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The lysosome-targeting chimera (LYTAC) strategy provided a very powerful tool for the degradation of membrane proteins. However, the synthesis of LYTACs, antibody-small molecule conjugates, is challenging. The ability of antibody-based LYTACs to penetrate solid tumor is limited as well, especially to cross the blood-brain barrier (BBB). Here, we propose a covalent chimeric peptide-based targeted degradation platform (Pep-TACs) by introducing a long flexible aryl sulfonyl fluoride group, which allows proximity-enabled cross-linking upon binding with the protein of interest. The Pep-TACs platform facilitates the degradation of target proteins through the mechanism of recycling transferrin receptor (TFRC)-mediated lysosomal targeted endocytosis. Biological experiments demonstrate that covalent Pep-TACs can significantly degrade the expression of PD-L1 on tumor cells, dendritic cells and macrophages, especially under acidic conditions, and markedly enhance the function of T cells and tumor phagocytosis by macrophages. Furthermore, both in anti-PD-1-responsive and -resistant tumor models, the Pep-TACs exert significant anti-tumor immune response. It is noteworthy that Pep-TACs can cross the BBB and prolong the survival of mice with in situ brain tumor. As a proof-of-concept, this study introduces a modular TFRC-based covalent peptide degradation platform for the degradation of membrane protein, and especially for the immunotherapy of brain tumors. LYTAC strategies often face challenges in solid tumor penetration and synthesis. Here, the authors introduce Pep-TACs, a modular TFRC-based covalent peptide degradation platform that effectively degrades membrane protein PD-L1. This approach significantly suppresses both anti-PD-1-responsive and -resistant tumor growth, particularly in brain tumors. [ABSTRACT FROM AUTHOR]

Genomic DNA is assembled into chromatin by histones, and extruded into loops by cohesin. These mechanisms control important genomic functions, but whether histones and cohesin cooperate in genome regulation is poorly understood. Here we identify Phf2, a member of the Jumonji-C family of histone demethylases, as a cohesin-interacting protein. Phf2 binds to H3K4me3 nucleosomes at active transcription start sites (TSSs), but also co-localizes with cohesin. Cohesin depletion reduces Phf2 binding at sites lacking H3K4me3, and depletion of Wapl and CTCF re-positions Phf2 together with cohesin in the genome, resulting in the accumulation of both proteins in chromosomal regions called vermicelli and cohesin islands. Conversely, Phf2 depletion reduces cohesin binding at TSSs lacking CTCF and decreases the number of short cohesin loops, while increasing the length of heterochromatic B compartments. These results suggest that Phf2 is an 'epigenetic reader', which is translocated through the genome by cohesin-mediated DNA loop extrusion, and which recruits cohesin to active TSSs and limits the size of B compartments. These findings reveal an unexpected degree of cooperativity between epigenetic and architectural mechanisms of eukaryotic genome regulation. Synopsis: Genomic DNA is extruded into loops by cohesin and regulated by histone-modification binding proteins, but how these architectural and epigenetic regulatory mechanisms cooperate is poorly understood. This study describes and functionally characterizes interactions between cohesin and the epigenetic reader protein Phf2. The PHD-finger and JmjC-domain protein Phf2 binds a conserved essential sequence (CES) on cohesin. Phf2 co-localizes with H3K4me3-nucleosomes and cohesin in the genome. Cohesin contributes to genomic positioning of Phf2, possibly via loop extrusion. Phf2 limits the size of heterochromatic B compartments and contributes to the formation of short cohesin loops. Phf2 recruits cohesin to active transcription start sites. Phf2 interacts directly with cohesin, promotes cohesin binding at transcription start sites, and limits the size of chromosomal B compartments. [ABSTRACT FROM AUTHOR]

The N6-methyladenosine binding protein YTHDF1, often upregulated in cancer, promotes tumor growth and hinders immune checkpoint blockade treatment. A comprehensive understanding of the molecular mechanisms governing YTHDF1 protein stability is pivotal for enhancing clinical response rates and the effectiveness of immune checkpoint blockade in cancer patients. Here, we report that USP5 interacts with YTHDF1, stabilizing it by removing K11-linked polyubiquitination. Insulin activates mTORC1, phosphorylating USP5 and promoting its dimerization, which binds to and protects YTHDF1 from degradation. Conversely, the CUL7-FBXW8 E3 ligase promotes YTHDF1 degradation. Deficiency in YTHDF1 or USP5 increases PD-L1 expression and suppresses immune-related gene expression, facilitating immune evasion. Combining USP5 inhibition with anti-PD-L1 therapy enhances anti-tumor immunity, suggesting USP5 as a potential biomarker for patient stratification. This study reveals a ubiquitination-dependent regulation of YTHDF1, proposing USP5 inhibition alongside PD-(L)1 blockade as a promising cancer treatment strategy. The m6A binding protein YTHDF1 is frequently upregulated in cancer. Here the authors report that USP5 deubiquitinates and stabilizes YTHDF1, associated with cancer growth and immune evasion, and that targeting USP5 potentiates response to anti-PD-L1 in preclinical models. [ABSTRACT FROM AUTHOR]

Nanobodies (Nbs) hold great potential to replace conventional antibodies in various biomedical applications. However, conventional methods for their discovery can be time-consuming and expensive. We have developed a reliable protein selection strategy that combines magnetic activated cell sorting (MACS)-based screening of yeast surface display (YSD) libraries and functional ligand-binding identification by Tat-based recognition of associating proteins (FLI-TRAP) to isolate antigen-specific Nbs from synthetic libraries. This combined process enabled isolation of three unique Nb clones (NbT15, NbT21, and NbT22) that all bound specifically to a target antigen, namely proprotein convertase subtilisin/kexin type 9 (PCSK9) as well as a gain-of-function PCSK9 mutant (D374Y). All three clones bound to PCSK9 and blocked the interaction between the low-density lipoprotein receptor (LDLR) and either wild-type PCSK9 or the D374Y mutant. Overall, our combined protein selection method enables rapid and straightforward identification of potent antigen-specific Nbs in a manner that can be executed in a basic laboratory setting without the need for specialized equipment. We anticipate that our strategy will be a valuable addition to the protein engineering toolkit, allowing development of Nbs or virtually any other synthetic binding protein for a wide range of applications. [ABSTRACT FROM AUTHOR]

Glycosylation changes of circulating proteins carrying the CA19-9 antigen may offer new targets for detection methods to be explored for the diagnosis of epithelial ovarian cancer (EOC). Search for assay designs for targets initially captured by a CA19-9 antigen reactive antibody from human body fluids by probing with fluorescent nanoparticles coated with lectins or antibodies to known EOC associated proteins. CA19-9 antigens were immobilized from ascites fluids, ovarian cyst fluids or serum samples using monoclonal antibody C192 followed by probing of carrier proteins using anti-MUC16, anti-MUC1 and, anti STn antibodies and seven lectins, all separately coated on nanoparticles. Compared to reference CA19-9 and CA125 immunoassays, nanoparticle aided detection using MUC16, Ma695 and STn antibodies and lectin WGA provided, both separately and combined, improved discrimination of EOC and borderline cancers from benign samples when applied to 60 cyst fluid specimens. When applied to a panel of 44 serum samples (EOC N = 24, healthy and benign samples N = 20) two assays, CA19-9Ma695 and CA19-9MUC1, stood out with equally superior separations (p-values < 10–8) of the two groups compared to conventional CA19-9 immunoassay (p-value 0.03).Eu+3 -NP based CA19-9MUC16, CA19-9Ma695, CA19-9STn and CA19-9WGA show promise for improved EOC detection when applied to ascites & cyst fluids. When applied to circulation-derived samples, the two MUC1 based assays, CA19-9Ma695 and CA19-9Ma552 outperformed other assay constructs. Our results call for further validation in larger EOC cohorts preferentially with early stage ovarian cancers and all major histotypes against commonly occurring benign conditions. [ABSTRACT FROM AUTHOR]

Europinidin is a novel anthocyanidin found in the petals of Plumbago europea that exhibits several physiological effects. Research was conducted to assess europinidin's cardioprotective efficacy in a diabetic and myocardial infarction (MI) experimental model. Rat was injected through the intraperitoneal administration of 45 mg/kg of streptozotocin (STZ), while MI was induced by subcutaneously administering 85 mg/kg of isoproterenol (ISP) at 24 and 48 h prior to the sacrifice procedure. Europinidin 10 and 20 mg/day was administered orally for 4 weeks after validation of diabetes (glucose > 250 mg/dl) on the 7th day. Experimental rats were randomly allocated to control, STZ-ISP control, STZ-ISP + europinidin-10 mg, STZ-ISP + europinidin-20 mg and europinidin 20 mg perse group. Biochemicals parameters including anti-diabetic (Glucose, HbA1c, serum insulin), cardiac markers (hs-CRP, CPK-MB), dyslipidaemia (lipid analysis), anti-inflammatory (IL6, TNF-α and IL-β), oxidative stress (MDA) and antioxidant (SOD, CAT and GSH), kidney function (creatinine), liver function (AST) and pancreatic function (lipase) along with apoptosis markers (Bcl-2, caspase-3) were evaluated. In addition, histopathological indices of heart injury were investigated. In addition, molecular docking (AUTODOCK Tools 1.5.6.) and dynamics were performed. Europinidin (10 and 20 mg/day) reduced blood glucose, HbA1c, hs-CRP, and CPK-MB. It improved serum insulin, blood lipid profile and reduced inflammatory cytokines (IL-6, TNF-α, IL-β), oxidative stress and increased antioxidant enzymes (SOD, CAT and GSH). Europinidin also protected renal, hepatic functions and restored apoptosis markers (increased Bcl-2, decreased caspase-3 levels). Histopathological analysis demonstrated a reduced extent of myocardial necrosis and fibrosis. Europinidin binds in silico to proteins 1NME, 1I0E, 3I2Y and 4AQ3 with energies of -7.038, -6.682, -8.6 and − 8.761 kcal/mol, respectively. While molecular dynamics simulation studies supported the interactions of europinidin with important therapeutic target proteins. Europinidin demonstrates significant cardioprotective and anti-diabetic potential in a diabetic MI experimental model. [ABSTRACT FROM AUTHOR]

