Forkhead box (Fox) family, an evolutionarily conserved family of transcription factors carrying the “Forkhead” motif, plays an indispensable role in human health and disease. Fox family genes are involved in cell differentiation, proliferation and apoptosis, embryonic development, aging, glucose and lipid metabolism, and immune regulation. The regulatory role of the Fox family in the context of bone metabolism and orthopedic diseases is an emerging research hotspot. In this review, we highlight the major molecular mechanisms underlying the regulatory role of Fox factors in bone metabolism, bone development, bone homeostasis, and bone diseases associated with inhibition or upregulation of Fox factors. In addition, we discuss the emerging evidence in the realm of Fox factor-based therapeutics. Fox family, identified in 2000 , is a grThe role of Fox in the regulation of bone metabolism was first recognized in 2001 when a defective reproductive phenotype was identified as a molecular marker of prospective rib cartilage . Since tStudies have demonstrated differential expression of Fox factors in osteoblasts in the setting of skeletal disease compared with normal osteoblasts; these differentially expressed factors have been shown to promote or suppress the development of osteoporosis by regulating bone metabolism . Bone meAlthough the close relationship between Fox factor and skeletal disease is well established, the role of Fox factors in promoting or inhibiting skeletal disease and the associated underlying mechanisms are highly controversial and perplexing. In this review, we summarize the available evidence of the functional role of the Fox family in the context of bone-associated cell metabolism and various bone diseases . In addiOsteoporosis is a metabolic bone disease  caused bFoxO1 was found to inhibit the expression of osteogenic markers, such as Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN), even in the presence of strong osteogenic stimulants, resulting in decreased culture calcification  . Besidesin vitro, and the bone degradation attenuated by tamoxifen-induced FoxM1 deletion was partly reversed by the overt transfer of FoxM1+/+ CX3CR1+ monocytes in vivo  die shortly after birth and exhibit skeletal abnormalities and defects  promotes osteogenic differentiation of BMSCs partially activating the ERK pathway  and over67 .There is inconsistency in the reported effects of FoxD3 and FoxF1 on osteoblast differentiation . HoweverThe above evidence demonstrates that Fox factor plays a key role in osteoporosis pathology by acting on both osteoblast differentiation and osteoclast differentiation or by influencing osteoblast differentiation alone to regulate bone metabolism.in vitro experiment, upregulation of FoxO increased the expressions of autophagic genes , which could prevent aging, protecting cartilage by mediating apoptosis, and elimination of ROS  is a fibrocartilaginous tissue that lies between two vertebrae and functions as a shock absorber. It includes the jelly-like nucleus pulposus, the surrounding fibrocartilaginous annulus fibrosus, and the cartilaginous endplate anchoring the IVD to the corpus vertebrae . Interveral tube . Recentlral tube .2+ (Osteosarcoma (OS) is the most common primary malignant tumor of bone, and it occurs mainly in children and adolescents . Recentl2+ .Second, FoxM1 is associated with tumor cell proliferation, migration, invasion, and angiogenesis, suggesting that FoxM1 is an oncogenic factor . FoxM1 iThird, FoxC2, a transcription factor involved in epithelial–mesenchymal transition (EMT), is defined as a carcinogenic factor . SilenciWAF1/CIP1 107. Upregulation of miR-491-5p suppressed proliferation of human OS cells and induced apoptosis by targeting FoxP4  is a rare and highly aggressive cancer that occurs primarily in the bones and surrounding tissues of children and adolescents. It is the second most common primary malignant bone tumor in children and adolescents . The patFoxO1, as previously mentioned, is a tumor-suppressor factor with low expression in ES caused by the repressive effect of EWS/FLI1 binding to the FoxO1 promoter . BesidesFoxM1, an oncogenic factor, is highly expressed in Ewing sarcoma and cell lines. Reduction of FoxM1 expression impairs the ability of Ewing cell lines to grow in an anchorage-independent manner . Based oHigh expression of FoxQ1 was detected in human ES lines KH and EWS. FoxQ1 may be associated with enhanced activation of downstream target genes through interaction with EWS–FLI1 at the N-terminal end of EWS, thereby promoting ES proliferation .PTHLH gene , development of new and more effective treatments is a key imperative. The role of the Fox gene family represents an entirely new area of research in bone metabolism that is expected to address this challenge.Although Fox factors have been identified to play a pivotal role in the maintenance of bone homeostasis, much of the contemporary research is limited to cellular and animal studies. The use of drugs targeting Fox factors in the treatment of bone tumors has been attempted; however, there are few reports of clinical efficacy. Apart from this, development of targeted drugs for Fox factors in the clinical treatment of other skeletal diseases is still at a theoretical stage. Therefore, clarifying the prospects for clinical application of the Fox factors and development of Fox-targeted drugs represent key areas of future research.


Embolization is a well-known and accepted form of treatment for bleeding caused by a multitude of renal procedures. We present a case of a 66-year-old woman who had a history of left nephrectomy for clear cell carcinoma seven years previously and now presented with a 6 cm tumor involving the solitary kidney. She underwent partial laparoscopic nephrectomy with removal of the tumor on the right kidney. In the immediate postoperative period she had important and persistent hematuria associated with tachycardia, hypotension, and lumbar pain. After showing signs of hemodynamic instability, she was taken to the catheter laboratory where selective angiography of the right kidney was performed. Superselective embolization with controlled release of fibrous microcoils was performed. The superselective renal embolization technique performed on an emergency basis to control hemorrhage after a urological procedure is effective and achieves lasting resolution of symptoms. Development of innovative materials and improved surgical techniques have allowed it to be employed in a preventive manner in vascular defects, cancer, and even for therapeutic embolization of iatrogenic and spontaneous hematuria or after urological procedures.2-5The main criteria for choosing the technique are minimal invasiveness, low morbidity and mortality rates, few early or late complications, lack of need for general or intrathecal anesthesia, and low interference in renal function.3The objective of this article was to report the effectiveness of superselective angioembolization of the renal artery in a case of hemorrhage in the urinary tract after partial nephrectomy in a patient with a solitary kidney.We report the case of a 66-year-old woman who was hypertensive, dyslipidemic, and an ex-smoker who had undergone left nephrectomy for clear cell carcinoma seven years previously, and then presented on with a 6 cm tumor involving the solitary kidney affected and a 1.4 cm aneurysm of the middle third of the renal artery . She undIn view of this presentation, the following treatment options were immediately considered: conventional surgical treatment with residual nephrectomy or endovascular treatment with embolization of the segmental renal artery with detachable coils.Micromola fibrada Concerto – EV3) was released into the affected branch. The degree of occlusion was determined with angiography after each microcoil deployment (three units). After total occlusion of the bleeding, a post-embolization renal angiography was performed to reveal a small avascular segment, preserving all vascularization to the remaining kidney (The patient was taken to the hemodynamics department and underwent intervention. Briefly, the right common femoral artery was punctured with the aid of ultrasound and a 5-F introducer sheath was inserted. Right segmental renal artery angiography identified active bleeding at a site suggestive of a pseudoaneurysm and a 2.4-F superselective coaxial catheter was advanced as close as possible to the hemorrhagic branch for selective and superselective renal angiography . Repeateg kidney . The pro,2,4Treatment of renal vascular lesions depends on the etiology and clinical course of the disease. Hemorrhagic complications after urological procedures have been preferably treated by endovascular access since the 1970s, with applications in several clinical scenarios.1Laparoscopic urological interventions have made it possible to perform numerous minimally-invasive diagnostic and therapeutic renal procedures, enabling “nephron-sparing” management. Nevertheless, the risk of renal vascular lesions with active bleeding, pseudoaneurysm, arteriovenous fistula, or a combination of these has remained unpreventable.-5-7Endovascular control by angioembolization is a minimally invasive and safe method with proven efficacy for management of hemorrhagic urological emergencies.1,4-3,4Progressive advances in interventional radiographic techniques, such as enhanced imaging and the introduction of smaller devices, as well as more accurate embolic agents, have made superselective embolization of the renal artery an effective approach to diagnosis and treatment of renal hemorrhage.1-2,6,7,9Various embolic agents have been described in the literature for controlling bleeding in the renal region, but most series report embolization with coils as the preferred technique for renal artery embolization in several clinical scenarios.6-5Reported technical success rates vary between 71 and 100%.4Sam et al.,8In summary, the superselective renal embolization technique performed on an emergency basis to control hemorrhage after a urological procedure is effective, with immediate and lasting resolution of symptoms, aiding in maximum renal preservation particularly in patients with a solitary kidney.


