This is achieved via selective activation of effectors embedded in multiprotein complexes, or signalosomes, that reside at distinct subcellular locations. cAMP is also one of many pathways known to operate within the primary cilium. Dysfunction of ciliary signaling leads to a class of diseases known as ciliopathies. In Autosomal Dominant Polycystic Kidney Disease (ADPKD), a ciliopathy characterized by the formation of fluid-filled kidney cysts, upregulation of cAMP signaling is known to drive cystogenesis. For decades it has been debated whether the primary cilium is an independent cAMP sub-compartment, or whether it shares a diffusible pool of cAMP with the cell body. Recent studies now suggest it is a specific pool of cAMP generated in the cilium that propels cyst formation in ADPKD, supporting the notion that this antenna-like organelle is a compartment within which cAMP signaling occurs independently from cAMP signaling in the bulk cytosol. Here we present examples of cAMP function in the cilium which suggest this mysterious organelle is home to more than one cAMP signalosome. We review evidence that ciliary membrane localization of G-Protein Coupled Receptors (GPCRs) determines their downstream function and discuss how optogenetic tools have contributed to establish that cAMP generated in the primary cilium can drive cystogenesis. PDEs are a super-family of enzymes that includes 11 families (PDE1-11). Of these, PDE4, PDE7, and PDE8 selectively hydrolyse cAMP. PDE1, PDE2, PDE3, PDE10, and PDE11 hydrolyse both cAMP and cGMP, whereas PDE5, PDE6, and PDE9 are cGMP selective. Several families of PDEs include multiple genes and each gene can generate differently spliced transcripts or be transcribed from different initiation sites, resulting in a large number of PDE isoforms. These isoforms display different affinities for cAMP and enzyme kinetics, their activity can be regulated by different mechanisms  pathway  and its Gongation . PGE2 isongation , suggestAnother study demonstrated that cAMP and PKA regulate both cilia length and function in vascular endothelial cells. In these cells, activation of PKA significantly increased cilia length through a pathway involving cofilin and protein phosphatase-1 (PP-1)-mediated restructuring of actin stress fibers into cortical filamentous actin. The change in cilia length upon cAMP upregulation also led to enhanced flow sensing activity .Consistent with a role of cAMP in promoting cilia length, higher levels of the second messenger are found in cystic tissue from ADPKD patients where the primary cilia of renal cells are abnormally elongated, , althoug0 and ciliary vesicles begin to dock at distal sites of the basal body  is a GPCR involved in food consumption and mutations in this receptor are the most common cause of monogenic obesity . MC4R waKisspeptin is another GPCR localised to the cilium in gonadotropin-releasing hormone (GnRH) secreting neurons. Kisspeptin signalling is involved in the onset of puberty and mature reproductive function . In adulTGR5, MC4R, NPY2R, β2AR and Kisspeptin are examples of how the same GPCR at the cilium can result in one outcome while a different response is triggered when the same receptor operates from the plasma membrane. How the primary cilium can achieve such feats is still a mystery. One hypothesis would look to the cilium as a highly compartmentalised organelle. Perhaps the complexes that interact with the receptor, such as AKAPs and G-proteins, are distinct at the two locations. Further, the downstream effectors of GPCRs and cAMP at the cilium could be different to effectors localised in the cell body. GPCR in the cilium could also be in closer proximity to downstream functional components and a temporal aspect could be at play whereby cilium elements could be engaged more rapidly than those in the cytosol. Finally, enhanced isolation of the cAMP signal, due to the primary cilium structure, might likewise contribute to the distinct function of these GPCRs. Whatever explanation turns out to be correct, it is likely that compartmentalisation of cAMP at the cilium plays a role.2+ inhibited AC5/AC6  localises to the primary cilium of the epithelial-like pig kidney cell line (LLC-PK) where it couples with Ca AC5/AC6 .  sensor localising to the AKAP150 complex in the primary cilium. In the final section we will discuss the evolution of imaging techniques that have been used to study cAMP signalling in the primary cilium.Another set of proteins has been shown to form a complex with AC5 and AC6 in the primary cilium. Choi et al. found AC5/AC6 to interact with PDE4C, AKAP150, PKA, as well as Polycystin 1 and Polycystin-2 . As aforogenesis . The linEver since “ciliopathies” such as ADPKD were recognised as a severe class of diseases caused by dysfunction of the primary cilium, understanding ciliary signalling, including cAMP signalling, emerged as a priority. However, measuring cAMP signalling within the cilium, or even imaging the organelle, with its inherent reactional movements and transient nature, is challenging. FRET-based reporters for cAMP have been developed by several groups to narrow in on GPCR signalling in the cilium and, while some findings support the notion that the cilium is an independent cAMP subdomain, others serve to reject this hypothesis. The cAMP response to the serotonin receptor, 5-hydroxytryptamine (5-HT6R), was also measured using several different FRET sensors with somewhat contrasting results. In one study, 5HT6 was found to localise to the cilium of primary neuronal cells. Over-expression of the serotonin receptor in the cilium of primary neurons elongated the organelle and increased dendritic branching, with inhibition of the GPCR causing the reverse phenotype. Rescue of 5HT6 in 5HT6R-KO mice was enough to increase cilia length and dendritic expansion, but primarily in neurons where 5HT6 localised exclusively to the cilium rather than also to the plasma membrane . The samin situ (cADDis) reporters  as well as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) and a recent series of elegant experiments used these reagents in combination with FRET imaging to manipulate cAMP production in a more targeted manner.A ground-breaking study carried out by αs -coupled receptors which are strictly activated by cognate designer drugs such as clozapine-N-oxide (CNO). Truong et al., used DREADDs to test their hypothesis that ciliary GPCRs regulate SHh signalling and not plasma membrane receptors. Arl13B was used to localise DREADDs to the cilium and the complex tagged with GFP. Activation of cilia-DREADD with CNO, in the presence of SAG, inhibited Gli1 expression, but no attenuation of Gli1 was observed when cytosolic-DREADD was stimulated in the same fashion in NIH/3T3. These results suggest ciliary activation of GPCRs is responsible for regulation of SHh signalling, although they contradict findings by αs signalling does not control the SHh pathway.DREADDs are designer Gin vitro culture system to emulate the in vivo cyst environment, only photo-activation of ciliary-bPAC, and not photo-activation of cytosolic-bPAC led to cyst formation (The optogenetic approaches described above are powerful tools and promise to provide novel insight that can be exploited for the treatment of ciliopathies. In a recent study, optogenetics was used in combination with RNA sequencing to better characterise the role of ciliary-cAMP signaling in ADPKD. With the aid of ciliary and cytosolic bPACs ormation . (Elbergormation  The grouormation , and PDEormation . Consistormation . Treatmeormation . TogetheFRET imaging using cilia-targeted reporters alongside the application of optogenetic tools provides strong support to the notion that the cilium can handle cAMP independently of the cytosol. Furthermore, many examples have highlighted how ciliary localization of GPCRs leads to a distinct cAMP response compared to stimulation of the same GPCRs when it is localized to the cell body plasma membrane. These findings indicate that the cilium is a vital center for signaling control, and suggest it may be possible to manipulate selectively cAMP within the cilium to treat ciliopathies such as ADPKD with subcellular precision. However, many questions about the regulation and role of ciliary cAMP remain unanswered. For example, how many cAMP signalosomes exist in the primary cilium and how is their activity coordinated? Why do GPCRs localized to the cilium behave differently when localized to other parts of the plasma membrane? What PDEs control ciliary cAMP from diffusing into the cell body, and could PDE isoforms exclusively localized to the cilium be directly targeted for precision medicine? Could ciliary cAMP signaling be altered without disturbing cytosolic cAMP? With the help of current tools and development of future technology, the full characterization of ciliary cAMP signaling will be possible, with the potential of revealing new insights into the many functions of this mysterious organelle.