RNA helicase DEAD-box helicase 1 (DDX1) forms a complex with the RNA ligase 2´,3´-cyclic phosphate and 5´-OH ligase (RTCB), which plays a vital role in non-spliceosomal splicing of tRNA and X-box binding protein 1 (XBP1) mRNA. However, the importance of DDX1 in non-spliceosomal splicing has not been clarified. To analyze the functions of DDX1 in mammalian cells, we generated DDX1 cKO cells from the polyploid human U2OS cell line and found that splicing of intron-containing tRNAs was significantly disturbed in DDX1-deficient cells, whereas endoplasmic reticulum (ER) stress-induced splicing of XBP1 mRNA was unaffected. Additionally, the enforced expression of DDX1, but not of its helicase-inactive mutant, rescued the splicing defects of tRNAs in DDX1-deficient cells. These results indicate that RTCB is required for the splicing of both tRNA and XBP1 mRNA, whereas the DDX1 enzymatic activity is specifically required for tRNA splicing in vivo. RTCB is required for the splicing of both tRNA and XBP1 mRNA, whereas the DDX1 enzymatic activity is specifically required for tRNA splicing in vivo. [ABSTRACT FROM AUTHOR]

Group A Streptococcus (Strep A) is a human-exclusive bacterial pathogen killing annually more than 500,000 patients, and no current licensed vaccine exists. Strep A bacteria are highly diverse, but all produce an essential, abundant, and conserved surface carbohydrate, the Group A Carbohydrate, which contains a rhamnose polysaccharide (RhaPS) backbone. RhaPS is a validated universal vaccine candidate in a glycoconjugate prepared by chemical conjugation of the native carbohydrate to a carrier protein. We engineered the Group A Carbohydrate biosynthesis pathway to enable recombinant production using the industry standard route to couple RhaPS to selected carrier proteins within Escherichia coli cells. The structural integrity of the produced recombinant glycoconjugate vaccines was confirmed by Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry. Purified RhaPS glycoconjugates elicited carbohydrate-specific antibodies in mice and rabbits and bound to the surface of multiple Strep A strains of diverse M-types, confirming the recombinantly produced RhaPS glycoconjugates as valuable vaccine candidates. [ABSTRACT FROM AUTHOR]

The CD225/Dispanin superfamily contains membrane proteins that regulate vesicular transport and membrane fusion events required for neurotransmission, glucose transport, and antiviral immunity. However, how the CD225 domain controls membrane trafficking has remained unknown. Here we show that the CD225 domain contains a SNARE-like motif that enables interaction with cellular SNARE fusogens. Proline-rich transmembrane protein 2 (PRRT2) encodes a SNARE-like motif that enables interaction with neuronal SNARE proteins; mutations in this region disrupt SNARE binding and are linked to neurological disease. Another CD225 member, interferon-induced transmembrane protein 3 (IFITM3), protects cells against influenza A virus infection. IFITM3 interacts with SNARE proteins that mediate late endosome-late endosome (homotypic) fusion and late endosome-lysosome (heterotypic) fusion. IFITM3 binds to syntaxin 7 (STX7) in cells and in vitro, and mutations that abrogate STX7 binding cause loss of antiviral activity against influenza A virus. Mechanistically, IFITM3 disrupts assembly of the SNARE complex controlling homotypic fusion and accelerates the trafficking of endosomal cargo to lysosomes. Our results suggest that SNARE modulation plays a previously unrecognized role in the diverse functions performed by CD225 proteins. Synopsis: Proteins of the CD225 superfamily regulate membrane trafficking events in diverse tissues, but the mechanism remains unclear. This study reveals that CD225 proteins contain a conserved SNARE-like motif that binds canonical SNARE proteins and regulates membrane fusion in mammalian cells. Genes in the CD225 family encode an R-SNARE-like motif. Mutations in the R-SNARE-like motif of the CD225 protein PRRT2 disrupt neuronal SNARE binding and are linked to neurological disease. Another CD225 protein, IFITM3, interacts with endosomal SNARE proteins, inhibits homotypic late endosome fusion, and promotes the delivery of endosomal cargo to lysosomes. CD225 proteins regulate membrane transport in mammalian cells by binding SNARE proteins through an R-SNARE-like motif. [ABSTRACT FROM AUTHOR]

Protein citrullination modification plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA), and anti-citrullinated protein antibodies (ACPAs) are extensively employed for clinical diagnosis of RA. However, there remains limited understanding regarding specific citrullinated proteins and their implications in the progression of RA. In this study, we screen and verify insulin-like growth factor-2 mRNA binding protein 1 (IGF2BP1) as a novel citrullinated protein with significantly elevated citrullinated level in RA. Autoantibodies against citrullinated IGF2BP1 are further detected in serum and synovial fluid samples from RA patients, which are positively correlated with erythrocyte sedimentation rate (ESR) and disease activity score 28 (DAS28). Transcriptomic sequencing and functional verification show that citrullination at the R167 site of IGF2BP1 promotes the proliferation, migration, and invasion of RA fibroblast-like synoviocytes (RA-FLSs) by improving the mRNA stability of Semaphorin 3D (SEMA3D). Experiments in collagen-induced arthritis (CIA) mice, the classical animal model of RA, show that IGF2BP1 R176K point mutation (Igf2bp1R167K/R167K) mice exert reduced inflammatory response, clinical scores, and joint destruction. At a molecular level, citrullination of IGF2BP1 promotes the stability of SEMA3D mRNA by promoting the interaction between IGF2BP1 and its cofactor ELAV-like protein 1 (ELAVL1), thereby promoting the invasiveness of RA-FLSs. In this study, a new citrullinated protein of IGF2BP1 is discovered, and the molecular mechanism of its citrullinated modification promoting the progression of RA disease is elucidated, which provides theoretical basis for the diagnosis and treatment of RA. Citrullinated IGF2BP1 enhances the aggressiveness of fibroblast-like synoviocytes by increasing the mRNA stability of SEMA3D, autoantibodies against citrullinated IGF2BP1 are detected in patients with rheumatoid arthritis and positively correlate with disease severity. [ABSTRACT FROM AUTHOR]

Background: Variants in SLC34A1 and SLC34A2 genes, which encode co-transporters NaPi2a and NaPi2c, respectively, can lead to hypophosphatemia due to renal phosphate loss. This condition results in hypercalcitriolemia and hypercalciuria, leading to formation of kidney stones and nephrocalcinosis. Phenotype is highly variable. Management includes hyperhydration, dietary modifications, and/or phosphate supplementation. Thiazides and azoles may be used, but randomized studies are needed to confirm their clinical efficacy. Methods: We conducted a retrospective study in the pediatric nephrology unit at Grenoble University Hospital from January 2010 to December 2023. The study aimed to describe clinical and biological symptoms of patients with confirmed SLC34A1 and SLC34A3 gene variants and their outcomes. Results: A total of 11 patients (9 females) from 6 different families had variants in the SLC34A1 (5 patients) and SLC34A3 (6 patients) genes. Median age at diagnosis was 72 [1–108] months. Average follow-up duration was 8.1 ± 4.5 years. Presenting symptom was nephrocalcinosis (4 cases), followed by renal colic (3 cases). At diagnosis, 90% of patients had hypercalciuria and 45% had hypercalcitriolemia. Management included hyperhydration and dietary advice. All patients showed favorable outcomes with normal growth and school attendance. One patient with an SLC34A3 variant showed regression of nephrocalcinosis. Kidney function remained normal. Conclusion: Clinical and biological manifestations of SLC34 gene variants are highly variable, even among siblings; therefore, management must be personalized. Hygienic and dietary measures (such as hyperhydration, a low sodium diet, and age-appropriate calcium intake) result in favorable outcomes in most cases. Use of azoles (e.g., fluconazole) appears to be a promising therapeutic option. [ABSTRACT FROM AUTHOR]

Background: Furosemide stress test (FST) is a novel functional biomarker for predicting severe acute kidney injury (AKI); however, pediatric studies are limited. Methods: Children 3 months to 18 years of age admitted to the intensive care unit (ICU) of a tertiary care hospital from Nov 2019 to July 2021 were screened and those who developed AKI stage 1 or 2 within 7 days of admission underwent FST (intravenous furosemide 1 mg/kg). Urine output was measured hourly for the next 6 h; a value > 2 ml/kg within the first 2 h was deemed furosemide responsive. Other biomarkers like plasma neutrophil gelatinase-associated lipocalin (NGAL) and proenkephalin (PENK) were also evaluated. Results: Of the 480 admitted patients, 51 developed AKI stage 1 or 2 within 7 days of admission and underwent FST. Nine of these patients were furosemide non-responsive. Thirteen (25.5%) patients (eight of nine from FST non-responsive group) developed stage 3 AKI within 7 days of FST, nine (17.6%) of whom (seven from non-responsive group) required kidney support therapy (KST). FST emerged as a good biomarker for predicting stage 3 AKI and need for KST with area-under-the-curve (AUC) being 0.93 ± 0.05 (95% CI 0.84–1.0) and 0.96 ± 0.03 (95% CI 0.9–1.0), respectively. FST outperformed NGAL and PENK in predicting AKI stage 3 and KST; however, the combination did not improve the diagnostic accuracy. Conclusions: Furosemide stress test is a simple, inexpensive, and robust biomarker for predicting stage 3 AKI and KST need in critically ill children. Further research is required to identify the best FST cut-off in children. [ABSTRACT FROM AUTHOR]

