Around 9% of the quantified proteins were differentially expressed, pinpointing aberrant proteostasis involved in synaptic transmission, nucleoside monophosphate and carbohydrate metabolism, and neuron projection regeneration. In addition, network-driven proteomics revealed a modulation in cell-survival related pathways such as ERK, AKT, and the PDK1-PKC axis. Part of the differential OB protein set was not specific of MixD, also being deregulated across different tauopathies, synucleinopathies, and tardopathies. However, the comparative functional analysis of OB proteome data between MixD and pure AD pathologies deciphered commonalities and differences between both related phenotypes. Finally, olfactory proteomics allowed to propose serum Prolow-density lipoprotein receptor-related protein 1 (LRP1) as a candidate marker to differentiate AD from MixD phenotypes.The most common form of mixed dementia (MixD) is constituted by abnormal protein deposits associated with Alzheimer’s disease (AD) that coexist with vascular disease. Although olfactory dysfunction is considered a clinical sign of AD-related dementias, little is known about the impact of this sensorial impairment in MixD at the molecular level. To address this gap in knowledge, we assessed olfactory bulb (OB) proteome-wide expression in MixD subjects ( Alzheimer’s disease (AD) and vascular dementia (VaD) are the most common causes of dementia in the elderly . In mediAlthough AD can be frequently diagnosed with a considerable accuracy, the distinction between MixD, isolated AD and VaD remains controversial, being a difficult diagnostic challenge .Particularly relevant to neurologists is the fact that olfactory dysfunction can be considered a clinical, or in some cases a preclinical, sign of different dementias like AD and VaD ,13. AlthGiven the global prevalence of MixD-associated cognitive impairment and the lack of therapeutic strategies, there is a clear unmet need for vascular therapies targeting mechanisms that precipitate the neurodegenerative process. It is well known that the molecular homeostasis of olfactory structures is deeply altered in the context of AD pathology ,22,23,24The following reagents and materials were used: The antibodies for pMEK (#9154), MEK (#9126), pERK (#4370), ERK (#9102), pPDK1 (#3061), PDK1 (#3062), pPKCpan (#9379), pAKT (#4060), AKT (#4685), pSEK (#9156), SEK (#9152), pSAPK (#9255), SAPK (#9252), pPKAc (#5661), PKAc (#4782), pP38 (#9211), P38 (#9212), pNFκB (#3033), NFκB (#8242) were purchased from Cell Signaling. Antibody for PKCpan (SAB4502356) was purchased from Sigma Aldrich. The antibody for pPI3K (PA5-104853) was purchased from Life Technologies. Antibody for PI3K (ab86714) was purchased from abcam. Electrophoresis reagents were purchased from Biorad and trypsin from Promega.n = 19) and AD (n = 31) patients together with serum samples from healthy subjects (n = 32) were collected  were obtained for research purposes from relatives of patients included in this study. Post-mortem fresh-frozen olfactory bulbs of 6 Mixed dementia (MixD) patients, and 7 controls were obtained from the Brain Bank of IDIBAPS  following the guidelines of Spanish legislation. The control group was composed of elderly subjects with no histological findings of any neurological disease. The study was conducted in accordance with the Declaration of Helsinki and all assessments, post-mortem evaluations, and procedures were previously approved by the Local Clinical Ethics Committee (PI_2019/108). All human brains considered in the proteomics and follow-up phases had a post-mortem interval (PMI) lower than 19 h . On the ollected . In all w/w) at 37 °C for 16 h as previously described [Whole OB specimens (70–80 mg) derived from controls and MixD cases were homogenized in lysis buffer containing 7 M urea, 2 M thiourea, 50 mM DTT. After ultracentrifugation, protein extracts were precipitated, pellets were dissolved in 6 M Urea and Tris 100 mM pH 7.8 and Bradford assay kit (Bio-Rad) was used for protein quantitation. Whole proteomes were concentrated in the stacking/resolving SDS-PAGE gel interface. After staining, protein digestion (10 ug) was carried out with trypsin . Hence, proteins with ratios below the low range of 0.77 were considered downregulated whereas those with higher range than 1.33 were considered up-regulated. MS data and search results files were deposited in the Proteome Xchange Consortium via the JPOST partner repository  [Mass spectrometry raw data were processed using the MaxQuant software (v.1.6.3.3)   followinil 2021)  with theil 2021) , Ingenuiil 2021)  tools.Equal amounts of OB protein (10 µg) were resolved in 4–15% stain free SDS-PAGE gels (Bio-rad) and electrophoretically transferred using a Trans-blot Turbo transfer system  (Bio-rad). Membrane probing, immunoreactivity visualization, equal loading, digitalization, and densitometric analysis was performed as previously described .p-value less than 0.05 was considered statistically significant.Serum Neurexin-3 (NRXN3), Tenascin-R (TNR) and Prolow-density lipoprotein receptor-related protein 1 (LRP1) concentrations were measured using enzyme-linked immunoabsorbent assay (ELISA) kits according to the manufacturer’s instructions . The data were analyzed using Graphpad Prism Software and Mann–Whitney U test was used to make group comparisons. A Unbiased omics approaches have been proposed as essential tools to increase our understanding of the AD pathogenesis subtype variety as well as the common presence of vascular effects present in mixed pathologies . SpecifiSince olfactory system is considered a potential gateway for the access of environmental insults and a prion-like propagation site in different forms of dementia ,41, we hSNAP25, PPP3CC, NDUFS4, GORASP2, GFAP, SGIP1, COX5B, DMTN, CAMKV, EEF1B2, UQCRB, PSMC1, GNAZ, PLCD1, PSAT1, APRT, AK1, SH3BGRL3, PLIN3, PPIA, MAT2B, CSRP1, MGST3, ARL8B, LRP1) whereas 6 DEPs  corresponds to Tau interactors. Five DEPs are part of the shared interactome between APP and Tau . Due to our olfactory proteotyping workflow is not able to differentiate specific cell layers, we have performed cell-specific enrichment analysis across olfactory DEPs using public single-cell RNA-seq data [GFAP, EZR, GPT, ENTPD2, MLC1, FABP1, TNC, MACF1) are considered highly enriched genes in astrocytes, PTK2B in microglia and COL1A2, ACAN, ITGB8, RCN1, DPP6, S100A1 in oligodendrocytes. In addition, part of the differential proteome tend to be specifically enriched in OB mitral/tufted cell layers , periglomerular cell layer  and granular cell layer   D. All th, HMGB1) E, being , HMGB1) F.MAPT) and APP were functionally interconnected with DEPs in MixD OBs. Proteome-scale interactome maps merging the OB DEPs were performed using the IPA software  and nucleocytoplasmic region . However, the deregulated olfactory APP interactome impacts across extracellular space , plasma membrane , and cytoplasm   .ATP5ME, ATP5F1D, ATP5MG, NDUFS4, COX5B, Cytochrome c oxidase, CYB5A, SOD1, GSTP1) suggested an impairment in mitochondrial function in the OB of MixD subjects. In accordance, mitochondrial dysfunction constitutes an early and well-known feature of neurodegenerative processes [In a wider scale, a whole proteome comparison revealed that signaling mediators like the nuclear factor kappa B (NFκB) and PI3K complexes and cell-stress related such as PKA appeared as principal hubs in functional interactome maps . As showrocesses  and our rocesses . On the rocesses , the recrocesses . Thereforocesses . On the rocesses . Likewisrocesses . Interesrocesses , suggestTo further study in detail the OB metabolic modulation in MixD, the differential proteome map was functionally characterized. As shown in BASP1, CALM, SOD1, ERP44, TPM4, ALAD) was similar across AD and MixD OBs   . To our le/male) A and neule/male) B, a signle/male) C. In thile/male) , no signAlthough this study has uncovered many intricacies in the OB homeostasis in the context of MixD, there are potential limitations that warrant discussion. Due to the technological approach used, we failed to accurately monitor many protein species with low expression levels. Both AD pathology and cerebrovascular disease independently are strongly related with cognitive decline and/or dementia. Frequently, they appear together, showing an additive or even a synergistic effect and the weight of each component may be different among patients. In those cases, it remains challenging to distinguish between AD and MixD. This difficulty in diagnosis limits the number of MixD patients available in our cohort. Furthermore, even in absence of cognitive impairment, it is difficult to find pure age-matched controls without any sign of amyloid pathology or cerebrovascular disease given the high incidence of these lesions in the elderly and the fact that both findings increase with age. This factor is the reason why our control group has decreased age compared with MixD group. Therefore, we could not exclude the possibility that part of the differences found between both groups could be influenced by other age-related factors apart from the MixD ocurrence. Regarding the vascular component of MixD, different cerebrovascular lesions and locations induce different phenotypes of dementia. Given a big enough cohort of subjects, OB proteomics may prove to be useful to discriminate different types of dementia according to different cerebrovascular lesions.Overall, the present study provides new clues regarding the molecular mechanisms concerning the olfactory dysfunction that occurs during MixD. Part of the differential OB protein set was not specific of MixD, being also deregulated across different tauopathies, synucleinopathies and tardopathies. However, functional analysis has unveiled OB commonalities and differences between pure AD and MixD. Based on olfactory proteomic data, LRP1 may be considered a potential serum biomarker to differentiate AD and MixD phenotypes.


