Retroviridae family, causes a neglected, silent, persistent infection affecting circa 5 to 10 million people around the world, with biology, immune pathology, clinical diseases, epidemiology, and laboratory issues still unsolved. Most of the infected subjects are asymptomatic, but severe clinical disorders appear as a neurodegenerative disease (HTLV-1 associated myelopathy—HAM) or a lymphoprolipherative disorder (Adult T Leukemia/Lymphoma—ATLL) and in other target organs of the human body. HTLV-1 infections are frequently asymptomatic, but there is a large spectrum of diseases that have been described along the years. The mechanisms by which the virus interacts with the host, the different modes of response of the host to the infection, and the immunogenic characteristics of the host are some of the interesting and unanswered questions that may direct the outcome of the disease. The most relevant published results dealing with the genetic variations of the host, the immune response to HTLV-1 infection, and the outcome of the infection are presented herein, including Human Leucocyte Antigen (HLA), Killer Immunoglobulin-like Receptors (KIR), interleukin 6, 10, 28, Fas and Fas ligand, IFN-gamma, TNF-A, and Mannose-binding lectin. In summary, there are still several unmet research needs in the field of useful biomarkers on HTLV-1 pathogenesis.Human T-lymphotropic virus 1, a member of the  Deltaretrovirus, endemic in Japan, Caribbean, South America, Sub-Saharan Africa, and Melanesia [Human T-lymphotropic virus 1 (HTLV-1) is a elanesia ,3.HTLV-1 is associated with several different types of diseases. Adult T-cell leukemia/lymphoma (ATLL) develops in up to 5% of infected people, and sometimes a chronic neurodegenerative inflammatory disease called HTLV-1-associated myelopathy (HAM) develops. Although a high percentage of people remain asymptomatic during their lifetime , a greatHAM is a chronic progressive demyelinating disease that affects the spinal cord and the white matter of the brain, leading to a severe clinical syndrome due to the motor limitations that affect the lower limbs associated with the autonomic dysfunction. The clinical picture begins and evolves insidiously, being difficult to establish when the first symptoms appeared. The main signs and symptoms of HAM may include gait disturbance, weakness, and stiffness of the lower limbs. The first manifestations of the disease occur in the fourth decade of life and there is a 2:1 woman/man ratio ,8. The iTwo important aspects of HAM development have been reported, including the efficiency of the cellular immune response and the rate of spontaneous expression of the viral antigen, due to the numerous immunological alterations, mediated mainly by the expression of Tax (regulatory protein of the virus) in the peripheral blood and in the cerebrospinal fluid of patients with HAM ,14.+ T lymphocytes mediate the main defense strategy of the host immune system [+ T cells, which are the central regulators of the acquired immune response, deregulating the host immune system [+ T cells, sometimes leading to ATLL [In order to control HTLV-1 replication and the proliferation of infected cells, CD8e system ,15. HTLVe system . To esta to ATLL  or to ch to ATLL ,19,20,21The absence of symptoms in most of the infected people raises the question of the multiple etiological roles of the virus-associated diseases and some of the interesting questions still unanswered includes: does the immunogenic characteristics of the host contribute to the upsurge of symptoms associated with HTLV-1 infection? The present manuscript presents the most relevant published results from several laboratories, including ours over the years regarding the associations between the genetic variations of the host related to the immune response and HTLV-1 infection, particularly those showing the close relationship between HTLV-1 infection and single nucleotide polymorphisms in genes of the immune system.+ T lymphocytes play an important role in limiting viral replication in vivo reducing the proviral load and the number of infected CD4+ T lymphocyte clones [The interaction between HTLV-1 and the host immune response appears to play an important role in the outcome of HTLV-1-induced diseases ,23, since clones . Thus, se clones ,25.I facilitates the replication of HTLV-1 and allows the virus to escape the host immune response, preventing the presentation of viral proteins by HLA class I molecules to cytotoxic T lymphocytes; p12I inhibits the expression of HLA-1 molecules on the surface of infected cells [Human leukocyte antigen (HLA) is of utmost importance in inducing the immune response against HTLV-1 and is a critical factor affecting the proviral load of the virus ,27; Tabl. The inted cells .Specific HLA alleles have been related to the protection of HAM development, while others appear to increase the risk for the development of ATLL . HLA-DRB+ T cells in patients with HAM, resulting in a higher viral burden and increased inflammatory response, which in turn indirectly intensifies the damage of the central nervous system [There are different mechanisms for each of the disease outcome. The HLA-DRB1*01 allele, like certain types of HLA class II, seems to present peptides more efficiently to virus-specific CD4s system .In Japan, HLA-A*02 was associated with protection against the development of HAM ,27. A stIn addition, patients with ATLL also presented this allele. Nucleotide sequence analysis indicated that these patients have variations in the tax gene, which generate a stop codon in the half of exon 5 or generate an amino acid change in the critical Tax epitope, which leads to the evasion of the immune response in HLA-A*02 with ATLL . IndividAt this point, it is relevant to stress that some HLA alleles that seem to be protective in one population may not have a similar effect in a different population ,35,36,42+ T lymphocyte immune response. This was in clear contrast to results observed by Tarokhian et al. [Different types of HLA class I were associated with the HBZ protein of HTLV-1, a regulatory protein which promotes T lymphocyte proliferation and is correlated with disease severity . Althougn et al.  that, inNon-classical HLA Class I, for instance, HLA-G, was associated with HTLV-1 infection. The 14 bp polymorphism (deletion/insertion) in the 3’-UTR region (HLA-G 3’-UTR) was not linked to diseases associated to HTLV-1, but it could be a risk factor for susceptibility to HTLV-1 infection . But forAnother important HLA class I activity is its binding to killer immunoglobulin-like receptors (KIR) expressed on the surface of NK cells. The HLA-KIR interaction may promote the inhibition or the activation of NK cell functions . Genes tThe initial approach to infer the influence of KIR on HTLV-1 infection was possible action of specific KIR genes and HLA-Cw groups in the development of HAM. Although there was a trend of association of genes with neuropathology and proviral load, in the final step, no significant associations were found between KIR genes and HLA-Cw groups in people with a clinical diagnosis of HAM .