigating the impact of accelerated brain atrophy on cognitive performance in healthy aging.

Unique and redundant roles of mouse BCMA, TACI, BAFF, APRIL, and IL-6 in supporting antibody-producing cells in different tissues

Antibody-producing plasma cells fuel humoral immune responses. They also contribute to autoimmune diseases such as systemic lupus erythematosus or IgA nephropathy. Interleukin-6 and the tumor necrosis factor (TNF) family ligands BAFF (B cell-activating factor) and APRIL (a proliferation-inducing ligand) participate in plasma cell survival. BAFF binds to three receptors, BAFFR (BAFF receptor), TACI (transmembrane activator and CAML interactor), and BCMA (B cell maturation antigen), while APRIL binds to TACI, BCMA, and proteoglycans. However, which ligand-receptor pair(s) are required to maintain plasma cells in different body locations remains unknown. Here, by combining mouse genetic and pharmacological approaches, we found that plasma cells required BCMA and/or TACI but not BAFFR. BCMA responded exclusively to APRIL, while TACI responded to both BAFF and APRIL, identifying three self-sufficient ligand-receptor pairs for plasma cell maintenance: BAFF-TACI, APRIL-TACI, and APRIL-BCMA. Together, these actors accounted for 90% of circulating antibodies. In BAFF-ko mice, the reduction of plasma cells upon APRIL inhibition indicated that APRIL could function in the absence of BAFF-APRIL heteromers. No evidence was found that in the absence of BCMA and TACI, binding of APRIL to proteoglycans would help maintain plasma cells. IL-6, alone or together with BAFF and APRIL, supported mainly splenic plasmablasts and plasma cells and contributed to circulating IgG but not IgA levels. In conclusion, survival factors for plasma cells can vary with body location and with the antibody isotype that plasma cells produce. To efficiently target plasma cells, in particular IgA-producing ones, dual inhibition of BAFF and APRIL is required.

To evaluate the role of mast cells on angiogenesis in various grades of oral squamous cell carcinoma: A histochemical study

Abstract Background: Oral cancer is the sixth most common cancer, and 90% of them are oral squamous cell carcinomas (OSCC). As most OSCC are asymptomatic and are only detected at an advanced stage, the 5-year survival rate is only 50%. Thus, using novel prognosticators can minimise mortality and morbidity associated with OSCC. This study aims to evaluate the relationship between mast cells and angiogenesis in different grades of OSCC to analyse their role in its progression. Material and Methods: A total of 45 cases were included, comprising 10 well-differentiated SCCs (WDOSCC), 10 moderately differentiated SCCs (MDOSCC), and 10 poorly differentiated SCCs (PDOSCC). Additionally, five normal buccal mucosae (NBM) samples served as negative controls for OSCC. Five cases of neurofibroma and pyogenic granuloma were used as positive controls for mast cells and angiogenesis, respectively. Results: The mean MCD in WDOSCC, MDOSCC, and PDOSCC were 3.2620 ± 2.65177, 3.0310 ± 1.38276, and 4.1580 ± 2.49482, respectively. The MVD in WDOSCC, MDOSCC, and PDOSCC were 10.2850 ± 4.35032, 9.9240 ± 2.72533, and 7.1520 ± 2.26966, respectively. Discussion: MCD was the highest in PDOSCC, followed by WDOSCC and MDOSCC. These results indicate a redundant role of mast cells in OSCC, or they might jumpstart malignancy but are retarded with OSCC progression. The MVD decreased with higher grades, in contrast to the prevalent literature. The correlation analysis between MVD and MCD revealed no significant correlation between them. Conclusion: We found a non-significant role of mast cells in tumour biology and a decrease in vascularity with advancing grades. These results indicate a lower need for mast cell activation to augment vascularisation. A study with a larger sample size is needed to confirm our results.

