The aim of this study was to validate the model of polygenic risk in Russian patients with dementia. A microarray-based assay was developed to identify 21 markers of polygenic risk and ɛ alleles of the APOE gene. This case–control study included 348 dementia patients and 519 cognitively normal volunteers. Cerebrospinal fluid (CSF) amyloid-β (Aβ) and tau protein levels were assessed in 57 dementia patients. PRS and APOE-ɛ4 were significant genetic risk factors for dementia. Adjusted for APOE-ɛ4, individuals with PRS corresponding to the fourth quartile had an increased risk of dementia compared to the first quartile . The area under the curve (AUC) was 0.559 for the PRS model only, and the inclusion of APOE-ɛ4 improved the AUC to 0.604. PRS was positively correlated with tTau and pTau181 and inversely correlated with Aβ42/Aβ40 ratio. Carriers of APOE-ɛ4 had higher levels of tTau and pTau181 and lower levels of Aβ42 and Aβ42/Aβ40. The developed assay can be part of a strategy for assessing individuals for AD risk, with the purpose of assisting primary preventive interventions.The polygenic risk score (PRS), together with the  The existence of familial forms of Alzheimer’s disease (AD) suggests that genetic factors play a crucial role in the pathogenesis of this disease. The heritability is 58–79% for late-onset AD and more than 90% for early-onset AD .ɛ4 allele of the APOE gene (APOE-ɛ4), which encodes apolipoprotein E. This allele increases the risk of AD by 3-fold in heterozygous carriers and 15-fold in homozygous carriers [APOE gene’s ɛ2 allele (APOE-ɛ2) have a lower risk of AD.The most studied but not exhaustive genetic risk factor for AD is the carriers . People APOE has been ongoing for some time. Large international consortia such as the International Genomics of Alzheimer’s Project (IGAP) and the Alzheimer’s Disease Genetics Consortium (ADGC) have been established to examine the genetic basis of Alzheimer’s disease. Genome-wide association studies (GWASs) have accumulated vast amounts of data on AD genetics using samples from tens of thousands of AD patients and healthy donors [The search for reliable genetic risk factors beyond y donors ,4. Thesey donors . This kny donors . Each SNAPOE-ɛ4 [APOE gene alleles, gender, age, and other social and physiological characteristics can also be incorporated.The first PRS model for assessing the risk of developing AD was released in 2005, preceding the large-scale GWAS. This model consisted of nine SNPs, incorporating APOE-ɛ4 . GWAS daAPOE-ɛ4 ,11,12,13Studies on PRS models have indicated their correlation with the age and risk of AD and dementia ,10,11,14Thus, polygenic models offer a promising approach to identifying individuals at high risk for developing AD. These tests may have practical applications in selecting personalized preventive measures and developing screening strategies. In addition, PRS models could be utilized in the design of clinical trials for AD therapy aimed at preventing disease progression. It is suggested that supplementing study cohorts with individuals who have a high PRS and thus an elevated risk for developing AD may enhance the probability of identifying effective treatment regimens ,16,26.It should be noted that most of the research on AD development with PRS has been performed on the European population. Therefore, further studies are required to extend the findings to other populations. In certain instances, polygenic risk models for AD have been investigated in Hispanics, Latin Americans, Asians, and other ancestry groups ,27,28,29ɛ alleles of the APOE gene. A multiplex assay utilizing hybridization on low-density hydrogel microarrays was the primary method for SNP genotyping. The genotype and allele frequency data based on the results of SNP identification are presented in APOE alleles were identified via real-time PCR using hydrolysis probes for all samples. The genotyping results obtained by various methods were consistent for 100% of the samples.The study included 348 patients with dementia and 519 cognitively normal volunteers. In total, 867 DNA samples were genotyped for 23 SNPs, including 21 polygenic risk markers according to the model of Tosto et al. [For each sample, the PRS was calculated. The PRS values ranged from −1.148 to 1.096. A PRS less than −0.268 corresponded to the first quartile, while a PRS greater than or equal to 0.1725 corresponded to the fourth quartile. The thresholds for the first and top deciles were −0.4598 and 0.3924, respectively.t-test, p-value = 0.001). The logistic regression analysis indicated an association between dementia and both PRS and APOE-ɛ4 for both homozygotes and heterozygotes, as shown in ɛ2-allele carriage was not a significant factor in this study, it was not included in the further analysis of how genetic factors impact the risk of dementia.The distribution of PRS in both patients with dementia and CN volunteers is displayed in APOE-ɛ4, individuals with a PRS falling within the fourth quartile or tenth decile had a higher risk of dementia compared to those in the first quartile/decile: OR = 1.85  and OR = 2.27 , respectively.Adjusted for APOE-ɛ4 as a risk factor in individuals with varying PRS values. Compared with non-carriers, carriers of APOE-ɛ4 with the fourth quartile of PRS had a greater risk of dementia, while the presence of the APOE-ɛ4 in individuals with the first quartile was not a significant risk factor in our cohort  analysis was conducted on logistic regression models utilizing various predictors. The impact of genetic factors was evaluated across the entire cohort (n = 867), and the ROC curves are displayed in APOE-ɛ4. When these variables were included, the AUC increased from 0.69 to 0.7343  who had available data regarding the presence of a family, higher education, and intellectual work. This analysis considered the impact of social factors in addition to the PRS and o 0.7343 b. The geo 0.7343 .APOE-ɛ4 were considered as predictors . The presence of this allele did not result in statistically significant differences in other CSF markers.It should be noted that individuals carrying the The polygenic risk scoring approach has become truly innovative in the study of the genetics of multifactorial diseases. Compared to individual SNPs, PRS models often yield highly reproducible results, which holds promise as a prognostic factor. It is advisable to investigate the information content of the developed models in the intended population before implementation. In this study, we employed the PRS model introduced by Tosto et al. , which wɛ alleles of the APOE gene, we developed a molecular approach using the established hydrogel microarray technology. The microarray analysis can be completed in less than 12 h. The software that executes the SNP identification algorithm, PRS calculation, and APOE allele determination automatically analyzes the microarray hybridization pattern. Compared to next-generation sequencing (NGS) assays, the cost of microarray analysis is significantly lower and comparable to PCR (less than USD 10 per sample). However, microarray-based assays are limited by the incomplete automation of all procedural steps and a lack of standardization that is critical for routine genetic testing in clinical laboratory settings. Nevertheless, several hydrogel microarray-based assays have been approved for in vitro clinical diagnostics by the Russian regulatory agency [To identify the SNPs included in the utilized PRS model and the y agency . In thisɛ alleles of the APOE on dementia risk among the residents of the Moscow region in the Russian population, utilizing a sample of 348 individuals diagnosed with dementia and 519 cognitively normal participants. For the first time, the AD polygenic risk model was tested on the European part of the Russian population, revealing its capacity to accurately assess the risk of dementia. The OR of 1.26 (per standard deviation) obtained in this study is equivalent to Tosto et al.’s OR of 1.29 for the European population. Additionally, we achieved similar AUC values (0.559 compared to 0.57) [The developed assay was applied to investigate the impact of PRS and the APOE-ɛ4 had the highest risk of dementia (OR = 7.98). The risk for individuals in the fourth quartile of PRS (OR = 1.85) was approximately equal to the risk for the heterozygous carriers of APOE-ɛ4 (OR = 1.81). Our data regarding the impact of PRS on disease development risk in APOE-ɛ4 carriers  and demonstrated an AUC of 0.72–0.84 in the original studies [Thus, there is compelling evidence that genetic factors, including PRS and  studies ,12, clin disease . Additio studies . HoweverAPOE-ɛ4 had higher levels of tTau and pTau181 and lower levels of Aβ42 and Aβ42/Aβ40 ratio in the CSF  for identifying dementia types . Studies the CSF , which aAPOE alleles significantly correlate with AD biomarkers, it