Human cell-based test methods can be used to evaluate potential hazards of mixtures and products of petroleum refining . Analyses of bioactivity and detailed chemical characterization of petroleum UVCBs were used separately for grouping these substances; a combination of the approaches has not been undertaken. Therefore, we used a case example of representative high production volume categories of petroleum UVCBs, 25 lower olefin substances from low benzene naphtha and resin oils categories, to determine whether existing manufacturing-based category grouping can be supported. We collected two types of data: nontarget ion mobility spectrometry-mass spectrometry of both neat substances and their organic extracts and in vitro bioactivity of the organic extracts in five human cell types: umbilical vein endothelial cells and induced pluripotent stem cell-derived hepatocytes, endothelial cells, neurons, and cardiomyocytes. We found that while similarity in composition and bioactivity can be observed for some substances, existing categories are largely heterogeneous. Strong relationships between composition and bioactivity were observed, and individual constituents that determine these associations were identified. Overall, this study showed a promising approach that combines chemical composition and bioactivity data to better characterize the variability within manufacturing categories of petroleum UVCBs. Regulatory agencies commonly categorize chemicals by the amount that is produced and/or imported into a particular jurisdiction; for example, substances whose aggregate quantities exceed some predefined amount per year are considered “high production volumes.” In the European Union, this would entail >1000 tons, and in the United States, the typical cutoff is >1 million pounds ~500 tons). Such substances receive heightened attention in terms of their hazard and risk evaluations and are typically subject to the most extensive testing requirements . While m00 tons. For regulatory registration and safety evaluation, petroleum UVCBs are grouped into categories of similar materials based on rather broad considerations about their composition and manufacturing methods . It is aTo overcome the challenges of grouping and read-across of petroleum UVCBs, two approaches have been recently proposed and tested. In the first approach, human cell-based in vitro studies have been conducted on a large number of substances and categories. These studies tested the hypothesis that in vitro biological activity signatures, both phenotypic and gene expression, can be used to support the grouping of UVCBs . As manyIndeed, efforts to provide more detailed compositional characterization constitute a second approach to refining the current read-across of petroleum UVCBs. While there are many analytical methods that have been used to characterize the composition of these substances , they arWhile both bioactivity and detailed chemical analyses have been used separately to evaluate similarity in petroleum UVCBs, a combination of the approaches has not been undertaken. Inclusion of PAC and other physio-chemical properties together with cell-based bioactivity did show advantages to data interpretation ,16,17; tAll lower olefin substances used in this study (assigned number identifiers) and their respective streams are detailed in Samples were stored at −80 °C until analyzed or otherwise processed. From each substance, an organic extract was prepared using DMSO and cyclohexane, a method that preferentially extracts PAC from petroleum-containing samples, according to the standard American Society for Testing and Materials (ASTM) IP 346 method . BrieflyAll substances were analyzed using an ion mobility spectrometry (IMS) instrument coupled to a quadrupole time-of-flight (QTOF) mass spectrometer (MS) . Neat and extracted samples were prepared for IMS-MS analysis as follows. A glass syringe was first used to add 100 µL of each sample to a glass vial. Substances were then diluted 3ive mode .m/z for each calibrant peak. Calibrated files for neat substances and extracts were then processed using Agilent Mass Profiler software to obtain two separate sets of detected compounds, or “features”, and their abundances in each sample. A library of compounds was then used to match identities to detected features based on m/z and collisional cross section (DTCCSN2) values for each compound  >95% of the constituents of a substance” [Second, the most recent regulatory guidance on substance chemical characterization  details bstance” . Thus, fThird, the composition of the constituents obtained using nontarget IMS-MS was compared to the typical constituents reported in REACH Category Identity Profiles ,49, infom/z compounds at a higher abundance than toluene (C7H8) for substances 83757, 83806, and 83946 were detected in this study. Subcategory II substances are expected to contain toluene, ethylbenzene (C8H10), and xylenes ; these were all detected by IMS-MS in relatively high amounts (though not as high as C9-C10 compounds), although ethylbenzene and xylene isomers could not be distinguished without analytical reference standards. Typical components for subcategory III include toluene, xylene isomers, styrene (C8H8), and naphthalene (C10H8). Naphthalene was the constituent detected by IMS-MS in the highest abundance for both samples belonging to subcategory III (2.4–2.7%), while the other component chemicals were detected at a lesser abundance (~0.05%). Subcategory V has no reported specific constituents apart from being described as “C9+ from o-xylene unit”; sample 83758 fit this description, and C9H8, C9H10, C10H8, C10H10, C10H12, and C18H20 were all listed constituents of highest abundance [Based on IMS-MS data, LBN substances exhibited similar relative abundances of the reported constituents, and little variation was observed between human health subcategories . Subcatebundance .The reported RO constituent list  includedCharacterizing the composition of UVCBs is critical to establishing structural similarity and the applicability domain of a category . Still, In the LBN category, subcategory II exhibited the greatest bioactivity similarity. Some of the substances had lower bioactivity  as compared to others . This result is concordant with the data on chemical composition; samples 84003 and 84070 are two of the least complex LBN substances tested, and they also exhibited few effects in vitro. Similarly, samples 84075 and 83979 were of comparable chemical complexity and elicited similar bioactivity profiles. iPSC-derived neurons and hepatocytes were the most affected cell types across the LBN substances.In the RO category, subcategory I exhibited the most similarity in bioactivity profiles; five out of the six samples assigned to subcategory I demonstrated bioactivity in all cell types tested. The sixth sample (E83955) was bioactive in four out of five cell types, albeit to a lesser extent. These results were also generally concordant with the chemical composition data in Human health evaluations for petroleum UVCBs are typically based on substance grouping using physio-chemical properties and manufacturing processes, followed by an assessment of possible hazards by several constituents. The bioactivity profiling described above  grouped The same analyses were conducted for RO substances . Three sData on PAC content with 3+ aromatic rings is conventional analytical chemistry-based information that is used to judge the potential health hazards of petroleum UVCBs; higher PAC content is assumed to have higher bioactivity ,52. Howeq-value <0.1) with the chemical profiles of both neat and DMSO-extracted samples  How much biological variability is to be expected within and between existing LBN and RO manufacturing categories? Additionally, (4) What constituents are potential drivers of bioactivity in complex petroleum UVCBs?This study is novel because it used new analytical and toxicological approaches to examine both the chemical composition and biological effects of complex petroleum UVCBs. Samples were from two HPV categories, and this study aimed to determine the extent of chemical and bioactivity similarity among substances that have been previously assigned to these categories using physio-chemical properties and manufacturing process information. The main questions of this study were four-fold: (1) To what extent can petroleum UVCBs be characterized using novel analytical methods such as IMS-MS to meet the most recent ECHA advice on substance characterization for read-across ? (2) HowFirst, it was found that in the DMSO extracts (but not in the neat substances) in the RO and LBN manufacturing categories, the sum of constituents present in amounts ≥1% of the overall substance was above the 80% ECHA threshold . This meSecond, broad chemical concordance was observed for substances belonging to the same category; however, considerable variability was observed between substances in the same category and even subcategory. This was likely a result of inherent substance variability or reaction byproduct impurities from manufacturing processes. While compositional variability is to be expected, recent advice from ECHA calls for the characterization of such variability. Not only is there a need to provide compositional characterization of the substances identified by different CAS RN but grouped into a category, but also characterization of the variability of the same product across manufacturing batches and refineries . The anaThird, similarity in bioactivity was observed within the overall LBN and RO categories; however, less concordance was evident within previously proposed HPV human health subcategories. This observation is similar to that from a larger study of other petroleum UVCBs, where 141 substances spanning 6 product categories were tested in 15 human organotypic cell types to investigate substance similarity using both bioactivity signatures  and tranFourth, this study is also informative in terms of the hazard evaluation of petroleum UVCBs. Due to the chemical complexity of petroleum UVCBs, there is no harmonized methodology for their risk assessment; both whole mixture and constituent-based approaches can be used ,59,60. T“at least five independent samples of the substance…from different production batches…as produced by all the registrants” must be included to characterize the variability [This study is not without limitations. The availability of samples, a common challenge in studies of large-volume produced substances, limited our ability to characterize the intra-category and sub-category variability. Even though we tested 25 samples that were representative of two manufacturing categories and multiple sub-categories within them, the desired replication was lacking. Prior studies showed that a single sample per category may not provide adequate information to capture the individual category characteristics . Updatediability . HoweverOur study used one analytical approach to characterize the chemical composition of tested substances; however, products of petroleum refining are highly complex, and both separation, ionization, and detection methods may affect the molecules that are identifiable using each technique ,19,71. TIn addition, DMSO extraction, a widely used method to enable testing complex petroleum substances ,72, captAnother well-recognized challenge of using in vitro bioactivity for hazard-based evaluations of chemicals is the translation of in vitro results to apical in vivo phenotypes. The complex composition of UVCBs makes it difficult to conduct traditional in vitro-to-in vivo extrapolation from bioactive concentrations to human exposures . It is sOverall, this study demonstrates the benefits of simultaneous assessment of both chemical composition and bioactivity when evaluating the potential hazard properties of petroleum UVCBs. We found that based on the samples analyzed herein, existing categories, based largely on the manufacturing considerations and intended future uses of these products, may be considered heterogeneous in terms of their composition and bioactivity. While additional work is needed to evaluate a larger compendium of substances, including different manufacturing batches of the same substance and testing alternative in vitro delivery methods for these “difficult to test” substances, we conclude that an approach that combines chemical composition and bioactivity data is sensible. These complementary data streams provide information that will enable a more comprehensive and confident characterization of similarities, differences, and variability between and within manufacturing categories of petroleum UVCBs.