+ T lymphocytes, both of which may exert better control in the development of clinical disease [KIR2DL2 binds to HLA-C molecules with different levels of affinities, and a high affinity binding to the C1 group containing asparagine at residue 80 has been described, as well as links with a low affinity to HLA-C2 molecules when there is a lysine at position 80 . In HTLV disease .+2+, KIR2DL2+C1+, KIR2DL2+C2+, KIR2DL3+C1+, KIR2DL1- or KIR2DL1+C2-, KIR2DL3- or KIR2DL3+C) showed that they all enhance HLA class I associations in HTLV-1 infection. These findings may be related to increased survival of CD8+ T cells in the presence of functional KIRs [The evaluation of the protective effect of HLA-B*57 on different types of KIR  and IFN-γ (+874T/A) gene polymorphisms among asymptomatic and symptomatic HTLV-1-infected subjects, and in control individuals, showed that genotype . The freTNFA −857C/T polymorphism, related to the high transcriptional activity of the gene. The polymorphic genotype was associated with a 2-fold increase in the risk of developing ATLL [TNFA 1031T/C and HTLV-1 was also reported for atherosclerosis-related disease [TNF-α is another significant cytokine in the development of symptoms induced by HTLV-1 infection. Higher TNF-α levels were associated with the  disease .IL10 −592A/C polymorphism was associated with HTLV-1 transcriptional activity mediated by the Tax protein, influencing both HTLV-1 proviral load and a 2-fold reduction on the chances of developing HAM [IL10 −592AA genotype is associated with higher proviral load. However, the investigation of this polymorphism, together with others located in the IL10 gene, showed that IL10 −819*C/T and −592*C/A polymorphisms may contribute to susceptibility to infection by HTLV-1, although it does not appear to be related to the protection against HAM development [A of IL-10 rs1800896 polymorphism to neurological symptoms (over-active bladder) among HTLV-1 patients, suggesting a regulatory role to IL-10.IL-10 can influence HTLV-1 infection through its immunoregulatory functions and inhibit the differentiation process of specific cytotoxic T cells. HTLV-1 infected individuals with low levels of IL-10 may allow inflammatory mechanisms that lead to HAM . The wilping HAM  the IL10elopment . In Brazelopment  reportedIL6 promoter region were associated with the development of HAM [IL6 gene promoter region are related to HTLV-1 infection and disease development. Among several investigated SNPs , IL6 −634G/C allele was described to be a significant predictor for the progression to HAM [IL-6 is an important pro-inflammatory cytokine of acute phase and in the defense against infectious agents . Changest of HAM . Howevern to HAM .IL6 −660G/−635C/−236G and IL10 −6653C/−1116G, independently of maternal provirus load and duration of breast-feeding, suggesting that host’s genetic variations, for both proinflammatory and anti-inflammatory cytokines genes, might be influencing the susceptibility to infection. The relationship of these cytokines to HTLV-1 infection is evidenced in the different activities of the immune response to infection. IL-6 induces the production of IL-2, a cytokine crucial for the activation, growth, and differentiation of cytotoxic T lymphocytes, important in establishing the inflammatory process of HAM. However, IL-10 may inhibit these activities. Therefore, polymorphisms in one of these genes may favor infection and the development of diseases related to infection [Susceptibility to HTLV-1 infection in children was linked to haplotypes nfection TNFA −308G/A, IL6 −174G/C, IFNG +874T/A, TGFB) and HAM patients from Brazil.In contrast, Schor et al. , in a caInterleukin 28B  plays an important role in immune defense against viruses, by inducing an “antiviral state” by intracellular signaling and activation of antiviral host factors in susceptible cells. The role of IL-28B in the context of infectious diseases may be influenced mainly by polymorphisms in the gene that encodes the cytokine .IL28B gene [IL28B expression levels are mainly associated with high levels of Tax protein [+ T lymphocytes leading to marked development of the inflammatory process characteristics, mainly HAM. Another study showed that patients with the CT/TT genotype of rs12979860 polymorphism have a greater proviral load than those with the CC genotype, thus suggesting that the presence of *T allele might be associated with the pathogenesis of infection and disease outcome contributing to the progression to HAM [There are few studies that seek an association of polymorphisms in the IL-28B gene with HTLV-1 infection, but they showed that polymorphisms of this gene are linked with the progression of HTLV-1 infection  and that28B gene . However28B gene . High IL protein  as a conn to HAM .IL28B polymorphisms should be investigated for all HTLV-1-infected subjects in order to monitor their risk for disease development [However, Sanabani et al.  failed telopment .IL28B gene and the development of HTLV-associated arthropathy (HAA), but no differences were observed in the allele and genotype frequencies between symptomatic and asymptomatic HTLV-1 infected people [+ T lymphocyte count and higher proviral load as compared to asymptomatic. Furthermore, high TNF-α and IFN-γ levels serum were observed in HAA patients carrying genotypes CC (rs12979860) and TT (rs8103142). On the other hand, HAA patients with genotype CT/TT (rs12979860) exhibited high IL-10 levels. These results suggest that haplotypes CCT and TTG might be associated with susceptibility to HTLV infection and progression to HAA, respectively.We investigated the association of the single nucleotide polymorphisms  in the d people . Seven hConsidering the role of cytokines in the signaling and activation of mediators of innate and adaptive immune response, it seems important to broaden the analysis of SNPs in genes of pro and anti-inflammatory cytokines in the search to identify possible haplotypes associated with the regulatory and activating functions of the immune response.Apoptosis mechanisms are highly complex and sophisticated, involving a cascade of energy-dependent molecular events . The proBoth pathways culminate in the activation of proteases known as caspases, which are responsible for a cascade of events that lead to the appearance of cellular changes characteristic of all cells in apoptosis, and the action of these proteases culminates in a common effector path independent of stimulant ,87.As death domain-containing member of the TNFR (tumor necrosis factor receptor) superfamily, the Fas (TNFRSF6/CD95/APO-1) and FasL (TNFSF6/CD95LG) molecules are involved in physiological regulation of programmed cell death ,89, beinFAS −670 A > G; rs 1800682) was identified in the FAS gene promoter region [FASLG gene, two of them include a T deletion in position −169 of intron 3  and an A to G substitution at nucleotide position −124 of intron 2  [A single nucleotide polymorphism (SNP) at position −670  ,93,94.FAS −670A/G SNP seems to favor the binding to activator of transcription (STAT1), leading to up and down regulation of the Fas gene expression. Farre et al. [The gene location of e et al.  showed tFAS levels were linked positively with lymphocyte activation among HAM patients, but the increased lymphocyte FAS expression was associated with: (i) decreased apoptosis, (ii) high lymphoproliferation in cell culture, and (iii) a strong increase in cellular Fas expression upon HAM progression .FAS −670 AA genotype was associated with high proviral load as compared to FAS −670 GG in HAM patients [FAS −670AA was more frequent among HAM patients as compared to the asymptomatic individuals [A being associated with high apoptosis activation, as reported by Khouri et al. [FAS −670A/G polymorphism [FAS −670A/G polymorphism seems to be a reliable biomarker associated to HTLV infection and further study involving larger numbers of other families should be conducted to confirm the association observed until now.Recently, ividuals , suggesti et al. , which li et al. . Furthermorphism . In thisGSTM1, GSTT1, and BCL2) were investigated, but no association with ATLL was observed in Japan [Polymorphisms in other genes related to apoptosis (in Japan .Mannose binding lectin (MBL) is an acute phase serum protein that recognizes structures of carbohydrates arranged in a particular geometry, found on the surface of microorganisms. MBL binds to serine proteases (MASPs) forming the complex, which promotes activation of the complement cascade via the lectin route .As previously mentioned, polymorphism in the major histocompatibility complex has shown a possible association with HTLV-1 infection and related pathologies . HoweverOur group investigated the association between MBL2 gene exon1 polymorphisms and the susceptibility to, as well as the outcome of, HTLV-1 and HTLV-2 infections .MBL*A, MBL*B, and MBL*D were similar between seropositive individuals and healthy controls, but genotype differences were statistically significant; where the genotype BB, related to low MBL production, was 9-fold higher among HTLV-infected individuals as compared to controls, suggesting a possible association with susceptibility to HTLV infection. The same difference was observed in genotype distribution between HTLV-1 and HTLV-2 infections, where it was attributed to the higher number of HTLV-1-infected subjects investigated [Regarding HTLV-1/2, the frequencies of alleles stigated .MBL2 gene promoter [Subsequently, we analyzed the frequencies of SNPs at position −550 and −221 of the promoter . No signSubsequently, it was demonstrated that combined genotypes associated with low or deficient production of MBL2  were significantly associated with susceptibility to HTLV-1/2, and more frequently in patients infected with HTLV-1/2 than in the controls. Moreover, the combination of genotypes with high protein production  were significantly associated with protection against HTLV-1. These results showed that the deficiency of the components of innate immunity may play a role in susceptibility to HTLV-1 infection .MBL2 gene has shown its relevance in the anti-HTLV response, especially in association with the proviral load. However, the small number of published articles and small sample sizes emphasizes the need for a broader evaluation, taking into account the ethnic differences of populations infected by HTLV-1 in order to better understand the relationship of the MBL2 gene with the infection.The investigation of the polymorphisms in the HTLV-1 infections are commonly thought to be asymptomatic, but there is a large spectrum of diseases which are currently being described along the years showing that the infection, by itself, cannot be the only cause for the progression towards HTLV-1 associated diseases. So far, there are no clear explanations why some people present a full-blown disease like HAM or ATLL, others develop milder diseases with few symptoms, and some are clearly asymptomatic.HTLV-1 is a genetically stable virus and individuals with the same virus strain have been reported with different clinical outcomes. There is strong evidence that HTLV-1 proviral load is a risk factor for disease progression. Proviral load is directly dependent on the strength of the host cytotoxic T lymphocyte response to HTLV-1, which in turn, is genetically associated with HLA class I type and cytokines signaling. The role of immunogenetic markers of the host is routinely investigated in our laboratory in order to find some explanation for the mechanism of the upsurge of symptoms in some point of the infection. Information regarding the action of polymorphisms and expression of the host genetic characteristics of IFN, TNF, Fas, FasL, IL-6, IL-8, IL-10, IL-28B, and MBL are clear indicators that the immunogenetic profile contributes to the appearance of symptoms as a consequence of HTLV-1 infection.It is extremely relevant to comment that most of the inconsistent results observed from the same biomarker in different human populations could be due to the complexity of epigenetic factors, population stratification effect, the small sample size, ethnic background, covariates, consistency of biomarkers described, and replication in different populations, which can interfere in case-control association studies producing false association results.It is important to stress that we are still trying to merge all the information of the few, but comprehensive, information so far generated by us and other laboratories to understand the synergism of the several biomarkers exploited. Recently, apart from this line of investigation, our laboratory has pointed into the direction to search for new biomarkers and found that the lowest expression of Annexin 1 gene and its serum levels act as a possible new biomarker associated to HAM clinical outcome .In summary, there are still several unmet research needs in the field of useful biomarkers that could help to answer questions such as: who is more easily infected with HTLV-1? Who preferentially becomes sick following infection? Which outcome of clinical disease(s) is linked to which biomarker(s)? In order to fulfill these and other answers, there should be an effort to increase the number of studies in search for genetic biomarkers in different human populations, expanding both the biodiversity of the human groups and the number of subjects investigated in order to confirm or reject the suggestions presented herein. HAM, ATLL, and all the other clinical manifestations of HTLV-1 infection are severe diseases of the poor population of the world, which remains silent and neglected.