can be assumed that the predictive value of genetic factors for AD may be higher. Secondly, this was a case–control study and not a cohort study. Therefore, some social characteristics could depend on the composition of groups of patients and healthy volunteers. Thus, we did not include sex and the presence of children as predictors in the models since they were not representative of the group of CN volunteers. Also, the age of onset of the disease was unknown for most patients; only the age of inclusion was noted.This study has some limitations. Firstly, the group of studied patients with dementia did not only include patients with AD. Since polygenic risks and Future directions lie in prospective cohort studies, which include patients with MCI. The influence of genetic factors needs to be evaluated while considering clinical, social, and biochemical data. These data will help us assess the impact of PRS on MCI patients, with the hydrogel microarray-based technique acting as an auxiliary tool for identifying high-risk individuals.The study was conducted in adherence to the guidelines of the Declaration of Helsinki. The procedures related to human subject experiments were carried out in accordance with the ethical standards outlined in Protocol No. 1, dated 25 January 2022 and established by the Local Ethics Committee of Mental-health Clinic No. 1 named after N. Alekseev of the Moscow Healthcare Department A. Data from the participants were collected between January 2022 and March 2023.Patients with dementia (N = 348) were recruited from the gerontological department of Mental-health Clinic No. 1, named after N. Alekseev of the Moscow Healthcare Department A. The inclusion criteria were as follows: patients aged 65 or older, diagnosed with one of the types of dementia  according to the International Classification of Diseases (ICD-10). The study included patients with multiple impairments of cortical functions in at least two areas: memory and one of the cognitive functions. These included thinking , speaking, practicing, and gnosis. Cognitive impairment causes difficulties in social and work adaptation and in performing everyday tasks. Symptoms manifested against the backdrop of clear consciousness. The duration of the disease was at least 6 months. The exclusion criteria included substance abuse and dependence as well as heavy comorbid severe somatic or neurological disorders such as cancer, cirrhosis, chronic lung failure or absence of a lung, tuberculosis, and viral hepatitis. Among the psychoneurological diseases, the exclusion criteria were also as follows: psychogenic pseudodementia, mental retardation, organic brain diseases, dementia due to schizophrenia, brain injury, epilepsy, tumor, HIV and syphilis, and normotensive hydrocephalus. Additionally, renal or liver failure was not permitted.CN volunteers (N = 519) were recruited from patients who received periodic medical check-ups in outpatient clinic No. 121 (Moscow), and they were matched by age. Participants were excluded if they had psychiatric disorders, a positive family history of psychiatric illness, substance abuse, or severe somatic diseases. All subjects signed a voluntary informed consent form to participate in the experiment.Some participants (88 CN volunteers and 259 patients with dementia) were evaluated using a structured clinical interview, including MMSE, CDT, and MoCA scales; they answered a detailed questionnaire and participated in neurological examination and neuropsychological testing. Other participants (431 CN volunteers and 89 patients with dementia) did not provide fully detailed descriptive characteristics.Genomic DNA was extracted from peripheral blood collected in EDTA-containing tubes using the QIAcube automated DNA extraction system  or the LumiPure genomic DNA Blood and Buccal Kit  following the manufacturer’s protocols.CR1 gene; rs6733839 in the BIN1 gene; rs35349669 in the INPP5D gene; rs190982 in the MEF2C gene; rs9271192 in the HLA-DRB5/1 gene; rs10948363 in the CD2AP gene; rs271 8058 in the NME8 gene; rs1476679 in the ZCWPW1 gene; rs11771145 in the EPHA1 gene; rs28834970 in the PTK2B gene; rs9331896 in the CLU gene; rs10838725 in the CELF1 gene; rs7274581 in the CASS4 gene; rs983392 in the MS4A6A gene; rs10792832 in the PICALM gene; rs112 18,343 in the SORL1 gene; rs17125944 in the FERMT2 gene; rs10498633 in the RIN-SLC24A gene; rs8093731 in the DSG2 gene; rs4147929 in the ABCA7 gene; and rs3865444 in the CD33 gene . Simultaneously, we analyzed rs429358 and rs7412, which determine the ɛ alleles of the APOE gene.In this study, we examined a panel of SNPs that were included in the PRS model proposed by Tosto et al. for AD . The panThe identification of 23 SNPs was carried out using a molecular technique that utilizes hydrogel-based oligonucleotide microarray technology developed at the Engelhardt Institute of Molecular Biology, Russian Academy of Sciences . The micAPOE gene’s ɛ2/ɛ3/ɛ4 alleles were determined based on whether it had homozygous or heterozygous variants of rs429358 and rs7412.The assay protocol involved multiplex PCR to generate mostly single-stranded, fluorescence-labeled DNA fragments, followed by the hybridization of the DNA fragments on a microarray and the acquisition and analysis of fluorescence images. The primer sequences employed for the multiplex PCR and the procedures for carrying out the PCR and microarray hybridization are outlined in https://www.pgscatalog.org/score/PGS000054/, accessed on 16 August 2023). PRS values were calculated by summing the coefficients multiplied by the number of corresponding effect alleles as follows:To calculate the PRS, the model proposed by Tosto et al.  was usedAPOE alleles and PRS are shown in The fluorescence image examples of the microarray after analyzing DNA samples from AD patients and determining the genotype at each SNP with ɛ alleles of the APOE gene, we utilized real-time PCR with hydrolysis probes, where probes and primers from Yi et al. were used [® 96 .To confirm genotyping results, Sanger sequencing and real-time PCR techniques, including PCR with hydrolysis probes and high-resolution melting analysis, were used. Sanger sequencing was performed using a 3730xl Genetic Analyzer from Applied Biosystems, Foster City, CA, USA. To determine the ere used . High-reCSF was collected from 57 patients via lumbar puncture between 9:00 a.m. and 2:00 p.m. and transferred to sterile polypropylene tubes. The samples were centrifuged at 1500 rpm for 15 min, and the resulting supernatant was divided into 0.5 mL aliquots and stored at −80 °C. The commercially available MILLIPLEX immunoassay  was utilized according to the manufacturer’s instructions to measure Aβ40, Aβ42, tTau, and pTau181 in the CSF samples. The corresponding commercial kits describe the ranges of detection for the inflammatory parameters, and the analysis was conducted in duplicate. The protein concentration was quantitated in pg/mL.APOE alleles with CSF biomarkers and MMSE scores. Statistical analysis and visualization were conducted using R version 4.3.1 , with the “pROC”, “psych”, and “ggplot2” packages. We checked for correspondence to the Hardy–Weinberg equilibrium using the online service SNPStats  [p-values using the Benjamini and Hochberg (BH) method. A comparison of logistic regression models, incorporating social and genetic factors, was conducted using JASP software (Version 0.16.3). Statistical significance was determined when the p-value was less than 0.05.PRS was analyzed as a quantitative or nominal variable, dividing it into quartiles (1st/2nd–3rd/4th) or deciles (1st/2nd–9th/10th). Logistic regression was utilized to analyze the role of genetic factors in the risk of dementia. ROC analysis was performed to assess the discriminative ability for genetic and social factors. Linear regression was applied to investigate the associations of PRS and il 2023)  and calc® Statistical Software version 22.013 . We hypothesized that the ROC curve for the studied PRS model will be different from the referenced 0.5, and the expected AUC will be 0.57, according to Tosto et al. [Statistical power was assessed using MedCalco et al. . Based oAPOE-ɛ4 with high PRS values exhibit a higher risk of AD development. Associations of the APOE-ɛ4 and a higher PRS with CSF biomarker levels reflect neurodegenerative changes that are linked to Alzheimer’s disease.The selected polygenic risk model for Alzheimer’s disease confirmed its ability to accurately assess the risk of dementia in patients from the European part of the Russian population. Individuals carrying Overall, this study demonstrates the potential of the microarray-based assay for Alzheimer’s disease risk assessment, which may in the future allow for the screening of individuals and the referral of carriers of risk-associated traits to specialized prevention programs.