The members of the transmembrane emp24 domain-containing protein (TMED) family are summarized in human as four subfamilies, α , β (TMED 2), γ  and δ (TMED 10), with a total of nine members, which are important regulators of intracellular protein transport and are involved in normal embryonic development, as well as in the pathogenic processes of many human diseases. Here we systematically review the composition, structure and function of TMED family members, and describe the progress of TMED family in human diseases, including malignancies , immune responses, diabetes, neurodegenerative diseases, and nonalcoholic fatty liver disease, dilated cardiomyopathy, mucin 1 nephropathy (MKD), and desiccation syndrome (SS). Finally, we discuss and prospect the potential of TMED for disease prognosis prediction and therapeutic targeting, with a view to laying the foundation for therapeutic research based on TMED family causative genes. Transmembrane emp24 domain-containing protein (TMED), also known as p24 protein, is made up of type I one-way transmembrane proteins that are common in the endoplasmic reticulum (ER), Golgi (Golgi), and ER-Golgi intermediate compartments of the early secretory route and found in all eukaryotes (ERGIC)Encapsulated vesicles and ancillary proteins mediate protein transport in the secretory pathwayA family of membrane proteins that are abundant in COPI- and COPII-coated vesicles as well as in ER and cis-Golgi membranes shuttles continually between organelles at the center of the early secretory pathwayAll members of the TMED family, while having minimal sequence homology, have the same structural arrangement, which consists of a brief cytoplasmic area at the C-terminus, a flanking region, a coiled-coil domain, a single transmembrane domain, and an N-terminal Golgi-dynamics (GOLD) domainTMED proteins dimerize and interact with COP protein complexes during cis- and retrograde transport to promote cargo choice and vesicle formation. The GOLD structural domain, assumed to be critical for molecular recognitionA conserved COP binding motif in the cytoplasmic tail is used to interact with COPI and COPIIMembers of the TMED family are involved in the synthesis of COPI vesicles43.Different mammalian p24/TMEDs are involved in different signaling pathwaysMembers of the p24/TMED protein family are expressed during organ development, with extensive co-expression of the same or different subfamilies of p24/TMED proteins; these proteins are involved in early embryonic development, insulin biosynthesis and subsequent pancreatic β-cell secretion, and amyloid precursor protein metabolismDiseases like cancer can result from abnormalities in the secretory pathway's modulation of protein transport. Particularly, deregulation of the immune system, cancer, liver disease, and pancreatic illness have all been associated with aberrant TMED protein expression. Although the symptoms of various illnesses vary greatly, they all emphasize the need of maintaining stable intracellular levels of TMED proteins. It is difficult to research the family since both too much and too little intracellular TMED might result in similar phenotypes.Current studies of TMED proteins suggest a function in cell proliferation and differentiation, specifically in malignant tumors. However, our knowledge of how changing TMED levels contribute to the majority of malignancies is still in its infancy. Determining how each TMED protein contributes will be crucial in understanding how many malignancies can form and advance while being promoted or inhibited by TMED proteins.Head and neck squamous cell carcinoma (HNSC) is the most frequent malignant tumor of the head and neck, occurring largely in the mucosal epithelium of the mouth, pharynx, and larynxResearchers' focus has been drawn to the significance of TMED family genes in the development of lung cancer. TMED proteins have been implicated in the regulation of proliferation and inflammation in lung adenocarcinomaIn vitro tests revealed that TMED3 inhibition has anti-tumor effects in naked mice. Overall, TMED3 stimulates the growth of NSCLC and improves Wnt/-catenin signaling through AKT regulation. The anti-tumor effects of TMED3 inhibition were both strong in vitro and in vivo, and it may be a promising therapeutic target for NSCLC in vitro was prevented by EZR knockdown, and TMED3 overexpression's ability to promote LUSC was lessened. Hence, via controlling EZR, TMED3 aids in the formation and progression of lung squamous cell carcinoma Feng et al. found that TMED2 knockdown increased apoptosis, inhibited tumor cell proliferation, reduced tumor volume, and decreased tumor biomarker and inflammatory factor levels via TLR4/NF-κB signaling, thereby significantly inhibiting the development of lung adenocarcinoma. Thus, TMED2 may regulate the inflammatory response of lung adenocarcinoma through TLR4/NF-κB signaling and promote the proliferation, development, and prognosis of lung adenocarcinoma by regulating the inflammatory response, providing a new strategy for treatment of lung adenocarcinomaBreast cancer is a common malignant tumor in women, with the continuous progress of breast cancer treatment, the survival rate has been greatly improved, but the tumor progression and metastasis are still not optimistic, there have been studies focusing on the role of the TMED family of genes in the process of breast cancer development and provide new biological reference indexes for prognostic assessment of breast cancer and the selection of targeted therapies.in vitro experiments revealed that High TMED9 expression in breast cancer patients encouraged the growth and migration of breast cancer cells and predicted a poor prognosisZhang et al. showed that TMED3 was much more expressed on the mRNA and protein levels in breast cancer tissues and cell lines as compared to healthy controls. TMED3 expression levels were substantially linked with clinicopathological characteristics in breast cancer patients and predicted a poor outcome. TMED3 knockdown prevented the proliferation, migration, invasion, and cell cycle advancement that TMED3 overexpression induced in breast cancer cell lines as compared to controls. TMED3 has also been demonstrated to facilitate breast cancer cell migration and proliferation through Wnt/-catenin signalingDifferential expression of TMED family genes in relation to the outcome for individuals with endometrial, and cervical cancers has attracted equal attention from researchers. Findings on the progression and prognostic function of different TMED family members in different female genital malignancies has provided a basis for exploring novel therapeutic targets.One study discovered that ovarian cancer patients had higher TMED2 levels, and that ovarian cancer cells that overexpressed TMED2 had greater cell migration and proliferation. These findings imply that TMED2 may have a role in the development of ovarian cancerin vivo tests supported the finding that TMED3 knockdown inhibits tumor growth. Through the upregulation of pro-apoptotic proteins and the targeting of the PI3K/AKT signaling pathway, TMED3 deletion may decrease cell viability, according to an investigation of the molecular pathways. The malignant phenotype of EC cells may be impacted by TMED3 knockdown, which could slow tumor growth and provide information for the creation of targeted drugs for the treatment of ECThe most common gynecological cancer in developed nations, endometrial cancer (EC), is growing every yearLatest evidence has shown that TMED5 promotes tumors by activating Wnt7b/-catenin signaling during the aggressive development of cervical cancerIn a multi-omics bioinformatics analysis, TMED2 was found to be a common diagnostic and prognostic biomarker in patients with different clinicopathological features of esophageal adenocarcinoma. It has been hypothesized that TMED2 promoter hypermethylation is closely associated with its overexpression in esophageal adenocarcinomain vitro studies have shown that serum leptin can induce proliferation of gastric carcinoma cells. However, to date, no in vivo tumorigenic effects of leptin on the stomach have been reported. Thus, the in vivo leptin-induced gastric carcinoma experiment designed by Isyraqiah et al. in rats, which showed upregulation of numerous potential driver genes such as TMED2, is of interestThe third most common cancer that leads to death worldwide is gastric carcinoma. It remains unclear why obese individuals have a higher chance of acquiring gastric carcinoma. In obesity, serum leptin levels are high and Previously, it was discovered that TMED3 inhibits metastasis in human colorectal cancer (CRC) cells via way of the WNT-TCF pathwayTMED2 is the only member of the mammalian β subfamily that shows cell type selectivity in cancerin vitro significantly affected cell migration, invasion, proliferation, and colony-forming abilityTMED9 levels in HCC tissues were found to be enhanced by proteomic analysis. In HCC tissues as opposed to healthy liver tissues or precancerous lesions, TMED9 mRNA and protein levels were greater. In individuals with HCC, TMED9 mRNA expression levels were substantially correlated with advanced stage and poor prognosis. Moreover, vascular invasion and disease-free survival were favorably linked with TMED9 protein expression levels. Manipulation of TMED9 expression in HCC cells Prostate cancer is a common malignancy in men. By analyzing 498 cases of differentially expressed prostate cancer genes in TCGA, Chen et al. found that TMED2 and TMED10 were downregulated in prostate cancer tissues and were biomarkers of good prognosis, as their high expression was linked to good OS in prostate cancer patients, revealing the potential role of TMED2 and TMED10 in prostate development, prognosis, and possible treatmentTMED3 differential expression was found between clear cell renal cell carcinoma (RCC)patients with low stage (I and II) and high stage (III and IV) in the TCGA and ICGC cohorts, as well as between RCC patients with low stage (I and II) and high stage (III and IV) in the TCGA cohort. TMED3 overexpression has been linked to a poor prognosis in RCC patients. TMED3 was further substantiated as a potential prognostic marker for RCC by Kaplan-Meier survival analysis, multivariate analysis, and time-dependent area under the curve (AUC) of the Uno c indexIn vitro studies revealed that TMED3 knockdown prevented the growth of osteosarcoma by reducing proliferation and migration and increasing apoptosis, whereas TMED3 knockdown prevented the formation of osteosarcoma in vivo. Furthermore, protein S15A (RPS15A) was discovered to be a downstream target of TMED3 that contributes to the development of osteosarcoma. Further research revealed that the inhibitory effect on osteosarcoma cells was enhanced by simultaneous knockdown of RPS15A and TMED3. Notably, the pro-moter impact of TMED3 overexpression in osteosarcoma cells was reduced by downregulating RPS15A. These results demonstrate the critical role of the TMED3/RPS15A axis in tumor growth and suggest a potential molecular target for osteosarcoma treatmentOsteosarcoma is a primary cancer that primarily affects children and young people. Xu et al. found that TMED3 expression was significantly higher in osteosarcoma tissues than in matched adjacent normal tissues. As the cargo and structural proteins between endoplasmic reticulum and Golgi apparatus, the abnormalities of TMED family genes will directly affect the process of protein transport and related pathways, which will lead to the development of malignant tumors. According to the previous literature, we have found that TMED family proteins participate in several classic tumor pathways, such as Wnt/-catenin pathway, PI3K/AKT signaling pathway, WNT-TCF pathway, and so on. The study of TMED family genes can provide us with new perspectives on the mechanism of tumor research, and lay the foundation for clinical translation and even targeted therapy research.Although it is well known that proteins with GOLD domains are involved in vesicular transport, a number of recent articles have proposed new functions for these proteins. Emerging data on the potential function of TMED family members in innate immune modulation is particularly intriguing.in vivo investigations have demonstrated that TMED1 is involved in IL-33 signalingVia influencing the translocation of ST2 receptors (members of the Toll-like/IL-1 receptor superfamily) to the cell membrane, TMED2 is required for the cellular interferon (IFN) response to viral DNA varies depending on MITA . plays a critical role in the innate immune response to cell-soluble viral double-stranded DNA. Assembly of COPII facilitates MITA translocation; TMED2 regulates MITA signaling and enhances the anti-DNA virus immune response in a number