However, the effects of long-term Ca2+ deficiency in orphan crops such as Tef [(Eragrostis tef) (Zucc.) Trotter], which accumulate high levels of Ca in the grains, remained unknown. Tef is a staple crop for nearly 70 million people in East Africa, particularly in Ethiopia and Eritrea. It is one of the most nutrient-dense grains, and is also more resistant to marginal soils and climatic conditions than main cereals like corn, wheat, and rice. In this study, tef plants were grown in a hydroponic solution containing optimum (1 mM) or low (0.01 mM) Ca2+, and plant growth parameters and whole-genome transcriptome were analyzed. Ca+2-deficient plants exhibited leaf necrosis, leaf curling, and growth stunting symptoms. Ca2+ deficiency significantly decreased root and shoot Ca, potassium (K), and copper content in both root and shoots. At the same time, it greatly increased root iron (Fe) content, suggesting the role of Ca2+ in the uptake and/or translocation of these minerals. Transcriptomic analysis using RNA-seq revealed that members of Ca2+ channels, including the cyclic nucleotide-gated channels and glutamate receptor-like channels, Ca2+-transporters, Ca2+-binding proteins and Ca2+-dependent protein kinases were differentially regulated by Ca+2 treatment. Moreover, several Fe/metal transporters, including members of vacuolar Fe transporters, yellow stripe-like, natural resistance-associated macrophage protein, and oligo-peptide transporters, were differentially regulated between shoot and root in response to Ca2+ treatment. Taken together, our findings suggest that Ca2+ deficiency affects plant growth and mineral accumulation by regulating the transcriptomes of several transporters and signaling genes.Calcium (Ca It is a structural component of the cell wall and membranes1 where it binds to an acidic group of the membrane lipids and crosslinks pectin2. Ca+2 also acts as a ubiquitous second messenger in environmental stress signaling3. Several environmental stimuli lead to the release of stored Ca2+, which binds to calmodulin. The Ca2+-calmodulin complex recruits several kinases and phosphatases that regulate transcription-dependent cellular processes4. Plant cells also possess other Ca2+-regulated proteins, including Ca+2 dependent protein kinase (CDPKs), calcineurin B-like (CBL) proteins, CBL interacting protein kinases (CIPKs), CDPK-related kinases (CRKs), etc., which have been well characterized6.Calcium (Ca2+ is taken up from the soil solution by roots and translocated to the shoot via the xylem4. Ca+2 enters plant cells through Ca2+ channels4. Plant glutamate receptor-like (GLR) channels, which are analogs to the mammalian ionotropic glutamate receptors (iGluRs), are implicated in Ca+2 sensing, signaling, and long-distance root to shoot Ca2+ wave propagation during wounding8. Several members of the AtGLRs have been characterized10. The role of AtGLR3.1 in C2+ sensing in Arabidopsis shoot has been reported11. Another class of Ca2+ channels known as cyclic nucleotide-gated channels (CNGCs) has also been implicated in Ca2+ transport13. Cellular Ca2+ concentration is tightly regulated to allow signaling role, control Ca2+ translocation to the shoot and prevent cation toxicity4. Antiporters, uniporters and Ca2+-ATPases localized in all major plant cellular membranes and rapidly expel Ca2+ from the cytosol. The Arabidopsis genome encodes five different Ca2+ efflux systems, called autoinhibited Ca2+-ATPases (ACAs), ER-type Ca2+ATPases (ECAs), and P1-ATPases (HMA1), the mitochondrial calcium uniporter complex (MCUC) and Ca2+exchangers (CAX)15. Similarly, Ca2+/Cation-exchangers have also been reported to play a role in cation homeostasis in plants17.In plants, Ca2+ deficiency is not commonly observed in most soils due to its relative abundance and regular liming practice18. However, developing tissues may experience Ca2+ deficiency due to lack of Ca2+ remobilization from older tissues, under conditions of low transpiration, in sandy soils untreated with lime (CaCO3 or Ca (OH)2), and inhibition of Ca2+ uptake due to competition from other cations18. Plants exposed to Ca2+ deficiency may experience necrosis of the meristems, shoot and root growth stunting4 and leaf carling. Ca2+-deficiency increases the level of reactive oxygen species, causing oxidative stress in high Ca-demanding plant species such as cabbage20 and broccoli21, Ca+2 treatment has been shown to enhance the antioxidant system and decrease oxidative stress in plants grown under high temperature22, drought23, salinity24 and heavy metals25.Ca+-deficiency26. The CBL1/9—CIPK23 has been implicated in the regulation of K+27 and Fe homeostasis (41). Similarly, CBL1/CBL9-CIPK11 interaction has been involved in regulating the bHLH transcription factor FIT, a crucial regulator of Arabidopsis Fe acquisition28. Fe deprivation has been linked to alteration of Ca2+ signatures evoked by exogenous ATP29, suggesting crosstalk between Ca2+ and Fe homeostasis. However, detailed studies on the interconnection between Ca2+ and Fe nutrition are missing. Recently, studies of factors modulating Ca2+-iron homeostasis are emerging30.Calcium-sensing networks, including the calcineurin B-like proteins (CBL)-CBL-interacting protein kinase (CIPK), has been implicated in plant abiotic stresses, including K32 and some other countries including the United Sates where it is grown primarily for animal feed33. Recently, tef has been getting increased attention in many laboratories due to its health benefits and potential roles in sustainable agriculture34. Its mineral nutrient composition has been studied35. Tef is one of the orphan crops reported to have higher mineral contents; mainly, the Ca2+ content of tef is higher than other crops such as sorghum, barley, wheat, and rice36. The Ca2+ content in the plant varies from 0.1 to 5% of the shoot dry weight4 and the average seed Ca2+ content may vary between 981 and 1811 mg/Kg in tef as compared to 33 mg/Kg in maize, 153 mg/Kg in rice and 533 mg/Kg in wheat35.Tef is an important orphan crop in the Horn of Africa2+ contents were higher than in other major crops such as wheat, rice, and maize37. Low Ca2+ consumption in the diet has been related to several disorders in humans, many of which can have significant health repercussions over time. Because most staple food grains are low in Ca2+, tef, which is an orphan crop with high Ca2+ content, has enormous promise as a nutritional security crop. However, orphan crops’ physiological and transcriptomic response to Ca2+ nutrition has not been studied before.Similarly, in millets such as finger millet, the Ca2+ deficiency and analyzed the physiological and transcriptomic responses. Our findings revealed that prolonged low Ca2+ treatment decreased shoot and root biomass, Ca, K, and copper (Cu) content while significantly suppressing Fe translocation from the roots to the shoot. Transcriptomic analysis using RNA-seq revealed that some members of Ca2+ transporters, including CNGC, GLR and CaUP, signaling genes such as CBP and CDPK and genes implicated in Fe homeostasis such as YSL, OPT, VIT and NRAMP are differentially regulated by Ca2+ treatment or between roots and shoots.This study exposed tef plants to long-term Ca2+ deficiency in tef, plants were grown in a modified Hoagland’s hydroponic solution supplemented with predetermined low (0.01 mM) or optimum (1 mM) Ca2+ for one month, and the plants were analyzed. As shown in Fig. 2+ exhibited typical Ca2+ deficiency symptoms, including leaf tip necrosis and growth stunting, compared to plants grown at optimum Ca2+ level . Plants grown in low Ca2+ media accumulated 0.5 g/Kg Ca+2 in the shoot (LCS) as compared to plants grown at optimum Ca+2 level (ConS), which accumulated 3 g/Kg Ca+2  and optimum Ca+2 (ConR). At the same time, there was a small but statistically significant difference between LCS and ConR , while in shoots, a small but significant decrease in Cu content was observed (0.01 g/Kg in ConS to 0.01 g/kg in LCS) . Complementary DNA libraries were generated from four independent biological replicates and sequenced using the paired-end sequenced using Illumina Hiseq 4000. A total of 476.5 million raw reads with an average of 30 million reads were obtained for each sample The total clean reads of the 16 libraries were aligned to the tef draft genome sequence38. On average 85.7% of the reads were uniquely aligned to the reference genome .To determine the integrity of RNA samples, we performed principal component analysis (PCA). The PCA shows no mark variation among most RNA samples from the same Cance Fig.  and mine2+ conditions. A large number of genes (4.654) were upregulated in roots and shoots of low Ca2+ relative to control, of which 366 and 4009 genes were uniquely upregulated in LCR and LCS relative to ConR and ConS, respectively. A total of 279 genes were commonly upregulated in LCR and LCS. We also detected 1279 DEGs that were downregulated in LCR (179) and LCS (1132) compared to ConR and ConS, respectively. A total of 32 genes were commonly downregulated in LCR and LCS as compared to ConR and ConS, respectively. Ca2+ deficiency also induced significant transcriptomic changes between roots and shoots. A total of 7045 DEGs were upregulated in roots, of which 4833 DEGs were upregulated in LCR relative to LCS, 6670 DEGs were upregulated in ConR relative to ConS, and 4458 DEGs were commonly upregulated in both LCR and ConR. Similarly, 7430 DEGs were downregulated in roots, of which 6,192 DEGs were downregulated in LCR relative to LCS, 6834 DEGs were downregulated in ConR relative to ConS, and 5596 DEGs were commonly downregulated in both LCR and ConR relative to LCS and ConS, respectively. Taken together, the number of transcripts that were upregulated in response to Ca2+ deficiency in both roots and shoots were higher than those whose transcript abundance was downregulated. Moreover, the number of DEGs that were upregulated in ConR versus ConS were higher than those DEGs that were upregulated in LCR versus LCS  enrichment analysis using the AgriGo database to determine the potential function of DEGs identified in our analysis. In the GO analysis, the DEGs were grouped into molecular function, cellular components, and biological process categories Fig. A for the2+ transport in plants. This study identified transcriptomic changes in some families of Ca2+ transporters, including GLRs, CNGCs, Ca2+ uniporters, cations/Ca2+ exchangers, and Ca2+-transporting ATPases. Our analysis identified Ca2+ transporters including Ca2+ uniporters, cation/Ca2+ exchangers and Ca2+ transporting ATPase , CNGC2 (R2 = 9626) and CaE1 (R2 = 9626), while a positive but weak correlation was observed for CNGC17 (R2 = 0.1689). These findings validate the RNA-seq data.To the RNA-seq data, we performed quantitative RT-PCR (qPCR) to analyze the expression of a few DEGs, including +2 in grains than other cereals43. However, the effect of prolonged Ca2+ deficiency on their physiology and transcriptome remained unknown. In the present study, we examined the response of tef to prolonger Ca2+ deficiency based on agronomic, physiological, and transcriptome changes in plants grown in hydroponics solution under optimum and Ca2+ deficient conditions.The Chloridoideae subfamily of Poaceae, including tef and finger millet, accumulate higher levels of Ca2+ deficient conditions showed typical deficiency symptoms in shoots, including leaf curling, tip necrosis, and growth stunting 1. Several Ca2+-permeable channels have been implicated in the uptake of Ca2+ from the soil solution to plant roots48. These include the GLRs, CNGCs, two-pore channels (TPCs), mechanosensitive-like channels (MSLs) etc.49. Ca2+ pumps are involved in maintaining homeostasis by controlling Ca2+ efflux from the cytosol to organelles and/or to the apoplast50 . In our dataset, we identified several DEGs including Ca2+ uniporters (CaUPs), cation/Ca2+ exchangers (CaE), Ca2+-transporting ATPases (CaTA) etc. 56.When plants undergo certain environmental stresses, Ca2+ transport against its concentration gradient and enhance the influx of monovalent cations 57. For example, overexpression of the apple cation/Ca2+ exchanger MdCCX1 in transgenic Arabidopsis and apple root cells has been shown to decrease Na+ accumulation and ROS levels and increase salt tolerance in transgenic cells56. In this study, Ca2+ deficiency decreased Ca and K content in roots. Therefore, upregulation of CaE1 in LCR and LCS could be a mechanism by which roots enhance K+ or Ca2+ acquisition from the external solution or facilitate their distribution between tissues. Transcripts of CaTA9-1, CaTA5-1, CATA9, and CaUP6 are downregulated by Ca2+ deficiency in the shoots. The CaTA5 was the only gene among the transporters that was downregulated by Ca2+ deficiency in roots. CaTA5 is a homolog of the plasma membrane Ca2+-transporting ATPases (PMCAs), which are high-affinity Ca2+-pumps that export Ca2+ ions from the cytosol into the extracellular solution58. CaTA5 may be a functional Ca2+-pump, and its downregulation under Ca2+ deficiency could be a strategy to suppress the cytosolic Ca2+ export to the extracellular solution. The autoinhibited Ca2+-ATPase ACA13 has been implicated in successful pollination in the Brassicaceae by exporting pollen to compatible pollen tubes59.The CaCA superfamily has been reported to facilitate Ca2+ and control plants. In Arabidopsis, Oligopeptide Transporter 1(AtOPT1) is a major nitrate/oligopeptide transporter family60. The Arabidopsis OPT3 is a phloem-specific iron transporter61 and its rice counterpart, OsOPT7, has been implicated in Fe loading into the phloem, the long-distance Fe transport, and loading to seeds63. OPT4 is involved in oligopeptide transport64. Furthermore, Arabidopsis Oligopeptide Transporter 6 (AtOPT6) was reported to transport glutathione65 and a possible connection between prolonged Ca2+ deficiency, redox status, and increased iron transport is a likely scenario as suggested in a recent review28. In this study, transcript levels of several transporters such as OPT1, EtNRAMP5, EtVIT5 and EtYSL2 were upregulated by several folds  across the root cell membrane, while the rice OsYSL2 is implicated in the long-distance transport of Fe associated with a PS precursor, nicotianamine (NA)68. The role of YSL2 in Fe uptake and distribution has been studied previously in maize69 and Arabidopsis71. The NRAMPs such as AtNRAMP3 and AtNRAMP4 function in transporting Fe out of vacuoles under Fe-limited conditions, while members of the VIT family mediate Fe transport into vacuoles72. For example, overexpression of the wheat VIT2 under the control of an endosperm-specific promoter increases Fe content in white flour fractions of wheat73. Some of these transporters are not specific to Fe but also transport other minerals, including Zn, Cd, Cu, and Mn74. Taken together, differential regulation of several mineral transporters in response to Ca nutrition suggests the involvement of Ca2+-signaling in the homeostasis of these minerals. The decrease in root to shoot Fe translocation in this study due to Ca2+ deficiency , promoting Fe uptake in Arabidopsis. Ca2+ promotes the interaction of C2-Domain Protein Enhanced Bending1(EHB1) and the root Fe-regulated transporter IRT1, inhibiting Fe acquisition in Arabidopsis76. However, further studies are needed to understand the crosstalk between Fe and Ca nutrition in Ca and Fe-rich cereals such as tef and finger millet.This study also identified various Fe/metal transporters, including OPTs, VITs, NRAMPs, and YSLs. All these transporters proteins were differentially expressed in the roots and shoots of low Ca2+-transporting channels were differentially expressed by Ca treatment. Our findings showed that transcripts of the CNGC, including CNGC2, CNGC4, and CNGC4-2, were more abundant in the shoots regardless of the Ca treatment 9, it has been shown that GLR1 encodes a non-selective ion channel that is permeable to Ca2+. Upregulation of GLR2.8 and GLR2.9 in roots compared to shoots in this study may suggest their involvement in Ca2+ uptake in the roots. Upregulation of GLR2.8 in LCR compared to ConR may suggest its role in Ca2+ acquisition under Ca2+-deficient conditions. However, the GLRs which were upregulated in the shoots under both high and low Ca2+, such as GLR3.4-1, GLR2.4, and GLR3.1, may play a role in Ca2+ distribution in the shoots, which remains to be studied in the future.Our study also revealed that several Caent Fig. B. This m2+ deficiency severely inhibits the growth of tef plants by disturbing the homeostasis of some minerals, including Ca, K, Fe, Mn, and Cu. Furthermore, our transcriptomic analysis revealed various genes, including those implicated in Ca2+-signaling, Ca2+- transporters, and Ca2+-permeable channels; Fe and metal transports which were differentially regulated by Ca2+ deficiency and/or between root and shoot tissues, suggesting their involvement in Ca2+ homeostasis. Heterologous expression and loss-of-function mutations studies of the DEGs identified in this study would elucidate their physiological functions in tef.In conclusion, we found that CaEragrostis tef) accessions obtained from the USDA-GRIN germplasm collection43. We used the accession with the highest seed Ca (1811 mg/kg) content in this study. Tef seeds were sterilized according to a method previously published with slight modifications80. Briefly, seeds were soaked in 70% ethanol for one minute, followed by soaking in 1% NaOCl solution containing 0.1% Tween-20 for 10 min with vortexing every 1 min. Subsequently, the seeds were washed with autoclaved high purity water (18.2 MΩ (megaohms)) five times. The seeds were then transferred to a petri-dish with a sterile wet filter paper disc. The plates were incubated in a growth chamber  for six days under 16 h/8 h day-night cycles at 28 °C. The seedlings were transferred to a modified Hoagland hydroponic solution81 in which the Ca(NO3)2 was replaced with NH4NO3 to investigate the effect of Ca2+. Plants were grown in the media containing either 1 mM Ca2+ (optimum or control) or 10 µM (low Ca or Ca-deficient). The nutrient solution was continuously aerated and was renewed every 4 days.We recently performed seed mineral profiling of diverse tef .One-month-old tef plants grown in 1/4th-strength Hoagland’s hydroponics solution was analyzed. Roots and shoots were separated and rinsed three times with Milli-Q (18.2 MΩ) water. Before weight measurements, the tissues were oven dried at 65 °C overnight and ground to fine powder using a Waring Laboratory spice blender. Briefly, 500 mg of ground tissue was digested using concentrated HNOFor RNA extraction, root and shoot samples were ground into powder under liquid nitrogen using a mortar and pestle. Total RNA was isolated using the GeneJET RNA purification kit following manufacturer’s procedure . The quality and quantity of the RNA were determined using a Nanodrop . mRNA-seq libraries were prepared using NEBNext® Ultra II RNA Library Prep Kit for Illumina  according to the manufacturer’s instructions. For each library, 500 ng total RNA was used. The mRNA-seq libraries were sequenced on an Illumina Hiseq platform using the 150 bp paired-end sequencing strategy.sickle with the options − q 12 and − l 15 38. All default parameters were used except –very-sensitive and –summary-file options passed to the hisat2 aligner83. The number of reads mapped to a genome feature was determined using htseq-count command line script from the HTSeq python package84(-s reverse -a 10 -t exon -I Parent -m union) and count data was imported to the R statistical computing environment for further analysis42. Initially, genes that have no count in at least 4 of the replicate samples were excluded from the analysis, and the remaining 28,796 features were used for downstream analysis. Data normalization based on dispersion and differential analysis were conducted using the DESeq2 package85. We fitted a generalized linear model (~ Treatment) between the expression count. Finally, differential expression analysis was performed between ConR verus LCR, ConS versus LCS, Con R versus ConS, and LCR versus LCS, and differential expressions were identified by applying multiple testing correction using Benjamin-Hockberg procedure86.Data analysis for RNA-Seq was performed by the Bioinformatics Core associated with the Center for Human Health and Environment at North Carolina State University. Quality control of read data was briefly evaluated with FastQC, and alignment trimming was performed using GLR3.1, 5′-tcatcttccatgctgccaaaggc-3′ and 5′-tctccagttgggccattgtgtc-3′ for CNGC2, 5′-gcgactcgaggtgtgaatgaagag-3′ and 5′-aatggaacccgccgaacaagac-3′ for CNGC17, and 5′-tgctgcaattccagagggcttg-3′ and 5′-tcacagttgtgcaacccaatgtc-3′ were used to amplify CaE1. The internal control, eukaryotic elongation factor (EF1A) was used as a housekeeping gene and amplified using sense 5′-catcaacatcgtggtcatcg and antisense 5′-gatgcctccaagcttgtaga-3′ primers. Gene expression in the untreated samples were used as a calibrator, and blank (without template) was used as a reference control. The qPCR parameters were as follows: 950C for 30 s, 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and a final incubation at 72 °C for 5 min followed by melting curve analysis. Relative expression levels based on fold changes were calculated using the ΔΔCT method87 available with the QuantStudio3 software  and in MS-Excel .To validate the RNA-seq data for representative genes, quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed using a QuantStudio 3 real-time PCR system and the PowerUP™ SYBR Green pre-formulated 2 × master mix . Total RNA was treated with DNase I  to remove contaminating genomic DNA before first-strand cDNA synthesis. First-strand cDNA was synthesized using 5 µg of total RNA and the SuperScript® III First-Strand Synthesis System  following the manufacturer’s instructions. First-strand cDNA was used in the real-time qRT-PCR reaction containing 2xSYBR Green Master Mix, 500 nM forward, and reverse gene-specific primers in a 20 uL reaction volume. Gene-specific sense and antisense primers 5′-tttggctcctgggactcctaac-3′ and 5′-ggccagattgcagtgtagagtttc-3′ for Eragrostis tef were annotated by comparing it to the genome sequences of Setaria italica (GCF_000263155.2), Oryza sativa88, and Sorghum bicolor (GCF_000003195.3). The CDS of E. tef were compared to S. italica, O. sativa, and S. bicolor using Blastx. A CDS was considered significant with e-value < 0.00001. Significant hits were first annotated by comparison to S. italica , then to O. sativa  and S. bicolor . A total of 94.6% of the CDS of E. tef had a significant hit to a CDS of these three genomes, and the corresponding gene annotations were used.Coding sequences (CDS) of 89. Signature genes from the RNAseq analysis were used as an INPUT, and significantly enriched pathways and/or families were selected based on molecular function, cellular structure, and biological process category.Go analysis was performed using the agriGO90. After the significant F-tests, the Tukey’s multiple comparisons92 were used to separate the means (P ≤ 0.05). Values represent mean ± SD of six (Figs. Treatments were replicated four times, and two independent experiments were conducted. Data were analyzed by the one-way ANOVA using the PROC GLM procedure in SASix Figs. B,C and fTef is primarily cultivated in East Africa and is becoming popular in several countries including South Africa, Australia, and the U.S. The tef accessions used in this study was obtained from Dr. John Cushman at the University of Nevada Reno. All methods are performed in accordance with relevant guidelines and regulations.Supplementary Information 1.Supplementary Information 2.