To detect such NO2 gas at low concentration, we fabricated a GNWs/NiO-WO3/GNWs heterostructure-based gas sensor using microwave plasma-enhanced chemical vapor deposition (MPECVD) and sputter, and we confirmed the NO2 detection characteristics between 10 and 50 ppm at room temperature. The morphology and carbon lattice characteristics of the sensing layer were investigated using field emission scanning electron microscopy (FESEM) and Raman spectroscopy. In the gas detection measurement, the resistance negative change according to the NO2 gas concentration was recorded. Moreover, it reacted even at low concentrations such as 5–7 ppm, and showed excellent recovery characteristics of more than 98%. Furthermore, it also showed a change in which the reactivity decreased with respect to humidity of 33% and 66%.Recently, as air pollution and particulate matter worsen, the importance of a platform that can monitor the air environment is emerging. Especially, among air pollutants, nitrogen dioxide (NO However, there are global issues of duplicity for industrial development, such as the destruction of natural ecosystems, depletion of fossil fuels, and lack of resources ,2,3. Theperature ,8,9. In e matter ,11. Thus2 have been reported using metal–oxide semiconductors (MOS), graphene-centered carbon materials, polymers, and transition metal dichalcogenide (TMD) as sensing layers [2) and tungsten disulfide (WS2), which provide low electrical noise, low detection limit, tunable band gap and high mobility, while they suffer from low selectivity and sensitivity as disadvantages [2 [The scientific techniques for detection of diverse harmful gas including NOg layers ,13,14,15g layers . In addig layers . In TMD vantages ,19,20. Atages [2 ,22,23. N3 layer on the surface of GNWs, where NiO-WO3 can serve as a metallic catalyst that elevates detection signal by NO2 gas molecules [3/GNWs structure for securing a larger adsorption area. Consequently, measurement of NO2 with several concentrations was achieved by the GNWs/NiO-WO3/GNWs heterostructure in this study.In this study, we used graphene nanowalls (GNWs) while keeping the advantages of graphene via microwave plasma-enhanced chemical vapor deposition (MPECVD). GNWs are three-dimensional materials with high porosity, which enable molecules of target gases to induce more absorption onto its surface and active sites ,25. Altholecules ,27,28. T3/GNWs heterostructure, as shown in 2:CH4 = 40:20 sccm), substrate temperature (600 °C), synthesis time (15 min), and working pressure (25 mTorr). In the formation of GNWs, the state of radicals affects the growth steps of GNWs, such as carbon nucleation, graphene domain, and growth rate. For these, two CVD reactions should be kept and accompanied: (i) CH, CH2, and CH3 radicals based on high energy and unstable state should stay until arrival onto the substrate under the appropriate microwave power, reaction gas ratio, synthesis time, and working pressure; and (ii) the radicals that have arrived on substrate should join the chemical reaction with dehydration under the proper temperature. Hence the chosen MPECVD condition is optimal for successful GNWs growth. Afterwards, the NiO-WO3 catalyst layer that can promote catalyst effects, such as ionization of oxygen species and spillover effect, was formed by the co-sputtering method via the RF magnetron sputtering system  on GNWs/Si substrate. In this process, in order to obtain the optimal composition and catalytic ability of NiO-WO3, we maximized mean free path (MFP), maintaining high vacuum for 10 h. As main sputtering after pre-sputtering for 5 min, the co-sputtering method was used under the following conditions: W target (99.95% purity), Ni target (99.95% purity), RF power (200 W), Ar flow rate (40 sccm), O2 flow rate (10 sccm), synthesis time (10 min), substrate rotation speed (1700 rpm), and working pressure (15 mTorr). Lastly, secondary GNWs were grown on the NiO-WO3/GNWs/Si substrate in the same conditions, except for the synthesis time .For preparation of the GNWs/NiO-WO3/GNWs heterostructure was observed via a field emission scanning electron microscopy  with 10 kV acceleration voltage and 9800 nA emission current. Raman spectroscopy  was used in the following conditions: excitation laser wavelength (~531 nm), excitation laser power (0.3 mW), objective lens , and spectral resolution (~1.9–2.1 cm−1). As shown in 3/GNWs heterostructure-based gas sensor was conducted using measurement equipment . Herein, the concentration of NO2 gas was adjusted by diluting it with dry air through a mass flow controller . For the certain injection and obstruction of dry air and gas, the air valve was placed between gas pipes or lines, which were operated by the air compressor . The NO2 gas produced by this gas-adjustment process was absorbed and desorbed at the sensing layer in the test chamber, thereby displaying a resistance change of sensing layer on the monitor.The morphology of the GNWs/NiO-WO3/GNWs heterostructure were displayed by FE-SEM. 2 molecules can sufficiently attach at sides of nanowalls through macropores, resulting in their suitability as sensing materials. The surface images of the NiO-WO3-based metallic catalyst are shown in 3 catalyst, tiny metal particles sputtered from respective targets  created agglomerate composites with diameters of 120–220 nm. In general, thin films synthesized on planar substrates such as Si wafer and glass are uniform. Nevertheless, uneven surfaces with valleys between the NiO-WO3 agglomerations in the SEM image are due to their intensive synthesis onto the top edges of the GNWs. In addition, the surface boundaries . This suggests the growth principal of GNWs based on radical reaction from nucleation, thereby forming a nanostructure. Consequently, using PECVD and the co-sputtering system, the GNWs/NiO-WO3/GNWs heterostructure was ideally synthesized.As shown in 3 layer depending on the co-sputtering method, energy-dispersive X-ray spectroscopy was employed. As shown in To analyze the composition ratio of the NiO-WO3/GNWs (red scatters), and the GNWs/NiO-WO3/GNWs (blue scatters). As crucial peaks, D (ca. 1350 cm−1), G (ca. 1580 cm−1), and 2D band (ca. 2700 cm−1) occurred in all samples. Typically, the high proportion of sp3 hybridization is significantly higher than the sp2 hybridization that was seen in the GNWs. In addition, the tops of the GNWs are similar to the graphene edges in their perpendicular structure. Hence, GNWs have a high D band and a low G band, unlike graphene and carbon nanotubes. The intensity ratio of the GNWs, NiO-WO3/GNWs, and GNWs/NiO-WO3/GNWs showed numerical variation. In this case, the ID/IG ratios of 2.14 (GNWs), 1.55 (NiO-WO3/GNWs), and 2.24 (GNWs/NiO-WO3/GNWs) were confirmed, where a notable change is a lowered value from 2.14 to 1.55. This decrease in intensity suggests that the NiO-WO3/GNWs was formed as composites. Nevertheless, the I2D/IG ratio was retained at less than 1, which means that the GNWs consist of multiple graphene layers.2 gas-sensing performance of the GNWs/NiO-WO3/GNWs heterostructure. First, as shown in 2 gas were injected at intervals of 300 s, thereby confirming the changing resistance value under a range of 10 to 50 ppm. The initial resistance of the GNWs/NiO-WO3/GNWs heterostructure-based gas sensor before gas injection was 363.21 Ω. As the NO2 concentration was increased, the resistance was reduced, while resistance increase occurred in air injection. This suggests that when the NO2 molecules are adsorbed onto the surface of the sensing layer, charge transfer was conducted depending on the amount of NO2 concentration, which directly caused the resistance to vary. Based on this result, change of resistance depending on NO2 concentration is shown in 2 concentration 11.97 (10 ppm) and 16.10 Ω (50 ppm), respectively. In the field of sensors, sensitivity is defined as slope . The sensitivity and coefficient of determination obtained by ΔR-versus-gas concentration were 0.1 Ω/ppm and 0.96, respectively in this study. The coefficient of determination is a factor signifying the linear correlation between the x-axis and y-axis variables, which shows a high reliability of linearity closer to 1. Herein, the coefficient of determination was 0.96, which indicates that these findings are very reliable. Moreover, initial resistance continuously decreased with repetition of the injection-recovery process. We have hypothesized that it is probably because target gas adsorbed on the surface of the sensing materials still remain a small minority during the recovery step. Another cause may be due to the vertical nanostructure of the GNWs. The vertical nanostructure has rather deep pores, in which deeply penetrated gas molecules, by Knudsen diffusion, can be limited on total desorption. Thus, to keep a stable pattern of sensing signal and maintain initial resistance, an additional platform with ultraviolet (UV) light or high temperature has been used, which can be chosen to improve overall sensing ability in three-dimensional sensing materials [aterials ,30.3/GNWs-based gas sensor under consistent concentration, measurements in 5, 7, and 10 ppm were conducted over several times, as shown in 2 molecules are highly stable at low concentration and room temperature. However, the repeatability test at 10 ppm showed an interesting phenomenon, drawing a pattern that does not fully recover at room temperature. This phenomenon is similar to the response characteristic according to 10–50 ppm observed previously. Herein, we can infer that NO2 molecules adsorbed from more than 10 ppm were not thoroughly desorbed in the recovery interval and at room temperature, thereby demonstrating a phenomenon to lower the resistance value before gas response by successive accumulation of NO2 molecules. To identify the repeatability of the GNWs/NiO-WO2 gas is diluted by humidity. Moreover, when there was no significant difference between the gas concentration and humidity, an ordinary response curve was showed, whereas in the case of a large amount of humidity, a response delay  was recorded at the initial stage, with a low response signal. 3/GNWs heterostructure-based gas sensor was greatly affected by the humidity, and it denotes that introduction of an ultraviolet irradiation system leading to enhanced stability in humidity during exposure to suitable UV wavelengths may be required to overcome the effects of humidity [On measurement of gas-sensing performance, since the relative humidity (RH) is a very important factor in room temperature, a humidity test was evaluated at RH 33% and RH 66%  it is specialized in good stability at low gas concentration (under 7 ppm); and (ii) it exhibited hardly any difference before and after gas exposure. However, issues such as the effect of relative humidity and variation of initial resistance must surely be improved. Through these results, we found that the potential of GNWs enable us to utilize them for detection of hazardous gases such as SOx, NOx, and VOCs with necessity of further research on humidity dependence, enhanced-sensing signal, and low limit of detection.In this study, the GNWs/NiO-WO