of ways, including enhanced MITA dimerization, translocation to the ER, and transport from the ER to the perinuclear region. Inhibition or deletion of TMED2 in cells significantly increases herpes simplex virus 1 (HSV-1) titer volume and results in impaired IFN-1 production upon HSV-1 infectionTMED7, a member of the γ subfamily, is important in the negative control of Toll-like receptor (TLR) 4 signaling, a receptor for the bacterial product endotoxin, which is regulated by sending signals, biogenesis, and transportation. TMED7 is necessary for TLR4 to form a stable complex with its outer structural domain during its translocation from the ER to the cell surface via the Golgi. TLR4 is involved in activating many downstream intracellular pathways, including the NF-κB pathway in response to cellular stressIn summary, these studies suggest that further analysis of the role of the TMED family in regulating the IL1R/TLR family may provide new insights into the control of innate immunity. The therapeutic implications of these data may lie in the control of clinical inflammatory immunity and improvement of vaccine adjuvants.TMED6 participates in protein transport and secretion and is specifically expressed in pancreatic islets. Thus, TMED6 has a potential role in the control of insulin vesiclesFinally, five new single nucleotide polymorphisms (SNPs) were identified using clinical and genetic risk factors to predict the incidence of type 2 diabetes in Koreans. One of the corresponding genes, SNP rs11057302, is located on an intron of TMED2, suggesting TMED2 may be involved in the involvement of type 2 diabetesIn conclusion, the above studies suggest that TMED family members TMED6 and TMED10 may play an important role in the development of diabetes mellitus, for the TMED family in the mechanism of diabetes mellitus in the future may bring new opportunities for the treatment of diabetes mellitus, in addition to the TMED2 may be a potential target for the study of diabetes mellitus, which deserves our attention.Amyloid β (Aβ) peptides accumulate extracellularly in the brains of people with Alzheimer's disease. The cleavage of the amyloid precursor protein by -secretase results in the production of Aβ, which impairs neurological function. TMED10 was previously known as a γ-secretase regulatory protein, and although TMED10 is expressed throughout the brain, it is most highly expressed in the hippocampus, an area of the brain essential for learning and memory and where AD pathology is typically observed. Interestingly, TMED10 levels are highest during embryonic development and decline with age. In humans, total TMED10 levels are lower in the frontal cortex and hippocampus of patients with Alzheimer's diseaseRecent research has linked abnormal post-translational modifications (PTMs) to the pathophysiology of schizophrenia. A sophisticated class of glycolipids known as GPI binds glycoproteins and surface proteins to the cell membrane. One of the most frequent PTMs is the binding of GPI to proteins, and GPI-associated proteins (GPI-APs) support a variety of cell surface activities, including the formation and maintenance of synapses. Mutations in GPI processing pathways are linked to intellectual disability, suggesting a possible involvement for GPI-APs in cognition and cognitive dysfunction in schizophrenia. TMED10 specifically recognizes and is essential for quality control of GPI-APs. Reduced TMED10 protein expression in schizophrenic patients suggests abnormal regulation of GPI-AP output by the ERTaken together, the available literature suggests that only TMED10 and TMED9 of the TMED family seem to be closely associated with neurodegenerative diseases, which is worthy of consideration as to whether it suggests that our research in the TMED family of genes in neurodegenerative diseases should be focused on these two genes.TMED2 is required for normal hepatocyte proliferationTMED2 may therefore contribute to the emergence of non-alcoholic fatty liver diseaseA Pakistani study identified a deleterious variant of TMED4 that resulted in gene overexpression in myocardial tissue of patients with dilated cardiomyopathy, suggesting a possible involvement of TMED4 in myocardial dilatationA shift mutation (MUC1-fs) in the MUC1 gene, which is therefore trapped in vesicles harboring cargo receptors in the TMED9-mediated early secretory pathway, causes Mucin 1 nephropathy (MKD), a neutrophil protein disease. When the tiny molecule BRD4780 attaches to TMED9, it releases MUC1-fs and sends it on its way to be degraded by lysosomes. By promoting lysosomal degradation and targeting TMED9, this small molecule reverses proteinopathies and demonstrates the potential therapeutic value of TMED9 in proteinopathies Disturbances in multiple signaling pathways involved in salivary secretion can cause xerostomia linked with Sjögren syndrome (SS), which is a complex condition. Patients with SS xerostomia have considerably lower levels of TMED10, PDIA4, and CanX gene expression compared to healthy controls, but not statistically different from SS non-xerostomia patients, demonstrating that TMED10 gene expression is reduced in xerostomia patients by a different mechanism than in Alzheimer's disease patients and that SS-associated xerostomia is not caused by increased autophagy activationMembers of the TMED family are type I transmembrane proteins that play important roles in Golgi-ER bidirectional transport. Recent studies of its members have shown that variations in TMED genes can lead to developmental abnormalities and disease progression in humans. Although targeted therapeutic agents for the TMED family have not yet been developed, studies suggest that members of the TMED family may provide a reference for prognostic prediction and therapeutic targets, laying the foundation for further translational medicine research.


Background: Electrospun fibers are widely studied in regenerative medicine for their ability to mimic the extracellular matrix (ECM) and provide mechanical support. In vitro studies indicated that cell adhesion and migration is superior on smooth poly(L-lactic acid) (PLLA) electrospun scaffolds and porous scaffolds once biofunctionalized with collagen. Methods: The in vivo performance of PLLA scaffolds with modified topology and collagen biofunctionalization in full-thickness mouse wounds was assessed by cellular infiltration, wound closure and re-epithelialization and ECM deposition. Results: Early indications suggested unmodified, smooth PLLA scaffolds perform poorly, with limited cellular infiltration and matrix deposition around the scaffold, the largest wound area, a significantly larger panniculus gape, and lowest re-epithelialization; however, by day 14, no significant differences were observed. Collagen biofunctionalization may improve healing, as collagen-functionalized smooth scaffolds were smallest overall, and collagen-functionalized porous scaffolds were smaller than non-functionalized porous scaffolds; the highest re-epithelialization was observed in wounds treated with collagen-functionalized scaffolds. Conclusion: Our results suggest that limited incorporation of smooth PLLA scaffolds into the healing wound occurs, and that altering surface topology, particularly by utilizing collagen biofunctionalization, may improve healing. The differing performance of the unmodified scaffolds in the in vitro versus in vivo studies demonstrates the importance of preclinical testing. Steadily turning into a major public health concern, the global skin and wound care market is expected to grow from USD 18.49 billion in 2017 to USD 25.98 billion by 2025 . HoweverCollagen is the key component of the ECM of several tissues including the skin. During the process of wound healing, collagen not only provides mechanical support for tissue repair, but influences the synthesis of other ECM proteins and plays a vital role in regulating the inflammatory response via the release of inflammatory cytokines as well as growth factors that are involved in the remodelling of the ECM ,16. CellWhile pure collagen-based scaffolds face the major limitations of a faster degradation rate and poor mechanical properties, blending collagen with other polymers  such as polyethylene oxide (PEO), PCL, poly (L-lactic acid) (PLLA), PVA, hyaluronic acid (HA), elastin (EL) and silk fibroin (SF) has significantly improved its mechanical properties and has been widely used in skin tissue engineering studies ,28,29,30In this study, we investigated how our different electrospun PLLA fiber scaffolds integrate into wounds and act to support healing in vivo using a mouse full-thickness excisional wound model. We hypothesized that application of these scaffolds could dramatically improve the healing of deep skin wounds and assessed not only the integration and infiltration of cells into the scaffold, but also their effects on wound closure, re-epithelialization and extracellular matrix deposition.w/v of PLLA solution using magnetic stirring for 4 h; this was followed by 4 h rest at room temperature (RT). The electrospinning process was performed at RT, a flow rate of 0.5 mL/h and an applied voltage of 15 kV. A grounded plate placed 12 cm from the tip of the needle was used as a collector. Porous fibers were fabricated using 11.5% PLLA solution with a binary solvent system of chloroform: dimethyl formamide (9:1) and stirred for 24 h. The solution was further allowed to rest for another 4 h before electrospinning. Electrospinning was performed at a potential difference of 15 kV with a flow rate of 2 mL/h. The grounded plate was placed 12 cm from the tip of the needle and was used as the collector. The air humidity and temperature conditions during the spinning process were about 40–50% and 25 °C, respectively. The mat from the collector was detached, dried to remove any residual solvents, and stored for further characterization studies.PLLA scaffolds were fabricated by an electrospinner  with PLLA,  . Smooth Smooth- or porous-surfaced polymeric scaffolds were subsequently functionalized by a simple drop casting method with 0.1% collagen type I , as described in Aswathy et al., 2020 . For colvol/vol). Full-thickness excisional wounds were created on the midline (wounds at 1.5 cm from the base of the skull and 0.5 cm either side of the midline) using a 10 mm diameter sterile biopsy punch . UV-sterilized circular scaffolds of 10 mm were inserted into the wound to act as dermal scaffolds within the full-thickness excisional wound; these were held in place by Tegaderm dressings that had an additional square of Tegaderm the size of scaffold (10 mm) placed over the sticky side to prevent the Tegaderm sticking to the scaffold itself whilst providing covering and protection of the scaffold within the healing wound  following the Australian Code of Practice for the Care and the Use of Animals for Scientific Purposes. Power studies showed that a sample size of 6  would give 80% power using a 5% test level and a one-tailed test. Male and female BALB/c mice of 10 weeks old were procured from Animal Resources Centre, Perth, Australia. Following acclimatization for 5 days, mice were administered pre-operative analgesia (Buprenorphine 0.05 mg/kg) via subcutaneous (SC) injection 30 min prior to wounding. Anaesthesia was induced by isoflurane inhalation, and the dorsal side of mice was prepared by removing hair with an electric shaver followed by Veet depilatory cream; the area was then cleansed with sterile water and ethanol  was used to measure the area and diameter across the midline of the wounds, which were calibrated against the 1 mm graduations of the ruler that was included in the frame of each image. Healing at day 7 and 14 calculated by normalization to the day 0 wound using the following formula: 2/h.High resolution images of the healed wound on day 14 were also obtained using the DermaScope unit of the DermaLab Combo (Cortex Technology) following the manufacturer’s instructions. Erythema within the wound area was also assessed using skin reflectance spectroscopy by the DermaLab Combo to determine the redness of vascularized or inflamed skin. To obtain the erythema measurement, the instrument was calibrated and the reading taken directly above the wound to obtain an open-ended index in CIELab colour values and normalized to an area of unwounded dorsal skin for each mouse. Transepidermal water loss (TEWL) as an indicator of restored skin barrier function was measured for 8 s per site using the Vapometer evaporimeter , following the manufacturer’s guidelines. Using an 11 mm adaptor, the Vapometer was placed directly onto the wounded or adjacent unwounded dorsal skin, and in the wound to obtain the TEWL in g/mThe entire wound at day 7 or 14, including the scaffold and adjacent normal skin, was excised from the