Biology subjects

Sen, S.

Publications and source records attributed to Sen, S..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

Whole-tissue deconvolution and scRNAseq analysis identify altered endometrial cellular compositions and functionality associated with endometriosis

The uterine lining (endometrium) exhibits a pro-inflammatory phenotype in women with endometriosis, resulting in pain, infertility, and poor pregnancy outcomes. The full complement of cell types contributing to this phenotype has yet to be identified, as most studies have focused on bulk tissue or select cell populations. Herein, through integrating whole-tissue deconvolution and single cell RNAseq, we comprehensively characterized immune and nonimmune cell types in endometrium of women with or without disease and their dynamic changes across the menstrual cycle. We designed metrics to evaluate specificity of deconvolution signatures that resulted in single cell identification of 13 novel signatures for immune cell subtypes in healthy endometrium. Guided by statistical metrics, we identified contributions of endometrial epithelial, endothelial, plasmacytoid dendritic cells, classical dendritic cells, monocytes, macrophages, and granulocytes to the endometrial pro-inflammatory phenotype, underscoring roles for nonimmune as well as immune cells to the dysfunctionality of this tissue. Teaser SentenceCell type deconvolution and single cell RNAseq analysis identify altered endometrial cellular compositions in women with endometriosis

bioinformatics↗

bioRxiv2021

The role of visual experience in brain inter-individual variability

Visual cortex organization is highly consistent across individuals. But to what degree does this consistency depend on life experience, in particular sensory experience? In this study, we asked whether visual cortex reorganization in congenital blindness results in connectivity patterns that are particularly variable across individuals, focusing on resting-state functional connectivity (RSFC) patterns from primary visual cortex. We show that the absence of shared visual experience results in more-variable RSFC patterns across blind individuals than sighted controls. Increased variability is specifically found in areas that show a group difference between the blind and sighted in their RSFC. These findings reveal a relationship between brain plasticity and individual variability in which reorganization manifests variably across individuals. We further investigated the different patterns of reorganization in the blind, showing that the connectivity to frontal regions, proposed to have a role in reorganization of the visual cortex of the blind towards higher cognitive roles, is highly variable. In a supplementary analysis, we link some of the variability in visual-to-frontal connectivity to another environmental factor - duration of formal education. Together, these findings show a role of sensory and socioeconomic experience in imposing consistency on brain organization. By revealing the idiosyncratic nature of neural reorganization, these findings highlight the importance of considering individual differences in fitting sensory aids and restoration approaches for vision loss. Significance statementThe typical visual system is highly consistent across individuals. What are the origins of this consistency? Comparing the consistency of visual cortex connectivity between people born blind and sighted people, we showed that blindness results in higher variability, suggesting a key impact of individual experience on brain organization. Further, connectivity patterns that changed following blindness were particularly variable, resulting in diverse patterns of brain reorganization. Individual differences in reorganization were also directly affected by non-visual experiences in the blind (years of formal education). Together, these findings show a role of sensory and socioeconomic experiences in creating individual differences in brain organization and endorse the use of individual profiles for rehabilitation and restoration of vision loss.

neuroscience↗

bioRxiv2021

Redox state and cellular uptake of copper is regulated by the N-terminus of human Copper Transporter-1

Copper(I) is essential for all life forms. Though Cu(II) is most abundant state in environment, its reduction to Cu(I) is prerequisite for bio-utilization, by a mechanism that is uncharacterized. We show that in human Copper Transporter-1, two amino-terminal methionine-histidine clusters and neighbouring aspartates distinctly binds Cu(II) and Cu(I) preceding its import. The endocytosis of hCTR1 from basolateral membrane of polarized epithelia to Common-Recycling-Endosomes is dependent on copper reduction and Cu(I) coordination by methionines. The transient binding of both Cu(II) and Cu(I) during the reduction process facilitated by aspartates acts as another crucial determinant of hCTR1 endocytosis. Mutating 7Met-Gly-Met9 and Asp13 abrogates copper uptake and endocytosis that is correctable by reduced and non-reoxidizable Cu(I). Histidines clusters are crucial for hCTR1 functioning at limiting copper. Finally, we show that two N-terminal His-Met-Asp clusters exhibit functional complementarity in regulating Cu(I)-induced hCTR1 endocytosis. We propose a mechanistic model where His-Met-Asp residues of amino-terminal hCTR1 coordinates copper and maintains its reduced state crucial for uptake.

cell biology↗

bioRxiv2021

Hypoxia induces early neurogenesis in human fetal neural stem cells by activating the WNT pathway.

