The purpose of this article is to present recent studies on the antimicrobial properties of linalool, the mechanism of action on cells and detoxification processes. The current trend of employing compounds present in essential oils to support antibiotic therapy is becoming increasingly popular. Naturally occurring monoterpene constituents of essential oils are undergoing detailed studies to understand their detailed effects on the human body, both independently and in doses correlated with currently used pharmaceuticals. One such compound is linalool, which is commonly found in many herbs and is used to flavor black tea. This compound is an excellent fragrance additive for cosmetics, enhancing the preservative effect of the formulations used in them or acting as an anti-inflammatory on mild skin lesions. Previous studies have shown that it is extremely important due to its broad spectrum of biological activities, i.e., antioxidant, anti-inflammatory, anticancer, cardioprotective and antimicrobial. Among opportunistic hospital strains, it is most active against Gram-negative bacteria. The mechanism of action of linalool against microorganisms is still under intensive investigation. One of the key aspects of linalool research is biotransformation, through which its susceptibility to detoxification processes is determined. It is worth noting that both enantiomers have different fragrance profiles: coriandrol is perceived as sweet, floral, herbal and petitgrain-like with citrus and fruity notes, while licareol has a woody, lavender aroma. The odor threshold of the (3R)-(−)-enantiomer is approximately nine times lower than that of the (3S)-(+)-linalool (0.8 vs. 7.4 ppb). It may also exist in racemic form . The racemate is also a product of the fermentation processes involved in the production of foodstuffs. In plants, linalool is made from geranyl pyrophosphate (GPP)—a universal monoterpene precursor, which is produced by the condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP) by GPP synthase [Escherichia coli strains were able to produce (3R)-(−)-linalool by overproducing its precursor, mevalonate. On the other hand, the use of bacterial linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus allowed linalool to be obtained in an amount of up to 500 mg/L. However, bLinS also accepts farnesyl diphosphate as a substrate that is naturally produced by E. coli; therefore, trans-nerolidol was formed as a by-product , making this technology difficult for commercial applications [S)-(+)-linalool synthase from Actinidia arguta was in turn introduced into Saccharomyces cerevisiae and Yarrowia lipolytica. In contrast, in Synechocystis spp. PCC 6803 cyanobacteria, the co-expression of the above (3S)-(+)-linalool synthase and E. coli farnesyl pyrophosphate synthase mutant S80F acting as GPP synthase was used. In the case of Pantoea ananatis, the solubility of the same (3S)-(+)-linalool synthase was improved by the N-terminal fusion of the halophilic β-lactamase from Chromohalobacter spp. 560 with hexahistidine [Linalool represents about 70% of the floral fragrances  and exissynthase . It may synthase . Thanks istidine . Since lSalmonella Typhimurium [R)-(−)-Linalool can interact with nitric oxide synthase (NOS) to inhibit nitric oxide production without reducing enzyme synthesis. NO regulates inflammatory and immune responses, contributing to the formation of edema, vasodilation, and the recruitment of immune cells at the site of infection [Linalool exhibits a number of other biological activities in addition to its antimicrobial activity, which will be discussed in detail in this publication. Thanks to its anti-inflammatory ,25 and ahimurium . Linaloohimurium . Linaloohimurium . (3R)--(−)-linalool in the treatment of anxiety disorders has been confirmed by clinical trials [In addition to its anti-inflammatory and antioxidant properties, linalool also exhibits anti-cancer ,37,38, sl trials .During inhalation, maximum transfer of linalool to the brain occurs after 90 min. Inhalation with racemic linalool restored the expression level of many stress-induced genes to normal. These genes were associated with synaptic transmission mediated by neurotransmitters, including anxiolytic neuropeptides such as oxytocin and neuropeptide Y, and contained MHC class I proteins that were required for proper brain neurodevelopment and plasticity. Additionally, Yoshida et al. suggested that inhalation of linalool may activate prolactin . HoweverBax).In turn, Mohamed et al.  investigLinalool also has an effect on vascular smooth muscle cells (VSMC). Long-term hypertension leads to changes in the structure and function of blood vessels, and abnormal VSMC proliferation and migration are important factors in these changes. Liang et al.  investigThe main purpose of the review is to present recent studies on the antimicrobial properties of linalool. Since every compound introduced into the organism undergoes transformations in it, the first part discusses the metabolism of linalool, with a special focus on the reactions of this compound catalyzed by CYP. In turn, when discussing the antimicrobial properties of linalool, we focused on potential mechanisms that may be responsible for this activity of linalool. New in our review is a detailed review of the microbial biotransformation pathways that linalool may undergo in the environment. Now more than ever, it is important to be aware of the human impact on the environment, and it is important not to contribute to increased pollution when bringing new products to market.v/v (3R)-(−)-linalool in propylene glycol solution), the compound appeared in the blood after 25 min and lasted for about 40–45 min [R)-(−)-linalool in peanut oil was applied to the skin, terpene alcohol was detected in the blood about 30 min after the massage. Interestingly, when applied to the skin, males showed better absorption than females. The longer time of penetration into the blood after application to the skin may be due to its accumulation in the subcutaneous fatty tissue [Although linalool has several beneficial effects on the human body, little data are available on its metabolism in humans. After inhalation for 45 min -furanoid-linalool oxide and (R/S)-pyranoid-linalool oxide, which are products of intramolecular rearrangement due to the attack of the hydroxyl group on the epoxy bond, has also been observed. It is worth noting that the enzymatic affinity of CYP2D6 for the enzymatic epoxidation of the double bond was approximately 3–10 times lower than the enzymatic affinity for the hydroxylation reaction. The catalytic yields of cyclic ether formation were approximately in the same range but approximately 3000–4000 times lower than that of the hydroxylation reaction [Linalool biotransformations were performed using human recombinant CYP2D6 and CYP2C19. Both CYP isoforms are present in human skin. The major product was reaction   have also been detected in significant amounts in urine. It was observed that when administered to rats, linalool induced hepatic microsomal cytochrome P-450 and UDP-glucuronyl transferase [b-naphthoflavone catalyzed the formation of 8-hydroxylinalool from linalool [In animals, the first attempts to study the metabolism of linalool in vivo were made at the beginning of the 20th century, when in 1904, Hildebrandt conducted research on rabbits. He showed that linalool is excreted as glucuronide . In 1974nsferase . It is wlinalool .R)-(−)-linalool induces CYP2A and does not affect 2B, 2C6, 2C11 and 3A [Further studies showed that in rats,  [Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Fusobacterium nucleatum, Aggregatibacter actonomycetemcomitans, Streptococcus mutans and Streptococcus sobrinus) [P. aeruginosa and Staphylococcus epidermidis) [Campylobacter spp.  and Salmonella spp. ) as well as Listeria monocytogenes [E. coli . However, the physical properties of linalool turn out to be a problem, mainly its solubility, stability and sensory properties, as well as its susceptibility to react with factors included in the food, i.e., water, fat, protein, salt and pH. This difficult to overcome barrier related to the bioavailability of this monoterpene in nutritional systems was solved by Soković et al. [S. Typhimurium ATCC 13311 than pure monoterpene. This is most likely due to the fact that it penetrates better into the bacterial cell. However, despite this, such a system is less effective at reducing the structure of the biofilm. More effective in this case, due to its small size, is pure linalool . To increase the antimicrobial activity, to eliminate negative organoleptic effects and to change the natural properties of food after adding the desired concentrations of linalool, it was combined with food ingredients acceptable for consumption, including vitamin C and copper (II) ions. This treatment significantly reduced the MIC values to 32 mM, 7.5 mM and 212 µM for V. fluvialis, S. enterica subsp. enterica serovar Typhi and E. coli ATCC 25922, respectively [There are many reports in the literature on the antimicrobial properties of linalool; however, especially those that prove that this monoterpene is effective against life-threatening strains, including multi-drug resistant strains, i.e.,  O157:H7  or Pseudruginosa , deserve aureus) ,59, as wobrinus)  and diseermidis) ,61. Addiytogenes  (soft anThymus vulgaris (thyme), Juniperus communis (juniper), Pelargonium graveolens (geranium), Citrus bergamia (bergamot), Citrus paradise (grapefruit), Lavandula angustifolia (lavender), Cinnamomum zeylanicum (cinnamon), Melaleuca alternifolia (tea tree) and Syzygium aromatum (clove) increase antimicrobial activity against both Gram-positive bacteria  and fungi , and Gram-negative bacteria , the treatment of which is very often problematic because they are characterized by a high level of resistance to antibiotics used in therapy [Lavandula angustifolia, La (27.2%) and Ocimum basilicum, Ob (69.3%), exhibit strong antimicrobial activity against Gram-positive strains: Bacillus subtilis (MIC = 4 µg/mL), S. epidermidis (MIC = 4 µg/mL) and S. aureus (MIC = 5 µg/mL for La and 4.5 µg/mL for Ob) [Linalool as a component of many essential oils (EO) is used not only in cosmetology but also in medicine. It has been shown that its addition to EO, i.e.,  therapy ,64. In a for Ob) .Shewanella putrefaciens (MIC = 1.5 µg/mL), a pathogen that poses a high risk to fish and seafood [Linalool also proved to be promising in combating infectious diseases not only in humans but also in, e.g., fish. This monoterpene has been shown to be very effective against  seafood .C. albicans [Trichophyton rubrum. De Oliveira Lima et al. [T. rubrum strain resistant to fluconazole, commonly used in the treatment of dermatophytosis.In addition to the action on bacterial pathogens, linalool also exhibits antifungal properties, both against yeast-like fungi, e.g., albicans , and dera et al.  demonstrAspergillus flavus. Li et al. [Recent antifungal research shows great promise in the treatment of diseases caused by i et al.  proved tOcimum sanctum [Ocimum basilicum [It is also interesting that there are already natural preparations on the market with proven antifungal properties that contain linalool, incl.  sanctum  and Ocimasilicum  used in In the publications we cited for antimicrobial activity, the essential oils whose main component is linalool was used for the study. In terms of biological properties, including participation in metabolic pathways, the molecule of linalool from natural sources or by organic synthesis shown no differences.Research described in the literature has shown that linalool can disrupt cellular function and even cell death. This monoterpene, after contact with a bacterial cell, first acts on the cell membrane, causing damage to its function by reducing the membrane