Roughly 3% of the human genome consists of microsatellites or tracts of short tandem repeats (STRs). These STRs are often unstable, undergoing high-frequency expansions (increases) or contractions (decreases) in the number of repeat units. Some microsatellite instability (MSI) is seen at multiple STRs within a single cell and is associated with certain types of cancer. A second form of MSI is characterised by expansion of a single gene-specific STR and such expansions are responsible for a group of 40+ human genetic disorders known as the repeat expansion diseases (REDs). While the mismatch repair (MMR) pathway prevents genome-wide MSI, emerging evidence suggests that some MMR factors are directly involved in generating expansions in the REDs. Thus, MMR suppresses some forms of expansion while some MMR factors promote expansion in other contexts. This review will cover what is known about the paradoxical effect of MMR on microsatellite expansion in mammalian cells. Tracts containing tandem arrays of short perfect- or near-perfect repeat units are common in the human genome Ref. . These sHTT) gene (Ref. FXN) gene (Ref. DMPK gene (Refs FMR1 gene (Refs Two major classes of human STR expansions are known: the first class is associated with genome-wide microsatellite instability (MSI), while the second class is associated with expansions of a specific microsatellite. Genome-wide MSI is associated with a predisposition to certain cancers including haematological malignancies as well as certain colon, urothelial, hepatobiliary, pancreatic, bladder, kidney, prostate, endometrial, ovarian and breast cancers Refs –20. In cene Ref. ; Friedreene Ref. ; myotoniene Refs , 24; andene Refs –28. Whilene Refs , this reDuring DNA replication two types of errors can be introduced into DNA: mismatches and insertions or deletions (INDELs). Mismatches arise from insertion of the incorrect base in the daughter strand by the DNA polymerase. Most of these mismatches are removed by the proofreading function of the polymerase, but those that escape this proofreading will cause point mutations if the daughter strand is replicated before the mismatch is removed Refs , 31. INDα complex and MSH2/MSH3 in the MutSβ complex. MutSα is primarily involved in the recognition of mismatches and 1 base INDELs 4E nuclease motif present in both PMS2 and MLH3 and its loss is not associated with increased MSI in mice  After ely Refs –46. MutLtSβ Refs , 48. Mutear Refs , 50. Whiice Ref. . Multiplins Refs , 52. A sair Ref. . Excisioair Ref. , 55 or Fair Ref. . Strand-−5 to 10−7 MSI events per locus per cell generation have been reported in human cells with functional MMR, orders of magnitude higher than the mutation rate seen in unique sequence  or the testing of candidate MMR gene polymorphisms suggest that functional MMR components are required for some, if not all, STR expansions that cause the REDs Refs –74. Thisels Refs , 76–79, els Refs , 73. Furels Refs . This suAs with cancer-associated MSI, the extent of expansion in the REDs is related in part to the length and purity of the repeat tract Refs –88. MathFMR1 gene that causes the FXDs only expands when it is on the active X chromosome indicates that STR expansion requires transcription or transcriptionally-competent chromatin  (Ref. Although MutLion Refs –105. Thutro Ref.  and MutLJs) Ref. . Loop ouJs) Ref. , 108. ClJs) Ref. , also plJs) Ref. . This raβ and MutLγ, MutLβ has also been shown to be required for expansion in embryonic stem cells from a mouse FXD model (Ref. β in generating expansions in HD is also suggested by the fact that PMS1 variants predicted to be deleterious are most frequently associated with a later age at onset/less severe phenotype in HD cohorts (Ref. β lacks a nuclease and is not required for MMR, how it contributes to expansions is unclear. This is not the only unresolved issue; although MutSα contributes to expansions in FXD and FRDA mice and FRDA iPSCs (Refs α levels also has different effects in various systems. In a mouse model of the FXDs, MutLα is required for expansions in ESCs (Ref. α only resulted in a 50% decrease in expansions (Ref. Pms2 showed an increase in expansions (Ref. PMS2 correlates with an earlier age at onset in HD (Ref. α and MutLα are able to act in an auxiliary capacity to promote expansions when the essential factors, such as MutSβ and MutLγ, and perhaps MutLβ, are limiting (Ref. In addition to MutSdel Ref. . An actirts Ref. . HoweverSCs Refs , 110, liSCs Refs  or in a SCs Refs . FurtherSCs Ref. , while ions Ref. . Reducedons Ref. , whereasons Ref. . The lat HD Ref. . These ding Ref. .Contractions are also seen in the REDs-associated STRs and their mouse models, although their aetiology is less well understood. A bimodal distribution of contractions is seen in the germline of a mouse model of the FXDs, with some contractions involving the loss of just 1–2 repeat units whilst others involve the loss of much larger numbers of repeat units Ref. . These lβ, OGG1 and NEIL1 that promote expansion, and low levels of proteins such as EXO1, that protect against them (Ref. Thus, MMR factors can both suppress and promote MSI. Which MSI pathway predominates is likely to depend on a variety of cell-type specific factors including the frequency of cell division and the relative levels of factors that promote or suppress each type of MSI. For example, while MMR is important for preventing genome-wide MSI in the colon, as evidenced by the high frequency of MSI-high colonic tumours in individuals with germline mutations in MMR proteins, MSI-high tumours originating in neurons are rare Refs –117. Thehem Ref. . The higFmr1 locus in embryonic stem cells from a mouse model of the FXDs. Consistent with the role of functional MMR components in generating expansions, a high rate of expansions is seen at this locus in MMR-proficient cells derived from these mice. For example, most alleles with ~280 repeat units gained an additional 19 repeats over a 52-day period in cells wildtype for MLH3 (Ref. The paradoxical effect of MMR proteins on MSI is particularly apparent at the LH3 Ref. . In contIn the case of MSI-high tumours, the expanded microsatellites themselves might be a source of vulnerability that could be exploited for therapeutic purposes. Since expansion results in a dependence on DNA helicases such as WRN Ref. , it may


TM) and its originator (HumiraTM) in IBD are lacking. We compared the efficacy and safety of GP2017 versus originator in IBD outpatients in an Italian real-life setting. This retrospective analysis enrolled consecutive IBD patients with complete clinical, laboratory, and endoscopic data. Clinical activity was assessed with the Mayo score in ulcerative colitis (UC) and the Harvey–Bradshaw Index in Crohn’s disease (CD). The primary endpoints were the induction of remission and the safety of GP2017 versus ADA originator. One hundred and thirty-four patients  were enrolled: 62 (46.3%) patients were treated with GP2017, and 72 (53.7%) with ADA originator; 118 (88.1%) patients were naïve to ADA. Clinical remission was obtained in 105 (78.4%) patients, during a median follow-up of 12 months, 82.3% and 75% in the GP2017 and ADA originator groups, respectively (p = 0.311). Treatment was well tolerated in both groups. This analysis of real-world data suggests that GP2017 and its originator are equivalent in terms of efficacy and safety in patients with IBD.The approval of adalimumab (ADA) biosimilars for inflammatory bowel disease (IBD) has reduced the cost of treatment. While several ADA biosimilars are currently available, comparative data on the ADA biosimilar GP2017 (Hyrimoz Inflammatory bowel diseases (IBD), which primarily include Crohn’s disease (CD) and ulcerative colitis (UC), are frequently observed in the Western world, with 6.8 million cases of IBD reported globally in 2017 . The pleiotropic proinflammatory cytokine tumor necrosis factor α (TNFα), performs a crucial role in the pathogenesis of CD and UC, and anti-TNFα agents were the first biological drugs approved to treat IBD . HoweverTM, Amgen Inc., Thousand Oaks, CA, USA), SB5 , MSB11022 , and GP2017  are currently limited  during treatment) of GP2017 and ADA originator.AEs occurring at the injection time were classified as early, while AEs occurring at least 1 week after the injection were defined late events. AEs were graded as mild (not required to stop treatment) or severe (requirement to stop treatment). The occurrence of opportunistic infections was also regarded as an AE . OpportuThe secondary endpoints assessed differences between GP2017 and ADA originator in terms of: (1) clinical response (defined as a decrease of at least 2 points in the Mayo score in UC patients and at least 3 points in the HBI in CD patients); (2) mucosal healing (defined as a Mayo subscore for endoscopy ≤ 1 in UC and SES-CD ≤ 2 in CD patients); (3) reduction of steroid use during the study period; (4) prevention of colectomy in UC and any disease-related surgical procedure in CD; and (5) optimization rate to reach remission for the biosimilar GP2017 during follow-up. To exclude any bias on mucosal healing evaluation in patients switched from ADA originator to biosimilar, we evaluated mucosal healing only in patients naive to GP2017 who had performed colonoscopy. Therapeutic optimization was allowed as follows: 40 mg every week or 80 mg every 2 weeks for both the ADA originator and biosimilar GP2017.Data analysis was perfomed with MedCalc® Release 14.8.1. p-values of <0.05 were considered to be significant.Continuous non-parametric variables were reported as median (interquartile range [IQR]), and categorical variables were reported as number (percentage). The Shapiro-Francia test was used to test for normal distribution. The Chi-square test was used to compare categorical variables, and the Mann–Whitney test was used to compare continuous variables. A total of 134 patients  were enrolled according to the inclusion criteria. Forty-one patients were diagnosed with UC and 93 patients with CD. Patients with UC had a median disease duration of 7 years (IQR 4–11), while for patients with CD, the median disease duration was 5 years (IQR 2–10). Thirty-five (85.4%) patients with UC and 83 (89.2%) patients with CD were naïve to ADA. p < 0.007). For both UC and CD, the main indication to use ADA biosimilar was “steroid dependency”; “steroid resistance” was the second most common indication in patients with UC, while this was “others” for patients with CD. Median FC levels were significantly higher in