Diabetic retinopathy remains as the leading cause of preventable blindness in working-aged people. The pathophysiology of this sight-threatening disease is complex and involves intricate interactions among metabolic, hemodynamic and epigenetic pathways, leading to molecular, structural, functional and genomic abnormalities in retinal vascular and nonvascular cells. Diabetes also results in differential expressions of several noncoding RNAs, including micro RNAs (miRNAs) and long noncoding RNAs (LncRNAs). Compared to about 2000 miRNA identified in human genome thus far, more than 30,000 LncRNA transcripts have been already identified, but the function and mechanism of action of most of the LncRNAs is still not fully characterized, and there remains a possibility that some LncRNAs could have diverse functions under different contexts. LncRNAs are mainly noncoding, but they have many regulatory functions, and regulate gene expression by interacting with DNA, RNA and protein. Aberrant expressions of several LncRNAs including MALAT1, MEG3, HOTAIR, MIAT1 and H19, is associated with metabolic abnormalities implicated in the pathogenesis of diabetic retinopathy. LncRNAs are also released into circulation and show high organ and cell specificity, and greater disease-associated differences compared to disease-associated mRNAs. Furthermore, LncRNAs maintain stable expression in the plasma and can be isolated from total RNA present in biological samples including blood, which makes them promising and reliable candidates for diagnostic or prognostic markers and therapeutic targets for various diseases. With continued improvement in innovative RNA modifications and delivery modalities, use of LncRNAs as possible biomarkers, and of LncRNA-based therapeutics, for diabetic retinopathy appears promising.

Malaria remains one of the leading causes of morbidity and mortality worldwide, mainly because of the emergence of drug resistance against current antimalarials. The Plasmodium falciparum food vacuole (FV) proteins, P. falciparum chloroquine (CQ) resistance transporter (PfCRT), PfMDR1 and the cytosolic protein PfKelch13 have been linked to CQ and artemisinin resistance, respectively. Here, we aimed to identify the associations of these resistance markers with mutations in other FV transporters in several field isolates. In this study, we isolated intact P. falciparum FVs and carried out detailed proteome analysis to identify new FV transporters. Furthermore, we carried out co-existing mutational analysis for these transport proteins identified in the FV-enriched fraction with known PfKelch13 and PfCRT polymorphisms via single-nucleotide polymorphism (SNP) data from the Pf3K and MalariaGEN databases. Proteome analysis identified 16 transporter proteins in Plasmodium FVs. Comparative amino acid analysis of these transporter proteins revealed a coassociation of mutations in several transport proteins identified in the FV-enriched fraction with mutations in the PfKelch13, PfCRT, and PfMDR1 proteins. SNP data analysis of the Pf3K and MalariaGEN databases for 2,517 samples revealed the coassociation of six mutations in four transporter genes, PfCRT, PfNT1, PfCTR2, and PfMDR2, with the PfKelch13 polymorphisms (P < 0.0001), suggesting the contribution of additional parasite transporters to the evolution of CQ and artemisinin resistance. Furthermore, functional complementation with the wild-type PfNT1 and PfMFR5 proteins and their mutant forms (PfNT1-F394L, PfMFR5-S278T, and PfMFR5-Y570F) in Saccharomyces cerevisiae resulted in resistance to mutant phenotypes in the presence of dihydroartemisinin, suggesting a possible role of these mutations in the acquisition of drug resistance. Together, the genome sequence data from field isolates and yeast complementation analysis of the mutant phenotypes identified novel loci related to PfKelch13-mediated antimalarial resistance and revealed unexplored contributions of transporters to artemisinin resistance.

