Biology subjects

Krishnamurthy, S.

Publications and source records attributed to Krishnamurthy, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Tumor emboli-associated adaptive stress response signatures identify aggressive disease features in inflammatory breast cancer

Inflammatory breast cancer (IBC) is an aggressive breast cancer subtype characterized by tumor emboli, lymphovascular invasion (LVI), and early dissemination. Herein, we establish adaptive stress response (ASR) as a biologic feature linking stress adaptation to tumor emboli survival, lymphatic dissemination, therapeutic response, and disparities. Using a previously defined 226 ASR-related genes, complementary preclinical models of tumor emboli and lymphatic circulating cell clusters, and independent patient cohorts, we identified ASR genes enriched for XIAP-NF{kappa}B, oxidative stress response, inflammatory, and immune pathways. CXCL8 emerged as one of the most highly upregulated transcripts in tumor emboli and was shared across both models; however, CXCL8, IL6, and PTGS2 were downregulated in lymphatic circulating cell clusters and LVI-positive triple-negative IBC patients, suggesting dynamic remodeling of inflammatory signaling during dissemination. CYP4B1 was associated with ER status, LVI, and therapeutic response across multiple cohorts, implicating metabolic stress adaptation in dissemination. IL6 and PTGS2 were elevated in self-reported Black patients with triple-negative IBC compared to White patients. Pharmacologic inhibition of XIAP-NF{kappa}B and oxidative stress pathways suppressed tumor emboli formation. Collectively, these findings identify ASR signaling as a framework linking tumor emboli survival, dissemination, and therapeutic vulnerability in IBC.

cancer biology↗

bioRxiv2026

Memory reactivation during sleep promotes structure abstraction

We readily detect structure in our environments, which in turn guides future learning. Disentangling this structure from the superficial features of a specific learning environment provides an especially strong basis for future generalization, but it remains unclear when and how this kind of abstraction occurs. Memory reactivation during sleep has been hypothesized to support such abstraction, but this has yet to be directly tested. Here we examined this hypothesis by teaching participants novel categories in which patterns of feature covariation were governed by different graph structures. Participants then learned a new category, defined by entirely different features, whose structure was either congruent or incongruent with a previously learned category. If structural knowledge is abstracted away from superficial features, it should facilitate transfer when structures are congruent. In Experiment 1, when two categories were learned in immediate succession, participants showed no transfer benefit, suggesting that structure understanding remained tied to the original features. In Experiment 2, we tested whether offline processing promotes abstraction. Participants either remained awake between learning phases spaced 3 hours apart, or took a nap during which a previously learned category was reactivated using targeted memory reactivation (TMR). Transfer benefits emerged only when the reactivated and target categories shared the same structure, and these benefits increased with the number of cues presented during slow-wave sleep. These findings provide the first direct evidence that memory reactivation during sleep promotes the abstraction of structure, enabling knowledge to transfer across learning episodes with no overlap in features.

neuroscience↗

bioRxiv2026

A framework for testing structural hypotheses of protein dynamics against experimental HDX-MS data

Protein dynamics determine biological function, yet extracting structural ensembles from Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) remains a challenging inverse problem. Current ensemble-fitting approaches often achieve good agreement with uptake curves but lack rigorous validation and uncertainty quantification, limiting structural confidence. We propose ValDX, a validation framework for quantitative integration of HDX-MS data with structural ensembles. ValDX combines overlap-aware data splitting, replicate-based uncertainty estimation, and uptake-independent "Work Done" metrics that quantify how much an ensemble must be modified to match experiment. Across 22 ensembles spanning six proteins (58-474 residues), we show that conventional error metrics fail to distinguish structurally representative ensembles from incorrect ones, whereas Work Done metrics robustly discriminate global and local conformational quality. We further demonstrate that clustering yields compact, interpretable ensembles with minimal loss of accuracy, and that staged optimisation enables reliable fitting of both ensemble weights and forward-model parameters without requiring a reference structure. Together, this framework establishes HDX-MS ensemble integration as a quantitative structural hypothesis-testing problem, enabling inference of protein dynamics from HDX-MS data.