Water pollution stands as a pressing global environmental concern, elevating the significance of innovative, dependable, and sustainable solutions. This study represents an extensive review of the use of photocatalytic zinc oxide nanoparticles (ZnO NPs) for the removal of emerging pollutants from water and wastewater. The study examines ZnO NPs’ different preparation methods, including physical, chemical, and green synthesis, and emphasizes on advantages, disadvantages, preparation factors, and investigation methods for the structural and morphological properties. ZnO NPs demonstrate remarkable properties as photocatalysts; however, their small dimensions pose an issue, leading to potential post-use environmental losses. A strategy to overcome this challenge is scaling up ZnO NP matrices for enhanced stability and efficiency. The paper introduces novel ZnO NP composites, by incorporating supports like carbon and clay that serve as photocatalysts in the removal of emerging pollutants from water and wastewater. In essence, this research underscores the urgency of finding innovative, efficient, and eco-friendly solutions for the removal of emerging pollutants from wastewater and highlights the high removal efficiencies obtained when using ZnO NPs obtained from green synthesis as a photocatalyst. Future research should be developed on the cost–benefit analysis regarding the preparation methods, treatment processes, and value-added product regeneration efficiency.The online version contains supplementary material available at 10.1007/s11356-023-30713-3. The significance of water quality underscores the formulation of innovative design concepts for advanced nanomaterials, specifically tailored to address emerging pollutants in water bodies and wastewater treatment facilities , or Fe2O3 exhibit outstanding photocatalytic and antibacterial activity, making them the most promising materials in this field for the removal of organic pollutants and bacterial disinfection , make good photocatalysts for the purification of water .TiO2 or CuO, ZnO in the form of nanoparticles is a cheap, stable, and quickly obtained material  identify green syntheses as alternative for classical syntheses for ZnO, (ii) offer a screening analysis on EP types, (iii) analyze the degradation performances on EPs, and (iv) offer future perspectives.For the present purpose, we extensively reviewed a large number of research papers as the most suitable methodological tool for conveying the correct data in the field to academic scholars. Thus, a comprehensive literature review ranging from theory to experimental results was analyzed. Besides these, our research included a number of existing guidelines referring to the literature reviews available today Snyder . Many ofThe literature review was based on a number of publications between 2000 and 2023, according to the search engine of Web of Science. The primary keywords “green synthesis, ZnO” were chosen, for which a number of 4679 publications were recorded. According to the citations and publication number, it could be observed that in the last 10 years, there has been a continuous interest in the field of green synthesis for ZnO preparation. Thus, from only two or three papers at the beginning of 2000, a number of 725 papers were published in 2022. The number of citations was significant from 2020 where over 10,000 citations were recorded. Figure Research design and methods of synthesisPhotocatalysis performances for emerging pollutantsCharacterization investigation for ZnO NPs in order to validate structure and material performancesThe selected publications, based on mentioned keywords, were collected and organized in accordance with the following criteria:From the presented publications, an important impact for our research was identified at a number of 342 references that was chosen for EndNote as reference manager, with high impact as scientific information regarding ZnO NPs obtained with green synthesis and high efficiency as photocatalyst. The results of these publications are presented in the next chapters of this paper.Based on conventional methods, ZnO NPs can be synthesized by applying controlled chemical and physical methods, such as sol–gel, sonochemical, solvothermal/hydrothermal, precipitation, microemulsion, and polyol   and about 80% phenol from water  and TiO2-Bi2WO6 composite was developed for Rhodamine blue degradation  , and decreasing precipitating agent volume per minute leads to the change of morphology from ZnO NPs to nanorods , paracetamol (PAR), diclofenac sodium (DCF), and ciprofloxacin (CIP) pharmaceuticals in water Through sonochemical synthesis, the crystallinity and size of ZnO NPs depend on the ultrasonic wave intensity, sonication time, and solvent type   nanocomposites showed an enhanced photocatalytic activity in the degradation of methylene blue (MB) and methyl orange (MO), attributed to the formation of a heterojunction, which facilitates effective hole extraction by BNQDx while simultaneously preventing the recombination of photoinduced charge carriers. The optical characteristics of both ZnO and ZnO/BNQDx nanocomposites were explored using UV–visible diffuse reflectance spectroscopy and photoluminescence (PL) analysis, as displayed in Fig. x  capable of absorbing visible light photons  through a one-pot microwave-assisted hydrothermal synthesis as a promising approach for polychlorinated biphenyl (PCB) degradation  synthesis method for ZnO nanoparticles offers advantages such as controlled size and enhanced properties but is accompanied by notable drawbacks  in which the surface is immersed  as an environmentally friendly, cost-effective, safe, byproduct-free, ecological solution , dendrimers, and block copolymers with extracts from plants, fungi, yeasts, bacteria, and algae , but they can also be stabilizing or coating agents. From certain parts of plants, from roots and fruits to seeds, reducing extracts and stabilizer properties for ZnO-type nanoparticles can be obtained, such as: Orthosiphon stamineus leaf extract peaked at 40 °C with 80% methanol and began to decline at 60 °C as a result of degradation , a source rich in phytochemicals, which also contains flavonoids and catechins, all with a reducing role for metal salts, but also other compounds of the nitro or ketone type  solution and grape extract in the presence of NaOH to maintain the pH of the mixture at a value of 8, the white color indicating the formation of ZnO NPs 2 is formed, and by calcination, ZnO  to zero valence, after which Zn oxide is formed by calcination. In the case of polyphenols from plant extracts, they produce complexation with ZnZnO Fig. . The forZnO Fig. .Fig. 7MeOne of the advantages of green synthesis is the integration of ZnO NP synthesis processes into the concept of sustainability and waste minimization 2·6H2O, in the presence of a solution of NaOH. After maintaining the temperature for 3 h and drying the precipitate, ZnO NPs of approx. 15–25 nm were obtained 2·6H2O solution, the precipitate formed being dried in an oven at 60 °C and calcined at 700 °C for obtaining crystalline ZnO NPs. In order to make the dye degradation process more efficient by irradiation with natural sunlight, NPs were anchored in multilayer graphene obtained from corn husk, thus obtaining nanocomposites with a crystallite size of about 40 nm  were created using three different green protocols: sonochemical, mechanochemical, and hydrothermal methods  demonstrated that the synthesis methods had a substantial influence on the morphology of ZnO, resulting in various nanostructures depending on the chosen method Fig. . SpecifiOlea europaea) fruit extract in the synthesis of zinc oxide NPs (ZnO@OFE NPs)  and methyl orange (MO) dyes when exposed to sunlight. To conduct a comprehensive analysis of their photocatalytic activity, a catalyst dose of 30 mg in 30 mL of solution was determined to provide optimal absorptions of 75% for MB and 87% for MO within 180 min, with photodegradation rate constants of 0.008 and 0.013 min−1, respectively. Additionally, the ZnO@OFE NPs displayed impressive antioxidant properties, combating DPPH, hydroxyl, peroxide, and superoxide radicals. In contrast, when sunlight was absent, the removal efficiency via adsorption onto the ZnO@OFE NPs’ surface was only 12% for MB dye and 10% for MO dye after 180 min.A different research paper documented the utilization of fresh olive  and MO . These outcomes were twofold compared to the performance of pure ZnO and CuO NPs.Bimetallic ZnO–CuO hetero-nanocomposite, ZnO, and CuO nanostructures were synthesized utilizing a hydrothermal synthetic procedure, employing leaf extract from Lactobacillus casei, prokaryotic bacteria, actinomycetes, Escherichia coli, etc. Yeasts can be successfully applied in green synthesis to obtain nanoparticles of silver, gold, zinc, and/or titanium  and multicellular algae (such as chlorophyll) serve as examples of photosynthetic organisms, notably including brown algae. Unlike conventional plants, algae lack typical plant structures like leaves and roots. Marine algae are categorized based on their pigments, with Rhodophyta, Phaeophyta, and chlorophytes characterized by red, brown, and green pigments, respectively. Algae have been extensively utilized for producing gold (Au) and silver (Ag) nanoparticles, but their application in synthesizing zinc oxide (ZnO) nanoparticles has been limited and is documented in relatively few studies , along with the identification of carbonyl and hydroxyl stretching in nanoparticles that exhibited substantial structural diversity. From some microalgae, ZnO NPs of about 36 nm can be obtained, whose stability has been demonstrated through structural investigations even after 6 months  under natural sunshine. This technology offers a cutting-edge, environmentally benign way to make zinc oxide nanoflowers with potential uses in water purification and dye deterioration.An eco-friendly, biologically mediated method for producing ZnO nanoflowers at low temperatures has been investigated in a recent study  and membrane separation processes combined with biological treatments   is essential in establishing the interactions between adsorbate and adsorbent at different pH values , which ends up in water through urine, after consumption  lead to the formation of reactive oxygen species (ROS), such as hydroxyl radicals (OH∙) and superoxide radicals (O2∙−). These ROS are highly reactive and can attack and degrade the pollutants, further enhancing the photocatalytic activity of ZnO. The degradation process that was based on this mechanism was successfully applied in the degradation of dyes and is presented in Fig. The photocatalysis process follows specific kinetic models as indicated in the literature, which serves as the basis for establishing the degradation mechanism. Adeola et al. indicate the first-order Langmuir–Hinshelwood model for the ZnO-CuAn efficient photocatalyst must provide good charge separation and efficient light absorption, especially in the visible