Chloroplasts are usually considered spheroid organelles, but this is not the only shape of chloroplasts. The chloroplast of Chlamydomonas has been typically described as cup-shaped. However, in old studies, it was also modeled as a complex shape with "perforations" or windows. Here, we reconstructed the cellular architecture of Chlamydomonas reinhardtii and C. applanata using an array tomography system installed on a field emission scanning electron microscope. C. reinhardtii chloroplasts resembled a baseball glove or a cup without a side, featuring numerous large and small holes that may facilitate the transport of metabolites and proteins produced in the Golgi apparatus fitted in the holes. In a lipid-accumulating, high-light condition, the chloroplast volume increased by filling the side cleft with an entire wall. Many accumulated large lipid droplets were accommodated within the chloroplast holes, which could have been considered as "chloroplast lipid droplets." Mitochondrial meshworks surrounded the chloroplast. C. applanata chloroplasts appeared like a folded starfish or a cup with many side clefts and a few holes. There was a single mitochondrion or two that branched in a complex form. Tight contacts of various organelles were also found in C. applanata. These reconstructions illustrate the complexity of chloroplast shape, which necessitates a revised understanding of the localization of lipid droplets and the evolution of chloroplasts: The prevailing image of the spheroid chloroplasts that reminds us of the similarity between chloroplasts and cyanobacteria is no longer tenable. [ABSTRACT FROM AUTHOR]

In forensic pathology, identifying causes of death in traumatic brain injuries (TBIs) devoid of observable signs presents a significant challenge. Post-mortem biochemistry plays a crucial role in forensic medicine, particularly in determining causes of death in TBIs that lack macroscopic or histopathological evidence. This study aimed to evaluate the utility of Neuron Specific Enolase (NSE) and S100 Calcium Binding Protein B (S100B) in post-mortem serum and cerebrospinal fluid (CSF) as markers for TBI. The relationship of these biochemical markers with survival time and post-mortem interval was also studied. The study sample consisted of 63 cases each from the TBI and the Non-TBI (NTBI) group. The NTBI group comprised of deaths due to mechanical asphyxia, myocardial infarction and isolated trunk trauma. While serum S100B and CSF NSE emerged as a promising marker for TBI, CSF S100B failed to differentiate TBI from the other causes of death. The absence of an association between the level of markers and survival time or post-mortem interval in TBIs highlights the limitations of these biomarkers in such contexts. This study underscores the potential of biochemical markers like serum S100B and CSF NSE in identifying TBI deaths, aiding forensic diagnoses where there are evidentiary limitations in traditional methods. Further research exploring additional markers and body fluids could enhance diagnostic precision in forensic neuropathology. [ABSTRACT FROM AUTHOR]

An electrochemiluminescence (ECL) immunosensor was developed for the highly sensitive and specific detection of heart-type fatty acid binding protein (H-FABP) and the rapid diagnosis of acute myocardial infarction (AMI). H-FABP is a biomarker that is highly specific to cardiac tissue and is associated with a range of cardiac diseases. Following myocardial injury, the rate of increase in H-FABP levels is greater than that observed for myoglobin and troponin. Therefore, the measurement of H-FABP is crucial for the early exclusion of AMI. Silver-cysteine nanorod (AgCysNR), which served as the ECL emitter, was produced with a one-step, green, simple, template-free aqueous phase method. The surfaces of AgCysNR displayed many amino and carboxyl groups that were connected to a large number of a secondary H-FABP-specific antibody. Ferrum-doped molybdenum oxide (FeMoOν), with a large specific surface area, was richly decorated with silver nanoparticle (AgNP), which increased the interfacial electron transfer rate of FeMoOν. The AgNP was used as a co-reaction accelerator to promote persulfate to produce more sulfate anion radical and then enhance the ECL intensity of AgCysNR. The linear range of the ECL immunosensor was 10 fg/mL to 100 ng/mL, and the detection limit was 2.3 fg/mL (signal/noise = 3). The sensor was determined to be stable, repeatable, and reproducible, and the method achieved recoveries of 101.0 to 102.6% with relative standard deviations of 1.4 to 2.0%. This immunosensor represents a promising tool for the early diagnosis of AMI. [ABSTRACT FROM AUTHOR]

Purpose: Hepatocellular carcinoma (HCC) frequently recurs after radical resection, resulting in a poor prognosis. This study assessed the prognostic value of Mac-2 binding protein glycosylation isomer (M2BPGi) for early recurrence (ER) in patients with HCC. Methods: Patients who underwent radical resection for HCC between 2015 and 2021. HCC recurrence within one year after curative resection was defined as ER. Results: The 150 patients were divided into two groups: non-ER (116, 77.3%) and ER (34, 22.7%). The ER group had a lower overall survival rate (p < 0.0001) and significantly higher levels of M2BPGi (1.06 vs. 2.74 COI, p < 0.0001) than the non-ER group. High M2BPGi levels (odds ratio [OR] 1.78, 95% confidence interval [CI] 1.31–2.41, p < 0.0001) and a large tumor size (OR 1.31, 95% CI, 1.05–1.63; p = 0.0184) were identified as independent predictors of ER. M2BPGi was the best predictor of ER according to a receiver operating characteristic (ROC) analysis (area under the ROC curve 0.82, p < 0.0001). Conclusions: M2BPGi can predict ER after surgery and is useful for risk stratification in patients with HCC. [ABSTRACT FROM AUTHOR]

Although various sensors specifically developed for target analytes are available, affordable biosensing solutions with broad applicability are limited. In this study, a cost-effective biosensor for detecting human epidermal growth factor receptor 2 (HER2) was developed using custom-made gold leaf electrodes (GLEs). A novel strategy for antibody immobilization on a gold surface, for the first time mediated by protein L and HER2-specific antibody trastuzumab, was examined using commercial screen-printed gold electrodes and GLEs. A self-assembled monolayer of 11-mercaptoundecanoic acid (MUA) was formed on the gold surface, which was used to covalently immobilize protein L. Further binding of trastuzumab to the protein L was employed and HER2 detection was achieved through electrochemical impedance spectroscopy (EIS). The HER2 detection was examined in phosphate-buffered saline (PBS) and supplemented cell culture medium. The modified GLEs showed good specificity and high sensitivity of HER2 detection without any enrichment steps, achieving a limit of detection (LOD) of 1 ng mL− 1 in PBS and 2.7 ng mL− 1 in cell culture medium, making the proposed immunosensor a cost-effective and sensitive solution for detection in complex biological matrices. [ABSTRACT FROM AUTHOR]

Dysfunction of the endo-lysosomal intracellular Cholesterol transporter 2 protein (NPC2) leads to the onset of Niemann–Pick Disease Type C (NPC), a lysosomal storage disorder. Metabolic and homeostatic mechanisms are disrupted in lysosomal storage disorders (LSDs) hence we characterized a cellular model of NPC2 knock out, to assess alterations in organellar function and inter-organellar crosstalk between mitochondria and lysosomes. We performed characterization of lipid alterations and confirmed altered lysosomal morphology, but no overt changes in oxidative stress markers. Using several techniques, we demonstrated that contacts between mitochondria and late endosomes/lysosomes are reduced in NPC2−/− HEK cells, we observed that the acidic compartments are swollen and lipid dense. Quantification of endogenous lipids in HEKNPC2−/− cells by mass spectrometry reveals accumulation of lipid species indicative of sphingolipid metabolic dysregulation within the lysosome. Specifically, HEK NPC2−/− cells exhibit marked elevation of glucosylsphingosine and glucosylceramides, substrates of beta glucocerebroside (GBA), as well as accumulation of sphingosine and sphingomyelins. Our studies suggest an involvement of NPC2 in the formation of contact sites between mitochondria and lysosomes and support the hypothesis of a role for NPC2 in the endo-lysosomal trafficking pathway and dynamic organellar crosstalk. [ABSTRACT FROM AUTHOR]

Introduction: Anti-MDA5-positive dermatomyositis (anti-MDA5-DM) is a rare autoimmune disease that often leads to rapid-progressive interstitial lung disease (RP-ILD). The lack of effective prediction and treatment methods makes RP-ILD a major risk factor for death in patients with this condition. S100A6 is a member of the S100 Ca2 + ‐ binding protein family, which plays important roles in inflammation, tumor, injury, and fibroblast reparation. This study aims to explore the correlation between serum S100A6 and RP-ILD in anti-MDA5-DM, and to determine whether S100A6 can be used as a specific biomarker to predict RP-ILD. Methods: The authors enrolled 80 participants, including 20 healthy volunteers, 20 patients with anti-synthase syndrome, and 40 patients with anti-MDA5-positive dermatomyositis. Serum samples were collected and the levels of S100A6 were measured using ELISA. Logistic regression was used to analyze the relationship between serum S100A6 levels and RP-ILD, along with other clinical and laboratory parameters. Results: Serum S100A6 levels were significantly lower in anti-MDA5-DM patients with RP-ILD than those without RP-ILD (odds ratio:0.393 (95% CI, 0.164–0.943, p = 0.036)). High serum S100A6 level was found to be a protective factor for RP-ILD. This study shows that high serum S100A6 level may be a protective factor for RP-ILD in anti-MDA5-DM patients. Serum S100A6 may be used as a specific biomarker to predict whether RP-ILD occurs in anti-MDA5-DM. Key Points • This research discovers and reports a biomarker (S100A6) for distinguishing potential RP-ILD in Anti-MDA5 positive dermatomyositis. [ABSTRACT FROM AUTHOR]