potential (MP) and structure, causing intracellular leakage of macromolecules . In addition, it inhibits the synthesis of pathways related to obtaining energy by bacteria. It also leads to metabolic dysfunctions and inhibits the activity of key enzymes, causing an abnormal structure of the bacterial cell and thus leading to its death ,65,71,72Bacterial cells exposed to stress from the external environment usually defend themselves by activating some pathways of amino acid biosynthesis . Such beMoreover, it has been proven that this monoterpene contributes to an increase in the concentration of histidine and methionine. These amino acids are involved in the structural phenotype of the bacterial biofilm. Thus, linalool directly contributes to the damage of the biofilm structure, i.e., the physical barrier that effectively protects microorganisms against the action of antibiotics, antiseptics and the host’s immune system .Linalool also contributes to disturbing the pH homeostasis of the bacterial cell by increasing the concentration of lysine while reducing the concentration of L-aspartate. Both of these amino acids, apart from L-glutamate, L-glutamine and L-arginine, play a huge role in the homeostatic mechanisms that determine the proper functioning of the body in prokaryotes .Under various conditions of environmental stress, microorganisms generally reduce their energy metabolism, aiming to save energy for the longest possible period of time . GlycolyIn addition, linalool has been shown to reduce the activity of pyruvate kinase (PK) and succinate dehydrogenase (SDH), enzymes that play a key role in the TCA cycle. When their activity is disturbed, the production of pyruvate and ATP (interference with the starting point of the TCA cycle) and the second mitochondrial complex of the electron transport chain are disrupted (the correct conversion of energy from nucleotides into ATP energy) .Bacteria are not indifferent to environmental stress. They respond to it by changing the fluidity of the cell membrane through changes in the ratio of C16 and C18 fatty acids and the ratio of saturated and unsaturated fatty acids .High levels of saturated fatty acids, i.e., linolenic, linoleic, oleic, arachidonic, myristic, octadecanedioic and palmitic acids, indicate low fluidity of the cell membrane. In the presence of linalool, the content of C16 and C18 fatty acids as well as saturated and unsaturated fatty acids increases. In addition, this monoterpene shows the ability to bind to phospholipids, thus damaging the structure of the membrane, because phosphadylcholine (PC) and phosphatidylethanolamine (PE), the main phospholipids present in the cell membrane, are responsible for the integrity and fluidity of the bacterial cell .In the pathogenic process of fungi, their adhesion to host tissues, germination of conidia, and the formation of hyphae as well as penetration into the cell are important. De Oliveira Lima et al. ,67 showe2−, peroxide H2O2 and hydroxyl •OH.A key enzyme in the biosynthesis of ergosterol, a sterol that is an integral component of the fungal cell wall involved in the modulation of electrolyte flow, is 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. It oversees the conversion of HMG-CoA to mevalonic acid. Mate et al.  showed tResearch shows that linalool, by directly or indirectly influencing the integrity and function of the plasma membrane in fungal cells, can inhibit their growth and induce significant morphological changes .Antibiotics are commonly used in the treatment of various infections and, when enter surface waters and soil, negatively affect the local ecosystems. For this reason, it is important to identify the possible degradation pathways for any new antimicrobial drug, including linalool. The first stage of degradation is often biotransformation processes, which are discussed in detail in this section.R)-(−)-isomer of linalool, the products can be trans--furanoid linalool oxide, cis--furanoid linalool oxide, and trans--pyranoid linalool oxide and cis--pyranoid linalol oxide. On the other hand, as a result of cyclization of the (3S)-(+)-isomer of linalool, the products may be trans--furanoid linalool oxide, cis--furanoid linalool oxide, trans--pyranoid linalool oxide and cis--pyranoid linalool oxide [One of the main reactions that linalool undergoes under the influence of many microorganisms is epoxidation of the double bond followed by cyclization to obtain cyclic ethers. In the case of the (3ol oxide .R)-(−)-linalool were used as substrate for biotransformation by Aspergillus niger ATCC 9142. Biotransformation was carried out for 20 days. At the time of inoculation, 50 µL of linalool was added to 100 mL of the medium; after 14 days, another 100 µL of linalool was added; and after 20 days, the transformation process was completed. In both cases, epoxidation of the double bond took place, followed by cyclization of the resulting epoxide. In the case of (±)-linalool, a mixture of cis-- and trans--furanoid linalool oxide (15–24% yield), and a mixture of cis-- and trans--pyranoid linalool oxide (5–9% yield) resulted. In turn, (3R)-(−)-linalool was transformed mainly into trans--furanoid linalool oxide with ee > 95% and trans--pyranoid linalool oxide [Both racemic (±)-linalool and (3ol oxide .A. niger DSM 821 was used to biotransform (3S)-(+)-linalool with 77% purity. The main products were cis--furanoid linalool oxide with a yield of 30% and an enantiomeric excess of 100%, and cis--pyranoid linalool oxide obtained with a yield of 14%. Moreover, it was found that the (3S)-(+)-isomer of linalool was transformed much better than the (3R)-(−)-isomer of linalool, which did not exceed 4% conversion [Another strain: nversion .R)-(−)-linalool by the Diplodia gossypina ATCC 10936 fungal strain. In this case, 10 μL of linalool was added to 100 mL of the grown culture and the transformation process was carried out for 24 h. As a result, trans--furanoid linalool oxide with an efficiency of 12% and ee = 97% and trans--pyranoid linalool oxide with an efficiency of 21% and ee = 98% were obtained [The same cyclic products were formed during the biotransformation of -(−)-enantiomer have shown that the formation of cyclic oxygen derivatives of linalool is highly stereospecific. The products obtained from (±)-linalool were trans- and cis--furanoid linalool oxide and trans- and cis--pyranoid linalool oxide. For the (3R)-(−)-enantiomer of linalool, the main products were trans--furanoid linalool oxide and trans--pyranoid linalool oxide [Studies using the ol oxide .Colletotrichum acutatum TQ058A and C. nymphaeae CBMAI 0864 strains were used for the biotransformation of linalool. Both strains converted linalool to cis- and trans-furanoid linalool oxide. In the case of the C. acutatum strain, the products were formed in the amount of 0.04 g/L and 0.03 g/L, respectively. In the case of the C. nymphaeae strain, these values were slightly higher and amounted to 0.05 g/L and 0.04 g/L, respectively was obtained [In another experiment, obtained .Oocystis pusilla, (3R)-(−)-linalool was transformed into a mixture of trans--furanoid linalool oxide with an efficiency of 8% and trans--pyranoid linalool oxide with an efficiency of 6% [Not only fungi but also microalgae can convert linalool into cyclic ethers. As a result of a 24 h biotransformation using the microalgae cy of 6% .S)-(+)-linalool was used as a substrate for biotransformation, it turned out that microalgae led to not only double bond epoxidation and subsequent cyclization of linalool but also a reduction in double bonds. Microalgae isolated from rice fields in Iran were immobilized in calcium alginate and incubated with substrate for 24 h. The Chlorella spp. MCCS 028 strain transformed the substrate into a mixture of cis-- and trans--furanoid linalool oxide (both with a yield of 1.4%) and into dihydrolinalool with a yield of 3.2%. On the other hand, the strains of Chlorella spp. MCCS 029 and Chlamydomonas spp. MCCS 036 were only able to reduce linalool to dihydrolinalool, which allowed for 4.1% and 3.3% of this compound to be obtained, respectively [On the other hand, when -(+)-linalool - and trans--furanoid linalool oxide, with efficiencies of 65% and 35%, respectively. For the first of these products, the yield was 160 mg/L. Similar products were obtained using the LB-2008 strain isolated from acerola for biotransformation. The LB-2077 strain, isolated from graviola, converted linalool to geraniol with a yield of 7 mg/L. The LB-2010 and LB-2050 strains, on the other hand, were able to convert linalool to α-terpineol with capacities of 150 and 130 mg/L, respectively [Fungi isolated from fruits found in Brazil were used for the biotransformation of , Torulaspora delbrueckii NCYC 696 and Kluyveromyces lactis IFO1267 were used to biotransform the racemic mixture of (±)-linalool. Twenty-five µg/mL of linalool was added to the grown cultures, and the biotransformations were carried out for 72 h. The only product of biotransformation was (±)-α-terpineol. It was obtained with yields of 12.2% for the S. cerevisiae strain and 20.8% for the T. delbrueckii strain. The K. lactis strain, which gave 31.4% of α-terpineol, turned out to be the most effective [The yeast ffective .Fusarium strains isolated from the fruits of the genus Citrus growing in the Amazon rainforest were also used for the biotransformation of (±)-linalool. After adding the substrate in the amount of 1 g/L to the grown culture, the transformation was carried out for 5 days. Different products were obtained depending on the strain used. The Fusarium spp. 1D2 strain transformed linalool via epoxy into a mixture of cis- and trans-furanoid linalool oxide . As a result of using the Fusarium spp. 1C5 strain, cis-furanoid linalool oxide was obtained with a yield of 32% and a mixture of cis- and trans-pyranoid linalool oxide . The Fusarium spp. 1DC5 and F. concentricum strains were able to epoxidate the terminal double bond in the linalool molecule to give 1-methyl-1-(4-methylpentyl)-oxiranemethanol with a yield of 42%. The F. fujikuroi strain was able to cleave the vinyl fragment from the linalool molecule, converting it into 6-methylhept-5-en-2-one with a yield of 49%. The second product (in the amount of 10%) was also observed in the post-reaction mixture, resulting from the reduction in the carbonyl group, i.e., 6-methylhept-5-en-2-ol [In turn, -en-2-ol  , A. niger DSM 821 (for 6 days), Botrytis cinerea 5901/2 (for 9 days), B. cinerea 02/FBII/2.1 (for 3 days) and C. cassiicola DSM 62475 (for 3 days). Three of them—A. niger DSM 821, B. cinerea 02/ FBII/2.1 and C. cassiicola DSM 62475—transformed linalool into a mixture of trans-- and cis--furanoid linalool oxide at efficiencies of 7.7, 24.9 and 81.6%, respectively, and a mixture of trans-- and cis--pyranoid linalool oxide at efficiencies of 17.4, 8.6 and 14.9%, respectively. In addition to the above-mentioned products, A. niger DSM 821, B. cinerea 02/FBII/2.1 strains carried out the hydroxylation of linalool to 8-hydroxylinalool with yields of 14.2 and 17.2%, respectively. In the case of A. niger ATCC 16404 strain, only cis--furanoid linalool oxide was observed with a yield of 5.3% and 8-hydroxylinalool was observed with a yield of 21%. On the other hand, the B. cinerea 5901/2 strain was unable to convert linalool to cyclic ethers, but it only carried out the substrate hydroxylation reaction to 8-hydroxylinalool with an efficiency of 60% [Such mixtures of various compounds that are both cyclic ethers and a hydroxyl derivative of linalool have been observed during the biotransformation of racemic (±)-linalool. In this case, the substrate was biotransformed in cultures of y of 60% .Novosphingobium aromaticivorans [8-hydroxylinalool was also obtained as a result of the biotransformation of (±)-linalool by the enzyme CYP111A2 