Background: The 14q32 cluster is among the largest polycistronic miRNA clusters. miRNAs encoded here have been implicated in tumorigenesis of multiple organs including endocrine glands. Methods: Critical review of miRNA studies performed in endocrine tumors have been performed. The potential relevance of 14q32 miRNAs through investigating their targets, and integrating the knowledge provided by literature data and bioinformatics predictions have been indicated. Results: Pituitary adenoma, papillary thyroid cancer and a particular subset of pheochromocytoma and adrenocortical cancer are characterized by the downregulation of miRNAs encoded by the 14q32 cluster. Pancreas neuroendocrine tumors, most of the adrenocortical cancer and medullary thyroid cancer are particularly distinct, as 14q32 miRNAs were overexpressed. In pheochromocytoma and growth-hormone producing pituitary adenoma, however, both increased and decreased expression of 14q32 miRNAs cluster members were observed. In the background of this phenomenon methodological, technical and biological factors are hypothesized and discussed. The functions of 14q32 miRNAs were also revealed by bioinformatics and literature data mining. Conclusions: 14q32 miRNAs have a significant role in the tumorigenesis of endocrine organs. Regarding their stable expression in the circulation of healthy individuals, further investigation of 14q32 miRNAs could provide a potential for use as biomarkers (diagnostic or prognostic) in endocrine neoplasms. MicroRNAs (miRNAs) are single-stranded, small (~17–22 nucleotide long), protein non-coding RNA molecules that regulate gene expression post-transcriptionally by RNA interference. According to the canonical miRNA biogenesis, the mature miRNA is generated from a hairpin RNA precursor molecule produced by RNA polymerase II or III [After biogenesis, the mature miRNA incorporates into a protein complex called miRISC (miRNA-induced silencing complex) . In the Approximately 30–50% of all protein-coding genes are thought to be controlled by miRNAs .MiRNAs have been shown to be involved in the control of many physiological and pathophysiological processes, such as proliferation, differentiation, metabolism and apoptosis through the modulation of target gene expression. Altered miRNA expression has been identified in several endocrine diseases including neoplasms ,6. DepenThe 14q32 miRNA cluster is among the largest polycistronic clusters comprising almost a hundred small non-coding RNAs, including a significant number of miRNAs . MiRNAs DLK1-DIO3 domain or DLK1-MEG3 cluster. Indeed, this cluster contains protein-coding (Delta-like 1 (DLK1), Deiodinase Iodothyronine Type III (DIO3) and Retrotransposon-like Gene 1 (RTL1)) and nonprotein-coding genes , Maternally Expressed Gene 8 (MEG8) and RTL1 antisense (RTL1-AS)), small nucleolar RNAs (snoRNAs) and miRNAs. This approximately 300 kilobase miRNA region can be divided into two parts: cluster A and cluster B. Cluster A includes MEG3 and RTL1 genes, while cluster B can be found 5′ from MEG9 and DIO3 genes , a DMR 1.3 kb upstream of the MEG3 transcription start site (MEG3-DMR) and a DMR found in the DLK1 promoter (DLK1-DMR) [The expression of 14q32 genes is regulated by genomic imprinting. The differential expression of alleles inherited from a mother or father at the genomic imprinted loci is realized by different methylation. The regulatory loci of the methylated nucleotides are called differentially methylated regions (DMRs). Differential methylation patterns at DMRs provide monoallelic expression from either maternal or paternal allele. Generally, imprinted genes have a key role in regulating growth and other physiological functions during embryonic development. Germline deletions and uniparental disomy of this locus in humans associate with developmental abnormalities and dysmorphism, suggesting that the 14q32 locus might have significant importance in development ,22,23,24 cancers ,27,28,29and RTL1 . From thxpressed . This imxpressed ,32: a DMLK1-DMR)   for the entire locus during development [MEG3-DMR serves as the principal regulator in adult tissues [DLK1 and MEG3.The elopment , whereas tissues . This imAccordingly, 14q32 miRNAs are also involved in the imprinting regulation ,35 and a14q32 miRNAs also influence prognosis of various cancers. Oshima et al. presented that the expression of 14q32 miRNAs was a favorable prognostic factor in patients with metastatic cancer . Based oDespite of the relevance of 14q32 miRNAs in other malignancies, regarding endocrine tumors, there has no comprehensive review published about the role of 14q32 miRNA cluster. In this work, the authors aimed to collect high-throughput miRNA studies performed in endocrine tumor samples, to extract the role and potential relevance of 14q32 miRNAs through investigating miRNA targets and to integrate the knowledge provided by literature data and bioinformatics predictions.Literature mining was performed using Pubmed database using the following keywords: “14q32” or “miRNA” or “DLK1-MEG3” and combined with either of “endocrine tumors”, “neuroendocrine tumor”, “pituitary adenoma”, “adrenocortical tumor”, “pheochromocytoma” or “thyroid cancer”. Publication focused on 14q32 miRNAs regarding endocrine tumors, and high-throughput miRNA profiling studies of endocrine tumors were selected to construct an expression heatmap . Downreghttp://www.mirbase.org/, access date: 25 February 2021). Pathway analysis for 14q32 miRNA targets by gene set enrichment analysis for KEGG pathways was performed using DIANA TOOLS miRPath v.3 following target prediction by microT-CDS algorithm . Gene ontology was assessed using miRDB Target Ontology Analysis module by selecting enrichment for Biological Process and Molecular Function .Also, miRbase MIMAT IDs of dominant mature 14q32 miRNAs were used for bioinformatics analysis (Neuroendocrine tumors (NETs) are a group of heterogeneous neoplasms arising from neuroendocrine cells throughout the body . Although gastro-entero-pancreatic NETs (GEP-NETs) represent less than 1% of all digestive system cancers it consists 7–21% of all neuroendocrine neoplasms . Lung NEIn GEP-NETs tissue, however, several miRNA studies have been published ,116, scaSeveral studies evaluated the role of miRNAs as prognostic markers . Among oAlso among 14q32 miRNAs, the expression of miR-127, miR-136, miR-154, miR-485, miR-770-5p showed negative correlation with tumor biology of lung NET, and miR-377* was identified, showing a significant impact on survival time .A recent study reported that among the most abundant miRNAs in lung NET types, miR-127 encoded at 14q32 showed high expression in typical carcinoids tumors . BesidesRET mutations, von Hippel Lindau syndrome due to VHL mutations, neurofibromatosis type 1 with NF1 mutations or hereditary PG syndrome caused by mutations of succinate dehydrogenase (SDH) genes, PPGL genes including KIF1b, PHD2, TMEM127, MAX, FH, MDH2, GOT2 and SLC25A11 [The rare pheochromocytomas (PCC) and paragangliomas (PGL)  arise from the same type of neural crest tissue of the sympathetic and parasympathetic paraganglia . While tSLC25A11 . UnfortuMAX-related PPGLs and a subset of sporadic PC samples as well [TMEM127-related cases overexpression of eight 14q32 miRNAs were detected [VHL-related PCCs vs. sporadic counterparts, and these miRNAs had a lower level of expression in recurrent tumors compared to primary PCC [DLK-MEG3 locus was reported in approximately 10% of PCC samples [DLK1-MEG3 cluster. In this study 15 of 17 tumors belonging to cluster Mi3 displayed loss-of-heterozygosity (LOH) at the 14q32 locus harboring DLK1-MEG3. The authors hypothesized that the loss of the maternal unmethylated allele might explain the repression of this imprinted miRNA cluster that was also supported by the methylation analysis of MEG3 promoter [Expectedly, miRNA profile in different genetic subtypes was also distinct and based on a high-throughput miRNA profiling ,72,124 s as well . In TMEMdetected . MiR-541mary PCC . As hype samples , the pat samples . Indeed, samples , while Mpromoter . Interespromoter . In linepromoter . The dowpromoter . In anotKCNJ5 mutant APA vs. non-KCNJ5 mutant samples [In aldosterone producing adenomas (APA), no differentially expressed miRNAs encoded at 14q32 were detected ,127,128. samples .MEG3 promoter methylation. The authors suggested that the loss of the maternal unmethylated allele resulted in silencing of the 14q32 miRNA cluster in Mi1 ACC tumors, suggesting that this region had a key role in ACC pathogenesis [Regarding adrenocortical carcinoma (ACC), a combined genomic approach classified tumor samples into 3 clusters (Mi1-3) based on miRNA expression pattern . ACC samogenesis . Özata aogenesis . Additioogenesis . Interesogenesis .ZEB1), a target of miR-431, was implicated in reversal of the epithelial-mesenchymal transition (EMT), leading to increased cell responses to adjuvant therapies in ACC [Regarding ACC behavior miR-370, miR-376a, miR-376b, miR-376c, miR-377, miR-379, miR-382, miR-411, miR-487a, miR-494, and miR-495 encoded at 14q32 miRNA cluster were downregulated in non-aggressive ACC as compared to aggressive ones . In anots in ACC .DLK1 was found as a marker of adrenal gland tumor, which was in line with findings that 14q32 miRNAs (except for Mi1 subgroup) were upregulated in ACC suggesting a common transcriptional regulation of the entire locus in ACC  proved to be a miR-432 target [Prolactinomas. In a work using next generation sequencing, no 14q32 miRNAs were reported differentially expressed in prolactinomas . However2 target . On the 2 target . Additio2 target , as wellPTTG1) was identified as a target of miR-126 and miR-381 encoded at 14q32 cluster [PTTG1 expression [PTTG1 targeting miRNAs, PTTG1 and p53 where p53 transcriptionally activated the expression of the four miRNAs, while PTTG1 inhibited the transcriptional activity of p53 [HMGA2 [Growth hormone (GH) producing tumors. Numerous underexpressed miRNA located at 14q32 were identified using NGS and PCR array ,82 Figu. However cluster . Also, Lpression . The auty of p53 . Among 13 [HMGA2 .MEG3 and DLK1 also showed underexpression in NFPA samples [DLK1-MEG3 locus in human NFPAs [MEG3-IG DMR [MEG3 was not expressed in NFPAs; therefore the authors suggested that the silencing of the DLK1-MEG3 locus played an important role in human NFPA pathogenesis [CCNB1) as target of miRNA-410 since its overexpression reduced CCNB1 at protein and mRNA levels, decreasing cell proliferation.Non-functioning pituitary adenomas (NFPA). Several miRNAs mapped to 14q32 showed significant underexpression compared to normal pituitary in NFPA detected by different platforms ,88,89. Ban NFPAs  also rep3-IG DMR ,140,141.ogenesis ,140,141.ogenesis . Bottoniogenesis . Others ogenesis . The funogenesis . Regardiogenesis . AuthorsDLK1 was found downregulated in corticotroph tumors [Corticotroph adenomas. Cheunsuchon et al. investigated 18 members of the 14q32 miRNA cluster, among which several miRNAs identified as significantly downregulated  and only miR-431 was found overexpressed in tumors compared to normal tissues . Although tumors . While Sh tumors , others h tumors .LGALS3, lectin, galactoside-binding, soluble, 3) and Runt-related transcription factor 2 (RUNX2) genes, [Pituitary carcinomas. In corticotroph carcinomas, miR-323-5p was downregulated in comparison to normal pituitary, and miR-493 was upregulated in carcinoma vs. adenoma . It was ) genes, ,144,145.Pituitary oncocytoma. In pituitary oncocytoma, numerous underexpressed miRNAs  compared to normal control were mapped to 14q32 region .RET proto-oncogene [Thyroid cancer is the most frequent malignant endocrine tumor. The majority of them (~95%) arise from follicular cells and classified as papillary , follicular  or anaplastic thyroid cancer  . Tumors oncogene . Most ofoncogene .Nikiforova et al. detected markedly different profiles of miRNA expression between MTC and all other thyroid tumors that derives from follicular cells, reflecting tissue-specific characteristics of miRNAs . Among tNumerous miRNAs were described in non-medullary thyroid cancer types, however, with controversial results. Therefore, a comprehensive re-analysis integrating 21 thyroid cancer miRNA studies by Saiselet et al. determined the commonly reported differentially expressed miRNAs in non-medullary thyroid carcinomas compared to normal tissues . Of the From a clinical point of view miRNAs are suggested as potential biomarkers, as cytology following fine-needle aspiration biopsy (FNAB) are interpreted as indeterminate without definitive diagnosis regarding thyroid tumors in 3–6% to 10–25% . HoweverMEG3 non-coding RNA located upstream [14q32 locus contains more than forty miRNAs, and previously it had been thought that they were generated from one polycistronic transcript containing the whole miRNA cluster under a coordinated regulation with the upstream ,12,151. upstream ,45,152.However, in several endocrine tumors, the pattern of 14q32 miRNAs were not so homogenous. Indeed, in other tissues and tumor types, similar findings were described ,153,154.methylation was the most obvious. Genomic imprinting imbalance could result in the differential modulation of paternally and maternally expressed genes from the 14q32 region that might serve as an explanation, at least in part, for the increased levels of DIO3 observed in some papillary thyroid cancer samples [DLK1 and MEG3 expression [However, several mechanisms have been identified in the context of this variable expression of the cluster members, among which  samples ,153,158. samples . Direct  samples . Moreove samples . In urotpression . The autpression .chromatin remodeling by lncRNA-mediated mechanisms, may also participate in regulating the expression of the 14q32-encoded miRNAs [Recent studies have shown that d miRNAs ,68.Additionally, Greife and colleagues demonstrated the loss of active and gain of repressive histone modifications at all regulatory sequences using chromatin immunoprecipitation .splicing, primary transcript processing or pre-miRNA cleavage and maturation were also reported related to 14q32 miRNAs [Differences in miRNA 2 miRNAs . Some su2 miRNAs ,160,161.2 miRNAs , therefo2 miRNAs ,163 was 2 miRNAs .RNA Binding Proteins (RBPs) were other post-transcriptional regulators of miRNA