Fetal neural stem cells (FNSCs) are present in the brain of human fetuses that differentiate into cells of neuronal and glial lineages. Difference in oxygen concentration between maternal and fetal circulation indicates that the developing fetus may be exposed to lower oxygen concentrations compared to adults. This physiological hypoxia may influence the growth and differentiation of the FNSCs. This study aimed to evaluate the effect of hypoxia on the differentiation potential of human FNSCs isolated from the sub-ventricular zone of aborted fetal brains (n=5). FNSCs were isolated, expanded, and characterized by Nestin and Sox2 expression, using immunocytochemistry and flowcytometry respectively. These FNSCs were exposed to 20% oxygen (normoxia) and 0.2% oxygen (hypoxia) concentrations for 48 hours, and hypoxia exposure (n=5) was evaluated by a panel of markers (CA9, PGK1 and VEGF). Whole human genome transcriptomic analyses (Genespring GX13) of FNSCs exposed to hypoxia (Agilent 4x44K human array slides), highlighted that genes associated with neurogenesis were getting enriched. The pathway analysis of these enriched genes (using Metacore) showed that WNT signaling played a role in determining the cell fate of FNSCs exposed to hypoxic environment. Microarray analyses was validated using neuronal and glial lineage commitment markers such as Ngn1, Ngn2, ASCL1, DCX, GFAP, Olig2 and Nkx2.2 using qPCR (n=9). This demonstrated upregulation of the neuronal commitment markers on hypoxia exposure, while no change was observed in astrocytic and oligodendrocyte lineage commitment markers. Increased expression of downstream targets of the WNT signaling pathway, TCF4 and ID2, by qPCR (n=9), indicated its involvement in mediating neuronal differentiation on exposure to hypoxia.

neuroscience↗

bioRxiv2021

Human fetal neural stem cell-derived astrocytes maintain glutamate transport after hypoxic injury in vitro

Astrocytes are the most abundant glial cells that play many critical roles in the central nervous system physiology including the uptake of excess glutamate from the synapse by Excitatory Amino Acid Transporters (EAATs). Among the EAATs, EAAT2 are predominantly functional, astrocyte-specific glutamate transporters in the forebrain. Hypoxic brain injury is a pathological phenomenon seen in various clinical conditions including stroke and neonatal hypoxic ischemic encephalopathy. Glutamate excitotoxicity is an important cause of neuronal cell death in disorders involving hypoxic brain injury. As findings from rodent models cannot always be reliably extrapolated to humans, we aimed to develop a homogenous population of primary human astrocytes to study the effect of hypoxic injury on astrocyte function, especially glutamate uptake. We successfully isolated, established and characterized cultures of human fetal neural stem cells (FNSCs) from aborted fetal brains. FNSCs were differentiated into astrocytes, and characterized by increased expression of the astrocyte markers, glial fibrillary acidic protein (GFAP), EAAT1 and EAAT2. A concomitant decrease in neural stem cell marker, Nestin, was observed. Differentiated astrocytes were exposed to various oxygen concentrations mimicking normoxia (20% and 6%), moderate and severe hypoxia (2% and 0.2% respectively). Interestingly, no change was observed in the expression of glutamate transporter, EAAT2 and glutamate uptake by astrocytes, even after exposure to hypoxia. Our novel model of human FNSC derived astrocytes exposed to hypoxic injury, establishes that astrocytes are able to maintain glutamate uptake even after exposure to severe hypoxia for 48 hours, and thus provides evidence for the neuroprotective role of astrocytes in hypoxic injury.

neuroscience↗

bioRxiv2021

The pitfalls of using Gaussian Process Regression for normative modeling

Normative modeling, a group of methods used to quantify an individuals deviation from some expected trajectory relative to observed variability around that trajectory, has been used to characterize subject heterogeneity. Gaussian Processes Regression includes an estimate of variable uncertainty across the input domain, which at face value makes it an attractive method to normalize the cohort heterogeneity where the deviation between predicted value and true observation is divided by the derived uncertainty directly from Gaussian Processes Regression. However, we show that the uncertainty directly from Gaussian Processes Regression is irrelevant to the cohort heterogeneity in general.