isolated from civorans .B. cinerea strains , which were grown on grape must of the Milller-Thurgau variety, were also used for the biotransformation of (±)-linalool. The flasks containing the sterilized must were inoculated with the microorganism and incubated at 25 °C for 2 weeks. Linalool was added to the culture prepared in this way. The major product of the biotransformation (greater than 90%) was found to be 8-hydroxylinalool [linalool  -(−)-Linalool (20 mg per flask) was added to the grown culture (40–60 g per flask) and then shaken at 25 °C in the dark for 7 days. 8-Hydroxylinalool was obtained in a yield of 16.5% [The same derivative of linalool was also obtained when of 16.5% .Vitis vinifera L. cv. Gamay Freau  in infections, the ability to persist in a hospital environment for long periods and chronic infections. Due to the fact that more and more often in the treatment of these infections, even the so-called last chance antibiotics, linalool seems to be a promising future. Its mechanism of action on microbes is multifaceted, which is important in combating superbugs that mutate at a rapid pace. Therefore, it is worth considering the use of combination therapy—administration of an antibiotic with a natural compound, which is linalool, in the case of synergistic action of both compounds. Such an approach may reduce the emergence of drug resistance to antibiotics of “last resort” of microorganisms previously sensitive to them. Therefore, it is puzzling that still little is known about the interactions of linalool with known drugs or with food ingredients. First of all, there are no unequivocal results of research on whether and to what extent linalool acts as an inducer for all CYP isoforms occurring in humans. Therefore, further research is needed in this area using such modern methods as transcriptomics and proteomics techniques, metabolomics, X-ray crystallography, spectroscopy and computer modeling. Clinical trials of the combination of antibiotics with linalool are also necessary, with a particular emphasis on severely ill patients, when factors such as organ failure, increased vascular permeability and renal replacement therapies may alter the pharmacokinetics of any medication used, which may significantly reduce the effectiveness of the treatment undertaken.Linalool has a number of biological activities as it has antioxidant and anti-inflammatory, antimicrobial, cardioprotective, neuroprotective, sedative, anxiolytic, anticonvulsant and anticancer effects. In terms of its antibacterial activity, it is active mainly against Gram-negative bacteria, as shown in A somewhat neglected area of research is the biotransformation of linalool. Finding strains of microorganisms capable of transforming compounds with antimicrobial activity is extremely tedious and laborious, which may explain the limited number of publications on this subject. However, research of this type is of particular importance in the context of a holistic view of the functioning of linalool in the environment.


A high incidence of cardiovascular disease (CVD) events and premature mortality is observed in patients with chronic kidney disease (CKD). Thus, new biomarkers that may help predict the development of CVD in early stages of CKD are being investigated along with other traditional risk factors.To investigate cathepsin S as an early biomarker for CVD in patients with CKD.A total of 64 patients with CKD were included and classified into 2 groups: CKD patients with established CVD and CKD patients with non-established CVD. All patients were submitted to routine investigations including complete blood count, random blood sugar, glycated hemoglobin (HbA1c), serum electrolytes, urea, creatinine, total protein, total albumin, calcium total, phosphorous, uric acid, vitamin D, parathormone, lipid profile, liver function test, measurement of serum cathepsin S (Cat S), and 2D Echo of the heart.The level of serum Cat S was increased in CKD patients with CVD (p <0.05) as well as in later stages of CKD (p <0.05). CVD was also more common in patients in early stage CKD. In early stages CKD, Cat S and CVD were positively correlated.These findings suggest that serum Cat S might be useful as an early biomarker for CVD in CKD patients. Active cathepsin S (Cat S) is co-localized with integrin 3 as a receptor on the surface of vascular smooth muscle cells (SMC), playing an important role in SMC-mediated extracellular matrix (ECM) degradation. It has been evidenced that cathepsins are implicated in arterial vascular diseases through their activation, liberation, and modification of angiogenic growth factors, cytokines, and proteases associated with degradation of lipid metabolism, cell events , angiogenesis and matrix protein remodelingThe aim of this study was to assess the association of Cat S with CVD in CKD. We hypothesized that Cat S might increase in earlier stages of CKD and predict the risk of CVD in patients with CKD.This was a cross-sectional retrospective study conducted within 1 year at a tertiary center hospital in northern India.2), IV (eGFR 29-15 mL/min/1.72m2), and V (eGFR < 15 mL/min/1.72m2), who were non-dialyzed and who signed an informed consent form. Pregnant or lactating women and patients with malignancy or severe infections were excluded.Inclusion criteria were patients with 18 to 65 years of age, with CKD of stage III  CKD patients with established CVD and (B) CKD patients with non-established CVD . 2D Echo2) calculation. Specific investigations included measurement of Cat S by ELISA kit and 2D echocardiography of the heart.All patients in the study were subjected to assessment of medical history and thorough clinical examination according to patient records. Routine investigations including complete blood count, random blood sugar, serum electrolytes, urea, creatinine, total protein, total albumin, total calcium, phosphorous, uric acid, vitamin D, parathormone, lipid profile, liver function test, and glycated hemoglobin (HbA1c) were done. The modification of diet in renal disease (MDRD) equation was used for eGFR  program version 16.0. Qualitative data are presented as frequencies and relative percentages. The Chi-square test was used to calculate difference between qualitative variables. Quantitative data are presented as mean ± SD (standard deviation). An independent T-test was used to compare differences between quantitative variables in two groups with normally distributed data. The ANOVA F test was used to compare differences between quantitative variables in more than two groups with normally distributed data. A p-value > 0.05 indicated non-significant results, < 0.05 indicated significant results, and < 0.01 indicated highly significant results.A total with 64 patients of CKD were enrolled of which 46 (71.88%) CKD patients were male and 18 (28.13%) were female. The majority of patients were in the age group of 41 - 60 years (47%). Forty-four percent of patients was diabetic, hypertension was present in 42.2%, and 39% of patients had diabetic kidney disease. Our results showed that as the stage of CKD increased, there was a significant decrease in hemoglobin and calcium with a significant increase in the level of uric acid, phosphorous, parathormone, and Cat S . HoweverAccording to stage, CKD patients with established CVD were 13 (65%), 14,(70%), and 16,(66.6%) in stages 3, 4, and 5, respectively.A cut off of 6.8 for serum Cat S showed the probability of CVD to be 55%, 70% and 83% in stages 3, 4 and 5 respectively. Hence Cat S, can be used as an early biomarker for CVD in patients with CKD.The high incidence of cardiovascular events in CKD warrants an accurate evaluation of risk aimed at reducing the burden of disease and its consequences. Several biomarkers have been used to identify patients at high risk in the general population.Chronic vascular inflammation combined with the imbalance in mineral bone disorder due to calcium phosphate metabolism increases the risk for cardiovascular disease due to accelerated atherosclerosis and arteriosclerosis. Cat S plays a crucial role in various conditions that involve large biological systems, such as autoimmune disease, cardiomyopathy, heart valve disease, and atherosclerosis. Among various cathepsins, such as cathepsin B, C, F, H, K, L, O, S, V, W, and Z, Cat S is thought to generate bioactive elastin peptides due to potent cysteine protease, which cleaves elastin and leads to the promotion of cardiovascular inflammation and calcification,Here we evaluated Cat S as a biomarker in CKD patients. We found that Cat S increased as the eGFR declined and the stage of CKD advanced. It was found that as the glomerular filtration rate decline, Cat S and markers of inflammation-related endothelial dysfunction increased and hence Cat S is suggestive of inflammatory-related endothelial dysfunction for prediction of cardiovascular morbidity and mortality. This indicated that Cat S activity increases with CKD progression, suggesting that Cat S may be a therapeutic target to prevent cardiovascular complications in CKDIn our study, the overall prevalence of CVD among 64 CKD patients was 67.19% (n=43), which was also found in other studies. It was also found that CKD is an independent risk factor for CVD and the majority of patients die due to CVD rather than due to progress to end-stage renal disease (ESRD),-Diabetes mellitus and hypertension were major risk factors for CVD in CKD, which is in accordance with other studiesAs age advanced, the risk for diabetes mellitus and its complications, such as atherosclerosis, also increased. In this subgroup, Cat S was significantly associated with CVD. The increase in serum Cat S level in atheroma patients was more significant than in non-involved patients-In this study, we found calcium, phosphorous, calcium phosphorous product, HDL, and LDL to be significant factors for CVD in patients with CKD,Cat S in cardiovascular calcification is associated with mineral imbalance found in diabetes and CKD. While looking at the role of fibroblasts in medial vascular calcification, it was found that calcified nodules are formed in the presence of elastin degradation products and transforming growth factor beta 1 (TGF-b1). It has been shown that elastin degradation peptides can induce calcification of mesenchymal cells in vitro. These findings suggest that elastin degradation could induce calcification of vascular SMCs and valvular myofibroblasts and thus mediate calcification,In our study, using a cut-off value of 6.8 for Cat S, a higher CVD risk was found when compared with CKD patients with and without established CVD . It has been shown that Cat S inhibition reduces the progression of atherosclerotic lesions, hence reducing risk of CVD.In our study, the values of Cat S, age, HBA1c, and RBS were found to be significantly increased in patients with CKD with established CVD and with risk factors. This is in agreement with another study,Circulating Cat S levels and mortality were correlated, as were Cat S activity and the development of CVD due to increase in the formation and destabilization of atherosclerotic plaques. This indicates that Cat S activity increases with CKD progression, suggesting that inhibition of Cat S may be a therapeutic target to prevent cardiovascular complications in CKDDiabetes mellitus and hypertension are major risk factors for CKD and CVD. Cat S increased as the eGFR declined and the stage of CKD advanced. In CKD patients with CVD and with risk factors such as age, HBA1C was found to be significantly increased with higher Cat S levels. Cat S showed a significant association with CVD and may be used as an early biomarker for CVD in CKD as evidenced by the increased level of Cat S in early stage CKD. Large multicentre prospective studies are needed to validate the findings of the present study.