The STAT3 pathway promotes epithelial–mesenchymal transition, migration, invasion and metastasis in cancer. STAT3 upregulates the transcription of the key epithelial–mesenchymal transition transcription factor SNAIL in a DNA binding-independent manner. However, the mechanism by which STAT3 is recruited to the SNAIL promoter to upregulate its expression is still elusive. In our study, the lysine methylation binding protein L3MBTL3 is positively associated with metastasis and poor prognosis in female patients with breast cancer. L3MBTL3 also promotes epithelial–mesenchymal transition and metastasis in breast cancer. Mechanistic analysis reveals that L3MBTL3 interacts with STAT3 and recruits STAT3 to the SNAIL promoter to increase SNAIL transcription levels. The interaction between L3MBTL3 and STAT3 is required for SNAIL transcription upregulation and metastasis in breast cancer, while the methylated lysine binding activity of L3MBTL3 is not required for these functions. In conclusion, L3MBTL3 and STAT3 synergistically upregulate SNAIL expression to promote breast cancer metastasis. The transcription factor SNAIL is regulated by STAT3 in a DNA binding-independent manner. Here the authors find that STAT3 is recruited to the gene promoter of SNAIL by the lysine methylation binding protein L3MBTL3, which is essential for epithelial–mesenchymal transition and metastasis of breast cancer. [ABSTRACT FROM AUTHOR]

The study aims to address the critical issue of toxic side effects resulting from drug combinations, which can significantly increase health risks, clinical complications, and lead to drug being withdrawn from the market. A model named TSEDDI (toxic side effects of drug-drug interaction) has been developed to improve the identification of drug pairs that may induce toxicity or adverse reactions. By utilizing drug chemical structures and diverse proteins, we employ a convolutional neural network (CNN) to extract features from molecular images, enzyme proteins, transporter proteins, and target proteins. Furthermore, we introduce a weighted binary cross entropy loss function to tackle class imbalance and integrate the multi-head attention mechanism with residual connections to enhance model performance. Our model outperformed advanced baseline models in predicting drug-drug interaction (DDI) side effects, achieving an accuracy of 0.9059 (± 0.0010) and consistently excelling across various evaluation metrics. The case study confirms the potential mechanisms by which four pairs of drugs cause side effects, thus demonstrating the effectiveness of our model in predicting DDI side effects. The TSEDDI model combines multiple attention mechanisms and residual connections, enhancing its ability to detect toxic and adverse effects related to DDIs. As a result, it becomes a valuable resource for promptly identifying adverse reactions in clinical trials. Future research could investigate drug substructures prone to toxic side effects. [ABSTRACT FROM AUTHOR]

The NS1 binding protein, known for interacting with the influenza A virus protein, is involved in RNA processing, cancer, and nerve cell growth regulation. However, its role in stress response independent of viral infections remains unclear. This study investigates NS1 binding protein's function in regulating stress granules during oxidative stress through interactions with GABARAP subfamily proteins. We find that NS1 binding protein localizes to stress granules, interacting with core components, GABARAP proteins, and p62, a protein involved in autophagy. In cells lacking NS1 binding protein, stress granule dynamics are altered, and p62 ubiquitination is increased, suggesting impaired stress granule degradation. Overexpression of NS1 binding protein reduces p62 ubiquitination. In amyotrophic lateral sclerosis patient-derived neurons, reduced NS1 binding protein and p62 disrupt stress granule morphology. These findings identify NS1 binding protein as a negative regulator of p62 ubiquitination and a facilitator of GABARAP recruitment to stress granules, implicating it in stress granule regulation and amyotrophic lateral sclerosis pathogenesis. The NS1 binding protein (NS1-BP) is involved in RNA processing, cancer, and nerve cell growth regulation. Here, the authors show that NS1-BP regulates stress granule (SG) dynamics under oxidative stress by interacting with GABARAPs and p62. Reduced NS1-BP in amyotrophic lateral sclerosis (ALS) neurons affects SG morphology, linking it to ALS. [ABSTRACT FROM AUTHOR]

Lipid metabolism is critical for male reproduction in plants. Many lipid-metabolic genic male-sterility (GMS) genes function in the anther tapetal endoplasmic reticulum, while little is known about GMS genes involved in de novo fatty acid biosynthesis in the anther tapetal plastid. In this study, we identify a maize male-sterile mutant, enr1, with early tapetal degradation, defective anther cuticle, and pollen exine. Using genetic mapping, we clone a key GMS gene, ZmENR1, which encodes a plastid-localized enoyl-acyl carrier protein (ACP) reductase. ZmENR1 interacts with β-hydroxyacyl-ACP dehydratase (ZmHAD1) to enhance the efficiency of de novo fatty acid biosynthesis. Furthermore, the ZmENR1/ZmHAD1 complex is regulated by a Maize Male Sterility 1 (ZmMS1)-mediated feedback repression loop to ensure anther cuticle and pollen exine formation by affecting the expression of cutin/wax- and sporopollenin-related genes. Intriguingly, homologous genes of ENR1 from rice and Arabidopsis also regulate male fertility, suggesting that the ENR1-mediated pathway likely represents a conserved regulatory mechanism underlying male reproduction in flowering plants. Authors identify a genic male sterility gene, ZmENR1, in maize and report that it encodes an enoyl carrier protein reductase that forms heterodimers with ZmHAD1 to ensure maize pollen and anther development through regulating lipid and ROS metabolism. [ABSTRACT FROM AUTHOR]

Background: Various biomarkers associated with sarcopenia have been identified. However, there is a scarcity of studies exploring and validating biomarkers in individuals with age-related sarcopenia. Aims: This study aimed to investigate the proteome and identify potential biomarkers for age-related sarcopenia. Methods: Proteomic analysis and experimental validation were conducted using plasma from hospitalized older adults. Sarcopenia diagnosis was based on the Asian Working Group for Sarcopenia 2019 criteria. Data-independent acquisition-based proteomics was performed on plasma from 60 participants, with 30 diagnosed with sarcopenia and 30 without sarcopenia. Differentially expressed proteins (DEPs) were selected and evaluated by Receiver Operating Characteristic (ROC) analysis. Biomarker candidates were further quantitatively validated by enzyme-linked immunosorbent assay (ELISA) utilizing plasma from 6 participants with sarcopenia and 6 without sarcopenia. Results: A total of 39 DEPs were identified and 12 DEPs were selected for ROC analysis. 8 DEPs were included for ELISA validation based on their predictive performance. Paraoxonase-3 (PON3) consistently showed down-regulation in the sarcopenic group across both methodologies. Insulin-like growth factor-binding protein-2 (IGFBP2) showed inconsistency in the sarcopenic group, with up-regulation observed in proteomic analysis but down-regulation in ELISA. Discussion: Decline in PON3 may result in an overload of oxidative stress in skeletal muscles and contribute to sarcopenia. Protein modifications of IGFBP2 might exhibit during sarcopenia pathogenesis. Conclusions: Plasma proteins are implicated in sarcopenia pathogenesis. PON3 is highlighted as a potential biomarker for patients with age-related sarcopenia. Further studies are imperative to gain an in-depth understanding of PON3 and IGFBP2. [ABSTRACT FROM AUTHOR]

RBM family proteins plays the critical role in the progression of numerous tumors. However, whether RBM family proteins involved in prostate cancer (PCa) progression is remain elucidated. In our study, an RNAi screen containing shRNA library targeting 54 members of the RBM family was applied to identify the critical RBM proteins involved in prostate cancer progression under docetaxel treatment, and RBM19 was selected. RBM19 was up-regulated in PCa specimens and correlated with the prognosis and Gleason score of PCa patients. Functionally assays revealed that RBM19 promoted PCa progression under docetaxel treatment both in vivo and in vitro. Mechanistically, RBM19 could bind to LncRNA SNHG21, thereby increased SNHG21 expression through enhancing its stability. Furthermore, SNHG21 bind to PIM1 proteins and prevented it from ubiquitin-protease dependent degradation and ultimately enhancing mitochondrial homeostasis. Overall, our study indicates the RBM19/SNHG21/PIM1 axis may be the encouraging target for docetaxel-tolerance PCa treatment. [ABSTRACT FROM AUTHOR]

