Pancreatic cancer is a malignant tumor of the digestive system with a very high mortality rate. While gemcitabine-based chemotherapy is the predominant treatment for terminal pancreatic cancer, its therapeutic effect is not satisfactory. Recently, many studies have found that microorganisms not only play a consequential role in the occurrence and progression of pancreatic cancer but also modulate the effect of chemotherapy to some extent. Moreover, microorganisms may become an important biomarker for predicting pancreatic carcinogenesis and detecting the prognosis of pancreatic cancer. However, the existing experimental literature is not sufficient or convincing. Therefore, further exploration and experiments are imperative to understanding the mechanism underlying the interaction between microorganisms and pancreatic cancer. In this review, we primarily summarize and discuss the influences of oncolytic viruses and bacteria on pancreatic cancer chemotherapy because these are the two types of microorganisms that are most often studied. We focus on some potential methods specific to these two types of microorganisms that can be used to improve the efficacy of chemotherapy in pancreatic cancer therapy. However, more than 80% of pancreatic cancer patients have already lost the opportunity to receive surgery when they are diagnosed. This is a possible explanation for the extremely low five-year survival rate (less than 9%) among pancreatic cancer patients Pancreatic cancer is one of the most destructive and lethal malignant neoplasms. According to data from the authoritative American Medical Association, the numbers of new male and female clinical cases of pancreatic cancer rank as the tenth and ninth highest, respectively, among all new tumor cases, and more critically, the number of deaths from pancreatic cancer ranks 4Many studies have demonstrated the potential role of microorganisms in pancreatic cancer carcinogenesis. Chronic inflammation primarily induced by microorganisms may be a vital mechanism, and microorganisms can also change the immune microenvironment and regulate the hallmarks of pancreatic cancer Many viruses are thought to be related to cancer carcinogenesis, including human papilloma virus , Epstein-Barr virus  and hepatitis virus (related to liver cancer). Interestingly, hepatitis virus may also be correlated with pancreatic cancer, particularly hepatitis B virus (HBV) and hepatitis C virus (HCV) Porphyromonas gingivalis  FusobacteriumH. pyloriP. gingivalis was correlated with a 2-fold increased risk of pancreatic cancer occurrence H. pylori was found to be positively related to pancreatic cancer, and Helicobacter DNA was confirmed in tumor tissues but not in normal tissues Bacteria are critical carcinogens and have been investigated in many tumors, including pancreatic cancer. Oral bacteria, including P. gingivalis and Fusobacterium, has been shown to be a critical risk factor for pancreatic cancer  p53 and K-ras mutations Bacteroidetes in obese mice is decreased and the level of Firmicutes is increased Actinobacteria abundance and upregulated Escherichia-Shigella abundance are observed, and the diversity and coordination of gut microbiota are decreased Some risk factors for pancreatic cancer may also interact with bacteria in the digestive tract, including periodontitis, type 2 diabetes mellitus, pancreatitis and obesity. Periodontitis, which is always caused by Malassezia spp.Malassezia spp. in the oral cavity, which also confirmed the important role of oral pathogens in the occurrence of pancreatic cancer Malassezia spp.Fungi, a component of the gut microbiota, have not received much attention from researchers. Although fungi are much lower than bacteria in terms of flora number and abundance, they may play an equally important role as bacteria in pancreatic cancer oncogenesis Viruses are pathogens that cause various detrimental effects on physical and mental health. Of the nearly one million vertebrate viruses, approximately 320,000 can infect mammalian cells E1B55K, E1B19K, and E1ACR2, and the majority of oncolytic adenoviruses are based on adenovirus serotype 5 (Ad5), which has been shown to be safe in cancer patients and to eliminate cancer cells with limited toxicity to healthy cells specifically E1B55K gene E1B55K gene is found within the E1B region of the viral genome. It can combine with the p53 gene and inactivate it. Due to its deletion, the oncolytic adenovirus can replicate in tumor cells but cannot replicate in normal cells. This property guarantees the safety of the oncolytic adenovirus  p53 expression, and some scientists found that infected pancreatic cancer cells can express viral antigens on the surface, resulting in a host immune response to reinforce antitumor immunity  E1B55K weakened the ability of the virus to replicate and spread E1B19K deleted), AdΔΔ (E1ACR2 and E1B19K deleted), AdΔCR2 (E1ACR2 deleted), dl312 (E1A and E3B deleted) and dl922-947 (E1ACR2 and E3B deleted) E1B19K gene deletion can achieve valid targeting at a dose lower than the clinically toxic dose when used in combination with chemotherapy. The enhanced antitumor effect is due to the enhanced drug-induced apoptosis induced by synergism between the virus and gemcitabine. Gemcitabine induces tumor cell death through classical apoptosis, and adenovirus activates tumor cell death through nonapoptotic pathways E1ACR2 was deleted, are more efficient than ONYX-015, but they are more toxic E1ACR2 and E1B19K deleted. Compared with wild-type adenovirus, ONYX-015 and dl922-947, AdΔΔ retains the ability to sensitize cancer cells in combination with cytotoxic drugs, and when combined with gemcitabine, it not only induces more apoptosis to effectively kill tumor cells but also has lower toxicity than when it is used as a monotherapy E3gp19k, resulting in a new mutant, Ad-5-3Δ-A20T, which was more lethal than AdΔΔ against pancreatic cancer when combined with gemcitabine. Its primary mechanisms lie in its increased recruitment of immune cells to infected tumor cells and its enhancement of gemcitabine-dependent apoptosis EIACR2 region to improve tumor selectivity and has an E2F binding site inserted upstream of E1A to control virus replication; in addition, its most critical feature is an insertion of the trimerized, membrane-bound human CD40 ligand (TMZ-CD40L) and the full-length human 4-1BB ligand (4-1BBL) Adenoviruses are small, non-enveloped double-stranded icosahedral DNA viruses, and there are >57 serotypes of adenoviruses that are classified into subtypes A-G based on their respective agglutination properties 134.5γ gene and an insertion of the E. coli. lacZ gene in the infected cell polypeptide 6 (ICP-6)-coding region E. coli lacZ gene enables G207 to replicate more efficiently than HSV-1 in nondividing cells in vitro. Their study confirmed the infection, replication and killing abilities of G207 and was the first to demonstrate the great potential of G207 for treating pancreatic cancer 134.5γ is driven by the B-myb promoter in vitro and is much more effective than the use of gemcitabine alone Herpes simplex viruses (HSVs) can be divided into type 1 and type 2. HSV-1 is an enveloped double-stranded DNA virus that has the following merits and can be used as an oncolytic virus  in vivo and in vitro models of pancreatic cancer. More specifically, many scientists have discovered their beneficial synergistic effects when combined with gemcitabine for treating pancreatic cancer. ING4 is a protein that can inhibit angiogenesis and increase the sensitivity of cancer cells to chemotherapy; undoubtedly, it is a highly effective protein that can be used to treat pancreatic cancer in vitro experiments, they significantly inhibited the replication of SW1990 and PANC-1 pancreatic cancer cells. This result may have occurred because VV-ING4 improves chemotherapy sensitivity and promotes the penetration of gemcitabine Vaccinia viruses have been widely known since their application in preventing smallpox. Recently, due to their tropism for cancer cells, easy genetic modification and other biological characteristics, vaccinia viruses have become a popular choice for use as oncolytic viruses Interestingly, in research on the combined application of GLV-1h68 (an oncolytic vaccinia virus) and chemotherapy drugs in pancreatic cancer, different scientists have obtained slightly different results. Yu et al. found that the tumor-killing effect was significantly enhanced when GLV-1h68 was combined with gemcitabine to treat PANC-1 tumors In addition to the commonly used oncolytic viruses that have been introduced above, there are several oncolytic viruses that have shown to have increasing potential for treating pancreatic cancer, including Newcastle disease virus, measles virus, myxoma virus and vesicular stomatitis virus.In vivo and in vitro studies have shown that the use of myxoma virus and gemcitabine sequential therapy on the Hs766T and Pan02 pancreatic cancer cell lines improves the overall survival rate of mice. In Hs766T tumor cells, treating with gemcitabine followed by oncolytic virus is more effective than either agent as a monotherapy. In the Pan02 cell line, treating with oncolytic virus followed by gemcitabine had better results than either agent as a monotherapy Newcastle disease virus belongs to the Paramyxoviridae family and is a naturally occurring negative-sense single-stranded RNA virus that has been studied for many years. Its genome contains 6 genes encoding eight proteins that can efficiently and selectively kill many types of human tumor cells Although substantial evidence indicates that oncolytic viruses can have a good synergistic effect with chemotherapy, the specific molecular basis has not yet been summarized. In this section, we prioritize concrete mechanisms that may involve direct oncolysis, increasing the sensitivity of cancer cells to chemotherapy and activating antitumor immunity and the apoptosis of tumor cells. These important mechanisms are summarized in Fig. P53 and RBThe oncolytic effect of oncolytic viruses is their foremost property. Oncolytic viruses can selectively invade pancreatic cancer tumor cells, leading to their cracking and death, which in turn leads to increasing positive feedback for lysis. Normal cells have complete antiviral immunity, and oncolytic viruses are quickly cleared when they try to invade normal cells. However, the antiviral capabilities of tumor cells are defective, and they may have defects in the PI3K/AKT signaling pathway or tumor suppressor genes such as E1B19K gene can make pancreatic cancer cells sensitive to chemotherapy-induced death The biggest flaw of chemotherapy is its easily induced tolerance, and oncolytic viruses solve this drawback to a certain extent. Deleting the Over the past few years, the immune response has been considered to be an obstacle to oncolytic virotherapy. However, we are now aware of the great importance of the immune system in oncolytic virotherapy despite its clearing of oncolytic viruses. Typically, pancreatic cancer generates an immunosuppressive microenvironment that includes immune suppressive cytokines such as interleukin-10 (IL-10) and transforming growth factor β1 (TGFβ1) and immunosuppressive cells, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), leading to the inhibition of the immune response P53 and RB pathways are two classic pathways that ensure the normal progression of the cell cycle. The occurrence of P53 mutations in pancreatic cancer cells is the key factor in their unlimited replication capacity. Oncolytic viruses often act on the P53 pathway to promote the upregulation of Bax and the downregulation of Bcl2 and further activate caspase8/9/3 to cause apoptosis Chemotherapy drugs such as gemcitabine can induce apoptosis in pancreatic cancer tumor cells by damaging DNA and terminating chain synthesis Recently, many studies have shed light on the potential link between the microbiota and pancreatic cancer Gammaproteobacteria, which can express the long form of CDD (cytidine deaminase) and can metabolize the active form of gemcitabine  into the inactive form, 2′,2′-difluorodeoxyuridine. Interestingly, scientists have observed a similar phenomenon in mycoplasma, which can encode the CDD to inhibit the antitumor activity of gemcitabine in mycoplasma-infected mouse tumor cells, and this inhibition can be relieved by tetrahydrouridine (a cytidine deaminase inhibitor) or antibiotics such as tetracycline Escherichia coli (E. coli) and Listeria welshimeri on the efficacy of commonly used chemotherapeutics