patients with UC than CD .For the total population, 62 (46.3%) patients received GP2017 while 72 (53.7%) patients received ADA originator. Significantly more patients with CD than UC had undergone a previous appendectomy (The characteristics of the study group are reported in Patients’ median follow-up time was of 12 (IQR 6–12) months. p = 0.311). Overall, 105 of 134 (78.4%) patients reached clinical remission, with no significant difference between treatment groups. Specifically, clinical remission was obtained in 51 of 62 (82.3%) patients treated with GP2017 and 54 of 72 (75.0%) patients treated with ADA originator (p = 0.207). In patients naïve to biologics clinical remission was obtained by 93 of 118 (78.8%) patients, including 39 of 46 (84.8%) in the GP2017 group and 54 of 72 (75.0%) in the ADA originator group  patients reached clinical remission, all from the GP2017 group. Only 2 (4.9%) patients with UC and 3 (3.2%) with CD experienced an AE . TreatmeNo cases of malignancy, tuberculosis, or death were reported during the study. p = 0.692 and p = 0.910, respectively). Mucosal healing was obtained by a significantly higher proportion of patients after therapy with GP2017 than ADA originator . There were no surgeries performed in either treatment group during follow-up.The outcomes of secondary endpoints are reported in p = 0.051).Optimization was required by 9 (6.7%) patients in total; 7 (11.3%) patients in the GP2017 group and 2 (2.8%) in the ADA originator group, with a trend toward difference  of IBD patients treated with GP2017 in a separate real-life Italian study [ of IBD pImportantly, the biologic status of patients did not affect clinical remission in our study. In ADA-naïve patients, who represented almost 90% of the study population, clinical remission was obtained in more than 75% of patients with no significant difference in terms of biological treatment. Interestingly, GP2017 was also effective in most patients with prior exposure to TNFα inhibitors, although this patient population was limited in size.Our study also found that GP2017 was similar to its ADA originator in clinical response and steroid reduction. Interestingly, the rate of mucosal healing was significantly higher in GP2017-treated patients than in the ADA originator group: it could be hypothesized that patients who switched from the ADA originator to GP2017 were already in remission. However, the optimization rates show that more patients treated with GP2017 switched to the weekly dose than those receiving ADA originator. To explain these results, we hypothesize that patients already treated with ADA would still need an optimization rate. On the other hand, we cannot exclude a “nocebo” effect that could negatively influence the performance of the ADA biosimilar , requiriFinally, GP2017 seems to be as safe as the ADA originator in real-life clinical practice, with only five AEs reported, and no between-group significance for AE-related treatment discontinuation.Our study has strengths and limitations. One strength is the multicentric organization, which allow collection of data from several Gastroenterology Units, making the study population representative of a range of geographical areas across Italy thus limiting single center clinical bias. However, the main limitation is the retrospective design which did not allow the definition “a priori” of the patient number to power the analysis, leading to the small sample size. In addition, retrospective design did not allow enrolled patients to have the same timing throughout follow-up . Moreover, the follow-up period was limited to 12 months and we recognize that a longer follow-up would add insightful information. Nevertheless, it should be acknowledged that ADA biosimilar GP2017 was approved in Italy in January 2019 and had only been in clinical practice for approximately 20 months at the time of our study. The ongoing nature of our study will allow for a longer follow-up period in future analyses.This observational study is the first analysis to show that the ADA biosimilar GP2017 is as safe and effective as its originator ADA in managing IBD patients in real-life clinical practice in Italian IBD centers. This results strongly support a wider use of ADA biosimilars in the routine clinical practice also for IBD patients. This would reduce the total costs for the hospitals without affecting therapy efficacy and patient safety. To confirm these promising results, randomized, double-blind, prospective studies with higher patient numbers and a longer follow-up are necessary.


Alzheimer’s disease (AD) and type 2 diabetes mellitus (DM2) are chronic degenerative diseases with complex molecular processes that are potentially interconnected. The aim of this work was to predict the potential molecular links between AD and DM2 from different sources of biological information.In this work, data mining of nine databases  was performed to identify gene and protein information that was shared in AD and DM2. Next, the information was mapped to human protein-protein interaction (PPI) networks based on experimental data using the STRING web platform. Then, gene ontology biological process (GOBP) and pathway analyses with EnrichR showed its specific and shared biological process and pathway deregulations. Finally, potential biomarkers and drug targets were predicted with the Metascape platform.A total of 1,551 genes shared in AD and DM2 were identified. The highest average degree of nodes within the PPI was for DM2 (average = 2.97), followed by AD (average degree = 2.35). GOBP for AD was related to specific transcriptional and translation genetic terms occurring in neurons cells. The GOBP and pathway information for the association AD-DM2 were linked mainly to bioenergetics and cytokine signaling. Within the AD-DM2 association, 10 hub proteins were identified, seven of which were predicted to be present in plasma and exhibit pharmacological interaction with monoclonal antibodies in use, anticancer drugs, and flavonoid derivatives.Our data mining and analysis strategy showed that there are a plenty of biological information based on experiments that links AD and DM2, which could provide a rational guide to design further diagnosis and treatment for AD and DM2. Alzheimer’s disease (AD) and type 2 diabetes mellitus (DM2) are chronic degenerative human diseases with complex molecular processes . WorldwiThe relationship of DM2 with AD has attracted the attention of the scientific community due to its possible link derived from epidemiological research . Other sβ) cell development, negative regulation of PI3K/AKT signaling pathway, β-amyloid and insulin degradation. Other studies have used these tools to identify new genes and possible pharmacological targets in AD , signaling pathways, and hub proteins that potentially connect both diseases. The molecular interactions described here could contribute to the elucidation of the pathophysiological processes underlying the AD—DM2 association. Furthermore, potential biomarkers and drug targets for its diagnosis and treatment were identified.https://www.disgenet.org/), Ensembl (https://www.ensembl.org/index.html), OMIM (https://omim.org/), Protein Data Bank (https://www.rcsb.org/), The Human Protein Atlas (http://www.proteinatlas.org/), UniProt (https://www.uniprot.org/), Human Cell Atlas (https://www.humancellatlas.org/), PubMed (https://pubmed.ncbi.nlm.nih.gov/), and Gene Expression Omnibus . The key words “AD”, “DM2”, “Alzheimer”, “type 2 Diabetes Mellitus” were used to search on each database.The workflow for gene and protein data mining is shown in https://pubmed.ncbi.nlm.nih.gov/) was retrieved with the PubTator application (https://www.ncbi.nlm.nih.gov/research/pubtator/)  were considered. In GEO, the data sets GSE5281, GSE122063 were chosen for AD, and the GSE7014, GSE29221 for DM2. Those files comprise 297 patients for AD and 60 for DM2; the differentially expressed genes (DEGs) were determined with the GEO2R tool (https://www.ncbi.nlm.nih.gov/geo/geo2r/)  (http://bioinformatics.psb.ugent.be/beg/) to identify the genes shared between AD and DM2. The intersected information was considered as the AD-DM2 association.Further data processing was performed for the following databases: The PubMed gene information (btator/)  and the btator/) , with a /geo2r/) . DEG selry 2022) . The finhttp://string-db.org) to determine PPI in humans. The parameters to predict PPI were experimental evidence which include only biochemical or biophysical data produced from yeast two-hybrid experiments, and a minimum required interaction score >0.900, which represents the approximate probability that a link exits between two proteins in the same metabolic map in the KEEG database to reduce the rate of false positives   and KEGG 2021 pathway analysis through the EnrichR platform (nrichr/) . These aanalysis , only thhttps://maayanlab.cloud/Enrichr/) was used. The Expression2Kinases program  were taken for analysis, as previously did for GOBP.To determine the proteins of high biological value within the PPI networks groups (hub proteins), the hub option from the EnrichR platform ( program  was used program . Only prhttps://apps.cytoscape.org/).To determine if the hub proteins could be possible biomarkers or drug targets, the protein names were submitted to the Metascape platform to match our data with the available options “plasma” (protein atlas) and “drug bank” . The obtn = 6, 069) and the information in the scientific literature (PubMed) was scarce (n = 77) . The shared data was considered as the AD-DM2 association group.To identify data shared by both AD and DM2, nine databases were mined . After e(n = 77) . Venn di(n = 77) , but botThe gene lists for the AD, DM2 and AD-DM2 groups were assigned to PPI in humans on the STRING platform . BecauseTo understand the biological significance of the PPI data, analysis of GOBP and signaling pathways were performed for each group. The most significant  GOBP associated with AD was related to the “nuclear-transcribed mRNA catabolic process (GO:0000184)”, followed by transcriptional and translation processes related to the neuron’s cells. On the other hand, GOBP for DM2 were associated with “mitochondrial respiratory chain complex I assembly (GO:0032981)”, followed by related bioenergetics terms. For DM2 the most significant pathway was “Thermogenesis”. The GOBP analysis for the AD-DM2 association was related to the “cellular response to cytokine stimulus (GO:0071345)”, followed by the “cytokine-mediated signaling pathway (GO:0019221)” that connects all group information . The sigHub analysis was performed to identify the main proteins that interact within the PPI networks. EnrichR showed SRC (a tyrosine-protein kinase) shared with the DM2 and AD-DM2 data . Two proThe Metascape platform showed that of the ten hub proteins identified in the association AD-DM2, seven  were matched as proteins present in plasma, except UBC, MAPK3, and ESR1. All of them could be inhibited by multiple drugs . For exaAD and DM2 are complex diseases for which a link have been suggested . The larFunctional analyses based on GOBP for AD were found related to specific terms that affect the vital genetics processes of the cells. For example, the most significant were transcriptional and translation terms, which were also observed in gene expression studies specific to AD , indicatThe hub proteins within the networks showed that some TFs, kinases, and ubiquitin proteins are shared between the study groups. Among the most relevant were UBC, SRC, ESR1, BRCA1, and SLC2A4. UBC, also known as ubiquitin C, has been associated with protein ubiquitination processes . This prβ-amyloid has been used (β-amyloid as the cause of Alzheimer’s (α (HIF-1) found related to the AD-DM2 association (Among the hub proteins related to the AD-DM2 association, some of those were targets of drugs already in use. The EGFR protein is a target of mAbs. This class of immunotherapy has been proposed for the treatment of AD; for example, mAbs against the A een used . In DM1 een used . For exaeen used . Moreoveheimer’s  obligateociation , 5 are iociation , a microociation . In the ociation . In relaociation . As microciation . Anotherociation  as well ociation . TherefoIn conclusion, we found considerable biological information that links AD and DM2. Prediction of PPI guided the inference of the potential dysregulated GOBP and pathways shared for both diseases or specific to each of them, highlighting the inflammatory deregulation for the AD-DM2 association. Analysis of hub proteins allows the identification of anticancer drugs and flavonoid nutraceuticals already in use, underlining potential drugs and targets for further drug repurposing efforts. In addition, those hub plasma-predicted proteins could be potential blood biomarkers that could lead to improved diagnostic strategies. Also, our data mining strategy to study the complex interactions underlying AD and DM2 could be adapted to other diseases where an epidemiological or molecular link has been recognized.10.7717/peerj.14738/supp-1Supplemental Information 1Table S1: Raw data obtained from the databases; Table S2: HUGO curated gene lists; Table S3: PPI experimental data obtained from STRING; Table S4: Gene ontology biological process and signalling pathways for the study groups. Table S5: EnrichR predicted HUB proteins for the study groups; Table S6: Metascape results for the HUB proteins of the AD-DM2 group.Click here for additional data file.


Propolis has demonstrated wound healing effects. Propolis' effects vary based on its composition and geographical origin. However, there are few reports on the effects of propolis on oral wound healing. The aim of this study was to evaluate the antioxidant and in vitro wound healing were determined by MTT, modified Boyden chamber, and scratch assay, respectively.  The crude ethanol extract of propolis was obtained by maceration with 95% ethanol that was sequentially fractionated with hexane, ethyl acetate, and distilled water. The chemical profiles of the samples were assessed by thin-layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). Antioxidant activity was determined using DPPH and FRAP assays. The effects of the propolis fractions on human gingival fibroblast (HGF) proliferation, migration, and  We found that solvent polarity greatly affected the extract yield and TLC profiles. The highest extract yield was found in HEP (38.88%), followed by EEP (19.8%) and AEP (1.42%). TLC revealed 7 spots in the crude ethanol extract (Rf 0.36–0.80), 6 spots in HEP (Rf 0.42–0.80) and EEP (Rf 0.36–0.72), and 4 spots in AEP (Rf 0.17–0.79). GC-MS analysis revealed a high amount of triterpenoids in HEP (82.97%) compared with EEP (28.96%). However, no triterpenoid was found in AEP. The highest antioxidant activity and stimulation of HGF proliferation were observed in HEP, followed by EEP and AEP. HEP and EEP, but not AEP, enhanced HGF migration. However, all propolis fractions induced wound closure. in vitro wound closure effects were found in HEP, EEP, and AEP fractions. HEP contained a large amount of triterpenoids. Antioxidant and  Oral wound healing is a complex process involving a cascade of well-orchestrated biological events that regulate the repair of oral tissue lesions. Healing consists of four phases, inflammation, proliferation, remodeling, and homeostasis. Although oral and dermal wound healing contain similar stages, oral wounds are characterized by rapid healing with minimal scar formation, mediated in part via an enhanced oral fibroblast and oral epithelial cell repair response .Successful wound healing is accomplished by a series of coordinated processes that include cell migration and proliferation, extracellular matrix deposition, and remodeling. Fibroblasts are the most abundant cell type in connective tissue. These cells are involved in producing and remodeling the extracellular matrix . GingivaIt has been demonstrated that oxygen is required to disinfect a wound; therefore, oxygen-dependent redox-sensitive signaling is an important process for regulating oxygen levels during the wound healing cascade. Formation of free radicals in tissues can directly impair the function of some aspects of the cell membrane or intracellular organelles or can initiate an inflammatory signaling pathway that leads to the production of numerous mediators of cell injury. The process of wound healing is largely impeded by excessive oxidative stress in a wound. The excessive reactive oxygen species (ROS) accumulated in the wound induce strong inflammatory reactions that can inhibit wound healing. Moreover, ROS inhibit the functions of endogenous stem cells and macrophages, slowing wound tissue regeneration , 5.in vitro, in vivo, and ex vivo studies, as well as in human clinical trials [The interest in evidence-based complementary and alternative medicine has increased, resulting in many studies on the use of natural products for wound healing. Propolis, or bee glue, is a natural resinous substance collected by honeybees from tree buds and exudates. It consists mainly of resins, balsams, beeswax, essential oils, pollen, and other organic compounds. Propolis has antimicrobial, antioxidative, immunomodulatory, antiulcer, and wound healing effects. Therefore, it has been extensively used in the health industry worldwide , 7. Propl trials , 9.The components of propolis vary. The characterization of the phenolic and flavonoid contents in propolis is needed to obtain the best therapeutic and medicinal benefits in dentistry and medicine . Althougp-anisaldehyde, dimethyl sulfoxide (DMSO), 1,1-diphenyl-2-picrylhydrazine (DPPH), and pyridine (HPLC grade) were purchased from Sigma-Aldrich . Dichloromethane  and formic acid were purchased from Thermo Fisher Scientific . Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) was purchased from Cerilliant Corporation .95% ethanol , methanol , ethyl acetate , and n-hexane  were purchased from Apex Alco Co., Ltd. . Quercetin, gallic acid, galangin, naringenin, apigenin, Tetragonula fuscobalteata was harvested from an orchard in Chiang Mai province in Northern Thailand in January 2018 and kindly provided by Professor Somnuk Boongird, Faculty of Science, Ramkhamhaeng University. The preparation and extraction process was modified from Szliszka et al. [Raw propolis 163 g) from 3 g from n-hexane, ethyl acetate, and water) in order of increasing polarity with a separating funnel. The methanol solution was partitioned with n-hexane (2 × 200 mL). The n-hexane layers were pooled and dried using a rotary evaporator. The methanol layers were pooled, dried, and dissolved with 100 mL of distilled water to obtain the aqueous-methanol extract. The aqueous-methanol extract was further partitioned with ethyl acetate (2 × 200 mL). The ethyl acetate and aqueous layers were collected separately. Each extract was pooled and dried using a rotatory evaporator. The dry weight of each partition was recorded as total yield. After separation, 63.37 g of the n-hexane extract of propolis (HEP), 32.34 g of the ethyl acetate extract of propolis (EEP), and 2.33 g of the aqueous extract of propolis (AEP) were obtained , placed 1 cm from the lower edge of the plate. Preliminary TLC separation of the extracts was performed using different solvent systems; the solvent front was allowed to travel to at least 75% of the TLC plate height. Two solvent systems, n-hexane : ethyl acetate : formic acid (30 : 70 : 1) as a combination of nonpolar solvents and ethyl acetate : methanol : formic acid (50 : 50 : 1) as a combination of polar solvents, were used to elute the compounds in the extracts [Thin-layer chromatographic (TLC) plates, composed of a 0.2 mm thick precoated silica gel 60F-254 layer , received 1 extracts . The spoextracts . FinallyμL) were injected and analyzed by GC-MS.The propolis extracts were derivatized prior to gas chromatography-mass spectrometry (GC-MS) analysis as previously described . The drim-coumaric acid, ferulic acid, caffeic acid, galangin, naringenin, apigenin, and quercetin) were characterized by GC-MS analysis. Each standard compound (2 mg/mL) was prepared in pyridine mixed with 1% TMCS BSTFA solution. An Agilent 7890 B/5977A Series Gas Chromatograph/Mass Selective Detector System with an autosampler model GC Sampler 80  was used in this study. The MS was operated in the electron ionization mode (70 eV). Helium was employed as the carrier gas, and its flow rate was adjusted to 0.8 mL/min. The chromatographic separation of the chemical compositions was performed on a GC capillary column HP-5MS . The initial temperature of the column was set at 60°C, then increased by 5°C /min to 300°C, and held for 10 min. The injector temperature was set at 320°C in the split mode (split ratio 10 : 1). The temperature of the GC-MS interface and ion source was set at 300°C and 230°C, respectively. The MS was operated in the full scan mode, and the MS scan range was 35–800 m/z. Interpretation of the GC-MS mass spectrum was performed using the Wiley Registry® 10th Edition/NIST 2014 Mass Spectral Library database. The mass spectrum of the unknown compounds was compared with the spectrum of specific standards and reference compounds in the library. An MS match score in the range of 70–100% was reported as the identified compounds in our propolis extracts [The chemical compositions of the propolis extracts and standard compounds  and ferric reducing antioxidant power (FRAP) assays were performed. Quercetin was used as a positive control in these assays.