Bisphenol A (BPA) is a well-known synthetic compound that belongs to the group of chemicals that disrupt the endocrine system in humans and animals. Although bones represent a potential target for these compounds, studies investigating BPA-related effects in bones in large farm animals are limited. We exposed young rams aged 9–12 months to BPA through feed for 64 days and investigated the effects of BPA on bone length, mass, microscopic structure, mineral content, strength, and serum bone parameters. We discovered that BPA had no significant effects on most of the parameters studied. Only manganese was decreased, and copper increased in the femurs of the BPA-exposed rams. These results suggest that a 2-month, low-dose exposure to BPA in growing rams did not affect the macro- and microstructure, metabolism, and biomechanical behavior of femur bones; however, it did affect the composition of microelements in bone, which could affect the bone in the long term.p ≤ 0.05) and increased copper content  in the femurs of BPA-exposed rams. These results suggest that a 2-month, low-dose exposure to BPA in growing rams did not affect the histomorphology, metabolism, and biomechanical behavior of femurs; however, it affected the composition of microelements, which could affect the histometric and biophysical properties of bone in the long term.Bisphenol A (BPA) is a well-known synthetic compound that belongs to the group of endocrine-disrupting chemicals. Although bone tissue is a target for these compounds, studies on BPA-related effects on bone morphology in farm animals are limited. In this preliminary study, we investigated the effects of short-term dietary BPA exposure on femoral morphology, metabolism, mineral content, and biomechanical behavior in rams aged 9–12 months. Fourteen rams of the Istrian Pramenka breed were randomly divided into a BPA group and a control group (seven rams/group) and exposed to 25 µg BPA/kg bw for 64 days in feed. Blood was collected for determination of bone turnover markers , and femurs were assessed via computed tomography, histomorphometry, three-point bending test, and mineral analysis. BPA had no significant effects on most of the parameters studied. Only mineral analysis showed decreased manganese (50%;  BPA is ght (bw) . In 2015ght (bw) , whereasght (bw) . Conjugaght (bw) , human aght (bw) .The endocrine-disrupting activity of BPA has been described through several pathways with an affinity for binding to receptors, such as estrogen (ER) ,13,14, aTherefore, the aim of this preliminary oral toxicity study, which lasted 64 days using an acute, repeated daily dose of BPA, was to investigate the possible BPA-related effects on bone tissue and its metabolism in young, growing male sheep. It was the first study on the effects of BPA on bone tissue in sheep. The selection of a particular animal model was based on its presumed suitability for translational studies. Sheep are suggested as a suitable model for experimental orthopedic studies in humans, given their similarities in bone biochemistry and mineral composition, trabecular bone structure, modeling, and biomechanics. Most ossification centers of long bones close between 12 and 30 months of age  ,43,44. T(Ovis aries) of the autochthonous Slovenian sheep breed Istrian Pramenka in the western part of Slovenia with a moderate continental climate. At the Infrastructure Centre for Sustainable Recultivation (ICSR) in Vremščica, Slovenia, the rams were born (average body mass at birth of 4.36 ± 0.7 kg), individually marked with conventional sheep ear tags, selected for the study, and housed in a roofed stable with concrete paving for 9 months until the start of the procedure, which lasted from October to December. The sample size for the procedure was determined according to guideline VICH GL48 [ad libitum with hay and once daily with 550 g of commercial pellets  from stainless steel bowls. They had unlimited access to drinking water from concrete rainwater tanks provided in enamel bowls (50 L). Feeding management was the same for all rams. A keeper monitored the animals’ condition thrice daily. The animals entering the procedure were clinically healthy. Basic blood analysis was performed before the procedure; blood was collected in the morning before pellets were administered [To evaluate the effects of BPA on bone parameters, an in vivo animal study was performed on 14 rams ICH GL48 . Rams wenistered . Vital snistered .Chemicals and their preparation were previously reported by Cerkvenik-Flajs et al. . BrieflyThe rams were randomly divided into two experimental groups , a BPA group and a control group (7 rams/group). One day before the first BPA administration at 9–10 months of age, the rams weighed 34.5–54 kg (mean body mass of 43.0 ± 4.9 kg). During the procedure, the animals of the two groups were housed in the stable mentioned previously, separated by 2 m with a wooden/stainless steel fence to prevent physical contact but still allow visual and auditory contact between the two groups. The rams had hay and straw bedding cleaned weekly and were exposed to a natural light/dark cycle at a temperature of 6 to 15 °C and relative humidity of 45–55% . Rams were weighed once per week during the BPA treatment. Each morning, all rams were sorted into individual wooden boxes for pellet feeding. The daily amount of BPA for individual rams was prepared according to body mass. After complete BPA impregnation of the pellets, the pellets were offered to the rams, and it was ensured that all rams had eaten their entire ration. After the meal, rams were released back to group housing, where the water regime was the same as mentioned before. On Day 31 of the study, blood samples were collected in the morning before pellet administration. The treatment lasted 64 days. Before anesthesia and euthanasia (age 11–12 months), the rams were weighed, and blood was collected. The rams were premedicated with xylazine i/v  and euthanized after 7–10 min with pentobarbital . After euthanasia, we dissected the left and right femur bones for further analysis and collected the liver and kidneys for the study of edible tissues ,51. The Throughout the procedure, extreme care was taken to ensure that the animals were not exposed to uncontrolled BPA exposure from products, i.e., glass or BPA-free materials were used . The absThe animal procedure was approved by the Administration of the Republic of Slovenia for Food safety, Veterinary, and Plant protection . Animal husbandry and animal handling procedures complied with the Slovenian Animal Protection Act  , CouncilPreviously published studies on these rams demonstrated the average maximum plasma concentration of total BPA in the blood plasma of rams from the BPA group was 10.93 µg/L, measured after the first administration . In the g for 10 min), and stored at −20 °C during transport and at −80 °C until analysis. Serum biochemistry to assess renal and hepatic function, i.e., levels of urea, creatinine, cholesterol, triglycerides, aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT) activity, as well as levels of calcium (Ca) and inorganic phosphate (PO4), were analyzed in addition to bone turnover markers, including C-terminal telopeptide of type I collagen (CTX-I) and P1NP. Serum basic biochemistry was determined using an automated chemical analyzer . Bone turnover markers were selected as previously reported [Blood was collected from the jugular vein in the morning (9–10 AM), before pellet feeding , and thrreported ,56, and The right hind legs were disarticulated at the hip. The femur was dissected, and soft tissues were removed with a knife and a scalpel. The distal extremity of the right femur was cut with a saw at the metaphysis in a transverse plane approximately 1 cm proximal to the distal EGP, and the limb extremity was longitudinally halved and immediately fixed in 5% buffered formalin. The left hind leg was shaved and removed through the hip joint, and the thigh was dissected  and frozen at −20 °C until computed tomography (CT) scanning.−1 was used to define cortical bone, and 300–600 cm−1 was used to define trabecular bone. Femur length was measured in the sagittal plane from the femoral head to the medial condyle, and femur width was measured in the axial plane at 50% of the femur length  as previously described [© 4.6.9). Trabecular bone density was determined 1 cm proximal to the distal EGP, and cortical bone density was determined at 50% of the femoral length. Other trabecular microarchitecture parameters were analyzed proximal to the distal EGP using the BoneJ plug-in for ImageJ software [The geometry of the femur was determined by scanning frozen left thighs using a Siemens Somatom Scope CT scanner . Acquisition and reconstruction protocols were established before scanning and were the same for all specimens: the voltage used was 130 kV, automatic tube current modulation was 31 mAs, detector collimation was 16 × 0.6 mm, slice thickness was 1 mm with a 0.8-mm recon increment, and reconstruction core kernel B70s sharp was used. The threshold of 1700 to 2000 cmescribed . The medescribed  at two dsoftware : bone vo® solution  for 6–7 weeks at room temperature until the bone tissue was sufficiently demineralized and then embedded in paraffin blocks . Tissue sections of 5 µm  that were cut longitudinally through the EGP and metaphysis were stained with hematoxylin and eosin stain (HE) and cover slipped . Comparable longitudinal sections were used for histomorphometric analysis (n = 1 section/sample). Images were obtained by a light microscope  and a digital camera  and quantified with the imaging software program NIS-Elements BR 4.6 . In analyzing the zones of the distal EGP, the relative thickness of the zones of the reserve, proliferative, hypertrophied, and calcified cartilage  were morphologically analyzed, i.e., measured in µm (n = 7 measurements/zone per sample), and calculated to the total thickness of EGP , as previously described [After fixation in formalin, the longitudinally bisected distal extremities of the right femurs were decalcified in Osteosoftescribed . A repreAfter CT, the left femurs were cleaned of soft tissues and weighed . Biomechanical properties were determined by a three-point bending test using a universal hydraulic testing machine MTS . Each femur was loaded with a press head (diameter 20 mm) perpendicular to its longitudinal axis at the mid-diaphysis at a loading rate of 0.2 mm/s. The femurs were placed on two supports (diameter 5 mm) with a span of 120 mm