Sterna hirundo) preliminarily named tern atadenovirus 1 (TeAdV-1). TeAdV-1 genome is a linear double-stranded DNA molecule, 31,334 base pairs which contain 30 methionine-initiated open reading frames with gene structure typical for Atadenovirus genus, and the shortest known inverted terminal repeats (ITRs) within the Atadenovirus genus consisted of 25 bases. The nucleotide composition of the genome is characterized by a low G + C content (33.86%), which is the most AT-rich genome of known avian adenoviruses within Atadenovirus genus. The nucleotide sequence of the TeAdV-1 genome shows high divergence compared to known representatives of the Atadenovirus genus with the highest similarity to the duck atadenovirus 1 (53.7%). Phylogenetic analysis of the protein sequences of core genes confirms the taxonomic affiliation of the new representative to the genus Atadenovirus with the degree of divergence from the known representatives exceeding the interspecies distance within the genus. Thereby we proposed a novel TeAdV-1 to be considered as a separate species.Discovery and study of viruses carried by migratory birds are tasks of high importance due to the host’s ability to spread infectious diseases over significant distances. With this paper, we present and characterize the first complete genome sequence of atadenovirus from a tern bird (common tern,  Atadenovirus, Aviadenovirus, Ichtadenovirus, Mastadenovirus, Siadenovirus, and recently accepted Testadenovirus . As noted earlier, in addition to a solid pool of phylogenetic evidence, the TeAdV-1 virus does not share a host with any other described Atadenovirus species, has a different nucleotide composition from the most related species , and also contains genes unique to its genome.It should be noted that according to the current criteria approved by the ICTV, species designation in the Atadenovirus (or even higher taxa) undergoes rapid gene evolution throughout the evolutionary history under consideration.Pervasive selection, which we found in the core genes of all tested representatives of AdVs, confirms that the genus Genes and specific sites under pressure from the long-term positive selection can be significant in the arms race. Indeed, the phenomenon of pervasive selection is generally most prevalent in pathogen evolution and any biological system influenced by evolutionary arms race dynamics , including adaptive immune escape by viruses . This efAtadenovirus representatives: DNA replication machinery  and capside proteins (III (penton base), pIIIa). Such sites are evolutionary hotspots under the constant pressure of adaptive selection and, summarizing all of the above, can be directly involved in antagonistic communication between the virus and host cells  in the genes, encoding the following proteins of the TAtV-1: DNA replication machinery , DNA packaging machinery (pIVa2), and 100 K protein. The specific sites we discovered are under the pressure of positive selection (ω > 1) in the TeAdV-1 genome, while in the genomes of other representatives of Atadenovirus genus the sites mentioned above are under the pressure of negative selection (ω < 1) or evolve neutrally (ω = 1). Such independent events of adaptive evolution might be associated with the speciation process and adaptation to a new host. It has already been shown that positive selection can be associated with crossing the species barrier. As an example, episodic events of positive selection in the molecular evolution of bats rabies virus were detected during the repeated host shifts [Atadenovirus in the phylogenetic tree are species that infect birds . It is well known that host shifts tend to occur between related species [The episodic positive selection that we found in the TeAdV-1 genome confirms that the virus underwent rapid evolution. We found sites under the pressure of episodic positive selection (t shifts . Our ass species .


With the improvements of sanitation and nationwide safe water supply the occurrence of bacterial diarrhea declined remarkably, while viruses became the leading causes of acute gastroenteritis (AGE). Of these viruses, noroviruses (NoVs) are responsible for a considerable burden of gastroenteritis, especially in children < 2 years and elderly ⩾ 65 years. NoVs circulating in the Chinese population are antigenically highly diverse with the genotype GII.4 being the dominant strain followed by GII.3. Given the widespread contamination in environmental sources, and highly infectious nature of NoVs, vaccination would be the desirable strategy for the control of NoV infections. However, a better understanding of acquired immunity after infection, and a reliable immunological surrogate marker are urgently needed, since two vaccine candidates based on virus-like particles (VLPs) are currently moving into clinical evaluations in China.


The most common scoring system for critically ill patients is the Sequential Organ Failure Assessment (SOFA) score. Little is known about specific molecular signaling networks underlying the SOFA criteria. We characterized these networks and identified specific key regulatory molecules. We prospectively studied seven patients with sepsis and six controls with high-throughput RNA sequencing (RNAseq). Quantitative reverse transcription PCR (RT-qPCR) confirmation was performed in a second independent cohort. Differentially and significantly expressed miRNAs and their target mRNA transcripts were filtered for admission SOFA criteria and marker RNAs for the respective criteria identified. We bioinformatically constructed molecular signaling networks specifically reflecting these criteria followed by RT-qPCR confirmation of RNAs with important regulatory functions in the networks in the second cohort. RNAseq identified 82 miRNAs (45% upregulated) and 3254 mRNAs (50% upregulated) differentially expressed between sepsis patients and controls. Bioinformatic analysis characterized 6 miRNAs and 76 mRNA target transcripts specific for the SOFA criteria. RT-qPCR validated miRNA and mRNAs included IGFBP2 (respiratory system); MMP9 and PDE4B (nervous system); PPARG (cardiovascular system); AKR1B1, ANXA1, and LNC2/NGAL (acute kidney injury); GFER/ALR (liver); and miR-30c-3p (coagulopathy). There are specific canonical networks underlying the SOFA score. Key regulatory miRNA and mRNA transcripts support its biologic validity. Since its development in the 1990s, the Sequential Organ Failure Assessment (SOFA) score has become a standard instrument in the ICU to quantify acute morbidity of critically ill patients  and the SOFA is based on six different sub scores, one each for the respiratory, cardiovascular, hepatic, coagulation, renal, and neurological systems, which span from 0 to 4, and a final summary score is then calculated ranging from 0 to 24 points . IncreasDespite the widespread use and clinical validation of the SOFA score, it merely reflects a numeric simplification of morbidity. There appears to be a missing link between the SOFA criteria  and the specific molecular signaling patterns underlying these criteria.We addressed this issue by high-throughput and unbiased sequencing (RNAseq) of small non-coding miRNAs and protein-coding mRNAs from blood cells in patients with sepsis and healthy controls. For both types of RNA, transcripts significantly regulated between patients and controls were first identified by differential gene expression analyses. Bioinformatic methods were then applied to construct gene networks and to identify key regulatory molecules that specifically reflect the SOFA criteria from the RNAseq data set. We assumed that this novel approach could help to validate the score on a molecular level and may also be applied to the development of more refined scoring systems combining numeric indicators of organ dysfunctions with molecular findings.n = 20) and healthy controls (n = 5) was then matched to the original RNAseq group from an independent and prospectively recruited sample of patients with septic shock . For the initial characterization of SOFA-related molecular networks by RNAseq, we used blood cell-derived RNA in seven patients with septic shock and six healthy controls. mRNA sequencing was performed, while miRNA expression data were already available from this group . We idenSepsis was diagnosed according to the 2016 Sepsis-3 definition [SOFA calculation for the study was performed at admission to the ICU. Blood sampling and recording of clinical data were performed at admittance to the ICU, along with the calculation of the admission SOFA. Details on blood sampling, sample collection, RNA processing, high-throughput sequencing, and RT-qPCR confirmation are given in the We included patients with sepsis fulfilling the following criteria: (1) age ≥ 18 years and (2) onset of signs of infection within the last 24 h severe enough to require ICU admission. Exclusion criteria were (1) pregnancy, (2) known infection by virus , (3) malignancy, (4) pre-existing immunologic disorders , (5) immunosuppressive medication , and (6) severe pre-existing cardiovascular disorders.Ingenuity pathway analysis  was used to identify functional interactions between the observed miRNAs, mRNAs, and protein functions.For network analysis and identification of significant pathways in relation to the SOFA symptom criteria, we used the file containing all significantly regulated miRNAs between sepsis patients and controls and proceeded stepwise: (1) differentially expressed miRNAs were filtered to experimentally observed and findings in humans by the IPA microRNA target filter and (2) paired with the differentially expressed mRNAs from the mRNA data setting by IPA expression pairing to miRNA up to mRNA down, and vice versa . The resulting data set was then filtered to match SOFA criteria by selecting the compounds and conditions evaluated by SOFA  and the disorders typically associated with the respective criteria  to increase specificity. The overall disease state was set to “infectious disease” to exclude findings in cancer and other non-infectious inflammatory disorders. mRNAs identified as significantly expressed and targeted by miRNAs using the IPA microRNA target filter option and that were part of the above-mentioned signaling pathways were included using the IPA pathway function to create an interactome. Genes of interest and regulatory miRNAs were identified as a subset with direct interactions with other genes and miRNAs identified by the QIAGEN Knowledge Base and used for network construction .Commonly used drugs with effects on SOFA criteria  and the Specific regulation of miRNAs and mRNAs was confirmed in an independent cohort of 20 patients and 5 controls by RT-qPCR validation. The selection of miRNAs and mRNAs for confirmation was based on their regulatory function in the networks (miRNAs) and their possible targets (mRNAs) of the pharmacologic interventions . Detailst-test. Data in the text and in tables are reported as median and interquartile range. Statistical calculations were performed using SPSS .Demographic and clinical characteristics between controls and sepsis patients from both the RNAseq and the RT-qPCR group were compared using the Wilcoxon rank sum test for nonparametric continuous variables and the Chi-square or Fisher’s exact test for comparison of categorical