can be used to support this conjecture partially. The tumor volumes of mice treated with gemcitabine plus bacteria were significantly larger than those of mice treated with gemcitabine alone, indicating that the presence of bacteria had an adverse effect on gemcitabine chemotherapy Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans have been detected in patients receiving pancreatic cancer treatment. More specifically, these two bacteria can also express CDD, which indicates that they may be related to chemotherapy resistance in pancreatic cancer Fusobacterium nucleatum (F. nucleatum), a bacterium that can cause colorectal cancer to be resistant to oxaliplatin and 5-fluorouracil by affecting the TLR4/MyD88 pathway, has been found to be more abundant in the cancer tissues of patients with pancreatic cancer than in nonpatient controls  F. nucleatum leads to the loss of miR-18a* and miR-4802 through the TLR4/MYD88 pathway, which leads to the activation of ULK1 and ATG7 and further induces the autophagy pathway, thereby triggering drug resistance in colon cancer. F. nucleatum can also cause the immunosuppression of the pancreatic cancer microenvironment through the same MyD88 pathway. Thus, some scientists speculate that this bacterium may also be related to drug resistance in pancreatic cancer based on the similar signaling pathways involved Previous studies on the relationship between bacteria and pancreatic cancer have focused on the mechanism by which bacteria give rise to the occurrence and progression of pancreatic cancer Salmonella typhimurium improved the therapeutic effect in pancreatic cancer-bearing mice Lactobacillus paracasei is a gram-positive lactic acid bacterium located in the human intestine. Adding Lactobacillus paracasei to a mouse model treated with gemcitabine can improve the efficacy of the chemotherapy and increase tolerance Lactobacillus paracasei can inhibit the proliferation of pancreatic cancer cells and restrain pancreatic cancer cells resistant to 5‑fluorouracil in a mouse model, indicating that the antitumor activity of probiotics may come from the active ingredients in probiotics Enterococcus faecalis and Clostridium butyricum seem to have fewer infection complications The role of bacteria in pancreatic cancer chemotherapy is not always deleterious, and bacteria can sometimes have a beneficial effect. In a mouse model of pancreatic cancer treated with gemcitabine and bevacizumab, adding Firmicutes (from approximately 39 to 17%) and gram-negative Bacteroidetes (from 38 to 17%) in mouse intestines in the tumor-bearing control group decreased significantly, while the proportions of Proteobacteria (E. coli and Aeromonas hydrophila) and Verrucomicrobia (Akkermansia muciniphila) increased dramatically Firmicutes and gram-negative Bacteroidetes are usually the dominant species in the gut of tumor-bearing control mice, while Proteobacteria and Verrucomicrobia are usually minor components Proteobacteria and Verrucomicrobia was related to inflammation of the intestine The physical condition of cancer patients is generally poor, and the addition of chemotherapy undoubtedly aggravates the patient condition Research on the relationship between microbes and tumors has made noticeable progress, which will surely bring new promise for the treatment of pancreatic cancer. Oncolytic viruses, which are microorganisms that can be manipulated by humans, have unpredictable clinical prospects in pancreatic cancer treatment. As we mentioned above, many oncolytic viruses have significant synergistic effects when used in combination with chemotherapy to treat pancreatic cancer. However, there are still many directions worth exploring. For example, how can genetic modification achieve greater tumor selectivity and immunogenicity? How can we minimize the toxicity and maximize the activity of oncolytic viruses? How can we reduce virus loss before reaching the target site? When we combine chemotherapy and oncolytic viruses, which order of administration and dosage have the best effect? In the future, oncolytic viruses that interfere with the tumor microenvironment may reduce the number of resistant cases and the number of cancer stem cells. At present, most oncolytic viruses are delivered by intratumoral injection, and some experiments are guided through endoscopic ultrasound (EUS) Pancreatic cancer is a malignant neoplasm with poor prognosis, and gemcitabine is the primary chemotherapy drug for advanced pancreatic carcinoma. Currently, plenty of evidence suggests that there is an intricate interplay between microorganisms and pancreatic cancer. However, attention is primarily focused on how microorganisms lead to the occurrence and development of pancreatic cancer, and few studies have focused on elaborating on and summarizing the impact of microorganisms on pancreatic cancer chemotherapy. In this review, we provide new insights into the interplay of microorganisms and chemotherapy, summarizing some meaningful views and guidance for the development of new treatment methods and patterns. Our results indicate that the combination of oncolytic viruses and chemotherapy has great potential utility, and its mechanism possesses four characteristics: apoptosis of tumor cells, activation of antitumor immunity, increased sensitivity of cancer cells to chemotherapy and direct oncolysis. In addition, the use of bacteria in the treatment of pancreatic cancer has beneficial and negative effects on patient prognosis; chemotherapy may also interact with bacteria and cause various results, and antibiotics might have therapeutic potential in pancreatic cancer treatment. Surprisingly, fungi can also contribute to the occurrence of pancreatic cancer, but their influence and application during chemotherapy still require investigation. In addition, it should be noted that most of the studies in the above discussion were conducted in mouse models. Because there will likely be different effects during applications in humans, the results should be interpreted with caution. Overall, microorganisms are of paramount importance to pancreatic cancer chemotherapy, although the complex relationship between microorganisms, chemotherapy and pancreatic cancer remains unclear. In the near future, microorganisms will surely become highly important in pancreatic cancer treatment and new drug development.


XACT associated with X chromosome erosion in human pluripotent stem cells and recent findings from non-human primate post-implantation embryos. I focus on the experimentally challenging peri- and post-implantation stages of human development when the timing of XCI is prolonged and imprecise in humans. Collectively, this research has raised some important unanswered questions involving biased sex ratios in human births and the male bias in the incidence of ASD.The etiology of autism spectrum disorders (ASD) is complex, involving different combinations of genetic and environmental factors. My lab’s approach has been to investigate DNA methylation as a tractable genome-wide modification at the interface of these complex interactions, reflecting past and future events in the molecular pathogenesis of ASD. Since X-linked genes were enriched in DNA methylation differences discovered from cord blood from newborns later diagnosed with ASD, this has prompted me to review and revisit the recent advancements in the field of X chromosome inactivation (XCI), particularly in humans and other primates. In this Perspective, I compare XCI mechanisms in different mammalian species, including the finding of the noncoding transcript  Autism spectrum disorders (ASD) is a collective term representing an etiologically diverse group of neurodevelopmental disorders characterized by deficits in social behaviors, deficits in language, and a gain of restrictive interests and repetitive behaviors . ASDs hain utero exposure to a multitude of maternal exposures, including air pollution, pesticides, Rubella infection, and obesity increase risk for ASD in the offspring  that is poorly understood . In addiffspring . Such in fetuses . Epigene fetuses , this Petory RNA , I will Because females have two X chromosomes compared to only one in males, X chromosome inactivation (XCI) is predicted to reduce the level of X linked transcripts in females as a mechanism of dosage compensation . In euthXIST. XIST acts in cis by localizing to and coating the inactive X chromosome and recruiting polycomb repressive chromatin factors, resulting in a more compact and heterochromatic chromosome  mammals in its imprinted pattern of XCI pre-implantation  between rodents and primates are likely responsible for the timing and imprinting differences. Specifically, while a romosome . JPX is cription . Similar tissues . Interesman Xq23 . Like XIem cells .XIST and a gain of XACT localization and a partial expression of genes from the previously established inactive X chromosome  and histone H3 Lys27 trimethylation (H3K27me3) and chromosome compaction lasted beyond the loss of XIST localization on the future active X chromosome. By embryonic days 13–16, roughly 4–7 days post-implantation, XCI as measured by monoallelic expression and X to autosomal expression ratio was complete  clinics have suggested that sex differences exist based on sex chromosome content in the timing of recognizable stages, with males showing faster cell division and progression to blastocyst stage than females, although some of the results have not been replicated , which is equal  and Early Autism Risk Longitudinal Investigation (EARLI) are both longitudinal studies of pregnancies at enriched risk for ASD because of recruitment from mothers with at least one child diagnosed with ASD . We haveXACT were replicated in both sexes and both cohorts. We also replicated prior candidate-based findings for the syndromic ASD genes MECP2 and FMR1. In addition, of the X-linked genes mapping to cohort-replicated ASD DMRs, many were also implicated in ASD at three different levels  from other published studies   (DNMT3L and an increase in DNMT3B (25). XACT showed relatively low uniform expression throughout all lineages, unlike XIST expression (left scatterplots) which varied across cells and lineages and modestly correlated with DNMT3B expression. In contrast, the cells with the highest expression of DNMT3B epiblast (colored blue) showed lower expression of X-linked genes PTCHD1 and MID1  . Howeverryo.org) . InteresID1 from . These phylation . DNMT3B deletion . DNMT3L  regions , a chrom regions .XACT is particularly intriguing because it is primate-specific and appears to be involved in the phenomenon of X chromosome erosion in vitro, the prolonged delay of XCI that is observed in primates, and the timing of neuronal differentiation. This Perspective is therefore intended to raise new questions about this elusive stage of peri-implantation life in humans and if differentially DNA methylation patterns identified at birth may be reflecting gene by environment interactions in the etiology of ASD. First, is the sex bias in ASD somehow related to a higher likelihood that genetically susceptible female embryos may fail to successfully implant? Are human female peri-implantation embryos, because of their suspended state of poised chromatin and lack of absolute X-linked dosage compensation, epigenetically more plastic and therefore the sex more selectable by environmental variables, both positively and negatively? Can some of the features of X chromosome erosion occur in vivo, and if so, are these epigenetic changes a potential etiology of neurodevelopmental disorders? When and where do the X-linked DNA methylation differences observed in ASD cord blood occur: the developing brain, the pre- or post-implantation embryo, or the oocytes and/or sperm of the parents (or even their PGCs from the parents’ in utero life)? Clearly, these are all very difficult questions to answer because of the challenges in getting the right human samples. But new technologies such as single cell transcriptomes and epigenomes are expected to provide important new information. There is also a clear need for non-human primate studies in this area to better understand this critical life stage of early post-implantation for both XCI and ASD research.Our recent discovery that DNA methylation differences in cord blood from newborns later diagnosed with ASD were enriched for X-linked locations and early developmental functions has prompted me to revisit the recent X chromosome inactivation literature for insights into how and when these epigenetic errors may occur.