Lysine acetylation is emerging as a key player in cellular regulation across species by controlling the fate of metabolic proteins as well as modulating gene expression via histone modification. Phosphoglycerate mutase, a conserved enzyme of the sole energy‐yielding pathway of glycolysis in the human malaria parasite Plasmodium falciparum , is indispensable for its growth. Here, we demonstrate that P. falciparum phosphoglycerate mutase PfPGM1 (phosphoglycerate mutase) is regulated via lysine acetylation. In mammalian cells, acetylation of phosphoglycerate mutase modulates its catalytic activity, although the acetyl transferase enzyme remains elusive. However, the parasites exhibit a unique way of regulating the fate of PfPGM1 via acetylation that modulates its stability, thus providing an increased protein pool for the rapid growth and proliferation of the parasites. We show that K100, a critical residue for PfPGM1 catalytic activity, is acetylated by the essential histone acetyltransferase PfGCN5. Downregulation of PfGCN5 through a knockdown approach in the parasites along with cycloheximide treatment indeed leads to a reduction of PfPGM1 protein. Additionally, PfGCN5 occupies the promoter of PfPGM1 in a stage‐specific manner, and downregulation of PfGCN5 protein leads to a reduced transcript level of PfPGM1. Collectively, our data highlight a dual regulation of PfPGM1 by PfGCN5 through acetylation of the protein as well as regulation of the transcription of the gene. Such dual control is not only rare but showcases the importance of the above two proteins and their potential as excellent targets against malaria.

Diabetic retinopathy (DR), a leading cause of vision impairment and blindness, is characterized by abnormal retinal vascular changes due to chronic hyperglycemia. TheTie-1signaling pathway, essential for vascular growth and remodeling, has emerged as a key therapeutic target, though its molecular mechanisms and interactome remain largely unclear. Through a protein-centric approach, we identified a novel lncRNA and named itTie1-associated angiogenic lncRNA (TAAL).TAALlncRNA regulates endothelial cell migration, proliferation, tube formation, and permeability by modulating ER-calcium homeostasis and cytoskeleton dynamics. In zebrafish,taalmodulation led to angiogenic defects, which were rescued by humanTAALorthologue. Our molecular studies further revealed thatTAALnegatively regulatesTie1protein via ubiquitin-mediated degradation. Notably,TAALexpression is upregulated in the blood of DR patients and downregulated in endothelial DR cell models. Overexpression ofTAALrestored endothelial permeability and VE-cadherin surface expression. These findings establishTAALas a novel regulator ofTie1protein turnover, with potential therapeutic implications for diabetic retinopathy.

BackgroundDiabetic eye disease is a highly prevalent and sight-threatening disorder. It is a disease of neuro-vascular unit of the retina, if left untreated can cause blindness. Therapeutic approaches followed for its treatment can only restrict the progression of the disease with highly variable results. There is no known biomarker for an early diagonsis of this disease, therefore by the time it is detected it goes beyond repair. This creates a massive demand for development of such biomarkers that help detect disease in its earlier stages.MethodsPUBMED (https://pubmed.ncbi.nlm.nih.gov/) was searched for articles relevant to the topic published till November 2023. The search was made using keywords such as Diabetic Retinopathy, inflammation, tear, biomarker, proteomics etc. The studies providing relevant information to prove the importance of biomarker discovery were chosen. After compiling the data, the manuscript writing was planned under relevant headings and sub-headings.ResultsThe review provides a comprehensive overview of all the tear protein biomarker studies in the field of DR and DME. Briefly, their potential in other diseases is also elucidated. While there are many studies pertaining to DR biomarkers, the identified markers lack validations which has restricted their usage in clinics. In case of DME, there was no such study towards biomarker discovery for its diagnosis and prognosis.ConclusionsThe review highlights major studies and their lacunae in the field of biomarkers discovery for DR and DME.