back of the mice and fixed in a formaldehyde-buffered solution  and embedded in paraffin wax for histological analysis. Histological sections of 4 μm were stained with haematoxylin and eosin (H&E) or Masson trichrome using previously described methods , and imaInfiltration of cells into the scaffold within the wound was represented as a score from 0 to 5 (none to high) based on H&E staining and visualization of blue/purple nuclei within the scaffold. The images were then analyzed using cellSENS Microscope Imaging Software (Olympus) to quantify the number of cells per area by counting the nuclei within the scaffold. Matrix deposition was also given a score from 0 to 5 by monitoring the density of the extracellular matrix within the wound in each test group from H&E images. For quantitative morphometric analysis of collagen deposition, RGB images of Masson Trichrome-stained section were analyzed in Image J software  using a macro written by Kennedy et al., 2006 , wherebyp < 0.05 is considered statistically significant. All statistical analysis was performed using GraphPad Prism version 8.0 All data are displayed as mean ± standard error of mean (SEM). The one-way analysis of variance test was used to assess the statistical comparisons. A value of The morphology of smooth and porous PLLA electrospun fibers before and after collagen modification and subsequent coculture with keratinocytes and fibroblasts was assessed by SEM a. UnmodiWounds were imaged on the day of surgery prior to scaffold application (day 0) and again on days 7 and 14 post-surgery a. No sigQuantification of the macroscopic wound area a and diaThe histological evaluation of wound healing was subsequently performed on H&E-stained tissue sections from wounds of mice treated with all four PLLA-based scaffolds at day 7 and 14 a. Wound The trends observed suggest that collagen coating impacts healing potential regardless of the surface topography of the original scaffold, which agrees with previous observations that synthetic scaffolds have limited cellular infiltration in their native form without modification . FurtherMasson’s Trichome staining, wherein collagen fibers, the primary ECM component,  are staiIn this study, electrospun PLLA scaffolds of different topologies (porous and smooth) were fabricated and functionalized with one of the major ECM proteins, collagen type I, and employed as wound dressings in mouse excisional wounds. Our previous in vitro studies showed that collagen modification enhanced major cellular activities including cell adhesion, spreading, proliferation and migration when skin cells were grown on the scaffolds, suggesting that collagen-modified electrospun scaffolds had the potential be used as promising wound-dressing materials to support a variety of cells and accelerate wound healing. Early indications confirmed that unmodified, smooth PLLA scaffolds may perform the worst in vivo, with these wounds having consistent trends towards delayed healing, appearing to be the largest in area, having the greatest wound width (including a significantly larger panniculus gape), and the lowest re-epithelialization. By day 14 however, no significant differences in healing were observed between the scaffolds. Nevertheless, wounds treated with the unmodified, smooth PLLA scaffolds still appear to have the least functional healing, with the lowest collagen deposition within the wound site. Taken together, the trends we have observed suggest that the limited incorporation of smooth PLLA scaffolds into the healing wound may be overcome by altering surface topology or biofunctionalization, and that in general, collagen biofunctionalization of PLLA scaffolds with either smooth or porous topology offers the greatest improvement to healing, with the collagen-functionalized smooth surfaces having a slightly decreased wound area and width compared to all other scaffolds, and collagen-functionalized porous scaffolds showing a decreased wound area and width when compared to the non-functionalized porous scaffold. Similarly, re-epithelialization at 14 days post-wounding was also highest in wounds treated with collagen-functionalized scaffolds. This trend towards faster healing in wounds in which scaffolds are modified prior to application may present some clinical advantages, such as protection from infection, due to a faster re-establishment of barrier function and reduction in the overall area; however, as no significant differences in the healing outcome of these uninfected wounds were observed, alternate avenues for improving the ability of electrospun scaffolds to enhance wound healing are required. Moreover, the opposing performance of the unmodified scaffolds in the in vitro versus this in vivo study demonstrates the importance of preclinical testing where unexpected results can occur.


Roxadustat is a novel agent with a distinct mechanism of action compared to erythropoiesis-stimulating agents (ESAs) and a potentially different combination of effects on iron parameters. This narrative review describes the effects of roxadustat on iron parameters and on hemoglobin levels in the context of iron supplementation in patients with anemia of non-dialysis-dependent (NDD) or dialysis-dependent (DD) chronic kidney disease (CKD). Roxadustat use was associated with a greater reduction in serum ferritin levels than seen with ESAs and an increase in serum iron levels compared to a decrease with ESAs. Decreases in transferrin saturation in patients treated with roxadustat were relatively small and, in the case of patients with NDD CKD, not observed by Week 52. These changes reflect the concomitant increases in both serum iron and total iron-binding capacity. Compared to placebo and an ESA, roxadustat improved iron availability and increased erythropoiesis while requiring less intravenous iron use. Hepcidin levels generally decreased in patients who received roxadustat compared to baseline values in all CKD populations; these decreases appear to be more robust with roxadustat than with an ESA or placebo. The mechanisms behind the effects of roxadustat and ESAs on iron availability and stores and erythropoiesis appear to differ and should be considered holistically when treating anemia of CKD. Hypoxia-inducible factor (HIF) prolyl hydroxylase (PH) inhibitors represent a novel class of orally active small molecule drugs that transiently inhibit the HIF-PH enzymes, which causes the cellular accumulation of HIFs and induces activation of the genes involved in erythropoiesis, even in the presence of normal oxygen tension  . HIF-PH Roxadustat has been evaluated for the treatment of anemia of chronic kidney disease (CKD) in sixteen phase 3 studies, including six studies in Japan ,6,7,8,9,Product labeling advises that patients should be iron-replete when roxadustat is initiated , a recomRoxadustat is a novel agent with a unique mechanism of action compared to ESAs and a different combination of effects on iron parameters . This naAnemia is a common complication of CKD, with a prevalence that increases with CKD severity and may be >50% in advanced stages of CKD ,24. MultIron is absorbed in the duodenum and bound to serum transferrin. Subsequently, iron is transported to the liver and spleen to be bound to ferritin for storage or to the bone marrow to be used in red blood cell production. Additionally, the iron regulatory hormone hepcidin regulates iron uptake from the gastrointestinal tract and release from macrophage and hepatocyte stores where iron is complexed with ferritin . FerropoErythropoietin production is stimulated by hypoxia through gene expression induced by HIFs ,30. At nBeginning in 1997, the Kidney Disease Outcomes Quality Initiative recommended that serum ferritin and TSAT be the primary tools for assessing iron management in patients with anemia of CKD . A TSAT While the Kidney Disease: Improving Global Outcomes (KDIGO) guidelines and European Renal Best Practices (ERBP) statement for when a trial of intravenous (IV) iron may be suggested are similar , the decision in an individual patient may be affected by a variety of factors ,28,29,33Hepcidin is the master regulator of systemic iron metabolism . Hepcidip = 0.208), whereas with DA, a significant increase in dosing was observed with decreasing ferritin (p < 0.001) [Serum ferritin levels were evaluated in most of the clinical studies with roxadustat. Mean serum ferritin values decreased in patients with NDD and DD CKD randomized to receive roxadustat ,17,18,19< 0.001) . In a si< 0.001) .Regional differences in baseline serum iron values were observed  due to international differences in the standard of care ,17,18,19In patients treated with roxadustat, TIBC increased absolutely and relative to placebo and ESA comparators in the NDD and DD CKD populations ,17,18,19In the pooled analysis performed by Pergola et al., the adjusted least squares mean (LSM) change in hemoglobin levels from baseline averaged over Weeks 28–52 was greater with roxadustat versus placebo in the NDD CKD population , with a less-pronounced difference in the DD CKD population when compared to ESA  . This fiAdditionally, the correction and maintenance of serum hemoglobin is affected by iron supplementation. To increase hemoglobin 1 g/dL, approximately 170 mg of iron is required based on a total blood volume of 4.9 L in a 70 kg man . Across In general, hepcidin levels decreased in patients who received roxadustat compared to baseline values in all CKD populations ,17,18,19The rate of decline in ferritin levels has previously been shown to correspond to the extent of erythropoiesis stimulation, reflecting the shift of iron from stores into erythrocyte hemoglobin  Figure . RoxadusWhile the decreases in TSAT in patients treated with roxadustat were relatively small and, in the case of patients with NDD CKD, not observed by Week 52, these changes reflect the concomitant increases in both serum iron and TIBC. In particular, TIBC rose more quickly than serum iron, which remained generally stable through Week 8 in patients with NDD CKD, suggesting that a relative iron deficiency does not commonly develop in patients receiving roxadustat because of iron being mobilized and utilized following roxadustat initiation . ObserveIncreased erythropoiesis exerts negative feedback on hepcidin synthesis, but hepcidin is not a direct target of HIF stabilization. Nevertheless, roxadustat does appear to have some additional direct and/or indirect effects on hepcidin beyond those seen with ESAs . A potenThe effects from oral and IV iron supplementation and blood transfusions throughout the studies must be considered. While these findings may suggest improved iron availability and increased erythropoiesis while requiring less IV iron use with roxadustat versus comparators and are consistent with the mechanism of action for roxadustat, they should be interpreted cautiously. Most notably, in the ROCKIES study, IV iron was restricted to rescue therapy in the roxadustat group, whereas IV iron was administered per local guidelines in the ESA arm . HoweverRoxadustat’s mechanism of action on erythropoiesis differs from that of ESAs, resulting in unique and potentially beneficial effects on iron stores and availability. These differences should be considered holistically while treating patients with anemia of NDD and DD CKD. The use of supplemental IV iron is generally decreased in patients receiving roxadustat, likely due to an increased promotion of iron availability and utilization. This finding should not preclude clinicians from following best practices for monitoring of iron parameters or providing iron supplementation. However, this reduced use of IV iron, particularly in the NDD CKD population, may spare veins that could be viable vascular access sites in the future if the patient progresses to requiring dialysis. The outcomes in the average iron-deplete patients receiving roxadustat are encouraging; however, current product labeling and expert opinion advise that patients should be iron-replete when roxadustat is initiated and continued . AdditioLess IV iron supplementation is generally used in patients with NDD CKD treated with roxadustat, an oral medication, than in patients who received placebo, and in patients with DD CKD treated with roxadustat than in patients treated with an ESA, while achieving noninferior (vs. ESA) and superior (vs. placebo) effects on hemoglobin change from baseline. The distinctive effects of roxadustat on iron physiology were generally similar in direction though not necessarily in the extent of change in patients with NDD and DD CKD  and when compared to ESAs may decrease the use of iron to support erythropoiesis. The extent to which factors such as iron supplementation and study drug dose affected iron parameters requires further investigation.