ASD genetic diagnosis has dramatically improved due to NGS technologies, and many new causative genes have been discovered. Consequently, new ASD phenotypes have emerged. An extensive exome sequencing study carried out by the Autism Sequencing Consortium (ASC) was published in February 2020. The study identified 102 genes which are de novo mutated in subjects affected by autism spectrum disorder (ASD) or similar neurodevelopmental disorders (NDDs). The majority of these genes was already known to be implicated in ASD or NDDs, whereas approximately 30 genes were considered “novel” as either they were not previously associated with ASD/NDDs or very little information about them was present in the literature. The aim of this work is to review the current literature since the publication of the ASC paper to see if new data mainly concerning genotype–phenotype correlations of the novel genes have been added to the existing one. We found new important clinical and molecular data for 6 of the 30 novel genes. Though the broad and overlapping neurodevelopmental phenotypes observed in most monogenic forms of NDDs make it difficult for the clinical geneticist to address gene-specific tests, knowledge of these new data can at least help to prioritize and interpret results of pangenomic tests to some extent. Indeed, for some of the new emerging genes analyzed in the present work, specific clinical features emerged that may help the clinical geneticist to make the final diagnosis by associating the genetic test results with the phenotype. The importance of this relatively new approach known as “reverse phenotyping” will be discussed. With the recent development of NGS technologies, ASD genetic diagnosis has dramatically improved, and many new genes associated with ASD have been discovered. Consequently, new ASD phenotypes have emerged. Therefore, it seems that we are entering a new era, in which the ASD diagnostic path definitely benefits from a genetic work-up, as it can lead to a precise definition of etiology, family counselling, prognosis forecasting, and gene-based stratification, hoping that targeted therapies are eventually developed. We need to be aware that the clinical presentation of ASD  in some instances drives crucially the genetic testing, which is often a multi-step process. Bearing this in mind, and by reviewing the emergent ASD genes and phenotypes, we will outline some practical guidelines that can be useful for geneticists and neuropsychiatrists to implement the genetic diagnosis of patients with ASD.https://www.omim.org/ (accessed on 1 July 2021)), but six of them were listed in OMIM afterwards. The aim of this paper is to review the current literature for new information and data concerning these “truly novel” defined genes and their associated phenotype. Indeed, our ultimate goal is to delineate genotype–phenotype correlations for these newly emerging genes in order to help the clinical geneticist to address the (a) appropriate genetic test if possible and above all (b) to interpret pangenomic results in light of the phenotype in patients with non-syndromic ASD.In February 2020, the most extensive exome sequencing study on autism spectrum disorders (ASD) and related neurodevelopmental disorders (NDD) was published by Satterstrom and colleagues . A totalWe introduced the following key words in Pubmed for each of the 30 truly “novel genes”: (i) name of the gene; (ii) name of the gene AND autism; (iii) name of the gene AND neurodevelopment; (iv) name of the gene AND intellectual disability. We selected all papers not mentioned by Satterstrom and colleagues  as they We found new information concerning the correlation between the “novel genes” and emerging phenotypes for 6 out of the 30 novel genes. The list of the 30 novel genes and new data concerning emerging genotype–phenotype correlations are summarized in RORB gene (OMIM*601972) located on chromosome 9q21.13 is a transcription factor expressed in immature neurons where it regulates neural differentiation and migration. Two isoforms are known, RORß1 and RORß2, which are differentially expressed: the first one predominately in the retina and pineal gland, whereas the other one in more central nervous system (CNS) regions including the cortex, spinal cord, and pituitary [RORB gene have been described to be associated with epilepsy and mild intellectual disability by Baglietto and colleagues [RORB was considered the strongest candidate. The causal role of RORB was recently confirmed by the identification of missense, nonsense SNVs (single nucleotide variant) [RORB in patients with epilepsy , sometimes in comorbidity with intellectual disability. A recent report [RORB SNV in a patient affected by intellectual disability, attention deficit hyperactivity disorder (ADHD), and eyelid myoclonia with absences  encompassing an intragenic duplication of the RORB gene [RORB which arose de novo. Indeed, the two brothers were affected by mild NDDs: the first one had an expressive language impairment, whereas the second one displays an unusual form of ASD, characterized by very intense visual sensory self-stimulation and stereotypic behaviors. A recent report [RORB variants in fourteen individuals belonging to four families: eleven of them were affected by epilepsy, one had intellectual disability, and two were unaffected. Epilepsy was focal or generalized in five patients, whereas six patients had both types. Cognitive difficulties ranging from individuals having learning difficulties at school to mild or severe intellectual disability were reported in seven out of eleven patients. In summary, it appears that RORB variants are mainly associated with different forms of epilepsy , often in comorbidity with neurodevelopmental disorders such as intellectual disability, ADHD, and ASD. However, sporadic cases of RORB variants have also been described in association with severe forms of ID only [The lleagues ; intellelleagues  and develleagues . The smavariant)  and intrabsences . The latORB gene . At the  ID only .PRR12 gene (OMIM*616633) encodes a proline-rich protein nuclear factor involved in neural development, and it has a possible DNA binding activity and a chromatin remodeling role. Cordova-Fletes and colleagues [PRR12 gene in a girl affected by intellectual disability and neuropsychiatric alterations. More recently, de novo loss of function SNVs have been reported in three patients affected by neurodevelopmental disorders and iris abnormalities [PRR12 affected by unilateral or bilateral complex microphtalmia without a neurodevelopmental phenotype. The cohort described by Reis and colleagues consisted of five individuals with asymmetric ocular phenotypes [PRR12 SNVs are associated to a new multisystemic disease characterized mainly by eye abnormalities and almost always by a neurodevelopmental phenotype.The lleagues  reportedmalities . All patmalities  who descene OMIM*16633 encSATB1 gene (OMIM *602075) encodes for a transcription factor involved in development and in T cell maturation. Recently SATB1 disrupting de novo pathogenetic variants has been identified in patients affected by NDD, suggesting a role for this gene also in neurodevelopment [SATB1 variants. Of these variants, twenty-eight were de novo, three were inherited from an affected parent, two were inherited from unaffected parent (reduced penetrance), five were probably due to parental mosaicism, and for the last four, inheritance could not be established. Thirty individuals carried missense variants that clustered in the two CUT1 and CUT2 DNA-binding domains. On the other hand, PTVs were present in 10 subjects  and were localized throughout the entire gene. The remaining two patients had two small gene deletion variants. Interestingly, the authors observed a clear genotype–phenotype correlation: individuals carrying missense variants were more severely affected than individuals carrying protein-truncating variants (PTVs). Indeed, 57% of patients carrying a missense variant were affected by severe intellectual disability, which was not observed for any individuals carrying PTVs. Other common clinical features were (i) visual abnormalities such as myopia, amblyopia, divergent squint, and strabismus, and (ii) dental anomalies such as small, malpositioned, or widely spaced teeth. In addition, other severe clinical features as spasticity, hypotonia, and epilepsy were significantly more common in patients with missense variants than in patients with PTVs. These observations were confirmed by the authors using a partitioning around medoids clustering algorithm [SATB1 NDDs. Indeed, functional assays  using cells transfected with some of the identified missense variants localized in the CUT1 and CUT2 DNA binding domains clearly demonstrated a stronger binding of the proteins encoded by SATB1 missense variants to their downstream targets compared to the wild type protein. Some of the identified PTVs localized in the last exon of SATB1 were predicted to escape non-sense mediated mRNA decay (NMD), a quality control mechanism that selectively degrades mRNAs harboring premature termination. The prediction was also confirmed by functional assays, and the selected PTVs were further assessed with the same functional assays used for missense variants, showing remarkable differences between the two distinct variant classes with respect to protein localization and their ability to repress transcription. Protein stability and protein SUMOylation were assessed only in some of the selected PTVs. In summary, this work demonstrated the existence of two different classes of SATB1 pathogenetic variants (missense and PTVs) associated with two clinically distinct NDDs and distinct pathophysiological mechanisms . Furthermore, this work highlights the importance of combining clinical data, functional assays, in silico models, and genetic data in order to characterize and fully understand the pathophysiological mechanisms underpinning SATB1-associated NDDs. The  OMIM *6075 encodelgorithm  based onSATB2 variants have been previously associated with SAS, SATB2-associated syndrome (OMIM#612313), a similar neurodevelopmental disorder. Indeed, Satb1 and Satb2 are both transcription factors involved in the processes of cortex development and maturation of neurons. However, unlike for SATB1, no differences could be discerned in the range or severity of phenotypes between individuals with clear loss-of-function mutations and those with missense variants, supporting haploinsufficiency as the common pathogenic mechanism for SATB2 [SATB1 and SATB2 patients display very strong overlapping phenotypes. However, subtle differences in the neurological phenotype have been reported: absence of speech and drooling seem to be more common in patients with SATB2 variants, whereas epilepsy seems to be more common in patients with SATB1 variants, especially in patients with missense variants, as previously discussed [or SATB2 . As expeiscussed .RFX3 gene (OMIM*601337) is listed among the 31 novel genes related to ASD and associated NDDs. This gene belongs to a family of transcription factors highly expressed in the developing and adult brain. Other members include RFX1, RFX4 (OMIM*603958), and RFX7 (OMIM*612660) genes. De novo and inherited deleted CNVs encompassing RFX3 have been identified in a girl and in seven other patients all affected by ID, ASD, and behavioral problems [RFX3 has also been reported in a patient affected by schizophrenia [RFX4 and RFX7 variants. Indeed, a very recent report by Harris and colleagues [RFX4 and RFX7 genes in addition to the already known RFX3 causal gene. Variants in all three genes included missense variants (thirteen), frameshift variants (eight), nonsense variants (five), splicing variants (two), and deletions (five) . All identified variants except for one