neuroscience↗

bioRxiv2021

IMMUND: an Early Stage Diagnostic and Therapeutic Frame for Neurodegenerative Diseases and Multiple Sclerosis based on Immunological Markers

For neurodegenerative diseases, the impact of immunological markers is one of the modern research areas. It has been observed that neuroinflammation increases the cellular precipitation of some of the key proteins associated with neurodegenerative diseases. Therefore, the possibility of functional loss can be enhanced due to neuroinflammation which leads to the initiation of any related diseases. In this regard, autoantibodies, which are known for their autophagy nature, can be considered as key elements for early diagnostic as well as early therapeutics. In this article, we have proposed a comprehensive framework to unveil the diagnostic as well as the therapeutic possibility of the autoantibodies which are largely associated with Mild-Moderate Alzheimers Disease, Early-Stage Parkinsons Disease, and Multiple Sclerosis. Here, we have introduced a new concept of average p-value where multiple p-values of an autoantibody in a singular disease have been considered as a multi-occurrence of that sample in cellular systems. Also, multiple proteins from a single protein family under a differentially expressed range have been prioritized. As a result, the top ten autoantibodies have been selected for further study and also considered as diagnostic markers. Interestingly, most of the selected autoantibodies are either cytokines or immunoglobulins. Subsequently, we have performed an evolutionary sequence-structure space study to identify the druggable structural facet for the selected autoantibodies. To make the therapeutic perspective more robust, we have introduced the concept of protein moonlighting. Hence, it provides more robustness in therapeutic identification. Finally, two autoantibodies i.e., Q9NYV4 and P01602 are identified as a novel marker.

neuroscience↗

bioRxiv2021

Identification of Differentially Expressed Genes and associated pathways common to Eyelid and Non-Ocular Basal Cell Carcinoma to understand the Molecular Biology of BCC

BackgroundEyelid BCC accounts for more than 90% of Eyelid malignant neoplasms. Various aberrant signalling pathways and genes in Non-Ocular BCC have been found whereas Eyelid bcc remains elusive. ObjectiveThis study aims to find the common DEGs of Eyelid and Non-Ocular BCC using bioinformatic analysis and text mining to gain more insights into the molecular aspects common to both BCC non-ocular and Eyelid BCC and to identify common potential prognostic markers. Material and methodThe Gene Expression profiles of Eyelid BCC (GSE103439) and Non-Ocular BCC (GSE53462) were obtained from the NCBI GEO database followed by identification of common DEGs. Protein-Protein interaction and Pathway Enrichment analysis of these screened genes was done using bioinformatic tools like STRING, Cytoscape and BiNGO, DAVID, KEGG respectively. ResultsA total of 181 genes were found common in both datasets. A PPI network was formed for the screened genes and 20 HUB genes were sorted which included CTNNB1, MAPK14, BTRC, EGFR, ADAM17. Pathway enrichment of HUB genes showed that they were dysregulated in carcinogenic and apoptotic pathways that seem to play a role in the progression of both the BCC. ConclusionThe result and findings of bioinformatic analysis highlighted the molecular pathways and genes enriched in both Eyelid BCC as well as Non-Ocular BCC. The identified pathways should be studied further to recognise common molecular events that would lead to the progression of BCC. This may provide a window to explore the prognostic and therapeutic strategies common to both BCC.

bioinformatics↗

bioRxiv2021

Genomic heterogeneity affects the response to Daylight Saving Time

Circadian rhythms drive the timing of many physiological events in the 24-hour day. When individuals undergo an abrupt external shift (e.g., change in work schedule or travel across multiple time zones), circadian rhythms become misaligned with the new time and may take several days to adjust. Chronic circadian misalignment, e.g., as a result of shift work, has been shown to lead to several physical and mental health problems. Despite the serious health implications of circadian misalignment, relatively little is known about how genetic variation affects an individuals ability to shift to abrupt external changes. Accordingly, we use the one-hour advance from the onset of daylight saving time (DST) as a natural experiment to comprehensively study how individual heterogeneity affects the shift of sleep-wake rhythms in response to an abrupt external time change. We find that individuals genetically predisposed to a morning tendency adjust to the advance in a few days, while genetically predisposed evening-inclined individuals have not shifted. Observing differential effects by genetic disposition after a one-hour advance underscores the importance of heterogeneity in adaptation to external schedule shifts, and these genetic differences may affect how individuals adjust to jet lag or shift work as well.