μg/mL and 10 μg/mL, respectively. The mixture of DPPH and propolis extracts or quercetin was added to a 96-well plate (100 μL/well). The plate was incubated at 25°C for 30 min in the dark, after which the absorbance was read at 512 nm using a spectral scanning multimode reader (Varioskan® Flash Type 3001) . DPPH in DMSO was used as control. The DPPH radical scavenging activity was calculated as follows:The DPPH assay was employed to evaluate the stable free radical scavenging activity by modifying previous methods , 23. BriThe experiment was performed in triplicate, and three independent experiments were performed.3.6H2O at a ratio of 10 : 1 : 1) was freshly prepared. Working FRAP reagent (175 μL) was added to 25 μL propolis extracts or quercetin in each well of 96-well plates. The plate was incubated at 37°C for 4 min. The absorbance was read spectrophotometrically at 593 nm. The percentage of ferric radical scavenging activity was calculated as shown follows:The FRAP of the propolis extracts was determined using a previously described method based on the reduction of ferric tripyridyltriazine complex to ferrous tripyridyltriazine . BrieflyThe experiment was performed in triplicate, and three independent experiments were performed.Human gingival fibroblasts (HGFs) were isolated from the explants of healthy gingival tissues from patients who underwent minor oral surgery at Thammasat University Hospital. The protocol for human tissue acquisition was performed according to the International Conference on Harmonization-Good Clinical Practice (ICH-GCP) and was approved by the Institutional Review Board of Thammasat University (IRB number 043/2562). The HGFs were isolated and cultured as previously described [3 cells/well) for 24 h. The cells were treated with HEP , EEP , or AEP  for 24 h. The cell proliferation in each treatment group was determined using the thiazolyl blue tetrazolium bromide (MTT) (Sigma) as previously reported [HGFs were cultured in 96-well plates (3 × 10reported . The abs4 cells) were resuspended in HEP (15.62 μg/mL), EEP (40 μg/mL), or AEP (400 μg/mL) diluted in DMEM containing 0.1% BSA and seeded into the upper well of the chamber. The cell without propolis extract treatment was used as control. The cells were incubated for 5 h, and the filters were fixed and stained with crystal violet for 30 min. The migrated cells were counted by three investigators using a microscope at 400x magnification. The migrated cell counts were averaged from five randomly selected fields. Three chambers were used in each treatment. Three independent experiments were performed.The modified blind-well Boyden chamber migration assay was performed to evaluate the effect of the propolis extracts on HGF cell migration. The modified Boyden chemotaxis chamber  was used based on a chamber with two medium-filled compartments by modifying a previous method . Briefly6 cells/ well) for 24 h. When the cells were 80–90% confluence, the medium was replaced with serum-free DMEM to minimally maintain HGF growth. After 24 h, artificial wounds were created in the monolayers by making a linear scratch in the center of each well using a 200 μL pipette tip. Any cellular debris created from scratch was removed by gently washing the wells with PBS. The HGFs were treated with HEP (15.62 μg/mL), EEP (40 μg/mL), or AEP (400 μg/mL) for 24 h. Cells without propolis extract treatment were used as the control. The images of five randomly selected areas on each well were captured using a microscope camera . A mark was placed on the back of each random area of each well as a reference point for subsequent imaging. The area measurements were calculated using Image J software  by measuring the gap that was closed by the cells. The percent closure of the scratch width was calculated using the following equation:T0 is the area of wound measured immediately after scratching and AT24 is the area of wound measured 24 h after scratching. Three independent experiments were performed.The scratch assay was performed to test the effect of the propolis extracts on wound closure by HGFs as previously described . HGFs weP ≤ 0.05 was considered statistically significant.The data analyses were performed using GraphPad Prism 7.04 software . The data are expressed as means and standard error of the mean (SEM) as previously recommended . The mean-hexane, ethyl acetate, and distilled water were evaluated to determine their effects on the extraction yield of propolis. The results demonstrated a difference in the extraction yield using different solvents. Among the solvents tested, HEP resulted in the highest extraction yield (38.88%), followed by ethyl acetate (19.84%) and distilled water (1.42%)  . This inn-hexane, ethyl acetate, and formic acid solvent systems   . The ethfor AEP) . Therefofor AEP) .The HEP, EEP, and AEP extracts were analyzed using GC-MS to determine their chemical composition. The 7 compounds that are commonly found in propolis, (1) m-coumaric acid, (2) ferulic acid, (3) caffeic acid, (4) galangin, (5) naringenin, (6) apigenin, and (7) quercetin, were used as standard compounds in our study. The chemical profiling of the standard compounds and the 3 propolis extracts is presented as chromatograms in The chemical component in each propolis extract was identified based on the Wiley Registry® 10th Edition/NIST 2014 Mass Spectral Library. The compounds in each extract were reported when their mass spectral match score was higher than 70. Forty-four compounds met the criteria and were characterized as triterpenoids, tetraterpenoids, phenolics, flavonoids, alkaloids, sugars, and other compounds in HEP, EEP, and AEP . The resμg/mL) was used as a positive control in the DPPH and FRAP assays. All propolis extracts exhibited antioxidant activity but significantly lower than that of quercetin (P < 0.05). However, the HEP and EEP exhibited significantly higher antioxidant activity than that of AEP in the DPPH assay (P < 0.05)  . The FRA < 0.05) .μg/mL compared with that of control (P < 0.05). In contrast, significantly inhibited cell proliferation was found when the concentration was increased to 250 μg/mL (P < 0.05) (μg/mL compared with that of control (P < 0.05); however, significantly inhibited cell proliferation was found when the concentration was increased to 320 μg/mL (P < 0.05) (μg/mL) compared with that of control (P < 0.05); however, there was no significant difference when the concentration was increased to 1,000 μg/mL (P > 0.05)  . EEP rev < 0.05) . AEP sig > 0.05) . The minμg/mL HEP, 40 μg/mL EEP, or 400 μg/mL AEP, and cell migration was evaluated using a modified blind-well Boyden chamber migration assay. The migrating cells were stained with crystal violet after incubating with or without propolis extracts (P < 0.05) (P > 0.05).The HGFs were treated with 15.62 extracts . The mig < 0.05) . HoweverP < 0.05)  .n-hexane was the most effective method of raw propolis extraction to obtain the most chemically complex product. These results did not agree with the findings of the other studies that used ethanol extraction for processing raw propolis [The present study analyzed the phytochemical profile and the antioxidant activity of sequential hexane, ethyl acetate, and aqueous extracts of Thai propolis and determined their effects on human gingival fibroblast migration and proliferation. We found that the extraction yield of Thai propolis was the highest in the nonpolar solvent and gradually decreased with increasing polarity of the extract solvent. This suggests that the differential distribution of extracted compounds in the polar and nonpolar solvents was based on their polarity. Based on the polarity scale, water has the highest polarity of 9.0, and ethyl acetate has a medium polarity of 4.3, while hexane has the lowest polarity of 0.00 . Thus, tpropolis , 33.The TLC of the crude ethanol extract, HEP, EEP, and AEP demonstrated that the resolution of the nonpolar eluent was greatest compared with the polar-mobile solvent. In addition, we found that the crude ethanol extract, HEP, and EEP had spots that reflected blue fluorescent in UV366 nm and became purple after staining with an anisaldehyde-sulfuric agent. Therefore, it is likely that most compounds in the crude ethanol, HEP, and EEP are terpenoids ,34.β-steroids. Flavonoids have been reported to be the main compounds in propolis. Terpenoids, which represent only 10% of the content, are responsible for propolis' odor because they are volatile components in plants and contribute to their biological properties [Tetragonula fuscobalteata species. Our propolis chemical profile is similar to that of propolis from tropical areas that are enriched in triterpene. Triterpene-rich propolis was found in propolis samples from Malaysia [To date, numerous studies have been published regarding the chemical composition and biological effects of propolis. However, analysis of a large number of samples from different geographic regions has revealed that its chemical composition is highly variable and difficult to establish because it depends on several factors, including the vegetation and season at the collection site and the bee species. The main chemical groups present in propolis comprise phenolic compounds, such as esters, flavonoids, terpenes, aromatic aldehydes and alcohols, fatty acids, stilbenes, and operties . Surprisoperties , 36. TheMalaysia , Yemen [Malaysia , EthiopiMalaysia , BoliviaMalaysia , and CamMalaysia . In contMalaysia ,43. ThisMalaysia . Our datSanghuangporus sanghuang [Reissantia indica [α-amyrin, and β-amyrin that are classified as triterpenoids in the extract may have a synergistic effect on the antioxidant activity of HEP [The antioxidant activity of triterpenoid was observed in propolis from Cameroon , a medicanghuang , and thea indica . It is py of HEP –49. In cy of HEP , 51.The term antioxidant covers a wide range of different molecules; however, a common feature is their ability to readily donate electrons while remaining stable, thus acting as reducing agents and minimizing the damage caused by