expression. RBPs bound precursor miRNAs and promoted or inhibited their maturation. For instance, Myocyte Enhancer Factor 2A (MEF2A) was such an RBP regulating miR-329 and miR-494 encoded at 14q32 chromosomal region [CIRBP) and hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit β (HADHB) were also RNA binding proteins that regulated 14q32 miRNA expression [l region . Cold-inpression .CCCTC-binding factor (CTCF) which was implicated in transcriptional activation/repression and imprinting [The different expressional pattern regarding this miRNA cluster was also observed by Manodoro et al., who attributed it to the presence of the binding sites of printing ,157,166.printing ,168. IntAltogether, these data suggest that multiple mechanisms other than genetic mutations or chromosomal loss might be involved in the regulation of 14q32-encoded miRNAs.By analyzing function of individual 14q32 miRNAs, besides several molecular functions and biological processes, TGFβ and Wnt signaling were also identified, which are frequently involved in tumor development .However, as 14q32 miRNAs more or less function in cooperation, we performed target prediction and gene set enrichment analysis to investigate the net effect of their co-expressional pattern. Several cancer-related pathways , pathways involved in invasiveness and metastasis development  or influencing pluripotency and stemness were identified as a potentially functional role .MEF2A regulated the expression of 14q32 miRNAs being a direct target of miR-329 [Literature/experimental data also suggested the regulation of axon guidance, actin cytoskeleton, focal adhesion, mammalian target of rapamycin, calcium, mitogen-activated protein kinase and ErbB signaling pathways by 14q32 miRNAs . Liu et  miR-329 . Uppal a miR-329 ,170,171. miR-329 .CDK5) and Twist Family BHLH Transcription Factor 1 (TWIST1) have been reported to increase metastasis through regulating cell cycle and EMT and they were found to be upregulated in osteosarcoma tumors with low levels of 14q32 miRNAs [TK1), that expressed at high levels in proliferating cells and appeared to correlate with high risk in multiple cancer types, was negatively correlated with 14q32 miRNA expression [In osteosarcoma, the decrease of 14q32 miRNA levels stabilized c-MYC protooncogene expression and consequently increased the level of oncogenic miR-17-92 miRNA cluster . Cell cy2 miRNAs ,174,175.pression ,177,178.CDH1) and catenin α 1 (CTNNA1), and decreasing the expression of Snail Family Transcriptional Repressor 2 (SNAI2) [In thyroid cancer, the role of 14q32 miRNAs was particularly investigated. Geraldo et al. showed that 14q32 miRNAs contributed to tumor progression and metastasis by targeting key regulators of cell adhesion, migration, proliferation, hypoxic response and wound healing . The rei (SNAI2) . Also, i (SNAI2) .VEGFA) as a target of miR-494 [CCL2), through which it affects proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) [FOXO1) influencing endothelial growth and proliferation [Angiogenesis and neovascularization were also significantly regulated by 14q32 miRNAs. This was confirmed by identifying vascular endothelial growth factor A  . Forkheaferation , wound cferation ,182,183.feration ,169.Altogether, 14q32 miRNAs have an important role in development and tumorigenesis. The importance of this miRNA cluster regulatory function is represented by the finding that their expression is stable in healthy cells, and even in cell-free serum samples. Their expression is also independent of the most common confounding factors, such as age, sex or BMI. Several studies have shown downregulation of miRNAs from the 14q32 region in different types of cancer; however, 14q32 miRNAs are overexpressed in some cancer types reflecting the tissue-specificity of miRNA function.Among different endocrine tumors in pituitary adenoma and oncocytoma, papillary thyroid cancer and a particular subset of pheochromocytoma and adrenocortical cancer are characterized by the downregulation of almost all miRNAs encoded by the 14q32 cluster. In the subgroups of ACC and PCC, the silencing of the imprinted 14q32 cluster due to LOH of chromosome arm 14q or 14q32 locus. In other tumor types including NFPA and/or gonadotroph pituitary adenomas increased methylation of the region DMRs could explain the orchestrated downregulation of the coding and non-coding gene expression of the entire region.Interestingly, pancreas NET, most of the adrenocortical cancer cases and medullary thyroid cancer are particularly distinct, as 14q32 miRNAs are overexpressed in these tumors. The role of these overexpressed miRNAs should be further investigated in relation to tumorigenesis.In the third group of endocrine tumor types such as pheochromocytoma and growth-hormone producing tumors, and based on the expression pattern of 14q32 miRNAs, however, both increased and decreased expression of 14q32 miRNAs cluster members were observed. In the background of this phenomenon, methodological, technical and biological factors can be hypothesized as well. Different researcher groups applied different study design, different sample numbers, RNA extraction and miRNA quantification methods which all could lead to controversial results. Also, as detailed above, several biological explanations have been revealed in the context of the various expressional pattern of the different members of 14q32 miRNA clusters; e.g., different methylation pattern, chromatin remodeling, histone modifications, alteration of miRNA biogenesis, the effects of RNA binding proteins and transcription factors. These factors await being further investigated in pheochromocytoma and growth-hormone producing pituitary adenomas.PTTG1 and HMGA2. In PTC, the significance of another 14q32 miRNA, miR-654-3p, was demonstrated in in vivo experiments as it influenced proliferation, migration, metastasis-related gene expression and EMT.Unfortunately, the function of 14q32 in endocrine tumors is not so broadly investigated compared to other tumor types. However, in different types of pituitary adenoma cell lines, 14q32 miRNAs proved their tumor suppressor role by inducing cell cycle arrest and cell growth inhibition by targeting 14q32 miRNAs are also associated with disease prognosis. In endocrine tumors several 14q32 miRNAs were identified as prognostic markers in pancreatic, small intestinal and lung NET. Some of these miRNAs are also linked to patient survival in lung NET. In PCC 14q32 miRNAs indicated metastatic cases compared to non-metastatic cases, assisting the discrimination of benign and malignant tumors. Also, the higher expression of miRNAs encoded at 14q32 were associated with aggressive ACC cases. Finally, while in MTC, 14q32 miRNA miR-375 has not only been reported as a strong prognostic factor of poor prognosis, but its higher level was associated with reduced overall survival, while on the contrary, the decreased 14q32 miRNA in PTC was associated with long-term progression.Based on data presented in HMGA2 was proposed as an attractive candidate to be one of the original targets because its 3′ regulatory region contained multiple predicted target sites for 14q32 miRNAs, with some of these target sites evolutionarily older than the 14q32 miRNA cluster [HMGA2 have special role in this context as it has reported to be overexpressed in several cancer type including endocrine tumors and often regulated by miRNAs [The evolutional role and constraint can be considered another point of view of imprinted miRNAs ,185,186. cluster . HMGA2 hy miRNAs ,189, whiSimilar to other cancer types, 14q32 miRNAs have a significant role in the tumorigenesis of endocrine glands. In different endocrine tumor types this miRNA cluster reflects the general tissue specificity of miRNAs regarding expression pattern, tumor suppressor or oncogene function, and they have a significant impact on prognosis as well. Regarding the stable expression of 14q32 miRNAs in healthy individuals in circulation, the further investigation of this miRNA cluster could provide an option to use them as diagnostic or prognostic biomarkers in endocrine neoplasms.


Atherosclerosis remains the underlying process responsible for cardiovascular diseases and the high mortality rates associated. This chronic inflammatory disease progresses with the formation of occlusive atherosclerotic plaques over the inner walls of vascular vessels, with oxidative stress being an important element of this pathology. Oxidation of low-density lipoproteins (ox-LDL) induces endothelial dysfunction, foam cell activation, and inflammatory response, resulting in the formation of fatty streaks in the atherosclerotic wall. With this in mind, different approaches aim to reduce oxidative damage as a strategy to tackle the progression of atherosclerosis. Special attention has been paid in recent years to the transcription factor Nrf2 and its downstream-regulated protein heme oxygenase-1 (HO-1), both known to provide protection against atherosclerotic injury. In the current review, we summarize the involvement of oxidative stress in atherosclerosis, focusing on the role that these antioxidant molecules exert, as well as the potential therapeutic strategies applied to enhance their antioxidant and antiatherogenic properties. Atherosclerosis constitutes a chronic inflammatory disease affecting the middle and large arteries, which mainly initiates by infiltration and deposition of low-density lipoproteins (LDL) into the arterial intima, where they undergo oxidation . This, iOxidative stress mediated by reactive oxygen species (ROS) plays a key role in the atherosclerotic process. ROS not only oxidize LDL, they also promote several pro-atherogenic effects, including inflammation, apoptosis, and alteration of vascular tone ,6. The iAtherosclerosis mainly affects two of the three artery layers: the endothelial and the middle layer. The endothelium is composed mainly of endothelial cells (ECs), representing the innermost layer of the artery. The endothelium constitutes a selectively permeable barrier responsible for maintaining arterial homeostasis by controlling the exchange of substances and the release of vasoconstrictor/vasodilator factors. This layer also regulates vascular tone, cell adhesion, and smooth muscle cells (SMCs) proliferation ,17. The Under pathological conditions, many of the biological functions regulated by the endothelium are compromised, and such endothelial dysfunction constitutes the first step in the development of atherosclerosis A. OveralECs are also susceptible to high concentrations of different compounds, including ox-LDL or high glucose levels, among others B. LDLs aLDL molecules are recognized in the middle layer by scavengers receptors (SR) on the cell-surface of macrophages and SMCs, whose expression is also induced by ROS  Figure B. ox-LDLLipids and foam cells not only act as initiators of the atherosclerotic process, they also maintain inflammation, and aggravate it. For instance, lipids bind to toll-like receptors, which activates the synthesis of inflammatory chemokines, promoting the recruitment of additional macrophages together with B and T cells towards the injured area ,23. MoreThe deposition of lipid-rich cell debris within the damaged area takes place as result of apoptosis of foam cells and immune cells, building-up the “necrotic core” ,29. VSMC2.₋, .OH) and non-free radicals , reactive nitrogen, copper, iron and sulfur species are also considered as oxidative stress related molecules, since they promote or participate through different mechanisms in the synthesis of ROS ..39].2 to superoxide (O2.₋) by transferring one electron from NADH or NADPH to O2. Superoxide radicals react with NO producing peroxynitrite (ONOO₋), a cytotoxic molecule that contributes to EC injury. This reaction decreases NO bioavailability, together with its vasorelaxant effect [₋/₋) [The NOX family is comprised of seven isoforms of NADPH oxidases, all transmembrane proteins with multiple cytosol and cell membrane subunits . Thet effect . NADPH ot effect . Thus, wt effect ,43, the ct [₋/₋)  and elevct [₋/₋) . On the ct [₋/₋) .2.₋, ONOO₋, and H2O2, therefore increasing oxidative stress products, which increases endothelial dysfunction and atherosclerosis initiation [NOX enzymes also promote the oxidation of tetrahydrobiopterin (BH4), the main cofactor of eNOS. As a result, eNOS, principal producer of endothelial NO during phy-siological conditions, becomes dysfunctional, reducing the levels of this vasodilator and antiatherogenic agent . Moreoveitiation .2 as electron donor, producing NADH, H2O2, and O2.