Purpose Diabetic retinopathy (DR) is a neurovascular complication of diabetes (DM) causing the loss of neurons (ganglion cells) in the retina. The disease etiology and potential pathogenic mechanisms in this disease remains unclear. In the present study, we aimed to further understand the key and novel pathogenic mechanisms involved in DR pathogenesis by taking cues from our global proteomics data. Methodology The study was approved by the institutional review board (IRB) of LVPEI, Hyderabad, India. Vitreous humour samples (PDR; n=3, DM; n=3, Control; n=3) were collected from patients undergoing vitrectomy and subjected to LC-MS-MS analysis. The acquired raw data were searched against the human vitreous proteome and was further analysed by various bioinformatic and proteomic tools. Western blotting and IHC was performed to validate crucial pathways. Blood samples from patients (DM, PDR & NPDR) and controls (n=50); cadaveric retinas from diabetic and non-diabetic donors (n=10) and epiretinal membranes (ERM, n=10) from DR cases and controls were collected and RNA was isolated. Quantitative expression of genes involved in autophagy were performed. ɅɅCT was compared across different categories and significance estimated using a student t-test. Result A total of 1079 proteins were identified with 16 completely novel proteins in eye proteome. Top pathways identified were autophagy, inflammation, LXR/RXR activation (lipid metabolism), ROS generation by macrophages, apoptosis and protein degradation. Regulatory proteins identified were associated with cell death, phagocytic activation, angiogenesis and apoptosis. Autophagy inducers such as ROS was found to be accumulated in the DR vitreous. TREM2, microglial receptor was identified as a novel protein in PDR vitreous. The expression of TREM2, an autophagy-associated gene was significantly (p-value = 0.05) upregulated in all categories as compared to control (NDM and/or NDM/No-DR). TREM2 protein also seemed to colocalise with microglial marker F4/80 in retinal tissues and intense expression was observed near the blood vessels in case of PDR retina. Other autophagy-associated markers were also differentially regulated in DR as compared to controls. Conclusion This study emphasises on the strong role of autophagy pathways and its associated genes in the development of DR. ### Competing Interest Statement The authors have declared no competing interest.

In spite of 150 years of studying malaria, the unique features of the malarial parasite, Plasmodium, still perplex researchers. One of the methods by which the parasite manages its gene expression is epigenetic regulation, the champion of which is PfGCN5, an essential enzyme responsible for acetylating histone proteins. PfGCN5 is a ∼170 kDa chromatin-remodeling enzyme that harbors the conserved bromodomain and acetyltransferase domain situated in its C-terminus domain. Although the PfGCN5 proteolytic processing is essential for its activity, the specific protease involved in this process still remains elusive. Identification of PfGCN5 interacting proteins through immunoprecipitation (IP) followed by LC-tandem mass spectrometry analysis revealed the presence of food vacuolar proteins, such as the cysteine protease Falcipain 3 (FP3), in addition to the typical members of the PfGCN5 complex. The direct interaction between FP3 and PfGCN5 was further validated by in vitro pull-down assay as well as IP assay. Subsequently, use of cysteine protease inhibitor E64d led to the inhibition of protease-specific processing of PfGCN5 with concomitant enrichment and co-localization of PfGCN5 and FP3 around the food vacuole as evidenced by confocal microscopy as well as electron microscopy. Remarkably, the proteolytic cleavage of the nuclear protein PfGCN5 by food vacuolar protease FP3 is exceptional and atypical in eukaryotic organisms. Targeting the proteolytic processing of GCN5 and the associated protease FP3 could provide a novel approach for drug development aimed at addressing the growing resistance of parasites to current antimalarial drugs.