variables. Statistical significance of RNA expression changes between sepsis patients and controls were assessed using Student’s The study was approved by the Ethics Committee of the Medical Faculty of the University of Munich (protocol #551-14) and carried out according to the World Medical Association Declaration of Helsinki. All study samples were pseudonymized during analysis.Written informed consent for study participation including publication of blinded individual personal data were obtained from each participant or the patient’s legal representative.There were no significant differences in SOFA scores, biometric data, source of infection, or number and type of organ failure between patients from the RNAseq and the RT-qPCR confirmation groups. p-values for miRNA and mRNA regulation from the RNAseq data set. Further detailed results of RNA characterization according to subgroups, differential gene expression analysis, and RT-qPCR validation of selected miRNAs and mRNAs are shown in the The resulting network for SOFA criteria 1 is shown in Molecules identified for this criterion from the RNAseq dataset with a known or possible involvement in encephalopathy and delirium included miR-320a-3p, ACHE, TSPO, and TIMP1, which interacted with MMP9. Potential targets of LPS in the network included highly upregulated LNC2 (NGAL), MMP9, and TSPO . Upregulated MMP9 was targeted by miR-320-3p and let-7a-5p and was—along with PDE4B—unique for this pathway . PropofoThe SOFA criterion 3  reflectiFor this criterion, two networks were constructed with the disease status set at increase in serum creatinine a and acuSignificantly upregulated transcripts in this network were TLR4, which was activated by LPS and by the downregulated and validated miRNA let-7a-5p. Upregulated TLR4 was also shown to interact with the validated and downregulated miR-92-3p and with miR-125-5p targeting SCID. SCID was shown in the network to have a direct effect on bilirubin. Other upregulated transcripts in the network were ABCG2 and EPAS1; both had a regulatory function on bilirubin plasma levels a. No intUpregulated transcripts identified in the network included PF4 indirectly targeting GATA 1 with a direct relationship to thrombopenia. Downregulated and validated BCL2 was also directly associated with thrombopenia. Upregulated miR-30c-5p  targeted downregulated PDGFB. Other molecules directly associated in the network with thrombopenia included THBS1 , ITGA 2b, and JAK2, all unique to this pathway b.In this study, we used RNAseq data and bioinformatic analysis to identify key regulatory molecules and constructed signaling networks to characterize molecular mechanisms underlying the clinically validated SOFA criteria.When applying this approach to SOFA criterion 1 reflecting hypoxia (low PaO2/FiO2 ratio), we selected the disease state ARDS to increase network specificity, as hypoxia is also seen in other conditions . A unique molecule for this criterion was downregulated and validated IGFBP3 with a direct relationship in the network to ARDS. The IGFBP3 pathway was involved in acute lung injury and ARDS in a proteomics study  and in fOne major target identified in the network of SOFA criterion 2  was the highly upregulated transporter molecule LCN2. Interestingly, LCN2 has been characterized as the most substantially elevated protein in the brain of mice after an LPS challenge and showed neuroprotective effects during systemic inflammation . The modInterestingly, the translocator protein TSPO was also significantly regulated in the hypoxia pathway of SOFA criteria 1. TSPO is mainly found on the outer mitochondrial membrane  and is iWhen constructing the signaling network for SOFA criterion 3, reflecting circulatory failure, upregulated IL1RN was identified as directly involved in hypotension and septic shock and in the production of lactic acid, indicating tissue hypoperfusion. Upregulation of IL1RN protein levels was described in patients with sepsis  and septThe networks for SOFA criterion 4  identified numerous molecules. Among these, PARP1 appeared to have protective renal effects in knock-out mouse models of ischemic injury . In contThe network reflecting AKI centered around MYC. Its central role as a regulator of gene expression was demonstrated in a pathway study comparing high-quality microarray studies of gene expression in AKI . MYC alsThe network representing an increase in bilirubin showed a direct effect of LPS on bilirubin in experimental animals . GFER, aThe complex network constructed for SOFA criterion 6 (low platelet count) included downregulated PDGFB signaling by the upregulated miR-30c-5p (PDGFB knock-out mice showed thrombocytopenia ). The moThe major aim of our study was to construct signaling networks directly related to SOFA sub scores using high-throughput RNAseq in patients with sepsis. We detected many significantly regulated RNAs with several miRNA and mRNA transcripts specific to the respective criteria. For construction of the networks, we used a broad bioinformatic approach. This methodology is unbiased, but often includes cellular and animal studies not primarily related to sepsis, but identified experimentally proven signaling networks only potentially underlying the SOFA sub scores. Furthermore, RNAseq was performed in RNA isolated from blood cells, whereas many molecules identified in the networks were investigated in different cell models or organs, but their presence in blood cells was confirmed by our sequencing data, a limitation that applies to almost all biomarkers used in critically ill patients.Given the pronounced impact on the immunologic response to sepsis, one may have expected a much higher number of regulated molecules including interleukins and chemokine transcripts in each network reflecting any given SOFA sub score. The reason for the relatively few regulated molecules in our analysis is stringent filtering to clinical conditions and the most important bioinformatic correlate to the SOFA criteria under study . In addition, only molecules with an experimentally confirmed relationship to the SOFA context were included in the networks in order to focus on the most important and scientifically robust signaling pathways. This resulted in a further reduction of the number of regulated molecules in the networks and made their graphical representation clearer.Our study identified specific signaling networks underlying the SOFA score and provides evidence for the biologic validity of this scoring system both for the assessment of critically ill patients and as inclusion criteria or endpoint for clinical studies. Further and more refined ICU disease severity systems could include expression values of key molecules identified in our study to increase their validity for the identification of patients with an increased risk for negative outcomes, direct therapeutical decisions, and lead to novel endpoints in sepsis trials.


MECP2. The disorder mainly affects girls and it is associated with severe cognitive and physical disabilities. Modeling RTT in neural and glial cell cultures and brain organoids derived from patient- or mutation-specific human induced pluripotent stem cells (iPSCs) has advanced our understanding of the pathogenesis of RTT, such as disease-causing mechanisms, disease progression, and cellular and molecular pathology enabling the identification of actionable therapeutic targets. Brain organoid models that recapitulate much of the tissue architecture and the complexity of cell types in the developing brain, offer further unprecedented opportunity for elucidating human neural development, without resorting to conventional animal models and the limited resource of human neural tissues. This review focuses on the new knowledge of RTT that has been gleaned from the iPSC-based models as well as limitations of the models and strategies to refine organoid technology in the quest for clinically relevant disease models for RTT and the broader spectrum of neurodevelopmental disorders.Rett syndrome (RTT) is an X-linked neurodevelopmental disorder, mostly caused by mutations in  Since the discovery that mutations in the MECP2 gene were the underlying cause of RTT, a great amount of work has been undertaken to understand the molecular function of MeCP2 . MeCP2 wFrom a cellular point of view, it is known that MeCP2 loss affects different cell types in the brain. Region specific knockouts in mouse brains have shown several different phenotypes; for instance, removal of MeCP2 from dopaminergic and noradrenergic neurons induced motor incoordination  and remoRett syndrome (RTT) is a rare neurodevelopmental disorder that manifests between 6 and 18 months of age after a period of apparently normal prenatal and postnatal development. Symptoms vary amongst individuals and include loss of fine and gross motor skills, abnormal social behavior, growth retardation, seizures, breathing dysregulation and stereotypic hand wringing movements . RTT hasde novo mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene, which plays a critical role in normal brain development, specifically in the maturation of the central nervous system (CNS) and synapse development and function (The majority of RTT cases are caused by function .MECP2 as well as X chromosome inactivation (XCI). As the mutations observed in RTT girls are predominantly hemizygous, XCI renders a mosaic expression of the variant with some cell populations expressing the mutant MECP2 allele and others, the wild type allele. While these two populations are normally present in nearly equal (1:1) proportion, the X inactivation pattern can be skewed, favoring the expression of one cell population over the other. In contrast, RTT males present with a homogenous population of mutant cells and are thus more severely affected.Substantial phenotypic variability is observed in girls with RTT, which is attributed to the type of mutation  and its location on MECP2 is ubiquitously expressed, its expression is particularly high in post-mitotic neurons in the brain. Mutations that result in the dysregulation of MECP2 cause a range of abnormalities at the anatomical, cellular, and molecular levels that is well characterized. There is an apparent reduction in brain volume in both humans and mouse models correlating with the severity of the phenotype  and induced pluripotent stem cells (iPSCs) to the discipline of precision medicine and advanced therapeutics has revolutionized the pre-clinical research of neurodevelopmental disorders (NDDs) such as RTT. hESCs are derived from blastocyst-stage human embryos whereas iPSCs are derived from somatic cells such as fibroblasts, blood and exfoliated urinary epithelial cells by cell reprogramming through transgenesis or foot-print free genetic modulation . Both tyThe use of iPSCs has been particularly useful as a complementary approach to existing disease models, such as murine and post-mortem tissue, as they offer a more human-centered approach in which to test therapies given the inaccessibility to patient brain tissue. Encouragingly, disease modeling has been achieved for neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and Huntington’s disease, extending recently to neuropsychiatric, monogenetic, and chromosomal aberration disorders.MECP2, with the majority lying in exons 3 and 4 in the methyl binding domain (MBD), transcriptional repressor domain (TRD) and the C-terminal functional domain (CTD) regions, disrupting the normal function of the MeCP2 protein  and g.67072_67200del (p.?) (∗487Trpext∗27) and a female cell line with a frameshift mutation that results in a truncated transcript (p.V247∗) showed significantly reduced dendritic branching. Interestingly, a reduction in expression of the tumor suppressor protein p53 was also observed  . Consistel (p.?)  and a 20el (p.?)  comparedel (p.?) , which hel (p.?)  and maleel (p.?)  cell linobserved . p53 is observed .