free radicals . AntioxiThe DPPH-free radical scavenging by antioxidants is due to their hydrogen-donating ability. Therefore, DPPH radicals are scavenged by antioxidants through the donation of hydrogen, forming reduced DPPH . In the 3+) to the ferrous ion (Fe2+) [The FRAP assay is based on the rapid reduction in ferric-tripyridyltriazine by antioxidants present in the samples forming ferrous-tripyridyltriazine, a blue-colored product . In contn (Fe2+) . In our n (Fe2+) , 41, 60.n (Fe2+) , 61. Then (Fe2+) , 62, 63.n (Fe2+) –67. Thesn (Fe2+) , 60. Thin (Fe2+) , its actin vitro model. Therefore, the effects of HEP, EEP, and AEP on HGF proliferation, migration, and in vitro wound healing were evaluated in the present study. Here, we demonstrated that Thai propolis extracts stimulated HGF proliferation and migration. HGF cells are known to play an essential role in wound healing. Cell migration is one of the most essential steps during the proliferative phase, which is responsible for wound closure [in vitro oral wound closure. This indicates that the propolis extracts, especially HEP and EEP, stimulate the proliferative phase of oral wound healing, especially oral fibroblast migration and proliferation. Our findings are in line with a previous study [The purpose of this study was to test the hypothesis that the antioxidant compounds in Thai propolis extracts stimulate HGF cell behaviors that increase wound healing in an  closure . HEP demus study  that demWound healing requires a fine balance between the positive and deleterious effects of reactive oxygen species (ROS), a group of extremely potent molecules that are rate limiting in successful tissue regeneration. A balanced ROS response will debride and disinfect a tissue and stimulate healthy tissue turnover, suppressed ROS will result in infection, and increased ROS will destroy otherwise healthy stromal tissue . The homin vitro wound closure. Therefore, our results confirm those of previous studies that reported that antioxidant compounds in the propolis extract play an important role in wound healing [Antioxidants are proposed to control wound oxidative stress and thereby accelerate skin and oral wound healing , 71. Pot healing , 13.Two systematic reviews revealed that triterpenes induce wound healing , 76. TheThe results of this study have some implications for the clinical use of propolis extracts in oral wound healing. However, wound healing has various stages and is facilitated by antioxidant, antimicrobial, and anti-inflammatory activities. Therefore, further investigations need to be performed on the effect of this propolis on the other processes of wound healing. In addition, experiments should be performed using different cell types involved in oral wound healing.in vitro oral wound healing model. This model may not fully reflect the overall oral wound healing processes. Therefore, this study should be repeated using an animal model. Another limitation is that this study did not identify the exact bioactive components that regulate our observed HGF behaviors. To overcome these limitations, future studies are necessary to identify the bioactive components involved in oral wound healing activation from Thai propolis.This study has some limitations that need to be considered when interpreting the results. The present study was performed using an However, in line with the previous reports, our data support that propolis at a defined concentration is cytocompatible and, thus, may be used as an adjuvant therapeutic supplement that has the potential to enhance oral wound healing.n-hexane extract and ethyl acetate extracts of Thai propolis enhance the proliferative phase of oral wound healing. The extracts also stimulate the proliferation of human gingival fibroblasts. However, the effect of the aqueous extract of propolis on antioxidation and cell migration was scant compared with the n-hexane and ethyl acetate extracts. Furthermore, the effect of bioactive compounds identified in our propolis on in vivo oral would healing is needed to be addressed.Within the scope of this study, we have demonstrated that the


This study aimed to evaluate the efficacy and adverse events of esophageal variceal ligation (EVL) vs. EVL combined with endoscopic injection sclerosis (EIS) in the therapy of esophageal varices.Patients from January 2017 to August 2021 who received EVL alone (control group) or EVL plus EIS (intervention group) were enrolled in this retrospective study. Efficacy, including rebleeding , variceal recurrence rate (the presence of esophagogastric varices which is needed to be treated again) the number of sessions performed to complete eradication of varices, and safety (adverse events) were compared. The variceal recurrence-associated factors were derived by univariate and multivariate logistic regression analyses.P = 0.006 and 20.7% vs 37.5%, P = 0.029, P = 0.006, respectively, in the 12-month follow-up). The adverse events  showed no significant difference between the two groups (P > 0.05). Further research showed that the efficacy of the intervention group was better than the control group only achieved in prophylactically endoscopic treatment patients. The diameter of esophageal varices and gastric varices co-exist showed significant effects on variceal recurrence in intervention group .The variceal recurrence and rebleeding rate in the intervention group showed significantly lower than the control group  and similar incidence of adverse events, especially for prophylactically treatment. Among the intervention group, the diameter of esophageal varices and gastric varices were closely associated with variceal recurrence. Esophageal variceal bleeding (EVB) is one of the most severe adverse events of liver cirrhosis . Seven pIn recent years, some evidence suggests that EVL combined with EIS can significantly reduce bleeding and recurrence of esophageal varices , 11, witn = 136) or EVL plus EIS  at Tianjin Second People's Hospital between January 2017 and August 2021 were included in this retrospective study. Patients included in the study were those with cirrhosis and esophageal varices with or without gastric varices or those who experienced acute bleeding related to esophageal varices. Despite this, their cirrhosis-related complications were not severe . Exclusion criteria were as follows: (1) patients with hepatocellular carcinoma (HCC); (2) patients treated with splenectomy; (3) patients treated with transjugular intrahepatic portosystemicstent-shunt (TIPS); (4) patients treated with subtotal gastrectomy; and (5) patients taking propranolol or other NSBBs. Clinical data  classification, a diameter of esophageal varices, presence of gastric varices, prophylactic or acute bleeding, and whether the gastric varices were injected with tissue adhesives) and laboratory data  at the initial treatment were collected before. Medical history was also collected, including hypertension, diabetes, and coronary heart disease. The Ethics Committee approved this retrospective cohort study of Tianjin Second People's Hospital, and written consent was obtained from the patients before the study.Three hundred and ten patients who received EVL . Variceal ligation was performed starting from the nearest position near the gastroesophageal junction, releasing five to seven bands depending on the variceal condition.For patients receiving EVL plus EIS, EVL was performed first, in the same way as in the EVL group. Then a puncture needle (23-gauge) was inserted into varicosity 1–2 cm proximal to the upper part of the band and injected with lauromacrogol  on the same day, 2–8 ml at a time, as a sclerosant .After the initial treatment, the treated esophageal varices are examined endoscopically 2 or 3 weeks later. Repeated treatments were then performed until the esophageal varices were eradicated. The primary follow-up outcome was the number of sessions until eradication of esophageal varices and rebleeding and variceal recurrence assessed at 6, 12, and 18 months after the eradication of esophageal varices. Rebleeding was defined as clinical hematemesis or melena which was confirmed by endoscopy as esophagogastric varices bleeding. Variceal recurrence was defined as the presence of varicose veins requiring retreatment. The number of sessions was defined as the number of sessions performed to eradicate the varicose veins.For the first objective, the primary outcomes were efficacy and adverse events between control and intervention groups. For the second objective, we further estimated the efficacy difference between prophylactic and therapeutic endoscopic treatment patients in the two groups.t test and χ2 test. P-values <0.05 were considered statistically significant.In the case of normal distribution, the data were presented as mean ± standard deviation, or amount and percentage. Predictors of varicose vein recurrence were identified by logistic regression analysis. Propensity scores were selected from propensity score matching (PSM) analysis to overcome the bias introduced by the different distributions in each group. All data were analyzed by R software v3.6.1 . The “Matchlt” package was used in the PSM matching. Comparisons of baseline parameters and outcomes between the two groups were performed using Student's n = 136) and intervention group (n = 174) participated in this study after a 6-month follow-up for varicose vein recurrence, rebleeding rates, number of sessions, and adverse events. Of these patients, 252 patients  completed the 12-month follow-up and 206 patients  completed the 18-month follow-up. Before PSM matching, there were significant gender differences, esophageal variceal diameter, tissue adhesive injection or not, INR, ammonia, ascites, and CTP classification between the control and intervention groups at 12-month follow-up (P < 0.05). P < 0.05). Among patients at 18-month follow-up, the proportion of men was significantly higher in the intervention group than in the control group before PSM (P = 0.006). At the same time, the esophageal variceal diameter was significantly larger in the intervention group (P < 0.001). Statistically, the intervention group had a statistically significant proportion of tissue adhesive injection (P < 0.001), a higher INR (P < 0.001), and higher ammonia (P = 0.001). Statistically significant findings were also found for ascites and CTP classification in the control and intervention groups (P = 0.016 and P = 0.031). After PSM, there were no differences between the two groups in terms of gender, esophageal variceal