Mirror-image proteins, composed of d-amino acids, are an attractive therapeutic modality, as they exhibit high metabolic stability and lack immunogenicity. Development of mirror-image binding proteins is achieved through chemical synthesis of d-target proteins, phage display library selection of l-binders and chemical synthesis of (mirror-image) d-binders that consequently bind the physiological l-targets. Monobodies are well-established synthetic (l-)binding proteins and their small size (~90 residues) and lack of endogenous cysteine residues make them particularly accessible to chemical synthesis. Here, we develop monobodies with nanomolar binding affinities against the d-SH2 domain of the leukemic tyrosine kinase BCR::ABL1. Two crystal structures of heterochiral monobody-SH2 complexes reveal targeting of the pY binding pocket by an unconventional binding mode. We then prepare potent d-monobodies by either ligating two chemically synthesized d-peptides or by self-assembly without ligation. Their proper folding and stability are determined and high-affinity binding to the l-target is shown. d-monobodies are protease-resistant, show long-term plasma stability, inhibit BCR::ABL1 kinase activity and bind BCR::ABL1 in cell lysates and permeabilized cells. Hence, we demonstrate that functional d-monobodies can be developed readily. Our work represents an important step towards possible future therapeutic use of d-monobodies when combined with emerging methods to enable cytoplasmic delivery of monobodies. In this work, the authors develop mirror-image monobodies (Mb) made of D-amino acids against the BCR::ABL1 SH2 domain with high binding affinities. The heterochiral Mb-SH2 structures reveal an unusual binding mode. The D-Mbs are protease-resistant, inhibit BCR::ABL1 kinase activity and bind BCR::ABL1 in cell lysates. [ABSTRACT FROM AUTHOR]

This study aims to comprehensively analyze the role of the exportin (XPO) family in the development and progression of cancer. These nuclear transport proteins have been increasingly recognized for their involvement in oncogenic processes and tumor growth. We utilized updated public databases and bioinformatics tools to assess the expression levels of the XPO family and their associations with key oncological markers including patient survival, immune subtypes, tumor microenvironment, stemness scores, drug sensitivity, and DNA methylation across various cancers. Expression levels of XPO family proteins varied significantly across different cancer types, indicating cancer-specific roles. Specific XPO proteins were linked to adverse prognosis in particular cancers. Additionally, expression levels were correlated with classifications of immune subtypes and tumor purity; notably, lower expression levels were often found in tumors with elevated stromal and immune scores. A marked correlation was observed between XPO proteins and RNA stemness scores, whereas the correlation with DNA stemness scores varied. Furthermore, XPO expression levels significantly influenced cancer cell drug sensitivity and generally showed correlations with gene methylation patterns, although these correlations differed among cancer types. Our findings underscore the distinct roles of XPO family members in cancer, linking them to immune infiltration, the tumor microenvironment, and drug sensitivity. These insights not only enhance our understanding of the prognostic and therapeutic potentials of XPO proteins in cancer but also lay the groundwork for further studies into their mechanisms and applications in cancer diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Microtubules, composed of conserved α/β-tubulin dimers, undergo complex post-translational modifications (PTMs) that fine-tune their properties and interactions with other proteins. Cilia exhibit several tubulin PTMs, such as polyglutamylation, polyglycylation, detyrosination, and acetylation, with functions that are not fully understood. Mutations in AGBL5, which encodes the deglutamylating enzyme CCP5, have been linked to retinitis pigmentosa, suggesting that altered polyglutamylation may cause photoreceptor cell degeneration, though the underlying mechanisms are unclear. Using super-resolution ultrastructure expansion microscopy (U-ExM) in mouse and human photoreceptor cells, we observed that most tubulin PTMs accumulate at the connecting cilium that links outer and inner photoreceptor segments. Mouse models with increased glutamylation (Ccp5−/− and Ccp1−/−) or loss of tubulin acetylation (Atat1−/−) showed that aberrant glutamylation, but not acetylation loss, disrupts outer segment architecture. This disruption includes exacerbation of the connecting cilium, loss of the bulge region, and destabilization of the distal axoneme. Additionally, we found significant impairment in tubulin glycylation, as well as reduced levels of intraflagellar transport proteins and of retinitis pigmentosa-associated protein RPGR. Our findings indicate that proper glutamylation levels are crucial for maintaining the molecular architecture of the photoreceptor cilium. Synopsis: Tubulin post-translational modifications (PTMs) are highly enriched in the cilia and are important for photoreceptor architecture and function. This study reveals the nanoscale localization of tubulin PTMs along the cilium of mouse and human photoreceptor cells and implicates glutamylation in correct establishment of ciliary molecular architecture. Ultrastructure expansion microscopy reveals that most tubulin PTMs are enriched in the connecting cilium and bulge region of photoreceptor cilia. Hyperglutamylation upon loss of deglutamylases CCP1 or CCP5 leads to architectural defects of the axoneme and loss of the bulge region. Hyperglutamylation causes mislocalization or loss of ciliary components, including intraflagellar transport proteins. Hyperglutamylation is associated with extended connecting cilium. Super-resolution ultrastructure expansion microscopy reveals the nanoscale localization and role of post-translational tubulin modifications in photoreceptors. [ABSTRACT FROM AUTHOR]

Due to the complex pathophysiology of AD (Alzheimer's Disease), there are currently no effective clinical treatments available, except for acetylcholinesterase inhibitors. However, CREB (cyclic AMP-responsive element binding protein) has been identified as the critical factor for the transcription in memory formation. Understanding the effect of potential drugs on the CREB pathway could lead to the development of new therapeutic molecules. Rufinamide has shown promise in improving memory in animal models, and these effects may be associated with modulation of the CREB pathway, however, this has not been previously reported. Thus, the present study aimed to determine the involvement of the CREB pathway in the cognitive improvement effects of rufinamide in STZ (streptozotocin) induced mouse model of dementia. Administration of STZ [3 mg/kg, i.c.v. (intracerebroventricular) bilaterally] significantly impaired cognitive performance in step-down passive avoidance and Morris water maze tests in animals, reduced brain endogenous antioxidant levels (GSH, superoxide dismutase, and catalase), and increased marker of brain oxidative stress [TBARS (thiobarbituric acid reactive substances)] and inflammation [IL-1β (Interleukin-1 beta), IL-6 (Interleukin-6), TNF-α (Tumor necrosis factor alpha) and NF-κB (Nuclear factor kappa B)], along with neurodegeneration. These effects were markedly reversed by rufinamide (50 and 100 mg/kg) when administered to STZ animals. However, the pre-treatment with the CREB inhibitor (666-15) in STZ and rufinamide-administered animals neutralized the beneficial influence of rufinamide. Our data suggest that rufinamide, acting via CREB signaling, reduced oxidative stress and inflammatory markers while elevating anti-oxidant levels. Our study has established that rufinamide may act through CREB signaling in an investigational AD model, which could be crucial for developing new treatments beneficial in progressive neurological disorders. [ABSTRACT FROM AUTHOR]

The identification of host factors with antiviral potential is important for developing effective prevention and therapeutic strategies against SARS-CoV-2 infection. Here, by using immortalized cell lines, intestinal organoids, ex vivo intestinal tissues and humanized ACE2 mouse model as proof-of-principle systems, we have identified lipolysis-stimulated lipoprotein receptor (LSR) as a crucial host defense factor against SARS-CoV-2 infection in the small intestine. Loss of endogenous LSR enhances ACE2-dependent infection by SARS-CoV-2 Spike (S) protein-pseudotyped virus and authentic SARS-CoV-2 virus, and exogenous administration of LSR protects against viral infection. Mechanistically, LSR interacts with ACE2 both in cis and in trans, preventing its binding to S protein, and thus inhibiting viral entry and S protein-mediated cell–cell fusion. Finally, a small LSR-derived peptide blocks S protein binding to the ACE2 receptor in vitro. These results identify both a previously unknown function for LSR in antiviral host defense against SARS-CoV-2, with potential implications for peptide-based pan-variant therapeutic interventions. Synopsis: Host cell factors that affect the interaction of SARS-CoV-2 Spike (S) protein with its receptor ACE2 can modulate SARS-CoV-2 infection. This study shows that lipolysis-stimulated lipoprotein receptor (LSR) acts as a defense factor against SARS-CoV-2 by impairing S protein binding to ACE2. LSR suppresses SARS-CoV-2 infection in the small intestine. LSR blocks viral entry and restricts S protein-mediated cell–cell fusion. LSR interacts with ACE2 both in cis and in trans, preventing its binding to S protein. Lipolysis-stimulated lipoprotein receptor (LSR) and its peptide derivative inhibit SARS-CoV-2 entry into host cells by blocking interaction between viral Spike protein and its ACE2 receptor. [ABSTRACT FROM AUTHOR]

A series of novel pyrazole-based 1,2,3-triazole hybrids 7a–7h were synthesized and tested for in vitro cytotoxicity against MCF-7, IMR-32, HeLa, and HER293 cell lines. The compounds 1-(4-methylphenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde O-[(1-phenyl-1H-1,2,3-triazol-5-yl)methyl]oxime (7a), 1-(4-bromo-phenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde O-[(1-phenyl-1H-1,2,3-triazol-5-yl)methyl]oxime (7e), and 1-(4-nitrophenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde O-[(1-phenyl-1H-1,2,3-triazol-5-yl)methyl]oxime (7g) displayed potential cytotoxic activity against the tested cell lines. The in silico molecular docking study of pyrazole-based 1,2,3-triazole hybrids 7a–7h on the EGFR receptor revealed that compounds 7a, 7e, and 7g strongly bind to the protein, and the calculated binding energies were in excellent agreement with the corresponding IC50 values. [ABSTRACT FROM AUTHOR]