∗ and p.N126I showed no difference in NPC morphology compared to their respective paternal control  and control iPSC-derived neurons has demonstrated a significant reduction in synapse number (measured according to VGLUT1 puncta), pointing to glutamate transport defects in RTT iPSC differentiated neurons   . In addi∗) . Functio∗) .Synaptic function and plasticity have been studied in RTT derived iPSCs and controls using several methods including electrophysiology, gene expression and imaging. However, each method has its own challenges. Whole cell electrophysiological recordings provide high temporal and spatial localization, but the restriction of measurements to individual neuronal cells limits its efficacy in understanding complex network interactions . On the in vitro by treatment with candidate drugs has been an obvious area of exploration  and control iPSCs (HUES6) by measuring the global gene expression profiles of RTT-iPSCs and control lines at different stages of differentiation. This in vitro study showed that the thyroid hormone receptor alpha 3 gene , which encodes a hormone receptor critical for brain development, displayed a different expression profile in the mutant cells. It was demonstrated that treatment with IGF1, mediated by TRalpha3, was able to enhance neurite growth in MeCP2-deficient cells, suggesting IGF1 as a treatment option (A deficiency in the expression of potassium chloride cotransporter 2 (KCC2) in RTT iPSC-derived neurons has been demonstrated to result in a delayed GABA functional switch from excitation to inhibition . MeCP2 rt option , 2019.Motor and cognitive impairments in RTT mouse models may be related to abnormalities in the cholinergic system  and thusThe development of therapeutic strategies like the ones listed in this review  needs toin vitro studies demonstrated that co-cultured mutant mouse astrocytes, and the supplementation of mutant astrocyte conditioned medium, failed to support normal dendritic morphology of wild type and mutant hippocampal neurons   . To undep.R306C) . Neuronap.R306C) . Short-tp.R306C) .∗) in MECP2 revealed abnormal growth patterns and gene expression in differentiated mutant astrocyte cells compared to their isogenic controls. In particular, the glial marker GFAP  was shown to be dysregulated in MeCP2-deficient neural cells, suggesting that abnormal astrocytic differentiation is involved in the pathogenesis of disease and a likely therapeutic target for RTT . In comparison to iPSC lines from unaffected fathers , mutant lines showed significantly reduced levels of expression of astrocytic markers  is a phenomenon of female (XX) somatic cells, where one X chromosome undergoes inactivation in a stochastic manner such that genes of only one X-chromosome are expressed in each cell, thus balancing the dosage of X-linked genes with the male (XY) cells. This is critical to RTT as Female iPSCs have been shown to retain the inactive X-chromosome , in contThe activation status of the X chromosome is critical when using iPSCs as cellular models for RTT research and thus warrants careful investigation. The XCI status has been studied in RTT iPSCs by several groups . Interes2 levels result in the expression of both alleles of the X-linked gene  (fs∗ mutation (6:4 to 98:2), despite the originating fibroblasts showing equal expression of both alleles (55:45)  and experimental iPSC lines (expressing mutant MECP2). Recommendations to ensure that the iPSCs and derived cells retain the XCI status of the parental patient cell lines, whether they originate from fibroblasts, urine or blood, require validation by mRNA expression of which X chromosome is being expressed, and selection of iPSC clones that only express the same X chromosome and skewing as the parental line.As observed in RTT patients, skewed XCI has also been reported in neuronally differentiated iPSCs (p.R294p.V247∗) . A drast (55:45) . Extremein vitro. The discovery that neural rosettes  can be generated from embryonic stem cells has powered the development of cerebral organoids  and the paternal controls in the presence of dual SMAD inhibition and Y-27 (ROCK inhibitor) in a spinning orbital shaker. To enhance the growth of the organoids and initiate neuroepithelium differentiation, the culture was initially supplemented with fibroblast growth factor (FGF), epidermal growth factor (EGF) and then BDNF, GDNF, NT-3, L-ascorbic acid, and dibutyryl cAMP. Organoids were then matured in an orbital shaker for 10 months (Two recent studies using RTT patient iPSC-derived brain organoids have characterized the morphology and synaptic function of mutant neurons  and the 0 months . In cont0 months .MECP2-deficient organoids showed significant reductions in cell size, neural protrusions, spine-like density and synaptic puncta. In addition, reduced neural activity indicated an absence of network oscillations, which was not found in the isogenic controls at the same age in vitro. Not only were significant differences in neuronal morphology and function observed between RTT and wild type organoids, but this work also provides a cortical organoid model that recapitulates the synaptic function of the in vivo prenatal brain, the incremental emergence of glial cells, and the formation of inhibitory neurons by 6 months in culture ( culture . As RTT Human brain organoids offer the opportunity to better understand neural circuits, enabling elucidation of the mechanisms that underlie the properties of human circuits. However, despite considerable progress in the field, there has been little evidence to demonstrate the establishment of reliable cortical circuits. One of the obvious obstacles is that brain organoids lack critical cell types of the human cortex, including endothelial cells, and microglia and oligodendrocytes. Recently, efforts have been made to implement vascular structure in human brain organoids by adjusting cultivation protocols, introducing microfluidic devices, and transplanting organoids into immunodeficient mice . AnotherOne caveat in the utility of iPSCs is related to their variability in lineage propensity and this has a confounding effect on the consistency and reproducibility of the cellular composition of the organoid. iPSC derivation and differentiation is a multi-step processes and experimental procedure variations can produce different outcomes . Often, in vitro models for vascularization are also being devised including endothelial cells cultured on microfluidic channels made with degradable materials to resemble the blood vessels in tissues (Another known and reported limitation is the inadequate functionality of the tissue in the organoid such as the lack of vascularization. Vascularization supports efficient transport of oxygen and nutrients to the core of the brain organoids, which are critical for proper growth and differentiation, because non-vascularized organoids often display tissue necrosis. An appealing approach is the xenograft transplantation of brain organoids into mice, allowing the murine blood vessels to transport oxygen and nutrients to support the growth of the transplanted organoids . Concurr tissues .in vivo. A single cell RNA sequencing study has shown that directed patterned organoids have a higher rate of reproducibility over self-patterned whole brain organoids (The most critical limitation is related to the ability to mimic the normal development trajectory of the human brain. iPSC-derived patient neurons and brain organoids are imperfect in that they are highly heterogenous, only replicate a specific brain region, and are not connected to the peripheral nervous system, amongst other shortcomings. Directed patterned and self-patterned models have been developed to replicate the anatomically relevant spatial organization found rganoids . Howeverrganoids  midbrainrganoids  forebrairganoids  and cererganoids  with no MECP2 is tightly regulated both temporally during development and spatially throughout the brain. As MECP2 is an X-linked disorder, it is subjected to X-chromosome inactivation, resulting in the skewing of either the wildtype or mutant allele. All these unusual characteristics make it very challenging to study the pathophysiology of disease in in vitro cellular expression systems and in mouse models as they do not completely recapitulate the disease that is observed in human subjects.Currently, brain organoids do not reproduce the requisite tissue complexity of the human brain, and models have yet to recapitulate the developmental stages of the brain of a young child for studying the postnatal manifestation of neurodevelopmental disorders. RTT patients develop normally from 6 to 18 months after birth, which is followed by a developmental trajectory regression where they lose their ability to control their hand movements, they stop walking and talking, and they develop phenotypes such as microcephaly, seizures, autism, prolonged QT intervals, and breathing problems including apneas. Symptoms range in severity with limited phenotype-genotype correlations. The expression of Rett syndrome patient iPSC-derived neurons and brain organoids are effective models to address several specific phenotypes such as synaptic dysfunction, glutamatergic neuron morphology, electrophysiological and synaptic dysfunction, GABA-ergic dysregulation, non-cell autonomous effects of astrocytes, transcriptional dysregulation, neurogenesis, and miRNA target sites. In addition to this, therapeutic molecules have also been tested, broadening our understanding of iPSC-derived models as well as the pathophysiology of RTT. However, one of the critical questions in the field is how developmental delay and regression can be interrogated using these models, and whether they can inform us about how to reverse RTT in humans. The contributions by Both the cellular model and the organoid model have their specific utility and limitations. There may be no ideal models, but rather appropriate models that fulfill the requirements of the research question. This review outlines the major phenotypes that have been modeled in RTT-iPSC-derived cells and organoids and their application for drug screening. Impending optimization in modeling technology will enable the creation of high-fidelity disease models for the human brain, which in addition to modeling pediatric neurodevelopmental disorders, will allow the characterization of normal and abnormal neurogenic differentiation and tracking of the disease trajectory through pre and postnatal development.FH wrote the manuscript. BC summarized studies used in the review. LR checked the genetic nomenclature. LCC, LR, PPLT, and WAG contributed to the drafts and revisions. PLTT and WAG framed