were absent from the gnomAD human database (https://gnomad.broadinstitute.org (accessed on 1 July 2021)). In addition, the majority of variants (20 out 33) identified were predicted to cause protein truncation or gene deletion, supporting a loss of function or haploinsufficiency model. In relation to the remaining missense variants, eleven out of the 13 were predicted to be deleterious by at least four of six algorithms and two by two algorithms. Functional analysis of five non truncating variants using HeLa cells resulted in a consistent decrease in RFX3 expression level affecting protein stability for the majority of them. Individuals affected by pathogenic or likely pathogenic variants in one of the three RFX transcription factor genes identified in this study shared a common and peculiar behavioral phenotype characterized by sensory auditory hypersensitivity and impulsivity. This unique behavioral phenotype was present in nearly all individuals. In addition to such distinct behavioral phenotype, RFX-affected individuals have ID/global developmental delay with ASD and/or ADHD. In general, individuals with RFX4 or RFX7 are more severely affected than individuals with RFX3 variants. Furthermore, RFX7 affected individuals all have language delay and ID and are less commonly affected by ASD or ADHD. On the other hand, dysmorphic facial features, seizures, and neuroimaging findings, though widely reported in affected patients, do not seem to be specific for any of the three RFX genes. The mechanism by which RFX variants affect neurodevelopment and cause a neurodevelopmental phenotype is not clear yet. A study by Sun and colleagues [The problems . A deletophrenia . On the s (five) . For alllleagues  has showGRIA2 gene (OMIM*138247) encodes for one of the four subunits (GluA1-A4) of the AMPA glutamate receptor, a ligand-gated ion channel playing an important role in excitatory synaptic transmission in the central nervous system. Specifically, the GluA2 subunit encoded by GRIA2 regulates calcium permeation and voltage rectification. Few reports have described an association between GRIA2 variants and neurodevelopmental disorders. Currently, it is listed in OMIM as responsible for the NEDLIB phenotype . Hackmann and colleagues [GRIA2 gene. A very recent report by Alkelai and colleagues [GRIA2 variant (p.Glu508Ter). Additional clinical features included autism spectrum disorder, epilepsy, and obsessive–compulsive disorder. The most extensive work on GRIA2 genotype–phenotype correlations has been recently carried out by Salpietro and colleagues [GRIA2 variants. In total, they found 24 de novo GRIA2 SNVs  and three de novo 4q32.1 microdeletions. The authors performed functional assays in order to characterize several missense variants found in their cohort. When they expressed missense variants in HEK293T cells, they found a decrease in agonist current mediated by these “mutant subunits” compared to wild-type channels. Furthermore, co-expression of mutant or wild-type GluA2 subunits together with wild-type GluA1 showed significant differences in KA (kainic acid)-evoked current amplitude. Specifically, five missense variants significantly decreased the KA-evoked current amplitude compared to the wild-type variant, and some also increased calcium permeability. The functional characterization of GRIA2 variants explain the neurological phenotypes observed in most patients very well. These include Rett-Syndrome-like features , gait abnormalities including ataxia and dyspraxia, abnormal sleep rhythm, and irregular breathing patterns with hyperventilation episodes. Progressive microcephaly was also observed (in 14% of patients), though it was not as common as in Rett syndrome patients. In addition, seizures or developmental epileptic encephalopathy (DEE) were also reported in about 40% of patients, usually arising very early in life (within the first six months of life). These same neurological features are also reported in patients with variants in other genes encoding for AMPAR/NMDAR subunits as GRIA1, GRIA3, and GRIA4. With respect to the main core phenotype, all patients had language impairment, which was severe in most cases. Other frequently reported neurodevelopmental disorders included ID and ASD. Some patients with GRIA2 variants also display progressive brain atrophy (mainly cerebellar) and white matter anomalies [The lleagues  describelleagues  describelleagues , who desnomalies . In summnomalies  aimed atTAOK1 gene (OMIM*610266) encodes for the serine/threonine protein kinase TAO1, which is highly expressed in the brain, where it regulates many cellular processes. The evidence for this gene in NDDs was very limited before the release of Satterstrom’s paper in May 2020 [TAOK1 variants and their associated phenotypes have been published recently [TAOK1 variants: four missense variants , three nonsense variants , and one frameshift (p.Leu790Phefs*3) variant. All eight individuals were affected by developmental delay, six by muscular hypotonia, and four by intellectual disability, and five had dysmorphic facial traits. The authors further functionally characterized the frameshift variant p.Leu790Phefs*3. For this variant, they showed decreased mRNA levels compared to controls in blood cells as well as in fibroblast-derived cells from the same patient. Additionally, the mutant fibroblasts were unable to express phosphorylated TAO1 kinase and tau protein and displayed altered mitochondrial morphology. Knock-down experiments of the ortholog gene Tao 1 in Drosophila confirmed a role for this gene in neurodevelopment: mutant flies exhibited altered morphology of the ventral nerve cord and of neuromuscular junctions. Additionally, a decrease in the size of motor neurons’ axons and altered mitochondria distribution were reported in the mutant flies. The recent work by van Woerden and colleagues [TAOK1 variants in the nervous system, demonstrating a crucial role for this gene in neuronal morphology and migration. The authors described a cohort of 23 patients with 19 SNVs and 4 deleted CNVs ranging in size from 807 bp to 2 Mb. Of the nineteen SNVs, five were missense variants , seven nonsense variants , four indels , and three splice site variants. The phenotypic characteristics of their cohort overlap with those described by Dulovic-Mahlow and colleagues [TAOK1 carriers. In addition, other significant characteristics reported in many individuals were joint hypermobility, growth anomalies , feeding difficulties during the neonatal period, and recurrent ear and airway infections. To further define the functional role of TAOK1 in neurodevelopment, the same authors performed functional studies in vivo and in vitro. They performed in utero electroporation in mouse embryos and found that shRNA-mediated knockdown of TAOK1 at a critical time for neurodevelopment resulted in neuronal migration deficit. Hence, the reduction of TAOK1 levels affects brain development, implying haploinsufficiency as the likely pathogenetic mechanism for TAOK1 associated NDDs. They also tested five missense variants identified in their cohort  is associated with “developmental delay with dysmorphic facies and dental anomalies”, and GRIA2 (#618917) is associated with “neurodevelopmental disorder with language impairment and behavioral abnormalities”. We expect that, in the coming years, new literature data will be added to the existing one. Furthermore, we would like to highlight that some of these studies  have alIn addition, these studies also show the importance of performing a fully clinical assessment with particular attention to the neurological and the psychiatric characterization of the patient. Differentially specialized clinicians  must collaborate together to characterize the patient phenotype in-depth.RORB variants; eye anomalies  are commonly found in patients with PRR12 variants; dental anomalies and visual abnormalities are reported for SATB1-affected carriers; a unique behavioral phenotype characterized by sensory auditory hypersensitivity and impulsivity is typical of RFX-affected patients; and a neurological phenotype characterized by Rett-Syndrome-like traits, gait abnormalities, and abnormal sleep is found in GRIA2 carriers. Unfortunately, these gene-specific phenotypic features cannot be used by the clinical geneticist to address a specific genetic test, as they are not unique to a single NDD gene but are often shared by different NDDs. Nevertheless, the reverse phenotyping data from patient cohorts and the functional data on disrupted alleles can be of great benefit for the clinicians, not just for an appropriate prescription of a genetic test but also for interpretation of the genetic results in terms of severity, prognosis, and natural history of the condition. In fact, in the current era of high-throughput technologies, it is easier (and even cheaper) to test multiple genes simultaneously rather than going gene-by-gene, regardless of the phenotype: therefore, the true challenge is not to identify the causative gene but to understand how it could be related to the clinical issues of our patients. Additionally, it is becoming frequent to detect rare variants in young children with NDD using genome or exome analysis: when the identified gene is new and there is no information about severity and prognosis, the clinical impact of the genetic results is quite low and disappoints the physician as well as the parents, because it is impossible to make any prediction about patient outcome. In this regard, clinical cohort studies focusing on a single novel gene as those reported in the present work represent a precious resource for the clinical geneticist during phenotype reevaluation as they can be of fundamental importance to reach a clinical diagnosis and make the final genotype–phenotype association. In summary, we can conclude that, with the exception of a relatively small number of conditions (such as for example Fragile X or Prader Willi syndrome), in the majority of cases, the causative diagnosis of ASD/ID cannot be reached simply by “gestalt” recognition but rather by the knowledge of specific clinical issues (see Some specific phenotypic features are associated with at least five of the six novel genes discussed in this paper : epilepssues see  reported