Brassica juncea is a crucial oilseed crop, and its seeds possess high economic value as they are a source of edible oil. In order to understand the role of long non coding RNAs (lncRNAs) in the regulation of seed development, we carried out computational analysis using transcriptome data of developing seeds of two contrasting genotypes of B. juncea, Pusajaikisan (PJK) and Early Heera 2 (EH2). The seeds were sampled at three stages, 15, 30, and 45 days after pollination. We identified 1,539 lncRNAs, of which 809 were differentially expressed. We also carried out extensive characterization and functional analysis of seed lncRNAome. The expression patterns were analysed using k-means clustering, and the targets were analysed using pathway, transcription factor, and GO enrichment, as well as ortholog information. We shortlisted a total of 25 robust lncRNA candidates for seed size, oil content, and seed coat color. We also identified 4 lncRNAs as putative precursors of miRNAs regulating seed development. Moreover, a total of 28 miRNA-lncRNA-mRNA regulatory networks regulating seed traits were identified. We also developed a comprehensive database, (BrassIca juncea database or “BIJ” ( https://bij.cuh.ac.in/ ), which provides seed omics as well as other functional genomics and genetics data in an easily accessible form. These candidate lncRNAs are suitable for including in crop improvement programs through molecular breeding, as well as for future validations through genome editing. Together, the knowledge of these candidate lncRNAs and availability of BIJ database shall leverage the crop improvement efforts in B. juncea.

Extremely preterm infants are at risk of developing retinopathy of prematurity (ROP), characterized by neovascularization and neuroinflammation leading to blindness. Polyunsaturated fatty acid (PUFA) supplementation is recommended in preterm infants to lower the risk of ROP, however, with no significant improvement in visual acuity. Reasonably, this could be as a result of the non-consideration of PUFA metabolizing enzymes. We hypothesize that abnormal metabolism of the arachidonic acid (AA) pathway may contribute to severe stages of ROP. The present study investigated the AA-metabolizing enzymes in ROP pathogenesis by a targeted gene expression analysis of blood (severe ROP = 70, No/Mild = 56), placenta (preterm placenta = 6, full term placenta = 3), and human primary retinal cell cultures and further confirmed at the protein level by performing IHC in sections of ROP retina. The lipid metabolites were identified by LC-MS in the vitreous humor (VH; severe ROP = 15, control = 15). Prostaglandins D2 (p = 0.02), leukotrienes B5 (p = 0.0001), 11,12-epoxyeicosatrienoic acid (p = 0.01), and lipid-metabolizing enzymes of the AA pathway such as CYP1B1, CYP2C8, COX2, and ALOX15 were significantly upregulated while EPHX2 was significantly (0.04) downregulated in ROP cases. Genes involved in hypoxic stress, angiogenesis, and apoptosis showed increased expression in ROP. An increase in the metabolic intermediates generated from the AA metabolism pathway further confirmed the role of these enzymes in ROP, while metabolites for EPHX2 activity were low in abundance. Inflammatory lipid intermediates were higher compared to anti-inflammatory lipids in VH and showed an association with enzyme activity. Both the placenta of preterm infants who developed ROP and hypoxic retinal cultures showed a reduced expression of EPHX2. These findings suggested a strong involvement of EPHX2 in regulating retinal neovascularization and inflammation. The study results underscore the role of arachidonic acid metabolism in the development of ROP and as a potential target for preventing vision loss among preterm-born infants.image Polyunsaturated fatty acids, including arachidonic acid (AA), form a major constituent of the retina providing energy for retinal cells. We investigated the role of lipid-metabolizing enzymes in the AA pathway in causing retinopathy of prematurity (ROP). Genes coding for the key enzymes in the AA pathway were significantly upregulated except EPHX2, which was downregulated. Significant alterations in their activities under hypoxia, as confirmed by the vitreous humor metabolomics, further demonstrated that a reduced synthesis of dihydroxyeicosatrienoic acid activates notch signaling, leading to neovascularization and inflammation. Targeting PUFA metabolism could serve as better therapeutics for the prevention of blindness because of ROP. Created with .image

Worldwide, diabetes is a chronic disorder that affects millions of people. One of the main complications of diabetes, diabetic retinopathy, can lead to blindness if left untreated. Diabetic retinopathy must be diagnosed and treated at an early stage in order to protect the eyes from long-term damage. In this paper, we analyze the most recent findings in the study of diabetes diagnosis based on retinopathy. We discuss the many imaging techniques for detecting retinopathy, such as fundus photography, optical coherence tomography, and fluorescein angiography. Furthermore, we explore the application of artificial intelligence and machine learning techniques for the automated diagnosis and classification of diabetic retinopathy.