biophysics↗

bioRxiv2025

Thermodynamic principles govern evolutionary tradeoffs by regulating allostery

Allostery governs biological activities by signaling environmental cues at distal sites, however, the molecular basis for the orchestration and evolution of "the second secret of life" is evanescent. Seminal work sheds light embracing structure- and function-centric approaches, overlooking the widely accepted ensemble allosteric model based on a proteins free-energy landscape and the thermodynamic nature of allostery. Here, we unraveled allosteric regulation and its evolvability by examining energetic funnels of proteins harboring a highly evolvable ancient scaffold. We uncover intricate allosteric connectivities and their coordinated cross-talk to enable the statistical thermodynamic coupling. We decipher universal molecular determinants for the emergence of functions and environmental adaptability. Our integrative biophysical/statistical/evolutionary analysis ties the evolutionary forces via thermodynamic principles and decrypts how tradeoffs are settled at the molecular level. One-Sentence SummaryWe reveal how physical and evolutionary laws shaped the biophysical properties of proteins

biophysics↗

bioRxiv2025

Soluble E-cadherin Drives Brain Metastasis in Inflammatory Breast Cancer

The brain is a common site of relapse in inflammatory breast cancer (IBC), an E-cadherin positive, aggressive form of breast cancer. We found that elevated serum levels of soluble E-cadherin (sEcad), an 80-kDa fragment of E-cadherin, in patients with metastatic IBC correlated with poorer outcomes and increased rates of brain metastases. In our effort to understand the underlying mechanism, we discovered that sEcad binds to XIAP, an inhibitor of cell death, activating the pro-survival NF-k{beta} signaling in tumor cells. We also discovered that sEcad affects the tumor cell microenvironment by enhancing cancer cell adhesion to endothelial cells and inducing reactive astrocytosis in the brain. In addition, we found that sEcad-mediated reactive astrocytosis relies on the CXCL1/CXCL8-CXCR2 axis and treatment with a brain-permeable CXCR2 antagonist reduced brain metastatic burden and prolonged survival. These findings implicate sEcad in brain metastasis and provide new insights into potential therapeutic targets for IBC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/660428v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@10a7c47org.highwire.dtl.DTLVardef@1653278org.highwire.dtl.DTLVardef@d4787corg.highwire.dtl.DTLVardef@132489b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIHigh serum sEcad levels correlate clinically with poor survival outcomes and development of brain metastasis C_LIO_LIsEcad drives IBC brain metastasis growth in mouse models C_LIO_LIsEcad binds XIAP to activate NFkB and promote anoikis resistance and invasion of IBC cells C_LIO_LIsEcad activates reactive astrocytes and induces CXCR2 expression on tumor cells in vitro and in vivo C_LIO_LICXCR2-IN-1, a brain-permeable CXCR2 antagonist, reduces metastasis and improves survival in IBC brain metastasis models C_LI

cancer biology↗

bioRxiv2025

The RNA-binding protein RSRC2 promotes mitotic fidelity by interacting with the lncRNA C1QTNF1-AS1

Mitotic fidelity depends on proper chromosome alignment at the spindle equator, a process known as chromosome congression, driven by well-established protein networks. Whereas RNA-binding proteins and noncoding RNAs have been implicated in cell division, their interplay during this process remains unknown. Here, we discover that RSRC2, an arginine/serine-rich RNA-binding protein, plays an essential role in cell division by interacting with the long non-coding RNA C1QTNF1-AS1. The loss of either RSRC2 or C1QTNF1-AS1 results in defects in chromosome congression and mitotic progression. We show that RSRC2 interacts with distinct sets of proteins involved in splicing and centrosome biogenesis, contributing to the fidelity of cell division through two different mechanisms: one linked to the splicing of mitotic regulators and the other by localising to mitotic centrosomes for which the interaction with the C1QTNF1-AS1 RNA is required. Our study uncovers RSRC2 as a new regulator of cell division and illustrates how RNA-protein complexes promote error-free mitosis.

cell biology↗

bioRxiv2023

Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model". However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFN{gamma}-activated human fibroblasts, likely due to ITCHs role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses. IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasites replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFN{gamma}. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

immunology↗

bioRxiv2023

TREX reveals proteins that bind to specific RNA regions in living cells

Different regions of RNA molecules can often engage in specific interactions with distinct RNA-binding proteins (RBPs), giving rise to diverse modalities of RNA regulation and function. However, there are currently no methods for unbiased identification of RBPs that interact with specific RNA regions in living cells under endogenous settings. Here, we introduce TREX (Targeted RNase H-mediated extraction of crosslinked RBPs), a highly sensitive approach for identifying proteins that directly bind to specific RNA regions in living cells. We demonstrate that TREX outperforms existing methods in identifying known interactors of U1 snRNA, and reveals endogenous region-specific interactors of NORAD lncRNA. Using TREX, we generated a comprehensive region-by-region interactome for 45S rRNA, uncovering both established and novel interactions that regulate ribosome biogenesis. With its applicability to any RNA in any cell-type, TREX is the first RNA-centric tool for unbiased positional mapping of endogenous RNA-protein interactions in living cells.