Canine osteosarcoma (OSA) is an aggressive malignancy that frequently metastasizes to the lung and bone. Not only has there been essentially no improvement in therapeutic outcome over the past 3 decades, but there is also a lack of reliable biomarkers in clinical practice. This makes it difficult to discriminate which patients will most benefit from the standard treatment of amputation and adjuvant chemotherapy. The development of reliable diagnostic biomarkers could aid in the clinical diagnosis of primary OSA and metastasis; while prognostic, and predictive biomarkers could allow clinicians to stratify patients to predict response to treatment and outcome. This review summarizes biomarkers that have been explored in canine OSA to date. The focus is on molecular biomarkers identified in tumor samples as well as emerging biomarkers that have been identified in blood-based (liquid) biopsies, including circulating tumor cells, microRNAs, and extracellular vesicles. Lastly, we propose future directions in biomarker research to ensure they can be incorporated into a clinical setting. Osteosarcoma (OSA) is an aggressive neoplasm of the bone that is characterized by the production of osteoid . In caniTreatment for canine OSA involves removal of the primary tumor through either limb-spare or limb amputation surgery. Patients that undergo surgery alone have a short median survival time of 101–177 days (~6 months) due to the development of lung metastases, the most common site of OSA metastasis . Other rThe variability in the time it takes dogs to develop detectable metastasis after treatment, as well as variability in the overall survival (OS) time, suggests that patients respond differently to treatment; yet current practice effectively lumps all these patients into the same category of “poor prognosis.” Biomarkers could be particularly informative in this regard and are defined by the NIH Biomarkers Definitions Working Group as a “characteristic that can be objectively measured and evaluated as indicator of biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention” . BiomarkAlkaline phosphatase (ALP) status, body weight, and location of the primary tumor are examples of proposed biomarkers. Greater weight at the time of diagnosis has been associated with metastasis; while primary lesions in the proximal humerus, distal femur, or proximal tibia and age are associated with an increased mortality . Two graMolecular profiling of tumor tissue is an invaluable way to understand what signaling pathways and genes may be contributing to cancer progression. Gene expression microarray analysis found 51 genes to be differentially expressed between short survivors (<6 months) and long survivors (≥6 months). A majority of these genes were upregulated in the short survivors group and mediate drug resistance, proliferation, or metastasis. Pathways that were overactive in short survivors included Wnt, chemokine/cytokine, and integrin signaling . A similThe hyperactivation of several pathways have been suggested to promote the growth of OSA cells. The increased expression of receptor tyrosine kinases (RTK) can lead to enhanced downstream signaling and pro-tumourigenic phenotypes. OSA tumors have significantly higher levels of epidermal growth factor receptor (EGFR) mRNA compared to normal bone tissue. Analysis in a tissue microarray (TMA) found that patients with high levels of EGFR protein had shorter OS and DFI, compared to patients with low or negative levels . In the Insulin growth factor receptor 1 (IGFR1) and platelet derived growth factor (PDGF) signaling promote cancer cell proliferation and survival by activating PI3K/AKT and mitogen-activated kinase (MAPK) signaling. High levels of IGFR1 were found to be associated with reduced OS but no association was observed between IGFR1 levels and DFI . Neithervia activated SMAD2 and yes-associated protein (YAP), as high levels of phosho-SMAD2 and YAP in canine OSA tissue associated with reduced DFI and OS , which were previously found to be mutated in OSA, were assessed in a canine OSA TMA comprised of 150 cases . DifferePrevious studies have shown that developing an infection post-limb-spare surgery is a positive predictor for recurrence and survival, suggesting that an effective immune response could provide an anti-tumor effect , 74. How+ macrophages within the primary tumor associated with a longer DFI (+ (T-lymphocytes), and FOXP3+ (T-regulatory) cells were positively correlated between the two sites. To determine if there were significant differences in immune cell infiltrates between the primary and metastatic lesion, CD3+, PAX-5 (B cell) and CD204+ (macrophage) levels were compared. All three cell types were significantly increased at the metastatic lesion, compared to the primary  and lymphocytes (> 1 × 103/μL) before SOC treatment was associated with a significantly shorter DFI  and weakly positive for osteocalcin (OCN). In an analysis of 3 patients, it was found that the number of CTCs declined after amputation but increased before metastatic disease or euthanasia .10 EVs/1 mL of plasma  class III component C6 were both able to distinguish the OSA by their various stages with 77% accuracy . In relaThe studies reviewed herein provided valuable information on OSA biology, but multiple barriers prevent the clinical application of the proposed biomarkers. Some of the studies compared only a handful of patients, leading to problems with statistical power . Almost The feasibility of evaluating some of the above markers in a clinical setting will also need to be improved. Isolating miRNA and EVs and detecting CTCs and blood cells require specialized equipment and reagents which makes the pipeline from sample acquisition to data acquisition cumbersome. Certain technologies, such as microfluidic devices  or synthGiven these obstacles, it is imperative that future biomarker research follows a critical discovery, validation, and clinical translation pipeline. The study populations included in the discovery and validation studies need to be well-documented, ideally with genetic background of the patient's tumor (given the chaotic nature of OSA), adequate history of treatments received, and complete dates of defined clinical endpoints. If the biomarker proves useful, focus should then be directed to developing technologies to allow its use in a clinic setting. As blood-based (liquid) biopsies are easier to access, permit serial sampling, and potentially have an easier clinical translation, future studies should focus on blood-based (liquid) biomarkers.Canine OSA lacks reliable diagnostic, prognostic, and predictive biomarkers. Several tissue-based and liquid-based biomarkers have been proposed in the literature. These studies showcase the potential of biomarkers to predict patient response to therapy, determine the presence of metastasis, predict patient outcomes, and allow for personalized medicine. Confirming the results in larger cohorts through collaboration and increasing the feasibility of evaluating these molecules in a diagnostic laboratory setting will bring biomarkers one step closer to becoming a clinical reality for canine OSA patients.AL and AV-P conceived the ideas to develop the manuscript. AL drafted the manuscript and prepared all figures and tables. AV-P and GW revised the manuscript, figure, and table. All authors edited and approved the final manuscript.AV-P studies on canine OSA biomarkers are funded by OVC Pet Trust Grant #053666. GW studies on canine OSA biomarkers are funded by OVC Pet Trust Grant # 053527.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


With the advent of increasingly sophisticated fluorescence imaging approaches and methods for spatially-resolved light stimulation in the heart, the zebrafish represents an experimental model with unrealized potential for cardiac optogenetic studies. In this review we summarize the use of zebrafish for optogenetic investigations in the heart, highlighting their specific advantages and limitations, and their potential for future cardiac research.Optogenetics, involving the optical measurement and manipulation of cellular activity with genetically encoded light-sensitive proteins (“reporters” and “actuators”), is a powerful experimental technique for probing (patho-)physiological function. Originally developed as a tool for neuroscience, it has now been utilized in cardiac research for over a decade, providing novel insight into the electrophysiology of the healthy and diseased heart. Among the pioneering cardiac applications of optogenetic actuators were studies in zebrafish, which first demonstrated their use for precise spatiotemporal control of cardiac activity. Zebrafish were also adopted early as an experimental model for the use of optogenetic reporters, including genetically encoded voltage- and calcium-sensitive indicators. Beyond optogenetic studies, zebrafish are becoming an increasingly important tool for cardiac research, as they combine many of the advantages of integrative and reduced experimental models. The zebrafish has striking genetic and functional cardiac similarities to that of mammals, its genome is fully sequenced and can be modified using standard techniques, it has been used to recapitulate a variety of cardiac diseases, and it allows for high-throughput investigations. For optogenetic studies, zebrafish provide additional advantages, as the whole zebrafish heart can be visualized and interrogated  Here we provide an overview of the use of zebrafish for cardiac optogenetic studies, highlighting their advantages, limitations, and future potential  and used for in vivo voltage mapping in 2–3 dpf embryos under normal conditions and after application of the histamine H1 receptor blocker astemizole . Measurements showed that astemizole disrupted the normal sequence of cardiac excitation, causing retrograde propagation from the atrioventricular boundary to the atrium  promotor [Tg(myl7:chimeric VSFP-butterfly CY) and Tg]. The hearts of 3 dpf and 14 dpf zebrafish were imaged after administration of drugs targeting the sympathetic nervous system or various cardiac ion channels to assess effects on electrical activation, action potential morphology, and intracellular Ca2+ dynamics  IKr block (with E-4031) increased action potential duration; (iii) L-type calcium current  block (with nifedipine) prevented Ca2+ transients, increased ventricular action potential duration, and disrupted atrioventricular conduction; and (iv) differences exist in atrial and ventricular Ca2+ recovery dynamics between 3 and 14 dpf zebrafish (but not in the Ca2+ upstroke).Since that time, a variety of new GEVIs have been developed with improved sensitivity and kinetics. In a recent study using zebrafish  with the GECI GCaMP5) under control of the heart-specific cmlc2 promoter [Tg(cmlc2:Arch(D95N)-GCaMP5G)] -eYFPs1990t)] and the chloride-specific ion channel halorhodopsin from Natronomonas pharaonis (NpHR) ], to locate and control cardiac pacemaker cells in intact 1–5 dpf zebrafish embryos. In NpHR-expressing zebrafish, maps were generated at each day post-fertilization by sequentially illuminating small, overlapping regions of the heart, and measuring the heart rate response, or the incidence of cardiac arrest or arrhythmia (Optogenetic actuators are light-activated proteins that generate a transmembrane ion flux. The discovery and cloning of the cation-selective ion channel channelrhodopsin-2 (ChR2) from the green alga rhythmia . It was Guillardia theta anion channelrhodopsin 1 and 2 (GtACR1 and GtACR2) ], which in combination with experiments in genetically transfected single rabbit ventricular myocytes demonstrated that GtACR1 activation causes depolarization of ventricular myocytes when applied during the resting (diastolic) phase of the AP , but causes repolarization when applied during the (systolic) plateau (resulting in shortening of the AP). This biphasic response relates to the reversal potential of chloride in ventricular myocytes, which is somewhere between −40 and −33 mV , the Heart and Stroke Foundation of Canada (G-18-0022185 to TQ), and the Canadian Institutes of Health Research (MOP 342562 to TQ).