diameter, tissue adhesive injection, INR, ascites, and CTP classification   .P = 0.006). The rate of variceal recurrence was lower in the intervention group  than in the control group  (P = 0.029). The mean number of sessions in the control group were 31 for one time, 55 for two times, 44 for three times, and 6 for four times. However, the number of sessions were 55 for one time, 34 for two times, 24 for three times, and 3 for four times in the intervention group (P = 0.001). After PSM-matched analysis, two patients (2.9%) in the intervention group had rebleeding, similar to the control group (P = 0.101). Variceal recurrence occurred in 12 (17.4%) patients in the intervention group and 27 (39.1%) patients in the control group (P = 0.008). Of the patients in the intervention group, 37 were treated one time, 19 for two times, 13 for three times, and 0 for four times. Fifteen for one time, 31 for two times, 19 for three times, and 4 for four times in the control group (P = 0.001). There was also no significant difference in rebleeding rates between control and intervention groups after PSM matching patients at 6-month follow-up (P = 0.612). Similarly, the rate of variceal recurrence proved to be equal in both matched groups (P = 0.076). At 12-month follow-up, rebleeding occurred in 3 patients (2.6%) in the intervention group, much lower than in 14 patients (10.3%) in the control group (P = 0.042); 14 (17.1%) patients in the intervention group experienced variceal recurrence, much lower than 33 (34.4%) patients in the control group (P = 0.015) during prop hylactically treatment. The number of sessions in the control group compared with the intervention group was significantly different (P < 0.001)  than in the control group  (< 0.001) .P = 0.016] and gastric varices  were statistically associated with variceal recurrence in patients who received EVL plus EIS. Meanwhile, based on multivariate logistic regression analysis, the diameter of esophageal varices  and gastric varices  were shown to be predictors of variceal recurrence in patients in the intervention group  = 15.856; 95% confidence interval (CI), 1.709–160.143; on group .P = 1), chest pain (P = 0.676), and swallowing (P = 0.79) in the intervention group were similar to those of patients in the control group. In all patients, there were no patients with esophageal stricture. After the PSM matching analysis, the adverse events  also showed no significant difference between the intervention group and control groups. For the patients after 6 months and 12-month follow-up before and after the PSM matching, the incidence rate of adverse events was also similar between the two groups (In the 12-month follow-up patients, the incidence rate of fever (o groups .In our study, we observed the variceal recurrence and rebleeding after the eradication of esophageal varicose for the follow-up of 6 months, 12 months, and 18 months, respectively. Because many clinical factors influence variceal recurrence and rebleeding, we balanced the baseline clinical data by PSM. Prior to PSM, our study showed that patients treated with EVL plus EIS had significantly less varicose recurrence and rebleeding than patients treated with EVL alone, whether at 6-, 12-, or 18-month follow-up. Our results agree with Mansour et al. and Wang et al. , 14, 15.EVL is the recommended option for treating esophageal varices and esophageal variceal bleeding . HoweverOur study also has certain limitations. It was a single-center retrospective study. The regression analysis did not include more clinical parameters, and long-term follow-up of patients' deaths was impossible. Further large-scale studies need to be organized in the future.EVL plus EIS has lower variceal recurrence and rebleeding, compared with EVL alone, and has no obvious adverse events in our study. But we still need prospective, multicenter, randomized controlled studies to further confirm, in order to provide better treatment for esophageal varices.


HTT gene and striatal pathologic grade have yet to be formally established. We immunohistochemically studied 65 brains with a pathologic diagnosis of Huntington disease to investigate the cortical distributions and densities of HTT inclusions within the calcarine (BA17), precuneus (BA7), motor (BA4) and prefrontal (BA9) cortices; in 39 of these brains, a p62 immunostain was used for comparison. HTT inclusions predominate in the infragranular cortical layers (layers V-VI) and layer III, however, the densities of HTT inclusions across the human cerebral cortex are not uniform but are instead regionally contingent. The density of HTT and p62 inclusions (intranuclear and extranuclear) in layers V-VI increases caudally to rostrally (BA17 < BA7 < BA4 < BA9) with the median burden of HTT inclusions being 38-fold greater in the prefrontal cortex (BA9) than in the calcarine cortex (BA17). Conversely, intranuclear HTT inclusions prevail in the calcarine cortex irrespective of HTT CAG length. Neocortical HTT inclusion density correlates with CAG repeat expansion, but not with the neuropathologic grade of striatal degeneration (Vonsattel grade) or with the duration of clinical disease since motor onset. Extrapolation of these findings suggest that HTT inclusions are at a regionally-contingent, CAG-dependent, density during the advanced stages of HD. The distribution and density of HTT inclusions in HD therefore does not provide a measure of pathologic disease stage but rather infers the degree of pathogenic HTT expansion.Huntington disease is characterized by progressive neurodegeneration, especially of the striatum, and the presence of polyglutamine huntingtin (HTT) inclusions. Although HTT inclusions are most abundant in the neocortex, their neocortical distribution and density in relation to the extent of CAG repeat expansion in the  Hallmarks of many neurodegenerative diseases are the relative selective vulnerability of specific cell types to injury and the accretion of pathologic protein inclusions in the central nervous system (CNS). In Huntington disease (HD), the cardinal pathologic features are the worsening, topographic, degeneration of the neostriatum , in which the medium spiny neuron is the most vulnerable cell type, and the appearance of huntingtin inclusions due to abnormal polyglutamine (polyQ) expansion of the huntingtin protein (HTT) , 63, 64.N-terminal region of HTT have revealed visible pathologic HTT aggregates (inclusions) within the HD brain and in murine model systems of HTT expansion [Wild-type huntingtin has a myriad of functions throughout life (reviewed in ) and is xpansion , 37, 63.xpansion , 21, 58.xpansion , 18, 42.xpansion . Conversxpansion . The EM4xpansion . Becher Attempts to address any differential neurodegeneration in the HD neocortex have provided mixed results. Three morphometric studies that examined the macroscopic thickness of the cortex of coronal sections of the cerebral hemispheres demonstrated diffuse, even, and symmetric cortical thinning, except for relative sparing of the medial temporal lobe by Halliday et al. , 20, 61.The entire neocortex sends corticostriatal glutamatergic projections to the neostriatum in a topographic manner, a subset of which synapse onto the medium striatal spiny neurons , 33. GivWith this in mind, we sought to characterize the neocortical distribution of pathologic inclusions in 65 HD brains. These were selected to encompass the spectrum of severity in HD, including juvenile- and adult-onset individuals with CAG repeat expansions ranging from 38- 71 repeats and ages at death between 24 and 89 years-of-age. HTT inclusions and their relationship to CAG repeat expansion, metrics of disease burden  score, length of time from motor onset to death) were investigated. The selected distribution of HTT inclusions in the brain, correlation with CAG repeat expansion and relationship to grade and symptom duration are discussed.All brain donors were consented for autopsy and brain donation. All procedures performed were in accordance with the ethical standards of the institution and with the 1964 Helsinki declaration and its later amendments.HTT CAG repeat size. All brains were processed using the standardized protocol in place at the New York Brain Bank as outlined previously [The 65 brains of this study had a pathologic diagnosis of HD and known eviously , 62. Theeviously . FormaliParaffin sections were immunostained on an automated immunostaining platform using a DAB with or without alkaline phosphatase dual staining system on a Roche Ventana staining platform Table . HematoxHTT CAG repeat length, clinical disease duration, and neuropathologic grade before examination and analysis. All slides were reviewed by one board-certified neuropathologist (RAH). HTT and p62 inclusions were defined as a protein aggregate visible by light microscopy that were immunolabeled by their respective antibody (anti-HTT or anti-p62). Intranuclear inclusions were defined as inclusions localized to within the hematoxylin-counterstained nucleus; extranuclear inclusions were defined as any inclusion outside of the nucleus, i.e. within the visible perikaryal cytoplasm of a cell or as inclusions that appear within the neuropil. Since HTT inclusions predominate in the infragranular layers, quantification of inclusions was performed in cortical layers V–VI. For HTT quantification, inclusions labeled with brown chromogen with the huntingtin/ p62 double stain were analyzed. For p62 quantification, inclusions labeled with brown chromogen with the p62 single stain were analyzed. For both HTT and p62 quantification, three photomicrographs per cortical region  of layers V–VI were obtained at an original magnification of 200x. Then, using ImageJ 1.52P and the Cell counter plugin, intranuclear and extranuclear inclusions were manually counted per image field (RAH) [2).Immunohistochemically stained slides were blinded by demographics, ld (RAH) . The totHTT at the end of life. For individuals with a known age-of-onset of HD motor signs (n = 22), the duration of clinical disease was calculated by subtracting the age of clinical onset of motor signs from the age at death.Several measures have been proposed to assess disease severity. One reliable and widely used measure of neurodegeneration within the neostriatum is the Vonsattel grading system; all brains studied were diagnosed and graded either by JPV or RAH . AnotherP value < 0.05 was deemed significant (*) and a P value < 0.01 was considered highly significant (**); P values < 0.001 and < 0.0001 were