Acanthamoeba is an emerging pathogen, infamous for its resilience against antiprotozoal compounds, disinfectants and harsh environments. It is known to cause keratitis, a sight-threatening, painful and difficult to treat corneal infection which is often reported among contact lens wearers and patients with ocular trauma. Acanthamoeba comprises over 24 species and currently 23 genotypes (T1-T23) have been identified.Acanthamoeba species and genotypes recovered from patients with Acanthamoeba keratitis (AK), determine the presence of endosymbionts in ocular isolates of Acanthamoeba and review the clinical presentations.This retrospective study was designed to examine the Acanthamoeba isolates were identified by sequencing the ribosomal nuclear subunit (rns) gene. Acanthamoeba isolates were assessed for the presence of bacterial or fungal endosymbionts using molecular assays, PCR and fluorescence in situ hybridization (FISH).Thirteen culture-confirmed AK patients treated in a tertiary eye care facility in Hyderabad, India from February to October 2020 were included in this study. The clinical manifestations, medications and visual outcomes of all patients were obtained from medical records. The A. culbertsoni  was the most common species, followed by A. polyphaga and A. triangularis. Most of the isolates (12/13) belonged to genotype T4 and one was a T12; three sub-clusters T4A, T4B, and T4F were identified within the T4 genotype. There was no significant association between Acanthamoeba types and clinical outcomes. Eight (61.5%) isolates harboured intracellular bacteria and one contained Malassezia restricta. The presence of intracellular microbes was associated with a higher proportion of stromal infiltrates , epithelial defect  and hypopyon  compared to 50% (2/4), 25% (1/4) and 25% (1/4) AK cases without intracellular microbes, respectively.The mean age of the patients was 33 years . Six (46.2%) cases had AK associated risk factors; four patients had ocular trauma and two were contact lens wearers. Acanthamoeba clinical isolates in this study harboured intracellular microbes, which may impact clinical characteristics of AK.Genotype T4 was the predominant isolate in southern India. This is the second report of T12 genotype identified from AK patient in India, which is rarely reported worldwide. The majority of the The online version contains supplementary material available at 10.1186/s12879-022-07741-4. Acanthamoeba keratitis (AK) is a rare eye disease accounting for 2% of global corneal infections . Smear and inoculation of all media were done (as is standard) by the attending clinician at the slit lamp. Dehydrated culture mediawere supplied by HiMedia, Mumbai, India and freshly prepared in-house for use. Bandage contact lenses (2/13) were aseptically cut into small pieces and inoculated into BA, CA, SDA, NNA and BHI in the microbiology laboratory. All media were incubated aerobically at 37 °C except for SDA and PDA at 27 °C and chocolate agar which was incubated in 5% CO2 at 37 °C. The media were observed for 14 days for any growth. Acanthamoeba isolates from corneal scraping of 13 patients were confirmed by observation of active trophozoites and double-walled polygonal cysts; confirmed isolates were preserved on NNA and transported to School of Optometry and Vision Science, Faculty of Medicine and Health, UNSW, Sydney, Australia. The thermotolerance of isolates was evaluated by growing isolates at 28 °C, 37 °C, 40 °C and 42 °C. Following initial isolation, the Acanthamoeba strains were grown axenically in peptone yeast glucose (PYG) medium [During the study period, as per institutional protocol, all patients presenting with clinical features of microbial keratitis underwent a comprehensive slit-lamp examination for investigation of the corneal epithelial defect, size of infiltrate (if present), size of hypopyon, involvement of sclera, absence/presence of foreign particles and documentation of the ocular adnexal status . The micAcanthamoeba strains were further identified by PCR assay. Amoebal cells grown on NNA were collected into 500µL of 1X PBS  using a sterile scraper . Genomic DNA (gDNA) was extracted using 10% v/v Chelex lysis solution  in 0.1% v/v Triton X-100 (Sigma-Aldrich) and Tris buffer, pH 8.0 , as previously explained [xplained . The quarns gene region of 18S rRNA was amplified in a PCR reaction using Acanthamoeba genus specific primers, JDPFw (5’-GGCCCAGATCGTTTACCGTGAA-3’) and JDPRv (5’-TCTCACAAGCTGCTAGGGGAGTCA-3’), which encode the highly variable DF3 region and give amplicons of ~ 450 bp [2; Thermo Fisher Scientific), 1µL of each primer (10 µM), 6.5µL of molecular grade water and 4µL of DNA template. PCR amplification was carried out in a 96-well T100 thermal cycler  using the following thermal cycling conditions: 95 °C for 5 min for initial denaturation, followed by 35 cycles of amplification  and a final extension at 72 °C for 10 min [The ~ 450 bp . Each PCr 10 min . PCR ampAcanthamoeba genotypes and species. Additionally, isolated Acanthamoeba strains were morphologically characterized to the species based on the classification scheme of Pussard and Pons [Acanthamoeba genotypes were retrieved from the NCBI as reference strains for phylogenetic analysis  software  . The triand Pons . All theAcanthamoeba were grown axenically and maintained in PYG  [2+ ions [ pH 6.5) . All iso[2+ ions . To enhaE. coli ATCC 10,798 and nuclease free water were included as positive and negative controls, respectively. The presence of intracellular fungi was assessed using fungus-specific forward ITS1Fw (5’- TCCGTAGGTGAACCTGCGG-3’) and reverse ITS4Rv (5’-CCTCCGCTTATTGATATGC-3’) primers targeting the conserved sequences of 18S and 28S rDNAs [Candida albicans was included as a control and intracellular fungus was identified by Sanger sequencing of PCR amplicon.The 16S rRNA gene (V4 region) of intracellular bacteria was amplified using eubacteria 16S rRNA primer; 515Fw/806Rv: Fw (5′- CCTACGGGNGGCWGCAG-3′) and Rv (5’ – GACTACHVGGGTATCTAATCC-3′) . Genomic8S rDNAs . A cliniTable In order to visualise the intracellular bacteria or fungi, FISH in combination with confocal microscopy was performed following a previous protocol . BrieflyDemographic and clinical features of all 13 AK patients were retrieved in a customised datasheet from hospital records and were reviewed for the following features: initial and final visual acuity (VA); reported symptoms; duration, size and characteristics of infiltrates and epithelial defects ; prior treatment (defined as medicines used before the first presentation of patients at LVPEI); clinical treatment regimens and duration under care at LVPEI; need for surgery; treatment outcome; patients’ occupation; and reported risk factors for AK .P value < 0.05 for 2-tailed tests.Data analysis was performed in SPSS software version 26.0 . Proportions were presented as percentage and mean ± standard deviation was calculated for continuous data. A 95% confidence interval (CI) was calculated for demographic and clinical data using proportion (p) ± 1.96* SEM (standard error of mean). Fisher’s exact and chi square were used for comparison of demographic and clinical data with endosymbiont status with the level of significance of Among the 13 AK patients, the highest number of cases  were observed in February . Figure Acanthamoeba, of which eight  harboured intracellular bacteria and one isolate (L-2429/20) contained fungus. No bacteria grew from the PYG aliquots cultured on trypticase soy agar, suggesting that any bacteria identified in FISH experiments were very likely to be located intracellularly.Identification of the ate L-248/20. FiguA. culbertsoni; L-579/20, L-1326/20: A. polyphaga; L-1137/20, L-2429/20: A. triangularis, and L-604/20, L-1765/20: Acanthamoeba spp. T4) and one isolate (L-2391/20) being genotype T12 , cyst morphology was not obvious to a particular species of Acanthamoeba but the NCBI nucleotide blasting, and phylogenetic analysis confirmed both strains belonged to genotype T4. Genotype T12 was detected from a farmer from Maharashtra with no history of ocular trauma; two other cases from the same state were infected with genotype T4. The remaining ten isolates of T4 variants were recovered from AK patients from Andhra Pradesh, Rajasthan, Telangana, Tripura, and Uttar Pradesh  was with T4A Acanthamoeba spp.  were positive for intracellular microbes with eight harbouring bacteria  or contact lens wear (n = 2) and three were farmers. A severe form of AK with stromal infiltrate  and hypopyon  was noted in the presence of amoebal endosymbionts and five cases had both stromal infiltrate and hypopyon. However, these differences in stromal infiltrate (p = 0.2) and hypopyon (p = 1.0) were not significantly more common in AK patients with amoebal endosymbionts compared to those without the endosymbionts. Interestingly, a longer medical treatment duration  was observed in patients without Acanthamoeba endosymbionts (p = 0.2). In the presence of endosymbionts, the proportion of patients with a healing ulcer (66.6%) or an improvement in final VA (44.4%) were lower compared to those without endosymbionts  (p > 0.05). Two patients  infected by Acanthamoeba with bacterial endosymbionts had received antibiotics along with antiamoebic drugs and the ulcers of both cases were resolved after medication.The mean symptom duration of AK patients infected by p > 0.05) in BCVA at final presentation compared to cases treated only with PHMB and chlorohexidine. Overall, the median duration of medical treatment was 38 days (IQR = 23—90). Of six cases with surgical treatment, 4 cases had therapeutic penetrating keratoplasty , including one case that had an amniotic membrane transplant (Table Acanthamoeba strains with intracellular bacteria.Most of the AK patients were farmers 5/13) or students (5/13) and the reported risk factors associated with AK were ocular trauma  and contact lens wear . Unilateral AK was reported in 12 (92.3%) cases. The majority of AK patients had decreased vision (84.6%) as a major symptom followed by eye pain (69.2%), redness (69.2%), watering (53.8%), and white spot on the cornea (15.4%). A typical AK characteristic ring infiltrate (> 4 mm) was observed in 3 (23.1%) cases. Epithelial defect was noted in 6 cases , stromal infiltrates in 10 cases , and hypopyon in 6 cases . The median duration of symptoms onset was 20 days (IQR = 15—30) and the final visual acuity was not improved by ≥ 2 lines among patients with farming background , age > 32 years  and patients showing AK symptoms more than 20 days  , as has been reported globally [A. culbertsoni  was the predominant species among the T4 genotype, and this has been previously reported in India [Thirteen clinical isolates of globally –48. A. cin India . One isoin India .A. healyi T12 genotype from corneal infection in India [Acanthamoeba isolates recovered from Chilean AK patients, followed by T4B, T4G, T4C and T4D [The current study supports that the predominant keratitis causing T4 genotype is also prevalent in multiple states  of India. To the best of the authors’ knowledge, this is only the second report of in India . A severin India  where thin India . T4A , patients aged > 32 years (80%) and cases