This review discusses chromatin-based regulation mechanisms as determined in the fungal plant pathogen Verticillium dahliae and relates the importance of epigenetic transcriptional regulation and other nuclear processes more broadly in fungal plant pathogens.Through the association of protein complexes to DNA, the eukaryotic nuclear genome is broadly organized into open euchromatin that is accessible for enzymes acting on DNA and condensed heterochromatin that is inaccessible. Chemical and physical alterations to chromatin may impact its organization and functionality and are therefore important regulators of nuclear processes. Studies in various fungal plant pathogens have uncovered an association between chromatin organization and expression of in planta Arguably, one of the most important transition towards eukaryotic evolution has been cell compartmentalization, which allowed physical separation of diverse cellular processes . One of Regulation of DNA-templated processes involves the histone code, made up of posttranslational chemical modifications to DNA-interacting histone proteins that help regulate genome functionality ,3. EukarNeurospora crassa . Ad. AdV. datromeres . The crutromeres . However and 5mC ,157, sugfunction ,157. TheIn this review, we have highlighted the importance of epigenetics in the regulation of nuclear processes in fungal plant pathogens. Moreover, we describe experimental evidence that genomic regions containing effector genes are characterized by the presence of heterochromatic features, but it remains to be seen if in planta transcriptional activation requires chromatin de-condensation or epigenome remodeling. As we show that differential gene expression in vitro for only a subset of genes located in H3K27me3 domains involves local H3K27me3 depletion, we postulate that gene expression in planta may display local H3K27me3 depletion, but that it is not strictly required. To demonstrate this, future studies will require a functional and reliable procedure to perform in planta chromatin immunoprecipitation assays or other types of nuclear capture or single-cell interrogation.Histone proteins are not only found in eukaryotes, but are also common in archaea ,159, ind


Circulating vaccine-derived poliovirus (cVDPV) outbreaks can occur when oral poliovirus vaccine strains  undergo prolonged circulation in undervaccinated populations, resulting in genetic reversion to neurovirulence. A novel type 2 oral poliovirus vaccine (nOPV2) has been developed, which has been shown in clinical trials to be less likely than mOPV2 to revert to paralytic variants and to have limited genetic modifications in initial field use  samples were detected in six countries, all in Africa: Burundi, Central African Republic (CAR), Democratic Republic of the Congo (DRC), Nigeria, Tanzania, and Zambia . The iso*Circulation of four of the seven cVDPV2 emergences derived from nOPV2 use has been detected only in subnational areas of the countries in which they originated , two have spread to other areas of the originating country (CAR and DRC), and one has spread from the originating country (DRC) to neighboring countries . Three emergences spreading within or outside of the originating country  might be ongoing, with most recent detections in July 2023. The largest and widest-spreading emergence, RDC-SKV-1, was first detected in DRC’s South Kivu province in September 2022; viruses from this emergence have been identified in six provinces of DRC and in neighboring Burundi, Tanzania, and Zambia. RDC-SKV-1 has been detected as recently as July 2023 in Tanzania. The potential for mutation and reversion to neurovirulence is a rare but recognized risk for all live attenuated oral poliovirus vaccines; extensive use of nOPV2 worldwide since March 2021 suggests that reversion occurs less frequently than with mOPV2 (


Several viruses including human herpes viruses (HHVs), human polyomavirus JCV, and human papilloma virus (HPV) have been implicated in brain cancer, albeit inconsistently. Since human leukocyte antigen (HLA) is centrally involved in the human immune response to viruses and has been implicated in brain cancer, we evaluated in silico the immunogenicity between 69 Class I HLA alleles with epitopes of proteins of 9 HHVs, JCV, and HPV with respect to a population-based HLA-brain cancer profile. We found that immunogenicity varied widely across HLA alleles with HLA-C alleles exhibiting the highest immunogenicity, and that immunogenicity scores were negatively associated with the population-based HLA-brain cancer profile, particularly for JCV, HHV6A, HHV5, HHV3, HHV8, and HHV7. Consistent with the role of HLA in foreign antigen elimination, the findings suggest that viruses with proteins of high HLA immunogenicity are eliminated more effectively and, consequently, less likely to cause brain cancer; conversely, the absence of highly immunogenic HLA may allow the viral antigens to persist, contributing to cancer. Still other viruses, particularly other human herpes viruses (HHV) such as HHV1 and HHV2 (herpes simplex viruses), HHV4 (Epstein–Barr virus), and HHV6 (Roseolovirus) have been cited as possibly linked to brain cancers although research is relatively sparse and inconsistent9. Some of the inconsistencies with regard to the associations of viruses with brain cancer might be partially attributable to population11 and individual12 variation in Class I human leukocyte antigen (HLA) genes, an essential component of the human immune response to viruses and other foreign antigens.Investigation into the role of viral infections on brain cancer have supported an influence of several common viruses on brain cancer risk and prognosis. For example, human cytomegalic virus (HHV5), human polyoma JC virus (JCV) and human papilloma virus (HPV) have been widely implicated in brain cancers and have been detected in brain tumors, albeit inconsistently12; consequently, subtle differences in the amino acid residues of the binding groove influence antigen elimination13. Exposure to viral or other foreign antigens in the absence of high affinity HLA-antigen binding or insufficient immunogenicity may result in antigen persistence and downstream deleterious health effects15. Indeed, HLA has been implicated in a wide variety of conditions including cancers31 and, specifically, has been implicated in brain cancer risk and protection39.Class I HLA genes code for cell-surface proteins located on nucleated cells that bind and export small peptides derived from proteolytically degraded cytosolic viruses and other foreign antigens to the cell surface for presentation to CD8+ cytotoxic T cells, thereby signaling cell destruction. The success of this process rests on both the binding affinity of an HLA molecule with a foreign antigen epitope and immunogenicity, or the ability to mount an immune response. HLA is the most highly polymorphic region of the human genome with nearly all of the variability located in the binding groove31. Here, we evaluated HLA-virus immunogenicity with respect to a population-based HLA-brain cancer profile. Specifically, we computed an HLA profile for brain cancer characterized by the covariance between the population frequency of HLA alleles and the population prevalence of brain cancer in 14 Continental Western European countries. Next, we used an in silico approach to evaluate the binding and immunogenicity of viral antigens linked to brain cancer with common HLA Class I alleles and to assess the association between those HLA-antigen epitope immunogenicity scores and the HLA-brain cancer profile.Taken together, separate lines of research have linked several viruses and HLA to brain cancer. We have suggested that HLA moderates the influence of exposure to viruses on long-term health effects including cancer such that HLA-facilitated antigen elimination promotes health and, conversely, the inability to eliminate foreign antigens due to weak binding affinity and/or immunogenicity, contributes to cancer risk–6).The BC-HLA PScov scores are given in Table I score (12.81) and A*03:01 had the lowest score (3.45), a 3.7 × differential. An analysis of variance (ANOVA) was used to evaluate the effect of HLA Gene on the log-transformed immunogenicity score  = 51.9, P < 0.001). It can be seen in Fig. Immunogenicity scores varied appreciably among HLA alleles Fig. . Allele I score 1.81 and AI score 1.81 and AA main objective of this study was to assess in silico the possible association between BC-HLA PScov scores and the HLA-immunogenicity of the proteins of various viruses that have been implicated in brain cancer. Our rationale was that viruses with proteins of high HLA immunogenicity would be eliminated more effectively from the body and the brain, and hence less likely to cause cancer: this hypothesis would predict that BC-HLA PScov scores would be negatively correlated with HLA virus immunogenicity. Indeed, the immunogenicity scores With respect to specific viruses, the key measure was the percent of the BC-HLA PScov scores explained by the virus immunogenicity . The results are shown in Table The total, combined PVE of the 11 viruses was 44.52%, which means that of the BC-HLA PScov variance, 44.52% was accounted for by the immunogenicities of the 11 virus proteins studied  which focus on the epitope-HLA allele binding affinity, which is a necessary but not sufficient step for immunogenicity.Here we employed the iNeo-Epp algorithm40 see “” that usPrevious findings regarding the influence of specific viruses on brain cancer have been somewhat inconclusive. Here, we evaluated the association of 11 viruses and the population risk of brain cancer with respect to Class I HLA, a system that is critically involved in the human immune response to viruses and other foreign antigens. Specifically, we evaluated correspondence between HLA-viral antigen immunogenicity and the brain cancer HLA protection/susceptibility profile based on the premise that variability in HLA binding and immunogenicity with viral antigens influences cancer risk via differential ability to eliminate potentially oncogenic viruses. The findings demonstrated a negative association between brain cancer-HLA protection/susceptibility profile and all 11 HLA-viral antigen immunogenicities, with substantial effects (> 4% PVE) by 6 viruses  and negligible effects (< 1% PVE) by 5 viruses . This suggest that Class I HLA alleles that bind with, and eliminate, the former 6 viruses in particular may protect against brain cancer risk; conversely, in the absence of antigen elimination due to weak binding and/or low immunogenicity, these viruses may be involved in brain cancer risk. Since both BC-HLA and immunogenicity scores refer to the same HLA Class I alleles, and since the potential presence (and associated effects) of the various viruses are not mutually exclusive, the PVE contributions of the 6 viruses above can be added and their sum would be an estimate of how much of the variance in BC-HLA scores could be accounted for by PVE contributions of these viruses. This sum amounts to 44.5%, which leaves 55.5% of the variance of BC-HLA scores to be accounted for. It is reasonable to suppose that this would be the contribution of HLA Class II alleles, involved in the formation of antibodies against those viruses. This hypothesis remains to be investigated. Finally, it should be noted that only negligible contributions were found by HHV1, HHV2, HHV4, HHV6B, and HPV.42. Although not indicative of causality in and of itself, the co-localization of viral components and malignancy provides compelling evidence supporting an association. The case for VZV/HHV3, however, is not quite as clear. The majority of studies investigating VZV/HHV3 in relation to brain cancer have been serological studies