Molecular characteristics of CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) have been well documented in Western, but not in Chinese, populations.BRAF/KRAS mutation, and microsatellite instability (MSI) in a Chinese population with CRC (n = 401) and analysed associations between CIMP status and clinicopathological and molecular features.We investigated the incidence of CIMP, BRAF mutation in adenomas (2%) and CRC (0.7%), and a relatively low incidence of KRAS mutation (24.9%) compared with that in other populations. We also detected a relatively low incidence of CIMP-high (10.2%), which was significantly associated with younger age (≤49 years of age), female sex, and proximal tumour location.A total of 41 cases, 310 cases, and 40 cases were classified as CIMP-high, CIMP-low, and CIMP-negative, respectively. We detected a significantly low incidence of This study revealed unique characteristics of CIMP in a Chinese population with colorectal cancer. Developing specific CIMP markers based on unique populations or ethnic groups will further help to fully elucidate CIMP pathogenesis. BRAF mutation, and/or somatic KRAS mutation status [KRAS, BRAF, and CIMP for personalised medicine [Based on global estimates in 2012, colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females, with an estimated 1.4 million cases and 693,900 deaths occurring annually [n status . Over thBRAF mutation [CIMP characterises a subset of CRCs exhibiting a very high frequency of aberrant DNA hypermethylation at “type C” loci, which are defined as loci methylated in cancer, but not in normal, tissues . The CIMmutation , 14–17. mutation , 18, 19,BRAF/KRAS mutation status) have diagnostic, therapeutic, and prognostic significance in CRC [BRAF mutation, and CIMP, vary considerably among different ethnic groups [BRAF mutation in different populations varies widely, from 1.1% in Taiwan to 19.8% in the Netherlands [BRAF mutation has been observed in most Asian populations, from 1.1% in Taiwan to 7% in North China [MINT1, MINT31, p16, MLH1, MGMT, APC, and RUNX3) are not very sensitive and specific for CIMP diagnosis [Although accumulating evidence indicates that these molecular characteristics  in Chinese populations, we sought to utilise a population-based CRC cohort to more accurately determine the prevalence and characteristics of these features in a Chinese CRC population. We comprehensively investigated the incidence of MSI, CIMP, and BRAF and KRAS mutations in a unique ethnic Chinese CRC population and analysed associations between CIMP status and clinicopathological and molecular features. We also aimed to elucidate the aetiological factors and pathogenesis of CIMP-high CRC in this unique ethnic Chinese population of CRC patients.Due to the lack of data on the molecular characteristics of CRC . Fresh colorectal tumour and surrounding normal tissues were collected at surgery from 84 patients, and representative sections for research were removed by a pathologist. These patients had undergone curative surgery at the First Affiliated Hospital of Kunming Medical University between 2014 and 2016. Patients gave a written informed consent for the use of their bowel tissue for research. Selection was based solely on the availability of archival tissue blocks for the study, and we did not exclude patients with a family history of CRC. Clinicopathological information, including age, sex, tumour location, and tumour stage, was obtained from all 401 patients  for formalin-fixed, paraffin-embedded archival tissues and the QIAamp DNA Mini Kit (QIAGEN) for fresh tissues. Bisulphite modification was carried out using an EpiTect Fast DNA Bisulphite Kit (QIAGEN) according to the manufacturer’s instructions.DNA methylation analyses were performed using MethyLight, as previously described , 32. TheCACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1), as these have been shown to be sensitive and specific for CIMP diagnosis [CACNA1G, CDKN2A, CRABP1, MLH1, and NEUROG1), described by Ogino and colleagues [CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1), described by Weisenberger and colleagues [BRAF mutation, and MSI status.Although several marker panels have been proposed to standardise the classification of CIMP-positive , 36, 37,iagnosis . These elleagues ; and CIMlleagues , 33, 38.lleagues . For theBRAF codon 597 and 600 mutations and KRAS codons 12 and 13 mutations in 98 adenomas and 401 CRC samples. Mutations of BRAF (nucleotides 1790 and 1799) and KRAS (nucleotides 35 and 38) were analysed by genotyping assay on the MassARRAY platform . PCR and extension primers for these mutations were designed using MassARRAY Assay Design 3.0 software (Sequenom) and applying default single-base extension settings and default parameters  to classify fresh tumour tissues as MSI-high (MSI-H), MSI-low (MSI-L), or microsatellite stable (MSS). MSI-H was defined as ≥2 markers demonstrating novel alleles compared to non-tumour tissues, MSI-L was defined as 1 marker with a novel allele, and MSS was defined as no markers with novel alleles.For determination of MSI status, we used a panel of 5 microsatellite markers (2 test or Fisher’s exact test (for categories with n < 10) was performed on categorical data using the IBM SPSS Statistics 22.0 software. All P values were two-sided, and statistical significance was set at P ≤ 0.05.For statistical analysis, the χOf the 401 patients, the proportion of males (57%) was slightly higher than that of females (43%), with a male to female ratio of 1.32:1. Patient age at presentation ranged from 20 to 87 years . There were 309 patients (77.1%) that presented with stage II or III disease, while 77 patients (19.2%) were diagnosed with stage I disease and 15 patients (3.7%) were diagnosed with stage IV disease. There were 214 patients (53.4%) whose primary tumours were derived from the colon, while the tumours in the remaining 187 patients (46.6%) were derived from the rectum. Detailed sample information is summarised in Table BRAF and KRAS mutations using the MassARRAY platform, BRAF mutation was observed in three cases, with an incidence of 0.7% (3/401); KRAS mutation was detected in 100 cases, with an incidence of 24.9% (100/401) , and KRAS mutation was detected in 23 cases, with an incidence of 23.5% (23/98). All five BRAF mutations were V600E mutations, while KRAS-mutated cases showed mutations at codon 12 (67%) and codon 13 (33%). Repeat Sanger sequencing was conducted for 229 specimens, including all of the BRAF- and KRAS-mutated cases. The results of repeat Sanger sequencing were in accordance with MassARRAY analyses, with no new mutations identified.Of the 401 CRC specimens analysed for MSI status was determined in 82 CRC patients due to inadequate DNA, lack of paired normal tissues, or technical issues with the remaining specimens. The incidences of MSI-H, MSI-L, and MSS were 6.1% (5/82), 23.2% (19/82), and 70.7% (58/82), respectively  using MethyLight technology. Methylation frequencies were 20% for CACNA1G (80 cases), 57.6% for CDKN2A (231 cases), 49.6% for CRABP1 (199 cases), 30.7% for IGF2 (123 cases), 3.7% for MLH1 (15 cases), 31.2% for NEUROG1 (125 cases), 7.2% for RUNX3 (29 cases), and 17% for SOCS1 (68 cases). A summary of the clinicopathological and molecular characteristics of CRC cases according to each of the three CIMP panels  is provided in Fig. P < 0.01) and significantly higher in cases with proximal tumour locations (14.9%) than in those with distal tumour locations . CIMP-2 positivity was significantly more frequent in women (17.3%) than in men  and was associated with a BRAF mutant type (66.7%) rather than BRAF wild type . For the CIMP-3 panel, of particular note is the fact that the frequency of CIMP-high varied by age, with a significantly higher rate in patients ≤49 years of age (16.3%) compared to that in patients 50–59 years of age (7.5%) and ≥ 60 years of age . Moreover, CIMP-high was significantly more frequent in women (16.2%) than in men  and in proximal tumour locations (17.8%) than in distal tumour locations . No significant differences were observed in other clinicopathological characteristics among the CIMP phenotypes for the three panels of CIMP markers. Because previous studies suggested that the CIMP-3 panel outperformed the CIMP-1 and CIMP-2 panels both in Western CRC populations and in Asian CRC populations [We obtained 401 colorectal cancer specimens and successfully quantified DNA methylation in eight CIMP-specific gene promoters  in determining CIMP-1 status. Similarly, CRABP1 and NEUROG1 demonstrated high sensitivity (≥90%) in determining CIMP-2 status, and CDKN2A, CRABP1, and NEUROG1 demonstrated very high sensitivity (≥97%) in determining CIMP-3 status. For all three panels, MLH1 and RUNX3 exhibited superior specificity (≥96%).To compare the performance of the eight individual methylation markers for the determination of panel-specific CIMP status, the sensitivity and specificity were calculated for each of the eight markers among all 401 tumours Table . As showBRAF mutation, KRAS mutation, and MSI status) have diagnostic, therapeutic, and prognostic significance in CRC personalised medicine and incidences that vary considerably among different ethnic or geographic populations, this study determined the frequency of CIMP, BRAF/KRAS mutation, and MSI in a unique ethnic Chinese population-based CRC cohort. Surprisingly, we detected a significantly low incidence of BRAF mutation, both in adenomas (2%) and in CRC (0.7%), and a relatively low incidence of KRAS mutation (24.9%) compared with that in other populations [BRAF mutation, KRAS mutation, or MSI status. In addition, by comparing the accuracy of the associations of the three CIMP marker panels with previously known clinicopathological features of CIMP-positive CRC, our data indicated that the CIMP-3 panel outperformed the CIMP-1 and CIMP-2 panels in most comparisons. Therefore, consistent with analyses in American and South Korean CRC populations [Due to accumulating evidence indicating that certain molecular characteristics  in Chinese CRC populations remains unclear due to very low incidences of BRAF mutation. Although very low incidences of BRAF mutation were observed in Saudi Arabia and Israel, with frequencies of 2.5 and 5%, respectively [BRAF mutation varies widely among CRC populations even within the same region or country [BRAF mutation in CRC populations from different areas of China. A BRAF mutation frequency of 2.3% (5/220) was observed in Shanghai [BRAF mutation was observed in a Yunnan CRC population may be due to differences in ethnic populations and the associated variation in underlying genetic and epigenetic backgrounds, as well as environmental influences such as food habits, smoking, drinking, and other unknown factors.e in CRC , 42. In ulations , 9, 10. ulations , 42, butectively , 43, andectively  to 2.3–7ectively , 29, 44,n 3.7%, 1/401 in Cectively , and 4.1ectively , 10, 42. country , 45, 46,Shanghai , with thMLH1, MGMT, p16, APC, MINT1, MINT31, and RUNX3 as CIMP panel markers and classified 13.12% (37/282) of patients as CIMP-high [p14ARF, hMLH1, p16INK4a, MGMT, and MINT1 as CIMP markers and identified 12 CIMP-positive cases (24%) in 50 CRC specimens [The CIMP-1 panel was first developed by Ogino and colleagues ; in thei 13.12% 3/282 of ppecimens . TherefoBRAF mutation, and wild-type KRAS [BRAF mutation, or wild-type KRAS. Notably, this is the first study to report an association between CIMP-high and younger age. A possible reason for the lack of an association between CIMP-high and MSI may be the limited sample size (n = 82) included in the MSI analysis. The lack of an association with BRAF mutation may be due to the very low incidence of BRAF mutation observed in this population (0.7%). Alternatively, just as accumulating evidence has demonstrated that differences in CIMP marker panels may contribute to discrepancies in CIMP frequency, even in the same CRC population, the same CIMP marker panel may not be suitable for the diagnosis of CIMP among different populations. For example, using the same CIMP-3 marker panel with the same CIMP-high criterion, Ogino and colleagues found that CRABP1, IGF2, and NEUROG1 demonstrated very good sensitivity (≥95%), whereas CACNA1G, MLH1, RUNX3, and SOCS1 showed superior specificity (≥90%) in 920 American CRC cases [CDKN2A, CRABP1, and NEUROG1 demonstrated very good sensitivity (≥97%), while MLH1 and RUNX3 exhibited superior specificity (≥96%) when using CIMP-3. In contrast, the sensitivity of IGF2 was 83%, and the specificities of CACNA1G and SOCS1 were 87 and 86%, respectively, in our CRC population, while the sensitivity of CDKN2A was 87% among American CRCs [BRAF mutation frequency detected in this study, we speculate that different ethnic populations with different underlying genetic and epigenetic backgrounds and environmental influences, such as food habits, lifestyle habits, and environmental exposures, may contribute to the varied CIMP characteristics and prevalence observed. The development of specific CIMP markers based on unique CRC populations or ethnicities will further help to fully elucidate the pathogenesis of CIMP.Studies on American CRC populations have indicated that CIMP is significantly associated with female sex, older age, proximal tumour location, MSI, ype KRAS , 36, 48.RC cases . Howevercan CRCs . Thus, tcan CRCs , 32, 36,can CRCs  or ethniBRAF mutation [BRAF mutation is much higher in serrated adenomas than in conventional adenomas. For example, the frequency of BRAF mutation was found to be 67% among 200 traditional serrated adenomas, but no BRAF mutations were identified in 50 control tubulovillous adenomas [BRAF mutation was very rare among CRC cases (only 0.7%). Similarly, the BRAF mutation frequency was only 2% in adenomas. Therefore, as the acknowledged precursors of CIMP-positive CRC, the contributions of advanced serrated adenomas to the incidence of CIMP-high CRC should be very limited in our CRC population. We believe that the unique ethnic population and the associated underlying genetic and epigenetic backgrounds may contribute to the unique molecular characteristics of CIMP-high CRC in our population. Associations between CIMP-positive CRC and environmental exposures have been carefully investigated, and associations of smoking and obesity with CIMP-positive CRC were evident only for females in an American population [CIMP-positive tumours are generally thought to develop through the serrated neoplasia pathway and are associated with mutation , 49. Furpulation . TherefoBRAF mutation in adenomas (2%) and CRC (0.7%), and a relatively low incidence of KRAS mutation (24.9%) compared with that in other populations. We also detected a relatively low incidence of CIMP-high (10.2%), which was significantly associated with younger age (≤49 years of age), female sex, and proximal tumour location. To our knowledge, this is the first study to suggest an association between CIMP-high and younger age, while most previous studies have associated CIMP-high and older age. We speculate that different genetic backgrounds and lifestyle habits may contribute to the unique pathogenesis of CIMP-high CRC among the ethnic Chinese population. Developing specific CIMP markers based on unique populations or ethnic groups will further help to fully elucidate CIMP pathogenesis.This study detected a significantly low incidence of Additional file 1: Table S1.KRAS and BRAF primers used for MassARRAY analyses and Sanger sequencing in this study.