showing keratitis symptoms for > 20 days (50%). A study from the UK has also reported worst clinical outcomes for AK patients aged > 34 years . Cornealrn China .Acanthamoeba isolates, 8 (61.5%) possessed intracellular bacteria and this is very similar to the 59.4% of Acanthamoeba possessing intracellular bacteria in a study from the USA [Pseudomonas, Legionella, Chlamydia, and Mycobacterium have been detected in Acanthamoeba isolates recovered from AK cases in the USA [Mycobacterium and Parachlamydia spp. were detected in ATCC strains isolated from human nasal swab, cornea, and lake sediment, respectively [Acanthamoeba from different locations. Co-occurrence of phylogenetically diverse intracellular microbes within a Acanthamoeba cell has been observed [Aspergillus spp., P. aeruginosa, and HAdV (human adenovirus) were detected in a clinical isolate of Acanthamoeba in Iran [M. restricta as an endosymbiont of Acanthamoeba. The patient was a farmer with a history of eye trauma with a chemical agent and the corneal ulcer was not resolved with medical treatment using PHMB and chlorhexidine for a month. Acanthamoeba and Malassezia coinfection has not been described in AK patients but M. restricta has infrequently caused fungal keratitis [Acanthamoeba with fungi such as Cladosporium, Fusarium and Curvularia [Among the 13  the USA , but gre the USA  or an ol the USA  while Riectively . Fritschobserved ; Aspergilates, 8 .5% posseeratitis . Other srvularia , 63, 64.Acanthamoeba may enhance corneal epithelial damage as has been shown in a clinical study and a cell model [Intracellular bacteria of ll model . AlthougAcanthamoeba with bacterial endosymbionts had received antibiotics (ciprofloxacin and doxycycline) along with topical antiamoebic drugs and the ulcer of both cases resolved after treatment. The remaining cases of AK with intracellular bacteria healed when treated only with PHMB and chlorhexidine. This is expected as these antiseptics are also effective against bacteria [Acanthamoeba keratitis. Future studies are necessary to examine the impact of amoebic endosymbionts on host pathogenicity, drug susceptibility, clinical outcome, and benefits of adding antibacterial adjuvants to standard antiamoebic chemotherapy with higher cohort and extended follow-up of AK patients.In the current study, two cases 25%, 2/8) affected by bacteria . Additiobacteria . On the bacteria  and the %, 2/8 afAcanthamoeba isolates were mainly of T4 genotype followed by T12, with three sub-genomic variants T4A, T4B, and T4F identified within the predominant T4 genotype. There was no significant association between Acanthamoeba species and/or genotypes and a patient’s clinical outcome. In the current study, a high number of Acanthamoeba strains with endosymbionts were observed and although there was no clear relationship with clinical outcome, Acanthamoeba endosymbionts may be involved in shaping virulence, survival, and drug susceptibility of amoebal host.This study confirms that the keratitis causing Additional file 1: Table S1: Reference Acanthamoeba strains used in this study for phylogenetic analysis. Table 2: Genotype and species identification of Acanthamoeba isolates recovered from AK patients. Map 1: Map showing the states of the AK patients from different states of India with sample codes and identified genotypes of Acanthamoeba from AK patients. The map was created using ArcGIS . Fig. S1. Monthly distribution of AK cases during study period. Fig. S2. Sequence alignment of Acanthamoeba 18S rDNA DF3 region using ClustalW. Table S3. Overall clinical presentation of the keratitis patients infected with Acanthamoeba spp. Fig S3. Phylogenetic tree inferred from the 18S (ITS1) rDNA sequence of fungi; tree was created using the neighbour-joining approach with the Kimura 2-parameter based on 1000 replicates bootstrap values. Fig. S4. Agarose (1%) gel image of PCR amplicons of 13 Acanthamoeba isolates (18S rRNA), PCR assay was performed using Acanthamoeba genus specific primer pair JDPFw and JDPRv which yielded ~450bp amplicons. Fig. S5: Agarose (1%) gel image of PCR amplicons of 13 Acanthamoeba isolates targeting intracellular bacteria 16S rRNA, primer pair 515Fw and 806Rv  was used which yielded ~293bp amplicons.


Musculus rhomboideus of piglets isolated on days 5  and 20  of age were used. Our expression analyses highlighted differentially expressed genes in porcine myoblasts cultures under heat or cold induced stress. These gene sets showed enrichment for biological processes and pathways related to organelle fission, cell cycle, chromosome organization, and DNA replication. Culture at 35°C resulted in increased metabolic flux as well as a greater abundance of transcripts of the cold shock protein-encoding gene RBM3 and those of genes related to biological processes and signaling pathways, especially those involving the immune system . For cultivation at 39°C, differences in the expression of genes related to DNA replication and cell growth were identified. The highest glutathione index ratio was also found under 39°C. Meanwhile, cultivation at 41°C induced a heat stress response, including the upregulation of HSP70 expression and the downregulation of many biological processes and signaling pathways related to proliferative ability. Our analysis also identified differentially expressed genes between cells of donors with a not yet (P5) and already fully developed (P20) capacity for thermoregulation at different cultivation temperatures. When comparing P5 and P20, most of the changes in gene expression were detected at 37°C. At this optimal temperature, muscle cells can develop to their full capacity. Therefore, the most diverse molecular signaling pathways, including PI3K-Akt signaling, Wnt signaling, and EGFR tyrosine kinase inhibitor, were found and are more pronounced in muscle cells from 20-day-old piglets. These results contribute to a better understanding of the mechanisms underlying the adaptation of skeletal muscle cells to temperature stress in terms of their thermoregulatory ability.Rapid climate change is associated with frequent extreme heat events and the resulting thermal stress has consequences for the health, welfare, and growth of farm animals. The aim of this study was to characterize the transcriptional changes and the effects on energy metabolism in proliferating porcine myoblasts derived from piglets of different ages, representing differences in thermoregulatory abilities, and cultivated below (35°C) and above  the standard cultivation temperature (37°C). Satellite cells originating from  The natural environment of the muscle fiber type and its origin play an important role in controlling satellite cell properties . Additioin vitro , while tin vitro . Muscle in vitro . Low temin vitro . Relativin vitro  and diffin vitro  stages.We hypothesize that satellite cell-derived cell cultures are able to mimic muscular adaptation to temperature stress as well as exhibit distinct gene expression patterns that reflect their developmental commitment and influence their responsiveness to thermal stress. Therefore, we cultured proliferating myoblasts below (35°C) and above  the standard cultivation temperature (37°C) and evaluated the effects on the transcriptome, oxidative stress and energetic metabolism, using our well-established cell pooling approach . We alsoM. rhomboideus of 10 female five- and 20 days old piglets and the establishment and validation of two muscle cell pools  were performed as previously described  for microarray analysis. To explore mitochondrial and glycolytic functional changes, 2,000 cells/well and 20 wells per pool per replicate  were used. To estimate the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), 3,000 cells/well and 10 wells per pool per replicate were used . Three replicates were generated for each experiment.The isolation of satellite cells from the kursiv escribed . For pron = 12 per pool) were hybridized on the microarrays. Afterwards washing and scanning was performed according to manufacturer’s recommendations. Quality control was performed using Affymetrix GCOC 1.1.1. software. Expression Console software was used for robust multichip average (RMA) normalization and the detection above background (DABG) algorithm was used to detect the genes that were present. Probe sets with low signal and those that were present in less than 80% of the samples within each temperature group were excluded. After filtering, 13,226 probe sets were finally used for further analyses.Total RNA was isolated from cells after 72 h of proliferative growth using TRIzol reagent (Sigma-Aldrich) and a RNeasy Mini Kit  according to the manufacturer’s instructions. Porcine Snowball Microarrays  containing 47,880 probe-sets were used in this study. For cDNA synthesis, 500 ng of total RNA was used and subsequent biotin labelling was performed with the Affymetrix WT plus Expression Kit (Affymetrix) and Genechip WT terminal labeling and hybridization Kit (Affymetrix) according to the manufacturer’s instructions. A total of 24 label cRNA samples .Differential expression analysis was performed using mixed model analysis in JMP genomics . Temperature , pool (P5 or P 20) and the interaction of pool and temperature were used as fixed factors. Differences between least square means (LSMs) were analyzed using Tukey-Kramer tests Adjustments for multiple comparisons were performed using the p-values, while dot-plots generated using the R package ggplot2 were used to visualize the DAVID enrichment analysis results. p ≤ 0.05 was considered significant for biological processes and KEGG pathways.To identify relevant functional categories across temperatures, pools, and pools under specific temperatures, gene ontology (GO) and KEGG pathway enrichment analysis of differentially expressed genes (DEGs) was performed using WebGestalt 2019 [WEB-based Gene SeT AnaLysis Toolkit ] and DAVAREG, MYH3, TBP, ACTB, F - 5′ CTGGCACCACACCTTCTAC - GGGTCATCTTCTCACGGTTG 3′), proliferating cell nuclear antigen , by pool (p = 0.281) or by the interaction between temperature and pool (p = 0.656). The LSM ± standard errors (SE) of four genes AREG, PCNA, MYH3, and HSP70 have been published before  was used for the evaluation of the microarray results. RNA isolation, reserve transcription, and qPCR were performed as described by en PCNA, , hypoxand before  and was p < 0.05 were considered significant.Mitochondrial and glycolytic functions were analyzed using the Seahorse XFp Extracellular Metabolic Flux Analyzer, as described in DES, ACTB, LMNA), proliferation , immune responses , and prostaglandin biosynthesis (prostaglandin-endoperoxide synthase 2) PTGS2, IGF1, and AREG are growth factors and RNA-binding motif protein 3 (RBM3) is a cold-shock marker.The microarray-based expression profiles of myoblasts after 72 h of proliferation at 35, 39, and 41°C were compared with those cultured at the standard cultivation temperature of 37°C  and the We used DEGs from each comparison of cultured proliferating myoblasts  for GO and KEGG pathway enrichment analysis . For bioAt 35°C, myoblasts showed an enrichment of DEGs associated with the immune response, RNA processing, regulation of immune system process, and regulation of