genomics↗

bioRxiv2021

Identification of APOE4 modulators, targeted therapeutic candidates in Coronary Artery Disease, using Molecular Docking studies

A significant genetic suspect for coronary artery disease is the pathological adaptation of apolipoprotein E4 (APOE4) through intramolecular interaction. With the prevailing evidences on APOE4 genotype and its prevalence in coronary artery disease, the present study has investigated the protein-ligand binding affinity and unveil the receptor binding abilities of different classes of ligands for APOE4 through molecular docking studies. Structural basis of APOE4 involvement in CAD suggests that the intramolecular domain interactions to be a suitable target for therapeutic intervention. Various classes of ligands including known drugs used in the treatment of CAD, fragment-based stabilizers and their similar structures and molecules with known bioactivity against APOE4 were screened for their binding affinity and further investigated for their interactions with APOE4. Computational studies show the benzyl amide derived structures to be useful candidates in modulation of APOE4. The dynamics of the binding analysis can be further achieved with an in-depth understanding of drug-receptor interactions performing molecular dynamic simulation studies.

bioinformatics↗

bioRxiv2021

Pre-metastatic niche drives breast cancer invasion by modulating MSC homing and CAF differentiation

The extent to which cancer-associated alterations in extracellular matrix stiffness influences the crosstalk between cancer cells and mesenchymal stem cells (MSCs) remains unclear. By analyzing multiple singlecell RNA sequencing datasets, we establish the existence of a cell sub-population co-expressing MSC and cancer associated fibroblast (CAF) markers in highly aggressive triple-negative breast cancers in primary tumor, secondary sites, and in circulatory tumor cell clusters. Using hydrogels of varying stiffness corresponding to different stages of cancer progression, we show that on pre-metastatic stroma mimetic 2 kPa gels, MDA-MB-231 breast cancer cell secreted conditioned media drives efficient MSC chemotaxis and induces stable CAF differentiation in a TGF{beta}/contractility-dependent manner. In addition to enhancing cancer cell proliferation, 2 kPa CAFs maximally boost local invasion and confer resistance to flow-induced shear stresses. Together, our results suggest that homing of MSCs at the pre-metastatic stage and their differentiation into CAFs actively drives breast cancer invasion and metastasis.

cancer biology↗

bioRxiv2021

-actinin-4 drives invasiveness by regulating myosin IIB expression and myosin IIA localization

The mechanisms by which the mechanoresponsive actin crosslinking protein -actinin-4 (ACTN4) regulates cell motility and invasiveness remains incompletely understood. Here we show that in addition to regulating protrusion dynamics and focal adhesion formation, ACTN4 transcriptionally regulates expression of non-muscle myosin IIB (NMM IIB), which is essential for mediating nuclear translocation during 3D invasion. We further demonstrate association between NMM IIA and ACTN4 at the cell front ensures retention of NMM IIA at the cell periphery. A protrusion-dependent model of confined migration recapitulating experimental observations predicts a dependence of protrusion forces on the degree of confinement and on the ratio of nucleus to matrix stiffness. Together, our results suggest that ACTN4 is a master regulator of cancer invasion that regulates invasiveness by controlling NMM IIB expression and NMM IIA localization.

cell biology↗

bioRxiv2021

The novel leishmanial Copper P-type ATPase plays a vital role in intracellular parasite survival