Cetacean morbillivirus  causes epizootic and interepizootic fatalities in odontocetes and mysticetes worldwide. Studies suggest there is different species-specific susceptibility to CeMV infection, with striped dolphins , bottlenose dolphins (Tursiops truncatus), and Guiana dolphins  ranking among the most susceptible cetacean hosts. The pathogenesis of CeMV infection is not fully resolved. Since no previous studies have evaluated the organ-specific immunopathogenetic features of CeMV infection in tissues from infected dolphins, this study was aimed at characterizing and comparing immunophenotypic profiles of local immune responses in lymphoid organs , lung and CNS in CeMV-molecularly (RT-PCR)-positive cetaceans from Western Mediterranean, Northeast-Central, and Southwestern Atlantic. Immunohistochemical (IHC) analyses targeted molecules of immunologic interest: caspase 3, CD3, CD20, CD57, CD68, FoxP3, MHCII, Iba1, IFNγ, IgG, IL4, IL10, lysozyme, TGFβ, and PAX5. We detected consistent CeMV-associated inflammatory response patterns. Within CNS, inflammation was dominated by CD3+ (T cells), and CD20+ and PAX5+ (B cells) lymphocytes, accompanied by fewer Iba1+, CD68+, and lysozyme+ histiocytes, mainly in striped dolphins and bottlenose dolphins. Multicentric lymphoid depletion was characterized by reduced numbers of T cells and B cells, more pronounced in Guiana dolphins. Striped dolphins and bottlenose dolphins often had hyperplastic (regenerative) phenomena involving the aforementioned cell populations, particularly chronically infected animals. In the lung, there was mild to moderate increase in T cells, B cells, and histiocytes. Additionally, there was a generalized increased expression of caspase 3 in lymphoid, lung, and CNS tissues. Apoptosis, therefore, is believed to play a major role in generalized lymphoid depletion and likely overt immunosuppression during CeMV infection. No differences were detected regarding cytokine immunoreactivity in lymph nodes, spleen, and lung from infected and non-infected dolphins by semiquantitative analysis; however, there was striking immunoreactivity for IFNγ in the CNS of infected dolphins. These novel results set the basis for tissue-specific immunophenotypic responses during CeMV infection in three highly susceptible delphinid species. They also suggest a complex interplay between viral and host's immune factors, thereby contributing to gain valuable insights into similarities, and differences of CeMV infection's immunopathogenesis in relation to body tissues, CeMV strains, and cetacean hosts. Cetacean morbillivirus  has caused multiple outbreaks of lethal disease in odontocetes and mysticetes worldwide. Interepizootic or endemic morbidity and mortality is also recorded (Tursiops truncatus), striped dolphins , and Guiana dolphins  ranking among the most susceptible cetacean hosts, with fatal epizootics  involve initial replication in lymphoid tissues, followed by viral dissemination in infected lymphocytes through the lymphatics and blood stream (“leukocyte trafficking”), and eventual spread to epithelial and nervous cells , 7. ImmuSotalia guianensis,” “Stenella coeruleoalba,” “Tursiops truncatus,” “CeMV reverse transcription polymerase chain reaction (RT-PCR)-positive” “Toxoplasma gondii PCR-negative.” Only individuals in a “fresh” (code 2) post mortem preservation status, or in a “moderate post mortem autolysis” (code 3) condition , the “Laboratory of Aquatic Mammals and Bioindicators Profa. Izabel M. G. do N. Gurgel—MAQUA” , the “Institute for Animal Health and Food Safety—IUSA” , the “Department of Comparative Biomedicine and Food Science of the Faculty of Veterinary Medicine of the University of Padua ,” the “Laboratories of Histopathology and Immunohistochemistry of Istituto Zooprofilattico Sperimentale dell'Abruzzo e Molise G. Caporale,” and the “Faculty of Veterinary Medicine of the University of Teramo ,” were queried based upon the following criteria: “ondition , which csyn. major histocompatibility complex II, MHCII) [antigen presenting cell], ionized calcium binding adaptor molecule 1 (Iba1) [histiocyte/microglia], interferon gamma (IFNγ), immunoglobulin (Ig)-G, interleukin (IL)-4, IL10, lysozyme, transforming growth factor beta (TGFβ), and paired box protein (PAX)-5 [B-cell]. Most of this IHC panel was standardized with successful cross-reactivity in cetacean tissues (Pontoporia blainvillei) lymph node, spleen, lung, and brain were used as positive controls  and spleen, lung, and CNS  were subjected to IHC using the following primary antibodies (pAbs): cleaved caspase 3 (CAS3) [final apoptosis pathway], cluster of differentiation (CD)-3 [T cell], CD20 [B cell], CD57 [natural killer cell], CD68 [histiocyte], Forkhead Box (Fox)-P3 [regulatory T cell], human leukocyte antigen , lung, and CNS tissues according to following score: –, no; +, <10%; ++, 10–50%; +++, 51–90%, and ++++, >90% immunopositive cells, in 10 high-power (400x) fields. The histo-anatomical compartments analyzed were: lymph nodes ; spleen ; lung ; brain  , 14. Addn = 2), juveniles (n = 11), and adults (n = 14). All CeMV-negative dolphins were calves. Detailed gross and microscopic pathologic findings with most probable cause(s) of stranding and/or death for CeMV-positive are published elsewhere and for CeMV-negative dolphins are recorded in Twenty-seven CeMV-positive dolphins, including 13 striped dolphins, 11 Guiana dolphins, and 3 bottlenose dolphins met the inclusion criteria. Guiana dolphins were infected by GD-CeMV , 3, whilConsistent immunolabeling (with variations according to LIR) was detected for CAS3, CD3, CD20, CD57, CD68, FoxP3, MHCII, Iba1, IFNγ, IgG, IL4, IL10, lysozyme, TGFβ, and PAX5 in all organs/tissue sections examined. The following alterations in immunophenotypic profiles of LIRs are referred as to comparisons to “control” animals. “Normal” or “physiologic” antigen cell distribution and intensity for the pAbs aforementioned in control animals  were similar to those previously reported  Supplem.CeMV-LIR often overlapped focally with verminous lymphadenitis-associated LIR, especially in Guiana dolphins. The following changes had somewhat similar distribution patterns in all lymph nodes  and spleens evaluated . There wIn the spleen, there was increased CAS3 expression in MNCs of white pulp and to a lesser extent in red pulp . There wApproximately a third of the infected dolphins had increased IFNγ immunoreactivity in lymph nodes and/or spleen tissue compared to uninfected dolphins. IFNγ was variably expressed in MNCs , rare syncytia and extracellularly in lymph nodes, and spleen . Except Staphylococcus aureus) had abundant T cell and B cell infiltrates around bronchi/bronchioles, and alveoli ; thus, no IHC evident differences were noted based on the proposed semiquantitative approach. IFNγ was mainly expressed by exudating MNCs including alveolar macrophages and syncytia , and fewBrucella co-infection based on cellular inflammatory components and neuroanatomical distribution of lesions. Rare T cells were seen circulating and/or in meningeal perivascular spaces in three Guiana dolphins, likely representing early CNS inflammation. CAS3+ cells appeared to involve mostly T and B cells. MHCII was widely overexpressed, involving MNCs either circulating or expanding the VRS and/or infiltrating the neuroparenchyma, along with vascular endothelial cells ; however, CeMV-infected striped dolphins and bottlenose dolphins had consistent IFNγ  and occaComparative analyses of LIRs are of paramount relevance as they help unraveling local immunopathogenetic mechanisms of disease processes. While in humans and in given domestic and livestock animal species, as well as in laboratory animals there is a considerable body of knowledge, the immunopathogenetic bases of disease including LIRs in cetaceans remain largely unknown . We haveHumoral and cellular immune responses to MeV , 19 and CAS3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins and playing a major role in apoptosis . In MeV If the host survives, incipient MeV and CDV infections are generally followed by regeneration of lymphoid organs , 6. RepoMammals differ in their expression of MHCII molecules . In thisComparatively, fewer studies have addressed in detail the immune disarrangements in the spleen of MeV and CDV cases , 6. Typiin situ” cytokine expression. Th1 responses evoke cell-mediated immunity and phagocyte-dependent inflammation. Th2 cells evoke strong antibody responses and eosinophil accumulation, but inhibit several functions of phagocytic cells (phagocyte-independent inflammation). Furthermore, Th1/Th2 balance can be evaluated by the ratio of their polarizing cytokines , and animals with imbalanced Th1/Th2 response may be more susceptible to certain kinds of infections. Since cytokine imbalance is implicated in the pathogenesis and outcomes of MeV and CDV infections, we aimed at evaluating, for the first time, Th1 and Th2 in situ cytokine immunoexpression and potential associations with CeMV-AD. We employed a set of commercially available non-cetacean-specific but cross-reactive proinflammatory cytokines to evaluate Th1 cells (IFNγ-secreting) and Th2 cells   . In distcreting) , 41. Placreting) . This shcreting) . In the Mycoplasmataceae infection (“cuffing pneumonia”) in other Cetartiodactyla members. The etiopathogenesis of this finding remains unknown. Furthermore, we detected increased expression of MHCII chiefly involving circulating MNCs, including presumed PIVMs, and infiltrating inflammatory MNCs (lymphocytes and histiocytes) and exocytosing/exudating MNCs. This was more prominent in parasitic LIRs. Putative PIVMs appeared to consistently express Iba1, lysozyme and CD68; these results broaden the repertoire of immunomarkers for PIVMs in cetacean species  experimentally infected with wild type MeV. Both CD4+ and CD8+ T cells were sources of IFNγ . CDV-CNS disease may show distinctive manifestations: acute fulminant encephalopathy and encephalitis, post-vaccinal encephalitis, old dog encephalitis, inclusion body polioencephalitis, and demyelinating leukoencephalitis (CDV-DL) , 50. The measles  and dist measles ; however measles . Divergeolvement . ImmunopWe observed consistently increased numbers of CAS3+ in CNS tissue, mainly involving lymphocytes and histiocytes circulating and infiltrating the meninges, expanding the VRS and infiltrating the neuroparenchyma. Occasionally, neurons and neuroglia expressed CAS3 in areas of marked neurodegeneration. These findings are in agreement with previous observations in MeV- and CDV-neurologic disease , 56. MHCin vitro analyses may allow better assessment of the roles of these two “mutually inhibitory” cytokines in CNS-CeMV infection. The participation of IL10+ and TGFβ+ cells, detected in much smaller numbers in CNS tissue sections, remains unclear. Further studies are necessary to understand the neuroimmunopathogenesis of CeMV infections, with special emphasis on cytokine networks.Previous studies suggested there is a cytokine