Many studies have aimed to clarify the relationship between Notch1 signaling and papillary thyroid carcinoma (PTC), but the results have been inconsistent to date. In the present study, a systematic review and meta-analysis were performed to analyze the relationship between Notch1 signaling and the clinical characteristics of PTC.Literature databases, including PubMed (Medline), Embase and China National Knowledge Infrastructure, were searched for relevant studies from inception to April 2018. A total of five studies, including 421 patients with PTC from China and South Korea, were included in the meta-analysis.The results revealed that the upregulation of Notch1 signaling was positively correlated with lymph node metastasis in patients with PTC . Additionally, positive correlations were found between Notch1 signaling and tumor size , capsular invasion  and clinical stage of PTC .The Notch1 signaling pathway may play a catalytic role in the progression of PTC, and upregulation of Notch1 signaling may have significant predictive value for the clinical prognosis of PTC. Thyroid cancer is one of the most common malignant tumors of the endocrine organ and is the most common malignant head and neck tumor. The morbidity and mortality rates of thyroid cancer remain high among head and neck cancers and are increasing each year. Papillary carcinoma is one of the most common pathological patterns in thyroid cancer, accounting for 60%–80% of cases among Chinese adult patients and 79.3% of cases in the United States . This paWith the advancement of molecular biology, the evidence for the close relationships between tumors and certain genes has grown increasingly abundant. The Notch protein family plays an important role in cell differentiation, organ development and tumor genesis, progression, invasion and metastasis , 4. The Human medullary thyroid carcinoma (MTC) tissue and TT cell strains of MTC could be restrained by the activation of Notch1 signaling by injecting doxycycline into mouse models of MTC, the mouse tumors demonstrated significantly slower growth than the normal control group . AdditioThe present meta-analysis, based on retrieved documents, was conducted to evaluate the correlations between Notch1 signaling and lymphatic metastasis and other clinical features of PTC, which may provide useful information for evidence-based medicine for the prognosis, prophylaxis and treatment of PTC.The research material was collected from published clinical case–control studies, with or without blinding. The language was limited to English and Chinese. Retrieval was conducted via a computer or retrospective database, such as PubMed (Medline), Embase and China National Knowledge Infrastructure (CNKI), from inception to April 2018. The retrieval words were papillary carcinoma of thyroid gland, papillary thyroid carcinoma, Notch1, Notch signaling, lymph node, thyroid cancer, lymphatic metastasis, tumor size, capsular invasion and clinical staging.(1) One-off primary studies related to PTC, Notch signaling, lymphatic metastasis, tumor size, capsular invasion or clinical staging published and unpublished. (2) Patients with PTC staging criteria conforming to the 2015 thyroid cancer National Comprehensive Cancer Network (NCCN) guidelines. (3) The literature had raw data and provided the odds ratio (OR) and 95% confidence interval (CI), or these could be obtained from the data through calculation. (4) Original documents with objectives and complete pathological diagnosis. (5) No history of other malignant tumors.(1) Documents with nonrigorous experiments and incorrect statistical approaches. (2) Repeatedly published thesis or research. (3) No control group. (4) Lacking an objective or complete pathological evidence.We defined high and low Notch1 expression by immunohistochemical staining evaluation, as well as through detailed reading of the full text, contacting the author and other methods of collecting information, and after strict discussion, we excluded articles that cannot be merged, such as those with (1) unqualified primary antibodies; (2) serious interpersonal bias; (3) unscientific positive definitions; (4) grouping methods that could not be merged.Papers were retrieved and screened by reading their titles and abstracts, which was performed by two researchers independently. The abstracts were read to determine whether they met the criteria and eliminate unqualified literature. The information extracted included the name of the first author, year of publication, research design  and patient characteristics . The quality of the selected papers was assessed by another two evaluators based on the Newcastle–Ottawa scale (NOS) . The fin2 value, and only low-heterogeneity (I2 ≤ 25%) and middle-heterogeneity data (25 < I2 ≤ 75%), not high-heterogeneity data (I2 > 75%), were used for the meta-analysis.The meta-analysis was conducted using RevMan5.3 . The OR was used to represent the selected enumeration data, and the 95% CI of each OR was calculated and used to draw a forest map, which was named the dominance ratio map, to demonstrate differences between the results. Heterogeneity was determined by the IAfter the initial screening, 307 documents were retrieved; 212 remained after removing duplicates; and 109 papers were selected through the initial screening after reviewing abstracts and prefaces. By reading through the full text, we eliminated 23 unrelated documents according to our inclusion and exclusion criteria. There were 70 papers without usable data or pathological diagnostic evidence and 11 studies without case controls. Finally, five studies were chosen, including three in English and two in Chinese –24. All In the five studies, the association between Notch1 signaling and sex of patients with PTC was analyzed, and an OR of 1.24 (95% CI 0.69–2.23) was found . These data reveal that there was no significant correlation between Notch1 signaling and sex in PTC patients . There was no significant association between Notch1 signaling and age  , and the 95% CI of the OR fell to the right side of the vertical line in the forest map , which suggested that there was a significant correlation between Notch1 signaling and lymphatic metastasis of PTC. Therefore, Notch1 signal overexpression may accelerate lymphatic metastasis of PTC  . Figure A total of 309 patients from three articles were analyzed to assess the association between Notch1 signaling and capsular invasion of PTC. The OR was 3.49 (95% CI 1.90–6.41), and the 95% CI of this OR fell to the right side of the vertical line in the forest map . These findings demonstrate that upregulation of the Notch1 signaling pathway may contribute to the process of primary tumor capsular invasion in PTC Fig. .Fig. 7AsThe Notch signaling pathway is involved in not only the normal development of the nervous and endocrine systems but also tumorigenesis in multiple tissues, including hematologic, breast, prostate, cervix, lung, liver, and brain malignancies –34. In tAs the most important member of the Notch family, the role of the Notch1 signaling pathway in