Copper is essential for all life forms; however in excess it is extremely toxic. Toxic properties of copper are utilized by hosts against various pathogenic invasions. Leishmania, in its both free-living and intracellular forms, was found to exhibit appreciable tolerance towards copper-stress. To determine the mechanism of copper-stress evasion employed by Leishmania we identified and characterized the hitherto unknown Copper-ATPase in Leishmania major and determined its role in parasites survival in host macrophage cells. L. major Cu-ATPase, LmATP7, exhibits high homology with its orthologues at multiple conserved motifs. In promastigotes, LmATP7 localized to the plasma membrane with a fraction in intracellular puncta. Upon copper treatment, LmATP7 expression increases few folds. LmATP7 is capable of complementing copper transport in Cu-ATPase-{Delta} yeast strain. Promastigotes overexpressing LmATP7 exhibits higher survival upon copper stress indicating efficacious copper export compared to wild type and heterozygous LmATP7 knock-out parasites. We explored macrophage-Leishmanial interaction with respect to copper stress subjected by the host upon parasite and the parasites reciprocating response thereon to evade the stress. The Copper-P-type-ATPases ATP7A/7B serve as major copper exporters in mammals that maintain cellular copper levels. We found that Leishmania infection triggers ATP7A upregulation in macrophages. Additionally, as part of host response, ATP7A traffics from trans-Golgi network and transports copper to the endosomal and endolysosomal compartments harbouring the Leishmania amastigotes. Finally, we show LmATP7 overexpression in this parasite increased amastigote survivability within infected macrophages whereas knocking it down reduces that drastically, establishing its role in combating host-induced copper stress.

cell biology↗

bioRxiv2020

Bio-Physical Modeling of Galvanic Human Body Communication in Electro-Quasistatic Regime

Human Body Communication (HBC) is an alternative to radio wave-based Wireless Body Area Network (WBAN) because of its low-loss, wide bandwidth leading to enhanced energy efficiency. HBC also shows better performance in terms of physical security as most of the signal is confined within the body. To obtain optimum performance and usability, modeling of the body channel plays a vital role. Out of two HBC modalities, Galvanic HBC has the promise to provide lower loss compare to Capacitive HBC for shorter channel length. In this paper, we present the first lumped element based detailed model of Galvanic HBC channel which is used to explain the dependency of channel loss on the material property of skin, fat and muscle tissue layer along with electrode size, electrode separation, geometrical position of the electrodes and return path capacitance. The model considers the impedance of skin and muscle tissue layers and the effect of various coupling capacitances between the body and Tx/Rx electrodes to the Earth-ground. A 2D planner structure is simulated in HFSS to prove the validity of the proposed model. The effect of symmetry and asymmetry at the transmitter and receiver end are also explained using the model. The experimental results show that, due to the mismatch at the transmitter and receiver side, the loss increases gradually with channel length and saturates to a finite value as channel length becomes significantly longer compare to the transmitting or receiving electrode pair separation.

bioengineering↗

bioRxiv2020

Identification of Fibronectin 1 as a candidate genetic modifier in a Col4a mutant mouse model of Gould syndrome

Collagen type IV alpha 1 and alpha 2 (COL4A1 and COL4A2) are major components of almost all basement membranes. COL4A1 and COL4A2 mutations cause a multisystem disorder called Gould syndrome which can affect any organ but typically involves the cerebral vasculature, eyes, kidneys and skeletal muscles. The manifestations of Gould syndrome are highly variable and animal studies suggest that allelic heterogeneity and genetic context contribute to the clinical variability. We previously characterized a mouse model of Gould syndrome caused by a Col4a1 mutation in which the severities of ocular anterior segment dysgenesis (ASD), myopathy, and intracerebral hemorrhage (ICH) were dependent on genetic background. Here, we performed a genetic modifier screen to provide insight into the mechanisms contributing to Gould syndrome pathogenesis and identified a single locus (modifier of Gould syndrome 1; MoGS1) on Chromosome 1 that suppressed ASD. A separate screen showed that the same locus ameliorated myopathy. Interestingly, MoGS1 had no effect on ICH, suggesting that this phenotype may be mechanistically distinct. We refined the MoGS1 locus to a 4.3 Mb interval containing 18 protein coding genes, including Fn1 which encodes the extracellular matrix component fibronectin 1. Molecular analysis showed that the MoGS1 locus increased Fn1 expression raising the possibility that suppression is achieved through a compensatory extracellular mechanism. Furthermore, we show evidence of increased integrin linked kinase levels and focal adhesion kinase phosphorylation in Col4a1 mutant mice that is partially restored by the MoGS1 locus implicating the involvement of integrin signaling. Taken together, our results suggest that tissue-specific mechanistic heterogeneity contributes to the variable expressivity of Gould syndrome and that perturbations in integrin signaling may play a role in ocular and muscular manifestations.

genetics↗

bioRxiv2020

In-The-Wild Interference Characterization andModelling for Electro-Quasistatic-HBC withMiniaturized Wearables