region. Thus, semiconductors alone require a high intensity of light, induce charge carriers, and thus lead to limitations of their applicability. Doping with elements or obtaining a composite photocatalyst can provide solutions to these challenges  were synthesized using a hydrothermal method and characterized through SEM, XRD, and XPS analyses that confirmed the formation of wurtzite ZnO and anatase TiO2 in the tandem nanostructure , rapid recombination, and low charge-transfer rates of photoinduced electron–hole pairs, TiOThe successful chemical precipitation process was employed to synthesize spherical ZnO NPs of varying sizes using different solvents, namely, water (referred to as ZnOw) and ethanol (referred to as ZnOe) , high-resolution transmission electron microscopy (HRTEM), and selected area electron diffraction (SAED) were performed. The TEM images , (1 2 2)), and gC3N4 ((0 0 2)) were identified and marked.Novel nano-heterostructures consisting of gCges Fig. a and b rage Fig. c allowedern Fig. d display2O/ZnO/rGO heterojunction photocatalysts used for the photocatalytic degradation of some BPA pollutants under simulated sunlight. Doping with Ag led to the lowering of the band gap of ZnO, which led to about 80% removal efficiency of BPA using 5% Ag and 3% GO by weight. The reuse of Ag2O/ZnO/rGO is possible, the material showing good photostability and pH adaptability , gold (Au), and palladium (Pd) , denoted as CNi0.05 and CNi0.1 , (002), and (101), which are indicative of the presence of ZnO NPs within the composites , confirming the presence of this phase in the composite materials. The absence of any other XRD peaks, aside from those of ZnO and NiFe2O4, demonstrated the successful formation of single-phase ZnO/NiFe2O4 nanocomposites using this synthesis method.ZnO/NiFetes Fig. . AdditioIn the absence of a catalyst, the degradation of Rhodamine B only reached 10%, while the degradation of MB reached 30% after 3 h. Notably, the CNi0.05 nanocomposite showed the highest degradation percentages for both dyes, when the degradation percentage reached 98% for Rhodamine B and 97% for MB at using the CNi0.05 composite as the catalyst. Without exposure to light, the degradation percentage of the CNi0.05 composite remained low at 6–8% after 3 h. These findings provide clear evidence that the degradation of dyes by CNi0.05 occurred through photocatalysis, as the irradiation process substantially enhanced the degradation percentages.3O4@rGO@ZnO/Ag NPs (FGZAg) is synthesized by using graphene oxide and ZnO-coated Fe3O4 microspheres doped with Ag NPs, and its efficiency in metabolizing metformin (MTF) is examined both in the ultraviolet and visible spectra of light. The resulting samples were analyzed using a variety of analytical techniques, which revealed onion-shaped spheres with a hexagonal wurtzite crystal structure and a mesoporous texture, as well as Ag NPs that were securely stuck to the catalyst’s surface. When degrading MTF under visible light, FGZAg showed significantly improved photocatalytic activity when compared to pure ZnO, completing total degradation and 60% mineralization of 20 mg/L MTF in just 60 min.In another study Khavar et al. , a novel2O3) and ZnO (wurtzite form) in their crystalline phases  from wastewater, a microwave-prepared porous ZnO/SnSTo enhance even more the stability and photodegradation properties of ZnO NPs, studies have reported the preparation of novel matrices with eco-friendly supports like clays and carbon-based materials that are further discussed in detail in the next chapter.3N4), and graphene , graphene oxide (GO), carbon nitride , with efficiency in the photocatalytic mineralization of ofloxacin from aqueous solution. The literature indicates the obtaining of nanophotocatalysts in situ with different contents of GO  using zinc nitrate, with pH control of about 9, in the presence of ammonia liquid at 110 °C for 7 h /ZnO composite was used to degrade atenolol, a beta-blocker used to regulate blood pressure, by artificial irradiation, achieving a degradation efficiency of 85% at a catalyst dosage of 1.2 g/L, pH 4 after 60 min . The synthesis of the ZnO/CdO/rGO and ZnO/CdO composites took place in an autoclave by using specific metal precursors of the type Zn(N03)2·6H20 and respectively Cd(N03)2·4H20, in the presence of GO, in a basic environment  obtained from bacterial cellulose biomass (BC) for the removal of persistent pharmaceutical substances such as tetracycline and di-chlorophenol. The degradation efficiency of tetracycline was about 97%, with visible light.The literature also indicates photocatalysts obtained by green synthesis in the form of a quaternary nanocomposite of the P-doped graphitic carbon nitride (PGCN)/AgI/ZnO/CQD type, from bamboo leaves, used for photocatalysis assisted adsorption at pH 4 of 2,4-dinitrophenol, with an efficiency of about 98% in 2 h , and triangular silver nanoplates (T-Ag)/ZnO and ZnO/N,S-doped carbon quantum dots  nanoflowers showed high efficiency for the degradation of norfloxacin in light visible, due to the synergistic and surface plasmon resonance effect of T-Ag on ZnO nanoflowers as well as the transfer of photogenerated electrons from the ZnO conduction band to the N,S-CQD surface  was 86.7% after 50 min 2·6H2O, the obtained solutions being mixed in different proportions with N,S-CQD, then placed in an autoclave at 100 °C for 12 h   was possible for 5 consecutive cycles, showing excellent chemical stability. In this sense, it can be observed that after the 5th cycle, the nanophotocatalyst still shows 93.7% of its initial activity, due to the strong bonds created between the ZnO NPs and the ICLT support.The nanophotocatalyst ZnO/ICLT prepared by sonoprecipitation demonstrated high photocatalytic activity for the degradation of furosemide. The zinc acetate precursor in the presence of NaOH was introduced into a homogeneous suspension dispersed by zeolite, which after about 2 h led to the formation of a precipitate. This by calcination led to homogeneous nanoarchitectures  values indicate a removal of more than 50% at natural pH  effectively adsorbed and removed malachite green and Congo red dyes   morphology was also used in the composition of Ag@ZnONSt and Pd@ZnONSt photocatalysts for the degradation of methyl parathion (MPT), pendimethalin (PDM), and trifluralin (TFL). Pd@ZnONSt had 99.8% degradation efficiency and stability over six degradation cycles  when removing a high concentration of MB  in comparison with MO dye (k = 2.325 × 102 min−1), which was likely due to the latter’s simpler structure  was combined with PANI-ZnO hybrids created in this study, and the combined PANI-ZnO/GCE system was evaluated for its potential as a microbial fuel cell anode. Through the chemical oxidative polymerization of aniline, Eskizeybek et al. developed PANI/ZnO nanocomposites and looked into how MB and malachite green (MG) dyes in aqueous medium were degraded under both natural and UV light irradiation  almost 63 times faster than that of the pure ZnO photocatalyst’s (k = 0.04 × 102 min−1)  was emphasized. Improved visible light absorption and a decrease in charge carrier recombination were related to the photocatalytic activity under UV and visible irradiation. After 6 cycles, the photocatalytic effectiveness under UV and visible irradiations only decreased by 9% and 8%, respectively. As a result, the findings demonstrated that the ZnO/PANI nanocomposite had excellent stability and could be utilized repeatedly.The removal of metronidazole (MNZ), a synthetic antibacterial agent, from wastewater before it is released into the environment has drawn significant attention due to its non-biodegradability and high solubility in water. Asgari et al. recently looked into the ZnO/PANI nanocomposites’ photocatalytic capacity for degrading MNZ when exposed to UV and visible light. They found that the ZnO/PANI nanocomposite presented a rate of MNZ degradation (k = 0.986 min−1) than the PPy-ZnO 5:1 composite. The sensitizing impact of PPy was said to be responsible for this improvement. However, it was demonstrated that a high PPy to ZnO ratio could result in flaws on the PPy surface that act as recombination centers for electron–hole pairs and lead to lower photocatalytic activity. The study also showed that after three consecutive cycles of degradation, the PPy-ZnO 25:1 composite maintained its photocatalytic activity, showing its potential for reuse.The synthesis of composite photocatalysts using ZnO nanoparticles and polypyrrole (PPy) is also discussed in the literature. These composites were made using PPy to ZnO ratios of 5:1 and 25:1, respectively   in the synthesis of biogenic ZnO NPs  were developed  proved to be the most efficient composite achieving 97.2% photodegradation of tetracycline. As anticipated, chitosan and graphene significantly improved the results of the degrading process. Taking into account the used operational settings, this innovative photocatalyst is capable of treating pharmaceutical wastewater.By using sol–gel and ultrasound-assisted techniques, several nanocomposites based on metal oxides and chitosan  (Maximize Market Research). The most effective methods involve nanomembrane systems, essential in pollutant removal and softening, especially for pharmaceutical contaminants. In addition, nanoadsorbents are included in this report as efficient materials for a wide range of organic and inorganic pollutants. An ideal nanomaterial is the one with the smallest possible dimensions to exhibit catalytic potential and reactivity through a large specific surface area; systems that include metal oxides such as ZnO will represent the next generation of sustainable materials.Because the risk of reaching the environment, after being used as a photocatalyst, is quite high, due to the small size, the creation of some supports that incorporate these NPs represents a viable alternative to current techniques. The most used support materials are the carbon ones, with proven properties and which in turn can be obtained from waste. Currently, the photocatalytic activity of these NPs is recognized, and these applications will represent a potential nexus on the research market.The conventional preparation methods of ZnO NPs are associated with toxicity, which has been a restraining factor for the global ZnO NP market. However, the new trend in green synthesis is bringing attention to new perspectives regarding the acquisition and integration of these NPs. The investments in the ZnO NP sector are in expansion and linked with research and development in industry. Environmental concerns regarding EPs bring into question new industrial solutions and participants in the global market. Future researches should be developed on the cost–benefit analysis regarding the preparation methods, treatment processes, and value-added product regeneration efficiency.Supplementary file1 (PDF 24 KB)Supplementary file2 (PDF 30 KB)Supplementary file3 (PDF 181 KB)Supplementary file4 (PDF 29 KB)Below is the link to the electronic supplementary material.