The role of link redundancy and structural heterogeneity in network disintegration

While link redundancy has long been acknowledged as a critical factor in network robustness, current approaches frequently neglect the inherent heterogeneity of structure, thereby falling short of optimal network disintegration. This study introduces the novel Neighborhood Dissimilarity Community Heterogeneity (NDCH) framework, which systematically investigates redundancy elimination from a new perspective of neighborhood dissimilarity, while concurrently integrating structural heterogeneity derived from community structure. Extensive experiments on both synthetic and diverse real-world networks reveal that strategies developed under NDCH framework markedly outperform existing state-of-the-art methods in network disintegration, with performance improvements reaching up to 60.151% and 31.000% for Schneider R and the critical removal fraction fc, respectively. Notably, additional analysis consistently indicates low correlations (Kendall’s Tau < 0.6) and distinct value distributions between NDCH-based strategies and the state-of-the-art approaches. In summary, the novel framework underscores the significance of both redundancy and structural heterogeneity when devising network disintegration strategies, offering a substantial leap forward in enhancing network robustness against malicious attacks and various disruptions, especially in infrastructure networks.

TENT5-mediated polyadenylation of mRNAs encoding secreted proteins is essential for gametogenesis in mice

Cytoplasmic polyadenylation plays a vital role in gametogenesis; however, the participating enzymes and substrates in mammals remain unclear. Using knockout and knock-in mouse models, we describe the essential role of four TENT5 poly(A) polymerases in mouse fertility and gametogenesis. TENT5B and TENT5C play crucial yet redundant roles in oogenesis, with the double knockout of both genes leading to oocyte degeneration. Additionally, TENT5B-GFP knock-in females display a gain-of-function infertility effect, with multiple chromosomal aberrations in ovulated oocytes. TENT5C and TENT5D both regulate different stages of spermatogenesis, as shown by the sterility in males following the knockout of either gene. Finally, Tent5a knockout substantially lowers fertility, although the underlying mechanism is not directly related to gametogenesis. Through direct RNA sequencing, we discovered that TENT5s polyadenylate mRNAs encoding endoplasmic reticulum-targeted proteins essential for gametogenesis. Sequence motif analysis and reporter mRNA assays reveal that the presence of an endoplasmic reticulum-leader sequence represents the primary determinant of TENT5-mediated regulation.

CBP60b clade proteins are prototypical transcription factors mediating immunity.

Transcriptional reprogramming is critical for plant immunity. Several calmodulin (CaM)-binding protein 60 (CBP60) family transcription factors (TFs) in Arabidopsis (Arabidopsis thaliana), including CBP60g, Systemic Acquired Resistance Deficient 1 (SARD1), CBP60a, and CBP60b, are critical for and show distinct roles in immunity. However, there are additional CBP60 members whose function is unclear. We report here that Arabidopsis CBP60c-f, four uncharacterized CBP60 members, play redundant roles with CBP60b in the transcriptional regulation of immunity responses, whose pCBP60b-driven expression compensates the loss of CBP60b. By contrast, neither CBP60g nor SARD1 is inter-changeable with CBP60b, suggesting clade-specific functionalization. We further show that function of CBP60b clade TFs relies on DNA-binding domains (DBDs) and CaM-binding domains, suggesting that they are downstream components of calcium signaling. Importantly, we demonstrate that CBP60s encoded in earliest land plant lineage Physcomitrium patens and Selaginella moellendorffii, are functionally homologous to Arabidopsis CBP60b, suggesting that the CBP60b clade contains the prototype TFs of the CBP60 family. Furthermore, tomato and cucumber CBP60b-like genes rescue the defects of Arabidopsis cbp60b and activate the expression of tomato and cucumber SALICYLIC ACID INDUCTION DEFICIIENT2 (SID2) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) genes, suggesting that immune response pathways centered on CBP60b are also evolutionarily conserved. Together, these findings suggest CBP60b clade transcription factors are functionally conserved in evolution and positively mediate immunity.