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The aerial surfaces of plants are covered by a protective barrier formed by the cutin polyester and waxes, collectively referred to as the cuticle. Plant cuticles prevent the loss of water, regulate transpiration, and facilitate the transport of gases and solutes. As the cuticle covers the outermost epidermal cell layer, it also acts as the first line of defense against environmental cues and biotic stresses triggered by a large array of pathogens and pests, such as fungi, bacteria, and insects. Numerous studies highlight the cuticle interface as the site of complex molecular interactions between plants and pathogens. Here, we outline the multidimensional roles of cuticle-derived components, namely, epicuticular waxes and cutin monomers, during plant interactions with pathogenic fungi. We describe how certain wax components affect various pre-penetration and infection processes of fungi with different lifestyles, and then shift our focus to the roles played by the cutin monomers that are released from the cuticle owing to the activity of fungal cutinases during the early stages of infection. We discuss how cutin monomers can activate fungal cutinases and initiate the formation of infection organs, the significant impacts of cuticle defects on the nature of plant–fungal interactions, along with the possible mechanisms raised thus far in the debate on how host plants perceive cutin monomers and/or cuticle defects to elicit defense responses. The aerial surfaces of plants are covered by a lipophilic protective shield called the cuticle. The cuticle acts as a diffusion barrier and, therefore, influences the diffusion of an array of molecules such as water, gases, and solutes . Yet, ap20 to C40 very-long-chain-fatty-acids (VLCFAs), which are further modified to form corresponding alkanes, aldehydes, ketones, primary and secondary alcohols, and esters  land on the host plant cuticle surface, germinate via germ tubes that eventually become the primary hyphae that penetrate through the host cuticle (Magnaporthe oryzae) spores germinate on the cuticle surface and develop a specialized infection structure called appressorium, a flattened organ that pressures the host plant surface eventually penetrating it via a penetration peg. This stage is considered biotrophic as the bulged hyphae that colonize the infected cells do not kill it (Blumeria graminis) germinate on the cuticle surface and typically develop an appressorium. This structure penetrates through the host plant cuticle and colonizes the intercellular space via a feeding structure called haustorium, which invades the host cell without piercing the plasma membrane and killing it  and enoyl-CoA reductase (ECR) in wheat (Triticum aestivum), both of which are important for VLCFA biosynthesis and the elongation reactions required for cuticular lipid biosynthesis, attenuates B. graminis spore germination  emr1 mutant, which is depleted in the leaf surface waxes due to a mutation in KCS6  leaves affected spore adhesion and fungal penetration during the early stages of infection , significantly induced spore germination and germ tube elongation, but had no effect on appressorium differentiation. Yet, the epicuticular waxes of tall fescue grass (Festuca arundinacea), which represents a nonhost species of this fungus, hindered fungal spore germination and appressoria formation  aldehydes trigger the spore germination and appressorium differentiation of B. graminis  epicuticular waxes fully restored the spore germination of the fungus  fruit triggers the spore germination and appressorium differentiation in Colletotrichum gloeosporioides  enzyme, were able to suppress the growth and reproduction of Golovinomyces orontii (Sclerotinia sclerotiorum (Cucumis sativus) fruit with lower expression of CsWAX2, a homolog of the Arabidopsis WAX2 gene involved in the biosynthesis of VLC wax alkanes, resulted in an overall reduction of 50% in the wax content of the fruit surface, owing to major reductions in C29 and C31 alkanes. Inoculation assays with B. cinerea demonstrated that its pathogenicity was dramatically impaired only at the fruit surface of this mutant, but not on the fruit surfaces of WT and WAX2-overexpression plants (CER3/YORE-YORE (YRE)/FACELESS POLLEN1 (FLP1) and has a pleiotropic phenotype, including an altered wax composition  Tnt1 retrotransposon insertion lines followed by the authors found that the inhibitor of rust germ tube differentiation 1 (irg1) mutant failed to promote the pre-infection structural differentiation of two rust pathogens, Phakopsora pachyrhizi and Puccinia emaculata, on the abaxial leaf surface. The chemical analysis of the epicuticular wax composition revealed a >90% reduction in C30 primary alcohols and overaccumulation of C29 and C31 alkanes in the leaves of the irg1 mutant  protein AtABCG32, an ABC transporter localized to the plasma membrane of epidermal cells, was suggested to export cutin precursors from these cells to the surface  plants with silenced or mutated expression of OsABCG31, the homolog of the Arabidopsis ABCG32, displayed increased cuticle permeability and were more resistant to M. oryzae , lacerata (cyp86a8/lcr), and bodyguard (bdg), which surprisingly accumulate more cutin, even though they carry mutations in key cuticle biosynthetic genes  mutant is deficient in its ability to oxidize long-chain ꞷ-hydroxy fatty acids to ꞷ-oxo fatty acids and, therefore, has less α,ꞷ-dicaroxylic fatty acids and more ꞷ-hydroxy fatty acids. This results in a disordered cuticle membrane structure and increased leaf cuticle permeability  fruit skin due to reduced expression of cutin regulator SHINE3 leads to higher susceptibility to B. cinerea -motif lipase/hydrolase family protein cutin deficient1 , the most abundant cutin monomer in barley, was amongst the most active compounds. Interestingly, these substances did not seem to have any inhibitory effect on pathogenic fungi when added to their growing media, further suggesting that the resistance observed in these treated plants is associated with the induction of plant defense responses (Solanum tuberosum) cells, where, again, HESA was the most active compound in the induction of transient alkalinization of the culture medium, implying an induced defense response. The authors also demonstrated that the application of cutin monomers stimulated the production of the plant stress hormone ethylene and activated the expression of defense-associated genes such as phenylalanine ammonia-lyase (PAL), glutathione S-transferase (GST), and 3-hydroxy-3-methylglutaryl-coenzyme A reductase , tomato, and apple to the epidermal surface of gently abraded hypocotyls of etiolated cucumber seedlings resulted in the generation of H2O2. The authors concluded that the physiological significance of this might be that upon cuticle degradation by fungal cutinases, the cutin monomers may act as H2O2 elicitors to induce defense responses . A study performed by oduction . A follo cinerea . Finally cinerea . Even th cinerea . Unlike  defects . To summlcr, lacs2, and pec1/abcg32, and also the cutinase-expressing CUTE plants  gene was identified and shown to encode a nuclear protein of multiprotein RNA-processing complexes and to be epistatic to lcr and bdg .  and bdg . A link ic acids .In this review, we delineate the multifaceted roles played by epicuticular waxes and released cutin monomers as chemical signaling molecules in the interactions between host plants and pathogenic fungi. The early and recent key reports we present in this fascinating field of research accentuate how these interactions are presumably far more complex than currently assumed. It is evident that these interactions are multifactorial, are regulated simultaneously by many components derived from both the pathogenic fungi and the host plant, and are highly influenced by the biochemical, structural, and permeability properties of the cuticle. Evidence shows that certain wax components affect pre-penetration and infection processes of fungi with different life styles, yet the mechanisms underlying these types of relationships are not fully known. Efforts to elucidate the roles of epicuticular waxes in plant–fungal interactions have thus far mostly utilized mutants with altered wax compositions. However, this approach is still challenging, as in most cases, compositional changes in one biochemical group of wax compounds are typically accompanied by changes in other groups of compounds.How the cutin monomers released from the cuticle by fungal cutinases during the early stages of infection are recognized by the host plant to elicit defense responses and acquired resistance to pathogens remains a question to be explicated. Thus far, several possible mechanisms have been proposed involving the production of ROS, the accumulation of fungitoxic compounds at the cuticle surface and a primed defense-related transcriptional response, all of which were associated with cuticle defects. In their review, While the cuticle has been solely attributed to aboveground tissues, a recent pioneering study showed that a cuticle-like cell wall structure covers plant root caps and contributes to its protection against abiotic stresses . The autGCA, EM, SS, and HC drafted the manuscript. GCA and HC prepared the figures. AA and HC edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The insulin-sensitizer pioglitazone exerts its cardiometabolic benefits in type 2 diabetes (T2D) through a redistribution of body fat, from ectopic and visceral areas to subcutaneous adipose depots. Whereas excessive weight gain and lipid storage in obesity promotes insulin resistance and chronic inflammation, the expansion of subcutaneous adipose by pioglitazone is associated with a reversal of these immunometabolic deficits. The precise events driving this beneficial remodeling of adipose tissue with pioglitazone remain unclear, and whether insulin-sensitizers alter the lipidomic composition of human adipose has not previously been investigated. Using shotgun lipidomics, we explored the molecular lipid responses in subcutaneous adipose tissue following 6months of pioglitazone treatment (45mg/day) in obese humans with T2D. Despite an expected increase in body weight following pioglitazone treatment, no robust effects were observed on the composition of storage lipids  or the content of lipotoxic lipid species  in adipose tissue. Instead, pioglitazone caused a selective remodeling of the glycerophospholipid pool, characterized by a decrease in lipids enriched for arachidonic acid, such as plasmanylethanolamines and phosphatidylinositols. This contributed to a greater overall saturation and shortened chain length of fatty acyl groups within cell membrane lipids, changes that are consistent with the purported induction of adipogenesis by pioglitazone. The mechanism through which pioglitazone lowered adipose tissue arachidonic acid, a major modulator of inflammatory pathways, did not involve alterations in phospholipase gene expression but was associated with a reduction in its precursor linoleic acid, an effect that was also observed in skeletal muscle samples from the same subjects. These findings offer important insights into the biological mechanisms through which pioglitazone protects the immunometabolic health of adipocytes in the face of increased lipid storage. Adipose tissue is the primary site for fat storage and quantitatively the most important energy reservoir in the body. The coordinated expansion and breakdown of adipose lipid stores is crucial to the dynamic regulation of circulating nutrient availability and plays a central role in the control of whole-body metabolism. Excessive nutrient storage in adipose tissue  promotes adipocyte dysfunction, inflammation, and insulin resistance and is therefore strongly implicated in the etiology of type 2 diabetes T2D; .Pioglitazone is an insulin-sensitizing drug approved for the treatment of T2D. The molecular effects of pioglitazone are primarily mediated through the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ), a transcriptional regulator of adipocyte differentiation and lipid storage which is highly abundant in adipose tissue . AlthougRecent reports have suggested that the expansion of adipose tissue following pioglitazone treatment could be driven by the formation of new adipocytes  in subcutaneous adipose depots . This isThe lipid composition of human adipose tissue is dominated by triglyceride species , but alsThe objective of the present study was to determine the impact of pioglitazone treatment on molecular lipids in adipose tissue from obese type 2 diabetics, using a multi-dimensional mass spectrometry-based shotgun lipidomics approach , which f2; HbA1c 8.0±0.6%) treated with diet alone or diet plus metformin and/or sulfonylurea participated in the study, which was approved by the Institutional Review Board of the South Texas Veterans Healthcare System, University of Texas Health Science