₋. Different stimuli can activate the expression of XO, including shear stress, hypoxia, or proinflammatory factors such as IL-6 or TNF-α [XO catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid. In this reaction, XO uses Oor TNF-α . XO has or TNF-α . For insor TNF-α , while Xor TNF-α . Finally, mitochondrial dysfunction can also drive to excessive ROS formation, as well as mitochondrial DNA (mtDNA) damage. Remarkably, mtDNA damage promotes itself ROS production. Both processes have been found associated with the development of a-therosclerosis ,52. For Different antioxidant systems—such as glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (Cat), or thioredoxins (Trx)—can be found within the vascular wall. All these enzymes play an important role in neutralizing ROS production and reducing oxidative stress . In this₋/₋ and Nrf2₋/₋ or Nrf2+/₋ mice indicate a reduction of the atherosclerotic process in these animals, suggesting a deleterious role of the transcription factor [₋/₋ Nrf2₋/₋ mice [Nrf2, encoded by NFE2L2 gene, constitutes a transcription factor that has been closely linked to atherosclerosis, although its specific role in this pathology is not clear. Nrf2 might play antagonistic roles, both preventing and enhancing atherosclerotic development ,58,59. Fn factor ,61. Nrf2n factor , while in factor , promotin factor  or the mn factor . Nrf2 mon factor . In this₋/₋ mice ,65. ConvThe majority of studies, however, have focused on the athero-protective properties of Nrf2, mainly as a key agent against oxidative stress . Apart fNrf2 not only protects against oxidative stress in ECs through activation of different antioxidant genes, it also exerts anti-inflammatory and angiogenic effects on these cells . Indeed,Nrf2 belongs to “Cap’n’Collar” (CNC) family of transcription factors with a b-ZIP domain ,77 whichMany factors—such as ROS, ox-LDL, lipid peroxides and their metabolites, electrophiles, pro-inflammatory cytokines, and many other molecules related to oxidative stress—can induce an alteration of Keap1 conformation (by modification of its cysteines residues) ,64, promCurrently, the Nrf2/Keap1 system is considered the principal mechanism against o-xidative stress, which is widely conserved, mainly in mammals . This syThe Nrf2/Keap1 system can also be regulated by the autophagy regulator protein p62 . Phosphorylation of p62 in response to many factors such as oxidative stress  allows tApart from Keap1, other molecules can regulate Nrf2 activity through additional control points at transcriptional, post-transcriptional, translational, and post-translational level ,91 Tabl. SeveralAlternatively, several studies suggest that different microRNAs (miRNAs)  can also regulate, directly or indirectly, Nrf2 expression and moreover, to control the antioxidant activity of the Keap1-Nrf2 complex and Nrf2/ARE signaling pathways ,112. miRHO-1 has many important roles that contribute to the protection against atherosclerosis, such as antioxidant, anti-inflammatory, antiapoptotic, or immunomodulatory effects . HO-1 isHO-1 is a highly conserved enzyme—also known as 32-kDa heat shock protein—encoded by the hmox1 gene ,118. The2+ and biliverdin IXα [In mammals, heme oxygenase enzymes are located on the surface of the endoplasmic reticulum, anchored to it by hydrophobic amino acids at its COOH-terminal ends. Its ca-talytic part contains a “heme binding pocket” of 24 amino acids, oriented to the cytosol ,118, togrdin IXα   signaling pathway . Besidesree heme ,141.2. However, free heme can be toxic for several reasons [2+ from its porphyrin pocket [3+ through the Fenton reaction, an important source of ROS. Fe3+ can promote LDL oxidation in presence of superoxide anion (O2.₋) [The heme molecule itself is fundamental for many biological functions, due to its role as prosthetic group in hemoproteins like Hemoglobin (Hb), a major transporter of O reasons . For ins reasons ,143, and reasons . Moreoven pocket , which cn (O2.₋) . Moreoven (O2.₋) . For insn (O2.₋) . Notablyn (O2.₋) . Therefo2+ release. However, HO-1 stimulates the expression of ferritin, an iron storing protein , it also reduces the levels of free heme molecules. HO-1 upregulation might represent a pro-oxidant process due to the degradation of heme and Fe protein . Indeed,2+ derived from heme degradation. Overall, HO-1 antioxidant activity can be explained by three key processes derived from its heme catalytic activity: production of biliverdin and related bilirubin, release of CO, and finally, stimulation of ferritin synthesis, therefore ensuring an optimal disposal/storage of FeDue to the beneficial properties linked to HO-1, different therapeutic approaches have been launched seeking HO-1 upregulation as a strategy to protect against oxidative stress and, for instance, to reduce vascular injury and atherosclerotic progression ,149. IndCurcuma (Curcuma longa), has been shown to induce HO-1 expression by activation of the Nrf2/ARE pathway in a dose- and time-dependent manner [Alternatively, heme analogs and/or its derivatives also constitute effective inducers of HO-1 expression, mainly by binding and inactivating Bach1, which promotes Nrf2 nuclear translocation and specific HO-1 transcription ,154. Fort manner . Besidest manner . Unfortut manner  or activt manner . Remarkat manner . Althougt manner , severalt manner . In addit manner . Similart manner . Finallyt manner  or allevt manner . Moreovet manner . Overallt manner . www.clinicaltrials.gov, accessed on 25 August 2021). Alternative strategies for the delivery of this compound such as membrane-based controlled systems to release CO are being tested [Direct administration of biliverdin/bilirubin or CO and CO releasing molecules (CORMs) are other strategies explored to treat vascular injury . Indeed,g tested , togetheg tested . www.clinicaltrials.gov, accessed on 25 August 2021) [Considering that the modulation of HO-1 expression is linked to Nrf2, pharmacological drugs are also applied to directly activate Nrf2/HO-1 signaling pathway instead . It thisst 2021) . In addist 2021) . Dimethyst 2021) , and itsOf note, any pharmacological strategy targeting HO-1 should take into account that Nrf2 activation could also derive in undesired pro-atherogenic effects , and morOxidative stress represents a key player in the pathophysiology of atherosclerosis. The imbalance between pro- and antioxidant agents drives to an overproduction of ROS, which promotes DNA and lipid peroxidation, cell death, and endothelial dysfunction, initial steps in the development of atherosclerotic plaques. In order to reduce ROS production and tackle oxidative stress and atherosclerosis itself, many researchers have focused on identifying mechanisms to activate the Nrf2/HO-1 signaling pathway. Overall, both, Nrf2 and HO-1 have shown to protect against atherosclerotic injury through their antioxidant, anti-inflammatory, or even immunomodulatory properties. Different strategies based either in the modulation of Nrf2 by its interaction with Keap 1 or alternatively through other pathways involving Nrf2 phosphorylation or epigenetic modulation via miRNAs are currently under research. In addition, many natural compounds like resveratrol or sulforaphane are also being tested due to their potential to activate Nrf2/HO-1 pathway. Some of these molecules have shown promising results at the preclinical side. On the other hand, Nrf2 may also enhance atherosclerosis through additional mechanisms that need to be further characterized. Thus, further research in this field is required, in order to better understand the involvement of Nrf2/HO-1 in the pathophysiology of atherosclerosis and moreover, to identify therapeutic alternatives to trigger their antioxidant and anti-atherogenic effects.


Histologically, bladder cancer is a heterogeneous group comprising urothelial carcinoma (UC), squamous cell carcinoma, adenocarcinomas (ACs), urachal carcinomas (UrCs), and small cell neuroendocrine carcinomas (SCCs). However, all bladder cancers have been treated so far uniformly, and targeted therapy options are still limited. Thus, we aimed to determine the protein expression/molecular status of commonly used cancer targets (programmed cell death 1 ligand 1 (PD-L1), mismatch repair (MMR), androgen and estrogen receptors (AR/ER), Nectin-4, tumor-associated calcium signal transducer 2 , epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and fibroblast growth factor receptor 3 (FGFR3)) to give first insights into whether patients with SCC, AC/UrCs, and squamous-differentiated carcinomas (Sq-BLCA) of the bladder could be eligible for targeted therapies. In addition, for MMR-deficient tumors, microsatellite instability was analyzed. We completed our own data with molecular data from The Cancer Genome Atlas (TCGA). We present ratios for each drug and cumulative ratios for multiple therapeutic options for each nonurothelial subtype. For example, 58.9% of SCC patients, 33.5% of AC/UrCs patients, and 79.3% of Sq-BLCA patients would be eligible for at least one of the analyzed targets. In conclusion, our findings hold promise for targeted therapeutic approaches in selected patients in the future, as various drugs could be applied according to the biomarker status. Bladder cancer ranks as the tenth most common cancer worldwide . AccountSo far, bladder cancer has been treated similarly, almost regardless of its histological subtype. Only SCC patients routinely receive chemotherapy with platin and etoposit  and beneMoreover, targeted therapies and personalized treatments are becoming increasingly important in bladder cancer. In the last two years, the pan-FGFR Inhibitor Erdafitinib and the antibody-drug conjugates enfortumab vedotin and sacituzumab govitecan received accelerated approval from the Food and Drug Administration (FDA) . FGFR-inAs therapeutic options are now changing, the purpose of our study was to investigate special histological subtypes of bladder cancer for therapeutic marker expression in order to evaluate the possible use of targeted therapies in these subtypes.n = 18 SCCs, n = 37 AC/UrCs, and n = 128 squamous differentiated bladder cancers  were immunohistochemically stained with DAKO 22C3 anti-PD-L1 antibody , or mismatch repair-deficient (dMMR) solid tumors that have progressed following prior treatment ,17. We uanalysis B.MLH1, MSH2, MSH6, or PMS2 genes. We found missense or truncating mutations in 6 of 46 squamous differentiated bladder cancer cases (13%)  of the Sq-BLCAs showed a deficiency in the mismatch repair proteins . By PCR,es (13%) C, indicaThe protein expression of androgen receptor (AR) and estrogen receptor (ER) was investigated and compared in SCCs, Sq-BCLAs, and AC/UrCs . While ENectin-4 expression was determined  by usingIn each subgroup, most carcinomas showed expression of Nectin-4, including 100% (14/14) of the SCC cohort, 91% (20/22) of the AC/UrC cohort, and 82% (69/84) of the Sq-BLCA cohort. Considering only the samples with a particularly strong expression, i.e., those with an H-Score of greater than 200, 43% (6/14) of SCCs, 18% (4/22) of AC/UrCs, and 11% (9/85) of Sq-BLCAs fulfilled these criteria .With a membranous expression of Tumor-associated calcium signal transducer 2  of at least 10% of tumor cells, tumors are classified as positive. Expression was scored as weak (1+), moderate (2+), and strong (3+) . Thus, iTM)  of cases had an EGFR gene amplification.When scoring the protein expression of tyrosine kinase receptors, staining intensity plays a crucial role. A tumor is classified as epidermal growth factor receptor (EGFR)-positive at any immunohistochemical staining of the tumor cell membrane above the background level (Dako EGFR pharmDxTM) A. In ourTM) . AdditioERBB2) gene in the “squamous-like” cohort. Amplification of the ERBB2 gene was found in one patient (1/1) with adenocarcinoma of the urinary bladder  protein overexpression. Tumors are considered HER2-positive if at least 11% of tumor cells have weak-to-moderate membrane staining (≥DAKO Score 2+). In addition, 6% (1/18) of SCCs, 8% (2/25) of the AC/UrC cohort, and 2% (3/122) of Sq-BLCAs were scored as positive . A stron bladder B.FGFR3 gene could be detected in 13% (6/46) of cases, with five single nucleotide variants being missense mutations and thus representing putative driver mutations (Fibroblast growth factor receptor 3 (FGFR3) positivity was reported according to a semiquantitative scoring system developed by Tomlinson et al. . Tumors utations B.All proteins analyzed in this study are either direct or indirect targets of novel anti-cancer drugs; thus, the dysregulation of wildtype and/or mutated genes was used as predictive markers whose corresponding protein could indicate a putative treatment option, i.e., PD-L1/MMR for immune checkpoint inhibitors, AR/ER for anti-hormone receptor treatment, Nectin-4 for anti-Nectin antibody-drug conjugates, Trop-2 