Background: Cardiac surgery associated acute kidney injury (CS-AKI) is common. Urine response to loop diuretic and urine neutrophil gelatinase associated lipocalin (uNGAL) are separately associated with CS-AKI. We aimed to determine whether urine response to loop diuretic and uNGAL together were associated with postoperative day 2–4 CS-AKI. Methods: Two-center prospective observational study (ages 0–18 years). uNGAL (8–12 h after admission) (ng/mL) and urine response to loop diuretic (6 h for bolus furosemide and 12 h for infusion bumetanide) (mL/kg/hr) were measured. All diuretic doses were converted to furosemide equivalents. The primary outcome was day 2–4 CS-AKI. Patients were sub-phenotyped using a priori cutoffs (uNGAL + ≥ 100 ng/mL and UOP + < 1.5 mL/kg/hr) and optimal cutoffs (uNGAL + ≥ 127 ng/mL and UOP + ≤ 0.79 mL/kg/hr): 1) uNGAL–/UOP–, 2) uNGAL–/UOP + , 3) uNGAL + /UOP–, and 4) uNGAL + /UOP +. Multivariable regression was used to assess the association of uNGAL, UOP and each sub-phenotype with outcomes. Results: 476 patients were included. CS-AKI occurred in 52 (10.9%). uNGAL was associated with 2.59-fold greater odds (95%CI: 1.52–4.41) of CS-AKI. UOP was not associated with CS-AKI. Compared with uNGAL + alone, uNGAL + /UOP + improved prediction of CS-AKI using a priori and optimal cutoffs respectively (AUC 0.70 vs. 0.75). Both uNGAL + /UOP + (IQR OR:4.63, 95%CI: 1.74–12.32) and uNGAL + /UOP– (IQR OR:5.94, 95%CI: 2.09–16.84) were associated with CS-AKI when compared with uNGAL–/UOP–. Conclusions: uNGAL is associated with CS-AKI. The sub-phenotype association was largely driven by uNGAL. Future studies standardizing diuretic dose and timing may be needed to refine the combined performance for clinical decision making. [ABSTRACT FROM AUTHOR]

Background: Nephronophthisis (NPH) comprises a heterogeneous group of inherited renal ciliopathies clinically characterized by progressive kidney failure. So far, definite diagnosis is based on molecular testing only. Here, we studied the feasibility of NPHP1 and NPHP4 immunostaining of nasal epithelial cells to secure and accelerate the diagnosis of NPH. Methods: Samples of 86 individuals with genetically determined renal ciliopathies were analyzed for NPHP1 localization using immunofluorescence microscopy (IF). A sub-cohort of 35 individuals was also analyzed for NPHP4 localization. Western blotting was performed to confirm IF results. Results: NPHP1 and NPHP4 were both absent in all individuals with disease-causing NPHP1 variants including one with a homozygous missense variant (c.1027G > A; p.Gly343Arg) formerly classified as a "variant of unknown significance." In individuals with an NPHP4 genotype, we observed a complete absence of NPHP4 while NPHP1 was severely reduced. IF results were confirmed by immunoblotting. Variants in other genes related to renal ciliopathies did not show any impact on NPHP1/NPHP4 expression. Aberrant immunostaining in two genetically unsolved individuals gave rise for a further genetic workup resulting in a genetic diagnosis for both with disease-causing variants in NPHP1 and NPHP4, respectively. Conclusions: IF of patient-derived respiratory epithelial cells may help to secure and accelerate the diagnosis of nephronophthisis—both by verifying inconclusive genetic results and by stratifying genetic diagnostic approaches. Furthermore, we provide in vivo evidence for the interaction of NPHP1 and NPHP4 in a functional module. [ABSTRACT FROM AUTHOR]

Beta-lactam antibiotic cephalexin is poorly recognized by bacterial beta-lactam binding proteins and therefore cannot be reliably detected by a group-specific receptor assay. For effective and rapid monitoring of cephalexin concentration in food products, the test systems based on high-affinity and selective binding of this antibiotic to polyclonal antibodies have been developed. The principle of the method for the test systems of enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA) is based on the competitive interaction of (a) cephalexin, potentially contained in the samples, and (b) a cephalexin-protein conjugate immobilized in the wells of a microplate or on the membrane of a test strip, with (c) polyclonal antibodies against cephalexin conjugated with peroxidase or adsorbed on gold nanoparticles. The high specificity of the obtained polyclonal antibodies against cephalexin and the absence of cross-reactivity to a number of cephalosporins, penicillins, and antibiotics of other classes have been confirmed. In the ELISA system in a buffer solution and in a milk matrix, the detection limit, the IC50 value, and the range of detected concentrations were 0.02, 1.0 and 0.025–100 ng/mL respectively. The limit of detection for visual and instrumental determination of cephalexin in the LFIA system were 1.5 and 0.1 ng/mL, and the working range of quantitatively measured concentrations was from 0.05 to 1.5 ng/mL. For both systems, the coefficient of variation of measurements was in the range of 2.5–8.0%. The test systems allow to detect cephalexin in milk without sample preparation. The recovery of cephalexin added to the milk samples was 90.0–106.7%. The presented developments can serve as the basis for kits of reagents for monitoring the content of cephalexin in food products. [ABSTRACT FROM AUTHOR]

The CD8+ T cells mediated antitumor immunity plays a critical function on gastric cancer (GC) immunotherapy. However, the mechanism of N6-methyladenosine (m6A) and lactate in GC immune microenvironment are still unclear. Here, present research investigated the role of Insulin like growth factor II mRNA binding protein 3 (IGF2BP3) in GC and its in-depth mechanisms in the antitumor immunity. Data illustrated that high IGF2BP3 level was associated to GC poor prognosis and tumor infiltration. Functional assays demonstrated that IGF2BP3 overexpression could promote the lactate accumulation, and impair the CD8+ T cells' antitumor immunity activity in co-culture system. Correspondingly, IGF2BP3 silencing enhanced the CD8+ T cells' antitumor immunity activity towards co-cultured GC cells. Mechanistically, IGF2BP3 could bind the m6A site on LDHA mRNA, thereby promoting its mRNA stability. Rescue assays elucidated that IGF2BP3/LDHA axis impaired the CD8+ T cells antitumor immunity by triggering lactate excess tumor microenvironment. In conclusion, our findings demonstrate that IGF2BP3 impairs the CD8+ T cells antitumor immunity by targeting LDHA/lactate axis, providing a novel therapeutic insight for GC immunotherapy. [ABSTRACT FROM AUTHOR]

In this study, we evaluated the role of the gnathosoma (mouthparts) in chemosensing of the most devastating honey bee parasite, Varroa destructor mite. Through transcriptomic analysis, we compared the expression of putative chemosensory genes between the body parts containing the main chemosensory organs (the forelegs), gnathosoma and the rest of the body devoid of these two body parts. Furthermore, we checked the presence of chemosensory-related transcripts in the proteome of the gnathosoma. Our comparative transcriptomic analysis revealed the presence of 83 transcripts with known characteristic conserved domains belonging to eight chemosensory gene families in the three Varroa transcriptomes. Among these transcripts, 11 were significantly upregulated in the mite's forelegs, compared to 8 and 10 in the gnathosoma and body devoid of both organs, respectively. Whilst the gnathosoma and the forelegs share similar expression of some putative lipid carrier proteins, membrane-bound receptors, and associated proteins, they also differ in the expression profiles of some transcripts belonging to these protein families. This suggests two functional chemosensory organs that may differ in their chemosensory function according to specific characteristics of compounds they detect. Moreover, the higher expression of some chemosensory transcripts in the body devoid of forelegs and gnathosoma compared to the gnathosoma alone, may suggest the presence of additional function of these transcripts or alternatively presence of additional external or internal chemosensory organs. Insights into the functional annotation of a highly expressed gustatory receptor present in both organs using RNA interference (RNAi) are also revealed. [ABSTRACT FROM AUTHOR]

Root knot nematodes (RKNs) are obligate parasites present in soil which belongs to the genus Meloidogyne. Meloidogyne spp. consists of 90 species having a wide range of crop plants as their host. M. incognita is an important pest which drastically lowers crop quality and yield. A substantial portion of vegetables in Solanaceae family contribute to food security with high nutrient value. Eggplant (Solanum melongena) is one of the most important vegetables in India as it has numerous nutritional as well as medicinal properties. Eggplant, although being affected by a number of pests, has been reported to suffer significant losses by M. incognita attack. There are various interactions between RNKs and host plant which are responsible for the establishment and proliferation of disease. The specific proteins were secreted from the pharyngeal glands of RKNs which interact with the plant receptors to turn on gene expression cascades that alter cell development causing essential metabolic reprogramming of the cells. The molecular interactions between host and pathogen are mediated by calreticulin, which are calcium binding proteins and act as chaperone. There is a need to control the pathogen in the face of rising food demand, climate change and use of chemical nematicides. Furthermore, omics technologies is a group of efficient molecular techniques that have reformed biological research and permit researchers to have an in depth understanding of molecular mechanisms behind a wide range of biological processes. The utilization of omics technique could be the promising approach for the future management of RKNs. Hence, the current review emphasizes on host pathogen interactions and omics as an efficient technique that can help researchers to devise biotechnology-based tools to control RKNs. [ABSTRACT FROM AUTHOR]

The growing rates of obesity and diabetes emphasize the importance of developing functional foods that manage glucose absorption. Banana flour and peel, rich in phenolic compounds, hold promise in this area. This study investigated the effects of pre-gelatinized banana flour with peel powder (PGP50) and waffle products made with banana flour and peel (WF_NW) on glucose uptake and consumption in Caco-2 cells, a model for intestinal absorption. We further assessed changes in glucose transporter proteins (SGLT1 and GLUT2) and characterized phenolics in undigested PGP50. Following in vitro digestion, Caco-2 cells exposed to PGP50 and WF_NW showed significantly reduced glucose uptake compared to the control. Furthermore, both PGP50 and WF_NW significantly reduced glucose consumption in Caco-2 cells after 24 h. PGP50 cells utilized 23.96% of the initial glucose, while WF_NW utilized 45.57% of the initial glucose, compared to the control which utilized 70.26%. Additionally, SGLT1 protein expression exhibited a downward trend. These findings suggest that PGP50 and WF_NW have potential as functional food ingredients for glucose management, possibly through modulation of SGLT1. The presence of specific phenolic compounds like flavones, flavanones, and flavanols in PGP50 warrants further investigation into their role in glucose regulation. [ABSTRACT FROM AUTHOR]