multi-organism process. Specifically, the DEGs in myoblasts cultured at 35°C (low-temperature stress) were enriched in the KEGG pathways of cytokine–cytokine receptor interaction, interleukin 17 (IL-17) signaling pathway, cell cycle, DNA replication, signaling pathway, and TNF signaling pathway. GO enrichment analysis showed that, at 39°C, DEGs were enriched in the biological process of protein-DNA complex subunit organization and the cellular components ribonucleoprotein complex, vesicle, and Golgi apparatus. At 39°C, one KEGG pathway was identified, namely, DNA replication. The most enriched GO terms for the DEGs in myoblasts cultured at 41°C compared with those cultured at 37°C were heterogeneous and included regulation of signaling receptor activity, cell division, regulation of organelle organization, chromosome segregation, response to organic cyclic compound, reproduction, response to lipid, cytoskeleton, signaling receptor regulator activity, and kinase binding. For the highest temperature tested (41°C), three KEGG pathways were prominently represented—DNA replication, cell cycle, and pyrimidine metabolism.A total of 503 DEGs were detected between P5 and P20 after 72 h of proliferation at 35, 37, 39, or 41°C, 340 of which were upregulated and 163 downregulated . For theWhen comparing the transcriptomes of the donor cell pools (P5 and P20), the identified DEGs were found to be enriched in key biological processes that included regulation of signal transduction, regulation of protein metabolism, regulation of gene expression, regulation of biological processes, positive regulation of metabolic processes, nervous system development, developmental processes, cell differentiation, and biological regulation . Our anaWe also undertook a functional annotation analysis of the interaction of culture temperature with the two donor cell pools. Detailed information regarding the biological processes associated with the DEGs for each interaction between pool and temperature is shown in Important KEGG pathways affected by the donor piglet age (P5 and P20) under different cultivation temperatures were identified . For theMYH3, DES, and ACTB are involved in muscle structure; AREG, IGFBP5, and FST are growth factors or their binding proteins; and HSP70 is a heat shock protein. The TBP, HPRT1, and PCNA genes are associated with mitogenesis and proliferation and the HDC gene is associated with amino acid transport. The microarray and qPCR data showed a high correlation based on Pearson’s correlation coefficient (r), as follows: MYH3 , DES , ACTB , AREG , IGFBP5 , FST , HSP70 , TBP , HPRT1 , PCNA , and HDC .Eleven genes were selected for the validation of the microarray data by qPCR . The mRNp < 0.001) and pool (p < 0.01). The highest levels were detected at 35°C . Additionally, non-mitochondrial respiration levels were higher in P5 (1.053 ± 0.065 pmol/min/µg of protein) than in P20 (0.754 ± 0.064 pmol/min/µg of protein). Basal respiration levels were affected by temperature (p < 0.05) but not pool. Basal respiration at 35°C was higher than that at 41°C (p < 0.05) but not at 37°C or 39°C. Similarly, maximal respiration levels were affected by temperature (p < 0.01) but not pool (p < 0.10). The highest respiration levels were recorded at 35°C . Proton leak levels were affected by temperature (p < 0.01) but not pool, and were higher at 35°C than at 37 and 41°C (p < 0.05 for both), but not at 39°C. ATP production levels displayed the same trend as the proton leak levels (p < 0.01). The spare respiratory capacity was unaffected by temperature or pool. However, spare respiratory capacity at 35°C was higher than that at 41°C (p < 0.05). None of the above parameters were affected by temperature/pool interaction.For the evaluation of metabolic flux, we next measured the OCR of the myoblasts . The levp < 0.05) but not pool or their interaction. The levels of non-glycolytic acidification were significantly higher at 35°C than at 37°C or 41°C (p < 0.05) but not 39°C. For glycolytic capacity, glycolysis and glycolytic reserve were unaffected by temperature, pool or their interaction.For the characterization of glycolytic stress, ECAR levels were measured at different points . The levp < 0.05) but not pool or temperature/pool interaction. The only difference was found between the two highest culture temperatures, with a higher ratio being detected at 39°C than at 41°C (p < 0.05). The results are shown in GSH is an important scavenger of reactive oxygen species (ROS). The GSH/GSSG ratio is a valuable biomarker of oxidative stress and was found to be affected by temperature , which codes for a pro/anti-inflammatory enzyme (ACSL4) expression is also associated with sensitivity to ferroptosis , a known transcriptional co-activator involved in mitochondriogenesis and mitochondrial energy metabolism  were primarily assigned to the GO terms of cell cycle, chromosome, DNA packaging complex, ribonucleoprotein complex, chromosome organization, protein-DNA complex subunit organization, Golgi apparatus, and vesicle.PCNA mRNA expression levels observed in our previous study (EGFR) and insulin-like growth factor 1 receptor (IGF1R)) with cultivation at 39°C was also found in our former study  or already fully developed (P20) capacity for thermoregulation. Understanding the biological effect of temperature stress on muscle cells in aging is important, especially in new-born piglets, which are still sensitive to environmental temperatures. Satellite cell activity was reported to be affected by the origin of donor cells, such as those obtained following maternal nutrient restriction or intrauterine growth restriction . Previoup < 0.001). The GSH:GSSG index, an indicator of oxidative stress, tended to be higher in P20 (p < 0.08). These results suggested that the muscle cells of a 5-day-old donor piglet are more susceptible to ferroptosis when exposed to cold temperatures than those of 20-day-old piglets.Our study also focused on identifying differences in the transcriptomic profiles of porcine muscle cells derived from donor piglets of different ages and continuously cultured at 35, 37 (control), 39, or 41°C. Most of molecular pathways changes when comparing cells of P5 vs. P20 were found at 37°C. At this optimal temperature, the muscle cells can develop to their full capacity and show the most different molecular pathways including PPAR signaling, PI3K-Akt signaling, Wnt signaling pathways and EGFR tyrosine kinase inhibitor. Most of the transcripts enriched in these pathways were more highly expressed in P20 than in P5. However, only small changes between P5 and P20 were detected at temperatures above or below 37°C. We found that the positive regulation of the biological process, the positive regulation of the cellular process, and the developmental process were also found at 39°C, the physiological body temperature of the piglets, when comparing P5 and P20. Interesting, at 35°C, the identified DEGs were enriched in pentose phosphate pathway (PPP) as well as iron-dependent lipid peroxidation (ferroptosis), which mediates programmed cell death. The glutathione (GSH) system is the main ferroptosis-limiting pathway . We founIn this study, we focused on the transcriptional profile and energy metabolism of primary porcine muscles derived from piglets of different ages (P5 vs. P20) after continuous cultivation for 72 h at 35, 39, or 41°C compared with that at 37°C, the standard cultivation temperature.RBM3, which encodes a cold-inducible mRNA binding protein, but not a HSP-related response. At 39°C, in addition to cell growth, other GO terms related to protein-DNA complex subunit organization, ribonucleoprotein binding, vesicle, and Golgi apparatus were found to be enriched, suggesting that myoblasts were more developed and more highly structured at this temperature. Only cultivation at 41°C resulted in increased expression of HSPs, indicative of induced heat shock and DNA damage processing responses. The GO terms and pathways associated with pyrimidine metabolism, cell cycle, DNA replication, and cytoskeleton represent the termination of the proliferative ability and cytoskeletal reorganization in porcine myoblast after 72 h of continuous cultivation at 41 °C. When comparing cells from animals of different ages (P5 vs. P20), most molecular changes were found at the control temperature (37°C), which is the optimal physiological temperature. Although only subtle changes in transcript levels were recorded between P5 and P20 at temperatures both above and below 37°C, we nevertheless identified changes in gene expression patterns that reflect the developmental fate of the myoblasts and influence their responsiveness to thermal stress.Similar patterns of affected GO terms related to organelle fission, cell cycle or chromosome organisation and the KEGG pathway DNA replication were found at the three experimental temperatures compared with cultivation at the control temperature. Cultivation at 35°C stimulated transcriptional responses in immune-related pathways, such as cytokine–cytokine receptor interactions and the IL-17 and TNF signaling pathways. Furthermore, cultivation at 35°C leads to an increase in the expression of


Later expression was observed in the thymus, brain, retina and exocrine pancreas. Zebrafish crlf3 mutants generated by genome editing technology exhibited a significant reduction in primitive hematopoiesis and early definitive hematopoiesis, with decreased early progenitors impacting on multiple lineages. No other obvious phenotypes were observed in the crlf3 mutants.Cytokine receptor-like factor 3 (CRLF3) is an ancient protein conserved across metazoans that contains an archetypal cytokine receptor homology domain (CHD). This domain is found in cytokine receptors present in bilateria, including higher vertebrates, that play key roles in a variety of developmental and homeostatic processes, particularly relating to blood and immune cells. However, understanding of CRLF3 itself remains very limited. This study aimed to investigate this evolutionarily significant protein by studying its embryonic expression and function in early development, particularly of blood and immune cells, using zebrafish as a model. Expression of  Drosophila melanogaster) that acts as a classical cytokine receptor (Cytokines and their specific receptors represent key components of cell-to-cell communication in multicellular organisms . Togethereceptor . All usereceptor .Cytokine receptor like factor 3 (CRLF3) consists mostly of a class I CHD . This anvia a conserved program of development involving distinct waves  with DIG-labeled probes as described  methanol, 50% (v/v) methanol, 25% (v/v) methanol and PBS before 4 × 5 min washes in PBS containing 0.1% Tween-20 (PBS-T). Embryos were then incubated at room temperature in PBS-T containing 10 µg/mL proteinase K for 1-30 min before fixing for 20 min in 4% (v/v) PFA/PBS followed by 5 × 5 min washes in PBS-T. Embryos were incubated in hybridization mix (HM)  for 2-5 h at 70°C. Embryos were then bathed in probe solution (1/50–1/300 dilution of DIG-labelled RNA in HM+) and incubated at 70°C overnight. Probe solution was removed and embryos washed briefly in 100% HM–  and then sequentially for 15 min