imbalance in SSPE. INFγ and TNFα are overexpressed in endothelial and glial cells from SSPE-affected patients . Furtherpost-mortem autolysis/decomposition phenomena that could have negatively interfered with IHC reactions. Additionally, although we used pAbs standardized for their use in cetacean species and we based our judgment(s) of suitable cross-reactivities upon cellular morphologic features and cellular immunostaining patterns similar to human and mouse and other cetacean control tissues , (a) there was multicentric lymphoid depletion, characterized by reduced numbers of T cells and B cells in all three species infected by CeMV; however, lymphoid depletion phenomena were more pronounced in Guiana dolphins infected with GDCeMV; (b) striped dolphins and bottlenose dolphins, infected with DMV, often had hyperplastic (regenerative) phenomena involving the aforementioned cell populations, particularly chronically infected animals; (c) there was generalized increased expression of caspase 3 in all three species; and (d) no differences were detected regarding cytokine immunoreactivity. In the lung, (a) there was a mild to moderate increase in T cells, B cells, and histiocytes in all three species; and (b) no differences were detected regarding cytokine immunoreactivity. Concerning the CNS: (a) inflammation was a consistent feature in DMV-infected striped and bottlenose dolphins in contrast to Guiana dolphins infected by GDCeMV; (b) inflammation was dominated by T cells and B cells, accompanied by fewer Iba1+, CD68+, and lysozyme+ histiocytes; (c) there was increased expression of caspase 3; and (d) no differences were detected regarding cytokine immunoreactivity except for IFNγ in the CNS of infected dolphins of all three species.In conclusion, these novel results delineate the local immunophenotypic response during CeMV infection in three highly susceptible delphinid species. They also suggest a complex interplay between viral and host's immune factors, thereby contributing to gain valuable insights into similarities and differences of CeMV infection's immunopathogenesis in relation to body tissues, CeMV strains and cetacean hosts. Finally, the herein presented IHC investigation results may help elucidating the immunopathogenetic bases, including the kinetics of LIRs in other infectious and non-infectious disease processes in cetaceans, with major applications in ecotoxicological pathology.All data for this study are included in the manuscript and/or the supplementary files.JD-D, KG, and JC-D contributed conception and design of the study. JD-D, KG, ES, SS, ÓQ-C, MA, AF, ES-N, JI, RC, JL-B, LF, CC, SM, LD, GDF, GDG, and JC-D contributed to organization of the databases and/or collected samples for histopathological, immunohistochemical, and molecular analyses. JD-D, KG, RR, IR, CK, NF, and BC contributed to immunohistochemical analyses and laboratorial resources. JD-D, KG, ES, CC, LD, and GDF conducted molecular analyses. JD-D wrote the first draft of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antimicrobial peptides (AMPs) have been considered as potential therapeutic sources of future antibiotics because of their broad-spectrum activities and different mechanisms of action compared to conventional antibiotics. Although AMPs possess considerable benefits as new generation antibiotics, their clinical and commercial development still have some limitations, such as potential toxicity, susceptibility to proteases, and high cost of peptide production. In order to overcome those obstacles, extensive efforts have been carried out. For instance, unusual amino acids or peptido-mimetics are introduced to avoid the proteolytic degradation and the design of short peptides retaining antimicrobial activities is proposed as a solution for the cost issue. In this review, we focus on small peptides, especially those with less than twelve amino acids, and provide an overview of the relationships between their three-dimensional structures and antimicrobial activities. The efforts to develop highly active AMPs with shorter sequences are also described.  Antimicrobial peptides (AMPs) are endogenous polypeptides produced by multicellular organisms in order to protect a host from pathogenic microbes. AMPs are also defined as host defense peptides because of their essential role in constituting the innate immunity system ,2,3,4. AAMPs show broad-spectrum antimicrobial activities against various microorganisms, including Gram-positive and Gram-negative bacteria, fungi, and viruses . Of partAMPs can be classified into four groups based on their structures: α-helical peptides, β-sheet peptides, extended peptides, and loop peptides ,2,3,6. TThe β-sheet AMPs, such as α-, β-defensins, and protegrin, are stabilized by disulfide bridges, and form relatively rigid structures. Many of β-sheet AMPs exert their antimicrobial activities by disrupting bacterial membranes. They are perpendicularly inserted or tilted into the lipid bilayer to form toroidal pores, and hydrophilic regions of the peptides are associated with the polar head groups of the membranes . The extended AMPs, which are predominantly rich in specific amino acids such as proline, tryptophan, arginine, and histidine, have no regular secondary structure elements. Indolicidin is a tryptophan/proline-rich extended peptide and Bac5 and Bac7 are proline/arginine-rich peptides ,18. ManyUnderstanding the structure-activity relationships (SAR) of AMPs is essential for the design and development of novel antimicrobial agents with improved properties. In particular, the atomic level structures of AMPs can provide versatile information for all stages of drug development, including the peptide design and modification for pharmaceutical application. Pexiganan , a synthetic variant of magainin 2, is one of the best-investigated AMPs in terms of drug development ,20. Pexide novo approaches to design model amphipathic helical peptides are noteworthy. For example, certain 14- or 15-mer peptides (namely LK peptides) composed of two kinds of amino acids (leucines and lysines), have been characterized as possessing strong antimicrobial activity [de novo designed model peptides remain to be further improved by optimizing the amino acid sequence to enhance stability and to reduce potential toxicity. In addition, their activities need to be checked using clinically isolated bacteria. In the LKW peptide design, the tryptophan residue was specifically incorporated in expectation of structural role for conferring activity [Knowledge on the functional structures of AMPs also enables a rational design of synthetic model peptides. In this respect, minimalistic activity ,25. Thenactivity . Tryptopactivity ,28,29,30N11) could recover the antimicrobial activity by a single tryptophanyl substitution at the hydrophobic-hydrophilic interface of the amphipathic helix [AMPs are fascinating targets as novel antibiotics because of their broad-spectrum activity, which include drug-resistant bacteria. Since the isolation of magainins from frog skin in 1987 , there hic helix ,30. 3+ ion, but also interact with the bacterial membrane directly, which enables the LF to possess antibacterial activity [Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii strains [Candida albicans, and can be used with classic antibiotics for a synergistic effect. Preincubation of fluconazole-resistant C. albicans with hLF1-11 significantly enhances the candidacidal effect of fluconazole [Lactoferrin (LF) is an iron-binding glycoprotein which is responsible for part of the innate defense system. The LF can not only bind and trap Feactivity ,41,42,43 strains ,45. Moreconazole .The three-dimensional structures of several lactoferrin-derived peptides in membrane mimetic conditions have been solved by NMR spectroscopy ,48,49,50The safety and tolerability of hLF1-11 in healthy volunteers and haematopoietic stem cell transplantation (HSCT) recipients have been tested ,51. IntrCandida albicans and distinct in action mechanism from other natural AMPs. The candidacidal activity of the α-MSH peptides is caused by increased cyclic adenosine monophosphate (cAMP) in the C. albicans cells [C. albicans and the cAMP-activating effect of α-MSH interferes with the cAMP-mediated signaling pathway [The α-melanocyte stimulating hormone , obtained from the cleavage of pro-opiomelanocortin (POMC), is a neuropeptide hormone showing anti-inflammatory activity and antimicrobial activity ,53,54. Ins cells , whereas pathway ,57. 11-13; KPV) also has anti-inflammatory and antimicrobial activities similar to those of the full-length α-MSH [2, also named CZEN-002, was designed based on the KPV (α-MSH11-13) peptide. The peptide is a dimeric octamer peptide comprising two units of KPV peptide connected by a cysteine-cysteine linker, which is classified as loop peptide. The (CKPV)2 peptide revealed outstanding candidacidal activity against C. krusei and C. glabrata that are emerging as drug-resistant strains, but it showed very low toxicity to host cells [2 peptide also exerts anti-inflammatory effects as well as the candidacidal activity. It can inhibit TNF-α production with comparable activity to that of the potent α-MSH analogues [Interestingly, the C-terminal tripeptide 2 is being currently evaluated in clinical trials for treatment of vulvovaginal candidiasis [2 can be a valuable target for the design of other short antimicrobial peptides. Thus, the three-dimensional structure of the (CKPV)2 was determined by NMR spectroscopy, to identify a novel scaffold for the design of small compounds having candidacidal activity [2 adopts a symmetric dimer with an extended backbone structure, which resembles the model structure of natural α-MSH peptide [2 showed a β-turn-like fold, which may be critically related to the higher activity of (CKPV)2 than KPV monomer [Because of the simple sequence and small size as well as the excellent candidacidal activity, (CKPV)didiasis . In addiactivity . (CKPV)2 peptide . The ove monomer .C. albicans, C. glabrate, C. parapsilosis, and C. tropocalis that cause oral candidiasis. In addition to the broad spectrum of anticandidal activity, P-113 is also active against fluconazole resistant C. albicans and C. glabrata [Histatin 5, a parent molecule of P-113, is a small cationic peptide secreted into the saliva. Among the histatin family peptides, the histatin 5 exerts the most potent antimicrobial activity against bacteria and fungi ,65. The glabrata , which sStructural investigations of hsitatin 5 and P-113 in various solvents have been performed mainly by NMR spectroscopy ,68,69,70N-terminal region (from Ala1 to His5) and a less defined C-terminal region (from Gly6 to His12). Imidazole rings of all three His residues  are coordinating one Zn(II) ion. The involvement of His12 in metal coordination makes the peptide to have lower propensity to adopt an alpha-helical conformation in C-terminal region, therefore resulting in the decrease of antimicrobial activity. The antimicrobial activity of Zn(II)-P-113 against a variety of microorganisms, including S. aureus, E. faecalis, and C. albicans, is much lower than that of metal free P-113 [Structural properties of P-113, which are related to the antimicrobial activity, are also similar to those of histatin 5. Structural conversion of the P-113 from a disordered conformation in aqueous solution to an α-helical conformation in membrane mimetic environments was evidenced by a circular dichroism study ,67. The ee P-113 . N11 [2 [The serious problems caused by drug resistant bacteria have created an urgent need for the development of alternative therapeutics. In this respect, AMPs are considered as promising antimicrobial agents for producing new generation antibiotics. Additionally, atomic level structures of AMPs are prerequisite information for the generation of improved peptide antibiotic candidates. Although there are several obstacles to be overcome for clinical applications, natural and synthetic AMPs are still attractive sources to the pharmaceutical companies. In order to facilitate commercial development of peptide antibiotics, it is reasonable to focus on small peptides. Successful generation of short antimicrobial peptide molecules includes the A4W-GGN5N11 ,30, hLF1N11 ,49,50,51N11 [2 ,60,61,62N11 [2 ,67,71, aN11 [2 ,25,26,27