Center San Antonio. After providing fully informed consent and completing a routine health screening visit, eligible subjects reported to The Bartter Clinical Research Unit of the South Texas Veterans Healthcare System following an overnight ~10-h fast. A baseline blood sample was drawn for the measurement of fasting blood glucose, HbA1c, and triglycerides, and subcutaneous abdominal adipose tissue and vastus lateralis muscle biopsies were obtained under local anesthesia (1% Lidocaine) for lipid profiling and gene expression analysis. Baseline measurements were repeated after 6months of pioglitazone treatment (45mg/day). Due to biopsy sample availability, lipidomic analyses were carried out on six of the seven subjects for adipose tissue and five of the seven subjects for skeletal muscle tissue, such that lipidomic analyses in at least one tissue are presented for all subjects. Clinical and adipose gene expression data are presented for all seven subjects.Seven obese individuals with T2D  equipped with a Nanomate device  and Xcalibur system software were used as previously described  were homogenized in ice-cold diluted (10%) phosphate-buffered saline, and lipids were extracted by a modified Bligh and Dyer procedure in the presence of internal standards added based on total protein content, as previously described . A triplescribed . Brieflyescribed .FADS1, FADS2, ELOVL5, CDS1, CDIPT, PLA2G4A, PLA2G4C, PLA2G7, and PLA2G16. Data were normalized to the geometric mean of the reference genes ACTB and GAPDH and expressed as a fold change relative to the baseline (pre-treatment) mean.Target mRNA expression was determined in adipose tissue lysates by qRT-PCR as previously described  using tht tests. Overall lipidomics data patterns were initially visualized by principal component analyses (PCA) performed on raw data for quantified species (in nmol/mg protein). Lipid data were then scaled to the total molar content of all detected species in that lipid class. Compositional changes in lipid classes were subsequently evaluated by comparing individual species or groups of individual species (for acyl chain length and saturation profiles) with multiple paired t tests, controlling for a<10% false discovery rate (FDR) using the two-stage step-up method of Benjamini, Krieger, and Yekuieli (GraphPad Prism 9). Gene expression changes were assessed by multiple paired t tests controlling for FDR.Clinical data and lipid class totals are expressed as mean±standard error and were compared using paired p=0.085). Subjects gained an average of 3.2kg body weight following pioglitazone treatment  revealed that much of the variance in adipose lipid species was driven by between-subject differences . HoweverGlycerophospholipids are critical components of cell membranes and the preferential trafficking of polyunsaturated fatty acids (PUFA) toward these complex lipids plays an important role in the regulation of membrane fluidity . ChangesThe fatty acid desaturases (FADS1 and FADS2) catalyze the rate-limiting enzymatic steps in the generation of PUFA, and an index of the combined flux through these pathways can be estimated from the product-to-substrate ratio of arachidonic acid to linoleic acid in tissue lipids . Consistde novo lipogenesis (DNL), which can be similarly estimated from the ratio of non-essential fatty acids (which can be synthesized) to essential fatty acids (obtained exclusively from the diet) in the glycerophospholipid pool , 26 species  were found to be altered by pioglitazone treatment  are the most abundant glycerophospholipids in adipose tissue  and reprholipids , pioglitin in PE , as wellin in PE . In contreatment ,B.sn-2 position of phosphatidylcholine (PC), another major constituent of cellular membranes, consistent with the lower enrichment of AA in PC vs. PE , which are especially highly enriched in AA  and haveC vs. PE . The incC vs. PE .3) and therefore playing a vital role in the regulation intracellular insulin signaling  of the same subjects. Free linoleic acid concentrations in skeletal muscle were~15% lower following pioglitazone treatment , including phosphatidylinositols and plasmenylethanolamines. These changes were not dependent upon transcriptional activation of phospholipase genes but were associated with a decrease in adipose tissue levels of free AA and its parent precursor linoleic acid. Notably, despite weight gain, which is likely indicative of an expansion of the subcutaneous adipose tissue depots , the molin vitro models of adipogenesis  or from the suppression of AA synthesis and/or esterification. In alignment with the former possibility, one previous study demonstrated that acute incubation of fibroblasts with pioglitazone accelerated the release of AA from cell membranes . MoreoveAnother interesting finding from the current study was that levels of linoleic acid were reduced following pioglitazone treatment, both in adipose and skeletal muscle tissues. Since linoleic acid is an obligate precursor for the synthesis of other omega-6 PUFAs, it seems likely that its reduction contributed to the decrease in AA available for esterification into glycerophospholipids. Notably, the lower linoleic acid level was also mirrored by a significant reduction in adipose tissue cardiolipins, a mitochondrial membrane lipid highly enriched in linoleoyl side chains. Cardiolipin is often used as a marker of mitochondrial mass and, as such, this finding appears to contradict previous reports that thiazolidinediones increase mitochondrial content and respiratory complex expression in human adipose tissue . HoweverThe mechanism through which pioglitazone treatment lowered tissue linoleic acid is unclear. Since we did not monitor dietary patterns in the current study, we cannot exclude the possibility that the intake of linoleic acid, an essential fatty acid, declined over the six-month intervention period. However, alterations in dietary linoleic acid do not appear to influence the systemic availability of either linoleic acid or arachidonic acid , suggestIt should be acknowledged that the reduction in PUFA-containing lipids could be indicative of their selective metabolism by peroxisomal β-oxidation, since the master driver of peroxisomes PPARα  has beenin vitro evidence that free AA availability can influence its enrichment in phosphatidylinositol (2 and PIP3) in the insulin signaling pathway  male sample and thus warrant further validation in expanded populations, especially given the increasingly appreciated role of sexual dimorphism in adipose tissue metabolism and health . The lacThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board of the South Texas Veterans Healthcare System, University of Texas health Science Center San Antonio. The patients/participants provided their written informed consent to participate in this study.All authors contributed to the data analysis and interpretation of the data, drafting, and revising the manuscript and approved the final version of the manuscript. The study design was conceptualized by JP, DT, RD, and CS. Methodology was performed by JP, AC-V, DT, MP, and MF. Funding acquisition, RD and CS.CS is supported by an RL5 training scholarship from the San Antonio Claude D. Pepper Older Americans Independence Center.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mobile health  apps may provide an efficient way for patients with lower urinary tract symptoms (LUTS) to log and communicate symptoms and medication side effects with their clinicians.The aim of this study was to explore the perceptions of older men with LUTS after using an mHealth app to track their symptoms and tamsulosin side effects.Structured phone interviews were conducted after a 2-week study piloting the daily use of a mobile app to track the severity of patient-selected LUTS and tamsulosin side effects. Quantitative and qualitative data were considered.All 19 (100%) pilot study participants completed the poststudy interviews. Most of the men  reported that the daily questionnaires were the right length, with 32% (n=6) reporting that the questionnaires were too short. Men with more severe symptoms were less likely to report changes in perception of health or changes in self-management; 47% (n=9) of the men reported improved awareness of symptoms and 5% (n=1) adjusted fluid intake based on the questionnaire. All of the men were willing to share app data with their clinicians. Thematic analysis of qualitative data yielded eight themes: (1) orientation , (2) triggers (routine or habit and symptom timing), (3) daily questionnaire , (4) technology literacy, (5) perceptions , (6) self-management, (7) clinician engagement (communication and efficiency), and (8) improvement .We assessed the perceptions of men using an mHealth app to monitor and improve management of LUTS and medication side effects. LUTS management may be further optimized by tailoring the mobile app experience to meet patients’ individual needs, such as tracking a greater number of symptoms and integrating the app with clinicians’ visits. mHealth apps are likely a scalable modality to monitor symptoms and improve care of older men with LUTS. Further study is required to determine the best ways to tailor the mobile app and to communicate data to clinicians or incorporate data into the electronical medical record meaningfully. Lower urinary tract symptoms (LUTS) comprise a complex and heterogenous syndrome, including urinary urgency, urinary frequency, weak urinary stream, hesitancy, straining, incomplete bladder emptying, nocturia, and urinary incontinence . LUTS arMobile apps are increasingly used to monitor symptoms and side effects in a wide range of urologic conditions. For men with LUTS, mobile apps have been successfully used to administer questionnaires , increasTo our knowledge, the use of mobile apps to track symptoms and side effects in men with LUTS has not been studied using qualitative methodology. Qualitative studies are uniquely equipped to explore the patient perspective and help ensure that future intervention designs are grounded in the patient experience. Thus, we designed the Placebo-Controlled, Randomized, Patient-Selected Outcomes, N-of-1 Trials (PERSONAL) pilot study, a 2-week intervention to determine the feasibility and acceptability of daily LUTS severity and tamsulosin side-effect assessment through a mobile app among older men with LUTS receiving chronic tamsulosin therapy. Following this study period, we aimed to explore the men’s insights about using an mHealth app to track their symptoms and medication side effects via interviews. Here, we present the findings and implications of the first mixed methods study exploring the experience of men with LUTS after using an mHealth app to track their symptoms and medication side effects.A convenience sample of 19 men was recruited from an academic urology clinic for the PERSONAL pilot study. Recruitment was targeted at men with LUTS who may be unsure if the benefits of tamsulosin outweighed the harms, specifically older men who were both receiving chronic tamsulosin therapy and interested in tracking their daily urinary symptoms and tamsulosin side effects outside of regular clinic visits. Men were eligible if they were taking tamsulosin for LUTS for at least 12 months, received care from a urologist at our institution, and had previously consented to being contacted for research purposes via the electronic medical record. The sample size was based on feasibility of recruitment. Additional inclusion and exclusion criteria are outlined in To gain a more nuanced understanding of participants’ experiences using the PERSONAL mobile app following the 2-week data collection period, semistructured feasibility interviews were administered by telephone, ranging from 10 to 30 minutes. The interviewer (EYW) was a medical