in 75% HM–/25% 2×SSC, 50% HM–/50% 2×SSC, 25% HM–/75% 2×SSC and 2×SSC, then twice in 0.2×SSC 30 min, then sequentially for 10 min in 75% 0.2×SSC/25% PBS-T, 50% 0.2×SSC/50% PBS-T, 25% 0.2×SSC/75% PBS-T and PBS-T. Embryos were then incubated in PBS-T containing 2% sheep serum and 2 mg/mL BSA at room temperature for 3 h then in antibody solution  at 4°C overnight with agitation. Embryos were rinsed in PBS-T then 6 × 15 min washes in PBS-T at room temperature with gentle rocking and then 3 × 5 min washes in staining buffer  Tween 20) containing 25 µg/mL levamisol. Following this, embryos placed in staining buffer containing 0.225 mg/mL nitroblue tetrazolium (NBT) and 90.175 mg/mL 5-bromo,4-chloro,3-indolyl phosphate (BCIP) at room temperature in the dark for 4 h-3 d. Embryos were finally rinsed in PBS-T and fixed in 4% PFA/PBS when stained to an appropriate extent. In some experiments, embryos were injected at the 1-4 cell stage with standard control or lycat morpholinos as described  paraformaldehyde (PFA) made in phosphate-buffered saline (PBS). Fixed embryos were subjected to whole-mount escribed . To makeescribed . Embryosescribed . These wcrlf3 gene was targeted using genome editing with transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9  software. To determine the statistical significance of various treatments, the unpaired independent student’s Protein sequences obtained from GenBank were aligned with Clustal Omega (version 1.2.4) , with a crlf3 during zebrafish embryogenesis was examined by WISH with a full-length anti-sense probe in comparison to a control sense probe. The sense probe gave a small amount of diffuse background staining  gene  (rag1 (p < 0.001) (tcra (p < 0.001) (p = 0.022) and neutrophils (p < 0.001)  (rag1  (p < 0.01) . Moreover, heterozygotes of either mutant produced no discernible phenotype (data not shown). Therefore, homozygous crlf3 mutants were analyzed with respect to early hematopoiesis in comparison to wild-type individuals. During the primitive wave of hematopoiesis, the crlf3 mutants showed no alterations in the expression of markers for bipotential hemangioblast cells or vascular precursors, but those for hematopoietic, myeloid and erythroid progenitors were reduced, as were markers for mature myeloid and erythrocytic cells. Collectively, these results indicate crlf3 likely impacts primitive hematopoiesis at the level of hematopoietic progenitors that affects downstream lineages. During definitive hematopoiesis, the markers for HSCs, mature myeloid and erythroid cells remained reduced, as did those for mature T lymphocytes – although not lymphocyte precursors. This suggests a broad role for crlf3 in definitive hematopoiesis, affecting HSCs and their downstream lineages as well as the production of mature lymphocytes. In each case, however, the impacts were relatively modest indicating it is not an essential regulator.Genome editing was used to generate two mutants of zebrafish CRLF3 variants have been associated with lymphocyte percentage in the blood .The broad embryonic expression of function , 56, wit tremors . HoweverCRLF3 observed during the early cancerous stage of actinic keratosis, cutaneous squamous cell carcinoma and non-melanoma skin cancer (UTP6-CRLF3 fusion in acute myeloid leukemia (crlf3 mutant will allow the further exploration of CRLF3’s mode of action.The mechanism of action for CRLF3 remains vague, although there is high conservation of the protein across bilaterians, including vertebrates and insects , suggestn cancer , and itsleukemia . Howeverleukemia , synaptileukemia  and the leukemia , while ileukemia . These eleukemia  and disrleukemia , 58, witleukemia . It is hThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by Deakin University Animal Ethics Committee.TT, KP and CL performed experiments; TT, KP, AW and CL analyzed the results and prepared figures; YG, AW and CL designed the research; TT, KP, AW and CL wrote the paper, which was read and approved by all authors.The authors recognize the support of funding from IMPACT at Deakin University. TT was supported by a Deakin University International Postgraduate Research Award.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Objective: To explore the effect and mechanisms of a traditional Chinese quadri-combination therapy  in treating recurrent spontaneous abortion (RSA).Methods: A clinical trial was conducted to study the effect of BYLY on RSA. Pharmacological network analysis and UPLC-Q/TOF-mass spectrometry (MS) were applied to investigate the key active component in BYLY and potential targets. Cellular experiments based on former results were performed to examine the mechanism of BYLY in the treatment of RSA.Results: Four hundred and eighty participants enrolled in the clinical trial. The results showed that, compared with the use of BYLY or duphaston alone, a combination of duphaston and BYLY could decrease the early abortion rate in RSA (p < 0.001). Network pharmacological analysis indicated that BYLY contained 132 active components and 146 core targets, and the quercetin maybe the key effective component. In vitro experiments found that pretreatment of quercetin at the correct concentration (2 μM) prevented hypoxia-induced viability and proliferation reduction, and apoptosis and mitochondrial dysfunction. Furthermore, quercetin could modulate mitochondrial fission/fusion balance in trophoblasts, and specifically decrease the expression of Drp1 by regulating miR-34a-5p.Conclusion: BYLY could improve pregnancy outcomes of RSA, based on multi-components and multi-targets. The protective effect of quercetin on trophoblasts, through decreasing Drp1 expression via regulating miR-34a-5p, might be one possible effective mechanism.  Recurrent spontaneous abortion (RSA), which affects ∼2.5% of women, involves a failure of two or more pregnancies before 20–24 weeks of gestation with the same sexual partner . Since mFor patients with RSA, growing evidence shows that the intervention of TCM might increase pregnancy success rate without obvious adverse events . In TCM,First, a preliminary clinical trial was conducted to observe the effect of BYLY in treating RSA. Then network pharmacology analysis and UPLC-Q/TOF-mass spectrometry (MS) were performed to explore the active components and the underlying mechanisms. Furthermore, cellular experiments based on former results were used to demonstrate the potential mechanisms of BYLY.This prospective clinical trial was conducted in the Obstetrics and Gynecology Hospital of Fudan University from 1 September 2018, through 30 December 2020 and was approved by the hospital’s Ethics Committee (No. 2019–57). The flow of patients through the study is shown in RSA patients diagnosed with clinical pregnancy were enrolled in this trial. RSA diagnosis was based on the European Society for Human Reproduction and Embryology (ESHRE) Guideline  and clinIn total, 480 patients were recruited and assigned to the BYLY group, duphaston group, and duphaston + BYLY group in a 1:1:1 ratio according to patient preference and clinician judgement. In the BYLY group, patients received BYLY treatment   and HERB (http://herb.ac.cn). Putative targets of active compounds were collected from TCMSP. The data of active components and their target genes were imported in Cytoscape 3.8.2 software to build a components-targets network.With the standards of oral bioavailability (OB) ≥30% and the drug-like (DL) ≥0.18, active components of BYLY were filtered from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform . RSA-related genes were obtained by searching “recurrent spontaneous abortion” on GeneCards (https://www.genecards.org). Putative gene targets of active components and RSA-related genes were compared to find the overlapped gene targets, which were considered to be the core regulatory genes of BYLY regulation on RSA.Since all putative targets of active components obtained from TCMSP were protein names, all protein names were coverted to gene names by the UniProt database (https://string-db.org). The species was “Homo sapien” and confidence was 0.4. The PPI network was then imported into Cytoscape 3.8.2 software to retrieve the crucial subnetwork. The Cytohubba and CytoNCA plugin was used to select the top 20 nodes and reconstruct the crucial network.A PPI network of core regulatory targets was conducted using the STRING database (https://metascape.org/) The results underwent visualization conversion using https://cloud.oebiotech.cn/.GO enrichment analysis and KEGG enrichment analysis were conducted to analyze the functions of these core regulatory genes using the Metascape database . Column temperature was 45°C and ultraviolet wavelength was 200–600 nm. The injection volume was set as 1 μl. The mobile phase was composed of phase A: 5 mM amine acetate +0.1% formic acid (in water) and phase B: 0.1% formic acid . Gradient elution at a speed of 0.4 ml/min was initiated from 5%B for 1 min, then increased linearly to 70%B over a period of 23 min, then increased linearly to 95% B within 2 min and maintained for 2 min before a final decrease to 5%B to equilibrate the column.m/z 554.2615).The MS experiment was conducted by Synapt G2-Si MS. The ionization mode involved negative electrospray. The source temperature and the desolvation gas temperature were set as 120 and 350°C, respectively. The capillary voltage and the cone voltage were set as 3.0 kV and 40 V, respectively. The desolvation gas was nitrogen and the collision gas was argon. Leucine Enkphalin was used as the lock mass  powder was dissolved with dimethyl sulfoxide (DMSO)  to 200 mg/ml for storage and diluted with culture medium to 2, 5, and 10 μM for cell treatments. The concentration of DMSO in the final culture medium was less than 0.01% (v/v). For hypoxia incubation, 70% confluency of HTR-8/SVneo cells were pretreated with a dose dependent quercetin (0/2/5/10 μM) for 48 h and were transferred to a hypoxia incubator chamber  with 1% O2, 5% CO2, and an N2 balanced atmosphere for 48 h. For normoxic incubation, cells were cultured with complete medium for 96 h.The HTR-8/SVneo cell line was obtained and conserved at the Institution of Obstetrics and Gynecology Hospital of Fudan University . Cells were cultured in RPMI 1640 Medium  supplemented with 10% (v/v) fetal bovine serum (FBS)  and 1% (v/v) penicillin-streptomycin solution  at 37°C in a 5% CO3 cells and 200 μL per well. After treatment, 20 μl CCK-8 were added into each well and cultured in the dark for 2 h. Absorbance at 450 nm of each well was measured using a microplate reader. The results were normalized to the hypoxia group without quercetin treatment.Cell viability was evaluated using a cell counting kit-8 (CCK-8) . HTR-8/SVneo cells were cultured in 96-well plates with 6 × 103 treated cells were washed twice with phosphate-buffered saline (PBS) and resuspended with binding buffer (Biolegend Inc.). After adding 5 μl Annexin V for 10 min and 3 μl PI for 5 min, cellular apoptosis rate was quantified by flow cytometry . Cells in the Annexin V +/PI + field and Annexin V +/PI - field were regarded as apoptosis events. Data were analyzed using Flowjo 10.4.0 software.Cell apoptosis was performed by labeling cells with Annexin V and propidium