for anti-Trop-2 antibody-drug conjugates, and tyrosine kinases (TK), including EGFR and HER2 for TK inhibitors (TKI).As the impact of the FGFR3 wildtype protein expression on the sensitivity of FGFR3 inhibitors has been controversially discussed , this taFirst, we correlated immunohistochemical results of predictive markers and clinico-pathological parameters  independFinally, we determined the cumulative ratios of a single and multiple therapy options for each nonurothelial subtype. Overall, 58.9% of SCC patients, 33.5% of AC/UrC patients, and 79.3% of Sq-BLCA patients could be eligible for at least one of the identified therapy options A. For boThus far, targeted therapies are an integral part of treatment of various types of cancer; however, the treatment options for histological subtypes of bladder cancer are still limited. For this reason, we evaluated potential treatment approaches for these subtypes by analyzing the protein expression and molecular status of potential targets that are commonly used in other entities, including immune response, ERBB, and hormone receptor pathways. In the following, we discuss the putative relevance of analyzed targets to indicate novel treatment options for nonurothelial cancer subtypes.Recent milestones in bladder cancer treatment were the implementation of immune checkpoint inhibitor treatment in first- and second-line therapy of metastatic bladder cancer . As an aIn hormone-dependent tumors such as prostate carcinoma or hormone receptor-positive breast cancer, anti-hormonal therapies, for example, by inhibiting AR or ER, are already an integral part of therapy ,28. ReceTargeting activated tyrosine receptor kinases and subsequent pathways is another possibility of targeted therapy. An effective therapeutic target, for example, in RAS wild-type colorectal cancer and EGFR-mutated non-small-cell lung cancer (NSCLC) ,36, is aIn recent years, the antibody-drug conjugates enfortumab vedotin and sacituzumab govetican received accelerated approval for the treatment of bladder cancer by the FDA regardless of their protein expression level on tumor tissues. Our data showed that both Nectin-4 and Trop-2 are expressed in all of the subtypes of bladder cancer studied here. Thus, 43% of SCCs, 18% of AC/UrCs, and 11% of Sq-BLCAs showed high Nectin-4 expression. Trop-2 was strongly expressed by 67% of Sq-BLCAs, 38% of Ac/UrCs, and 6% of SCCs and was further associated with advanced tumor stages independently of a given subtype. However, it is still unclear whether a high expression of the targets is associated with a better response to therapy as evaluation studies are missing. If this would be the case, evaluation of the expression level prior to the respective targeted therapy would be helpful, as expression differs among the subtypes of bladder cancer. As our study confirmed strong expression values of Nectin-4 and Trop-2, antibody-drug conjugate therapies should also be considered for nonurothelial subtypes of bladder cancer.Members of the fibroblast growth factor receptor family of kinases (FGFR1–4) are dysregulated in various cancers , increasn = 53, and pure squamous cell carcinomas, n = 77, were collected from collaborating Institutes of Pathology in Germany and the German Study Group of Bladder Cancers (DFBK e.V.)  samples from different urinary bladder cancer cohorts, including 18 SCCs, 37 AC/UrCs, and 128 cancers with squamous differentiation (Sq-BLCA), i.e., urothelial cancers with substantial (>75% tumor volume) squamous-differentiation, BK e.V.) . Tissue TMFLEX system (mouse or rabbit linker and horseradish peroxidase-conjugated polymer) for detection was applied. Reactions were visualized with the DAKO Liquid DAB Substrate Chromogen System and hematoxylin counterstain. Staining was evaluated by two independent investigators  according to the respective scoring system.For immunohistochemical staining, TMA sections were either pretreated with proteolytic enzymes or with heat retrieval with low pH (pH 6) or high pH (pH 9) . After incubation with primary antibodies , the DAKFor scoring EGFR staining results, the EGFR DAKO score was used. EGFR-positive staining is defined as any immunohistochemical staining of tumor cell membranes. Depending on the staining intensity, a score between 1 and 3 was assigned  . For HERPD-L1 expression was analyzed for tumor cells, immune cells, and combined  regardless of the staining intensity according to the PD-L1 IHC 22C3 pharmDx interpretation manual for urothelial carcinoma. Nectin-4 expression was determined by using the histochemical scoring system (H-score), which is defined as the sum of the products of the staining intensity (score of 0–3) and the percentage of cells (0–100) stained at a given intensity  . Trop-2 n= 6) was performed using the Maxwell RSC FFPE Plus DNA Kit according to the manufacturer’s instructions. For the evaluation of MSI by polymerase chain reaction (PCR), the Bethesda Panel, consisting of five microsatellite markers specific for two mononucleotide loci (BAT-25 and BAT-26) and three dinucleotide loci , was used [DNA extraction of FFPE tissue samples  network were used for in silico analysis of r cancer .p-values were equal or below 5% (≤0.05). Correlation analysis was performed by calculating the nonparametric Spearman’s rank correlation coefficient.Set overlaps were visualized using the “UpSet” function of the “ComplexHeatmaps” package v. 2.8.0. and the “VennDiagram” package v. 1.6.20. in R v. 4.1. Statistical analyses were performed using SPSS 25.0  and GraphPad Prism 5.0 . Differences were considered statistically significant if the two-sided In summary, we provided evidence for novel therapeutic strategies for rare histological subtypes of bladder cancer. For both SCCs and Ac/UrCs, anti-hormone therapies and anti-Nectin-4 therapy may be considered. As studies have shown that patients with bladder cancers with squamous differentiation have a poor response to standard chemotherapy , our fin


Heusler alloys are a material class exhibiting various magnetic properties, including antiferromagnetism. A typical application of antiferromagnets is exchange bias that is a shift of the magnetization curve observed in a layered structure consisting of antiferromagnetic and ferromagnetic films. In this study, a layered sample consisting of a Heusler alloy, Mn Heusler alloys have received growing attention for their rich physical properties, such as ferromagnetism, antiferromagnetism, half-metallic electronic structure, shape memory behavior, superconductivity, topological behavior, and so on ,2. Among2MnAl ,8,9, Ru2l [2MnGe ,11, Mn2VGe [2VSi , Fe2VAl Si [2VAl , Mn3Ga [VAl [3Ga , Mn3Ge [3Ga [3Ge , and so 3Ga [3Ge . Among t3Ga [3Ge . Howeverab initio calculation, MnIn the present study, Mn21 phase a and A2 21 phase b. The crrimagnet ,19,20,21rimagnet ,23. In arimagnet ,25. In ftructure . AlthougA2 phase . A possik sample ,25, for d in NiO , Pt-Mn [d in NiO , Ir-Mn [d in NiO ,29, and NiO [2O3  antiferrFilm samples were fabricated onto single-crystalline MgO (001) substrates using an ultra-high-vacuum (UHV) magnetron sputtering system. The stacking structure was a MgO substrate/MnFour samples were prepared with the different The wide-area cross-sectional TEM images are shown in chniques . For theTo characterize the distribution of the ordered phases of LXAS and XMCD measurements were conducted to probe site-specific magnetic moments at the Mnf Mn2VAl ,21,32, if Mn2VAl ,27. HaviH) for samples A to D. At H is found for the dip-peak feature around the H. The dip at 640 eV grows with increasing H, whereas the dip-to-peak amplitude at the H. Because the positive peak at 639 eV is observed only in Sample A, it may be considered to be responsible for the exchange bias. The XMCD spectra around the H. A double minimum feature is observed at 8 T. It should be noted that the dip feature at 639 eV for Sample C can be distinguished from the peak for Sample A at the same photon energy. The XMCD spectrum of Sample D is the most insensitive to the small H (≤2 T) among the four samples. The dip amplitude increases at 8 T, whereas it is quite small below 2 T. Element-specific XMCD hysteresis loops (ESMHs) were also measured to identify the magnetization process of the interface magnetic moments. The XMCD spectra provides us with further information on the interface magnetic moments. Regarding Sample A, both ESMHs of the Mn XMCD measured at 639 and 640 eV exhibit hysteresis with different signs a: The siH [H exhibit XMCD with peak-splitting around the Two factors are considered here as origins of the XMCD signals, that is, (a) the spontaneous magnetization that is characteristic of the ferro- or ferri-magnetic order, and (b) the magnetic-field-induced magnetization, which is typically observed for paramagnetic samples and antiferromagnets under sufficiently large magnetic fields. Factor I plays a dominant contribution to the XMCD signals of samples A and B, whereas Factor II affects all samples; however, it is dominantly observed in samples C and D. The details are described as follows: Among the present samples, the XMCD for Sample D is simply caused by Factor II, in which the XMCD signal originates from the paramagnetic Mn-moments aligned by the relatively large external H ,40,41,42agnetism ,24,25, t studies ,21,32. FHowever, for the other cases of Samples A and B, finite XMCD signals at low  in Ref.  was usedThe microstructures and magnetic moments at the interface between the Heusler alloy Mn


Starch is the main food source for human beings and livestock all over the world, and it is also the raw material for production of industrial alcohol and biofuel. A considerable part of the world’s annual starch production comes from crops and their seeds. With the increasing demand for starch from food and non-food industries and the growing loss of arable land due to urbanization, understanding starch biosynthesis and its regulators is essential to produce the desirable traits as well as more and better polymers via biotechnological approaches in cereal crops. Because of the complexity and flexibility of carbon allocation in the formation of endosperm starch, cereal crops require a broad range of enzymes and one matching network of regulators to control the providential functioning of these starch biosynthetic enzymes. Here, we comprehensively summarize the current knowledge about regulatory factors of starch biosynthesis in cereal crops, with an emphasis on the transcription factors that directly regulate starch biosynthesis. This review will provide new insights for the manipulation of bioengineering and starch biosynthesis to improve starch yields or qualities in our diets and in industry. As a fundamental commodity, starch was and is still widely used for human consumption in food and non-food industries ,2. SinceGBSSI, was partly mediated by tetrapyrrole intermediates, i.e., heme, in endosperm during early seed development in rice [Starch is composed of amylose and amylopectin glucan polymers, which are packaged to form the insoluble semi-crystalline starch granules . Starch  in rice , suggestThe process of starch biosynthesis in crops requires tight cooperation of various starch biosynthetic enzymes, and coordinates with other metabolisms. Until now, a great number of genes involved in starch biosynthesis have been identified in various cereals. However, despite these findings, the regulatory factors underlying starch synthesis are still poorly understood, especially regarding the regulation of starch biosynthetic genes. Therefore, the screening and identification of key regulators is vital to understanding the fundamental regulatory mechanism underlying starch synthesis. Here, we comprehensively provide an update on current knowledge on the pivotal spatiotemporal-dependent regulators of starch biosynthesis in cereal crops with a focus on transcription factors directly regulating starch biosynthesis, which will provide new insights into cereal starch yield or quality improvement on our diet as well as in industry.Starch biosynthesis is a complex process and requires various functional enzymes, which are regulated by transcription factors (TFs) and/or other regulators . TranscrTranscriptional regulation of starch biosynthesis varies with the environment, such as phytohormones and sucrose, and shows spatial-temporal gene targeting in different cereal crops. Based on the expression patterns in various tissues, genes involved in starch synthesis can be divided into two types ,20. The Oryza sativa L. [Zea mays L. [Hordeum vulgare L. [Triticum aestivum L. [bZIP factors are the first identified transcription factors to regulate starch biosynthesis ,22, and ativa L. ,24, Zea  mays L. , Hordeumlgare L. , and