Sulfadiazine (SDZ) and copper (Cu) are frequently detected in agricultural soils, but little is known on their single or combined impact on ammonia oxidizing microbial community and function across different soils.amoA gene sequences.In this study, a microcosm was conducted to distinguish the microbial ecotoxicity of SDZ and Cu across different soils by analyzing soil potential nitrification rate (PNR) and the amoA gene abundance, and diversity as well as the identified genus taxa of ammonia-oxidizing archaea (AOA) and bacteria (AOB). Meanwhile, only the specific genus taxa of AOA or AOB consistently corresponded to the variation of soil PNR across different treated soils. The further principal component analysis (PCA) exhibited that the variable influence of SDZ and Cu on ammonia oxidizing microbial community and function was greatly dependent on soil type.The results showed that the single spiking of SDZ caused a consistent decrease of soil PNR among three tested soils, but no consistent synergistic inhibition of SDZ and Cu was observed across these soils. Moreover, across three tested soils, the distinct responses to the single or joint exposure of SDZ and Cu were found in Therefore, in addition to ecological functionality and the specific prokaryotic taxa, soil microbial ecotoxicity of SDZ and Cu also was dependent on edaphic factors derived from soil types. This study proposes an integrative assessment of soil properties and multiple microbial targets to soil contamination management. The present study would offer an implication for the integrative management of soil contamination.2.2.1.Black soils, Brown soils, and Desert soils, three typical soil types of China, were sampled from three farmland zones along distinct climatic gradients . Surface2+ stock solution was prepared with CuSO4 in ultra-pure water. The reagents, except methanol, are all of the analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. .Sulfadiazine  was purchased from Shanghai Yuanye Biological Technology Co. Ltd.  and prepared in a stock solution (40 mmol/l) with ultra-pure water after being dissolved by methanol (HPLC grade). A total of 1,000 mg/l of Cu2.2.4-N and NO3-N) was extracted with 1 M KCl and analyzed by a flow injection analyzer . The clays were analyzed by a laser particle size analyzer  after being scattered with sodium hexametaphosphate. The cation exchange capacity (CEC) was measured by analyzing the concentration of exchangeable cations in ammonium acetate extract of soils with an inductively coupled plasma-atomic emission spectrometry . Cu in soils was determined with an inductively coupled plasma atomic emission spectrometer  after digested by HCl, HNO3, and HF  in a microwave digestion system 2SO4 and additional deionized water to reach soil moisture of 60% of WHC for soil potential nitrification rates (PNRs) analysis according to the ISO method  and then pre-incubated in dark at 25 ± 2°C for soil microbial recovery after adjusting water content to 50% of the field water-holding capacity . At theO method  with sliO method ,b; anoth2+, and C2S5 for the combined spiking of 5 μmol/kg SDZ and 200 mg/kg Cu2+. The soil treatments without any addition of Cu or SDZ were used as the control treatment designed as CK for each soil. Each soil treatment was conducted in triplicates.In this study, 5 μmol/kg of SDZ was chosen as the added rate according to the previous reports , but a 22.4.The fresh soil samples were transported in iced boxes to Lc-Bio Technology Co., Ltd.  for DNA extraction, PCR amplification, sequencing, and taxonomic assignment ,b.amoA and AOB-amoA were drawn as described previously  with the standard curves, respectively.In brief, the total DNA was extracted from 0.25 g of each soil sample with the PowerSoil™ DNA isolation kit . The fluorescence quantitative PCR  was conducted in triplicate on a real-time ABI7500  using the primer amoA-1F/amoA-2R for ammonia-oxidizing bacteria (AOB) and CrenamoA23F/A616R for ammonia-oxidizing archaea (AOA) . The staeviously ,b. QuantThe primers used for PCR amplification were the same as the qPCR as well as the amplification systems and conditions. After purification using the SanPrep® quick PCR purification kit, pyrosequencing working of the target PCR products was accomplished on the Illumina Miseq PE300 sequencing platform , and each treatment was performed in triplicate. The raw sequences were quality-filtered and processed using QIIME v2.0. Obtained sequences from different soil samples were, respectively, clustered into the operational taxonomic unit (OTU) at 97% sequence similarity using UPARSE (Version 7.1) after chimera detection using USTARCH7.0. The archaeal and bacterial OTUs were classified by using the ribosomal database project (RDP) classifier against the functional genomics resource (FGR) functional gene database.2.5.post hoc test at a value of p of <0.05 significant level. The principal component analysis (PCA) and the canonical correlation analysis (CCA) were used to account for the influence of soil properties on the variation of soil PNR and ammonia-oxidizers’ community in soils after being treated with SDZ and Cu.The software package SPSS 18.0  was used for data statistical analysis. The significant differences between soil treatments were identified by the one-way variance analysis (ANOVA) with Tukey’s HSD 3.3.1.F = 1031.84, p ≤ 0.05) inhibited in soils treated with SDZ and Cu singly or simultaneously; moreover, this inhibition was different across three tested soils. In detail, for the single exposure of SDZ, the highest decrease in soil PNR was observed in Brown soils, while the highest decrease of soil PNR was observed in Black soils when exposed to Cu. The combination of SDZ and Cu showed an antagonistic inhibition on soil PNR in Desert and Black soils, but there was a synergetic inhibition in Brown soils.As shown in amoA gene abundance  in Desert soils, while a significant inhibition  in Brown soils was observed when exposed to SDZ and Cu singly or simultaneously. In Black soils, amoA gene abundance was little changed with the single application of SDZ, while it was a synergetic inhibition to the combined exposure of SDZ and Cu. Similar results were also found for the individual AOA or AOB groups  proliferated in Desert soil, but it was inhibited in Black and Brown soils with the single application of SDZ or Cu, although the synergistic inhibition was all found in these three soils to the combined exposure of SDZ and Cu. In addition, both norank_Thaumarchaeota and norank_Crenarchaeota of the AOA group were mostly bolstered in three tested soils under the single or combined stress of SDZ and Cu  under Grant Nos. 42077129, 41877122, 41671322, and 42177403 and the Shandong Natural Science Foundation under Grant Nos. ZR2020ZD19 and ZR2022YQ34.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.


Bladder cancer (BLCA) typically has a poor prognosis due to high rates of relapse and metastasis. Although the emergence of immunotherapy brings hope for patients with BLCA, not all patients will benefit from it. Identifying some markers to predict treatment response is particularly important. Here, we aimed to determine the clinical value of the ribonuclease/angiogenin inhibitor 1 (RNH1) in BLCA therapy based on functional status analysis. First, we found that RNH1 is aberrantly expressed in multiple cancers but is associated with prognosis in only a few types of cancer. Next, we determined that low RNH1 expression was significantly associated with enhanced invasion and metastasis of BLCA by assessing the relationship between RNH1 and 17 functional states. Moreover, we identified 95 hub genes associated with invasion and metastasis among RNH1-related genes. Enrichment analysis revealed that these hub genes were also significantly linked with immune activation. Consistently, BLCA can be divided into two molecular subtypes based on these hub genes, and the differentially expressed genes between the two subtypes are also significantly enriched in immune-related pathways. This indicates that the expression of RNH1 is also related to the tumour immune response. Subsequently, we confirmed that RNH1 shapes an inflammatory tumour microenvironment (TME), promotes activation of the immune response cycle steps, and has the potential to predict the immune checkpoint blockade (ICB) treatment response. Finally, we demonstrated that high RNH1 expression was significantly associated with multiple therapeutic signalling pathways and drug targets in BLCA. In conclusion, our study revealed that RNH1 could provide new insights into the invasion of BLCA and predict the immunotherapy response in patients with BLCA. BLCA can be classified according to pathological and clinical manifestations as non-muscle invasive BLCA (NMIBC) and muscle invasive BLCA (MIBC)2. Transurethral resection of the bladder tumour (TURBT) is the mainstay therapy of those with NMIBC3, but they almost always recur and may progress to invasive BLCA. Radical cystectomy is the standard therapy for patients with MIBC4. However, because MIBC can rapidly metastasize to lymph nodes, liver, lungs, bone, and brain, approximately 50% of patients eventually develop distant disease due to disseminated micrometastases7. Although the combinations of radiotherapy, immunotherapy, and chemotherapy have improved survival9, most patients still experience relapse and metastasis, which leads to a poor prognosis. An important reason is the high functional heterogeneity of cancer cells. This underscores the urgent need to develop a method to decode the functional state of cancer cells and