the concept and structure of the review. All authors reviewed and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The present paper aims to compare the microstructural and mechanical properties of CK45 carbon steel plates, joined by friction stir (FSW) and tungsten inert gas (TIG) welding methods. Besides visual inspection, the welded joints and the base material were subsequently evaluated in respect of optical microstructures, hardness and tensile properties. Sound joints could be accomplished using both the FSW and TIG welding methods through proper selection of process parameters and the filler metal. The influence of a water-cooling system on the FSW and various filler metals on the quality of TIG welding were further assessed. Both the FS welded sample as well as TIG welded samples with two different filler metals ER70S-6 and ER80S-B2 exhibited brittle behavior that could be mitigated through optimized water cooling and use of R60 filler metal. A drastic reduction of brittle martensite phase constituent in the microstructure corroborated significant improvements in mechanical properties of the welded zones for both the FSW sample as well as TIG welded samples with R60 filler metal. Carbon steels are extensively used in both consumer as well as commercial applications. Joining them via welding methods, however, may encounter severe problems including the formation of brittle phases like martensite, hydrogen-induced cracks and solidification cracks, thus leading to the failure of components prematurely. Among the conventional fusion welding methods, tungsten inert gas (TIG) welding  with superb weld quality and precise control is extensively utilized in industrial applications. In this fusion welding process, an electric arc is generated between a non-consumable tungsten electrode and the workpiece to create an intense thermal energy, which melts the metals to be joined. Both the weld area and the electrode are protected from oxidation or any atmospheric contamination by shielding with an inert gas. In comparison with gas metal arc welding (GMAW), TIG welding is confined to weld thicker materials (more than 3 mm) in a single pass due to its shallow penetration ,2,3. TheA series of studies have been carried out on developing the FSW process for carbon steels, thus imparting grain refinement at the stir zone with a consequent improvement in mechanical properties. For example, Sato et al.  employedMedium-carbon steels are utilized for fabricating shafts, gears, axles, crankshafts, forgings, and couplings. Steels, possessing carbon from 0.4% to 0.6%, have been found to be suitable for applications in rails, rail axles and, railway wheels . ConsideThe nominal chemical compositions of the CK45 carbon steel plates and filler metals used in the study are shown in For the FSW process, a tungsten carbide tool consisting of a convex shoulder with a diameter of 16 mm and a 5 mm-in-upper-diameter conical pin was employed. In the first step of welding, the dwell time was considered at least 30 s for all the welds in order to enhance interface temperature. The rest of the input parameters for both the FSW as well as the TIG processes are given in The samples from transverse cross-section of joints were prepared for microstructural investigations using the standard metallographic practice comprising mechanical grinding and polishing. The mounted samples were mechanically ground up to 5000 grit and finally polished with 1 μm diamond paste. To reveal the microstructural features, the specimens were etched with 2% Nital (98 mL ethanol + 2 mL nitric acid) reagent. The samples were examined for microstructural features in a light optical microscope (LOM). According to the recommendations of ASTM E 384-05a standard , VickersCK45 carbon steel plates were successfully welded using TIG, FSW and cooling-assisted friction stir welding (CFSW) techniques and the surface morphologies of defect-free joints are given in In Low-magnification images of the welds taken on cross-sectional plane perpendicular to the weld direction are depicted in The microstructural observations of the nugget zone (NZ) in TIG welded plates, with and without the use of filler metals, are presented in The results obtained in respect of hardness data and tensile properties using both the friction stir welding as well as fusion welding methods revealed that sound joints with good mechanical properties would be attainable, if the process parameters are carefully selected, with an appropriate choice of filler metal in the case of TIG welding procedure.Sound joints could successfully be accomplished via CFSW procedure with the following parameters: rotational speed 950 (rpm), traverse speed 24 (mm/min), plunge depth 0.4 (mm), and tilt angle 0.3 (degree);The optimal mechanical property combination was accomplished in CFSWed plates, wherein the microstructure resembled BM. However, the SZ of CFSWed plates contained a finer ferrite-pearlite microstructure, owing to the lower heat input generated, in comparison to typical FSW;In TIG joints, the nugget zone (NZ) of welded samples with ER70S-6 and ER80S-B2 filler metals possessed essentially a martensite structure that was attributed to the presence of Mo in both BM and filler metals, which not only increased the hardenability of the materials, but also reduced the weldability of plates dramatically. In contrast, the NZ of a TIG welded sample using R60 filler metal consisted of a mix of bainite and Widmanstätten ferrite (WF) structure, thereby imparting reasonable mechanical properties;From the industrial application point of view, friction stir welding process stays highly competitive in this fast-changing world embracing new technologies due to both energy-saving as well as preventing the joints from getting riddled with defects, which are often seen in the case of fusion welding methods.An empirical study was performed on the evaluation of microstructures and mechanical properties obtained on CK45 carbon steel welded via FSW and TIG welding methods. Based on the results achieved, the following concluding remarks can be made:


Amid COVID-19, and other possible future infectious disease pandemics, dentistry needs to consider modified dental examination regimens that render quality care, are cost effective, and ensure the safety of patients and dental health care personnel (DHCP). Traditional dental examinations, which number more than 300 million per year in the United States, rely on person-to-person tactile examinations, pose challenges to infection control, and consume large quantities of advanced-level personal protective equipment (PPE). Therefore, our long-term goal is to develop an innovative mobile dentistry (mDent) model that takes these issues into account. This model supplements the traditional dental practice with virtual visits, supported by mobile devices such as mobile telephones, tablets, and wireless infrastructure. The mDent model leverages the advantages of digital mobile health  tools such as intraoral cameras to deliver virtual oral examinations, treatment planning, and interactive oral health management, on a broad population basis. Conversion of the traditional dental examinations to mDent virtual examinations builds upon (1) the reliability of teledentistry, which uses intraoral photos and live videos to make diagnostic decisions, and (2) rapid advancement in mHealth tool utilization.In this pilot project, we designed a 2-stage implementation study to assess 2 critical components of the mDent model: virtual hygiene examination (eHygiene) and patient self-taken intraoral photos (SELFIE). Our specific aims are to (1) assess the acceptance and barriers of mDent eHygiene among patients and DHCP, (2) assess the economic impact of mDent eHygiene, and (3) assess the patient’s capability to generate intraoral photos using mHealth tools .This study will access the rich resources of the National Dental Practice-Based Research Network to recruit 12 dentists, 12 hygienists, and 144 patients from 12 practices. For aims 1 and 2, we will use role-specific questionnaires to collect quantitative data on eHygiene acceptance and economic impact. The questionnaire components include participant characteristics, the System Usability Scale, a dentist-patient communication scale, practice operation cost, and patient opportunity cost. We will further conduct a series of iterative qualitative research activities using individual interviews to further elicit feedback and suggestion for changes to the mDent eHygiene model. For aim 3, we will use mixed methods  to assess the patient’s capability of taking intraoral photos, by analyzing obtained photos and recorded videos.The study is supported by the US National Institute of Dental and Craniofacial Research. This study received “single” institutional review board approval in August 2021. Data collection and analysis are expected to conclude by December 2021 and March 2022, respectively.The study results will inform the logistics of conducting virtual dental examinations and empowering patients with mHealth tools, providing better safety and preserving PPE amid the COVID-19 and possible future pandemics.PRR1-10.2196/32345 Amid the COVID-19 pandemic, dental health care personnel (DHCP) are at great risk of contracting the SARS-CoV-2 virus due to their close physical proximity to their patients, as well as the enhanced potential for transmission of airborne viruses in the dental setting [Aggressively converting traditional dental examinations  to virtual examinations could significantly reduce the exposure risk for patients and DHCP and preserve a large volume of PPE essential to the medical and dental communities. According to the American Dental Association, as of 2019, 200,419 dentists were practicing dentistry in the United States, with 158,331 (79%) general dentists (GD) providing a range of examination visits on a daily basis [In the current dental examination model , patientRationale for Developing the mDent eHygiene Model in a Digital EraOur long-term goal is to develop an innovative mobile dentistry  (mDent) This mDent model will fully engage relevant stakeholders  to conduct interactive oral health practices. The mDent model will also utilize a patient-driven mobile device to increase the accessibility of dental care. Moreover, in the era of COVID-19 risk, this remote virtual dental service model will lead to a well-planned dental service, better infection control, and reduced PPE consumption. As this eHygiene implementation study is a hypothesis-generating pilot study, our immediate objective is to assess the following 3 aims in the National Dental Practice-Based Research Network : (1) AimThis study will use a 2-stage implementation study to assess the acceptance of 2 components (eHygiene and SELFIE) of the mDent eHygiene model among patients and DHCP . The components are illustrated in This mDent eHygiene study will use mixed methods  to collect outcome measures and conduct data analysis. For details, see The mDent eHygiene study will be conducted in the Northeastern node of the National Dental Practice-Based Research Network in the United States. This mDent eHygiene study will enroll 144 patients and 24 DHCPs from 12 practices. Each practice will enroll 12 patients, 1 dentist, and 1 dental