Kinetic studies revealed that all tested compounds were found to be noncompetitive inhibitors against BACE1 and cholineseterases. In addition, molecular docking studies of these compounds demonstrated negative binding energies for BACE1, AChE, and BChE, indicating high affinity and tight binding capacity for the target enzymes. The present study suggested that the selected citrus flavanones could act together as multiple inhibitors of BACE1, AChE, and BChE, indicating preventive and therapeutic potential against AD.Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease, distinctively characterized by senile plaques, neurofibrillary tangles, and synaptic loss, finally resulting in neuronal death. β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) and cholinesterases have been identified as therapeutic targets for AD, and the discovery of their inhibitors is of critical importance for developing preventive strategies for AD. To discover natural multi-target compounds possessing BACE1, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) inhibitory properties, major citrus flavanones including hesperetin, naringenin, and hesperidin were evaluated. In vitro anti-AD activities were performed via BACE1 and cholinesterases inhibition assays, as well as enzyme kinetic predictions. For the design of potential inhibitors of AD-related enzymes, molecular docking analysis was performed. Based on the biological evaluation, hesperidin demonstrated the best inhibitory properties toward BACE1, AChE, and BChE, with IC Alzheimer′s disease (AD), characterized by the appearance of senile plaques and neurofibrillary tangles, as well as a loss of cholinergic neurons, is the most prevalent form of dementia . Senile Aβ peptide is generated by sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretase. Studies show that β-secretase  protein levels and activity are elevated in sporadic AD brains, and that BACE1 levels are upregulated under stress conditions such as oxidative stress, cerebral ischemia, and hypoxia, all of which are associated with increased AD incidence ,6,7. GivIn addition to BACE1, the cholinergic hypothesis has also played a large part in the development of AD therapy. The neurotransmitter acetylcholine (ACh) possesses an important role in the process of learning and memory in the hippocampus. Under normal physiological conditions, acetylcholinesterase (AChE) is the major enzyme carrying out the hydrolysis of ACh into choline and acetate, whereas butyrylcholinesterase (BChE) acts as a co-regulator of the activity of AChE . HoweverIn citrus fruits, flavanones comprise approximately 95% of the total citrus flavonoids, existing in both aglycone and glycosidic forms. The most abundant flavanone aglycones are hesperetin and naringenin. Hesperidin is the major glycoside with rutinose  . Various50, 16.99 ± 1.25 µM), followed by hesperetin  and naringenin . In addition, the IC50 value of hesperidin was similar to that of resveratrol, which was used as a positive control. The structures of hesperetin , naringenin , and hesperidin are shown in The potential causes in AD is the Aβ, which is generated through sequential cleavage by β- and γ-secretase. Cleavage by α-secretase produces a soluble N-terminal fragment, APPsα, and a membrane-bound C-terminal fragment APP (C83). The remaining C83 is then cleaved by γ-secretase to release a nontoxic p3, thereby precluding Aβ generation. Tumor necrosis-α converting enzyme (TACE) is a candidate of α-secretases belonging to the ADAM  family. To determine the BACE1 selectivity of citrus flavanones, all compounds were tested against TACE, trypsin, chymotrypsin, and elastase, and found to be inactive at up to 100 µM . 50 values of 22.80 ± 2.78 µM and 48.09 ± 0.74 µM, respectively. However, the inhibitory potential of hesperetin and naringenin was relatively specific to AChE, with IC50 values of 45.70 ± 2.69 and 42.66 ± 4.30 µM, but not BChE.Hesperidin exerted a strong inhibitory effect on AChE and BChE, with IC50 values, the inhibition modes and parameters of the three tested compounds on the targeted enzymes were investigated. The results indicated that the compounds noncompetitively inhibited BACE1 and cholinesterases. The Ki values for the inhibition of BACE1 by hesperidin, naringenin, and hesperetin were 39.4 μM, 47.1 μM, and 42.7 μM, respectively . On the other hand, several studies reported that hesperidin and hesperetin exhibited low inhibitory activity against cholinesterases [In the present study, the three citrus flavanones were investigated for their inhibitory activities on BACE1, AChE, and BChE. However, the results in our study showed that hesperidin, containing two sugar moieties, exhibited notable BACE1 and cholinesterases-inhibitory activity, when compared with hesperetin and naringenin. Similar results regarding the correlation between sugar moieties and AChE inhibitory activity was shown in a previous study . Rubrofusterases ,21.Docking results demonstrated that the lowest binding energies of glycosylated flavanone with the target enzyme including BACE1, AChE, and BChE were generally lower than that of the aglycone. Hesperidin probably increased the hydrophilicity of hesperetin and naringenin, thereby raising hydrogen interactions between hesperidin and its target proteins. The result of hesperidin is in accordance with the finding reported by Remya et al., that hesperidin has low binding energy with AChE .Hesperidin, a glycosidic compound has previously exhibited potent neuroprotective activity in vivo. Li and coworkers (2015) reported that oral administration of hesperidin (100 mg/kg) for 10 days recovered microglial activation, APP expression, Aβ deposition, and behavioral dysfunction in APP/PS1 Tg mice . In addiO-glucuronide (GLN) and hesperetin-3′-O-GLN were identified as the predominant hesperidin metabolites. In addition, hesperetin-5,7-,3′,7-O-diglucuronide, hesperetin-7-O-glucoside, and hesperetin-3′-O-sulphate were also found [O-GLN and naringenin-3′-O-GLN were detected. In general, metabolites of flavonoids showed reduced bioactivity compared to their parent compounds, though contrary results have also been reported. Hesperetin metabolites derived from hesperetin-administered rat serum were revealed to be potent antioxidants, and possess anti-inflammatory activity, when compared with hesperidin or hesperetin [To develop functional foods and dietary supplements preventing or treating AD, oral bioavailability, metabolic transformation, and the blood–brain barrier (BBB) permeability of active components is a cause of concern. When absorbed, hesperidin is hydrolyzed by the gut microflora into hesperetin, and conjugated by phase II drug-metabolizing enzymes into glucuronidated, sulfated, or sulfoglucuronidated forms . Hespereso found . The abssperetin . For central nervous system (CNS) penetration, the compounds must first cross the BBB, which ultimately controls the composition of extracellular fluid in the CNS, by firmly regulating molecular traffic and buffering against changes in the systemic circulation. An in vitro study by Youdim et al. discovered that naringenin and hesperetin cross the BBB in the ECV304/C6 co-culture model . Peng anHesperetin (>95% purity), naringenin (>95% purity), hesperidin (>97% purity), resveratrol (>99% purity), galanthamine, AChE, BChE, 5,5′-dithiobis-(2 nitrobenzoic acid) (DTNB), and acetylthiocholine iodide were purchased from Sigma-Aldrich . The BACE1 kit from Invitrogen  was used for the measurement of BACE1 activity. α-Secretase  and its fluorogenic substrate were purchased from R&D Systems . Elastase, chymotrypsin, trypsin, and their substrates were obtained from Sigma-Aldrich.2, N-benzoyl-l-Arg-pNA, N-benzoyl-l-Tyr-pNA, and N-succinyl-Ala-Ala-Ala-pNA as substrates, respectively. BACE1, TACE, chymotrypsin, trypsin, and elastase assays were carried out according to previously described methods . BACE1, AChE and BChE inhibitory activities were assessed by a modified version of the Ellman colorimetric method . Each reEnzymatic hydrolysis, mediated by AChE or BChE, was monitored according to the formation of yellow 5-thio-2-nitrobenzoate anions at 405 nm for 15 min, which were produced by the reaction of DTNB with thiocholine released from ACh or BCh. All reactions were performed in 96-well plates in triplicate, and recorded using a microplate spectrophotometer .To prove the kinetic mechanisms of hesperetin, naringenin, and hesperidin towards BACE1, AChE, and BChE, both Dixon plots and Lineweaver–Burk complementary kinetic methods were conducted. The inhibitory constant (Ki) was obtained by interpretation of the Dixon plot, and Vmax and Km were defined by Lineweaver–Burk plots, using the initial velocities obtained over a substrate concentration range. These kinetic parameters were calculated using the Enzyme Kinetic™ module of SigmaPlot™ version 12.3 .http://www.rcsb.org/), with the respective accession codes 2WJO, 4PQE, and 1P0I, respectively [The Autodock Vina program was used for molecular docking study to investigate structural complexes of the BACE1, AChE, and BChE with flavanones. The target proteins, BACE1, AChE, and BChE were obtained from the Protein Data Bank  version 9.3 .The present study suggests that hesperetin, naringenin, and hesperidin show noncompetitive inhibition against BACE1 and cholinesterases. In our docking study, hesperidin with hydroxyl groups of the sugar moiety, available for hydrogen bonding with amino-acid residues in target enzymes, were most effective. Although further in vivo studies of the tested compounds are required in order to confirm our present findings, these citrus flavanones might be useful as potentially functional compounds for the treatment and prevention of AD.