In this study, we aimed to study the mechanism of costunolide (COS) and dehydrocostus lactone (DEH) in the treatment of ulcerative colitis (UC) based on network pharmacology, molecular docking and animal experiments. Firstly, network pharmacology was used to predict the target proteins of compounds and diseases. Subsequently, the network analysis was performed, and the key target proteins were screened out. Finally, molecular docking and animal experiments were conducted to validate the network pharmacology analysis results. A total of 39 common target proteins of compounds and diseases were collected. The target proteins TLR4, PIK3R1, PTGS2, RELA, NOS3, MPO and CHUK were obtained from PPI network analysis, GO analysis, KEGG enrichment analysis, and the compound-target-pathway network. Molecular docking results showed that COS and DEH could bind to these target proteins. The results of animal experiments showed that dextran sulfate sodium (DSS) induced UC in mice, resulting in weight loss, colon shortening, and significantly upregulated expression of TLR4, PIK3R1 and RELA in colon tissue. COS and DEH could reduce these pathological changes in UC mice. And they could alleviate UC by downregulating TLR4, PIK3R1 and RELA. Our findings suggested that COS and DEH could exert protective effects on UC by downregulating TLR4, PIK3R1, and RELA. [ABSTRACT FROM AUTHOR]

During endosomal recycling, Sorting Nexin 17 (SNX17) facilitates the transport of numerous membrane cargo proteins by tethering them to the Retriever complex. Despite its importance, the mechanisms underlying this interaction have remained elusive. Here, we provide biochemical, structural, cellular, and proteomic analyses of the SNX17-Retriever interaction. Our data reveal that SNX17 adopts an autoinhibited conformation in the basal state, with its FERM domain sequestering its C-terminal tail. The binding of cargo proteins to the FERM domain displaces the C-terminal tail through direct competition. The released tail engages with Retriever by binding to a highly conserved interface between its VPS35L and VPS26C subunits, as revealed by cryogenic electron microscopy (cryo-EM). Disrupting this interface impairs the Retriever-SNX17 interaction, subsequently affecting the recycling of SNX17-dependent cargoes and altering the composition of the plasma membrane proteome. Intriguingly, the SNX17-binding pocket on Retriever can be utilized by other ligands containing a consensus acidic C-terminal tail motif. Together, our findings uncover a mechanism underlying endosomal trafficking of critical cargo proteins and reveal how Retriever can potentially engage with other regulatory factors or be exploited by pathogens. Endosomal recycling relies on SNX17 to transport membrane proteins via Retriever. Here, authors reveal the mechanisms of SNX17 autoinhibition and its interaction with Retriever, uncovering a broader family of factors with similar binding strategies. [ABSTRACT FROM AUTHOR]

Plasmodium vivax is the most widespread of the different Plasmodium species able to infect humans and is responsible for most malaria cases outside Africa. An effective, strain-transcending vaccine that alleviates or suppresses erythrocyte invasion would be a game-changer in eliminating vivax malaria. Recently, the binding of P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) to human Transferrin receptor (TfR1) has been described as essential for reticulocyte invasion, making this parasite protein an appealing vaccine candidate. Here, using P. vivax Cambodian clinical isolates in robust ex vivo invasion assays, we show that anti-PvRBP2b polyclonal and monoclonal antibodies that inhibit binding of PvRBP2b to TfR1 do not block P. vivax invasion into reticulocytes even at high concentrations. Anti-TfR1 antibodies do not inhibit P. vivax invasion either. Combinations at high concentrations of human monoclonal antibodies targeting different PvRBP2b epitopes do not inhibit invasion. Combinations of anti-PvRBP2b with anti-PvDBP do not enhance invasion inhibition caused by anti-PvDBP alone. We also show that the invasion of Cambodian P. vivax is trypsin-resistant while TfR1 is trypsin-sensitive, and we demonstrate that TfR1 is not recycled following trypsin treatment. We determined the PvRBP2b sequence of all isolates used in the invasion assays and analyzed polymorphism within epitopes recognized by anti-PvRBP2b antibodies. We show that polymorphism does not explain the absence of neutralization. Anti-PvRBP2b polyclonal antibodies recognized all four isolates tested in immunofluorescence assays while not inhibiting P. vivax invasion. Overall, our results demonstrate that PvRBP2b binding to TfR1 is not essential for invasion into reticulocytes of P. vivax Cambodian strains questioning the relevance of PvRBP2b as vaccine candidate. [ABSTRACT FROM AUTHOR]

Chronic alcohol consumption can lead to alcohol live disease (ALD). Steatosis is a critical hallmark of ALD, making it an important stage for therapeutic intervention. Saikosaponin A (SSa), a compound found in Radix Bupleuri, has previously shown promising hepatoprotective, anti-inflammatory, and antioxidant properties. However, its role in ALD remains understudied. We employ cell-based screening models and a chronic-plus-binge ethanol-fed mouse model to investigate the protective mechanisms of SSa and its metabolite Saikogenin A (SGA), against ethanol-induced hepatocyte injury. Our RNA-seq analysis in mice unveils that SSa primarily acts through the mTOR and PPAR-α signaling pathways in the liver. Biophysical assays and loss of function experiments confirm SGA directly binds to and modulates the activity of SIRT1 protein, mitigating ethanol-induced cell injury via the SIRT1-mTOR-PPAR-α axis. Furthermore, SGA displays a survival prolonging advantage compared to resveratrol for treating ALD. This suggests SGA holds promise as a potential therapeutic agent for ALD. Saikogenin A ameliorates ethanol-induced cellular damage likely by binding to the SIRT1 protein, activating PPAR-α and suppressing mTOR activity. [ABSTRACT FROM AUTHOR]

Vision and magnetoreception in navigating songbirds are strongly connected as recent findings link a light dependent radical-pair mechanism in cryptochrome proteins to signalling pathways in cone photoreceptor cells. A previous yeast-two-hybrid screening approach identified six putative candidate proteins showing binding to cryptochrome type 4a. So far, only the interaction of the cone specific G-protein transducin α-subunit was investigated in more detail. In the present study, we compare the binding features of the G-protein α-subunit with those of another candidate from the yeast-two-hybrid screen, cellular retinol binding protein. Purified recombinant European robin retinol binding protein bound retinol with high affinity, displaying an EC50 of less than 5 nM, thereby demonstrating its functional state. We applied surface plasmon resonance and a Förster resonance transfer analysis to test for interactions between retinol binding protein and cryptochrome 4a. In the absence of retinol, we observed no robust binding events, which contrasts the strong interaction we observed between cryptochrome 4a and the G-protein α-subunit. We conclude that retinol binding protein is unlikely to be involved in the primary magnetosensory signalling cascade. [ABSTRACT FROM AUTHOR]

Ghd7 is a central regulator to multiple growth and development processes in rice. While it is not clear how Ghd7 is regulated by upstream factors. To identify its upstream regulator, the truncated Ghd7 promoter fragments were used to screen cis elements binding to rice total nuclear proteins. Electrophoretic mobility shift assays screened one truncated fragment f3 binding to the proteins. Subsequently, the fragment f3 was employed to screen a yeast one-hybrid library, and a transcription factor OsIAA23 was screened as a direct upstream regulator of Ghd7. Dual-luciferase transient assay demonstrated the transcriptional repression effect of OsIAA23 on the activity of Ghd7, and the location of the cis elements binding to OsIAA23 in the region 1264 to 1255 bp upstream of ATG. Genetic analysis between the wild type Ghd7-OsIAA23 and single/double mutants further verified that OsIAA23 downregulated Ghd7 expression and led to a delayed heading under long day conditions. Moreover, natural variations in fragment f3 were associated with heading and geographic distribution in rice. This study sheds light on the direct regulatory mechanism of OsIAA23 on Ghd7, which enriches the understanding of the Ghd7 involved flowering regulatory network in rice. [ABSTRACT FROM AUTHOR]

Background: Heart-type fatty acid binding proteins (FABP3) constitute a family of lipid chaperone proteins. They are found in the cytosol and enhance cellular fatty acid solubilisation, transport, and metabolism. FABP3 is highly expressed in the myocardium and is released from myocytes during myocardial damage. As FABP3 content in the myocardium is closely related to the metabolic state of fatty acids, we hypothesised that targeting of FABP3 with a radiolabelled small organic compound would visualise myocardium. Results: The selective FABP3 inhibitor, 4-(4-fluoro-2-(1-phenyl-5-(2-(trifluoromethyl)phenyl)-1H-pyrazol-3-yl)phenoxy)butanoic acid (LUF), was radiolabelled via a two-step reaction comprising copper-mediated 18F-fluorination of an arylboronic precursor followed by alkaline hydrolysis of the ethoxy protecting group. [18F]LUF was successfully synthesised by automated synthesiser with sufficient activity yields (14.0 ± 1.8 GBq) and high quality (molar activity, > 250 GBq/µmol and radiochemical purity, > 99.6%). Biological assessment of [18F]LUF as an in vivo myocardial imaging agent included evaluations of biodistribution, metabolite analysis, and positron emission tomography (PET) imaging of small animals. [18F]LUF clearly visualised the myocardium with high contrast against background tissues such as the lung and liver. [18F]LUF also showed a high absolute myocardial uptake equivalent to that of the promising myocardial perfusion tracer [18F]flurpiridaz and excellent metabolic stability in the body. These properties are ideal for stable and noise-less imaging of the heart. PET imaging of rat surgical permanent myocardial infarction (MI) and experimental autoimmune myocarditis (EAM) was also performed. [18F]LUF successfully visualised lesions of permanent MI and EAM. Conclusion: Our results showed for the first time that the 18F-labelled FABP3 selective small organic compound clearly visualised myocardium with good quality. To determine the clinical utility of [18F]LUF for cardiovascular disease in clinical practice, it will be necessary to evaluate a greater variety of cardiovascular disease models and elucidate the accumulation mechanism, particularly in relation to fatty acid metabolism in the myocardium. [ABSTRACT FROM AUTHOR]