to identify some markers that affect specific functional states for predicting treatment response. In addition, it is vital to find a key molecule that can remodel the noninflamed tumour microenvironment (TME) into an inflamed TME and has the potential to be a combination therapy target10.Bladder cancer (BLCA) is one of the most common malignancies of the urinary system11. RNH1 interacts with angiopoietin (ANG) to inhibit its ribonuclease activity, and can also bind to the 40S ribosome and control translation12. For example, RNH1 binds to ribosomes and regulates erythropoiesis by controlling specific translation of the erythroid transcription factor GATA113. Additionally, several studies have identified that RNH1 is involved in various functional states such as cell differentiation, proliferation, invasion, migration, apoptosis, autophagy, and the cell cycle, apart from its ribonuclease inhibitor function. Rnh1 promotes differentiation and myelination via RhoA in oligodendrocytes in Rattus norvegicus14. RNH1 is essential for adult haematopoietic stem cell function and cell cycle regulation15. In cancer, RNH1 inhibits the proliferation of mouse melanoma16; overexpression of RNH1 in colorectal cancer cells HT29 resulted in increased autophagy17. Studies have also found that the upregulation of RNH1 inhibits cell proliferation, migration and invasion in BLCA, alters cell morphology and adhesion and leads to rearrangement of the cytoskeleton in vitro18. However, the application value of RNH1 in BLCA therapy has not been described and its role in immunotherapy is unclear.Ribonuclease/angiogenin inhibitor 1 (RNH1), a ribonuclease inhibitor (RI), is a ubiquitously expressed protein in the cytoplasm, nucleus, mitochondria as well as on endoplasmic reticulum19, we developed the R package FUNscore to assess tumour functional status and revealed that low expression of RNH1 was significantly associated with enhanced invasion in BLCA. In addition, this invasive effect is associated with immune activation and molecular subtypes of BLCA. Importantly, RNH1 can serve as a marker of invasion and has the potential to predict the therapeutic response to immunotherapy in BLCA.In the present study, RNH1 was found to be abnormally expressed in various cancers based on pancancer analysis. Based on the CRDscore algorithm developed by He et al.TCGAbiolinks (v2.20.0)20 in R  (accessed on 1 August 2021). For cancer types with no normal samples, or with less than ten normal sample number, we downloaded standardized data from the UCSC Xena data portal integrating the Genotype-Tissue Expression (GTEx) dataset. The abbreviations for various cancer types are given in Table The Cancer Genome Atlas (TCGA) data: the HTSeq–Counts data and the corresponding survival and clinicopathological information of cancer patients were downloaded from the TCGA data portal using the software package GEOquery (v2.64.2) in R. Detailed information on these datasets is available in Table Gene Expression Omnibus (GEO): Six BLCA GEO cohort datasets, namely GSE31684, GSE5287, GSE48057, GSE48277, GSE13507, and GSE69795, were downloaded using the software package IMvigor21021.Based on the Creative Commons 3.0 Licence, the complete expression data and detailed clinical information of a BLCA immunotherapy related IMvigor210 cohort (patients treated with atezolizumab) were obtained from the R data package 23. We also used UALCAN to analyze the promoter methylation level of RNH1. We utilized the cBioPortal web service25 for analysis the genetic alteration status of RNH1 based on the TCGA cohorts.The expression of the RNH1 gene in normal and tumour tissues was analysed in R software. To assess the expression of RNH1 protein in different cancers, we used data from Clinical Proteomic Tumor Analysis Consortium (CPTAC) in the UALCAN web service for analysis26, samples were stratified into high and low expression groups according to the median expression of the RNH1 gene in each cancer type, and OS and RFS analyses were performed. Similarly, we used cBioPortal to analyse the correlation between the genetic alteration status of RNH1 and disease-free survival (DFS) across cancers27. In addition, we used the survival package in R software to perform OS analysis for other specific groups.For the prognostic analysis of RNH1 expression in across cancers, we used the GEPIA2 tool to perform overall survival (OS) and relapse-free survival (RFS) analyses. In the ‘Survival Analysis’ module on the GEPIA2 web server28. Additionally, the collection of autophagy-related and ferroptosis-related genes was obtained from the HADb29 and FerrDb30 databases, respectively. A study identified gene signatures associated with tumour immune escape in mice31, and we converted these gene signatures to human orthologues. A total of 17 collected FUNGs  from the cancerSEA databaseGs Table  were use19 developed an algorithm that accounted for variation in the signal‐to‐noise ratio across genes and cells based on the expression profiles of circadian‐related genes to assess the level of circadian rhythm disruption. Here, we extend the algorithm to a broader field for assessing the functional status of tumour samples or cells. The specific steps were as follows:He et al.C of bulk RNAseq, microarray, or TPM for scRNA‐seq, we define the average expression Ei of gene i across N samples or cells as:First, for a given normalized expression matrix Ei, and the frequency of FUNGs in each bin was counted and designated BFUNGs. The signature genes K were randomly selected with the same number of FUNGs in each bin and iterated 1000 times. Subsequently, we define a centred gene expression matrix that can be interpreted as the data without an excessive migration signal:Xi,j represents the central expression of gene i in the sample or cell j.Next, a random sampling strategy was employed. All genes were divided into 50 expression bins according to their average expression K × 1000 random signatures sampled above and the FUNGs score, Finally, a random score, https://github.com/BioInfoNote/FUNscore.A cut-off of 75% based on quartiles was used as the threshold for p value < 0.01, Table WGCNA package32in R software was employed to execute the WGCNA analysis. In the process, we set the minimum module size (minClusterSize = 30) and soft-thresholding power (power = 6) for network construction. Subsequently, we calculated the correlation of each module with 17 i, ME). In this study, a gene with GS > 0.6 and MM > 0.8 was defined as a hub gene among the candidate gene modules.First, we obtained an expression data profile containing only genes encoding proteins significantly correlated with RNH1 expression . Gene Ontology (GO) enrichment analysis was performed using the clusterProfiler package (v4.0.2)34 in R.Differential expression analysis of RNA was performed using the DESeq22 to infer the consensus subtype of BLCA, which included a combined consensus subtype and six published molecular classifications (University of North Carolina (UNC), Baylor, Cancer Genome Atlas (TCGA), MD Anderson Cancer Center (MDA), Lund and Cartes d’Identité des Tumeurs (CIT)). Additionally, we inferred consensus clusters based on the 95 hub gene signatures using the R package ConsensusClusterPlus35. The optimal cluster number k was chosen depending on the elbow and CDF curve. Principal component analysis (PCA) was performed to evaluate the difference between the clusters. Molecular subtype information is available in Table We used the R package consensusMIBC36 developed and validated a pancancer T-cell-inflamed score, which could define preexisting cancer immunity, and predict the clinical response of immune checkpoint blockade (ICB) therapy. Here, we used a model to calculate the T-cell inflammation score. The calculation formula was as follows:Ayers et al.39 was used to evaluate the fractions of 28 tumour-infiltrating immune cell (TIIC) phenotypes in the tumour microenvironment. To avoid computational errors caused by a single algorithm and different sets of marker genes for TILs, we downloaded immune infiltrate data evaluated using the CIBERSORT40, CIBERSORT-ABS41, EPIC42, MCP-counter43, quantTIseq44, xCell45, TIMER46 and TIDE algorithms for the 33 cancer types from the TCGA database using the TIMER2 web server47.Single-sample gene set enrichment analysis (ssGSEA)48 (Table 2 (Table https://go.drugbank.com/) to compare their expression in RNH1 groups and BLCA subtypes.Gene signatures of several potential therapeutic pathways were collected from the study by Hu et al.48 Table . Twelve  2 Table . ssGSEA  2 Table  obtainedp < 0.05, and all statistical tests were two-sided. All statistical data analyses were performed using the R software or online analysis tools described in the relevant Materials and Methods subsections.Correlations between variables were explored using Spearman or Pearson coefficients. Continuous variables that conformed to the normal distribution were compared using independent t-tests for comparisons between binary groups, while continuous variables with skewed distributions were compared with the Mann–Whitney U test. Survival curves for categorical variable prognostic analyses were generated using the Kaplan–Meier method, while the log-rank test was used to estimate statistical significance. The significance level was set at Our study did not require an ethical board approval because this article does not contain any studies with human participants or animals performed by any of the authors.First, we explored the expression of RNH1 in human cancers based on the TCGA, GTEx, and CPTAC databases. We found