cell biology↗

bioRxiv2022

Evolutionary adaptation of the folding pathway for secretability

Secretory preproteins of the Sec pathway bear signal peptides and are targeted post-translationally to cross the plasma membrane or ER through translocases. After translocation and signal peptide cleavage, mature domains fold to native states in the bacterial periplasm or after further trafficking. During cytoplasmic transit, mature domains must remain non-folded for translocase recognition and translocation. Here, we sought the structural basis for the delayed folding mechanism of mature domains and how this is regulated by signal peptides. To address this, we compared how evolution diversified a periplasmic peptidyl-prolyl isomerase PpiA mature domain from its structural twin cytoplasmic PpiB. Using global and local hydrogen deuterium exchange mass spectrometry we showed that PpiA is a slower folder. We defined at near-residue resolution hierarchical folding initiated by similar foldons in the twins, that displayed different order and rates. Folding is delayed in PpiA by less hydrophobic/bulky native contacts, frustrated residues and a critical {beta} -turn in the early folding region and by signal peptide-driven disorder, which disrupts foldon hierarchy. When selected PpiA residues and its signal peptide were grafted onto PpiB they converted it into a slow folder with enhanced in vivo secretion. These data reveal the structural basis of non-folding in a secretory protein, that allows its trafficking.

biochemistry↗

bioRxiv2022

Stabilization of E-cadherin adhesions by COX-2/GSK3β signaling is a targetable pathway in metastatic breast cancer

Metastatic progression and treatment-resistance of breast cancer has been associated with epithelial-mesenchymal-transition including downregulation of E-cadherin (CDH1) expression, which can be initiated by inflammatory mediators such as COX-2. Recently, E-cadherin-mediated, cluster-based metastasis and treatment resistance has become more appreciated, though the mechanisms that maintain E-cadherin expression in this context are unknown. Through studies of inflammatory breast cancer and an in vitro tumor cell emboli culture paradigm, we identified a role for COX-2, a target gene of C/EBP{delta}, or its metabolite PGE2 in promoting protein stability of E-cadherin, {beta}-catenin and p120 catenin through inhibition of GSK3{beta}, without affecting CDH1 mRNA. The COX-2 inhibitor celecoxib downregulated E-cadherin complex proteins and caused cell death. Co-expression of E-cadherin and COX-2 was seen in breast cancer patients with poor outcome and, along with inhibitory GSK3{beta} phosphorylation, in patient-derived xenografts of triple negative breast cancer. Celecoxib alone decreased E-cadherin protein expression within xenograft tumors, reduced circulating tumor cells and clusters, and in combination with paclitaxel attenuated or regressed lung metastases. This study uncovered a mechanism by which metastatic breast cancer cells can maintain E-cadherin-mediated cell-cell adhesions and cell survival, suggesting that patients with COX-2+/E-cadherin+ breast cancer may benefit from targeting of the PGE2 signaling pathway.

cancer biology↗

bioRxiv2021

Met/HGFR triggers detrimental reactive microglia in TBI

The complexity of the signaling events, cellular responses unfolding in neuronal, glial and immune cells upon Traumatic brain injury (TBI) constitutes an obstacle in elucidating pathophysiological links and targets for intervention. We used array phosphoproteomics in a murine mild blunt TBI to reconstruct the temporal dynamics of tyrosine-kinase signaling in TBI and then to scrutinize the large-scale effects of the perturbation of cMet/HGFR, VEGFR1 and Btk signaling by small molecules. cMet/HGFR emerged as a selective modifier of the early microglial response, and cMet/HGFR blockade prevented the induction of microglial inflammatory mediators, of reactive microglia morphology and of TBI-associated responses in neurons, vessels and brain extracellular matrix. Acute or prolonged cMet/HGFR inhibition ameliorated neuronal survival and motor recovery. Early elevation of HGF itself in the CSF of TBI patients suggest that this mechanism has translational value in human subjects. Our findings identify cMet/HGFR as a modulator of early neuroinflammation in TBI with translational potential and indicate several RTK families as possible additional targets for TBI treatment. SummaryControlling neuroinflammation in neurotrauma is an important but unachieved goal. This study exploits a moderate TBI model and array-based proteomics to identify cMet as a new inducer of reactive microglia. A small-molecule inhibitor of cMet contains microglial reactivity, reduces neuronal and vascular alterations, limits behavioural disturbances and accelerates recovery. HighlightsO_LIMet is activated in microglia upon TBI and drives microglial reactivity. C_LIO_LIA Met inhibitor reduces motor dysfunction upon TBI and promotes recovery. C_LIO_LIBlockade of MET prevents the appearance of a reactive microglia. C_LIO_LIThe cMET inhibitor reduces the sub-acute neuronal loss after TBI. C_LI