the development of PTC is intriguing. The TNM system is the most widely adopted system for grading PTC, and large tumor size, lymph node metastasis, capsule invasion, and old age are high-risk clinicopathological characteristics in PTC. Because patients with these high-risk variables are prone to recurrence or metastasis even after operations, they are usually advised to undergo radioiodine remnant ablation (RRA) according to guidelines . TherefoWe pooled five studies and obtained a sample size of 421 cases of PTC in East Asia to yield more robust results. A systematic review and meta-analysis revealed that there was a significant positive correlation between lymphatic metastasis of PTC and activity of the Notch1 signaling pathway. Meanwhile, the trend of the relationship was highly consistent among the included studies, and four studies showed an estimated MHOR point of more than one. Therefore, we speculate that the activation of Notch1 signaling may promote the metastasis of PTC cells to lymph nodes. Grouping analysis was used to further evaluate the correlation between Notch1 signaling and other clinical features of PTC and revealed that Notch1 signaling was closely correlated with tumor size, capsule invasion and clinical stage in patients with PTC. Importantly, almost all the studies included in this meta-analysis suggested that higher Notch1 signaling would lead to poor prognosis in patients with PTC. The relationship trends in this analysis were highly consistent as well, and all the included studies showed a MHOR point estimate of more than one. Some previous studies reported that the downregulation of Notch1 signaling could reduce the proliferation of PTC cells . AnotherAlthough the incidence of PTC was higher among females according to the SEER investigation , we founIn summary, the results of the present study reveal that the Notch1 signaling pathway may play a catalytic role in the progression of PTC, and the activity of Notch1 signaling may be a significant predictive marker for the prognosis of PTC. Therefore, it can be suggested that PTC patients with higher Notch1 signaling activity may tend to have more tumor invasiveness and can benefit from more active treatment (such as RRA).At present, there has been no decisive breakthrough in the study of the Notch1 signaling pathway, but there are many recent studies supporting our view. In the process of tumor invasion, the epithelial–mesenchymal transition (EMT) occurs in some cells, and tumor cells lose cell–cell adhesion. EMT leads to tumor cytoskeletal rearrangement, reduced cell rigidity and cell–cell connectivity, supporting tumor metastasis and invasion. Studies have shown that Notch receptors can be linked to myosin, thereby affecting the mechanical properties and physical ability of cells . In addiSome pathological factors, such as histological subtype, serum thyroglobulin and several other protein levels, are important and may affect the progression of PTC . In the Here, we searched electronic databases for relevant studies, and enrolled 5 studies with a total of 421 patients for meta-analysis, drawing a conclusion that the Notch1 signaling pathway may play a catalytic role in the progression of PTC. Taken together, the results from our meta-analysis suggest that PTC patients with higher Notch1 signaling activity may tend to have more tumor invasiveness. More multi-center prospective cohorts are warranted to further validate the role of the Notch1 signaling in PTC.


Staphylococcus aureus infections place a huge burden on the healthcare sector and the wider community. An increasing rate of infections caused by methicillin‐resistant Staphylococcus aureus (MRSA) has necessitated the development of alternative agents. We previously reported that usnic acid (UA) has activity against MRSA; here, we report the effect of UA in combination with norfloxacin on the drug resistance of MRSA clinical isolates. We observed that the combination of UA–norfloxacin significantly reduces the bacterial burden in mouse models infected with S. aureus, without causing any detectable associated toxicity. Proteomic analysis indicated that UA–norfloxacin induces oxidative stress within cells, which leads to membrane damage and inhibits metabolic activity and biosynthesis of peptidoglycan and fatty acids. Collectively, this study provides evidence that UA in combination with norfloxacin may be a potential candidate for development into a resistance‐modifying agent for the treatment of invasive MRSA infections.Multidrug‐resistant  in vitro and in vivo conditions. These results may be helpful in the development of anti‐MRSA drug combinations from economical and non‐toxic natural products.This is the first report on the drug resistance‐modifying potential of usnic acid (UA) through inhibition of the multidrug resistance (MDR) efflux pump and down‐regulation of proteins involved in peptidoglycan and fatty acid biosynthesis. This compound may be helpful in the management of infection caused by MRSA through (a) lowering the prescribed amount of antibiotics, (b) decreasing MDR generation, and (c) intensifying the efficacy of antibiotics against MRSA/VRSA under both  CFUcolony forming unitsCLSIClinical and Laboratory Standards InstituteCTCp‐tolyl) tetrazolium chloride5‐cyano‐2,3‐di‐, which require urgent attention to prevent the spread of infections Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) Staphylococcus aureus, a Gram‐positive bacterium, is a ubiquitous member of the human microbiota and was recognized as a major pathogen in the late 19th century. It is the causative agent for a wide range of infections including skin infections, sepsis, toxic shock syndrome, endocarditis and osteomyelitis Staphylococcus aureus (VRSA) and multidrug resistance (MDR) strains which have acquired resistance to agents of last resort including vancomycin and linezolid. This scenario presents an urgent need to develop alternative approaches to combat such infections, which include modulating drug plasma concentration within the therapeutic window The infections caused by multidrug‐resistant pathogens pose a serious global public health challenge. The Centers for Disease Control and Prevention (CDC) has outlined the top 18 drug‐resistant threats, including methicillin‐resistant Plant‐derived natural products have gained global attention as novel antimicrobial compounds due to their being recognized as safe and their use in traditional medicine systems Usnea subfloridana led to the identification of usnic acid (UA), which has potent antibacterial activity against multidrug‐resistant clinical isolates of S. aureus with membrane damaging potential in vitro and in vivo assays. A gel‐based proteomic approach was further employed to understand the possible mechanism of synergy.The bioactivity‐guided fraction of 8 colony forming units (CFU)·mL−1.The details of clinical isolates used in the present study are presented in Table S. aureus were checked for the presence of the mecA gene, a marker for MRSA that impart resistance to β‐lactam antibiotics using PCR All the