in which the presence of VZV antibodies and self-reported history of varicella  have been associated with decreased risk of brain cancer45. Research evaluating the presence of VZV/HHV3 viral proteins in brain tumors is scant42; therefore, additional research is warranted regarding VZV/HHV3 viral presence in relation to brain cancer. Finally, with respect to CMV/HHV5, it is worth noting that the gB glycoprotein we used  they are practically ubiquitous; (2) initial infection typically occurs during childhood; 3) following initial infection, the viruses establish lifelong latency in the host; (4) the latent viruses can be reactivated by various triggers and/or waning immunity; and (5) they are all neurotropic. Moreover, the presence of JCV, HHV5/CMV, and HHV6 DNA and/or proteins have been fairly commonly detected in brain tumorssed see “” is “an gN Table , all of  followin48. These include direct mechanisms such as those affecting genomic instability, rate of cell proliferation, resistance to apoptosis and alteration in DNA repair mechanisms as well as indirect mechanisms including immunosuppression, chronic inflammation, and chronic antigenic stimulation. The mechanisms of carcinogenesis are fairly well characterized for JCV and CMV/HHV5 given the wealth of research on their role in oncogenesis and oncomodulation. CMV appears to indirectly promote oncogenesies via chronic inflammation, interference with signaling pathways, and influencing cell motility and proliferation factors whereas JCV directly promotes oncogenesis via expression of viral T-antigen which inhibits tumor suppression, prevention of DNA repair, and chromosomal alterations49. Considerably less research has evaluated the oncogenic mechanisms of HHV6 and HHV3/VZV. Several potential oncogenic and oncomodulataroy mechanisms have been proposed or preliminarily supported for HHV6 including disruption of apoptosis, cellular transformation, immunomodulatory mechanisms, and transactivation of other viruses50. A role of indirect oncogenic mechanisms including immune evasion and immunosuppression have been implicated for VZV2. Particularly for HHV6 and HHV3/VZV, additional studies are warranted to elucidate the mechanisms that may permit or promote brain cancer.With mounting research supporting a role of viruses in cancer, several viral oncogenic mechanisms have been identified50. The absence of findings for HHV6b and other herpes viruses studied here or HPV do not suggest that they are not implicated in brain cancer. Indeed, HPV has been shown to be associated with brain tumors3 and some evidence has linked HSV-1/HHV1, HHSV-2, and EBV with brain cancer8. Rather, the current findings suggest that their potential roles in brain cancer are likely unrelated to immunogenicity with common Class I HLA alleles. Whether that is potentially due to viral immune escape mechanisms including downregulation of Class I HLA molecules2 or the influence of other mechanisms remains to be seen.Of the 11 viruses studied, the immunogenicity of 4 of the herpes viruses and HPV were unrelated to the brain cancer-HLA protection/susceptibility profile. Interestingly HHV6A immunogenicity was clearly associated with the brain cancer-HLA protection/susceptibility profile but HHV6B was not. Despite high molecular homology, pathomechanisms (including carcinogenic mechanisms) of HHV6A and HHV6B differ52 in which case Class II HLA may be particularly relevant.Here we focused on Class I HLA due to its early immune response to viral infection. However, Class II HLA, which is involved in long-term immunity including antibody production and immunological memory, may also be associated with brain cancer risk and protection. HLA Class I  and HLA Class II  are complementary classes of cell surface proteins that are instrumental in binding and elimination of intracellular and extracellular foreign antigens , respectively. Widespread identification of CMV DNA in the peripheral blood of patients with CMV DNA-positive brain tumors has been suggested to reflect either systemic reactivation or viral shedding from tumor cells to the periphery54 and HHV855 has been reported in the brain. A major issue has to do with the large variety of brain tumors and the reasonable possibility that different viruses might be involved in different tumors, thus making the generalization of virus(es)-BC association difficult.In summary, across the 69 Class I HLA alleles investigated here, the immunogenicity of all 11 viruses studied were negatively associated with the BC-HLA profile such that HLA allele-epitope complexes with high immunogenicity were associated with protective  population brain cancer-HLA risk scores whereas those with low immunogenicity were associated with susceptibility  population brain cancer risk, accounting for 44.52% of BC-HLA variance explained. Of the 11 viruses tested, 6  had a substantial effect on BC-HLA scores, contributing 95% to the BC-HLA variance explained above, whereas the remainder 5 viruses  contributed only 5%. Although the association between brain cancer and JCV, HHV6A, HHV5 and HHV3 has been more investigated, the presence of genomic material from HHV711. Thus, the brain cancer-HLA profile used for analyses here may differ in other countries. Second, it is uncertain to what extent these population-based findings apply to individuals. The HLA region is the most highly polymorphic region of the human genome12. Each individual possesses 12 classical HLA alleles (two of each gene) that determine the repertoire of foreign antigens that can be bound and subsequently eliminated. Here our analyses focused on the association between the frequencies of single HLA alleles and the population prevalence of brain cancer; the combined effects of the 12 alleles carried by an individual with regard to viral immunogenicity and brain cancer occurrence remain to be investigated. Third, the brain cancer-HLA profile was determined here for overall brain cancer risk and may vary by brain cancer subtypes. Finally, we evaluated immunogenicity with respect to representative proteins of the 11 viruses and acknowledge that other proteins may differentially relate to the brain cancer-HLA profile. Despite these limitations, HLA binding and elimination of viral antigens that have been linked to brain cancer is an interesting avenue for continued investigation, particularly in light of ongoing research on viruses as candidates for oncolytic virotherapy58.Our findings speak to the interaction of viral exposure and host immunogenetics on brain cancer and may partially account for some of the conflicting findings linking these particular viruses to brain cancer risk. However, these novel finding must be considered within the context of study limitations. First, it is unclear if the current findings extend to other geographic locations outside of Continental Western Europe. Evolutionary pressure aimed at maximizing geographically-relevant pathogen elimination has shaped the distribution of HLA alleles at the population level59, in keeping with epidemiological evidence that higher population frequencies of HLA alleles found beneficial in ICB therapy are also associated with lower melanoma prevalence in those populations27. The results of this study form the basis of predictions with respect to outcomes of interventions against diseases caused by a virus among those we investigated (e.g. vaccines against HHV5/CMV48) or tumors where viral products have been found (e.g. glioblastoma60). It should be noted that the crucial role of HLA extends to any immunotherapy directed against tumor specific antigens 62. Given this fundamental, permissive role of HLA in enabling cancer immunotherapies, it would be ideal if the requisite HLA molecule for a particular tumor TSA or virus could be present. Indeed, we proposed recently such a procedure, namely the administration of mRNA of specific HLA Class I molecules with high binding affinities to specific TSA(s)63. Such an approach may be particularly useful when coupled with immune checkpoint blockade for glioblastoma64.The main focus of this study is on HLA, the common denominator for the epidemiologically derived BC HLA risk scores and the in silico derived immunogenicity scores for 11 viruses that have been implicated in nonmetastatic brain cancer. The main function of HLA is the elimination of foreign antigens, by killing the infected cell  and/or neutralizing the offending antigen by specific antibodies against it . The key and necessary step in this process is the formation of a complex between the HLA molecule and a peptide fragment (epitope) of the foreign antigen: it is this complex that is transported to the cell surface and engages the CD8+ and CD4+ lymphocytes. Therefore, the successful formation of a HLA molecule–antigen complex, and, hence, the successful lymphocyte engagement depends critically on the availability of HLA molecules which would bind to the foreign antigen. Indeed, the outcome of immune checkpoint blockade (ICB) therapy in melanoma depends on the HLA makeup of the patient65, a publicly available catalog of data from the Global Burden of Disease study, and divided those values by the total population of each country in 201965. Life expectancy was not included in the current analyses since it is virtually identical for these countries17.The population prevalences of brain cancer (BC) were computed for 14 countries in Continental Western Europe  occurred in 9 or more countries, with a minimum frequency (in any country) of 0.01. In this study we focused on the 69 HLA Class I alleles whose mean frequencies (over the contributing countries) are given in Table We obtained the population frequency in 2019 of 69 common HLA Class I alleles from 14 Continental Western European Countries ith country, respectively, andWe estimated the association between brain prevalence and allele frequency by computing the covariance between the prevalence of brain cancer and the population frequency of the 69 HLA Class I alleles of Table es Table  whose frH) and permuted (H’) PScov profiles were ranked and the absolute differences between the H and H’ ranks computed and summed: a sum of zero would indicate that the two profiles are the same. (4) Finally, we carried out this procedure 1,000,000 times and counted the number of times M that that sum was equal to zero, indicating that the ranks of the observed and permuted PScov scores are the same. (5) Then, the ratio In this analysis, we tested the null hypothesis that the BC-HLA PScov profiles (vectors) may be due to chance by performing the permutation test below. Let 60, we also estimated the HLA-related immunogenicity of the following 5 HHV5/CMV proteins  of proteins of 11 viruses that have been implicated in brain cancer, namely 9 human herpes virus species (HHV1-HHV9), human polyoma JC virus (JCV), and human papilloma virus (HPV). Details of the proteins analyzed are given in Table ns Table : pp65 (s40 was used for T-cell receptor (TCR) epitope prediction using the INeo-Epp web tool via the INeo-Epp web form interface67. For that purpose, we split a given virus antigen  and hierarchical tree clustering.The IBM-SPSS statistical package (version 28) was used for implementing standard statistical analyses, including descriptive statistics and regression analysis. Reported P-values are 2-sided, unless noted otherwise. For MDS, the PROXSCAL procedure was used . For hierarchical tree clustering, the average between-groups linkage was used as the method and squared Euclidean distance as the interval, implemented by the Hierarchical Cluster function. Finally, the permutations test was implemented using FORTRAN  and 64-bit Mersenne Twister random number generator with a large random double-precision odd-number seed.Supplementary Information.