Abstract B012: Predicting targetable paralog synthetic lethalities and functional redundancies in cancer genomes

Abstract Paralogs are prevalent in the human genome and are considered a rich source of synthetic lethality due to functional redundancy. For a cancer cell carrying a gene with Loss-Of-Function (LOF) mutation, inactivation of its paralog using gene editing can induce a selective decrease in viability, leaving normal cells that do not harbor the mutation unharmed. Previous studies have exploited this vulnerability of cancer genomes. However, the cancer-specificity of such synthetic lethality limits its application across different cancer types. Furthermore, the role of functional redundancy which can explain the cancer specificity has remained largely unexplored. In this study, we computationally predicted synthetic lethal paralogs along with mechanistically important functional redundancies between them in a cancer-specific manner. We applied our prediction method to publicly available data for an aggressive subtype of breast cancer called triple-negative breast cancer (TNBC), which lacks the expression key biomarkers and hence has the worst prognosis among other breast cancer subtypes. TNBC is characterized by the highest mutational load and the largest fraction of genome altered among the breast cancer subtypes providing a rich mutational landscape to identify LOF genes. Using the CRISPR inactivation screen data for cancer cell lines obtained from the Cancer Dependency Map (DepMap) project, we predicted sets of synthetic lethal paralogs that show a significantly greater viability decrease if a gene carries LOF and its paralog is inactivated, compared to the viability decrease due to the inactivation of only one of the paralogs. Consistent with previous findings of context-dependent synthetic lethality, we found that a relatively small fraction of TNBC-specific synthetic lethal paralogs overlapped with those found across other cancer types. To uncover the mechanistically important functional redundancies between paralogs, we analyzed the genomics and transcriptomics data from multiple sources: TNBC panel of primary tumors and patient-derived xenografts, Pan-Cancer Analysis of Whole Genomes (PCAWG), and the Cancer Cell Line Encyclopedia (CCLE). The functional redundancies varied across cancer types based on (1) mutual exclusivity of LOFs, (2) backup compensation of deleterious mutations, (3) backup upregulation, and (4) dosage balance. Overall, our findings show a strong context-dependency of synthetic lethal paralogs and estimates of functional redundancy, emphasizing the importance of such cancer-specific predictions in identifying targetable paralog synthetic lethalities. Collectively, the computational method and sets of targetable paralogs are a unique resource for developing precision oncology therapeutic strategies against TNBC and other cancers more broadly. Citation Format: Rohan Dandage, Elena Kuzmin. Predicting targetable paralog synthetic lethalities and functional redundancies in cancer genomes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B012.

Functional divergence of FTL9 and FTL10 in flowering control in rice

BackgroundFloral transition in cereals is a critical phenomenon influenced by exogenous and endogenous signals, determining crop yield and reproduction. Flowering Locus T-like (FT-like) genes encode a mobile florigen, the main signaling molecule for flowering.ResultsIn this study, we characterized two FT-like genes, FTL9 and FTL10, to study their functional diversity in flowering control in rice. We compared independent mutant lines of ftl10 with WT and observed negligible differences in the flowering phenotype, or agronomic traits implying potentially redundant roles of FTL10 loss-of-function in flowering control in rice. Nevertheless, we found that overexpression of FTL10, but not FTL9, substantially accelerated flowering, indicating the flowering-promoting role of FTL10 and the divergent functions between FTL9 and FTL10 in flowering. Besides flowering, additive agronomic roles were observed for FTL10-OE regulating the number of effective panicles per plant, the number of primary branches per panicle, and spikelets per panicle without regulating seed size. Mechanistically, our Y2H and BiFC analyses demonstrate that FTL10, in contrast to FTL9, can interact with FD1 and GF14c, forming a flowering activation complex and thereby regulating flowering.ConclusionAltogether, our results elucidate the regulatory roles of FTL9 and FTL10 in flowering control, unveiling the molecular basis of functional divergence between FTL10 and FTL9, which provides mechanistic insights into shaping the dynamics of flowering time regulation in rice.