hygienist. All 144 patients and 24 DHCPs will conduct the eHygiene session (1st stage) of the study. Among enrolled patients and DHCPs, 5 patients, 5 dentists, and 5 dental hygienists will be invited for a 30-minute recorded telephone interview for a qualitative session of the eHygiene study. In addition, 12 patients will be invited to conduct a SELFIE session to evaluate their capability for taking intraoral photos by themselves, with guidance from the dental hygienist.The dental hygienist will complete a routine hygiene visit, update x-rays, and perform a hard and soft tissue examination as part of routine clinical care. Intraoral photos of patients will be taken for research purposes following a recommended template See . IntraorThe dentist will then conduct a virtual visit with the patient lasting approximately 30 minutes at a later and suitable time within 14 days of the eHygiene intraoral photo visit, to review eHygiene findings and treatment plans.The self-taking of intraoral photos (SELFIE session) will be piloted by 12 (1 from each practice) of the 144 study patients who complete the eHygiene session. The intraoral camera and instructional video will be given to the patient when the patient leaves the hygiene visit. During a virtual visit with the dental hygienist, the patient will use an intraoral camera while being supervised by the dental hygienist to take a series of intraoral photos of the front and posterior teeth. This virtual visit session will be recorded.Patients will be encouraged to think-aloud  about thEach participating patient, dentist, and dental hygienist will complete baseline and post-eHygiene role-specific questionnaires  for virtual individual interviews lasting approximately 30 minutes. These 15 individuals will include those who rated above and below the average SUS score. The questions asked during the interview will address feedback and recommendations, perceived challenges, and suggestions for improvement of the mDent eHygiene model. The interviews will be standardized using an interview guide see , and intThe sample size calculation for the primary outcome (eHygiene—SUS) was based on the primary outcome of the SUS score from patients. Various studies ,14,15 hat test, a sample of 7 dentists or hygienists will achieve 90% power, at an alpha of .05. Considering potential dropouts, recruiting 12 dentists and 12 dental hygienists will achieve satisfactory statistical power for this aim.Dentists and dental hygienists will complete the SUS for each patient when the dentist and hygienist complete an eHygiene visit, which means each dentist and hygienist will evaluate the eHygiene model 12 times. Assuming a mean difference of 30 (SD 20) between the first and last patients evaluated by the dentists and hygienists, using a paired Using a cluster randomized design to calculate the sample size for the primary outcome of DPC and assuming a mean difference in the patient-evaluated DPC score between the current hygiene model and the eHygiene model of 8 (SD 10), a sample size of 48 patients from 12 practices (4 per practice) will achieve 85% power, at an alpha of .05. A sample size of 144 patients will satisfy the statistical power of the DPC outcome, while considering potential dropouts.For the sample size calculation of the tertiary outcome (SELFIE), we expect to reach data saturation   as rated by patients, dentists, and dental hygienists. The SUS score from the patients and DHCPs between practices will be compared. A linear mixed effects model will be used to examine factors that influence the SUS score as perceived by patients, including patient factors  and DHCP factors , while considering the clustering effects within practices and providers. The eHygiene SUS score as rated by dentists and dental hygienists from treating the first patient through the last study patient will be compared to assess whether the DHCP-determined SUS score is associated with a learning curve.We will calculate the DPC score as rated by patients, which assesses how well the patients understand the planned treatment and the quality of the communication between the patients and dentists who participate in eHygiene. We will use a linear mixed effects model to examine factors that influence the DPC score perceived by patients, including patient factors , DHCP factors , and time spent on the eHygiene visit, while controlling for the clustering effects within practices and providers. We will run separate models for patients and DHCP.Regarding qualitative data, the interviews will be standardized using interview guides, audio-recorded, transcribed, coded, and analyzed for thematic content. The audio recordings will be transcribed by the Temi  transcription service and further verified by 2 trained research personnel. Transcribed data will be analyzed using MAXQDA software . The data will be coded by 2 trained coders with predetermined open codes using a codebook with a description of the coding tree. Thematic content will be further analyzed using categorizing and contextualizing strategies to understand the factors associated with acceptance of and barriers to eHygiene among patients and DHCPs.We will conduct analysis for the following parameters: (1) PPE consumption and estimated cost and comparison between eHygiene and traditional hygiene examination models for each practice; (2) eHygiene chair time per study patient, learning curve–related fluctuations in chair time per practice, and comparisons between practices; (3) eHygiene virtual visit time and comparison between practices; and (4) DHCP  personnel cost related to eHygiene in-office and virtual visits, compared with traditional hygiene examination visits.Parameters to be evaluated include time spent on photo taking, number of photos, and readable photos by a dentist evaluator using a photo assessment from . FactorsThe “think-aloud” videos recorded during the SELFIE sessions will be reviewed by a trained study evaluator to analyze common themes of challenges patients encounter, using a SELFIE assessment form . The keyThe study has been peer-reviewed and funded by the US National Institute of Dental and Craniofacial Research. This study received single institutional review board approval from the University of Alabama at Birmingham (#300006506) and local context review from the University of Rochester (#6077). The eHygiene study is expected to launch in August 2021. Data collection and analysis are expected to conclude by December 2021 and March 2022, respectively.This study is innovative in several ways. First, conducting virtual dental examinations (the mDent model) using intraoral photos and x-rays is novel and potentially transformative to dental practice. Using smartphones and mobile devices to take photos of the mouth and teeth and conduct oral disease screening has been recently reported -21; howeSuccessful completion of this eHygiene pilot study will provide data on the acceptance and economic impact of virtual dental examinations during eHygiene visits, which will be a test vehicle for the future mDent model. The results will inform potential immediate modification of the dental service system to provide better safety and preserve PPE amid COVID-19 and other infectious disease outbreaks and beyond.


Fungal natural products (NPs) usually possess complicated structures, exhibit satisfactory bioactivities, and are an outstanding source of drug leads, such as the cholesterol-lowering drug lovastatin and the immunosuppressive drug mycophenolic acid. The fungal NPs biosynthetic genes are always arranged within one single biosynthetic gene cluster (BGC). However, a rare but fascinating phenomenon that a crosstalk between two separate BGCs is indispensable to some fungal dimeric NPs biosynthesis has attracted increasing attention. The hybridization of two separate BGCs not only increases the structural complexity and chemical diversity of fungal NPs, but also expands the scope of bioactivities. More importantly, the underlying mechanism for this hybridization process is poorly understood and needs further exploration, especially the determination of BGCs for each building block construction and the identification of enzyme(s) catalyzing the two biosynthetic precursors coupling processes such as Diels–Alder cycloaddition and Michael addition. In this review, we summarized the fungal NPs produced by functional crosstalk of two discrete BGCs, and highlighted their biosynthetic processes, which might shed new light on genome mining for fungal NPs with unprecedented frameworks, and provide valuable insights into the investigation of mysterious biosynthetic mechanisms of fungal dimeric NPs which are constructed by collaboration of two separate BGCs.  Fungi produce a plethora of chemically diverse natural products (NPs) with various bioactivities and a wide variety of applications in medicine and agriculture . BecauseIn fungi, the natural products are commonly synthesized by the genes arranged in a contiguous fashion as a biosynthetic gene cluster (BGC) . The corMyxococcus sp. SDU36, consists of N-ribityl 5,6-dimethylbenzimidazole and a linear fatty acid chain endowed with an isoxazole ring. Interestingly, a non-canonical PKS/NRPS biosynthetic pathway and the vitamin B12 metabolism pathway were proved to interwind together through the construction of isoxazole-benzimidazole hybrids . Th. ThecdA ompounds ,50,51. 40) is a fungal polyketide bearing an unprecedented 6/6/7/5/6-fused pentacyclic ring system . A bioinspired total synthesis of delitschiapyrone A has been achieved by simply stirring a heterogeneous mixture of two Diels−Alder substrates, which gave a hint that the intermolecular Diels−Alder reaction might be spontaneous. Recently, Houk and co-workers investigated the mechanisms and dynamics of biosynthetic formation of 40 by density functional theory (DFT) calculations and quasiclassical molecular dynamics simulations with DFT and xTB, and drew a conclusion that 40 is not formed from the proposed Diels−Alder/α-ketol rearrangement cascade but instead formed directly from a single cycloaddition reaction -cycloaddition in the assembly line of 43 [The fungal polyketide acautalide A (de model . The arc. H-JQSF ,65. Howene of 43 .One reason that fungi endow great potentials to produce natural products with complex structures and excellent biological activities might be ascribed to the complex metabolic regulatory networks and interaction of different biosynthetic gene clusters. Understanding how simple precursors are synthesized and assembled together to construct complex natural products in organisms may promote the development of new combinational and synthetic biology strategies to create new molecules. Over the course of evolution, fungi have evolved different strategies to increase the diversity of their NPs to protect themselves and acclimatize to the surrounding ecological environment . For exaIn general, more endeavors are needed to carry out the research for the discovery of fungal heterodimer NPs constructed by two building blocks, which not only provides an outstanding opportunity for investigation of the currently underestimated hidden biosynthetic crosstalk, but also facilitates the discovery of new BGCs, new regulatory mechanisms, and enzyme catalysts with novel catalytic mechanisms.