The present study is mainly to explore the mechanism that how Qigesan (QGS) affects the movement capacity of esophageal cancer (EC) cell. QGS incubates ECA109 and TE1 cell lines and detecting the motility of tumor cells by different experiments. Growth arrest-specific 6 (Gas6) and Anexelekto (Axl) were co-localized, and then detecting Gas6, Axl signaling pathway, and protein expression after QGS intervention. Similarly, Observing the signal localization and protein expression of P-phosphoinositide3-kinases (PI3K), P-AKT protein kinase B (AKT), P-nuclear factor-kappa B (NF-κB), matrix metalloproteinase-2 (MMP2), and matrix metalloproteinase-9 (MMP9). The results showed that the concentration of QGS was less than 200 ug/ml, and the cultured cells did not exceed 24 h, that no obvious cytotoxicity was observed. QGS significantly inhibited the mobility of ECA109 and TE1 cell lines in the concentration-dependent manner. In addition, QGS can regulate the Gas6/Axl pathway, inhibit the formation and localization of the Gas6/Axl complex, and reduce the protein activation of PI3K/AKT, NF-κB, MMP2, and MMP9. Experimental innovation shows that QGS can significantly slow down the mobility of EC cells by regulating the Gas6/Axl complex and downstream signaling pathways, and provides a theoretical basis for the pharmacological effects of QGS in the therapy of EC. Esophageal cancer (EC) is a severely upper gastrointestinal malignant tumor, and has a high mortality rate. The onset characteristics of EC have obvious regional differences in the world ,2. In WeQigesan (QGS) was created by Zhong-ling Cheng, a famous doctor during Qing Dynasty. In his book (Yi Xue Xin Wu), QGS was used to therapy ‘YeG’. EC belongs to the category of “YeGe” in Traditional Chinese Medicine (TCM) . StudiesStudies have shown that Gas6 and Anexelekto (Axl) are highly expressed in oral squamous cell carcinoma (OSCC) and other cancer tissues and cells and the binding of Gas6 and Axl affects cell migration and other functions ,15. ReceOur previous protein chip results showed that QGS can reduce the expression of Gas6 of esophageal cancer cells (unpublished data). It has been reported that Gas6 promotes the development of esophageal cancer, Gas6 expression is essentially the same as Axl and is maximally up-regulated in invasiveness and lymph node metastasis of esophageal cancer ,22. The http://lsp.nwu.edu.cn/tcmspsearch.php) and The Plan List (http://www.theplantlist.org/). According to the clinical daily dose of each patient, the above prescription formulas were used to treat an adult with an average body weight of 60 kg for 3 day. All herbs are soaked in 1000 ml of distilled water for 1 h, decocted twice. The two extracts are mixed, pressurized and filtered, concentrated in a rotary evaporator, and then freeze-dried. QGS freeze-dried powder was extracted and placed in the refrigerator at −20°C, diluted with conventional medium, and used after filtered. In our experiments,1 mg lyophilized powder, about 0.1% of a person’s daily dose, selecting the intervention dose based on principles of dose use of TCM [The Chinese materia medica of QGS come from Sinopharm Group Le-Ren-Tang Medicine Co., Ltd. . See e of TCM .Newborn Calf Serum (NCS), phosphate-buffered saline (PBS) , Trypsin-EDTA Solution . RPMI 1640 cell culture medium , Hochest, Mitotracker dye . Experimental dimethyl sulfoxide (DMSO) is derived from the Sigma . Cell Counting Kit-8 (CCK-8 kit ) .2, 37°C.Experimental esophageal cancer cell line Eca109, TE1 purchased from Shanghai Institute of Biosciences Cell Resource Center, Chinese Academy of Sciences. Cell culture (10% NCS-RPMI-1640) and passage according to protocol, Incubator condition 5% CO3 cells/well (96-well). Then incubating cells by different concentrations  of QGS for 24 h. Cytotoxicity was tested using. At the end of the treatment, removing the original medium, and adding the medium containing 10% CCK-8 solution to the cells, put in the incubator and continue to culture for 2 h. Measurement of cell absorbance using a microplate reader  at 450 nm. The optical density (OD) value of three wells, and the OD value of each group was statistically analyzed.The cells were digested by EDTA trypsin and counting, planking with 4 × 10Cells were plated in Perkin-Elmer (U.S.A.) 96-well plates overnight. The medium were removed, and the cells were stained with Hochest and Mitotracker  in the incubator for 30 min. Then remove the staining and incubating the cells with QGS , and placed in Operetta High-Content Analysis System (Perkin-Elmer) for 24 h. The results were analyzed using Harmony image (Perkin-Elmer) analysis software. Cell migration is expressed as mean speed and effective distance, monitoring cell motility by time-lapse image sequences, calculation method using manufacturer’s protocol (Perkin-Elmer). In order to assess cell status, the settings were made: hoechst channel for the nuclear; mitotracker channel for the cellular cytoplasm.Assessing Eca109, TE1 Cells migration with IncuCyte 3 Cell Migration & Invasion Assays  according to User Manual. A scratch was then made using the WoundMaker tool (Essen Bioscience). In short, cells were plated and grown overnight. Gently wash the cells twice, and treated with QGS  for 24 h. Then put the cells into the system and take pictures of the scratches at 0, 12, and 24 h (10× magnification). Evaluating Scratch closure rate by IncuCyte software, the result is expressed by relative wound density (RWD)%.4 cells in upper chamber , and adding calf serum-containing medium to the lower chamber. Cells are incubated overnight. Remove the matrigel and upper chamber cells with a cotton swab. The Transwell chamber was then put into the new plate, the upper compartment of the attached content and the lower compartment surface of the attached cells were fixed in 4% paraformaldehyde for 20 min and then coloring cells with crystal violet for 30 min. Finally, After washing with PBS, the cells were photographed under a microscope.Transwell assay for cell invasion. Transwell system from BD, U.S.A. . Incubat4 Cells/well, and pretreated with QGS  for 24 h. Then, 4% paraformaldehyde fixed, 0.3% Triton X-100 permeablilized, 10% bovine serum albumin (BSA) blocked and incubated overnight at 4°C in the refrigerators:anti-GAS6 , anti-AXL , anti-MouseAXL , anti-P-PI3Kp85 (Tyr607) , anti-P-AKT1 (Thr308) , anti-P-NF-κB p65 (Ser536) , followed by the appropriate FITC-conjugated or TRITC-conjugated secondary antibodies (KPL). Unbound antibodies were washed, and the cells were taken out and placed on slides, and cover with containing DAPI (CST). The cells were observed by Laser scanning confocal microscope .ECA109 and TE1 cells were planted on the coverslip, 5 × 10g, 5 min). The cells were lysed on ice for half an hour with lysis buffer, mixture is placed into centrifuge , extracting supernatant and protein quantification using the bicinchoninic acid (BCA) kit from Pierce, U.S.A. SDS-PAGE electrophoresis was performed, then transferred to the membrane, polyvinylidene fluoride (PVDF) membrane . PVDF membranes were pre-blotted with 5% BSA . Incubating PVDF membranes with primary antibody overnight: anti-GAS6 , anti-RabbitiAXL , anti-PI3K-P85 , anti-AKT1 , anti-NF-κB-P65 , anti-PI3K p85 (Tyr607) , anti-P-AKT1 (Thr308) , anti-P-NF-κB p6 5 (Ser536) , anti-RabbitiMMP-2 , anti-RabbitiMMP-9 , anti-β-ACT . The next day labeling fluorescent secondary antibody. Imaging PVDF membrane with Odyssey infrared imaging system (LI-COR Biosciences). The result is expressed by the ratio of protein/β-act.The cells were seeded into cell culture flasks and incubating cell with QGS  for 24 h. Cell lysate was prepared, and cells scraped. The suspension was put into the centrifuge  or unpaired two-tailed CCk-8 method  observinObserving the effect of QGS on migration of ECA109 and TE1 cells by Operetta CLS High content analysis system. The results showed that, in ECA109 cells, QGS showed significantly lower average movement speed of 200 μg/ml cells than the control group, and the effective movement distance of the cells was significantly shortened, while 100 μg/ml was not obviously A,C,D. SiTo demonstrate the inhibitory effect of QGS on the migration of ESCC cell lines ECA109 and TE1, we used a scratch test. We observed that the QGS dose of 200 μg/ml group compared with the control group, which obviously inhibits cell migration in TE1 cell at 12 h. More importantly, we observed that the QGS dose of 100 and 200 μg/ml group compared with the control group, which significantly inhibits cell migration at 24 h, but the effect of 200 μg/ml was significantly better than the 100 μg/ml group, the same trend was found in both cells. And as the dose of QGS increased, the cell area growth in the scratched area was significantly inhibited correspondingly as the dose and time increase A,B. Therin vitro cell invasion experiments were carried out. We observed that the QGS dose of 100 and 200 μg/ml compared with a blank group, gradually decreased the number of cells passing through the Transwell chamber and showed a concentration-dependence, the same trend was found in both cells, but the effect of 200 g/ml was significantly better than the 100 μg/ml group  of patients with esophageal cancer resection was significantly extended, and show a good trend to inhibiting metastasis . In ordeOur previous protein chip results showed that QGS can reduce the expression of Gas6 of esophageal cancer cells (unpublished data). Studies have shown that Gas6/Axl is highly expressed in various tumor tissues and cells . The GasAXL is an important member of the receptor tyrosine kinases, which is activated by its ligand GAS6 and triggers downstream signaling pathways according to different cell types, such as inositol PI3K/AKT, and nuclear transcription factor NF-κ B. It can perform many functions, including affecting cell migration, cell metastasis, and cell adhesion . StudiesThis research is mainly to observe QGS impact on the mobility of esophageal cancer, we chose the NF-κBp65 according to the relevant literature reports ,40. MMP2In recent years, TCM has carried out a lot of beneficial research in the treatment of cancer. TCM can improve the survival rate of patients with hepatocellular carcinoma after radiofrequency ablation . AdjuctiIn our hospital, we observed that QGS has a positive effect on controlling metastasis and improving symptoms in patients after esophageal cancer surgery . Clinica


Escherichia coli strain belonging to sequence type 617 (ST617), isolated from beach ghost shrimp from polluted coastal waters in Brazil. These data provide valuable information for comparative genomic analysis, related to the dissemination of MDR E. coli in marine ecosystems.Here, we present the draft genome sequence of a multidrug-resistant (MDR)  Escherichia coli strain belonging to sequence type 617 (ST617), isolated from beach ghost shrimp from polluted coastal waters in Brazil. These data provide valuable information for comparative genomic analysis, related to the dissemination of MDR E. coli in marine ecosystems.Here, we present the draft genome sequence of a multidrug-resistant (MDR)  Enterobacteriaceae in marine environments has been reported with particular concern about global public health  recovered from beach ghost shrimp  in an impacted ecosystem.The growing occurrence of multidrug-resistant (MDR) c health . Recentloastline –5, highlE. coli isolate (ECCO2) was identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF). DNA extraction was performed using the PureLink quick gel extraction kit . Next, DNA quality and quantity were checked with a NanoDrop spectrophotometer (Thermo Scientific) and Qubit 2.0 fluorometer (Life Technologies), respectively. Afterward, the genomic library was prepared using the Nextera XT DNA library preparation kit  and subsequently sequenced using the Illumina NextSeq 550 platform, with 2 × 75-bp paired-end reads and a genome coverage of 290.0×.Bacterial isolation and DNA extraction were performed as previously described , 7. Briede novo assembled using default settings in CLC workbench (such as automatic word [20] and bubble size [50]), a minimum contig length of 200 nucleotides, auto-detect paired distances, and mapping reads back to contigs.Resulting FastQ data were imported to the CLC genomic workbench 10 (Qiagen). Raw reads (∼29 million) were inspected for quality evaluation, and a