The protein phosphatase PP6 promotes RIPK1-dependent PANoptosis

BackgroundThe innate immune system serves as the first line of host defense. Transforming growth factor-β–activated kinase 1 (TAK1) is a key regulator of innate immunity, cell survival, and cellular homeostasis. Because of its importance in immunity, several pathogens have evolved to carry TAK1 inhibitors. In response, hosts have evolved to sense TAK1 inhibition and induce robust lytic cell death, PANoptosis, mediated by the RIPK1-PANoptosome. PANoptosis is a unique innate immune inflammatory lytic cell death pathway initiated by an innate immune sensor and driven by caspases and RIPKs. While PANoptosis can be beneficial to clear pathogens, excess activation is linked to pathology. Therefore, understanding the molecular mechanisms regulating TAK1 inhibitor (TAK1i)-induced PANoptosis is central to our understanding of RIPK1 in health and disease.ResultsIn this study, by analyzing results from a cell death-based CRISPR screen, we identified protein phosphatase 6 (PP6) holoenzyme components as regulators of TAK1i-induced PANoptosis. Loss of the PP6 enzymatic component, PPP6C, significantly reduced TAK1i-induced PANoptosis. Additionally, the PP6 regulatory subunits PPP6R1, PPP6R2, and PPP6R3 had redundant roles in regulating TAK1i-induced PANoptosis, and their combined depletion was required to block TAK1i-induced cell death. Mechanistically, PPP6C and its regulatory subunits promoted the pro-death S166 auto-phosphorylation of RIPK1 and led to a reduction in the pro-survival S321 phosphorylation.ConclusionsOverall, our findings demonstrate a key requirement for the phosphatase PP6 complex in the activation of TAK1i-induced, RIPK1-dependent PANoptosis, suggesting this complex could be therapeutically targeted in inflammatory conditions.

Arabidopsis Sar1b is critical for pollen tube growth.

Pollen tube growth is an essential step leading to reproductive success in flowering plants, in which vesicular trafficking plays a key role. Vesicular trafficking from endoplasmic reticulum to the Golgi apparatus is mediated by the coat protein complex II (COPII). A key component of COPII is small GTPase Sar1. Five Sar1 isoforms are encoded in the Arabidopsis genome and they show distinct while redundant roles in various cellular and developmental processes, especially in reproduction. Arabidopsis Sar1b is essential for sporophytic control of pollen development while Sar1b and Sar1c are critical for gametophytic control of pollen development. Because functional loss of Sar1b and Sar1c resulted in pollen abortion, whether they influence pollen tube growth was unclear. Here we demonstrate that Sar1b mediates pollen tube growth, in addition to its role in pollen development. Although functional loss of Sar1b does not affect pollen germination, it causes a significant reduction in male transmission and of pollen tube penetration of style. We further show that membrane dynamics at the apex of pollen tubes are compromised by Sar1b loss-of-function. Results presented provide further support of functional complexity of the Sar1 isoforms.

Combining multiple stressors unexpectedly blocks bacterial migration and growth.

In nature, organisms experience combinations of stressors. However, laboratory studies typically simplify reality and focus on the effects of an individual stressor. Here, we use a microfluidic approach to simultaneously provide a physical stressor (shear flow) and a chemical stressor (H 2 O 2 ) to the human pathogen Pseudomonas aeruginosa . By treating cells with levels of flow and H 2 O 2 that commonly co-occur in nature, we discover that previous reports significantly overestimate the H 2 O 2 levels required to block bacterial growth. Specifically, we establish that flow increases H 2 O 2 effectiveness 50-fold, explaining why previous studies lacking flow required much higher concentrations. Using natural H 2 O 2 levels, we identify the core H 2 O 2 regulon, characterize OxyR-mediated dynamic regulation, and dissect the redundant roles of multiple H 2 O 2 scavenging systems. By examining single-cell behavior, we serendipitously discover that the combined effects of H 2 O 2 and flow block pilus-driven surface migration. Thus, our results counter previous studies and reveal that natural levels of H 2 O 2 and flow synergize to restrict bacterial colonization and survival. By studying two stressors at once, our research highlights the limitations of oversimplifying nature and demonstrates that physical and chemical stress can combine to yield unpredictable effects.

Structural and functional mechanisms of actin isoforms.

Actin is a highly conserved and fundamental protein in eukaryotes and participates in a broad spectrum of cellular functions. Cells maintain a conserved ratio of actin isoforms, with muscle and non-muscle actins representing the main actin isoforms in muscle and non-muscle cells, respectively. Actin isoforms have specific and redundant functional roles and display different biochemistries, cellular localization, and interactions with myosins and actin-binding proteins. Understanding the specific roles of actin isoforms from the structural and functional perspective is crucial for elucidating the intricacies of cytoskeletal dynamics and regulation and their implications in health and disease. Here, we review how the structure contributes to the functional mechanisms of actin isoforms with a special emphasis on the questions of how post-translational modifications and disease-linked mutations affect actin isoforms biochemistry, function, and interaction with actin-binding proteins and myosin motors.