The natural aging process is carried out by a progressive loss of homeostasis leading to a functional decline in cells and tissues. The accumulation of these changes stem from a multifactorial process on which both external  and internal  risk factors contribute to the development of adult chronic diseases, including type 2 diabetes mellitus (T2D). Strategies that can slow cellular aging include changes in diet, lifestyle and drugs that modulate intracellular signaling. Exercise is a promising lifestyle intervention that has shown antiaging effects by extending lifespan and healthspan through decreasing the nine hallmarks of aging and age-associated inflammation. Herein, we review the effects of exercise to attenuate aging from a clinical to a cellular level, listing its effects upon various tissues and systems as well as its capacity to reverse many of the hallmarks of aging. Additionally, we suggest AMPK as a central regulator of the cellular effects of exercise due to its integrative effects in different tissues. These concepts are especially relevant in the setting of T2D, where cellular aging is accelerated and exercise can counteract these effects through the reviewed antiaging mechanisms. The natural aging process is carried out by a progressive loss of homeostasis entailing a variety of physiological changes in the function of cells and tissues. To systematically dissect the biological aging process, Lopez-Otin et al. characterized nine major hallmarks of aging that are divided as primary , antagonistic , and integrative hallmarks . Due to their functional characteristics, primary hallmarks are considered causes of damage, antagonistic hallmarks are responses to damage while the integrative hallmarks reflect the end results of the first two categories . The intExercise is a lifestyle intervention with known antiaging effects capable of counteracting several of the hallmarks of aging including senescence and age-associated inflammation –4. We prType 2 diabetes (T2D), a condition that integrates these concepts, is considered a disease of aging, affecting 30 million people in the United States, most of whom are over the age of 50 . MortaliFrom a pathophysiological point of view, accelerated cellular aging plays a role in T2D. Studies have shown that people with T2D have shorter telomeres and mitochondrial DNA depletion  and at aExercise is known to be an effective lifestyle intervention for T2D since it improves metabolic control. However, to consider the effects of exercise from a cellular aging point of view is a conceptual change in how physical activity is envisioned as a therapeutic tool for diabetes.Herein, the antiaging effects of exercise are reviewed from a tissue and cellular level, its effects upon the individual hallmarks of aging and how AMPK can integrate many of these effects. Finally, these concepts are applied to the setting of T2D to provide a novel view of how this disease can be approached from a cellular aging perspective.This review follows the guidelines of exercise and physical activity for older adults from the American College of Sports Medicine  where exThe aim of the present review paper is to survey the literature related to exercise and its association with longevity and aging. The rationale for conducting this review is that aging is often accompanied by declining cellular homeostasis which is crucial to the development of chronic diseases, but lifestyle interventions can slow down its effects. The literature was surveyed on MEDLINE through freely accessible PubMed as a search engine for the terms: “exercise”, “longevity” and “aging”; the most relevant studies were included as they related to the 9 hallmarks of aging. Additional searches were performed to elucidate the potential role of AMPK activation upon the hallmark of aging. Studies from animal models, human, meta-analysis and bibliographic reviews were consulted and cited accordingly.The aging process affects longevity and health span which are influenced by both genetic and environmental factors . To systThe complex relationship between factors that are accelerated by a sedentary lifestyle and those that are solely due to age, has been successfully addressed in various reviews , 20. TheRegardless of this limitation, numerous studies have shown that maintaining a minimum quantity and quality of exercise improves cardiorespiratory fitness and muscle function, flexibility and balance . CurrentThe beneficial effects of exercise upon longevity and health span are also evident in individuals that have a genetically determined longevity, such as centenarians. In this unique population, the decline in lung function and sarcopenia can be counteracted by exercise programs which increase their physical capacity and health span .When compared with other interventions directed at slowing aging, such as caloric restriction, some studies have shown that in mice, exercise lacks the adverse outcomes that were observed with time restricted feeding   and shouThe effects of exercise upon different organs and systems and its contribution to longevity and health span have been summarized in 2 max), is a strong and independent predictor of all-cause mortality [Cardiovascular (CV) disease is a major cause of mortality worldwide and sedentary lifestyle highly contributes to CV disease burden. Cardiorespiratory fitness, as measured by maximal oxygen uptake  which become vulnerable to injury with advancing age with resultant impaired vision, are also restored by exercise in mice. This is due to sustained levels of brain-derived neurotrophic factor (BDNF) levels in the retina underscoring the role of this critical factor in maintaining retinal health during aging of animal models .Within the peripheral nervous system, neuromuscular junctions modify their structure with age. These changes are characterized by axonal swellings, sprouting, synaptic detachment, partial or complete withdrawal of axons from some postsynaptic sites and fragmentation of the postsynaptic specialization. However, one month of voluntary exercise in 22-mo-old mice reversed age-related synaptic changes with no change on motor neuron number or muscle fiber turnover .Additionally, the psychological effects of exercise are profound and include relaxation and alleviation of anxiety and depression. These effects are strong enough that exercise can turn into an addiction . Given iSecondary aging is associated with the development of insulin resistance, increased adiposity, and accumulation of ectopic lipid deposits in tissues and organs; all of which contribute to metabolic dysfunction  increasiSeveral metabolic alterations accumulate over time along with a reduction in physical fitness, suggesting the existence of a "metabolic clock" that influences aging. The main features of the "westernized" lifestyle  accelerate the metabolic decline of secondary aging factors, such as insulin resistance, while the promotion of metabolic fitness leads to health span extension .The beneficial effects of exercise upon glucose metabolism are well known and have been thoroughly studied , convertIn summary, exercise has shown to have beneficial antiaging effects of many human organs and tissues either by reversing some of the aging phenotypes or by delaying their appearance .The consequences of exercise on the aging of specific organs and tissues can be studied at a cellular perspective and structured based on the changes it has upon the hallmarks of aging.Age is characterized by the accumulation of lesions in the DNA and defects in the nuclear architecture leading to genomic instability. These are the result of exogenous  and endogenous factors   that resExercise minimizes these lesions, partly through: reduction of the age-associated 8-hydroxy-2'-deoxyguanosine (8-OHdG) , increasTelomeres protect the integrity of chromosomal DNA during cellular division but are particularly susceptible to age-related deterioration . StudiesExercise is able to increase telomere length through changes in telomerase activity, inflammation, oxidative stress and skeletal muscle satellite cell content. Long-term exercise can activate telomerase reverse transcriptase (TERT) in leukocytes and also upregulate protective and DNA repair regulator proteins (such as telomeric repeat-binding factor 2 and Ku protein). This has important physiological consequences since a positive correlation has been shown between muscle regeneration processes and telomere length in older adults .Exercise is capable of inducing widespread epigenetic changes. General loss of histones, imbalanced histone modifications, transcriptional deregulations, changes in heterochromatin, breakdown of nuclear lamina, as well as DNA and histone methylation, are characteristics of aging .Physical activity increases DNA methylation, causes histone modifications and induces miRNA in muscle, brain and the cardiovascular system. Acute aerobic exercise decreases methylation of PGC-1α, mitochondrial transcription factor (TFAM), MEF2A, citrate synthase (CS) and pyruvate hydrogenase kinase isozyme (PDK4) . In addiSome age-related diseases are linked to impaired protein homeostasis – known as proteostasis. Cell autophagy is one of the mechanisms for degradation and recycling of damaged macromolecules and organelles, and its alteration can lead to disease. Although human data are still scarce, muscle autophagy markers are up-regulated after exercise training in older women  and coulThe target of rapamycin complex 1 (TORC1) - a central kinase involved in protein translation- is a negative regulator of autophagy and so may be an effector of exercise. TORC-1 is downregulated by exercise through modulation of IGF-1, Akt/mTOR, and Akt/FoxO3a signaling. This cascade has been shown to prevent loss of muscle mass and strength , 76. AddDeregulation of nutrient sensing pathways have been extensively involved in age-related phenotypes, and their downregulation is one of the most effective strategies to extend lifespan and health span. As humans age, the loss of muscle mass occurs due to acute changes in net protein balance, particularly in the myofibrillar protein fraction , 79, andInsulin like growth factor (IGF-1) acts as a key link between mechanical contraction and protein synthesis since it is acutely stimulated and promotes ribosomal biogenesis and translation to form new myofibril proteins. During exercise the mechanical loading and contraction cause the local release of IGF1 which activates IGF and leads to to muscle protein synthesis .2+ during muscle contraction can also directly activate AMPK and is implicated in the regulation of numerous