between them. The caudal surface of the femur was facing upward when it was on the supports. During the conduction of the bending test, the applied displacement and corresponding force were recorded. Based on the obtained data, work to fracture, stiffness, modulus of elasticity, and bending strength (ultimate stress) were calculated as previously described . The geoTo analyze the mineral content of the bone ash, the proximal extremity of the left femur was cut with a belt saw for bones at approximately 15% of the total length for further analysis. The analyses of dry matter and ash were performed by standard methods  with minp ≤ 0.05.Data were analyzed using SAS software  with the Proc Mixed function. In the statistical model, the observed group was considered a fixed effect, except for the serum biochemical parameters, where the group, the time of observation, and their interaction were used as fixed effects, whereas repeated measurements within the animal were considered. The least square means are presented, and differences were determined by a Tukey–Kramer multiple comparison test. The dispersion is expressed as the standard deviation (SD) or the standard error of the mean (SEM), except in the two serum bone markers, where relative values were used. Statistical significance was considered when p = 0.037), and AST was decreased in the BPA group on Day 31 (p = 0.022) but not Day 64. Creatinine was decreased and GGT was increased in the overall sample  on Day 31  and Day 64  compared to Day 0.In the BPA group, urea and creatinine as renal function parameters and cholesterol, AST, and GGT activity as hepatic parameters showed no change compared with the control group within each blood collection during the procedure , as shown in 4 value decreased over time until Day 64 . Thus, the ratio between Ca and P increased from Day 0 to Day 64 in the control group (p = 0.003). Serum concentrations of the markers CTX-I and P1NP in the rams were more heterogeneously distributed than suggested by the manufacturer .Analysis of Ca revealed no differences between the two experimental groups throughout the procedure, whereas the POn = 7) compared with controls . The relative femur mass (femur mass compared to bw), femur geometry, and histomorphometric parameters are summarized in Relative liver mass (liver mass compared to bw) was not changed in the BPA group  and substantially reduced Mn content  in the ash compared to the control group.The biomechanical and mineral properties of the left femur are summarized in This preliminary 2-month study investigated the effects of dietary exposure to an environmentally relevant dose of BPA on long bone metabolism. We analyzed bone turnover markers, femoral growth and geometry , and mineral content and investigated to what extent these parameters influence bone biomechanical behavior in young, growing rams, given the similarities of sheep bone anatomy, trabecular structure, and mineral metabolism to humans . A two-m4 concentration in all animals before BPA administration reflected their age and intense growth and possibly the daily administration of concentrated pellets. Therefore, the significant decrease in phosphate and increase in Ca:P ratio throughout the procedure—with increasing age—toward the Ca:P ratio common to adult animals, i.e., 1.37–1.86 [per se was difficult to interpret. To reduce sample variability, urine samples of bone resorption markers should be considered together with the interpretation of blood markers [The serum creatinine and urea concentrations were slightly decreased but still in the physiological range at the end of the procedure (urea in the control group only), although the feed (protein) intake and final body mass of these ruminants were not altered throughout the procedure. The high serum PO.37–1.86 , was exp.37–1.86 ), could .37–1.86 , althoug.37–1.86 . After B.37–1.86 , whereas.37–1.86 ,41. Even.37–1.86 , their v.37–1.86 ,43,62,63.37–1.86 . Studies.37–1.86 ,43,63. I.37–1.86 . Therefo markers .As mentioned above, the daily administration of 25 µg BPA/kg bw for a period of 2 months did not appear to affect the growth of the femurs or the bone biomechanical behavior of the growing rams. The fact that no change was observed in bone size, bone mass, or both types of bone tissue at three different sites, i.e., the cortical bone at mid-diaphysis, the EGP structure at the metaphysis, and the trabecular bone adjacent to the metaphysis, further confirmed this assumption. The EGP zones of the distal metaphysis were appropriately thick and aligned, and the chondrocytes followed the pattern of secondary ossification. The mineral content  showed a similar composition for all tested bones. Given that the mineral composition of the bones is one of the main parameters for bone biomechanical behavior, the stiffness and bending strength obtained with the three-point bending test were, therefore, similar. Similar to our observations in males, sex differences were found in several recent studies ,27,28,64Bacillus sp. from the human gut microbiota [The amount of ash and the content of macrominerals did not differ; however, Mn and Cu showed significant differences between the experimental groups even though Mn was added to commercial pellets . One of the possible explanations for the changes in Mn and Cu in bone could include the potential involvement of rumen microbiota in BPA metabolism. Future studies are, therefore, needed to elucidate the mechanism of BPA metabolism in the rumen and its impact on bone health in polygastric animals, as sheep represent a possible alternative model for orthopedic studies in humans.


Cancer is among the highly complex disease and renal cell carcinoma is the sixth-leading cause of cancer death. In order to understand complex diseases such as cancer, diabetes and kidney diseases, high-throughput data are generated at large scale and it has helped in the research and diagnostic advancement. However, to unravel the meaningful information from such large datasets for comprehensive and minute understanding of cell phenotypes and disease pathophysiology remains a trivial challenge and also the molecular events leading to disease onset and progression are not well understood. With this goal, we have collected gene expression datasets from publicly available dataset which are for two different stages (I and II) for renal cell carcinoma and furthermore, the TCGA and cBioPortal database have been utilized for clinical relevance understanding. In this work, we have applied computational approach to unravel the differentially expressed genes, their networks for the enriched pathways. Based on our results, we conclude that among the most dominantly altered pathways for renal cell carcinoma, are PI3K-Akt, Foxo, endocytosis, MAPK, Tight junction, cytokine-cytokine receptor interaction pathways and the major source of alteration for these pathways are MAP3K13, CHAF1A, FDX1, ARHGAP26, ITGBL1, C10orf118, MTO1, LAMP2, STAMBP, DLC1, NSMAF, YY1, TPGS2, SCARB2, PRSS23, SYNJ1, CNPPD1, PPP2R5E. In terms of clinical significance, there are large number of differentially expressed genes which appears to be playing critical roles in survival. High-throughput data is created at a large scale in order to understand complex diseases like cancer, and it has aided in research and diagnostic advancement6. However, extracting useful knowledge from such vast datasets for a complete and detailed understanding of cell phenotypes and disease pathophysiology remains a difficult task, and the molecular events that contribute to disease initiation and progression are still poorly understood9. The advancement of the post-genomics period has resulted in a huge amount of "big data" in biological sciences, which has led to a multitude of interdisciplinary applications in recent decades10. There are a number of biological databases that house various types of datasets. TCGA, oncomine, nephroseq, and GEO (gene expression omnibus) are the most widely used databases in biological sciences11. These databases mainly GEO store vast amount of datasets related with cancer, diabetes, and other biological problems16.Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults, responsible for approximately 90–95% of cases and it is one of the leading causes of cancer death. Its occurrence shows mainly male predominance over women with a ratio of 1.5:1. RCC, a kidney cancer originates in the lining of the proximal convoluted tubule which is the part of the very small tubes in the kidney and transport primary urine19. The improvement in tumor classification always helps in the improvement during therapeutic approaches21. In target specific therapy, effectiveness can be maximised while toxicity is reduced by using enhanced classification. To access biological datasets from these databases previously, a variety of tools/approaches were used. For molecular classification of cancer Golub TR et al.,22 have divided cancer classification into two challenges as class discovery and class prediction.The identification of pathogenetically distinct tumour types poses a significant challenge in the treatment of complex diseases 25. Copy number, gene sequencing, expression pattern, and methylation in primary RCC are all possible avenues for achieving this goal. With continued breakthroughs in omics technology, the application of molecular markers for early diagnosis and prognosis deserves further attention30.A number of oncogenes and tumour suppressor genes that are changed in RCC, resulting in pathway dysregulation, need to be identified and investigated further33. Healthy tissue usually contains many different types of cells grouped together. If the cancer looks similar to healthy tissue and contains different cell groupings, it is called differentiated or low-grade tumor and when the cancerous tissue looks very different from the healthy tissue, it is termed as poorly differentiated or high-grade tumor. The cancer’s grade may help the clinician to predict how quickly the cancer will spread. In general, the lower the tumor’s grade, the better the prognosis. Different types of cancer have different methods to assign a cancer grade37. In general, it is very hard to detect most of the cancers at early stage so the main focus was on exploring the gene expression pattern alterations and its functional consequences and further to avoid biasedness, we have incorporated TCGA dataset also which have the samples from all the grades.We have selected RCC dataset with samples from two stages (stages I