Redundancy can hinder adult L2 grammar learning: evidence from case markers of varying salience levels.

Grammatical redundancy is a widespread feature across languages. Although redundant cues can be seen to increase the complexity and processing burden of structures, it has been suggested that they can assist language acquisition. Here, we explored if this learning benefit can be observed from the very initial stages of second language (L2) acquisition and whether the effect of redundancy is modulated by the perceptual salience of the redundant linguistic cues. Across two experiments, three groups of adult native speakers of English were incidentally exposed to three different artificial languages; one that had a fixed word order, Verb-Object-Subject, and two in which thematic role assignment was additionally determined by a low-salient (Experiment 1) or a high-salient (Experiment 2) redundant case marker. While all groups managed to learn the novel language, our results pointed towards a hindering role of redundancy, with participants in the non-redundant condition achieving greater learning outcomes compared to those in both redundant conditions. Results also revealed that this impeding effect of redundancy on L2 learners can be attenuated by the salience of the redundant cue (Experiment 2). In conjunction with earlier findings, the present results suggest that the effect of redundancy on L2 acquisition can be differentially manifested depending on the stage of L2 development, learners' first language biases and age.

A protective role of CD4+ T cells during Staphylococcus aureus craniotomy infection

Abstract Craniotomy is a neurosurgery performed to resolve severe neurological complications. Despite prophylaxis, nearly 1-3% of craniotomies are complicated by infection, with approximately half attributed to Staphylococcus aureus (S. aureus). Using our novel mouse model, we established a protective role for CD4+ T cells during S. aureus craniotomy infection. Rag1 knock out (KO) mice or animals treated with anti-CD4 Ab showed significantly elevated bacterial burden, suggesting an important role for adaptive immunity in controlling infection. These findings were corroborated in mice treated with VLA-4 and LFA-1 Abs, underscoring the importance of peripheral CD4+ T cell responses. scRNA-seq revealed phenotypic heterogeneity within brain CD3+ infiltrates with CD4+ cells being most predominant that were typified by robust inflammatory and glycolytic profiles. CD4+ T cells displayed attributes of both Th1 and Th17 phenotypes, whose functional importance was validated in vivo as adoptive transfer of either Th1 or Th17 cells prevented S. aureus outgrowth in Rag1 KO mice. Interestingly, milder phenotypes were observed in IL-17A/F or IFN-γ KO mice, suggesting a redundant and cooperative role of both Th1 and Th17 driven responses. This was confirmed by treatment of IFN-γ KO mice with IL-17A/F neutralizing Ab, which recapitulated phenotypes of Rag1 KO animals. Collectively, our results implicate a critical role for CD4+ T cells in S. aureus containment during craniotomy infection.

Piezo1 and Piezo2 collectively regulate jawbone development.

Piezo1 and Piezo2 are recently reported mechanosensory ion channels that transduce mechanical stimuli from the environment into intracellular biochemical signals in various tissues and organ systems. Here, we show that Piezo1 and Piezo2 display a robust expression during jawbone development. Deletion of Piezo1 in neural crest cells causes jawbone malformations in a small but significant number of mice. We further demonstrate that disruption of Piezo1 and Piezo2 in neural crest cells causes more striking defects in jawbone development than any single knockout, suggesting essential but partially redundant roles of Piezo1 and Piezo2. In addition, we observe defects in other neural crest derivatives such as malformation of the vascular smooth muscle in double knockout mice. Moreover, TUNEL examinations reveal excessive cell death in osteogenic cells of the maxillary and mandibular arches of the double knockout mice, suggesting that Piezo1 and Piezo2 together regulate cell survival during jawbone development. We further demonstrate that Yoda1, a Piezo1 agonist, promotes mineralization in the mandibular arches. Altogether, these data firmly establish that Piezo channels play important roles in regulating jawbone formation and maintenance.