Purpose: Human ocular tissue banking plays an important part in the advancement of translational research for identifying the molecular processes involved in disease etiology and pathogenesis. Timely obtaining a good-quality ocular tissue from a cadaveric donor is exceedingly difficult, especially in remote areas, with a variable transportation time (within 12–24 h), raising concerns about RNA quality and its subsequent applications. Therefore, we assessed the utility of retinal tissues from cadaver donor and enucleated eyes based on the RNA quality and gene expression by real-time polymerase chain reaction (PCR). Settings and Design: Prospective study. Methods: Retina tissues were separated from the donor/enucleated eyes received in the eye bank within 24 h of death (n = 15) and within an hour from OR (n = 3), respectively, and stored immediately at -80 degree. RNA was isolated using trizol, and the quantity and quality were assessed using Qubit and agarose gel electrophoresis, respectively. QPCR was performed for measuring the expression of different retinal-specific genes. The cellular viability of the retina was assessed by establishing explant primary cell cultures. Statistical Analysis: The data were calculated as an average of normalised Ct values ± standard error of the mean. Results: RNA obtained from cadaveric tissues despite being partially degraded showed a uniform strong gene expression of several retinal-specific genes such as PAX6, RHO, TUBB3, CRX , and ALDH1L1 . The primary cultures established from cadaveric tissues showed viable cells. Conclusion: The cadaver donor tissues collected within 24 hours of death can be effectively utilized for gene expression profiling.

Abstract Tumor-derived Extracellular Vesicles (EVs) are emerging as potential liquid biopsy tool in the field of cancer diagnosis and therapeutic targets. The functionality of EVs depends on its diverse cargo, which is known to alter host-receipient cells. Nevertheless, extracting EVs while preserving its integrity and functionality of its contents is challenging, specifically in Low-middle Income countries (LMIC) countries. Our study focussed on extraction of serum derived EVs analysis from brain-tumor samples encompassing Oligodendrogliomas, Astrocytomas and Glioblastomas. The stability of EVs were examined in serum stored for 2 years at -80°C to understand the effect of preservation and storage in different conditions, that were left exposed to room temperature for variable periods of time before freezing. To explore the optimal suitable conditions, EVs were isolated from serum samples using IZON column size-exclusion based-method, and evaluated for structural integrity and stability through nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) technology. Interestingly, the majority of isolated vesicles exhibited round, intact structure with bilayer membrane of size varying between 30 - 250nm. Moreover, protein content of EVs positive markers (CD63, CD81 and CD9) was also detected with minimal contaminations evidenced by weak bands for negative marker (ApoB), as determined by Western blot analysis. Further, column-based EV-derived RNA demonstrated optimal yield and purity was confirmed by RNA integrity number (RIN) using Qubit. While cryopreservation had minimum detrimental effects on mRNA sample quality, leaving it exposed to room temperature before freezing had a significant effect on EV isolation, as samples were often haemolyzed. This will provide insights in optimizing pre-analytical and analytical procedures for more effective outcomes in the clinical setting

This review summarizes the impact of systemic and ocular inflammatory disorders on diabetes mellitus (DM) and diabetic retinopathy (DR). Local inflammation is a key pathology in diabetic retinopathy (DR) and is also an evolving target for clinical therapy. The legacy effects of local inflammation at the intracellular level make DR a persistent self-driven vicious process. Ocular inflammation is accompanied as well as incited by systemic inflammation due to diabetes mellitus (DM) itself. Over the years, a multitude of studies have evaluated the impact of systemic inflammatory disorders (SIDs, like rheumatoid arthritis, lupus, psoriasis, etc.) and anti-inflammatory drugs prescribed for managing them on manifestations of DM. Recent studies have indicated increased insulin resistance to be a result of chronic inflammation, and the anti-inflammatory drugs to have a protective effect towards DM. Very few studies have evaluated the impact of SIDs on DR. Furthermore, the evidence from these studies is conflicting, and while local anti-inflammatory therapy has shown a lot of clinical potential for use in DR, the results of systemic anti-inflammatory therapies have been inconsistent. The impact of local ocular inflammation due to uveitis on DR is a crucial aspect that has not been evaluated well at present. Initial pre-clinical studies and small-sized clinical reports have shown a strong and positive relationship between the presence of uveitis and the severity of DR as well as its progression, while larger cross-sectional patient surveys have refuted the same. The long term impact of ocular inflammation due to uveitis on DR needs to be studied while adjusting for confounders.