and II) for the purpose of understanding how gene expression patterns vary and how altered gene expression patterns lead to possible changes in the respective inferred functions as tumour stage I to II changes and from affymetrix platforms (U133A to U133B). Different cancer stages help in describing where a cancer could be located, how far it has spread, and whether it is affecting other parts of the bodyHere, we have selected a dataset from gene expression omnibus (GEO) where the samples are from human with two tumor stages (I and II). We have organized the samples in the order such as stage I normal versus tumor and stage II normal versus tumor for the affymetrix platforms U133A and U133B and analyzed the tumor samples with respect to their respective controls  for the gene expression alterations and evolved functions with the increase in tumor percentage. Based on our work, we conclude that irrespective of the tumor stage PI3K-Akt, Foxo, endocytosis, MAPK, Tight junction, cytokine-cytokine receptor interaction pathways and the major source of alteration for these pathways are MAP3K13, CHAF1A, FDX1, ARHGAP26, ITGBL1, C10orf118, MTO1, LAMP2, STAMBP, DLC1, NSMAF, YY1, TPGS2, SCARB2, PRSS23, SYNJ1, CNPPD1, PPP2R5E. In addition, we have also studied the clinical significance and observe that there are large number of differentially expressed genes which appears to be playing critical roles in for survival such as ARHGAP6, TGM4, CD248, SLC13A3, EPO, PARD6A, CLCA2, UBE2S, ERAL1, FGFR1, MRVI1, DYNC1I2, CDCA7.38, organized the samples in the order such as stage I normal versus tumor and stage II normal versus tumor for the affymetrix platforms U133A and U133B and processed it until normalization and log2 values for all the mapped genes as mentioned in the workflow Fig. In the first step, we have selected the data of our interest (raw expression dataset) GSE6344In this study, the initial focus of our goal was to understand the gene expression pattern between the different stages for normal versus tumor samples. For this purpose, the total number of the DEGs, up, and down regulated genes have been calculated Fig. b and theAfter analyzing the number of DEGs and the enriched pathways, we have analyzed the enriched pathways and the genes which are altered in different RCC tumor stages Table . We obseBased on the venn diagram of the enriched pathways, we have prepared the list of the pathways in five groups (commonly enriched) and matched the genes with these pathways lists from all the four DEGs list . In Fig. 39. We observe that most of the top-raked genes (from selected 30 DEGs) mainly up-regulated genes show very high significance on the patients survival  which contains the samples for adjacent normal, low grade, and high grade and compared the differentially expressed genes and the enriched pathways with each other  and analyzed the patients survival (Kaplan–Meier plot) for the patient samples from TCGA database and the dataset was TCGA kidney renal clear cell carcinoma (source data from GDAC Firehose) which contains 538 samplesval Fig. . In thisher Fig. –S6. More41. In terms of therapy and diagnosis, therapeutic and clinical outcomes differ between the individuals with even close similarity in clinical and pathological characteristics  and despite tremendous efforts to identify molecular biomarkers (prognostic and predictive) and with improved precision compared to clinical and pathological predictors only few molecular tests have been introduced into oncological practice29. So it is important to understand and unravel different levels  of diversities in cancer43. We gathered the previously published dataset for this purpose and conducted a detailed and precise study ranging from gene expression profiling to functional changes, including networks mapped from the human protein network database.Renal cell carcinoma is one of the most common cancers, and it is one of the leading causes of cancer deathOur work leads to the conclusion that irrespective of the tumor stage PI3K-Akt, Foxo, endocytosis, MAPK, Tight junction, cytokine-cytokine receptor interaction pathways and the major source of alteration for these pathways are MAP3K13, CHAF1A, FDX1, ARHGAP26, ITGBL1, C10orf118, MTO1, LAMP2, STAMBP, DLC1, NSMAF, YY1, TPGS2, SCARB2, PRSS23, SYNJ1, CNPPD1, PPP2R5E. Networks of DEGs for the enriched pathways show that there are large number of genes from few specific pathways are altered such as Ras signaling pathways and patients' survival for changes in gene expression, with Kaplan–Meier plots indicating clinical significance. We conclude that a large number of differentially expressed genes tend to be potentially important in terms of survival, with ARHGAP6, TGM4, CD248, SLC13A3, EPO, PARD6A, CLCA2, UBE2S, ERAL1, FGFR1, MRVI1, DYNC1I2, CDCA7 among the genes chosen. Using the publicly available datasets, we have investigated the gene expression profiling for renal cell carcinoma. In the previous work, it has been focused on selected genes and pathways. Here, we have investigated the list of critical pathways and the genes which appear to be clinically highly significant in case of renal cell carcinoma. These clinically significant genes lead to potential alteration in PI3K-Akt, foxo, endocytosis, MAPK, tight junction, cytokine-cytokine receptor interaction pathways. Our work will help in diagnosing the renal cell carcinoma patients because here, we have presented the differentially expressed genes, their inferred pathways, and the clinical impact of the selective genes. Since, our finding is from overall perspective including clinical relevance so this study will help in future for diagnostic also.37 and the different tumor stages could help in describing the severeness, tumor propagation speed, and its impact on the other organs33.. In general, it is very hard to detect most of the cancers at early stage so the main focus was on exploring the gene expression pattern alterations and its functional consequences and further to avoid biasedness, we have incorporated TCGA dataset also which have the samples from all the grades. Further, we have also investigated the expression of these clinically relevant genes by using protein atlas (https://www.proteinatlas.org/)48. We observe that most of these genes are expressed in case of RCC and act as biomarkers and only TGM4 and GGN were not expressed. This study will be an important step for the understanding of early stage tumor propagation and also will be helpful for clinical aspect.This work also appears to be more unique in comparison to the previous study that we potentially explored grade I and II of RCC and further explored the clinical relevance. Healthy tissue usually contains many different types of cells grouped together and if the cancer looks similar to healthy tissue and contains different cell groupings, it is called differentiated or low-grade tumor and when the cancerous tissue looks very different from the healthy tissue, it is termed as poorly differentiated or high-grade tumor. The cancer’s grade may help the clinician to predict how quickly the cancer will spread. In general, the lower the tumor’s grade, the better the prognosis. Different types of cancer have different methods to assign a cancer gradeBased on our findings, we conclude that PI3K-Akt, Foxo, endocytosis, MAPK, Tight junction, and cytokine-cytokine receptor interaction pathways are among the most commonly altered pathways in renal cell carcinoma, and that MAP3K13, CHAF1A, FDX1, ARHGAP26, ITGBL1, C10orf118, MTO1, LAMP2, STAMBP, DLC1, NSMAF, YY1, TPGS2, SCARB2, PRSS23, SYNJ1, CNPPD1, and PPP2R5E are the major sources of alteration for these pathways. Wnt, hippo, actin cytoskeleton control, ECM, infection and inflammation, metabolic, and other cancer-related pathways are among the most important pathways altered in RCC. ARHGAP6, TGM4, CD248, SLC13A3, EPO, PARD6A, CLCA2, UBE2S, ERAL1, FGFR1, MRVI1, DYNC1I2, CDCA7 are some of the genes that were chosen after survival study.38. In the first step, we selected the raw expression dataset GSE6344 and processed it until normalisation and log2 values of all mapped genes were achieved, as shown in Fig. Here, GSE6344 dataset was used for the study which contains the samples of stage I and II of gene expression for tumor kidney cancer51, GCRMA56, RMA, and EB are the most commonly used approaches. Here, we have used EB for raw intensity normalization. After normalization, we proceed for our goal which is to understand the gene expression patterns57 and its inferred functions58.In short the basic steps involved for the entire study are raw file processing, intensity calculation and normalization. For normalization61 have been used for pathway analysis and for which there is our own code designed62. In summary, for differential gene expression prediction and statistical analysis, MATLAB2017 functions  were applied and further for pathway analysis, we used KEGG61 database65.To prepare the list of DEGs and analysis, we have our own in-built codes. The samples were placed into two groups such as COVID-19 positive and negative and then normal and the tumor samples. The selection criteria were placed by the fold change and p-values which have been calculated and for the selection of genes as differentially expressed the threshold of fold changes and p-values applied were ± 2 and 0.05, respectively and then KEGG database66 has been used for all the networks throughout the work and cytoscape67 has been used for network visualization. For most of our coding and calculations MATLAB has been used65. Furthermore, FunCoup2.066 database and cytoscape and its applications68 were used for network visualization to understand the network and the connectivity of the genes within the network of DEGs70. The basic concept of FunCoup network database is that it predicts four different classes of functional coupling or associations such as protein complexes, protein–protein physical interactions, metabolic, and signaling pathways66. MATLAB 2017b codes and the command line codes have been used for figure plotting and during analysis. For the network level-analysis such as the number of connectivity per gene and the genes belonging to different number of pathways, the codes have been written in MATLAB and finally it has been plotted also by the codes written in MATLAB65. For venn diagram plotting, freely available webserver (http://bioinformatics.psb.ugent.be/webtools/Venn/) was used74.For generating DEGs network, FunCoup2.0Supplementary Figures.Supplementary Table S1.