Non redundant roles of group 2 innate lymphoid cells and T-helper 2 cells on anti-helminth immunity

Non redundant roles of group 2 innate lymphoid cells and T-helper 2 cells on anti-helminth immunity

Abstract 1039: Endothelial Deletion Of Nck1 Reduces Atherosclerosis In Apoe-/- Mice Through The Nck1-atf3 Axis

The Nck family of signaling adaptors, including both Nck1 and Nck2, classically regulate cell motility during vascular development and postnatal angiogenesis. While Nck1 and Nck2 play redundant roles in this regard with deletion of both isoforms required to affect tissue remodeling, we demonstrated that global Nck1 deletion was sufficient to reduce atherosclerotic plaque formation and selective Nck1 inhibition reduces proinflammatory endothelial activation in response to oscillatory shear stress. Therefore, we sought to determine the role of endothelial Nck1 in atherosclerosis and ischemic angiogenesis using novel endothelial-specific Nck1 knockout mice. Endothelial-specific Nck1 deletion significantly reduced Western diet-induced atherosclerotic plaque formation at multiple sites compared to mice expressing endothelial-specific Cre alone or to endothelial-specific Nck2 knockout mice. This reduced plaque formation was characterized by diminished macrophage and smooth muscle incorporation despite enhanced weight gain in endothelial Nck1 knockout mice. RNA Sequencing analysis of endothelial cells in culture showed that Nck1 depletion reduces the expression of a variety of proinflammatory pathways but enhances the expression of genes involved in central carbon metabolism and proliferation. While Nck1 deletion is sufficient to blunt atherogenic inflammation, Nck1 inhibition did not affect angiogenesis or limb perfusion in the femoral artery ligation model of hind limb ischemia. To further characterized Nck1’s proinflammatory role, we utilized affinity precipitation-mass spectrometry and identified Activating Transcription Factor 3 ATF3 as a Nck1-selective binding partner. While no studies to date have examined endothelial ATF3 signaling in atherosclerosis, our preliminary data show that Nck1 is required for ATF3 expression in response to disturbed flow in vitro and in vivo , but the mechanism by which disturbed flow stimulates ATF3 expression remains unknown. ATF3 knockdown reduces disturbed flow-induced proinflammatory gene expression, suggesting that the Nck1-ATF3 signaling axis is a novel regulator of flow-induced inflammation.

Abstract LB187: Investigating the roles of ROR1/2 in non-small cell lung carcinoma NSCLC and potential therapeutic applications

Abstract Lung cancer is the leading cause of cancer mortality worldwide, representing an unmet medical need. Receptor tyrosine kinase-like orphan receptors (RORs) are oncofetal antigens that act as receptors/co-receptors of WNT5A and other related noncanonical WNT proteins, in transducing β-catenin independent WNT signaling. Dysregulated ROR/WNT have been linked to cancer progression and therapy resistance. Recently, ROR receptors have drawn increasing attention as anti-cancer targets of antibody-drug conjugates (ADCs) due to their selective expression on the tumor cell surface. The ROR family contains ROR1 and ROR2, which share 58% sequence homology. However, their functional interactions between ROR/WNT5A and whether RORs have distinct or redundant roles in tumor phenotypes remain unclear. In this study, we sought to address these biological questions in NSCLC, and investigate the potential application of anti-ROR therapies. To this end, we used CRISPR/Cas9 to generate clones of ROR1 or ROR2 single knockouts (KOs) in the NSCLC LCLC-103H cell line and implemented an array of assays to characterize the effect of single ROR KOs. ROR KOs were confirmed by both western blot and flow cytometry. We found reduced ROR2 mean fluorescence intensity (MFI) on ROR1 KOs and increased ROR1 MFI on ROR2 KOs in flow cytometrical analysis. Phenotypically, ROR1 KOs did not affect tumor doubling time, but ROR2 KOs promoted cell proliferation in 2D cultures. Unlike 2D cultures, tumor 3D spheroid proliferation was significantly inhibited in both ROR1 and ROR2 KOs, implying that each ROR is indispensable in maintaining tumor growth featured by cell-cell/extracellular matrix interactions. Mechanistically, ROR1 KOs tended to decrease ROR2 and WNT5A/B protein expression, while ROR2 KOs caused compensatory upregulation of ROR1 and WNT5A/B. Both ROR1 and ROR2 KOs decreased SNAIL expression, which is a key driver of epithelial-to-mesenchymal plasticity (EMP). Furthermore, alterations in ROR1/2 signaling pathways were observed. We have identified pathways downstream of ROR1/2 including WNT, EMP, and survival, which may shed light on the biological roles of RORs in cancer and potential therapeutic intervention. Finally, the therapeutic application of ROR1/2 was evaluated by monomethyl auristatin F (MMAF) containing anti-ROR ADCs. ROR1 KOs demonstrated the on-target cytotoxicity of anti-ROR1 ADC, while ROR2 KOs showed increased sensitivity to anti-ROR1 ADC due to the increased ROR1 expression in ROR2 KOs. In vivo analysis is currently underway. Collectively, our results demonstrate that RORs have both distinct and compensatory functions in regulating tumor proliferation. We believe a greater understanding in ROR1/2 biology may lead to better targeted therapeutic applications. Citation Format: Wen-An Pan, Xin Guo, Christina Galang, Mira Ko, Jeff Kucharski, Nicole Miller, Haim Moskowitz, Jeff Watkins, Brian Lannutti, Katti Jessen, Hao Liu. Investigating the roles of ROR1/2 in non-small cell lung carcinoma NSCLC and potential therapeutic applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB187.