neuroscience↗

bioRxiv2021

Spatial charting of single cell transcriptomes in tissues

Single cell RNA sequencing (scRNA-seq) methods can profile the transcriptomes of single cells but cannot preserve spatial information. Conversely, spatial transcriptomics (ST) assays can profile spatial regions in tissue sections, but do not have single cell genomic resolution. Here, we developed a computational approach called CellTrek that combines these two datasets to achieve single cell spatial mapping. We benchmarked CellTrek using a simulation study and two in situ datasets. We then applied CellTrek to reconstruct cellular spatial structures in existing datasets from normal mouse brain and kidney tissues. We also performed scRNA-seq and ST experiments on two ductal carcinoma in situ (DCIS) tissues and applied CellTrek to identify tumor subclones that were restricted to different ducts, and specific T cell states adjacent to the tumor areas. Our data shows that CellTrek can accurately map single cells in diverse tissue types to resolve their spatial organization.

genomics↗

bioRxiv2021

Preproteins couple the intrinsic dynamics of SecA to its ATPase cycle to translocate via a catch and release mechanism

Protein machines undergo conformational motions to interact with and manipulate polymeric substrates. The Sec translocase promiscuously recognizes, becomes activated and secretes >500 non-folded preprotein clients across bacterial cytoplasmic membranes. Here, we reveal that the intrinsic dynamics of the translocase ATPase, SecA, and of preproteins combine to achieve translocation. SecA possesses an intrinsically dynamic preprotein clamp attached to an equally dynamic ATPase motor. Alternating motor conformations are finely controlled by the {gamma}-phosphate of ATP, while ADP causes motor stalling, independently of clamp motions. Functional preproteins physically bridge these independent dynamics. Their signal peptide promotes clamp closing; their mature domain overcomes the rate limiting ADP release. While repeated ATP cycles shift the motor between unique states, multiple conformationally frustrated prongs in the clamp repeatedly catch and release trapped preprotein segments until translocation completion. This universal mechanism allows any preprotein to promiscuously recognize the translocase, usurp its intrinsic dynamics and become secreted.

biochemistry↗

bioRxiv2021

Lipocalin 2 promotes inflammatory breast cancer tumorigenesis and skin invasion

Inflammatory breast cancer (IBC) is an aggressive form of primary breast cancer characterized by rapid onset and high risk of metastasis and poor clinical outcomes. The biological basis for the aggressiveness of IBC is still not well understood and no IBC-specific targeted therapies exist. In this study we report that lipocalin 2 (LCN2), a small secreted glycoprotein belonging to the lipocalin superfamily, is expressed at significantly higher levels in IBC versus non-IBC tumors, independently of molecular subtype. LCN2 levels were also significantly higher in IBC cell lines and in their culture media than in non-IBC cell lines. High expression was associated with poor-prognosis features and shorter overall survival in IBC patients. Depletion of LCN2 in IBC cell lines reduced proliferation, colony formation, migration, and cancer stem cell populations in vitro, and inhibited tumor growth, skin invasion, and brain metastasis in mouse models of IBC. Analysis of our proteomics data showed reduced expression of proteins involved in cell cycle and DNA repair in LCN2-silenced IBC cells. Our findings support that LCN2 promotes IBC tumor aggressiveness and offer a new potential therapeutic target for IBC.

cancer biology↗

bioRxiv2020

PyHDX: Derivation and visualization of protection factors from Hydrogen-Deuterium Exchange Mass Spectrometry at near residue resolution

Hydrogen Deuterium Exchange Mass Spectrometry is a powerful monitor of protein intrinsic dynamics, yet the interpretation, visualization and cross-comparison of HDX-MS datasets is challenging. Here we present PyHDX, an open-source python package and web server, that batch-extracts the universal quantity Gibbs free energy at residue level over multiple protein conditions and homologues. {Delta}G values relate to protein normal modes and together provide a universal measure of protein flexibility. AvailabilityPyHDX source code is released under the MIT license and can be accessed on GitHub.