The emergence of Human Body Communication (HBC) as an alternative to wireless body area networks (WBAN) has led to the development of small sized, energy efficient and more secure wearable and implantable devices forming a network in and around the body. Previous studies claim that though HBC is comparatively more secure than WBAN, nevertheless, the electromagnetic (EM) radiative nature of HBC in >10MHz region makes the information susceptible to eavesdropping. Furthermore, interferences may be picked up by the body due to the human body antenna effect in the 40-400MHz range. Alternatively, electro-quasistatic (EQS) mode of HBC forms an attractive way for covert data transmission in the sub 10MHz region by allowing the signal to be contained within the body. However, there is a gap in the knowledge about the mechanism and sources of interference in this region (crucial in allowing for proper choice of data transmission band). In this paper, the interference coupling modality in the EQS region is explained along with its possible sources. Interferences seen by the wearable in the actual scenario is a non-trivial problem and a suitable measurement EQS HBC setup is designed to recreate it by employing a wearable sized measurement setup having a small ground plane. For the first time, a human biophysical interference pickup model is proposed and interference measurement results using a wearable device are presented up to 250kHz in different environmental settings.

bioengineering↗

bioRxiv2020

Advanced Biophysical Model to Capture Channel Variability for EQS Capacitive HBC

Human Body Communication (HBC) has come up as a promising alternative to traditional radio frequency (RF) Wireless Body Area Network (WBAN) technologies. This is essentially due to HBC providing a broadband communication channel with enhanced signal security in the physical layer due to lower radiation from the human body as compared to its RF counterparts. An in-depth understanding of the mechanism for the channel loss variability and associated biophysical model needs to be developed before EQS-HBC can be used more frequently in WBAN consumer and medical applications. Biophysical models characterizing the human body as a communication channel didnt exist in literature for a long time. Recent developments have shown models that capture the channel response for fixed transmitter and receiver positions on the human body. These biophysical models do not capture the variability in the HBC channel for varying positions of the devices with respect to the human body. In this study, we provide a detailed analysis of the change in path loss in a capacitive-HBC channel in the electroquasistatic (EQS) domain. Causes of channel loss variability namely: inter-device coupling and effects of fringe fields due to bodys shadowing effects are investigated. FEM based simulation results are used to analyze the channel response of human body for different positions and sizes of the device which are further verified using measurement results to validate the developed biophysical model. Using the bio-physical model, we develop a closed form equation for the path loss in a capacitive HBC channel which is then analyzed as a function of the geometric properties of the device and the position with respect to the human body which will help pave the path towards future EQS-HBC WBAN design.

bioengineering↗

bioRxiv2020

Understanding The Role of Magnetic and Magneto-Quasistatic Fields in Human Body Communication

With the advent of wearable technologies, Human Body Communication (HBC) has emerged as a physically secure and power-efficient alternative to the otherwise ubiquitous Wireless Body Area Network (WBAN). Whereas the most investigated nodes of HBC have been Electric and Electro-quasistatic (EQS) Capacitive and Galvanic, recently Magnetic HBC (M-HBC) has been proposed as a viable alternative. Previous works have investigated M-HBC through an application point of view, without developing a fundamental working principle for the same. In this paper, for the first time, a ground up analysis has been performed to study the possible effects and contributions of the human body channel in M-HBC over a broad frequency range (1kHz to 10 GHz), by detailed electromagnetic simulations and supporting experiments. The results show that while M-HBC can be successfully operated as a body area network, the human body itself plays a minimal or negligible role in its functionality. For frequencies less than[~] 30 Hz, in the domain of operation of Magneto-quasistatic (MQS) HBC, the human body is transparent to the quasistatic magnetic field. Conversely for higher frequencies, the conductive nature of human tissues end up attenuating Magnetic HBC fields due to Eddy currents induced in body tissues, eliminating the possibility of the body to support efficient waveguide modes. With this better understanding at hand, different modes of operations of MQS HBC have been outlined for both high impedance capacitive and 50{Omega} termination cases, and their performances have been compared with EQS HBC for similar sized devices, over varying distance between TX and RX. The resulting report presents the first fundamental understanding towards M-HBC operation and its contrast with EQS HBC, aiding HBC device designers to make educated design decisions, depending on mode of applications.

bioengineering↗