iodide (PI) (Biolegend Inc.). In brief, 6 × 10Cell proliferation detection was performed using a Cell-Light EdU Kit  according to the manufacturer’s instructions. Edu-positive cells were scanned and calculated using fluorescence microscopy  from at least three random fields.4 treated cells in each group were stained with JC-1 following the manufacturer’s protocol. The fluorescence intensity of JC-1 aggregates (red fluorescence)/monomers (green fluorescence) were detected by flow cytometry . Data were analyzed using Flowjo 10.4.0 software.A JC-1 assay kit  was purchased to assess MMP. In total, 1 × 104, dehydrated, embedded, underwent polymerization, and ultrathin sectioning according to the TEM staining protocol provided by Servicebio. Samples were observed under TEM  and images were taken. At least three random fields of each sample were observed.Cells were fixed with fixative for transmission electron microscope (TEM) . Briefly, samples were prepared through post-fix with 1% OsOhttps://starbase.sysu.edu.cn), miRDB (http://mirdb.org), TargetScan (https://www.targetscan.org), and miRWalk . Gene target was set as DNM1L and species was human. The binding sites of miR-34a-5p on wild-type (wt) sequence of Drp1 were muted to generate a mutant (mut) sequence of Drp1. The Drp1-wt and Drp1-mut sequences were cloned to pSI-Check2 vectors, generating Drp1-3′UTR-wt and Drp1-3′UTR-mut, respectively . Drp1-3′UTR-wt or Drp1-3′UTR-mut luciferase report vectors and miR-34a-5p mimics or NC mimics were co-transfected into 293 T cells. Luciferase activity was analyzed by the dual-Luciferase system .The upstream miRNAs were obtained from starBase  following the manufacturer’s instructions.RNA from treated cells was extracted using RNAiso Plus  Co., Ltd.). RNA concentration quantification was performed by NanoDrop one (Thermo Fisher Scientific Inc.). Extraction of cDNA was obtained using PrimeScript™ RT reagent Kit  (Takara) and Veriti 96 well thermal cycler (Thermo Fisher Scientific Inc.) following the manufacturer’s instructions. For miRNAs expression detection, miRNA cDNA Synthesis Mix and miRNA qPCR Mix (WcGene Biotech Technology Ltd.) were used for reverse transcription and PCR. PCR was conducted by Quantstudio six Flex (Thermo Fisher Scientific Inc.) to evaluate the expression level of mRNA and miRNA. β-actin and U6 acted as the loading control. The primers designed are shown in 2 test. Data analysis was conducted using SPSS v.26. For cell experiments, all data values of at least three independent experiments were represented as ‾x ± SD. The Shapiro-Wilk test was employed for normal distribution. Data were analyzed with Student’s t-test and ANOVA by GraphPad Prism v.8. Asterisks indicate statistical significance of corresponding comparisons.For clinical trials, continuous variables were analyzed in normal distribution by the Kolmogorov-Smirnov test. Data were expressed as mean ± standard deviation (‾x ± SD) and analyzed by one-way analysis of variance (ANOVA) for normal distributions, and were expressed as median (interquartile range) and analyzed by Mann-Whitney test if not. Categorical variables were expressed as percentages and analyzed by Pearson χ. Compared with the BYLY and duphaston groups, the combination of duphaston and BYLY therapy showed a decreased early abortion rate (p < 0.001).Between 1 September 2018, and 30 December 2020, a total of 480 RSA outpatients met the inclusive and exclusive criteria and were enrolled into the trial. In total, 130 patients failed to finish the medication and follow-up  . The bas. ComparOur preliminary clinical trial demonstrated the effect of BYLY in RSA therapy to some extent. To uncover the mechanisms of BYLY, network pharmacological methodology was applied. After filtering with the standard OB ≥ 30% and DL ≥ 0.18, 132 active compounds were retrieved from the 15 herbs in BYLY . FifteenALB, AKT1, TP53, TNF, IL6, JUN, CASP3, CTNNB1, EGFR, ESR1, MYC, IL1B, HIF1A, PTGS2, MMP9, CCND1, FN1, PPARG, FOS, and HSP90AA1  (green bars), cellular component (CC) (blue bars), and molecular function (MF) (red bars). The top 20 enrichment data in BP, CC, and MF were retrieved respectively . ResponsFOS, HIF1A, IL1B, and MYC were also in the top 20 genes. One reduplicative gene, FN1, was found in particular targets of wogonin. Taken together, these results further emphasized the role of quercetin in BYLY, suggesting that it may be a key molecule in the treatment of RSA. Also, FOS, HIF1A, IL1B, and MYC might be potential targets. Potential mechanism of quercetin on RSA was further investigated through pharmacological network. Ninety-six core targets were retrieved and the top 20 targets in PPI analysis includes AKT1, TP53, TNF, IL6, JUN, CASP3, IL1B, EGFR, PTGS2, MYC, HIF1A, MMP9, PPARG, HSP90AA1, CXCL8, CCND1, MMP2, FOS, CCL2, and ERBB2 , apoptotic signaling pathways and cell proliferative regulation might be involved. Thus, we explored whether quercetin affected the apoptosis or proliferation of a human villous trophoblast cell line, HTR-8/SVneo, under hypoxic conditions.During early pregnancy, extravillous trophoblasts (EVTs) invade the wall of uterine spiral vessels and form temporary plugs, which make the maternal-fetal interface experience a physiological hypoxic condition . Remodel effects  in BYLY  effects . Accordi effects  and GO e effects , apoptosp < 0.001), and cell apoptosis was significantly increased (p < 0.001) after a 48-h hypoxic incubation. HTR-8/SVneo pretreated with 2 or 5 μM quercetin showed higher cell viability and lower cell apoptosis compared with cells without quercetin treatment under hypoxia (p < 0.001) (p < 0.001) compared with hypoxia without quercetin pretreatment, but not in a dose-dependent way  . A concedent way . Our resp < 0.001). Pretreatment of HTR-8/Svneo cells with 2 μM quercetin could prevent the hypoxia-induced the red/green ratio declination (p < 0.001). However, 5/10 μM quercetin pretreatment had no effect on red/green ratio. Thus, a concentration of 2 μM was considered as the effective dose and the ability of 2 μM quercetin pretreatment to prevent mitochondrial morphology change under hypoxia was further studied. TEM pictures of mitochondria are shown in As shown in As shown in our former results, quercetin might regulate mitochondrial function in trophoblasts and mitochondrial dynamic change was observed, which corresponded to the GO enrichment results. Mitochondrial fission and fusion, the equilibrium of which was highly associated with mitochondrial function, were studied to further explore the regulatory role of quercetin. Dynamin-related protein 1 (Drp1) is involved in mitochondrial fission. Mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) orchestrate the fusion of the outer mitochondrial membrane. Corporately, optic atrophy 1 (Opa1) is responsible for the fusion of the inner mitochondrial membrane . In the p < 0.001) and decreased Mfn1 (p < 0.001), Mfn2 (p < 0.001), and Opa1 (p < 0.05) mRNA levels. Quercetin at 2 and 5 μM concentrations could decrease the mRNA level of Drp1 and increase the mRNA level of Mfn1 compared with the hypoxia group (p < 0.001)  . Howeverp < 0.01) or with 2 μM quercetin pretreatment (p < 0.001).Abundant evidence verified the role of miRNAs as post-transcriptional regulatory factors . To furtp < 0.001) (We hypothesized that Drp1 was the downstream target of miR-34a-5p in quercetin regulation. The binding sites of miR-34–5p and Drp1 are shown in < 0.001) . Howeverp < 0.05) and promoted after knocking down miR-34a-5p (p < 0.05). These results indicated that quercetin inhibited Drp1 expression by regulating miR34-a-5p.To study whether quercetin inhibited Drp1 expression via miR-34a-5p, HTR-8/Svneo cells were transfected with NC-agomir, miR-34a-5p agomir, NC-antagomir, and miR-34a-5p antagomir to overexpress or knockdown miR-34a-5p. The expression of miR34a-5p and Drp1 were studied to validate the regulation of miR34–5p on Drp1 by qRT-PCR . MoreoveBushen, Yiqi, Lixue, and Yangtai. Our preliminary clinical trial validated BYLY as a useful assisting therapy for RSA. The coaction and interaction of various active molecules in BYLY make it difficult to study through in vitro research. Thus, highlighted by the study of pharmacological networks, quercetin was considered to be one of the major components for treating RSA and its effect on trophoblasts were further analyzed.Pregnancy is an event shared by both mothers and fetuses and a disorder of either can induce spontaneous abortion. The concept of BYLY innovatively emphasizes the holistic health of the maternal-fetal organism by combining and balancing Shoutai Pill is a classic TCM for threating RSA. Li HF et al.  are overlapped with the regulatory targets of RSA . Thus, w+/CD8+ ratio and IFN-γ/IL-4 ratio, indicating a Th2 shift in the Th1/Th2 balance /oxidized glutathione (GSSG) ratio, reducing caspase3/7 generation and inhibiting JNK/p38 activation (a) and beta (ß) subunits, and HIF-α contains three isoforms: HIF-1α, HIF-2α, and HIF-3α. Unlike HIF-β, HIF-α is sensitive to hypoxia and is stabilized in the maternal-fetal interface. Our work hypothesized that HIF-1α might be a target of BYLY and showed the protective effect of quercetin on HTR-8/SVneo cells under hypoxia. However, whether quercetin directly targets HIF-1α should be studied in future work. Quercetin is considered to be a phytoestrogen as it can interact with estrogen receptor (ER)-α -α . Howeverr (ER)-α . High dor (ER)-α . Meanwhir (ER)-α . The effMitochondria, the cellular energy centers, are important organelles involved in various biological functions. Pregnancy-relevant events including fertilization, implantation, and fetal development can be affected by mitochondrial function . The plap < 0.05), indicating the potential regulation of quercetin on miRs-34 (MiRNAs are a group of small non-coding RNAs, functioning as post-transcriptional regulators by inhibiting transcription or degrading mRNA. Although most miRNAs are in the cytoplasm or nucleus, a small amount of miRNAs are found within mitochondria called mitomiRs . These m miRs-34 . The resQi, promoting circulation, and supporting the fetus. The effect of BYLY relies on the interaction and coaction of various active components. However, our experimental study only observed the effect of quercetin, which is just the tip of the sophisticated network of BYLY. Owing to the difference between in vivo and in vitro pharmacological activities, the cellular experiments with quercetin cannot fully mimic its effect in the body. Thus, studies on the effect and mechanisms of other pivotal active components and well-designed animal experiments should be emphasized in future work.The limitations of our study are worthy of remark. The success of BYLY is a combination and balance of nourishing the kidney, reinforcing kidney-The results in this study showed the clinical efficacy of BYLY, uncovered its potential mechanisms and suggest that quercetin has a role in RSA therapy. Quercetin might protect trophoblasts under hypoxia, manifesting as increased cell viability and proliferation, inhibited apoptosis, and improved mitochondrial function. Quercetin suppression of Drp1 expression via regulating miR-34a-5p might be the underlying mechanisms.