trimming/cleanup step was applied to avoid a contamination of reads with barcodes, adapter sequences, and a large presence of Ns before assembly. In this regard, the software has ensured that the read length is in the appropriate size of 300 bp and a G+C content of around 50% to ensure accurate assembly. Finally, reads were N50 value of 86,325 bp, as well as a G+C content of 50.6%, which was annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/). The genome of ECCO2 was 4.8 Mb in size, containing 56 tRNAs, 2 rRNAs, 9 noncoding RNAs (ncRNAs), 284 pseudogenes, and 2 CRISPR arrays.The generated assembly showed a total of 5,133 genes with 5,060 protein-coding sequences. A total of 197 contigs were obtained, with an http://genomicepidemiology.org/). In this regard, the resistome revealed the presence of genes conferring resistance to β-lactams (blaCMY-2 and blaTEM-1B), quinolones (qnrS1), aminoglycosides [aph(6)-Id and aph(3″)-Ib], tetracycline (tetB and tetD), sulfonamide (sul2), and trimethoprim (dfrA8). In fact, ECCO2 displayed an MDR profile to cefoxitin , enrofloxacin , levofloxacin , nalidixic acid , ciprofloxacin , amikacin , gentamicin , tetracycline , and trimethoprim-sulfamethoxazole , determined by disc diffusion and Etest methods (gyrA (S83L), gyrB (D87N), and parC (S80I) genes, which confer resistance to fluoroquinolones (sugE) and heavy metals  were also identified. The ECCO2 isolate was assigned to the serotype O89:H9, and virulome analysis detected gad (glutamate decarboxylase) and iss  genes.The resistome, plasmid replicons, multilocus sequence type (MLST), serotype, and virulome were identified using ResFinder 3.1, PlasmidFinder 2.0, MLST 2.0, SerotypeFinder 2.0, and VirulenceFinder 2.0 databases (95% of identity and 60% of minimum length), respectively . ST617 belongs to clonal complex 10 (CC10), which is widespread internationally and related to clinical strains found in environmental, human, and food samples, and mostly in association with a broad-spectrum cephalosporin-resistant phenotype promoted by the acquisition of plasmid-mediated blaCMY-2 and blaCTX-M-type genes (10E. coli ST617 has been reported in urban lakes in Brazil (Leucophaeus pipixcan) in Chile (IncFIB, IncFIC, IncFII, and IncX3 incompatibility group plasmids were identified. Genomic analysis confirmed the presence of the  genes 1012. In thn Brazil  and in FE. coli strain displaying an extended resistome and recovered from beach shrimp. Our findings suggest that benthic animals living on polluted sand bottoms could become new hosts and vehicles for transmissions of MDR bacteria to marine predators, adding valuable information in the routes of transmission of MDR E. coli in the marine environment. Therefore, genomic surveillance studies in coastal habitats are urgently required.In summary, we present the first draft genome sequence of an MDR PIZJ00000000. The described version used in this paper is PIZJ01000000, and the SRA run number is SRR8186051.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number


The C3, C5, and C6 dibenzoates performed as well as or better than DEHP, with the exception of torsional modulus, further supporting their use as green plasticizers. For blends with 1,4-butanediol dibenzoate, differential scanning calorimetry and torsional temperature sweeps suggested that the compound partly crystallizes within PVC blends over the course of two days, thereby losing the ability to effectively plasticize PVC. However, upon heating to temperatures above 60 °C, effective plasticization was again observed. 1,4-Butanediol dibenzoate is thereby a reversible heat-activated plasticizer or processing aid with excellent plasticizer properties at mildly elevated temperatures.Several linear alkyl diol dibenzoate compounds, ranging from C3 to C6 in central diol length, were evaluated for their plasticizing effectiveness in blends with poly(vinyl chloride) (PVC). The results were compared to blends of PVC/di(2-ethylhexyl) phthalate (DEHP), the most commonly used commercial plasticizer. DEHP has come under scrutiny, due to its suspected endocrine-disrupting behaviour, and the proposed diol dibenzoates have previously been shown to have the potential to be green, safe candidates for DEHP replacement. The thermal and mechanical properties of PVC/dibenzoate blends were determined, and include glass transition temperature (T In terms of maximum observed stress, statistically significantly different values were observed for all blends, with the exception of those containing 1,5-PDB and 1,6-HDB .Similar results were obtained for the apparent modulus at 10% EL . As expected, 1,4-BDB exhibited higher surface hardness than all other blends , and a polarized light filter was used to image the surface A. For coThis study demonstrates that three diol dibenzoate compounds with alkyl central linker lengths of C3, C5, and C6  are as eThe C4 compound 1,4-butanediol dibenzoate  showed interesting behavior, in that the plasticizer seemingly loses its plasticization effectiveness over time, likely due to plasticizer crystallization within the blend. This behavior could be exploited for use in PVC processing as a reversibly heat-activated plasticizer, to shape the material at temperatures above 50 °C and subsequent material hardening at room temperature or below.g reduction, elongation at break, maximum tensile stress, apparent modulus, torsional modulus (G’ and G”), and surface hardness. These results confirm and expand the scope of reported material properties [et al., 1,5-PDB resembles the commercial diethylene glycol dibenzoate (DEGDB), except that the oxygen atom of the ether linkage in the central part of DEGDB is replaced with a carbon atom . The discrepancy in surface hardness observed between 1,3-PrDB and 1,5-PDB compared with 1,6-HDB could be due to a surface specific effect, such as reduced 1,6-HDB surface mobility. Differences between bulk volume properties (tensile testing) and surface properties (surface hardness) in polymers are not uncommon and have been described previously—for example, for polypropylene (PP) [The only structural difference between 1,3-PrDB, 1,6-HDB, and 1,5-PDB is the length of the central diol linker, yet the reported results suggest that the linker length is only marginally important; notably, blends containing 1,3-PrDB, 1,5-PDB, and 1,6-HDB produced stress–strain curves of almost identical shapes , resultiene (PP) . Differeg determination . This process would also be reversible, by simply heating the material again. It should be noted that another positive aspect of 1,4-BDB is that the central diol linker can be produced renewably by microbial fermentation, or by reducing microbially-produced succinic acid , therebyFor any plasticizer to truly qualify as a suitable replacement for problematic commercial plasticizers such as DEHP, many aspects beyond plasticizer performance need to be addressed, and the principles of green chemistry can provide a good framework for such an assessment ,17,43. TThis work further outlines the suitability of several diol dibenzoate compounds, ranging in central diol linker length from C3 to C6, to effectively plasticize PVC. Three of the four compounds were as effective, or indeed, more effective than commercially-available DEHP in all but one tested material property. In combination with previous studies that showed rapid environmental degradability ,24,30 an


Panax ginseng and Panax notoginseng, two well-known medical plants with economic value, have a long history of use for managing various diseases in Asian countries. Accumulating clinical and experimental evidence suggests that notoginsenosides and ginsenosides, which are the major bioactive components of the plants, have a variety of beneficial effects on several types of disease, including metabolic, vascular, and central nervous system disease. Considerable attention has been focused on ginsenoside Rb1 derived from their common ownership as an anti-diabetic agent that can attenuate insulin resistance and various complications. Particularly, in vitro and in vivo models have suggested that ginsenoside Rb1 exerts various pharmacological effects on metabolic disorders, including attenuation of glycemia, hypertension, and hyperlipidemia, which depend on the modulation of oxidative stress, inflammatory response, autophagy, and anti-apoptosis effects. Regulation of these pathophysiological mechanisms can improve blood glucose and insulin resistance and protect against macrovascular/microvascular related complications. This review summarizes the pharmacological effects and mechanisms of action of ginsenoside Rb1 in the management of diabetes or diabetic complications. Moreover, a multi-target effect and mechanism analysis of its antidiabetic actions were performed to provide a theoretical basis for further pharmacological studies and new drug development for clinical treatment of type 2 diabetes. In conclusion, ginsenoside Rb1 exerts significant anti-obesity, anti-hyperglycemic, and anti-diabetic effects by regulating the effects of glycolipid metabolism and improving insulin and leptin sensitivities. All of these findings suggest ginsenoside Rb1 exerts protective effects on diabetes and diabetic complications by the regulation of mitochondrial energy metabolism, improving insulin resistance and alleviating the occurrence complications, which should be further explored. Hence, ginsenoside Rb1 may be developed as a potential anti-obesity, anti-hyperglycemic, and anti-diabetic agent with multi-target effects. Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia caused by insufficient insulin secretion and/or insulin action. The increased prevalence of DM, especially type 2 diabetes (T2DM) ,2,3, is Recently, there has been a shift in the drugs prescribed for T2DM from agents that stimulate insulin secretion, such as sulfonylureas, towards agents that increase insulin sensitivity, such as biguanides (BGs)  and thiaGlucose transporter type 4 (GLUT4) also plays an important role in maintaining blood glucose homeostasis and its presence can reduce insulin resistance. The primary function of GLUT4 is partial glucose transport, which is mediated by the PI3K and Akt signaling pathways. In addition, many mechanisms initiate and sustain damage to the vasculature, including the classical polyol pathway, glycosylation end products pathway, protein kinase C pathway, and hexosamine pathway ,20.Conventional diabetes treatments include SUs, BGs, TZDs , and alpPanax notoginseng (Burk.) F.H.Chen [Panax ginseng C.A.Mey [P. notoginseng and its extracts have many functions, such as anti-inflammation [P. notoginseng and P. ginseng, as an anti-diabetic is widely publicized [Traditional Chinese medicines and their natural active ingredients have a variety of hypoglycemic effects and effectively control and delay the development of diabetes and complications by scavenging oxygen free radicals, improving blood hypercoagulability, inhibiting protein non-enzymatic glycation, inhibiting aldose reductase, correcting fat and protein metabolism disorders, and inhibiting platelet aggregation ,29,30,31F.H.Chen  and Pana C.A.Mey   [P. notoginseng. All ginsenosides have the same four-ring hydrophobic structure, but different numbers of sugar moieties. Ginsenoside Rb1 and Rg1 are the most abundant in P. notoginseng and the quality standard is also based on the total amount of ginsenoside Rb1, Rg1 and notoginsenoside R1, which is not less than 5.0% as the standard for measuring the P. notoginseng quality. Ginsenoside Rb1, the most important active factor [Panax species, including Panax quinquefolius (American ginseng) and Panax ginseng (Asian ginseng). It is thought that the variability of the sugar component may be associated with the specific action of each ginsenoside [e family . The vasculation ,35. Sapooginseng ,66 and P ginseng ,35. So f seeds.) ,66 and m seeds.) ,35. These factor , is alsosenoside . More imsenoside ,33,35.Insulin resistance is an abnormal physiological state and the main cause of type 2 diabetes (insulin-independent diabetes), meaning that target cells are not sensitive to insulin and require relatively more insulin to maintain normal blood glucose levels, which can be caused by abnormal structure of insulin, the abnormal binding of insulin receptors, insulin degradation, insulin receptor defects, metabolic signal transmission error, etc. Recently, the