eight clinical isolates of The fractional inhibitory concentration (FIC) of UA with different antibiotics was quantified through a modified broth checkerboard method S. aureus. First, the MIC of UA alone and in combination with norfloxacin was determined in MTCC‐96 as per the broth microdilution assay, in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Following this, serial passaging was carried out by transferring the bacterial cells growing at sub‐MIC of the compound/antibiotic and subjecting them to another MIC assay. After 24 h of incubation, cells at sub‐MIC of the test compound/antibiotic were further transferred and assayed for MIC determination. The process was carried out for 20 passages and the fold increase in MIC was plotted against the number of passages to evaluate the propensity of the bacteria to develop resistance. The post‐antibiotic effect of UA alone and in combination with norfloxacin against the clinical isolate MRSA‐2071 was measured using a previously described method The propensity of bacteria to develop resistance and the mutation prevention concentration of the UA and norfloxacin combination was evaluated following methodology describe earlier 10 decrease in CFU count at a specified time while a decline of less than 3‐log10 CFU·mL−1 was interpreted as bacteriostatic.To assess the bactericidal potential of UA and norfloxacin against MRSA‐2071, a time–kill kinetic study was performed at different concentrations alone and in combination as per the CLSI methods n = 5 per group) were infected with 106 CFU·mL−1 of S. aureus (MTCC‐96) through an intravenous route to evaluate therapeutic efficacy. The treatment groups comprised combinations  along with norfloxacin alone  and UA alone . Oral administration of treatments once daily was initiated 16 h after infection and continued till day 7. Blood was collected from retro‐orbital plexus 24 h after the last dose for toxicity assessment and spleen to check the bacterial load −1 tissue and presented as the mean ± SEM. The reduction in the bacterial load within the tissues was assessed by comparing the load in the treatment groups with respect to infection control.Swiss Albino mice  efflux was performed as per the earlier reported method The effect of the UA–norfloxacin combination on membrane permeability in MRSA‐2071 was assessed following propidium iodide (PI) uptake method described earlier and the fluorescence was measured at excitation and emission of 544 nm and 620 nm, respectively 3‐5, according to the earlier reported method D600 = 0.6) were collected and washed once with buffer  and resuspended in same buffer to achieve the D600 = 0.05. The cell suspension was incubated with 0.4 μm diSC3‐5 until dye uptake was maximal (approximately 90%), after which 100 mm KCl was added to equilibrate the cytoplasmic and external K+ concentration. Staphylococcus aureus cells (D600 = 0.3) were treated with phytomolecules and carbonyl cyanide m‐chlorophenylhydrazone as a positive control at 37 °C. Culture without treatment served as a negative control. The fluorescence reading was monitored with a microplate reader (FLUOStar Omega) at an excitation wavelength of 622 nm and an emission wavelength of 670 nm. The blank with only cells and the dye was used to deduct the background. The experiments were performed thrice and data expressed as mean values ± SEM .Membrane depolarization was studied in MRSA‐2071 using membrane‐potential‐sensitive cyanine dye diSCht analyzer 2d  software for comparative analysis. Spots present in all the gels were considered for detailed multivariate analysis and differentially expressed spots were excised and stored at 4 °C for subsequent mass spectrometric analysis.The mode of anti‐staphylococcal activity of the UA–norfloxacin combination was determined through a gel‐based proteomic approach. Protein extraction was carried out from bacterial cells (MRSA‐2071) exposed to sub‐lethal concentrations of UA, norfloxacin and UA–norfloxacin combination using a TRIzol extraction protocol biotools 3.0 software . The TOF spectra were recorded in positive ion reflector mode with a mass range from 700 to 3500 Da. Only proteins matched by a minimum of two unique peptide sequences were included in the results list. Sequence coverage was also considered to evaluate protein identification.In‐gel digestion of the excised spots was performed following the earlier described protocol https://david.ncifcrf.gov/) S. aureus using default settings https://string-db.org/) with default parameters and S. aureus subsp. aureus as the organism of interest The differentially expressed proteins identified in two‐dimensional electrophoresis were subjected to DAVID  database version 6.7  as per the method described by Wang and Joseph The analysis of intracellular levels of reactive oxygen species (ROS) was carried out using the ROS‐sensitive probe CM‐HP < 0.01 vs control: Dunnett's test).The nitrite level within the MRSA‐2071 cells exposed to UA and norfloxacin alone and in combination was quantified by spontaneous oxidation of nitric oxide (NO) −1, was used and the fluorescence intensity was measured at 590 nm for 30 min at 15 s intervals in a microplate reader (FLUOStar Omega). The experiment was performed in triplicate and data expressed are mean values ± SEM (**P < 0.01 vs control: Dunnett's test).A resazurin assay was performed in MRSA‐2071 cells exposed to UA and norfloxacin alone and in combination at different concentrations to check the cell viability and metabolic activity p‐tolyl) tetrazolium chloride (CTC) was performed as described previously m was added, followed by fixing the cells with 2.8% formaldehyde and 0.04% glutaraldehyde and counterstaining with 4′6‐diamidino‐2‐phenylindole. A negative control was prepared by disrupting the membrane prior to addition of CTC. Further, the samples were analyzed through flow cytometer using the FL‐1 channel  and cell quest 3.3 analysis software .Respiratory activity assay using 5‐cyano‐2,3‐di‐ followed by sonication, and cell debris was removed by centrifugation at 11 269 g for 10 min at 4 °C. Following the estimation of proteins using the Bradford assay, aliquots of protein (80 μg) were loaded on 12.5% SDS/PAGE gels and blot transferred onto polyvinylidene difluoride membrane. After blocking with 5% skimmed milk, MurA was probed with anti‐MurA antibody at 1 : 2000 dilution, followed by incubation with secondary alkaline phosphatase conjugated goat anti‐rabbit antibody at 1 : 5000 dilution. The blot was further developed using nitro blue tetrazolium/5‐bromo‐4‐chloro‐3‐indolyl‐phosphate substrate with glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) as an endogenous control.The overnight grown culture of MRSA‐2071 in MHB at 37 °C with agitation was diluted in MHB and again grown at 37 °C to a The qRT‐PCR analysis of identified genes was analyzed in MRSA‐2071 cells in the presence of UA and norfloxacin alone and in combination. The list of all the primers is presented in Table The protocols (CIMAP/IAEC/2016‐19/06 and CIMAP/IAEC/2016‐19/01) were duly approved by Institutional