Primary congenital glaucoma (PCG) occurs in children due to developmental abnormalities in the trabecular meshwork and anterior chamber angle. Previous studies have implicated rare variants in CYP1B1, LTBP2, and TEK and their interactions with MYOC, FOXC1, and PRSS56 in the genetic complexity and clinical heterogeneity of PCG. Given that some of the gene-encoded proteins are localized in the centrosomes (MYOC) and perform ciliary functions (TEK), we explored the involvement of a core centrosomal protein, CEP164, which is responsible for ocular development and regulation of intraocular pressure. Deep sequencing of CEP164 in a PCG cohort devoid of homozygous mutations in candidate genes (n = 298) and controls (n = 1757) revealed CEP164 rare pathogenic variants in 16 cases (5.36%). Co-occurrences of heterozygous alleles of CEP164 with other genes were seen in four cases (1.34%), and a physical interaction was noted for CEP164 and CYP1B1 in HEK293 cells. Cases of co-harboring alleles of the CEP164 and other genes had a poor prognosis compared with those with a single copy of the CEP164 allele. We also screened INPP5E, which synergistically interacts with CEP164, and observed a lower frequency of pathogenic variants (0.67%). Our data suggest the potential involvements of CEP164 and INPP5E and the yet unexplored cilia-centrosomal functions in PCG pathogenesis.

Diabetic retinopathy (DR), a leading cause of vision impairment and blindness, is characterized by abnormal retinal vascular changes due to chronic hyperglycemia. The Tie-1 signaling pathway, essential for vascular growth and remodeling, has emerged as a key therapeutic target, though its molecular mechanisms and interactome remain largely unclear. Through a protein-centric approach, we identified a novel lncRNA and named it Tie1-associated angiogenic lncRNA (TAAL) . TAAL lncRNA regulates endothelial cell migration, proliferation, tube formation, and permeability by modulating ER-calcium homeostasis and cytoskeleton dynamics. In zebrafish, taal modulation led to angiogenic defects, which were rescued by human TAAL orthologue. Our molecular studies further revealed that TAAL negatively regulates Tie1 protein via ubiquitin-mediated degradation. Notably, TAAL expression is upregulated in the blood of DR patients and downregulated in endothelial DR cell models. Overexpression of TAAL restored endothelial permeability and VE-cadherin surface expression. These findings establish TAAL as a novel regulator of Tie1 protein turnover, with potential therapeutic implications for diabetic retinopathy. ### Competing Interest Statement The authors have declared no competing interest.

The response of retinal pathology to interventions in diabetic retinopathy (DR) is often independent of the glycated hemoglobin (HbA1c) values at the point of care. This is despite glucose control being one of the strongest risk factors for the development and progression of DR. Previous preclinical and clinical research has indicated metabolic memory, whereby past cumulative glucose exposure may continue to impact DR for a prolonged period. Preclinical studies have evaluated punitive metabolic memory through poor initial control of DM, whereas clinical studies have evaluated protective metabolic memory through good initial control of DM. In this narrative review, we evaluate the preclinical and clinical evidence regarding metabolic memory and discuss how this may form the basis of preventive care for DR by inducing "metabolic amnesia" in people with a history of uncontrolled diabetes in the past. While our review suggested mitochondrial biology may be one such target, research is still far from a possible clinical trial. We discuss the challenges in such research.