Tritivum L. ,27. bZIPtivum L. ,28,29, wtivum L. . Furthertivum L. , barley tivum L. , and maitivum L. , but it ADP-glucose pyrophosphorylase large subunit 3 (OsAGPL3), OsGBSSI/Wx, starch synthase IIa (OsSSIIa), BEI, OsBEIIb, and Isoamylase-type starch debranching enzyme 2 (ISA2) [BEI and GBSSI during starch synthesis [starch synthase III (SSIII) and indirectly regulate SSIIa and BEI via interaction with prolamin-box-binding factor (PBF) to mediate starch biosynthesis at transcriptional levels. Moreover, ZmbZIP91 [pAGPS1, pSSI, pSSIIIa, and pISA1).bZIPs show broad affinities with different motifs in the process of regulating starch biosynthesis. A well-studied bZIP in rice, OsbZIP58  ,29, has 2 (ISA2) ; and (ii2 (ISA2)  by bindi2 (ISA2) , whereasynthesis . Similarynthesis  could diZmbZIP91  binds toTriticum aestivum, TabZIP28 [Besides, the functions of bZIPs depend on tissue types to some extent. OsZIP58 shows highly endosperm-dependent expression during seed development ,28, but TabZIP28 , were alAGPS1, SSI, SSIIIa, and ISA1 during starch synthesis [Interestingly, ZmbZIP91  and O2  are the ynthesis , while Oynthesis  usually Oryza sativa EREBP clone 89) protein to regulate the Wx gene expression in rice [Wx promoter [In addition to the heterodimers of bZIPs  and DOF family factors , the MYC proteins also often interact with the ethylene-responsive element-binding protein (EREBP) family in their regulatory functions during starch biosynthesis. For example, one MYC-like protein, OsBP-5, has been reported to interact with the OsEBP-89 , has been reported to negatively regulate starch synthesis-associated genes both in wheat  and riceSub1C, are reported to promote starch accumulation by depressing the α-amylase gene RAmy3D in rice internodes [Multiple AP2/ERF factors usually regulate the starch synthesis genes through the induction of phytohormones, including ethylene, abscisic acid (ABA), and gibberellin GA; . Two subternodes  and leavternodes . These pternodes .ZmSSIIIa to positively mediate starch synthesis [ZmSSI promoter and indirect regulation of ZmSh2 and ZmGBSSI during seed development [GAMYB and SSI genes, respectively. However, ABI3, also named viviparous-1 (VP1), was previously characterized as one regulator of seed development in maize [Hor2 (For B-hordeins) expression in developing endosperm and activates the GAMYB-mediated Amy6.4 expression in post-germinative reserve mobilization [ZmSSI [ABA-induced AP2/ERF in starch synthesis includes at least two TFs, EREB and ABI , ATAF1/2, cup-shaped cotyledon (CUC2)) represent one of the largest families in plants and are involved in the stress-induced response during development periods ,62. Yet,Brittle 2 (BT2) and six other hypothetic genes, including Zpu1 (encoding Zea mays pullulanase-type starch debranching enzyme), GBSSI, Sh2 (for AGPase large subunit), SSV, ISA2, and SSIIa  expression and interaction with TaGAMyb, and (iii) by adjustment of the accumulation of starch by the regulation of SSIIa and Susy1.Recently, a novel NAC-type transcription factor was revealed to regulate starch biosynthesis in wheat. TaNAC019 is specifically expressed in endosperm, and functions during starch synthesis through the following ways : (i) by As the widely distributed transcription factors, MYBs are vital for a variety of biological functions. However, the functional studies of MYBs on starch biosynthesis are still scarce in cereal crops. So far, only one MYB factor has been reported as a regulator during starch biosynthesis .ZmBT1 for ZmMYB14 to function as a regulator of starch synthesis may not be through the MBSI site (TAACTG) but other sites or mechanisms, while another is that there are spatial-temporal differences between type I starch synthesis-associated genes and ZmMYB14 [Although many MYBs, i.e., ZmMYB73, ZmMYB127, ZmMYB155, and ZmMYB14, seemed to play roles in starch biosynthesis, only ZmMYB14  has been ZmMYB14 , which mRPBF was negatively regulated by SERF1, displaying contrasting expression patterns [RAmy1A was enhanced in the presence of RPBF [Hordeum vulgare. GAMYB and BPBF in the developing endosperm activates the full Hor2 expression controlled by the expression pattern of HvVP1 (VIVIPAROUS1), orthologous to ABI3 from Arabidopsis thaliana, whereas the activation of Amy6.4 in post-germinative reserve mobilization was mediated by GAMYB [DOFs show endosperm-specific expression in the regulation of starch biosynthesis . Regulatpatterns ,35, whil of RPBF ,65. One by GAMYB . So, it by GAMYB .ZmGBSSI and ZmISA1 through the combination with DOF core elements in their promoters both in vitro and in vivo [SSIIa [DOFs also mediated the synthesis of starch and seed storage proteins (SSPs) through recognition of the AAAG motif . Several in vivo , while Zo [SSIIa .GRAS transcription factors are involved in plant growth and development processes, especially for endodermis specification . BesidesOne maize TF of GRAS, ZmGRAS20, plays regulatory roles in starch biosynthesis of rice endosperm . Throughisoamylase (ISO), and thereby mediates carbohydrate anabolism [SUSIBA2 produced a rice variety with a high starch content but low methane emission [ISO1 and BEIIb, and confers tissue-specific sugar responsiveness [ISO and BE.Sugar signaling in barley2 (SUSIBA2) is a plant-specific WRKY TF that regulates gene expression in the presence of sugar, thereby mediating the communication of source-sink ,70 via aemission , suggestemission . SUSIBA2siveness ,70. Ther2-responsive CONSTANS, CONSTANS-like, and time of chlorophyll a/b-binding protein1 (CRCT).Some regulatory factors cannot be listed in any of the above groups, and thus are included independently in this section , includiFLOURY ENDOSPREM is named based on its well-recognized but viable endosperm phenotypes, which usually display a chalky and soft endosperm. So far, 16 rice floury mutants [ZmFloury3 [ mutants  and thre mutants  have bee mutants  and FLO7 mutants , as wellmFloury3  serve asmFloury3 ,75,76, tFLO2 acts as a regulatory protein that controls the biosynthesis of seed storage substances, possibly through its tetratricopeptide repeat (TPR) motifs for protein–protein interactions . At leasN-terminal transit peptide and an unknown function 1338 (DUF1338), during starch synthesis and amyloplast development [FLO7 prefers to specifically function in the endosperm periphery, and harbors two essential domains of an elopment .Floury3 (named FL3), a plant AT-rich sequence and zinc-binding (PLATZ) protein, acts as a specific regulator in maize starchy endosperm cells . FL3 plaOsCRCT displays diurnal oscillation and is mainly focused on the vegetative organs. Therefore, OsCRCT serves as a positive regulator of several starch biosynthetic genes, including AGPS1, AGPL1, Glc-6-phosphate translocator2 (GPT2), plastidial α-glucan phosphorylase1 (Pho1), and BEIIa, and contributes to starch biosynthesis in rice leaves [OsCRCT is a potential approach to improve yields for food and biofuel [The CRCT protein has been reported to be a regulator of starch biosynthesis in rice but does not seem to belong to a zinc finger motif , and cane leaves . The eff biofuel , dependiAside from the direct regulators of starch biosynthesis, many other factors also participate in the regulation of starch synthesis, partially through the post-transcriptional regulation of TFs, i.e., micro RNAs (miRNAs), or via regulators of the formation of the upstream precursors and/or downstream products of starch.miRNAs can negatively mediate gene expression at the post-transcription levels , and havSome transcription factors have been reported to mediate starch accumulation in cereal endosperms, but they participate in regulation via control of the synthesis of the downstream and/or upstream products of starch, i.e., sugar and proteins, instead of directly regulating starch synthesis. Here, we mainly concentrate on some of them , includiα- and β-zein [b-32 [pyruvate orthophosphate dikinase 1 (PPDK1) [γ-zein and 22-kDa α-zein [α-zein and 50-kDa γ-zein through the binding of the CATGT motif in their promoters.Aside from direct regulation of starch synthetic enzymes, some bZIPs are also involved in the regulation of starch synthesis via indirect ways . For exad β-zein , b-32 [2in [b-32 , and pyr (PPDK1) ,22 via ra α-zein ,87 throua α-zein  to combia α-zein , is esseO2, PBF1, ZmbZIP22, NAC130, and O11.Recently, one B3 domain-containing AP2/ERF transcriptional factor, ZmABI19 , was wel16-kDa γ-zein genes [Regulation of NAC on starch synthetic enzymes is often accompanied with a simultaneous regulation of storage protein synthesis-associated genes . Althougin genes . Howeverin genes  do not din genes . TaNAC01in genes  has beenα-zein and γ-zein [27-kDa γ-zein [γ-zein [Nkd1 (naked endosperm 1) promoter to regulate aleurone cell differentiation.Some DOF TFs have similar effects on the regulation of starch synthesis. PBF specifically binds with P-box (TGTAAAG) to mediate the synthesis of proteins , i.e., αd γ-zein . PBF cand γ-zein  to regula γ-zein . Therefoa γ-zein  during s [γ-zein , while Z [γ-zein  can bindGBSSI, in mature seeds. Another MADS TF, ZmMADS47, through the interactions with OPAQUE2, directly regulates prolamin in maize [OsGIF1 during grain filling stages [MADS-box transcription factors have attracted increasingly more attention in the studies of seed development ,90. For in maize . Interesin maize . Howeverg stages . Thus, tNKD2 and ZmDOF3 during endosperm development. Besides, it is also involved in the regulation of nutrient and carbohydrate metabolism via O2 and PBF, as well as in response to environmental stresses. Thus, OPAQUE11 may work as the focus hub in the regulatory network during seed formation [TFs of basic helix–loop–helix act as important regulators of starch synthesis during seed formation ,94. For Arabidopsis thaliana [Arabidopsis [ZHOUP1 (ZOU) is involved in the seed formation of angiosperm plants, i.e., thaliana ,96 and mthaliana . ZmZOU ithaliana . Similarthaliana  and in Abidopsis , via theStarch was and remains an essential commodity in food and non-food industries. Cereal seeds provide a great account of starch production, and thereby, starch synthesis and its regulation in seed endosperm have been investigated by researchers of breeding. The enzymes involved in starch synthesis have been well identified in cereals, but, until now, the regulatory mechanism underlying starch synthesis has not been fully understood, especially regarding the regulators on starch synthetic genes. As the key regulators mediating gene expression, the working models of transcriptional factors perform complicated networks during starch biosynthesis , and maiThe race between the increasing starch demand and the shrinking arable land has resulted in the urgency to further understand starch biosynthesis and its regulation, which is vital to produce a rational design of agronomic traits and more and better polymers with biotechnological approaches. Besides, improved cereal grain quality is also essential to consumers; however, the formation of seed quality is a highly complex process. These processes are largely adjusted with various regulators, which perform precision guidance on the cooperation of starch synthesis-related proteins and lipids. Therefore, it is insufficient for the selection of any single trait in crop breeding.Arabidopsis, only dozens of regulators, especially for TFs, have been reported in cereal crops. Thus, the traditional mutagenesis technology, combined with advanced gene editing technology , is a commonly used way to explore novel TFs, whose binding motifs can be determined with DAP-seq [Currently, the main application of cereal starch is to feed the increasing human population as a vital food source, and thus, high yields of cereals or improvement of states of starch shortage are still the key breeding targets. However, the enzymes involved in starch synthesis have been well identified in cereals, but the efforts to improve starch synthesis have failed using the simplistic one-enzyme strategy . This wa DAP-seq  and/or C DAP-seq  technoloHomo sapiens [SUSIBA2 in rice [FTO, one specific Homo sapiens gene associated with obesity and fat mass, into rice and potato largely increased the productivity and biomass [Besides, the building of regulatory networks can also help to design a rational strategy to improve starch biosynthesis. In order to achieve this, the selection and visualization of homogenized cells is key to the transcriptomic technologies  with the sapiens . The het in rice  produced biomass . Since F biomass , it is fCollectively, this review summarized the regulators underlying starch synthesis in cereal crops, and will help to provide potential applications in crop breeding and engineering efforts.