that the mRNA expression level of RNH1 was low in tissues of 15 cancer types  compared with normal tissues (Fig. Next, we used the cBioPortal tool to analyse the genetic alteration status of RNH1 in different cancer types from the TCGA cohorts. We observed the highest RNH1 alteration frequency among patients with seminoma, followed by glioma (Fig. The aberrant expression pattern of RNH1 in human cancers prompted us to explore its prognostic value. Therefore, we performed a pancancer survival analysis concerning OS and RFS. We found that RNH1 expression was significantly associated with OS or RFS in several cancer types. As shown in Fig. Taken together, we determined that RNH1 is aberrantly expressed in multiple cancers but is associated with prognosis in only a few types of cancer. The relationship between this aberrant expression and human cancers requires further exploration.Pancancer analyses were performed to explore whether there are commonalities in the function of RNH1 in human cancers and to explore its functional role. Therefore, we evaluated the 17 functional status scores for each tumour sample. We found that the effect of RNH1 on the functional status of most cancers was not significant Fig. . NotablyTo further uncover the most representative and correlated hub genes affecting functional status, we performed WGCNA based on an expression profile containing only 3416 genes encoding proteins significantly correlated with RNH1 expression to find modules of genes highly correlated with functional status. Eight gene modules were obtained using the dynamic tree cut algorithm, and the RNH1-related genes were determined to predominantly link the brown, turquoise, and blue modules Fig. . InteresclusterProfiler package. Figure Molecular subtypes of BLCA have been constructed in several studies. Here, we wondered about the potential of RNH1 for predicting molecular subtypes. ConsensusClusterPlus was used to perform consensus clustering on the samples according to the 95 FUN-related hub genes. The optimal number of clusters was determined based on the CDF. According to the CDF curve and delta area under the CDF curve Fig. A,B, clusBased on the above analysis, RNH1 might be associated with the immune response. Next, we further explored the immunological role of RNH1 in BLCA. We found that RNH1 was positively correlated with a majority of immunomodulators in BLCA based on TCGA data, but only a few in other cancer types Fig. . This im49. Therefore, we calculated the correlation between the expression of RNH1 and the inflammatory score using a pancancer T-cell inflammation score model developed and validated by Ayers et al.36. Consistently, RNH1 expression in BLCA was negatively correlated with T-cell inflammatory scores (Fig. 50. Thus, we further evaluated the correlation between the expression of RNH1 and 20 typical inhibitory immune checkpoints. As expected, RNH1 expression in BLCA was positively correlated with the expression of multiple immune checkpoints, such as PD-L1, CD80, CD86, HAVCR2, IDO1, LAG3, LAIR1, and PD-1 (Fig. High immune cell infiltration is linked to an immune-inflamed TMETo further explore the potential of RNH1 for predicting treatment candidates in BLCA, we first analysed the relationship between RNH1 expression and different BLCA-related signalling pathways. Surprisingly, changes in RNH1 expression levels significantly affected BLCA-related signalling pathways Fig. A. For ex18. This suggests that multiple important roles of RNH1 in cancer deserve further exploration.BLCA is one of the most common malignant tumours of the urinary system. NMIBC patients often relapse after TURBT, whereas MIBC patients eventually develop distant disease due to disseminated micrometastases. This brings great challenges to the treatment of BLCA, and there is an urgent need to develop more accurate and effective treatment strategies. Identifying effective markers of therapeutic response is a viable strategy. It is generally known that RNH1 plays a role in tumorigenesis by affecting the functional status of tumours in terms of processes, such as cell differentiation, proliferation, invasion, migration, apoptosis, and cell cycleIn this study, pancancer analysis indicated that RNH1 was aberrantly expressed in a variety of cancers. Nevertheless, the impact of RNH1 expression on OS and RFS varied depending on the cancer type. It is worth mentioning that our analysis revealed higher methylation levels of RNH1 in multiple cancer tissues than in normal tissues. Although DNA methylation is an important factor affecting mRNA transcription, we found that the methylation level of RNH1 is very low in various cancer tissues. Therefore, the methylation of RNH1 affects its expression, but more experimental evidence is required to support this claim. Additionally, we found that RNH1 exhibits a high rate of deep deletion among genetic alterations in most cancers, but we noted that the total frequency of RNH1 alteration is not high in various cancers. This may be the reason why RNH1 alterations are not associated with OS or DFS.19. We found that RNH1 affects multiple functional states in BLCA based on a pancancer analysis. The affected functional status has been demonstrated in several studies. Additionally, we confirmed this finding based on other independent GEO datasets. Importantly, EMT and invasion have coincident results in several GEO datasets. This suggests that RNH1 plays an important role in BLCA by affecting EMT and invasion. Previous studies have shown that metastasis and invasion of bladder malignant tumours are important causes of death in patients51. Although studies have found that RNH1 inhibits the migration and invasion of BLCA cells, the underlying molecular mechanism has not been thoroughly studied18. Here, we found that RNH1 was downregulated in BLCA compared to NBM or BMSC. In addition, we also observed a higher FUN score for both invasion and EMT in BLCA. More importantly, high expression of RNH1 in MIBC was associated with higher FUN scores for EMT, invasion, and metastasis. These results confirm the reliability of the FUNscore method. This results also suggest that RNH1 may have significant clinical value in BLCA. Consistently, RNH1-related genes were found to be significantly associated with differentiation, invasion, and EMT based on WGCNA. We obtained 95 hub genes related to EMT, invasion, and differentiation. Interestingly, GO enrichment analysis revealed that these genes were significantly enriched in immune activation related pathways. This finding links RNH1 implicated EMT, invasion, and metastasis to immune activation.The high functional heterogeneity of cancer cells presents a major challenge to cancer therapy. In this study, we quantified 17 functional states in tumours based on the CRDscore algorithm developed by He et al.50. Additionally, high immune cell infiltration was linked to an immune inflamed TME49. Our analysis also confirms that RNH1 shapes an inflammatory TME. Surprisingly, both the RNH1 high expression group and C1 group exhibited higher immune activation scores for the immune cycle. These findings establish for the first time a relationship between RNH1 and immunity. They also suggested that RNH1 plays an important role in immunotherapy.In this study, we classified BLCA into two clusters based on 95 hub genes. There was a significant difference in OS between the two clusters. Interestingly, the DEGs derived from the differential expression analysis between the two clusters were also significantly enriched in immune response related pathways. This again confirms that RNH1 mediated EMT and invasion may influence the activation of immune responses. Indeed, based on different algorithms for the assessment of immune cell infiltration, we found a significant positive correlation between RNH1 expression and multiple immune cell infiltrates. In addition, other independent GEO dataset analyses showed that RNH1 expression was significantly correlated with multiple TIICs. In further analysis, we found a significant positive correlation between high RNH1 expression and the expression of multiple immune checkpoints. Meanwhile, RNH1 was significantly positively correlated with the immunotherapeutic response in the BLCA cohort IMvigor210. This is consistent with the most effective outcome of ICB therapy in patients with higher expression of immune checkpoints and T-cell infiltration52. We found that RNH1 expression was significantly associated with multiple BLCA-related signalling pathways. Notably, RNH1 expression was positively related to the enrichment scores of therapeutic signalling pathways. Furthermore, RNH1 expression predicted the response to therapeutic options in BLCA. In terms of drug response, high expression of RNH1 could make cancer cells more sensitive to cetuximab. There was a notably better response to ERBB therapy in the high-RNH1 group. This has important guiding significance for exploring the neoadjuvant combination therapy for BLCA53.Molecular subtypes can predict the response to immunotherapy, radiotherapy, neoadjuvant chemotherapy, and several targeted therapiesIn conclusion, our study revealed that RNH1 affects multiple functional states of BLCA, especially EMT and invasion, and can predict BLCA invasion and metastasis. Moreover, we found that RNH1 expression is significantly associated with multiple therapeutic signalling pathways and drug targets in BLCA. We believe that RNH1 can provide new insights into the invasion and migration of BLCA and predict the treatment response in patients with BLCA.Supplementary Figures.Supplementary Tables.