Objective: Mac-2 binding protein glycosylation isomer (M2BPGi) is produced in the extracellular matrix and serves as an indicator of hepatic stellate cell activation. Assessing M2BPGi levels could aid in predicting hepatocellular carcinoma (HCC) in individuals with hepatitis C virus (HCV). The objective of this study was to evaluate the usefulness of M2BPGi as a biomarker for HCC in HCV patients and its association with disease severity and progression. Methods: This study included patients who were cured of chronic hepatitis C virus. The patients were divided into three subgroups: HCV without cirrhosis, HCV with cirrhosis, and HCV with HCC. These subgroups were then compared to a subgroup of healthy volunteers. In addition to routine laboratory investigations, M2BPGi levels were measured in all the enrolled subjects. Results: The level of serum M2BPGi was significantly greater in the HCV with cirrhosis and HCC groups than in the control group (P < 0.001). Additionally, it was significantly greater in multifocal HCC than in those with unifocal HCC (P < 0.001), and it was directly proportional to the size of the focal lesion of HCC (P = 0.001). The cutoff for serum M2BPGi in diagnosing HCC was ≥ 0.869 (C.O.I), with an AUC of 0.762, a sensitivity of 78.6%, and a specificity of 61.9% (P = 0.004). Furthermore, the cutoff for predicting multifocality was > 0.93 (C.O.I), with an AUC of 0.73, sensitivity of 66.7%, and specificity of 63.8% (P = 0.03). Although the AFP level was still superior in predicting cirrhosis and HCC, the M2BPGi level was better at predicting the size and diagnostic value of HCC when the AFP level was normal. The cutoff for M2BPGi in this case was 0.903(C.O.I), with a sensitivity of 80%, specificity of 75%, and an accuracy of 76.25%. M2BPGi was independently associated with the CRP level (β = 0.484, P = 0.001) and the size of the HCC focal lesion (β = 1.422, P = 0.001). Conclusion: M2BPGi can be used as an effective marker to assess the biological behavior and aggressiveness of HCC. Further studies are warranted on a large scale of patients to confirm our findings. [ABSTRACT FROM AUTHOR]

The 4CMenB (BexseroR) vaccine contains detergent-extracted outer membrane vesicles (OMVs) from a Neisseria meningitidis (Nm) group B strain NZ98/254 and three recombinant Nm protein antigens: Neisseria adhesin A (NadA), Factor H binding protein (FHbp, as the C-terminal protein in the GNA2091-FHbp fusion), and Neisserial Heparin Binding Antigen (NHBA, as the N-terminal protein in the NHBA-GNA1030 fusion). Previous work has shown that 4CMenB generates serum antibodies to Nm and Neisseria gonorrhoeae (Ng) OMV proteins and lipooligosaccharide (LOS). Mounting evidence indicates 4CMenB can partially protect against mucosal infections with Ng. The immunologic basis for Ng cross protection remains to be fully elucidated. Ten paired human sera obtained pre- and post-immunization with 4CMenB (1 month after a third vaccine dose) were used in ELISAs and in Western blots to determine IgG and IgA serum responses to OMVs from Nm strain NZ98/254 (OMVNm) and two Ng strains, 1291 and CNG20 (OMVNg), and gonococcal recombinant NHBA (rNHBANg) proteins. Post 4CMenB sera, but not pre-sera, showed strong IgG and variable IgA responses to the OMVNm but lower (2–11-fold difference in signal intensity) recognition of OMVNg. All post (not pre) 4CMenB sera showed strong IgG, but variable IgA, recognition of rNHBANg by ELISAs and Western blots. Three post 4CMenB sera at 10% (v/v) concentration had serum bactericidal activity (SBA) against Ng strains 1291 and CNG20 (~30–40% killing), not seen in paired pre-sera. These data confirmed 4CMenB-induced cross-reactive functional antibody responses to Ng. In competitive SBA assays, in which sera were pre-incubated with rNHBA, minimal SBA against Nm strain NZ98/254 was titrated away. However, most of the SBA against Ng strains 1291 and CNG20 required NHBA-specific antibodies, and the Δnhba mutants were resistant to killing by post 4CMenB sera. Removing NHBA-specific and LOS-specific OMV antibodies simultaneously decreased SBA significantly more than the sum of removing individual antibodies alone, suggesting synergy between anti-NHBA and anti-OMV antibodies. Anti- NHBANm antibodies induced by 4CMenB vaccination cross react with NHBANg and substantially contribute to the bactericidal response toward Ng induced by the vaccine. [ABSTRACT FROM AUTHOR]

The goal of designing safer, more effective drugs has led to tremendous interest in molecular mechanisms through which ligands can precisely manipulate the signaling of G-protein-coupled receptors (GPCRs), the largest class of drug targets. Decades of research have led to the widely accepted view that all agonists—ligands that trigger GPCR activation—function by causing rearrangement of the GPCR's transmembrane helices, opening an intracellular pocket for binding of transducer proteins. Here we demonstrate that certain agonists instead trigger activation of free fatty acid receptor 1 by directly rearranging an intracellular loop that interacts with transducers. We validate the predictions of our atomic-level simulations by targeted mutagenesis; specific mutations that disrupt interactions with the intracellular loop convert these agonists into inverse agonists. Further analysis suggests that allosteric ligands could regulate the signaling of many other GPCRs via a similar mechanism, offering rich possibilities for precise control of pharmaceutically important targets. Ligands that activate GPCRs generally do so by stabilizing a particular conformation of the transmembrane helices. Here, the authors reveal a distinct activation mechanism where a ligand instead stabilizes a particular intracellular loop conformation. [ABSTRACT FROM AUTHOR]

Identification of PIN exporters for auxin, the major coordinative signal in plants, some 25 years ago, signifies a landmark in our understanding of plant-specific mechanisms underlying development and adaptation. Auxin is directionally transported throughout the plant body; a unique feature already envisioned by Darwin and solidified by PINs' discovery and characterization. The PIN-based auxin distribution network with its complex regulations of PIN expression, localization and activity turned out to underlie a remarkable multitude of developmental processes and represents means to integrate endogenous and environmental signals. Given the recent anniversary, we here summarize past and current developments in this exciting field. In this perspective, the authors summarize some 25 years of research on PIN auxin transport proteins and reflect on consequences for the understanding of cellular processes underlying plant development and adaptation. [ABSTRACT FROM AUTHOR]

Plasmodium vivax malaria poses a major global health challenge, fueled by the parasite's ability to establish chronic infections via dormant liver hypnozoites that enable immune evasion and show transmission resilience. A key virulence determinant of P. vivax blood-stage infection is the ligand-receptor interaction of infected erythrocytes mediated by the Duffy Binding Protein (PvDBP) ligand. Gene duplication events leading to increased PvDBP copy numbers have been documented in parasite populations in Cambodia, Brazil, and Sudan, but whether such changes exist in Indian isolates is not known. India shoulders a disproportionately high P. vivax burden in the world. DNA extracted from malaria-positive samples from a multisite survey was subjected to diagnostic PCR to evaluate PvDBP duplication. We identified PvDBP duplication at 18.6% frequency across various regions in India via diagnostic PCR. Both 'Cambodian-type' and 'Malagasy-type' duplication patterns were detected. PvDBP copy number variations associated with specific Duffy antigen receptor genotypes were correlated in the patient cohort. While PvDBP duplication was widespread, its geographic distribution varied, occurring most prevalently in northeastern regions of India. The presence of Duffy binding protein gene duplication in nearly one-fifth of P. vivax isolates in India may have significant epidemiological and clinical implications. This genetic variation could potentially impact, parasite fitness and invasion efficiency, possibly leading to higher parasitemia levels and immune evasion capabilities, which may affect the efficacy of natural immunity and vaccine development efforts and / or transmission dynamics if the duplication confers any advantage in mosquito stages. To fully understand these implications, we propose longitudinal studies tracking patients with and without PvDBP duplications, comparing clinical outcomes, parasitemia levels, and transmission rates. Additionally, spatial and temporal analyses of duplication frequency across India could reveal patterns of spread and potential selective pressures driving this genetic change. Highlights: 18.6% frequency of PvDBP duplication found across various Indian regions. Both 'Cambodian-type' and 'Malagasy-type' duplication patterns were detected. PvDBP copy number variation was associated with specific ACKR1 antigen receptor genotypes in patients. Geographic distribution of PvDBP duplication varied, with highest prevalence in northeastern India. [ABSTRACT FROM AUTHOR]