also designated on graphs as *** and ****, respectively.Statistical analyses were performed on GraphPad Prism version 9.1.2. Normality tests were performed on all numerical datasets using the D’Agostino Pearson test. For data that was not normally distributed, non-parametric tests were utilized. Average values are presented as means ± standard deviation (SD) if normally distributed, or as median ± interquartile range (IQR) if non-normal. Multiple comparisons of median inclusion densities between brain region were initially assessed by pairwise comparison using the Freidman test, followed by post-hoc Dunn’s multiple comparison test for regional differences. Correlational analyses between CAG repeat length and inclusion density were performed using Spearman’s rank correlational coefficient. Regression analyses assume linearity and significant differences in slopes of regression lines were assessed between BA9 and BA17 using Prism’s built-in function that is equivalent to the ‘analysis of covariance’ (ANCOVA) analysis [The mean age at death of the 65 brain donors was 56.3 years ± 14.1 years (SD) and 31 (47.7%) individuals were men. The Vonsattel grade of HD neuropathologic changes ranged from 1–4 with a mean grade of 3 ± 0.83 (SD). The median CAG repeat expansion was 45 (IQR: 4).As previously described, both intranuclear and extranuclear HTT and p62 inclusions predominated within neocortical layers V-VI and III, with some inclusions present in the immediate subcortical white matter , 18. In P < 0.0001, Kruskall Wallis test; BA17 vs. BA9, P = 0.007; BA7 vs. BA9, P = 0.013, post-hoc Dunn test, Fig. The ratio of intranuclear inclusions/total inclusions increases in more caudal cortices .We next quantified total HTT inclusion density (i.e. both intra- and extranuclear HTT inclusions) in the infragranular layers (layers V/VI) of neocortical areas BA17, BA7, BA4, and BA9 of the 65 brains. We found that the median density of HTT inclusions was least in the calcarine cortex (BA17) and that this density sequentially increased in a caudal to rostral direction with a nearly 38-fold difference in median inclusion density between BA17 and BA9 Figs. e-h, 3a. P < 0.0001; BA7: r = 0.60, P < 0.0001; BA4: r = 0.44, P = 0.0003; BA9: r = 0.53, P < 0.0001, n = 65, Spearman’s rank correlation coefficient, Fig. P < 0.0001, see methods for statistical analysis). Extrapolation of the regression lines demonstrated an incremental decrease in the x-intercept of the regression lines from caudal to rostral cortices .We next examined the relationship between p62 and CAG repeat expansion in a subset of cases (n = 39). Significant correlations were present between p62 inclusion density and CAG repeat expansion in BA17, BA7, and BA9 but not with BA4  that is irrespective of cortical cytoarchitecture . HTT inclusion density correlates with CAG repeat expansion and rostral cortices have greater increases in inclusion density per CAG repeat than caudal cortices. Although intranuclear inclusions are overall more frequent in the juvenile HD cortex with higher CAG repeat expansions than in adult-onset HD, we found that the ratio of intranuclear HTT inclusion density / total HTT inclusion density increases in more caudal cortices in HD brains regardless of the size of CAG repeat expansion and can predominate in the calcarine cortex in adult-onset HD brains , 18. TheHTT gene increases the length of the polyglutamine tract of the HTT protein and results in aberrant splicing of the HTT transcript in a CAG repeat length dependent manner [HTT1a transcript that encodes the highly aggregation-prone and pathogenic exon 1 HTT protein and may initiate aggregation within certain compartments of the cell, depending on its subcellular concentration [Pathogenic trinucleotide repeat expansion of the t manner . This prntration , 46. In ntration . In thisntration , 43. TheThe burden of HTT inclusions predominate in the infragranular layers of the cortex (layers V/VI), which was our rationale to focus our study on these layers , 18. StuHTT expansion and amyotrophic lateral sclerosis (ALS) [HTT gene CAG expansion that does not necessarily signify clinical disease or pathologic grade. This contrasts with other more common neurodegenerative diseases , where pathologic inclusions have a tendency to accumulate over time and involve successive brain regions allowing for reliable disease staging [Pathologic grading of HD is widely used to assess the severity of neurodegeneration for an individual postmortem. The general finding that the HTT inclusion burden does not differ across grade (except for one instance in BA7) suggests that there is a steady burden during the clinical disease. Ubiquitinated and HTT inclusions are indeed present in prodromal HD gene carriers, as well as in non-HD individuals with pathogenic is (ALS) , 26, 27. staging , 6, 29.There are several limitations to this study. Firstly, there was limited clinical annotation to many of these brains, partly because HD is rare and the brain bank relies upon referrals from across the nation to acquire this large collection often with limited clinical information. Secondly, only one HTT immunostain was employed in this study and may not have bound all HTT species. However, the p62 immunostain confirmed the presence of an increasing caudal to rostral gradient of inclusions and essentially supported the data gained from the HTT immunostains. Thirdly, like other autopsy studies, this work is correlative and cross-sectional thus preventing the intravitam study of HTT production and clearance, i.e., turnover, within individuals.HTT gene expansion. The spectrum of pathologic effects of pathogenic HTT gene expansion has now expanded to include not simply the HD neurodegenerative phenotype, but also to include abnormalities during neurodevelopment, including a greater propensity for developmental malformations in the brain, as well as ALS [HTT gene expansion.In summary, we have demonstrated a wide variation in HTT inclusion density within the human HD neocortex and a variable propensity for aggregation for different neocortices. The uncertainty remains as to whether these inclusions are toxic to the CNS. A higher burden of HTT inclusions in the neocortex did not unequivocally associate with higher grades of disease. Instead, these data suggest that the presence and distribution of HTT inclusions is a gain-of-function phenotypic expression of l as ALS , 28, 30.


Bassarona dunya is a butterfly species commonly found in the tropical regions of Asia, America, and Africa. This butterfly is a member of the subfamily Limenitidinae and the classification within this subfamily has been unstable. Here, we report the first complete mitochondrial genome (mitogenome) of B. dunya sampled from Malaysia. The mitogenome is 15,242 bp long, comprising a set of 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs), and an A + T rich region. All PCGs were initiated by the typical ATN codon, except for COX1 which started with a CGA start codon. Nine PCGs were terminated with a TAA or TAG stop codon, while COX1, COX2, NAD4, and NAD5 ended with an incomplete T. The 12S and 16S rRNAs were 716 bp and 1269 bp in length, respectively. Phylogenetic analysis supported the placement of B. dunya within Limenitidinae with a high support value.The Great Marquis or  Bassarona dunya  is a butterfly from the subfamily Limenitidinae and is distributed mainly in the tropical regions of Asia, America, and Africa . The specimen was deposited in Universiti Tun Hussein Onn Malaysia (UTHM)  with the voucher ID DIB031. Total genomic DNA was extracted from the hind leg tissue using the Qiagen Blood and Tissue Kit  following the manufacturer’s instruction, and fragmented via a Bioruptor® system. For library preparation, NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® was used prior to sequencing by Illumina NovaSeq 6000 system (PE150). The raw reads obtained were pre-processed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and then trimmed for sequencing adapters, as well as low quality reads using AdapterRemoval  to improve the annotation. Tablet , an updated version of the CGView web server (Grant and Stothard The adult Acraea zetes (NC029499), Argynnis sagana (NC037006), and Argynnis childreni (NC024415) from the subfamily Heliconiinae were used as the outgroups. Phylogenetic analysis was performed using IQ-Tree  tree. As we focus on the subfamily Limenitidinae, the combined dataset comprises three Heliconiinae and 32 Limenitidinae, including four recognized tribes (Dhungel and Wahlberg B. dunya (GenBank accession no.: ON165248) is 15,242 bp in length, encoding 13 PCGs, 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs), and a control region. The mitogenome has a depth coverage of 167X and is A + T biased (81.45%). It has a nucleotide composition of A (38.51%), T (42.93%), TC (10.96%), and G (7.59%). The PCGs have a total length of 11,226 bp, whereas the tRNAs are 1461 bp, ranging from 61 bp (tRNA-Ser) to 71 bp (tRNA-Lys). The length of the 12S and 16S rRNA genes are 716 bp and 1269 bp, respectively. Out of the 13 PCGs, 12 were initiated by the typical ATN start codon, while COX1 utilizes the CGA codon. Four PCGs  ended with an incomplete stop codon, while others were terminated either by TAA or TAG stop codon. Additionally, BLASTn analysis (McGinnis and Madden B. dunya COX1 sequence in GenBank (accession no.: GQ864742).The complete mitogenome of B. dunya formed a clade with the other Asian Adoliadini genera (Dhungel and Wahlberg Abrota ganga (NC024404) .Phylogenetic analysis showed that all tribes formed monophyletic clades. The ML tree recovered the relationship of Parthenini+(Adoliadini+((Cymothoini + Neptini)+)). The analysis also showed that the newly sequenced B. dunya and was deposited in GenBank (accession no.: ON165248). The newly sequenced B. dunya reported shared similar gene characteristics with the other genera in Limenitidinae. It has the shortest mitogenome length compared to other genera in the Adoliadini tribe, but it is still within the range of the typical Lepidoptera mitogenome. These differences could be influenced by the length of the control region (Cheng et al. B. dunya sequenced in this study provides useful information on the phylogeny of the Adoliadini tribe, and to further address their taxonomic issues, extensive taxon sampling should be considered.This study provided the complete mitogenome of