tendency of drug prescription for type 2 diabetics has moved from agents that stimulate insulin secretion toward agents that increase insulin sensitivity by activating the hepatic AMPK, PI3K, and Akt activity ,16,69,70The increased prevalence of obesity has focused attention on a worldwide problem that is not one of famine or infection, but one of surplus. Increasing research results show ginseng and ginsenoside Rb1 possess anti-obesity , anti-hyp < 0.01) [p < 0.01) [Current in vivo results  have sho < 0.01) ,71. Mean < 0.01) ,78, and  < 0.01) . All of p < 0.01) [p < 0.01) [First, ginsenoside Rb1, which was injected intraperitoneally  for 12 d, decreased body weight growth gain, food consumption, adipose tissue, and leptin levels in KK-Ay DM mice ( < 0.01) . Ginseno < 0.01) . db/db mice (p < 0.01) [db/db mice [Second, treatment with ginsenoside Rb1  for 14 d alleviated hepatic fat accumulation and increased insulin sensitivity; these effects were accompanied by reduced liver weight and hepatic triglyceride content in obese diabetic  < 0.01)  and upre < 0.01) . Treatme/db mice . The mec/db mice . p < 0.01) [Third, intraperitoneal injections of Rb1  for both 21 d  and 4 we < 0.01) ,71. Impo < 0.01) . These eGenerally, the underlying molecular mechanism is directly bound up with regulating glycolipid metabolism in muscle, liver, and adipose tissues. Ginsenoside Rb1 may regulate glucose and lipid metabolism, improve glucose tolerance and insulin sensitivity, and reduce inflammatory cytokines and the inflammatory response in adipose tissue and the liver, which is involved in regulation of the NF-κB pathway.In vitro experimental studies also indicate that ginsenoside Rb1 possesses insulin-sensitizing activity and regulates the effects of glucose metabolism; thus, this compound may be developed as a potential anti-diabetic drug ,74,75,76p < 0.05) [Adipocyte cell lines derived from mice, especially 3T3-L1 cells, are extensively used in adipose tissue metabolism studies ,81. It w < 0.05) . In C2C12 myotubes, incubation with ginsenoside Rb1 enhanced basal AdipoR1 and AdipoR2 expression in a time- (1–12 h) and dose-dependent  manner . In C2C1Furthermore, ginsenoside Rb1 acts as an insulin sensitizer and thus has a hypoglycemic function ,83. It aIn summary, as shown in Inhibit lipolysis and reduce free fats release and ectopic triglycerides deposition;Reduce hepatic fat accumulation and suppress adipocyte lipolysis;Decrease inflammatory stress in adipose and liver tissues;Promote adipogenesis and the 3T3-L1 adipocytes differentiation;Upregulate PPARγ and enhance GLUTs expression and translocation of GLUT1/ GLUT4;Upregulate perilipin in adipocytes and in fat cells;Decrease 11β-HSD1 and its mRNA in liver and adipose tissue;Decrease the expression of inflammatory markers ;Negatively regulate leptin signaling (SOCS3 and PTP1B);Inhibit HG-induced caspase-3 activation and apoptosis;Increase adiponectin receptor gene expression, upregulate perilipin adipocytes, upregulate PPARγ2, C/EBPα, and activate the adiponectin signaling pathway;Regulate PI3K and Akt activity;Activate AMPK, regulate energy metabolism, and promote glucose uptake, at least partially, by activating the insulin signaling pathway;Regulate NF-κB pathway (p-IKK and p-IκBα) and leptin–STAT3 signaling.Based on the above summary and analysis, ginsenoside Rb1 exerts significant anti-obesity, anti-hyperglycemic, and anti-diabetic effects by regulating the effects of glycolipid metabolism and improving insulin and leptin sensitivities, as showed in In summary, ginsenoside Rb1 acts as an insulin sensitizer and thus has a hypoglycemic function and this compound may be developed as an anti-obesity, anti-hyperglycemic, and anti-diabetic agent .Glycolipid metabolism disorder is one of the main physiological characteristics of T2DM, which can result in hyperlipidemia and various microvascular and macrovascular T2DM complications over the long term ,88. TherApoptosis is the main form of β-cell death in diabetes. Ginsenoside Rb1 has anti-hyperglycemic effects and can protect pancreatic β-cells by inhibiting apoptosis  and hype2+ concentration, as well as significantly increased GLP-1 and upregulated proglucagon; these results indicate that the protective effects on the islets were certainly involved in the enhanced GLP-1 secretion [Furthermore, Rb1 promoted glucagon-like peptide-1 (GLP-1) secretion, and increased the intracellular ATP to ADP ratio concentration and the intracellular Caecretion , as showIt is known that abnormal mitochondrial energy metabolism induces reactive oxygen species (ROS) and NO generation by oxidative stress . HydroxyP. notoginseng saponins (PNS) and Rb1 were produced partly via reducing the transforming growth factor β1 (TGF-β1) protein expression and blocking the TGF-β1-Smad2/3 signaling pathway [Glomerular filtration, extracellular matrix hypertrophy, and progressive expansion, which have been considered early features of DN, lead to extra cellular matrix accumulation and glomerular basement membrane thickening, followed by induction of renal fibrosis . Tubuloi pathway .Based on the fact that visceral epithelial cell (podocyte) injury plays a key role in the occurrence and development of DN, the early initiation of therapy that aimed at reducing oxidative stress or modulating ROS-sensitive signaling pathways may be used as a remedy for DN and is considered a new target for the treatment of kidney damage. Simultaneously, ginsenoside Rb1 directly decreases the overproduction of ROS , protectPatients with DM have a higher risk of developing Alzheimer’s disease (AD) and vascular dementia in the process of aging than non-DM control subjects; this specific disease is recognized as diabetic encephalopathy ,103. Metp < 0.01) (p < 0.01) [As known, ginsenoside Rb1 shows neuroprotective effects ,105. It  < 0.01) , suggest < 0.01) , inhibit < 0.01) , which pIt has been proven that ginsenoside Rb1 can not only inhibit food intake, reduce the diabetic patient’s weight, regulate blood lipids levels, and improve blood hypercoagulability in diabetic patients, but could also delay the occurrence of diabetic heart disease, including microangiopathy, myocardial lesions, and cardiac autonomic neuropathy ,95, as wGinsenoside Rb1 attenuated myocardial ischemia/reperfusion (MI/R) injury in non-diabetic myocardium and in diabetes models ,107. ComMoreover, the preventive and therapeutic effects of Rb1 on diabetic cardiovascular complications was determined and verified in the traditional Chinese herbal medicine and multi-component formula, such as the Shensong Yangxin Capsule. Although this is a traditional compound preparation, its constitution of bioactive components has been initially determined, including ginsenoside Rb1, sodium danshensu, chlorogenic acid, and paeoniflorin. It decreased the heart weight/body weight ratio, attenuated cardiac fibrosis and collagen deposition, and improved the impaired cardiac function of T2DM rats. Meanwhile, it upregulated Smad7, downregulated TGF-β1 and p-Smad 2/3 protein levels and TGF-β1, col-1, col-3, MMP-2, MMP-9, and α-SMA RNA levels, inhibited fibrosis, and improved cardiac function by suppressing TGF-β1/Smad signaling .These effects reveal that ginsenoside Rb1 may exert double-cardioprotective effects on both MI/R injury in diabetes and diabetic damage due to MI/R, partly by inhibiting oxidative stress and apoptosis, enhancing the eNOS expression, and increasing the NO concentration, as well as activating the PI3K/Akt pathway.d-glucosidase activity by modulating the gut microbiota and upregulating the transformation pathway of Rb1 in diabetic rat intestinal microflora (Rb1→Rd→F2→CK) [In addition to the above effects, PNS and ginsenoside Rb1 can modulate the gut microbiota. Ginsenoside Rb1 has significant hypoglycemic activity in the context of diabetes because its gut permeability is increased and its deglycosylation by intestinal microflora is inhibited ,108. On d→F2→CK) .In addition, ginsenoside Rb1 promoted the secretion of GLP-1, a brain gut peptide secreted by ileal endocrine cells. GLP-1 itself has a glucose concentration-dependent hypoglycemic effect and it stimulates islet β-cells to secrete insulin .Therefore, ginsenoside Rb1 could improve oral bioavailability, modulate gut microbiota composition/balance, increase gut permeability, and stimulate GLP-1 secretion to exert potential hypoglycemic and anti-diabetic effects.In addition, ginsenoside Rb1 not only lowers glucose, increases insulin sensitivity, and regulates lipid metabolism , but alsThese effects show the characteristics of multiple targets and joint regulation of multiple pathways as follows:Rb1 protects neurons from HG-induced cell damage and exerts neuroprotective effects against oxidative damage and apoptosis by inhibiting GSK-3β-mediated CHOP induction and reducing ROS production.Rb1 has protective effects against high glucose podocyte injury, kidney damage, and DN by inhibiting oxidative stress. It also decreases ROS overproduction, protects against mitochondrial dysfunction, restores cellular redox enzymes imbalances, and reduces fibronectin expression under diabetic conditions by regulating p44/4, p38-MAPK, JNK/SAPK, and Akt phosphorylation levels, reducing TGF-β1 protein expression, and blocking the TGF-β1-Smad2/3 signaling pathway.Rb1 inhibits fibrosis and improves cardiac function by suppressing TGF-β1/Smad signaling. It also exerts cardioprotective effects on both myocardial ischemia/reperfusion under diabetic conditions and diabetic damage from myocardial ischemia/reperfusion. Rb1 attenuates myocardial ischemia/reperfusion injury and reduces infarct size, cardiomyocyte apoptosis, and caspase-3 activity, partly by inhibiting oxidative stress and apoptosis, enhancing eNOS expression, and increasing NO concentrations, as well as activating the PI3K/Akt pathway.Rb1 has anti-hyperglycemic effects and protects pancreatic β-cells by inhibiting oxidative stress induced by hyperglycemia. It also reduces cell apoptosis and NO and 8-OHDG production, promotes GLP-1 secretion, enhances islet β-cell viability, increases the ratio of intracellular ATP to ADP concentration, downregulates iNOS expression, and decreases Bax, Fa, and caspase-3 expression via the caspase-3 pathway.Diabetes mellitus, a chronic metabolic disease, is a serious problem throughout the world. It is a metabolic disorder characterized by hyperglycemia caused by insufficient insulin secretion and/or insulin action. Type 2 diabetes is the insulin-dependent type and patients must be treated with insulin permanently. The main pathogenesis of T2DM involves islet β-cell dysfunction and peripheral insulin resistance. Insulin resistance is the main characteristic of DM. Currently, the number of DM patients increases each year due to changes in living standards and environment and this number is expected to reach 300 million by 2025 ,2,3. AftAccumulating evidence has shown that ginseng and its main active constituent ginsenoside Rb1 posse anti-diabetic and insulin-sensitizing properties, which may be partly achieved via their ability to regulate adipocyte development and functions. Meanwhile, ginsenoside Rb1 not only has the effects of lowering glucose, increasing insulin sensitivity, and regulating lipid metabolism , but it In the present review, we summarized the available reports on the therapeutic effects and the molecular mechanisms of ginsenoside Rb1 in diabetes and its complications. Ginsenoside Rb1 exerts significant anti-obesity, anti-hyperglycemic, and anti-diabetic effects by regulating the glycolipid metabolism and improving insulin and leptin sensitivities, as shown in Ginsenoside Rb1, a natural active ingredient, has various pharmacological activities. The current study revealed that Rb1 exerted anti-diabetic effects involved in multiple signaling molecules and multiple pathways, presenting the characteristic of multiple effects, multiple targets, and multiple pathways . Therefo