ERG) characteristics by dPCR. To calculate and quantify the content of GM canola using endogenous reference gene (ERG) characteristics, the suitability of several ERGs of canola, such as cruciferin A (CruA), acetyl-CoA carboxylase (BnAcc), phosphoenolpyruvate carboxylase (PEP), cruciferin storage (BnC1), oleoyl hydrolase (Fat(A)), and high-mobility-group protein I/Y (HMG-I/Y), was investigated by droplet dPCR. BnAcc and BnC1 were more specific and stable in copy number in the genome of Brassica napus L. than the other genes. By performing intra-laboratory validation of the suitability of ERG characteristics for the quantification of GM canola events, the ddPCR methods for BnAcc and BnC1 were comprehensively demonstrated in dPCR assays. The methods could provide technical support for GM labeling regulations.As an effective tool for genetically modified organism (GMO) quantification in complex matrices, digital PCR (dPCR) has been widely used for the quantification of genetically modified (GM) canola events; however, little is known about the quantification of GM canola events using endogenous reference gene ( Canola is widely cultivated in Canada, Europe, and Asia. It is one of the main sources of food and feed, providing edible vegetable oil and protein, and it is the second largest oil crop, accounting for more than 20% of the total edible vegetable oil worldwide, only exceeded by soybean. Genetically modified (GM) canola is one of the four major transgenic crops and is a vital part of genetically modified organism (GMO) safety supervision. As of 2019, more than 40 GM canola events had been authorized as food worldwide ,4. IdentPEP) . ddPCR data were analyzed using QuantaSoft software version 1.6.6 (Bio-Rad Laboratories). The GM content was calculated as (target DNA copy number)/(reference DNA copy number) × 100 .Brassica L. to verify whether there was a cross-reaction of endogenous genes or non-targeted specific genes of other genera. Intraspecific inclusion and species specificity are two basic requirements for reference gene selection. Six primers/probe sets  were useBrassica species: B. rapa, B.nigra, B. oleracea, B. juncea, B. napus, and B. carinata. All six endogenous genes had no homologous sequences in maize, soybean, rice, and other crops but had positive amplified fragments in all B. rapa, B. juncea, and B. napus varieties  showed similar amplification specificity in the test crops. Their fragments could be identified not only from the genomes of all six Brassica species but also from R. sativus. In the HMG-I/Y PCR reaction, the fragments could be amplified in B. rapa, B. juncea, B. napus, B. oleracea, and B. carinata. The BnAcc system exhibited a performance similar to that of BnC1 in the tested cultivars of B. rapa, B. juncea, and B. napus. Aside from the positive result of BnAcc in R. sativus, ERGs of BnAcc and BnC1 fragments were restricted to the B. rapa, B. juncea, and B. napus cultivars , PEP, HMG I/Y, BnAcc, and BnC1 , and CruA exhibited greater variability than that of the other genes in B. napus cultivars, with an RSD of 7.90%, 7.21%, and 9.18%, respectively , and BnAcc using the same samples and ddPCR reactions , and BnAcc measured by dPCR was close to the theoretical copy number of a single copy in the B. napus genome, which was consistent with the results of previous studies [B. napus genome, with deviations of less than 5%. The copy number of eactions . The cop studies ,12,17. TFat(A) assay. Setting the threshold manually may affect the accuracy of a low copy number determination. For CruA and HMG I/Y assays, there were some droplets in the amplification plot that interfered with the accuracy of the quantitative detection, whereas there was a clear separation of positive and negative clusters in the BnC1 and BnAcc gene amplification. Therefore, the analysis of copy number stability and amplification showed that BnC1 and BnAcc were accurate and reliable. They could be candidate ERG assays for the quantitative detection of GM canola using ddPCR.The observations of ddPCR amplitude plots for the six ERGs are shown in CruA, BnC1, and BnAcc was 100%; thus, these three ERGs have a lower LOD with about five copies. The LOD of Fat(A) and PEP was higher than that of the above three genes, with 10 copies. RSD analysis was performed on these low copy samples using copy number values obtained in the digital PCR. The RSD increased, whereas the copy number decreased. Thus, aLOQ can be estimated as the lowest copy where the RSD of the measurements is below 25%. When the copy number was above ten, the RSD values of each ERG were below the criteria, whereas in five copies for sample amplification, BnC1 and BnAcc exhibited 24.8% and 22.5%, respectively. That means the aLOQ of BnC1 and BnAcc could reach five copies. Considering the LOQ was for the ERGs instead of specific genes, this was a satisfactory result, as the content of the exogenous targets was not less than that of the ERGs in the GM detection. Therefore, BnC1 and BnAcc have stronger specificity in B. napus and higher sensitivity than the other four genes as ERGs for GM canola detection.aLOD can be defined as the minimum amount of analyte in a test sample that can be detected reliably , whereasB. napus endogenous genes of BnAcc and BnC1, the linear relationship between the theoretical and actual copy numbers of both reference genes was evaluated using ddPCR. Rapeseed DNA samples were diluted to different concentrations in the absolute amount in the reaction system, with 10,000, 2500, 625, 156, 40, and 10 copies. The theoretical copy number of BnC1 was double that of BnAcc in the rapeseed genome. The R2 coefficient represents a measure of the estimative capacity of the linear regression line in relation to the experimental data used. The result indicated that the copy numbers of both genes were linear along the entire copy range , with R2 > 0.99   E. We assBnC1 or BnAcc). The error indicated as bias (%) of all quantitative values was within ±25% of the European Network of Transgenic Laboratories recommended values [BnAcc as the ERG, whereas the maximum error bias of real-time PCR was 22.17%, as for Topas 19/2 samples, with 0.1% content determined by BnC1 as the ERG. Both RT-PCR and dPCR methods can achieve accurate results for both ERGs in transgenic samples with content as low as 0.1%. The results indicated that either the BnC1 or BnAcc gene was appropriate for the quantitative assessment of GM canola events. Moreover, real-time PCR and ddPCR methods could achieve accurate and consistent results. Compared with those of reported methods [BnC1 and BnAcc assays used in ddPCR assays achieved satisfactory results in GM canola quantification. The single or double copies for reference genes did not affect the ddPCR evaluation of the GM content in these three GM canola events in B. napus. The applicability of dPCR targeting BnC1 and BnAcc genes was successfully demonstrated. Moreover, the dPCR methods using BnC1 and BnAcc as reference genes were suitable for the quantitative detection of transgenic rapeseed. The dPCR amplification plot indicated that the assays could achieve the expected result with the endogenous genes , PEP, HMG I/Y, and BnAcc) were compared and evaluated using ddPCR in this research. With their combined specificity, stability, dynamic ranges, and amplification observation, both BnC1 and BnAcc could be used as ERGs for the quantification of GM canola content in ddPCR assays. Using the ERGs, a ddPCR system was established with an aLOQ of 10 copies/reaction. This method can reliably quantify the GM content, even when it is as low as 0.1%. Different content of MS1, Topas 19/2, and Oxy235 GM canola events validated that BnC1 and BnAcc endogenous genes were reliable in both real-time PCR and ddPCR with the same primers and probes. Concluding from these results, we believe that this newly developed method will be a good candidate of using ERGs for quantifying the amount of GM canola by dPCR. These methods can provide reliable and reproductive results and perform as effective tools to implement labeling systems.The criteria of six canola genes (


Oryza sativa). In this study, we investigated the effect of MiZax on the growth and yield of three dicot horticultural plants, namely, tomato (Solanum lycopersicum), capsicum (Capsicum annuum), and squash (Cucurbita pepo) in different growth environments, as well as on the growth and development of the monocot date palm (Phoenix dactylifera), an important crop in the Middle East. The application of MiZax significantly enhanced plant height, flower, and branch numbers, fruit size, and total fruit yield in independent field trials from 2020 to 2021. Importantly, the amount of applied MiZax was far less than that used with the commercial compound humic acid, a widely used biostimulant in horticulture. Our results indicate that MiZax have significant application potential to improve the performance and productivity of horticultural crops.Global food security is a critical concern that needs practical solutions to feed the expanding human population. A promising approach is the employment of biostimulants to increase crop production. Biostimulants include compounds that boost plant growth. Recently, mimics of zaxinone (MiZax) were shown to have a promising growth-promoting effect in rice ( Naturally, the growth of crop plants relies on their ability to get nutrients from the soil, which is inevitably affected by unfavorable growth conditions, such as drought, salinity, or biotic stresses  performance, which indicated its biostimulant activity ] plants under greenhouse conditions by foliar application. Moreover, to extend its practical application, we conducted four independent open field studies with three different fruit crops, namely, green pepper [Capsicum annuum (C. annuum)], squash [Cucurbita pepo (C. pepo)], and date palm [Phoenix dactylifera (P. dactylifera)] during the planting year 2020–2021 in the desert climate of Kingdom of Saudi of Arabia. Application of MiZax significantly increased the plant height and branches of date palm, as well as the flower numbers, fruit size, and total fruit yield of green pepper and squash. Notably, we observed these effects with MiZax amounts that are far less than that used for the commercial biostimulant humic acid, a common used growth regulator with positive effects on sustainable agriculture  were sown in a 24-well plastic tray to raise nursery. 1-week-old uniform seedlings were then transferred to 3 L plastic pots. Each pot was sprayed with biostimulants at 5 μM concentration twice per week. The pots were irrigated with nutrient solution when needed. Data on tomato growth, yield, and yield components were collected.Tomato seeds  were sown on 10 October 2019 in Jiffy-7 peat pellets (44 mm) containing a peat-based substrate (peat moss) (manufactured by Norway). Before sowing, seeds were soaked into water overnight and pelleted for 2 h. Pellets were then placed into the trays with seeds into hole and covered very well. After the appearance of first true leaves, plants were watered as required. After 5 weeks, the seedlings were transplanted into the well-prepared plot to initiate the experiment.For raising seedlings, green pepper seeds (C. pepo) seeds of the variety Camila 625 F1 Hybrid  were obtained from the local agricultural market at Jeddah, Saudi Arabia.Squash , King Abdulaziz University (KAU), Jeddah, Saudi Arabia to evaluate the performance of plant biostimulants (zaxinone and MiZax) and humic acid applications on growth and yield of green pepper under freshwater and salty water and squash under freshwater. The soil texture of the experimental site was classified as sandy loam, soil pH 7.8, and EC 1.79 d Smr season .