Here, we evaluate the effect of the COVID-19 pandemic on utilization of cardiothoracic imaging studies.  We queried our radiology record system to retrospectively identify numbers of specific key cardiothoracic imaging studies for five years prior and during the COVID-19 pandemic. Statistical analysis was performed to evaluate changes in the number of exams in 2020 and 2021 compared to 2019. p < 0.0001). Portable radiograph utilization was increased in 2020 averaging 320 (SD = 68.2) films daily in 2020 compared to 266 (SD = 29.1) in 2019 (p < 0.0001). Utilization of thoracic CT was decreased during the pandemic, with 21.8 (SD = 12.9) studies daily compared to 52.0 (SD = 21.4) (p < 0.0001) studies daily in 2019. Cardiac imaging utilization was also substantially decreased in 2020 compared to 2019, averaging a total of 3.8 (SD = 3.2) versus 10.8 (SD = 6.6) studies daily and 0.88 (SD = 1.7) versus 2.5 (SD = 2.3) studies daily for CT and MRI, respectively. Evaluation of cardiothoracic imaging for the subsequent 18 months after New York's entry to phase I recovery in June 2020 demonstrated that by one year after the emergence of COVID-19 imaging utilization had recovered to prepandemic levels. Cardiac imaging continued to increase throughout the chronic phase of the COVID-19 pandemic, reaching almost twice the prepandemic levels by the end of 2021.  Five-year retrospective analysis demonstrated progressive increases in nearly all cross-sectional studies. In 2020, daily chest radiograph utilization decreased with an overall number of daily radiographs of 406 (SD = 73.1) compared to 480 per day in 2019 (SD = 82.6) ( COVID-19 has had far-reaching effects on medicine and public health. Here, we demonstrate decreases in all cross-sectional cardiothoracic imaging studies, closely mirroring findings in other fields during the height of the pandemic, which have since rebounded. Cardiothoracic imaging encompasses the most commonly ordered radiology studies and is a cornerstone of medical decision-making . CardiotThe COVID-19 pandemic has been an unprecedented event in modern healthcare, infecting millions worldwide , 11. TheThe overall effect of the COVID-19 pandemic on trends in cardiothoracic imaging has, to this point, been unclear; cardiothoracic radiologists practicing during the pandemic reported a significant decline in cardiac imaging, likely resulting from the prohibition on elective procedures and patient preference to remain home and avoid exposure . ConversHere, we sought to quantify imaging utilization for key cardiothoracic imaging studies during the pandemic and evaluate how they are related to trends over time in order to better understand the effect of the COVID-19 pandemic on trends in cardiothoracic imaging utilization.We obtained a Human Research Protection Office and Internal Review Board Letter of Exemption to retrospectively study imaging trends over time; exemption was granted given that no individual patient data were accessed. We queried the radiology record system “MModal Catalyst” for all reports with specific exam labels corresponding with the type of study being evaluated regardless of age or gender. Imaging studies, rather than billing data/ICD-10 data, were used given that diagnoses were not always known at the time of admission, and therefore these data were the most accurate representation of the studies obtained. Data were recorded as yearly totals from 2015 to 2019 and daily totals for the period of the pandemic spanning from 03/11/2020 to 06/08/2020. Subsequently, monthly data were obtained from 06/01/2020 to 12/31/2021 and normalized to average monthly values prior to the pandemic, collected from 06/01/2019 to 12/31/2019. Sample size for this study was not prespecified given that all exams in the time frame were included in the analysis.t-test was used to assess statistical significance between samples. For samples that did not follow a normal distribution, we tested for significant differences using a Wilcoxon signed-rank test. For yearly numbers, data were normalized to 2015 exam numbers and expressed as a percent change. Of note, for all types of examinations, there was substantial variation in the quantity of imaging studies obtained on weekend days compared to weekdays which contributed significantly to standard deviation.All statistical analyses were performed in Microsoft Excel and using online calculator “Statistics for the Social Sciences” . StatistWe first sought to establish trends in cardiothoracic imaging within our practice, comprised a 900-bed university hospital in northern Manhattan and two affiliate community hospitals. Given the wide variety in average numbers of each type of exam, trends are expressed as percent change compared to 2015. p < 0.0001). The average number of portable radiographs was 320 (SD = 68.2) in 2020 compared to 266 (SD = 29.1) in 2019 which also showed a statistically significant difference (p < 0.0001)  ; Table 1p < 0.0001). By calculating the difference in percent portable radiograph utilization in 2020 compared to 2019  (p < 0.0001). Analysis of contrast-enhanced CTA-PE demonstrated an average of 14.1 (SD = 4.4) studies obtained daily in 2019 compared to 7.5 (SD = 4.4) in 2020, also statistically significantly different (p < 0.0001). No high-resolution chest CTs were performed in 2020 during the dates queried; 17 were performed during this time period in 2019.Similar analysis was performed to evaluate CT utilization. Assessing total thoracic CTs, in 2020, the daily average number of CTs obtained was 21.8 (SD = 12.9) compared to 2019 with an average daily number of 52.0 thoracic CTs (SD = 21.4), a statistically significant difference ( 0.0001) . In 2019p < 0.0001). Given the large daily variation, we analyzed weekly trends in the number of studies obtained in 2020 compared to 2019 (p < 0.0001). A statistically significant decrease in the number of CTA studies for aortic dissection was also seen in 2020 with a total number of 8 studies obtained over the timeframe, compared to 142 in 2019 . Interestingly, the number of cardiac CTA studies obtained was similar in 2020 compared to 2019, with daily average exam numbers of 3.2 (SD = 2.5) and 3.7 (SD = 3.1), respectively (p = 0.22).Cardiac CT studies were far fewer in number, however showed similar diminution in use in 2020 compared to 2019. The average daily number of all cardiac CT studies included in this analysis  in 2019 was 10.8 (SD = 6.6) compared to an average of 3.8 (SD = 3.2) in 2020, a statistically significant difference ( to 2019 . The bigp < 0.0001). Similar to cardiac CT, weekly numbers were analyzed to normalize large daily variation  in 2019, compared to 0.88 (SD = 1.7) in 2020; while there was substantial variation in daily numbers, the difference was nonetheless statistically significant (ariation . Numbersariation .Given that the COVID-19 pandemic persisted beyond phase I recovery, we sought to characterize how cardiothoracic imaging recovered and to evaluate the continued effects of the COVID-19 pandemic on imaging trends. We considered four groups: total chest radiographs, total chest CTs, total cardiac CTs, and total cardiac MRI studies for analysis. Our analysis demonstrated that there was no lasting diminution of cardiothoracic imaging utilization for any of these groups. Chest radiograph and chest CT utilization took about 6 months after entry into phase I recovery to return to their prepandemic levels and then remained there until present. Cardiac CT and cardiac MRI utilization began to increase above prepandemic levels prior to entry into phase I of recovery in the New York region and continued this upward trend, ending 2021 with approximately twice as many exams as performed on average per month prior to the pandemic .Our data, consistent with other groups, demonstrate progressively increasing utilization of cardiothoracic imaging studies over time. Specific to our practice, exams have steadily increased over time due to an increased patient census as well as expansion of specialty programs such as an interstitial lung disease program and a robust lung cancer practice. Slightly decreased numbers in 2019 prior to the pandemic may have represented leveling off of new patients in these programs.The COVID-19 pandemic dramatically impacted healthcare, including cardiothoracic imaging utilization. Here, we present the first report to our knowledge that quantitates trends in cardiothoracic imaging during this time. While historic data from the prior five years demonstrated continued increases in the number of cross-sectional studies obtained at our institution, the onset of the pandemic coincided with dramatic decreases in cardiothoracic studies performed. Our data demonstrate decreased chest radiograph utilization in total and significantly increased portable studies. These data concur with recommendations against the regular use of radiography for assessment of COVID-19 , 13, butIn contrast, we found that numbers of cross-sectional modalities were significantly decreased in 2020 compared to 2019. For cardiac CT and MRI, these data corresponded to reports of decreased presentations for cardiac symptoms and decline in the use of cardiac catheterization . While tOur data demonstrate a decline in total thoracic CT utilization during the time of the pandemic, with the nadir of the 2020 trend line correlating with the first and second weeks of April. This concurs with the CDC recommendation against the use of chest CT for the diagnosis of COVID-19 infection . Given tThe long-term effects of COVID-19 will impact cardiothoracic imaging for years to come. Studies have demonstrated progression to fibrotic lung disease in patients who have recovered from acute COVID-19 infection , 27, andLimitations of this study included that it was performed at a single institution in a region of high community transmission. Improvements in patient management and available therapies will result in continued change in the response to COVID-19 infection and patient outcomes, which could not be accounted for in this retrospective study. Despite these limitations, these data provide a robust snapshot of cardiothoracic imaging during the pandemic and how it relates to trends in imaging over time.The COVID-19 pandemic affected nearly all aspects of medicine, including imaging utilization in cardiothoracic radiology. Here, we quantify these changes and suggest clinical correlates as to why the observed changes occurred. While the COVID-19 pandemic significantly affected cardiothoracic imaging, our data demonstrate that it has rebounded and continues to grow as it did before the COVID-19 pandemic.