Sorghum were identified via bioinformatics tools. Phylogenetic analysis of their protein sequences in comparison with those family proteins from Arabidopsis and rice indicated that these proteins could be clustered into five typical subfamilies. There are 12 SbPHT1 members, one SbPHT2, six SbPHT3s, six SbPHT4s, and two SbPHOs in Sorghum. Further analysis of the gene structure, conserved motifs, subcellular localization, and transmembrane domains suggested that these features are relatively conserved within each subfamily. Meanwhile, the qRT-PCR assay implied that SbPHT1;2, SbPHT1;11, and SbPHT4;6 were significantly upregulated in roots when exposed to low-phosphate conditions, suggesting that these genes might be involved in P uptake in low-phosphate conditions. Our study will increase our understanding of the roles of phosphate transporters in Sorghum.The phosphate transporter (PHT) family mediates the uptake and translocation of the essential macronutrient phosphorus (P) in plants. In this study, 27 PHT proteins in  Although P is abundant in the soil, the form of Pi, available for plant uptake, is relatively rare (0.1–10 μM). However, in the cytoplasm of plant cells, the Pi concentration is generally about 5–10 mM [Phosphorus (P) is an essential nutrient for plant growth and development, contributing about 0.2% of the dry weight. It serves as an important component of many biological macromolecules, such as nucleic acids, membrane lipids, and ATP. Meanwhile, multiple plant biological processes, including photosynthesis, respiration, and glycolysis, require this element . In plan 5–10 mM . Thus, t+ symporters. The first PHT protein identified in higher plants was AtPT1 from Arabidopsis thaliana [Arabidopsis thaliana [Oryza sativa [Triticum aestivum [Solanum tuberosum [PHT family genes has also been reported in Populus trichocarpa [Arabidopsis thaliana [Triticum aestivum [Solanum lycopersicum [Oryza sativa [Solanum tuberosum [Malus domestica [Phosphorus uptake from soil and translocation between plant tissues and organs is facilitated by phosphate transporters (PHT), which are Pi:Hthaliana . Since tthaliana ,6, Oryzaa sativa , Triticuaestivum , and Soluberosum . In addichocarpa ,11, Arabthaliana ,13, Tritaestivum , Solanumpersicum , Oryza sa sativa , Solanumuberosum , and Malomestica .PHT1, PHT2, PHT3, PHT4, and PHO, the first four of which are reported to localize in the plasma membrane, plastid inner membrane, mitochondrial inner membrane, and Golgi compartment, respectively [PHT1 genes are expressed in roots and responsible for Pi uptake from the soil under Pi-limited environments [Arabidopsis, there are nine PHT1 family genes , some of which are regulated in response to changes in Pi availability [PHT1 family (OsPT1-13) contribute to Pi uptake from the soil and internal translocation [ZmPht1;1-5 are expressed both in roots and in shoots, and are induced by Pi starvation [PHT1 family is composed of 14 members (GmPT1-14), encoding high-affinity transporters and contributing to Pi uptake in roots under low-Pi conditions [Medicago truncatula, five PHT1 genes are also induced by low Pi and are responsible for Pi uptake and translocation [Arabidopsis, and maize. AtPHT2;1 is a low-affinity transporter located in chloroplasts, responsible for phosphate-starvation responses in Arabidopsis [PHO1 gene family in Arabidopsis, required for Pi translocation [There are two Pi uptake systems in plants: a low-Pi-inducible high-affinity system and a constitutive low-affinity system . The forectively . Among tronments , many ofronments ,20. In Alability . In ricelocation ,22. In marvation . In soybnditions . In Medilocation ,26. In cbidopsis . In addilocation ,28.AtPHT1;6 is mainly expressed in floral organs, revealing the role of PHT1 transporters in flower development [AtPHT1s are all at least expressed in the roots, in line with their major role in Pi uptake from the soil [AtPHT1;1 and AtPHT1;4 are two high-affinity transporters and contribute to Pi uptake under both normal and low-Pi conditions [AtPHT1;5 is required for Pi translocation from roots to shoots [AtPHT1;8 and AtPHT1;9 is inducible by low-Pi concentrations and functions in Pi translocation within plants [Arabidopsis are detected in diverse tissues or organs and share overlapping expression patterns with other PHT1 transporters, suggesting the functional redundancy and complexity of roles [Aside from the PHT2 family, the other families are composed of more than one member. The Pi-affinity and expression pattern within the family vary greatly. For instance, elopment ; the oththe soil . AtPHT1;nditions . In addio shoots . The expn plants ,33. Takeof roles ,34.Sorghum (Sorghum bicolor (L.) Moench) is a C4 crop with high photosynthetic efficiency and environmental adaptability. As compared with its closely-related species maize, Sorghum is more resistant to harmful environments, including drought, high temperature, high salinity, and sterile soil. Although the whole-genome sequence of Sorghum has been published [Sorghum have not been systematically investigated yet. In this study, we identified 27 potential SbPHT family members via sequence alignment, classified these proteins into subfamilies, and further analyzed their sequences and expression patterns.ublished , the PHTSorghum genotype BTx623 was used in this study. For low-phosphate treatment, Sorghum seeds were first surface-sterilized with 70% ethanol for 2 min, followed by 5% NaClO for 20 min. Then, the seeds were washed with sterile water three times and planted individually on one-half-strength Murashige and Skoog (1/2 MS) medium with (control) or without K2PHO4 (low phosphate). Thereafter, the plants were placed in a greenhouse at 28 ± 2 °C under 12 h light of 100 μmol quanta m−2 s−1 irradiance and 12 h dark cycles.The PHT genes were isolated via homologous alignment. First, the PHT family genes from rice and Arabidopsis were obtained from the rice genome annotation project database (http://rice.plantbiology.msu.edu/) and the Arabidopsis Information Resource (TAIR) database (https://www.arabidopsis.org/), respectively. Then, the CDS query of each gene was blasted in the Phytozome database  to search for PHT homologs in Sorghum with the E-value cutoff set as 1.00E−10. The top three matched genes were considered as candidates. Thereafter, we collected all the candidates and further checked their annotation information in the MOROKOSHI database (http://sorghum.riken.jp/morokoshi/Home.html). Finally, all the candidate protein sequences were further checked using NCBI’s conserved domain database (http://www.ncbi.nlm.nih.gov/cdd) and the Pfam protein family database (http://pfam.xfam.org). The Sorghum PHT genes were located on the corresponding chromosomes by the MapGene2chromosome web v2 (MG2C) database (http://mg2c.iask.in/mg2c_v2.0/) according to their position information, available on the Phytozome database (Sorghum bicolor v3.1.1).The potential PHT genes from rice, Arabidopsis, maize, and Sorghum were used for phylogenetic analysis. These sequences were downloaded from the rice genome annotation project database, the TAIR database, and the Phytozome database. Thereafter, multiple alignments of the CDS and protein sequences of AtPHTs, OsPHTs, and SbPHTs were carried out using ClustalX v1.83 software. Then, the unrooted phylogenetic trees were constructed using the neighbor-joining method with 1000 bootstrap iterations in MEGA5.05 software.The coding sequences (CDS) and protein sequences of Sorghum PHT family genes were obtained from the Phytozome database (Sorghum bicolor v3.1.1). Then, the intron/exon distribution was determined by using the online gene structure display server program . The conserved motifs in the full-length Sorghum PHT proteins were analyzed using multiple em (expectation maximization) for motif elicitation (MEME) version 5.0.3 (http://meme-suite.org/). The following parameters were applied: 20 motifs in maximum, 6–50 residues (inclusive) in motif width, and E-values <1.00E-40.The mRNA sequences of the http://expasy.org/). The transmembrane helices (TMHs) of the SbPHT proteins were predicted by using three online databases with their default settings, including the TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), the transporter classification database (http://www.tcdb.org/), and the TMbase .The isoelectric points (pIs) and molecular weights (MW) of candidate SbPHTs were detected using the ExPASy proteomics server database (http://cello.life.nctu.edu.tw/), Plant mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/), WoLFPSORT (http://www.genscript.com/wolf-psort.html), and MultiLoc2 (http://abi.inf.uni-tuebingen.de/Services/MultiLoc2). Also, to obtain high confidence predictions, WoLFPSORT, MultiLoc2, and CELLO v 2.5 were set with cutoffs of confidence score >9, confidence score >0.7, and reliability score >4.0, respectively. Similar results were obtained from these databases.In this study, four online databases were used to predict the subcellular localization of each SbPHT protein, including CELLO v2.5 , Sb02g026490 (three), Sb01g014790 (three), Sb02g032440 (12), and Sb03g011370 (two)  family members in proteins . Moreove70 (two) . For thoSorghum PHT proteins into subfamilies, we constructed an unrooted neighbor-joining (NJ) phylogenetic tree by comparing the protein sequences of known PHT proteins from Arabidopsis and rice. In total, we downloaded 26 OsPHT proteins, including 13 OsPHT1s, one OsPHT2, six OsPHT3s, and six OsPHT4s. Also, we obtained 30 AtPHT proteins, including nine AtPHT1s, one AtPHT2, three AtPHT3s, six AtPHT4s, and 11 AtPHTO1s. The locus names of PHT proteins from Arabidopsis and rice are listed in Sorghum. Meanwhile, the SbPHT3 and SbPHT4 subfamilies both have six members, the same as in rice. In addition, the SbPHT2 subfamily has only one member, similar to that in Arabidopsis, rice, and maize.Previous studies have revealed that the plant phosphate transporters include four PHT subfamilies  and PHO . To gainSorghum PHT proteins can be clustered into known subfamilies, indicating that this protein family may confer conserved functions in plants. On the other hand, the number and protein sequence of SbPHTs are more similar to OsPHTs than AtPHTs, suggesting that PHTs from Sorghum have a closer relationship with PHTs from rice as compared with those from Arabidopsis. Considering that Sorghum and rice are both dicotyledons, this observation is in accordance with the current understanding of plant evolutionary history. Based on phylogenetic analysis and their chromosome location, we have divided SbPHT genes into five subfamilies named SbPHT1;1–SbPHT1;12, SbPHT2;1, SbPHT3;1–SbPHT3;6, SbPHT4;1–SbPHT4;6, and SbPHO1;1–SbPHO1;2, respectively  and Sb01g020580 , Sb01g046890  and Sb01g046900 , and Sb06g002540  and Sb06g002560  are located in a series on chromosomes  to 852  amino acids (aa), with an average of 518.9 aa, and their relative molecular mass varies from 34.7  to 97.2  kDa . MeanwhiSbPHT genes, we determined the intron/exon distribution using an online gene structure display server program . Our results showed that the SbPHT1 subfamily genes have no more than one intron; SbPHT2;1 has two introns, the SbPHT3 subfamily genes have five introns, and the SbPHO1 subfamily genes have more than 10 introns  were upregulated more than 2-fold in the leaves after being phosphate-starved for 14 days  has been sequenced [id areas . As a C4id areas . Sorghumequenced , which pPHT genes from the poplar, containing 14 PtPHT1s, two PtPHT2s, six PtPHT3s, eight PtPHT4s, and 12 PtPHT5s [PHT family members from the apple, including 14 MdPHT1s, two MdPHT2s, seven MdPHT3s, 11 MdPHT4s, and three MdPHT5s [StPHT genes could be grouped into eight StPHT1 homologs, one StPHT2 homolog, two StPHT3 homologs, five StPHT4 homologs, and four StPHO homologs [PHT family genes in rice and Arabidopsis, respectively. In this study, we identified 27 putative SbPHT members based on sequence alignments with the Arabidopsis and rice PHT families , which exhibits high similarity to other PHT1s, not only in sequence similarity , the transporter classification database (http://www.tcdb.org/), and the TMbase . For most SbPHT members, the results from the above three databases are similar. However, the TMHMM server predicted that the SbPHT3 members had no TMHs, while the transporter classification database and TMbase predicted that they had 5–8 TMHs (Sorghum.As phosphate transporters, PHT proteins are reported to be membrane-bound ,46. Thus5–8 TMHs . This miPHT family members is inducible or constitutive [The expression of titutive . Our qRTPHT family members in Sorghum; they could be clustered into five typical subfamilies. The protein features in each subfamily are very close, but their expression patterns are quite different.We have identified a total of 27