Animal Ethics Committee on 29 February 2016.P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001, and P > 0.05 was considered to be non‐significant.The data were subjected to a one‐way ANOVA to analyze the mean values obtained for the treatment and control. Dunnett's test was used to compare the treatment and control and statistical significance was set at *Staphylococcus aureus/ Vancomycin Resistant Staphylococcus aureus (VISA/VRSA) and MDR S. aureus, urging the need to identify new agents. Plant secondary metabolites have displayed a potential as antibacterial agents individually and are known to potentiate the activity of other antibiotics through different mechanisms and thus they could be employed for the treatment of resistant pathogens S. aureus (MRSA), which were found to be multidrug resistant and harbor mecA gene (Table S. aureus (MRSA‐2071) were not able to develop resistance against UA alone as well as in combination with norfloxacin even after 20 subsequent passages. A 2‐ to 4‐fold increase in the MIC of UA alone was observed after 12–20 subsequent passages. However, in the case of norfloxacin alone, more than 900‐fold increase in MIC was observed during the same period. The prolongation of the post‐antibiotic effect of norfloxacin in the presence of UA represents a continued suppression of growth after a short exposure to the antimicrobial agents. Also, UA not only increased the inherent susceptibility of S. aureus to norfloxacin, but also simultaneously reduced the emergence of norfloxacin resistance .Synergy and the resistance‐modifying potential of UA with clinically used antibiotics were investigated and are presented in terms of fold reduction and FICI Table . In combin vivo efficacy of the UA–norfloxacin combination  of UA in combination with ED25 (4 mg·kg−1) of norfloxacin could reduce the bacterial load up to 50% (P < 0.01), whereas up to 80% (P < 0.001) reduction was recorded when the UA–norfloxacin combination was administered at 50% of the effective dose . Toxicity studies were also performed to evaluate the effect of the UA–norfloxacin combination on healthy animals and animals infected with MTCC‐96. No lethal effects were recorded post‐treatment with the combination in the biochemical parameters compared to vehicle control, with and without infection  indicated the presence of around 1200 spots among which 34 spots exhibited differential expression (P ≤ 0.05). The gel image with identified proteins is presented in Fig. N‐acetylglucosamine 1‐carboxyvinyltransferase (MurA), which catalyzes the first step in peptidoglycan biosynthesis. In addition, four proteins which were uniquely identified in the cells exposed to the UA–norfloxacin combination at sub‐inhibitory concentrations were mainly involved in transcriptional regulation, namely DEAD‐box ATP‐dependent RNA helicase CshA, transcriptional regulatory protein WalR, elongation factor Tu and UPF0355 protein MRSA252. Moreover, sequence level in‐depth analysis of the uncharacterized protein UPF0355 identified uniquely in the combination‐treated cells displayed a putative role in modulating the operon regulating the downstream expression of genes involved in oxidative stress responses. Another remarkable observation was a 0.95‐fold down‐regulation of d‐lactate dehydrogenase, encoded by ldhD, which has been implicated in imparting vancomycin resistance in S. aureus by incorporating d‐lactate at the terminal residue of peptidoglycan precursors fabH which may lead to inhibition of fatty acid biosynthesis.Multivariate analysis between the proteome of MRSA‐2071 control and the UA–norfloxacin combination  with UA and norfloxacin.Table S4. List of primers used in the present study.Fig. S1. PCR amplification of mecA gene from the clinical isolates of S. aureus. Fig. S2. (a) PAE of norfloxacin alone as well as in combination with UA against clinical isolate MRSA‐2071. (b) Propensity of development of resistance of S. aureus against UA and norfloxacin combination compared to norfloxacin alone.Fig. S3. Time kill kinetics of MRSA‐2071 at different concentrations of UA and norfloxacin alone as well as in combination. These data represent mean ± SEM of three independent experiments.Fig. S4. (a) Protein expression profiles of soluble proteins from MRSA‐2071 cells exposed to sub‐lethal concentrations of UA and norfloxacin alone as well as in combination. Proteins were resolved on 17 cm pH 4–7 IPG strips and 12.5% 20 cm SDS/PAGE gels. . (b) Temporal proteome changes of MRSA‐2071 under UA–norfloxacin treatment identified using classical two‐dimensional electrophoresis. Proteins were separated in first dimension isoelectric focussing on 17 cm IPG strips of pH 4–7 range followed by second dimension separation on 12.5% SDS/PAGE. Multivariate analysis was performed to identify the differentially expressing protein spots in response to UA–norfloxacin treatment compared to untreated control and UA and norfloxacin alone, all at sub‐inhibitory concentrations (0.5 MIC). Green arrows indicate up‐regulation, red indicate down‐regulation, blue indicate unique to UA exposed cells, brown indicate unique in Nor exposed cells and yellow indicate proteins unique in cells exposed to UA–norfloxacin combination.Fig. S5. Functional clustering and biological pathways using DAVID and PANTHER tools associated with the differentially expressed proteins identified in UA–norfloxacin treated MRSA‐2071 cells. Pie chart showing the protein classes (A), biological processes (B) and molecular functions (C).Fig. S6. Protein–protein interaction network of differentially expressed proteins. The differentially expressed proteins in MRSA‐2071 cells in response to UA–norfloxacin combination were searched for their protein–protein interactions using the web resource STRING v.10.0 (http://www.string-db.org/). Protein–protein interactions are shown in evidence view and proteins are linked based on neighborhood, gene fusion, co‐occurrence, co‐expression, experimental evidences, existing databases and text mining. Network analysis was set at high stringency (STRING score ≥ 0.7).Fig. S7. Time kill assay of different MRSA strains alone as well as in combination in three different clinical isolates namely MRSA‐1745, MRSA‐4627 and MRSA‐5944. These data represent mean ± SEM of three independent experiments.Fig. S8. Efflux pump modulatory potential of UA in clinical isolates MRSA‐1745, MRSA‐4627 and MRSA‐5944 using EtBr as marker, determined through spectrofluorometric analysis. Reserpine, an efflux pump inhibitor, was used as positive control in the experiment.Fig. S9. Prospecting membrane disruptive property of UA–norfloxacin combination against clinical isolates MRSA‐1745, MRSA‐4627 and MRSA‐5944 using PI through spectrofluorimetry. These data represent mean ± SEM of three independent experiments .Fig. S10. Membrane depolarization assay of UA–norfloxacin combination using diSC3‐5 against clinical isolates MRSA‐1745, MRSA‐4627 and MRSA‐5944 via spectrofluorimetry. These data represent mean ± SEM of three independent experiments .Click here for additional data file.