Measles virus (MeV) has a negative-sense 15 kb long RNA genome, which is generally conserved. Recent advances in high-throughput sequencing (HTS) and Dual RNA-seq allow the analysis of viral RNA genomes and the discovery of viral infection biomarkers, via the simultaneous characterization of the host transcriptome. However, these host–pathogen interactions remain largely unexplored in MeV infections. We performed untargeted Dual RNA-seq in 6 pharyngeal and 6 peripheral blood mononuclear cell (PBMCs) specimens from patients with MeV infection, as confirmed via routine real-time PCR testing. Following optimised DNase treatment of total nucleic acids, we used the pharyngeal samples to build poly-A-enriched NGS libraries. We reconstructed the viral genomes using the pharyngeal datasets and we further conducted differential expression, gene-ontology and pathways enrichment analysis to compare both the pharyngeal and the peripheral blood transcriptomes of the MeV-infected patients vs. control groups of healthy individuals. We obtained 6 MeV genotype-B3 full-genome sequences. We minutely analyzed the transcriptome of the MeV-infected pharyngeal epithelium, detecting all known viral infection biomarkers, but also revealing a functional cluster of local antiviral and inflammatory immune responses, which differ substantially from those observed in the PBMCs transcriptome. The application of Dual RNA-seq technologies in MeV-infected patients can potentially provide valuable information on the virus genome structure and the cellular innate immune responses and drive the discovery of new targets for antiviral therapy. H and P genes, but also non-coding sequences, have been used [Measles virus (MeV) infection causes worldwide mortality in children, as more than 100,000 fatal infections occur each year . Howevereen used . Whole-geen used ,8.The virus can be rapidly transmitted by dispersed respiratory droplets or through direct contact with infected objects. After approximately 10 days of incubation, the onset of high fever (>38.3 °C) is observed, accompanied by sneezing and coughing, symptoms that increase the transmissibility of the virus. The onset of a generalized maculopapular skin rash is observed 14 days post-infection, while patients are considered infectious 4 days before to 4 days after this time point, due to the higher replication rates of the virus in the respiratory tract during this period ,10. AfteSingle-analyte host biomarkers have been recruited as quantifiable indicators of inflammation; however, limited specificity and sensitivity of markers such as erythrocyte sedimentation rate and C-reactive protein remain a burden to their establishment, while probably the most useful is procalcitonin, which indicates the presence of bacterial infection in sepsis and lower respiratory tract infections ,20. In tWe retrospectively analyzed total nucleic acids (stored at −80 °C) extracted from pharyngeal swabs and PBMCs of 6 fully anonymized patients with MeV genotype B3 infection, as confirmed via routine real-time PCR testing for viral infections. In parallel, two sets of nucleic acid samples  were analysed from 6 clinically and laboratory healthy individuals, as controls . FollowiThe consensus genomes were generated after mapping alignment of the reads to the reference strain MeVs/London.GBR/3.14 (genotype B3-KT732219.1) using Bowtie2 . Samtool7 reads per library, while 2.29% of these reads were successfully mapped on the viral genome on average. The average genome coverage and depth was 99.87% and 1330.2×, respectively (Each of the 6 MeV (+) pharyngeal samples was analyzed twice, for both the reconstruction of the viral genome and the characterization of the host transcriptome. Poly-A enrichment yielded an average of 2.38 × 10ectively . The recDual RNA-seq analysis revealed the transcriptomic signatures of both pharyngeal epithelium and PBMCs of the patient specimens. By comparison to uninfected samples, we were able to identify all the differentially expressed genes  and to cRAD2 and OASL, known to be rapidly induced in response to viral infection, were observed [IFIH1 (MDA-5) gene, which has been shown to trigger the release of interferons (IFNs), was also upregulated [IFIT3, IFIT2, IFIT1, IFITM3, IFI44), as well as of the IVNS1ABP (NS-1) gene, which encodes Influenza virus NS1A-binding protein, an antagonist of IFNs I and III [TNFAIP3/A20, a gene that encodes a zinc finger protein, which acts as inhibitor of both NFκB activation and TNF-mediated apoptosis [CLEC7A (DECTIN 1), CLEC4E, and TNFAIP6 genes [AIM2 gene was upregulated, allowing the assumption that dsDNA-dependent AIM2-inflammasome activation is successful, leading to AIM2-mediated IL-1β and IL-18 production [Analysis of the pharyngeal epithelium transcriptome revealed several key genes known to be involved in the host immune responses. Among all differentially expressed genes, two upregulated genes, observed ,33. The egulated . Additio and III . Overexppoptosis . Innate P6 genes ,38. FinaP6 genes . The AIModuction   enrichment analysis using the differentially expressed genes, helicase . MCM2-7 CDC20, PTTG1, CDC25A, CCNB1, CCNB12), particularly linked to regulation of cell cycle checkpoints . “Cell division cycle 25 A”, a phosphatase encoded by the CDC25A gene, plays an important role in cell division, regulating the activities of cyclin-dependent kinases (CDKs) in a positive way, removing phosphorylation that acts as inhibitor [CCNB gene [PTTG1 gene, encoding a protein whose active role is to maintain genomic stability during mitosis, by controlling sister chromatids’ segregation [Moreover, GO analysis revealed that among the differentially genes there is a group related to cell division  encoding intracellular proteins. Elevated levels of these proteins are correlated with response to viral infection [RSAD2, acting as inhibitor of MeV release in infected cells [OASL and IFIT2 together with CXCL10 form a group of genes whose mRNAs levels can be considered as biomarkers, predicting respiratory virus infection with an accuracy of 97% [IFIT3, IFIT2, IFIT1, IFITM3, IFI44). This can be explained by the elevated levels of IFIH1 gene, encoding MDA-5 PRR, as well as by the action of the cytoplasmic helicase induced by IFN, encoded by the upregulated DDX60 gene. The helicase acts as guardian for viral RNA degradation and RIG-I activation in response to viral RNA and DNA [P (phosphoprotein) gene, affecting downstream RNA editing, resulting in the production of the IFN-antagonist, V. Ιn the lymphocytic cell line Granta-519, MeV variants negatively affect expression of functional V protein, while in the epithelial cell line H358, no variants are reported proximal to the editing site, V’s production is not affected, and the protein can act as a negative regulator of IFN-I signaling [Unbiased—dual RNA-seq transcriptomics can decipher the interactions between the host and pathogen genomes and transcriptomes at the same time. The aim of this study was the simultaneous reconstruction of the virus genome and the detailed characterization of the pharyngeal and the PBMCs’ transcriptome of MeV-infected patients. Differential expression RNA-seq analysis of the pharyngeal epithelium depicted upregulation of nfection ,48. Specnfection . OASL aned cells . Viperined cells ,51. Morey of 97% . In geney of 97%  is not r and DNA . Particu and DNA . Viral R and DNA . It shou and DNA . Finally and DNA . Specifi and DNA . Of note and DNA . During ignaling .IVNS1ABP (NS-1) gene, found overexpressed in the current study, encodes Influenza virus NS1A-binding protein and acts as an antagonist of host type I and III IFN production and signaling [Despite the abovementioned, IFN-I transcripts were not detected in our study, a finding in agreement with the literature. We report that the ignaling . Moreoveignaling . Like otignaling ,59.STAT1, STAT2, p53, IFIH1), considered as targets of MeV [IFIH1 gene was upregulated in our study. The cytoplasmic receptor encoded by this gene binds to dsRNA and undergoes structural rearrangements, triggering the release of proinflammatory cytokines, particularly IFNs by immune cells, thus inducing apoptosis of virus-infected cells [From another set of known key components  are upregulated, TNFAIP3 and TNFAIP6. In general, members of this protein family differ more than 75% at the level of amino acid residues, indicating different biological function and their gene expression is induced by TNF-α . The TNFpoptosis ,62. TNFApoptosis . During poptosis , thus blpoptosis . The secpoptosis ,64,65. HCLEC7A (DECTIN 1), a component of innate immune response, was also observed. It is known that the C-type lectin dectin-1 is a robust inducer of Th1 and/or Th17 responses. CLEC7A inhibits TLR4 signaling, downstream inflammation, and chemokines secretion, acting as a mixed blessing on the regulation of inflammation [In our study, overexpression of ammation ,66. Exceammation . It shouammation ,67. Inflammation . CXCL1 iammation ,68. Moreammation . These fCDC45, MCM2, ORC1, CDC25A), cell division  or act as regulators in cell cycle checkpoints . DNA replication and mitosis entry and exit are strictly regulated by a complex feedback system, which depends mainly on phosphorylation and dephosphorylation events by a number of kinases and phosphatases, respectively [The majority of overexpressed genes in PBMCs encode protein molecules that are activated during DNA replication  protein phosphatase plays an important role as it has dual specificity, in activation of CDKs ,76. Spec targets . Activat targets , and deg targets ,77,78. P complex . Another mitosis . PTTG1 iromatids . Moreoveromatids .In general, our results agree with what is reported on genetic reconstruction of immune cell pool after MeV infection. Infection of immune cells takes place after binding of viral hemagglutinin (H) to cellular receptors CD150, resulting in depletion of specific subsets of B and T lymphocytes ,80. In rThe major conclusion drawn from these observations is that massive infection of lymphocytes and depletion of effector cells and memory clones results in damaged adaptive responses and eventually induction of immune suppression ,84. Spec