–1) until end of growing season. The squash experiment followed a randomized complete block design (RCBD) with 3 replicates. Seedling of the green peppers were planted in plots each of 2.8 m × 1.8 m and the planting distance was 70 cm × 60 cm. The experiment was laid on a split-plot design where three levels of water salinity were assigned to the main plots, while mimics of zaxinone (MiZax3 and MiZax5) and humic acid were randomly distributed over the subplots following RCBD with 3 replicates. The water salinity of underground water was 3.51 d Sm–1 and used as control treatment. NaCl was added weekly to the other tanks to raise the water salinity to 8.04 and 11.71 d Sm–1 during the season. For watering squash and green peppers, a drip irrigation system was instilled to supply the plants with their water requirements twice per day for 10 min. The plants were weekly fertilized with nitrogen, phosphorus, and potassium (NPK) fertilizer (20:20:20) during vegetative growth and 10:10:40 (N:P:K) at flowering and fruit setting and maturity stages. The biostimulants MiZax3 and MiZax5 at 5 and 10 μM and humic acid at 1 and 1.5 g L–1 were foliar applied once per week and for 8 weeks to the plants and after 15 days of transplantation for green peppers and at the second true leaves for squash. Two control treatments received either acetone (as MiZax3 and MiZax5 dissolvent) at 1 ml per L or only water (dissolvent of humic acid). All the recommended agriculture practices for both the crops were applied and kept uniform across the treatments throughout the growing season. The growth and yield parameters of squash, including plant height (cm), numbers of leaves per plant, fresh and dry mass per plant (g), sex ratio , fruits per plant, and yield per plant (kg), were assessed. For green peppers, we measured plant height (cm) after 20, 40, and 60 days of transplanting, branches, leaves, and flowers per plant at end of season, roots length (cm) and mass (g), plant fresh and dry mass (g), fresh fruits per plant, and yield of fresh fruits per plant (kg). Pepper fruits quality were measured, including weight of single fruit (g), fruit length and diameter (mm), total soluble solid (TSS) (%), vitamin C (mg g–1), firmness (N), acidity (EC), and total phenol (mg g–1). Soil plant analysis development estimations were taken utilizing SPAD meter  from 9:00 to 11:00 a.m. in fully extended third to fifth leaf.Directing seeding was used to plant the squash seeds with the distance between rows of 1 m and 0.5 m between each two adjacent plants in the same row. The squash plants were irrigated with low saline water . After three successive plowing, the field was divided into plots and treatments were allocated by following randomized complete block design (RCBD) with three replications. The plot size was 3 m × 1.2 m and the planting distance was 50 cm × 60 cm. 4-week-old uniform-sized green pepper seedlings were transferred (8 plants per plot) into the field. MiZax3 and MiZax5 were sprayed at 5 and 10 μM concentration with 1 week interval up to 8 weeks. Humic acid was applied at 1 g l–1 and untreated plots were included to compare the treatments effect. The plots were irrigated and fertilized when needed and all the other agronomic practices were adopted uniformly by following the standard procedures. Data on plant growth, yield, and yield components were collected.A field experiment at the King Abdullah University of Science and Technology (KAUST)  was conducted to evaluate the growth-promoting activity of MZ3 and MZ5 on green pepper (P. dactylifera cv. ajwa) was conducted at the KAUST study field to investigate the effect of zaxinone and MiZax. Compounds were dissolved in acetone and prepared in a final volume mixed with 0.5% Tween-20. Zaxinone, MiZax3, and MiZax5 were sprayed at 5 μM concentration once per week on the 8-week-old uniform date palm plants. The pots were irrigated with nutrient solution once per week. Data on the shoot growth, branches, and leaves opening were collected.The field experiment of date palm . All the statistical elaborations were performed using GraphPad Prism version 8.3.0.Data are represented as mean and their variations as SD. The statistical significance was determined by one-way ANOVA with the Tukey’s multiple comparison test, using a probability level of Zaxinone and its mimics MiZax3 and MiZax5 showed to promote the growth on the monocot plant rice . Hence, Next, we applied the three stimulants to plantlets of date palm, an important regional fruit plant for sustaining current desert agriculture in the Middle East , to inveArabidopsis (ZAXINONE SYNTHASE (ZAS) gene (s) and can form a symbiosis with arbuscular mycorrhizal fungi (AMF). In contrast, ZAS is missing in most of the non-mycorrhizal plant, such as Arabidopsis thaliana  of MiZax3 and MiZax5 on the valuable crop, green pepper, in comparison to the commercial compound humic acid. We observed that MiZax significantly triggered shoot growth from 20 to 60 days after planting in the Agricultural Research Station of the King Abdulaziz University (KAU)  and clear plants , indicat–1), which represents a big advantage of MiZax. Moreover, the leaf single-photon avalanche diode (SPAD) values were higher after applying the high concentration of MiZax treatments  experimental field, a warmer and more moisture place next to the Red Sea. Following the study design in the KAU, we compared 1 g lplanting , while tplanting , compareplanting . Howeverplanting , the safFinally, to explore the suitability to other horticultural crops, we performed a trial on squash in the KAU field. Again, MiZax significantly boosted the height and biomass of squash plants, while humic acid failed to promote growth . AlthougIn conclusion, based on our three independent field studies, the amount of applied MiZax was much lower (at the micromolar or mg level) than the commercial biostimulant humic acid with a better bioactivity in promoting crop plant growth and development. Thus, MiZax are potential biostimulants enhancing the performance and production of horticultural plants (monocots and dicots).The original contributions presented in the study are included in the article/SA-B, IB, SA-S, and MM proposed the concept. JYW, MJ, and Md GH designed the experiments. JYW, MJ, Md GH, G-TEC, and LB conducted the experiments. JYW, MJ, Md GH, IB, MM, and SA-B analyzed and discussed the data. TO and TA synthesized and provided MiZax3 and MiZax5. JYW, MJ, and SA-B wrote the manuscript. All the authors have read, edited, and approved the final version of the manuscript.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.


Additionally, nephron formation does not continue after birth in humans. Therefore, a low glomerular number at birth is a lifelong burden on the glomeruli and increases the risk of developing CKD. Therefore, it is important to maintain the glomerular number at birth. Accurate glomerular counts are essential for conducting studies on the glomerular number. The dissector/fractionator method is the gold standard; however, it can only be performed at some institutions. Recently, methods have been developed to measure the glomerular number by combining computed tomography and pathological examination and measure the glomerular count using magnetic resonance imaging. Models of decreased and increased glomerular numbers have been developed. Moreover, research regarding the causes of decreased glomerular number and its relationship with development of lifestyle-related diseases and renal dysfunction has significantly progressed, furthering our understanding of the importance of glomerular number.Chronic kidney disease (CKD) prevalence is increasing worldwide, and reducing the number of patients with CKD is of utmost importance. The environment during the fetal, perinatal, and early childhood stages may influence CKD development . Under conditions of maternal malnutrition, the glomerular number of infants reduces, and the risk of developing CKD may increase. Nephron progenitor cells and ureteric buds interact with each other to form glomeruli at the tip of the ureteric bud. Thus, the number of glomeruli is determined by the number of ureteric bud branches, which are reportedly decreased due to maternal malnutrition, in turn reducing the glomerular number. Four possible mechanisms can explain the low glomerular number resulting from maternal malnutrition: 1) suppression of  Then, capillaries are induced to form glomeruli at the ends of branched tubular buds; therefore, the glomerular number is determined by the number of ureteric bud branches. In rats, the formation of nephrons begins on embryonic day (E) 12 and continues for 8–11 postnatal days . In mice3Brenner et al., in their study in 1989, proposed that “essential hypertension is caused by a decrease in the number of glomeruli and nephrons, which results from an undesirable prenatal intrauterine environment , genetic factors, and premature birth. Low glomerular number leads to renal glomerular hypertrophy, and eventually to hypertension, CKD, nephrosclerosis, and renal failure” (Brenner’s hypothesis) Figure 14While nephron formation does not occur after birth in term infants, it continues until 40 days after birth in preterm infants , after wThe glomerular number reportedly demonstrates racial variation: Caucasian Americans, African Americans, Aboriginals, and Japanese have a total glomerular number of approximately 900,000 , 950,000The glomerular number varies by sex, with a 12% lower glomerular number in women than in men , 19, 20.Furthermore, glomerular number is positively correlated with birth weight , 11. Forin vitro studies, the addition of RA to the culture medium increases the glomerular number , which is secreted from the mesenchyme, acts on the Wolffian ducts to germinate and elongate ureteric buds (Ret) and its co-receptor GDNF family receptor α1 (Gfra1) are expressed in the ureteric bud, and GDNF secreted in the mesenchyme transmits signals to the ureteric bud via Ret. GDNF-Ret/Gfra1 signaling is required for ureteric bud germination and elongation. Vitamin A-dependent c-Ret expression and nephron formation /Eyes absent homolog 1 (Eya1)/Paired box gene 2 (Pax2) form a complex that directly regulates GDNF expression and promotes ureteric bud branching (Pax2 gene expression via the AngII type 2 receptor (AT2R). Additionally, AT1R and AT2R expressions are strongly suppressed in a hypotrophic in utero environment  seems to be critically important in renal organogenesis. Hombox gene 11   4), Alagille syndrome , Axenfeld-Rieger syndrome , and CHARGE syndrome . Branchio-oto-renal (BOR) syndrome is caused by mutation in the EYA1, Sine oculis-related homeobox (Six) 1, Sall1, and Six 5 genes  . Disease 5 genes . This di 5 genes . In addian ducts .In addition, although the although the pathogenic mechanism is unclear, the frequency of CAKUT is approximately 4.5 times higher in children with Down syndrome than in those without it . FurtherIn Alport syndrome, which is caused by other type 4 collagen abnormalities, the glomerular basement membrane is abnormal and glomerular structure cannot be maintained, leading to end-stage renal failure at a young age.5Accurate measurement of the glomerular count is important to ascertain the glomerular number. Currently, five methods are used for glomerular counting, each with their own advantages and disadvantages Table 2.5.1via pipetting. Distilled water is added to the samples, which are then incubated overnight at 4°C. The sample is placed in a culture dish with a 2-mm grid, and the glomeruli are counted  that may develop later in adulthood .8An undesirable prenatal intrauterine environment, including maternal malnutrition, decreases the fetal glomerular number and increases the risk of CKD; therefore, an appropriate environment should be maintained to prevent the decrease in fetal glomerular number. Altogether, proper perinatal nutritional management and preventing the decrease of and increasing the glomerular number at birth may greatly reduce the incidence of CKD in the future Figure 3SF is the first author. Both authors contributed to the article and approved the submitted version.