trainee and research associate with formal qualitative research training, who had no interactions with the participants prior to the interviews. The interview guide is available in Continuous variables were reported using median and IQR, and categorical data were summarized using frequencies. Thematic analysis of open-ended questions yielded 22 codes, which were refined into eight themes. EYW completed initial coding; codes and themes were finalized in discussion with coauthors SRB and SAK. Qualitative data were integrated relative to quantitative responses . The stuAll 19 participants who participated in the 2-week PERSONAL pilot study completed poststudy interviews; their demographics and self-selected symptoms and side effects are reported in About two-thirds of participants felt that the questionnaires were just the right length, and about one-third felt that they were too short; the latter participants reported concerns about adequate detail or the appropriate gradations used in the questions and answer choices, commenting that those with milder symptoms might prefer additional gradations on the lower end of the spectrum. Some participants reported frustration about the side-effects questions, as they had difficulty parsing out which side effects to specifically attribute to tamsulosin. A few men were willing to log answers for seven to eight symptoms and side effects rather than the recommended limit of three. About two-thirds of participants set app notifications for mornings, which they found particularly useful for reporting nocturnal symptoms, such as nocturia.Some participants reported that they did not receive daily notifications via the mobile app due to technical difficulties, but the large majority of participants completed the questionnaire by “habit” at the same time every day—no participants logged responses purely as an immediate response to bothersome symptoms. Out of 19 participants, 9 (47%) felt that this app changed their perception of their health or LUTS management; 8 of these 9 patients (89%) reported increased awareness of symptoms and 1 participant (11%) was able to adjust his fluid intake to improve LUTS. All participants would be willing to share data with their clinician to complement their usual care.Almost half  of the participants reported prior use of other health apps or devices. When prompted to compare those experiences with the study app, most agreed that this app had a simpler interface than others they had encountered, though 1 participant (5%) noted that the app may still be inaccessible in populations with lower English proficiency.The qualitative analysis generated eight themes  and 22 codes organized into three categories  whose relationships are illustrated in In sum, mHealth apps may play an important role in the chronic management of LUTS. In this study, the key themes that emerged from our qualitative data were (1) orientation, (2) triggers, (3) daily questionnaire, (4) technological literacy, (5) perceptions, (6) self-management, (7) clinician engagement, and (8) improvement. Consistent with prior literature ,13, our Most American men and adults over the age of 65 years own a smartphone . Mobile Studies support the need for expert involvement in mobile app development, dissemination, and regulation ,16,18,19This study offers insights into the benefits and patient concerns associated with tracking symptoms via a mobile app among older men with benign urologic conditions, but there are some limitations. Traditionally, qualitative interviews are audio-recorded unstructured or semistructured interviews. Given the focus of the study, the homogeneous population, and the strong convergence of responses around a few common themes, we likely reached data saturation among 19 participants . We soliDespite the limitations, this is the first mixed methods study to examine the use of a mobile app to track symptoms in older men with LUTS. Importantly, these results suggest that mobile apps designed to improve symptom awareness and management may be adapted to benefit older men with LUTS due to BPH. As the use of mobile apps becomes increasingly popular, their usage in the health care setting will require further optimization. Tailoring to individual patients’ health and technology literacy levels, language proficiency, and symptom severity will be critical to maximizing the efficacy of these digital interventions. Incorporating the use of apps into clinical practice may play another important role, pending future study.


Cells and tissues in the human body are subjected to mechanical forces of varying degrees, such as tension or pressure. During tumorigenesis, physical factors, especially mechanical factors, are involved in tumor development. As lung tissue is influenced by movements associated with breathing, it is constantly subjected to cyclical stretching and retraction; therefore, lung cancer cells and lung cancer-associated fibroblasts (CAFs) are constantly exposed to mechanical load. Thus, to better explore the mechanisms involved in lung cancer progression, it is necessary to consider factors involved in cell mechanics, which may provide a more comprehensive analysis of tumorigenesis. The purpose of this review is: 1) to provide an overview of the anatomy and tissue characteristics of the lung and the presence of mechanical stimulation; 2) to summarize the role of mechanical stretching in the progression of lung cancer; and 3) to describe the relationship between mechanical stretching and the lung cancer microenvironment, especially CAFs. The main function of the lungs is to exchange oxygen and carbon dioxide with the outside world. The lung and thorax are important organs in the human respiratory system  Novak e. The resLung tissue is constantly influenced by breathing mechanics, and it is constantly exposed to a state of cyclical stretching and retraction. Therefore, lung cancer cells and lung cancer-associated fibroblasts (CAFs) are also influenced by mechanical load . FibroblThus, to better explore the progression of lung cancer pathogenesis, it is necessary to combine cell mechanics factors to obtain a more comprehensive analysis. The purpose of this brief review is: 1) to provide an overview of the anatomy and tissue characteristics of the lung and the presence of mechanical stimulation; 2) to summarize the role of mechanical stretching in the progression of lung cancer; and 3) to describe the relationship between mechanical stretching and lung cancer microenvironment, especially CAFs.The lungs are in the chest cavity, on either side of the mediastinum, above the diaphragm. The lungs are composed of two functional areas: 1) the airways through which air enters and passes the pharynx, larynx, trachea, main bronchi, bronchioles, and terminal bronchioles; and 2) the respiratory zone (gas exchange zone), including the alveolar tubes, bronchioles, and alveolar sacs .2) is continuously provided to the body and carbon dioxide (CO2) is discharged to maintain the body’s blood and gas balance and internal environment in equilibrium. The alveoli, which provide a large surface area for gas exchange, are lined with a thin layer of epithelial cells that form a tight single membrane on the basement membrane (1.66 ± 0.128 μm) in normal lungs  restrictive diseases, which include a reduction in the ability of the lungs to expand and 2) obstructive diseases, which cause increased airway resistance and restricted airflow. Restrictive or obstructive diseases a classified based on clinical measures, including the ratio of forced expiratory volume to full forced lung capacity in one second (FEV1/FVC) and total lung capacity percentage (TLC%) . The resin vivo model of bleomycin-induced pulmonary fibrosis to study the gene expression profile of fibroblast populations. They found that osteopontin is highly overexpressed in lung fibroblasts and can serve as a marker of CAFs activation. It has been reported that biomarkers commonly expressed by CAFs include α -smooth muscle actin (α-SMA), fibroblast specific protein-1 (FSP-1), fibroblast activating protein α (FAP-α), platelet-derived growth factor receptor -β (PDGFR-β), neural/glial antigen (NG2) (+/GPR77+ CAFs promoted cancer development and chemoresistance by sustaining cancer stemness (Pulmonary fibrosis (PF) is a chronic, restrictive lung disease in which excessive collagen deposition results from the accumulation of scar tissue in the lung caused by lung injury, inflammation, and/or long-term exposure to toxins or particles. Idiopathic pulmonary fibrosis (IPF) is a chronic progressive pulmonary fibrosis disease with unknown reasons. In recent years, more and more studies have shown that IPF is closely related to the occurrence of lung cancer. Also, IPF diagnosis and treatment guidelines in 2011 clearly indicate that IPF is prone to lung cancer, pulmonary embolism, pulmonary hypertension, and other lung diseases . Accordien (NG2) , and Gamen (NG2) . Su S etstemness . Interesstemness .via mechanoreceptors. These receptors often encounter the ECM, where external signals are converted into physiological responses that affect cell proliferation, differentiation, and migration  are detected and transmitted to cells igration .in vivo and with acute lung injury  cyclic uniaxial or biaxial, equi-biaxial strain for cell stretch stimulation (2+ levels in plasma (Many researchers have developed mulation  2) statimulation ; 3) micrmulation ; 4) gradmulation ; 5) cyclmulation ; 6) intemulation . The effmulation , cytoskemulation , and Ca2n plasma . Nitric n plasma . Endothen plasma .Like normal cells, cancer cells recognize the mechanical changes provided by the tumor microenvironment and convert them into signaling pathways through mechanical transduction pathways  Sporn a. Wang etThe lung is a complex organ, and types of mesenchymal cells are found in its tissues. Fibroblasts are one of the most important mesenchymal cells that contribute to maintaining the lung’s normal physiological function. The heterogeneity of fibroblasts is the key reason for their different phenotypes and functions . FibroblFibroblasts are cells that respond to mechanical stretching stimuli . They maAfter the lung tissue is slightly injured, fibroblasts in the lung parenchyma differentiate into myofibroblasts  to promoin vitro studies have shown that mechanical stretching with higher strain ranges (20–30%) activates the Ca2+ influx pathway independently of the actin cytoskeleton and the conventional stretch sensitive ion channel  family proteins, Piezo1, and Piezo2). Moreover, ATP is released in human lung fibroblasts after mechanical stretching ; thus, they are also known as CAFs. Compared with resting fibroblasts, CAFs present a larger morphology, nuclear depression, and more branching cytoplasm , and actCAFs are cells with mechanical ability and are the most abundant stromal cell type in the tumor microenvironment . They plThere is an inverse relationship between traction stress and metastatic capacity of breast cancer cells, and the generation of force decreases with the increase of metastatic potential and capacity . Studiesvia ECM modification and chemokine/cytokine secretion  in normal respiration. There are few studies on the mechanism of lung tumors causing changes in lung physical properties and then affecting lung cells.Due to the complexity of the lung cancer classification, there may be different types of lung CAFs, but once this role of mechanotransduction on lung cancer cells is recognized, this will be accompanied by further in-depth research. In the future, it will be extremely challenging and of practical significance to isolate different types of lung CAFs associated with different types of lung cancer, and elucidate the specific mechanisms involved in mechanical stimulation on tumor progression.