Renal artery Doppler sonography with resistive index (RI) determination is a noninvasive, fast, and reliable diagnostic tool increasingly used in the intensive care unit (ICU) to predict and assess the reversibility of acute kidney injury (AKI). However, interpreting the RI can be challenging due to numerous influencing factors. While some studies have explored various confounding factors, arterial blood gases have received limited attention. Therefore, our study aims to evaluate the impact of arterial blood gases on the RI in the ICU setting.This prospective observational study enrolled ICU patients who required blood gas analysis and had not experienced significant hemodynamic changes recently. The RI was measured using standardized Doppler ultrasound within an hour of the arterial blood gases sampling and analysis.2 , age , diastolic arterial pressure (DAP) , and SaO2 . Multivariate analysis confirmed that age > 58 years and PaCO2 were significant factors influencing the RI, with respective odds ratios of 18.67 (p = 0.0003) and 1.132 (p = 0.0267).A total of sixty-four patients were included in the analysis. Univariate analysis revealed a correlation between the RI and several variables, including PaCO2, age, and diastolic arterial pressure. Further studies are needed to develop a comprehensive scoring system that incorporates all these cofactors for a reliable analysis of RI levels.The interpretation of renal arterial RI should take into account thresholds for PaCOTrial registration This observational study, registered under number 70–0914, received approval from local Ethical Committee of Toulouse University Hospital. Acute kidney injury (AKI) is a significant complication that affects approximately 30–40% of patients in the intensive care unit (ICU) and 1% of patients undergoing major postoperative surgery . It servThe renal interlobar arterial resistive index (RI), initially introduced by Pourcelot for the study of renal perfusion, is increasingly utilized in various renal disorders, including graft dysfunction and obstructive nephropathies , 6. ThisThe relationship between renal macro hemodynamics and renal function is not linear. Multiple independent factors can influence the resistive index, including vascular resistance, vascular compliance, and mean arterial pressure . ConsequTherefore, the primary objective of this study was to evaluate the influence of arterial blood gases on the RI in ICU patients.This prospective, observational study included adult patients admitted to the ICU. Patients who required arterial blood gas analysis and had not experienced changes in catecholamine administration within the past hour were eligible for inclusion. The inclusion process commenced at the time of blood gas analysis.Exclusion criteria encompassed the following: individuals under 18 years of age, pregnant women, incapacitated adults, patients with a history of chronic kidney failure, kidney malformation, a single kidney, renal artery stenosis, or diabetic nephropathy. Patients who were undergoing dialysis or had received diuretic treatment within the past 4 h were also excluded. Furthermore, patients with hemodynamic failure necessitating an increased dosage of catecholamines in the hour before measurements, as well as those with therapeutic limitations, were not included in the study.This observational study, registered under number 70–0914, received approval from local Ethical Committee of Toulouse University Hospitals. The study was performed according to the declaration of Helsinki. No change in our current clinical practice  and no randomization was performed. As it was an observational study, in accordance with French law, oral informed consent was required.2, tidal volume, expiratory volume, respiratory rate, heart rate and blood pressure). The blood gas and ultrasound data were collected over a period of less than one hour . The other laboratory data were from daily assessments of the patient.At inclusion we collected the demographic data , and the ventilator and hemodynamic clinical data  and 160 mL/min/1.73 m2 (men)) [After a first descriptive statistical step and verification of the normal distribution of values (Kolmogorov–Smirnov test), the studied population was separated into three groups according to the glomerular filtration rate (decreased GFR for CrCl measured < 60 mL/min/1.73 m2 (men)) .The patient characteristics between the different groups were compared using non-parametric tests  due to the non-homogeneity of the group sizes. The results are expressed as median and 95% confidence intervals (95% CI).Then, we analyzed the relationship between the RI and the quantitative variables, including age, GFR, the hemodynamic parameters and those characterizing arterial blood gases (linear regression through Pearson’s r coefficient and/or Spearman’s rank correlation coefficient). Next, we integrated the significant variables in multiple regressions to describe a model that included the only factors of interest.As the limited population of patients with an alteration in renal function did not enable calculation of a statistically valid RI threshold, a RI threshold > 0.7 was chosen since it is found in most published studies to date , 19. TheA multi-dimensional analysis through logistic regression evaluated the association between the covariates for which the p value was < 0.2 and the dependent variable (RI > 0.7) through measurement of the odds ratio. We tested several models by choosing that for which the observed data was best adjusted to the model .To highlight patients at risk of having an RI > 0.7, a partitioning of the population was represented using a Classification and Regression Trees (CART) analysis. The advantage of this approach is to describe the means of distribution of the population in homogeneous groups according to RI level and the covariates selected from the multidimensional analysis.Statistical analysis was conducted using MedCalc® statistical software, version 15  for the majority of the analyses except for the CART method, which was carried out on SPSS® for Window version 24 . A p value < 0.05 was considered statistically significant.Among the 64 patients included in the study, the median interval between ICU admission and inclusion was 5 (3–7) days. 53% of the patients were under controlled mode ventilation, 30% were on non-invasive mode during the procedure, and 16% were on spontaneous ventilation. Table 2. Conversely, a significant negative correlation was found between the RI and measured creatinine clearance, diastolic arterial pressure (DAP), and SaO2 , and PaCOMultivariate analysis using multiple regressions was conducted, considering the integration or non-integration of measured creatinine clearance (CrCl) depending on whether the RI was regarded as a consequence or an indicator of glomerular filtration rate (GFR). In both models, it was found that the only significant factors influencing the RI level were age (p < 0.0001), PaCO2 (p = 0.0056), and DAP (p = 0.0416).Table Figure The ROC curve analysis determined an RI threshold of 0.77 (gray zone: 0.66–0.77) for detecting kidney function impairment. Notably, the discrimination ability was relatively low, with an AUC = 0.73) below the threshold of 0.8. Considering the limited sample size of patients with kidney failure and the wide “gray zone” of RI values within this population, we selected an RI threshold of 0.7 (previously used in the literature with a larger patient cohort for screening kidney function impairment). This threshold falls within the predefined gray zone. The results are presented in Table  below thThe most discriminatory variable appears to be age, with a threshold of > 58 years, demonstrating a sensitivity of 77%, specificity of 85%, positive predictive value (PPV) of 82%, negative predictive value (NPV) of 81%, and an area under the receiver operating characteristic (AUROC) curve of 0.82. However, for the other variables, the ROC curve analysis did not yield a statistically significant threshold (AUC not different from 0.5).Furthermore, the RI exhibits variations based on the underlying condition necessitating admission to the intensive care unit, as depicted in Fig. The RI values differ among patient groups, with a measurement of 0.62 (0.59–0.66) in multiple trauma patients, 0.72 (0.69–0.75) in postoperative patients, and 0.71 (0.65–0.74) in medical patients (p = 0.00504). The significant difference observed is primarily attributed to the multiple trauma group.The multivariate analysis of factors associated with an RI > 0.7, incorporating both continuous and categorical variables, is presented in Table However, multiple trauma was not included in the model due to its strong correlation with age. It was observed that 96% of multiple trauma patients were under the age of 58, and this variable lost significance when age was included in the analysis.2, although a specific threshold for PaCO2 could not be determined.The influence of age and PaCO2 on the RI level was confirmed. The RI tends to increase after the age of 58, and it also increases with higher levels of PaCO2 thresholds within different subgroups, along with a threshold for diastolic arterial pressure (DAP) for patients under the age of 58.The Classification and Regression Tree (CART) analysis was employed to define several thresholds based on the data. Figure 2 > 37.75 mmHg has a 100% likelihood of having an RI > 0.7. On the other hand, a patient under the age of 58 with a DAP > 56.5 mmHg and a PCO2 < 34.6 mmHg has a 100% chance of having an RI < 0.7.In summary, a patient above the age of 58 with a PaCO2 thresholds. The RI serves as a reproducible and user-friendly tool for early screening and prognostication of AKI in the ICU [Our study highlights that in the ICU setting, the interpretation of the RI should be based on age and PaCO the ICU . However the ICU , 24. Add the ICU , 25–29.2, on the RI in ICU patients under routine care conditions. Previous studies have suggested that severe hypoxemia in rats or patients on mechanical ventilation leads to renal vasodilation, while significant increases in PaCO2 result in vasoconstriction [2 (or SaO2) and variation in RI, which contradicts findings from other studies conducted on patients undergoing postoperative cardiac surgery or those with acute lung injury (ALI) [Our study stands out due to its focus on examining the influence of arterial blood gases, particularly PaCOtriction , 30. Howtriction , 17. Thery (ALI) .We selected an RI threshold of 0.7 based on relevant literature, as it has been demonstrated to be associated with the occurrence of organic acute kidney failure , 32. An 2 significantly influenced the RI (p = 0.0416) across patients with augmented renal clearance (ARC), normal GFR, or glomerular hypofiltration. In each of these groups, both PaCO2 and RI increased concurrently. The multivariate analysis further confirmed the impact of PaCO2 on the RI . Through multivariate analysis using classification and regression tree, we found that ICU patients aged over 58 years with a PaCO2 level exceeding 37.75 had a 100% risk of having an RI above 0.7. In certain conditions such as acute respiratory distress syndrome (ARDS), higher thresholds of PaCO2 are generally tolerated. Nevertheless, based on the results of our study, the actual effects of hypercapnia on renal parenchyma and its function still require further evaluation.Previous studies have not been able to establish a direct correlation between the resistive index (RI) and renal blood flow or glomerular filtration rate (GFR) , 33, 34.As evidenced in existing literature, age has a direct influence on the glomerular filtration rate (GFR), which was reduced in approximately 20% of the patients in our study . Additionally, there is a corresponding increase in the resistive index (RI) with age . This riWe also identified a correlation between diastolic arterial pressure (DAP) and the resistive index (RI). Previous studies have explored the relationship between global hemodynamics and the RI, indicating that an increase in mean arterial pressure (MAP) due to noradrenaline administration leads to an elevated RI with a threshold effect . This suThe resistive index (RI) has demonstrated its predictive value in determining the reversibility of acute kidney failure and its potential for guiding hemodynamic optimization, especially in cases of septic shock. However, its diagnostic performance cannot be utilized as a screening tool for augmented renal clearance (ARC) . AlthougOur study has several limitations. One key limitation is the complexity of interpreting the resistive index (RI) due to the influence of multiple factors, including mean arterial pressure, pulse pressure, vascular compliance, oxygenation levels, and intra-abdominal pressure –29, 38. 2 on the RI, particularly in hypercapnic patients. It highlights the importance of analyzing the RI alongside normalized PaCO2 levels and age and diastolic arterial pressure (DAP) thresholds. Further research is warranted to develop a scoring system that integrates these cofactors, enabling more reliable interpretation of the RI results.The resistive index (RI) serves as a noninvasive, fast, and reliable diagnostic tool that can help predict the reversibility of acute kidney injury (AKI) and tailor individualized hemodynamic management for patients. Our study demonstrates the positive influence of PaCO