TAK1-binding protein 2 (TAB2) and TAB3 are redundantly required for TLR-induced cytokine production in macrophages.

Transforming growth factor-β-activated kinase 1 (TAK1) plays a pivotal role in innate and adaptive immunity. TAK1 is essential for the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB pathways downstream of diverse immune receptors, including toll-like receptors (TLRs). Upon stimulation with TLR ligands, TAK1 is activated via recruitment to the lysine 63-linked polyubiquitin chain through TAK1-binding protein 2 (TAB2) and TAB3. However, the physiological importance of TAB2 and TAB3 in macrophages is still controversial. A previous study has shown that mouse bone marrow-derived macrophages (BMDMs) isolated from mice double deficient for TAB2 and TAB3 produced tumor necrosis factor (TNF)-α and interleukin (IL)-6 to the similar levels as control wild-type BMDMs in response to TLR ligands such as lipopolysaccharide (LPS) or Pam3CSK4, indicating that TAB2 and TAB3 are dispensable for TLR signaling. In this study, we revisited the role of TAB2 and TAB3 using an improved mouse model. We observed a significant impairment in the production of pro-inflammatory cytokines and chemokine in LPS- or Pam3CSK4-treated BMDMs deficient for both TAB2 and TAB3. Double deficiency of TAB2 and TAB3 resulted in the decreased activation of NF-κB and MAPK pathways as well as the slight decrease in TAK1 activation in response to LPS or Pam3CSK4. Notably, the TLR-mediated expression of inhibitor of NF-κB (IκB)ζ was severely compromised at the protein and messenger RNA (mRNA) levels in the TAB2/TAB3 double-deficient BMDMs, thereby impeding IL-6 production. Our results suggest that TAB2 and TAB3 play a redundant and indispensable role in the TLR signaling pathway.

Functional Redundant Microbiome Enhanced Anaerobic Digestion Efficiency under Ammonium Inhibition Conditions.

Revealing the role of functional redundancy is of great importance considering its key role in maintaining the stability of microbial ecosystems in response to various disturbances. However, experimental evidence on this point is still lacking due to the difficulty in "manipulating" and depicting the degree of redundancy. In this study, manipulative experiments of functional redundancy were conducted by adopting the mixed inoculation strategy to evaluate its role in engineered anaerobic digestion systems under ammonium inhibition conditions. The results indicated that the functional redundancy gradient was successfully constructed and confirmed by evidence from pathway levels. All mixed inoculation groups exhibited higher methane production regardless of the ammonium level, indicating that functional redundancy is crucial in maintaining the system's efficiency. Further analysis of the metagenome-assembled genomes within different functional guilds revealed that the extent of redundancy decreased along the direction of the anaerobic digestion flow, and the role of functional redundancy appeared to be related to the stress level. The study also found that microbial diversity of key functional populations might play a more important role than their abundance on the system's performance under stress. The findings provide direct evidence and highlight the critical role of functional redundancy in enhancing the efficiency and stability of anaerobic digestion.