Notably, a tight spatial and temporal correlation exists between local increases in neuronal activity and the subsequent changes in regional cerebral blood flow. The recognized concept of neurovascular coupling (NVC), also named functional hyperemia, expresses this close relationship and stands at the basis of the modern functional brain imaging techniques. Different cellular and molecular mechanisms have been proposed to mediate this tight coupling. In this context, astrocytes are ideally positioned to act as relay elements that sense neuronal activity through their perisynaptic processes and release vasodilator agents at their endfeet in contact with brain parenchymal vessels. Two decades after the astrocyte involvement in neurovascular coupling has been proposed, we here review the experimental evidence that contributed to unraveling the molecular and cellular mechanisms underlying cerebral blood flow regulation. While traveling through the different controversies that moved the research in this field, we keep a peculiar focus on those exploring the role of astrocytes in neurovascular coupling and conclude with two sections related to methodological aspects in neurovascular research and to some pathological conditions resulting in altered neurovascular coupling.The brain is a highly energy demanding organ, which accounts in humans for the 20% of total energy consumption at resting state although comprising only 2% of the body mass. The necessary delivery of nutrients to brain parenchyma is ensured by the cerebral circulatory system, through the exchange of glucose and oxygen (O The brain is populated by several cells, integrated in extracellular space and matrix to compose brain active milieu . All theNotably, the lack of energy storing in the cerebral tissue and the high level of energy consumption underlie the necessity of a continuous and controlled blood flow supply to the brain, in order to avoid permanent brain damage upon failure in the delivery of nutrients and oxygen. CBF regulation explicates at different levels. First, the flow needs to remain stable in spite of changes in the systemic blood pressure, a process known as cerebral autoregulation and mainly achieved through the myogenic reflex, in which smooth muscle cells modify cerebrovascular resistance to counterbalance pressure variations. This intrinsic regulation has been shown to be more efficient to compensate transient hypertension than hypotension . An addiThe initial evidence on NVC dates back to the end of 19th century, when the physiologist Angelo Mosso observed heartbeat pulsations in the exposed brain of two adults presenting head injuries. Thanks to his ingenious technical devices, he revealed a correlation between the magnitude of these pulsations and the grade of mental activities, concluding “We must suppose a very delicate adjustment whereby the circulation follows the needs of the cerebral activity. Blood very likely may rush to each region of the cortex according as it is most active” . 10 yearex vivo and by sensory stimulation in vivo, demonstrating a central role for neurotransmitter-dependent activation in astrocytes of metabotropic glutamate receptors (mGluRs). These G-protein coupled receptors (GPCRs) are linked to the intracellular pathway of Ca2+ release from endoplasmic reticulum. Consistently, perfusion with mGluR agonist or direct activation of Ca2+ increases in patched astrocytes was sufficient to trigger arteriole dilatation in cortical slices. All protocols used to induce vasodilatation ex vivo were significantly dependent on the release of cyclooxygenase (COX) products, most likely prostaglandin E2, which is released by astrocytes in a Ca2+-dependent process  activates inward rectifier potassium (Kir) channels in smooth muscle cells (SMCs), resulting in cell hyperpolarization and vasodilatation. Importantly, the concurrent inhibition of BK channels and of COX activity resulted in a complete blockade of neuronal activity-dependent vasodilatation  channels, large conductance calcium-activated Klatation . Figures+]o. On the other hand, the enzymatic reactions controlling the synthesis of vasoactive metabolites from arachidonic acid (AA) are sensitive to oxygen concentration, resulting in a shift of vascular response towards dilatation at physiological O2 levels and towards constriction at higher levels. It is noteworthy that standard procedures of O2 equilibration in slice experiments result in supra-physiological oxygenation, thus making dilatation more difficult to observe. Beyond affecting AA metabolism, oxygenation levels influence NVC also by regulating glycolysis, in that O2 consumption accompanying neuronal activity boosts anaerobic glycolysis. The consequent increase in the production and release of lactate leads to a reduction of prostaglandin clearance via the prostaglandin/lactate transporter, enhancing vasodilatation, while the lower production of ATP results in an increased availability of the vasodilator adenosine  of the GCaMP family, which allow to capture Ca2+ signals in the whole astrocyte territory  velocity, which increases during functional hyperemia, an approach particularly useful for small capillaries in which assessing diameter changes is less reliable. Both approaches, for instance, have been used in , which increase intracellular Ca2+ specifically in astrocytes by using an exogenous compound, typically clozapine N-oxide (CNO)  . Convers signals . In ordeFinally, technological advancements allow the study of brain dynamics in freely moving animals through the use of miniaturized microscopes, thus avoiding the need for the head restrained configuration required for awake 2P-LSM experiments. Recently, a first application of this approach has been shown in the context of cortical injury . StudiesEx vivo models of cerebral ischemia based on the application of hypoxic conditions revealed rapid depolarization of neuronal and glial cells resulting from changes in the extracellular ion concentration and resembling spreading depression  as a stroke model to investigate the capillary hyperemic response in brain regions outside the ischemic core. The results reveal an impairment in NVC in the peri-infarct cortex 1 day after MCAO, which could be reverted by inhibiting the synthesis of the vasoconstrictor 20-HETE. Consistently, cortical 20-HETE levels were increased after MCAO, in agreement with observations from stroke patients (Beside [Kpatients . The dysAlzheimer’s disease (AD) is the most common neurodegenerative disorder worldwide, characterized by progressive cognitive impairment and memory loss. Common hallmarks are extracellular amyloid-β (Aβ) oligomers and plaques, and intraneuronal accumulation of neurofibrillary tangles.Neurovascular dysfunction is an early event in the pathogenesis of AD, involved in a positive feedback loop described by the two-hit vascular hypothesis, in which vascular abnormalities favor AD pathogenesis and Aβ burden contributes to worsen CBF function and regulation . Along w2+ signaling (The accumulation of Aβ oligomers around cerebral vasculature has been known since the 70s . Recent ignaling . Consistignaling . Along tignaling .+ channels in initiating and propagating the hyperpolarization that drives vasodilatation. Recent studies on different AD mouse models report a reduction in the activity of endothelial Kir 2.1 channels, resulting in altered vascular responses to extracellular K+ (Another crucial NVC pathway affected in AD models relates to the role of Klular K+ . In the lular K+ , and tPAlular K+ . Intereslular K+  and in alular K+ , consistlular K+ .At the end of this journey across 20 years of NVC history and research, we believe that the controversies raised in this field stimulated a positive scientific debate that ultimately refined the comprehension of the underlying mechanisms. Science improves our knowledge as long as we are both open to doubts and rigorous in performing experiments dealing with them, underlining the importance of choosing the appropriate tools to study NVC.While this review focuses on the role of astrocytes in NVC, it is not meant to claim that these cells are the exclusive players involved. Similarly, the main pathways presented here do not exclude parallel mechanisms, which can have a modulatory or a primary role in different brain regions or in specific tissue conditions, with the role of NO derived from neurons or endothelial cells being an example of this concept. It is highly conceivable that the brain has evolved a redundancy of mechanisms to secure NVC through the involvement of different cell types and different molecular pathways. The ongoing research is expected to further deepen our understanding of these mechanisms, and hopefully to provide new strategies to face neurovascular impairment in different brain diseases.