intracellular proteins that mediate cellular transduction, including kinase C, calcineurin, and CaMKs [Another exercise-regulated nutrient sensing pathway is AMPK, which is activated in response to decreased intracellular ATP and changes in the NAD+/NADH ratio. Its function is to preserve ATP by inhibiting both biosynthetic and anabolic pathways while simultaneously stimulating catabolic pathways to re-establish cellular energy stores. The increased concentration of Cand CaMKs –83. Bothnd CaMKs  , followed by its cleavage to generate irisin –96, whicCellular senescence is characterized by lack of cellular proliferation in response to stressors and secretion of an array of proteins specific to each cell type. This array of proteins known as senescence-associated secretory phenotype (SASP) is part of the aging hallmark of altered intercellular communication.INK4a, and P53) shedding light on the beneficial impact of exercise on senescence [Cellular senescence is linked to other mechanisms of aging. ROS accumulation in mitochondria leads to single-strand DNA breaks that accumulate in telomere regions and result in telomere shortening and premature cellular senescence. Senescence has been linked to numerous age-related chronic diseases and risk factors, such as T2D , 97, 98.nescence .INK4a, a marker and effector of senescence, in cellular fractions of human whole blood exponentially increased with chronological age and associated significantly with sedentary life style [INK4a expression correlated with plasma interleukin-6 (IL-6) concentration, a marker of human frailty. Exercise induces increased IL-6 derived from muscle which has anti-inflammatory properties, whereas paradoxically, IL-6 resulting from TNF or NFkB activation relates to aging phenotypes [Expression of p16fe style . In addienotypes . In a seenotypes .In some settings, exercise-induced senescence is beneficial, such as the appearance of fibro-adipogenic progenitors in response to muscle damage, and leads to regenerative inflammation .A decline in the regenerative potential of tissues is expected with age. Specifically, a decline in satellite cells results in impaired repair of muscle fibers while decreased hematopoietic stem cells leads to immunosenescence –105. ExePro-inflammatory tissue, damage accumulation, cumulative dysfunction of the immune system and elevated levels of pro-inflammatory cytokines secretion underlie the development of inflammaging, a pro-inflammatory phenotype associated with progressive aging that affects intercellular communication . This isMoreover, exercise further suppresses inflammation via IL-6 released from muscle . Recent In summary, exercise attenuates all hallmarks of aging through different molecular pathways and effectors that seem independent and disconnected. We hypothesize there must be molecular regulatory nodes able to coordinate these responses and that AMPK can play such a role.+/NADH ratio [+/NADH causes activation of silent information regulator 1 (SIRT1) deacetylase activating LKB1.We propose that activation of AMPK plays a significant integrative role impacting the primary, secondary and integrative hallmarks of aging in response to exercise. In muscle, AMPK is a long known exercise effector that is activated by increased AMP/ATP and NADDH ratio . MammaliThus cellular energy balance effectively controls cellular responses via an integrated signaling network mediated by AMPK , which pPGC-1α is a critical regulator of gene transcription that controls energy homeostasis and is involved in mitochondrial biology . In mousSome of the effects of PGC-1α are mediated through its inhibition of NFκB. Ablation of PGC-1α led to activation of NFκB and upregulated pro-inflammatory cytokines  while inNrf2 is a basic leucine zipper protein involved in regulation of antioxidant proteins that protect against oxidative damage triggered by injury and inflammation. Studies using AICAR  showed stimulated expression of Nrf2 and upregulation of glutathione peroxidase 7 (Gpx7), leading to suppression of cellular senescence and SASP . In addiCrosstalk between AMPK activated signaling pathways is demonstrated by inhibition of Nrf2 by the p65 component of NFκB complex through kelch-like ECH-associated protein 1 (Keap1). This crosstalk can have an additive effect upon decreased cellular senescence and stem cell maintenance .Activation of the FoxO3a axis by AMPK increased stress resistance in long-lived animals . By mediCip1 and P16INK4a-RB signaling pathways [in vitro, it has been difficult to elucidate the in vivo functional role of P53 during aging. It is likely that the response partly depends on its cellular localization as well as the duration and intensity of the stimulus.Tumor protein P53 regulates the cell cycle and functions as a tumor suppressor. The effects of exercise-activated AMPK upon P53 are complex, with both activating and inhibiting effects. AMPK activation has been shown to induce phosphorylation of P53 and lead to cell cycle arrest. This promotes cellular survival in response to glucose deprivation (as might occur during exercise), however these cells can rapidly reenter the cell cycle upon glucose restoration. However, persistent activation of AMPK leads to accelerated P53-dependent cellular senescence, underscoring the importance of the timing and pulsatility of AMPK activation . Interespathways , 132. DuWhen activated simultaneously by AMPK, P53 and FoxO can induce the expression of sestrins, a family of highly conserved stress-response proteins with oxidoreductase activity that can protect cells from oxidative stress. Loss of sestrins has been linked to age related pathologies such as mitochondrial dysfunction, muscle degeneration and lipid accumulation. These effects are attributed to increased TOR activity and the associated decrease in autophagic uptake . These pC. elegans [Serine/threonine protein kinase mTOR was identified in mammalian cells as a target of the antiproliferative molecule rapamycin . It part elegans .Whereas autophagy declines during aging, AMPK activation can restore it by inducing the dissociation of mTORC1 from the ULK1 complex, directly binding and phosphorylating ULK1, an autophagy-initiating kinase, with the result of stimulating autophagy. . FurtherAutophagy and protein synthesis inhibition mediated by downregulation of mTOR have direct effects on proteostasis. In a mouse model of Parkinson’s disease, AMPK activation reversed behavioral impairments, reduced α-synuclein accumulation and enhanced LC3-II-mediated autophagy in dopaminergic neurons , 139. AMAMPK-activation can also exert antiaging effects through inhibiting the effects of AGEs . AGEs, mIn summary, AMPK activation through exercise can impact all the hallmarks of aging through different signaling pathways as summarized in T2D is a complex disorder that combines a genetic hereditary component and environmental risk factors, such as nutrition and lifestyle. Amongst the risk factors, age stands out with most patients being over 60 years old. There is evidence of accelerated cellular aging with hyperglycemia and in both Type 1 (T1D) and Type 2 (T2D) diabetes mellitus , 144.Hyperglycemia increases the hallmarks of aging, such as senescence of endothelial cells in atherosclerotic lesions and telomere shortening . The expPancreatic β-cells, which play a crucial role in the development of T2D, have also been shown to undergo cellular senescence in the setting of insulin resistance , T2D andExercise and physical activity are already cornerstones in the metabolic management of T2D . Randomi2+. Muscle cell contraction is ATP-dependent and an acute exercise bout increases AMP levels, activating the AMPK signaling pathway and leading the fusion of GLUT-4 containing vesicles with the plasma membrane [The benefits are mainly related to exercise improving blood glucose levels through both a reduction of peripheral insulin resistance –153 and membrane , 156 (Fi2+ATPase activity indirectly by formation of reactive aldehydes. Hence, ROS would hinder Ca2+ removal from the cell and encourage intracellular Ca+2 accumulation. Muscle contraction can also directly increase intracellular Ca2+, which promotes the membrane translocation of GLUT-4 [Muscle contraction increases ROS generation due to high oxygen consumption that takes place during mitochondrial activity, in fact, superoxide generation in skeletal muscle increases about 50-100-fold during exercise , 158. ROf GLUT-4 .Chronic exercise increases the number and activity of mitochondria in muscle , which cIn summary, exercise activates molecular signals that can bypass defects in insulin signaling in skeletal muscle and increase skeletal muscle mitochondria, which are associated with improved insulin sensitivity in skeletal muscle and therefore improve aging-associated effects of T2D.Exercise is an effective strategy to prevent aging and enhance longevity and health span both on a clinical and a cellular level due to its capacity to modulate all nine hallmarks of aging. Additionally muscle, one of the main systemic effectors of exercise, is recognized as an endocrine organ that produces and releases myokines, implying a complex cross talk between muscles and other tissues. The AMPK pathway , a well-This review has several limitations. As mentioned, the lack of deleterious effects of a sedentary lifestyle upon aging during exercise can sometimes be confused with antiaging effects of exercise. This conundrum can only be solved if aging studies are carried out in non-sedentary older populations. Unfortunately this rarely occurs and should be considered while interpreting the cited studies. Another limitation is the cross-sectional design of studies comparing an exercised and a sedentary population in spite of the knowledge that the rate of aging varies considerably amongst individuals. The ideal design for aging studies is a longitudinal follow up of non-sedentary individuals; this is rarely feasible due to constraints of time and resources.Although every attempt was made to include the most relevant studies for each subject, the vastness of publications in this area means that some important work may have been unintentionally omitted. We encourage readers to further their searches on specific subjects that have specially interested them.We propose that future studies should address the effects of exercise on tissues which are not considered its direct targets but do show accelerated aging in T2D, such as pancreatic β-cells. In these, the role of AMPK and its physiological control will become especially significant as exercise is considered a cellular antiaging strategy.