These nanoparticles were used for construction of potentiometric enhancement sensors employed for the estimation of ranitidine hydrochloride (RNT) in authentic powder and commercial products. The electroactive substance ranitidine-phosphotungstate (RNT-PT) was formed by combining RNT with phosphotungstic acid (PTA) in the presence of plasticizing material o-nitrophenyloctyl ether (o-NPOE). The outcomes showed that the enhanced MgO and Al2O3 nanosensors behaved linearly across the concentration ranges 1.0 × 10−9–1.0 × 10−2 and 1.0 × 10−10–1.0 × 10−2 mol L−1, respectively. However, the conventional sensor (RNT-PT) displayed a linearity over 1.0 × 10−6–1.0 × 10−2 mol L−1. Least square equations were calculated as EmV = (54.1 ± 0.5) log (RNT) + 762.33, EmV = (58.6 ± 0.2) log (RNT) + 696.48, and EmV = (52.2 ± 0.7) log (RNT) + 756.76 for enriched nanometal oxides modified and conventional sensors, respectively. The correlation coefficients of regression equations were 0.9997, 0.9995, and 0.9992 for the above suggested sensors, respectively. The recorded results showed excellent sensitivity and selectivity of the modified nanometal oxide sensors for the quantification of the analyzed drug in its authentic samples and commercial products.Two metal oxide nanoparticles, magnesium oxide nanoparticles (MgONPs) and aluminum oxide nanoparticles (Al Nanoscale materials have recently been identified as a possible key in sensors, material building, biomedical applications and targeting systems for cancer medication. Scientists are still attempting to expand their study field in which the advanced qualities of nanosized substances may be used to modify various sensitive sensors in our modern life . NanoscaLately, nanotechnology involves creating intelligent materials with novel features that have just been identified. This is extremely beneficial to analytical chemistry ,4. Magne2O3NPs) is a type of metal oxide nanoparticle used in various biomedical purposes due to their unique physicochemical and structural properties, including resistance to wear, chemicals, and mechanical stresses, as well as good optical properties and a porous large surface area. Another reason for Al2O3NPs extensive use is their inexpensive preparation cost and ease of handling [Aluminum oxide nanoparticles  between the surface of the indicator sensor and the reference one . Recentl2O3NPs have structures and sizes, allowing them to be used to build a variety of catalytic sensing systems with low detection limits, large concentration ranges, increased percentage of recoveries, good repeatability, and operation at room temperature [The synthesized MgONPs and Alperature ,19.Ranitidine hydrochloride RNT,  are H2 a2O3NPs that are very sensitive and selective. The results show that the created electrodes have several advantages, including simplicity, versatility, and cost-effectiveness.The use of modified metal oxide sensors to determine and quantify dose form pharmaceuticals is now gaining a lot of interest. The goal of this research is to create modified coated wire sensors enhanced with MgONPs and Alo-NPOE), phosphomolybdic acid, ethanol 99.9%, and polyvinyl chloride  were provided by Sigma Aldrich . The medication form of RNT HCl, Ranimax® tablets (150 mg ranitidine hydrochloride/tablet) were obtained from Jazeera Pharmaceutical Industries .Ranitidine hydrochloride (RNT-HCl), magnesium nitrate hexahydrate, aluminum nitrate hexahydrate, hydrochloric acid 37%, sodium hydroxide, acetone 99.9%, tetrahydrofuran (THF) 97.0%, methanol 99.9%, ortho-nitrophenyloctyl ether . Another pH meter (Metrohm-744) was used to adjust the pH of the analytical samples. The characterization of the synthesized nanomaterials was performed using spectroscopic devices including, Shimadzu-spectrophotometer , BX-spectrometer . The surface structure of the formed nanomaterials was examined under scanning electron microscopes . The size of the metal oxide nanoparticles was evaluated from the spectra of 6000-X-ray diffractometer . To confirm the presence of Mg and Al metals, Energy-Dispersive X-Ray Spectroscopy (EDX) analysis was performed using a SEM microscope connected with EDX.Salvia officials extract solution; 5.0 g of dried Salvia officials leaves were added to 500 mL deionized water and boiled for an hour at 100 °C. The formed extract was filtered using a filter paper (Whatman No. 1). The prepared extract was used for the synthesis of MgONPs. Approximately, 10 mL of Salvia officials extract was mixed with 50 mL of freshly prepared 0.1 mol L−1 of magnesium nitrate hexahydrate solution under magnetic agitation at 80 °C for 2 h. The pH was adjusted in the range of 10–12 using 2.0 mol L−1 sodium hydroxide solution. The MgONPs precipitate was collected after centrifugation of the solution for 10 min at 10,000 rpm. To remove the excess of Mg (NO3)2 and plant material from the MgONPs precipitate, the collected precipitate was washed three times with absolute ethanol, then oven-dried at 40 °C for 8 h. The resulting material was then finely powdered with mortar and pestle and then calcined in a muffle furnace at 450 °C for 30 min 3, 5 mL Cuminum cyminum seed extract was added; 2.0 mol L−1 NaOH was used to adjust the pH of the resulting solution to pH 8. A change in the color of the solution to brown indicated the production of Al2O3NPs. At normal temperature (25 °C), the reduction of aluminum ions to nano aluminum takes 2 h. The synthesized nanoparticle solution was centrifuged at 10,000 rpm for 10 min prior to being distributed in deionized water and left to dry  was obtained. RNT-PT was produced as a faint yellow precipitate. The produced precipitate was dried overnight at room temperature after being filtered and washed.By mixing 50 mL of 1.0 × 102O3NPs were designed by suspending PVC (190 mg), electroactive complex , and plasticizer  in 5 mL of organic solvent (THF). The blended solution was allowed to evaporate in a Petri dish with a diameter of 3 cm until an oily membrane solution developed. To create the conventional RNT-PT sensor, an Al wire that had been polished and cleaned with acetone was dipped into the membrane mixture numerous times. A plastic membrane mixture including MgONPs or Al2O3NPs (5 g), PVC (190 mg), RNT-PT-MgONPs or RNT-PT-Al2O3NPs ion pair (10 mg), and o-NPOE plasticizer (0.35 mL) in 5 mL of THF were performed in order to create the modified sensors. At room temperature, a well-homogeneous distributed membrane mixture was created by swirling constantly for 15 min. A thin layer on the surface of the sensors was simulated using the generated membrane mixture. After drying, sensors were repeatedly submerged in the coated membrane solution to create a thickly coated wire membrane  were finely crushed, and 0.53 g was dissolved in distilled water to make a standard solution of 1.0 × 10−2 mol L−1. To achieve varied concentrations of RNT in the range of 1.0 × 10−6–1.0 × 10−3 mol L−1, working solutions were created. To determine each concentration of the tested medication, the recommended sensors RNT-PT-MgONPs and RNT-PT-Al2O3NPs were utilized individually.Five Ranimax2O3NPs. Using field emission scanning electron microscopy (FESEM) showed the morphology and shape of the produced MgONPs and Al2O3NPs at 30,000× magnification. The micrographs of MgONPs and Al2O3NPs have been displayed in 2O3NPs.Microscopic methods such as SEM were used to characterize the synthesized MgONPs and Al−1 and 3449.49 cm−1 in the FT-IR spectra of the synthesized MgONPs were attributed to be associated to OH-stretching groups. A significant band at 2367.22 cm−1 verified the existence of CH-stretching. The bands at 1517 cm−1 and 1488 cm−1 and 1378 cm−1 were found to be associated to carboxylic acid C=O groups and equines C=C groups, respectively. The peak at 445.60 cm−1 .For Altigation b revealeo-NPOE was applied with the RNT-PT ion pair. The critical response characteristics of the fabricated sensors were determined and presented in RNT combines with PT to generate a stable RNT-PT ion pair that can be dissolved in organic solvents such as THF. In the presence of PVC, the solvent mediator −6–1.0 × 10−2, 1.0 × 10−9–1.0 × 10−2, and 1.0 × 10−10–1.0 × 10−2 mol L−1 for conventional RNT-PT and modified RNT-PT-MgONPs and RNT-PT-Al2O3NPs, respectively  belong to the nanostructures elements that gained excellent physical and chemical features that bulk their counterparts. The high surface area-to-volume ratio and semiconducting characteristics recognized them. The electroactive surface area is increased, and electron transport between the sensor membrane and the ions of analytes is improved [It is not surprising that the addition of metal oxide nanoparticles in the membrane composition or the introduction of a thin layer as an intermediate layer improved the stability, sensitivity, and selectivity of the modified sensors. The metal oxides  log (RNT) + 762.33, EmV = (58.6 ± 0.2) log (RNT) + 696.48 for enhanced nanometal oxides, respectively. The conventional type RNT-PT showed a potential response of EmV = (52.2 ± 0.7) log (RNT) + 756.76 with correlation coefficients 0.9997, 0.9995, and 0.9996 for the RNT-PT-MgONPs, RNT-PT-Al2O3NPs, and RNT-PT sensors, respectively.According to ICH requirements, the suggested analytical procedure was guaranteed and validated . The dev−10, 5.0 × 10−11, and 5.0 × 10−7 mol L−1 for the above described sensors, respectively. The devised potentiometric method was tested on nine samples, and the (mean SD) values for the aforementioned sensors were 99.040.7%, 99.490.4%, and 99.540.5%, respectively , suggesting high precision . The obtained results were found to be 4.9 × 10ectively . Throughrecision . By addi® 150 mg/tablet). The recorded readings were measured versus different concentrations of RNT samples, and the percentage of recoveries was evaluated.In order to determine the analytical applicability of the sensors that have been developed, RNT was detected in its pharmaceutical form (Ranimax2O3NPs was more sensitive to RNT determination than RNT-PT-MgONPs sensor. The greater dielectric constant of Al2O3 over MgO may explain how the conductivity of RNT-PT-Al2O3NPs is higher than that of RNT-PT-MgONPs. The results obtained were statistically analyzed using t-student’s test and F-test [The outcomes were 99.00 ± 0.26, 99.40 ± 0.37, and 99.45 ± 0.50 for the above-mentioned sensors, respectively. It was discovered that the modified sensor RNT-PT-Ald F-test . The resd F-test , which iAn analytical comparison was performed in Two coated wire membrane sensors modified with magnesium oxide and aluminum oxide nanoparticles were used in the proposed potentiometric investigation for ranitidine hydrochloride detection. The modified sensors’ potential readings were compared to those of a conventional sensor. Due to their sensitivity and selectivity, the created sensors proved to be superior to other traditional sensors. Furthermore, the employment of metal oxide nanoparticles as coated membrane modifiers resulted in good selectivity in measuring the chosen medication, with a wide linear concentration range and low limit of detection. Consequently, the metal oxide enriched membrane sensors can be utilized for regular ranitidine hydrochloride analysis in pharmaceutical companies, hospitals, and research labs.