bioinformatics↗

bioRxiv2020

Decorin, a novel negative modulator of E-cadherin in inflammatory breast cancer

ABSTRACTInflammatory breast cancer (IBC) is a clinically distinct and highly aggressive form of breast cancer with rapid onset and a strong propensity to metastasize. The molecular mechanisms underlying the aggressiveness and metastatic propensity of IBC are largely unknown. Herein, we report that decorin (DCN), a small leucine-rich extracellular matrix proteoglycan, is downregulated in tumors from patients with IBC. Overexpression of DCN in IBC cells markedly decreased migration, invasion, and cancer stem cells in vitro and inhibited IBC tumor growth and metastasis in vivo. Mechanistically, DCN functioned as a suppressor of invasion and tumor growth in IBC by destabilizing E-cadherin and inhibiting EGFR/ERK signaling. DCN physically binds E-cadherin in IBC cells and accelerates its degradation through an autophagy-linked lysosomal pathway. We established that DCN inhibits tumorigenesis and metastasis in IBC cells by negatively regulating the E-cadherin/EGFR/ERK axis. Our findings offer a potential therapeutic strategy for IBC, and provide a novel mechanism for IBC pathobiology.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2019

A genome-wide loss-of-function screen identifies Toxoplasma gondii genes that determine fitness in interferon gamma-activated murine macrophages

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFN{gamma}) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identified [~]130 Toxoplasma genes that determine parasite fitness in naive macrophages and [~]466 genes that determine fitness in IFN{gamma}-stimulated murine macrophages, seven of which we investigated and confirmed. We show that one of these genes encodes dense granule protein GRA45, which contains a putative chaperone-like domain, and which we show is critical in preventing other GRA effectors from aggregating. Parasites lacking GRA45 mislocalize GRA effectors upon secretion, are more susceptible to IFN{gamma}-mediated growth inhibition, and have reduced virulence in mice. Our results provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFN{gamma}-stimulated macrophages. IMPORTANCEThe intracellular parasite Toxoplasma gondii can cause congenital infections and severe disease in immunocompromised patients. The cytokine IFN{gamma} can block parasite replication by upregulating a variety of toxoplasmacidal mechanisms in many cells, including macrophages. Toxoplasma preferentially infects macrophages. Therefore, the parasite has evolved mechanisms to survive in these cells in the presence of IFN{gamma}. Here, we generated pools of Toxoplasma mutants for every gene and determined which mutants were specifically depleted in IFN{gamma}-stimulated macrophages, thus identifying parasite genes determining fitness in these cells. We show that one of these genes encodes for a dense granule protein (GRA45) that plays an important role in preventing other GRA effectors from aggregating. Parasites without GRA45 mislocalize GRA effectors upon secretion, have enhanced susceptibility to IFN{gamma}-mediated growth inhibition, and are avirulent in mice. Thus, our screen provides a resource to the Toxoplasma community to determine the function of Toxoplasma genes that affect its fitness in IFN{gamma}-stimulated macrophages.

microbiology↗

bioRxiv2019

Calcium Sensing Receptor Common Variants Influence the Effects of Serum Calcium on Coronary Artery Disease Risks

RationaleThe calcium-sensing receptor (CaSR) regulates serum calcium concentrations and common single nucleotide polymorphisms (SNPs) in a carboxyl terminal tri-locus haplotype block contribute to serum calcium variance in the general population. Altered serum calcium concentrations are associated with coronary artery disease (CAD), but direct role for CaSR in CAD remains to be determined.\n\nMethodsWe evaluated the associations of serum calcium and common CASR SNPs or the tri-locus haplotype block with major diseases including CAD in 51,289 patients from the DiscovEHR cohort derived from a single US health care system.\n\nResultsSerum calcium concentrations were positively associated with the risk of CAD, and this risk was modified by common CASR SNPs. The Ala986Ser SNP was positively associated with hypercalcemia. Carriers of Ala986Ser had a significantly increased CAD risk whereas Arg990Gly carriers had a reduced risk relative to the reference SNP, for those with albumin-corrected serum calcium from 8.5-9.5 mg/dL. In the context of the tri-locus haplotype, the reduced CAD risk conferred by Arg990Gly remained significant. Analysis of the association of common CASR SNPs with CAD risk factors showed Arg990Gly was negatively associated with the CAD risk factor of chronic kidney disease, but independent of alterations in lipids, hemoglobin A1c, or blood pressure.\n\nConclusionsThis study compares the common approach of single SNP analysis with the impact of a common variant haplotype block and refocuses attention on the CaSR Arg990Gly SNP which reduces the risk of CAD over a specific range of median albumin-corrected calcium concentrations.\n\nPrecisClinical data and whole exome sequences from a cohort of 51,289 individuals (DiscovEHR) were used to assess the independent contributions of serum Ca2+ and CASR common variants to cardiovascular diseases including CAD.

genetics↗