The highly pathogenic avian influenza virus H5N1 is a major threat to global public health and therefore a high-priority target of current vaccine development. The receptor-binding site (RBS) on the globular head of hemagglutinin (HA) in the viral envelope is one of the major target sites for antibody recognition against H5N1 and other influenza viruses. Here, we report the identification and characterization of a pair of human RBS–specific antibodies, designated FLD21.140 and AVFluIgG03, that are mutually complementary in their neutralizing activities against a diverse panel of H5N1 viruses. Crystallographic analysis and site-directed mutagenesis revealed that the two antibodies share a similar RBS-binding mode, and their individual specificities are governed by residues at positions 133a, 144, and 145. Specifically, FLD21.140 preferred Leu-133a/Lys-144/Ser-145, whereas AVFluIgG03 favored Ser-133a/Thr-144/Pro-145 residue triplets, both of which perfectly matched the most prevalent residues in viruses from epidemic-originating regions. Of note, according to an analysis of 3758 H5 HA sequences available in the Influenza Virus Database at the National Center for Biotechnology Information, the residues Leu-133a/Ser-133a and Ser-145/Pro-145 constituted more than 87.6 and 99.3% of all residues at these two positions, respectively. Taken together, our results provide a structural understanding for the neutralizing complementarity of these two antibodies and improve our understanding of the RBS-specific antibody response against H5N1 infection in humans. Chin–17–21–24–28–8–In influenza infection and vaccination, neutralizing antibody responses against HA play a critical role in protecting humans from infection and disease progression –37. Thro–in vivo (Table S1). Of the 18 diverse H5N1 strains, those sensitive to FLD21.140 were less sensitive or virtually resistant to AVFluIgG03 and vice versa. For example, FLD21.140 strongly neutralized strains from clades 0, 1, 2.2.1, 2.3.2.1, 2.4, 2.5, 3, 4, and 8, whereas AVFluIgG03 only neutralized those from the remaining clades 2.1.3.2, 2.3.4, 5, 6, 7, 7.1, and 9 . By contrast, AVFluIgG03 strongly bound the HA head of strains from clades 2.3.4 and 7 but weakly to those from clades 1 and 2.2.1 . Such dichotomy in their neutralizing and binding specificities was likely due to the distinct antibody response to the different infecting viruses in patients from whom the two antibodies were initially isolated. Indeed, FLD21.140 was isolated from the Vietnamese patient CL115, who was infected with the clade 1 strain A/Vietnam/CL115/2005, whereas AVFluIgG03 was from the Chinese patient AH06 infected with the clade 2.3.4 strain A/Anhui/1/2005 , and we compared it with the structure of AVFluIgG03 we published previously (C). FLD21.140 covered a total surface of 753 Å2 on the globular head, of which 654 Å2 were by the heavy chain and 112 Å2 were by the light chain. Similarly to several published antibodies such as AVFluIgG03 (42–D and E). Specifically, the hydrophobic residues Leu-103 and Leu-104 of FLD21.140, respectively, interacted with Val-135 and Ser-137 in the 130-loop of RBS through hydrophobic interactions and hydrogen bonds. The backbone carbonyl oxygen of Leu-104 in FLD21.140 formed two hydrogen bonds with Ser-137, one through the side chain hydroxyl oxygen and the other through the backbone amide nitrogen. Another hydrophobic residue, Pro-106, and the aromatic residue Tyr-107 of FLD21.140 interacted with HA residues Trp-153 and Leu-104 through hydrophobic and van der Waals interactions, respectively. Furthermore, residue Pro-106 inserted its side chain into the hydrophobic receptor-binding pocket surrounded by residues Trp-153 and Leu-104 in the 190-helix of RBS . Both residues were highly conserved, being present in 99.9 and 95.7% of 3758 influenza H5 HA sequences currently available in the Influenza Virus Database at the National Center for Biotechnology Information, respectively (Table S4). As shown previously . Furthermore, analogously to AVFluIgG03, FLD21.140 also used the HCDR1, HCDR2, HCDR3, LCDR3, and FR3 regions to interact with additional residues within and surrounding the RBS (A and B). For example, the HCDR1 loop contacted residues Ser-133 and Leu-133a in the 130-loop. The HCDR2 loop contacted residues Glu-131, Ser-133, and Leu-133a in the 130-loop, Lys-156, Asn-158, and Ser-159 in the 150-loop, and Lys-193 in the 190-helix. In addition to the specific interactions mentioned above, the HCDR3 loop also contacted residues Leu-133a and Ser-136 in the 130-loop, Lys-144 and Ser-145 in the 140-loop, Ile-155 in the 150-loop, as well as Glu-190 and Lys-193 in the 190-helix (C and D). The complex was further stabilized by these contacts.To study the structural basis for the abovementioned dichotomy between FLD21.140 and AVFluIgG03, we determined the crystal structure of FLD21.140 Fab bound to the globular head of A/Thailand/1(KAN-1)/2004 at a resolution of 2.33 Å  and their prevalence among 18 diverse H5N1 strains initially used for characterization of the two antibodies . Taken together, these results indicate that the residues at positions 133a, 144, and 145 play a critical role in determining the specificity of FLD21.140 and AVFluIgG03, whereas those at the remaining positions of the epitope were recognized by both antibodies with similar activity.To identify critical residues that differentiate the specificity and complementarity of FLD21.140 and AVFluIgG03, we generated a total of 52 single, 6 double, and 2 triple mutant HA envelope sequences on the backbone of clade 1 A/Vietnam/1203/2004 and clade 2.3.4 A/Anhui/1/2005 . The twotibodies . Pseudovtibodies . SimilarTable S4), we identified the degree of sequence variation at positions 133a, 144, and 145. Although dominated by two major residues at positions 133a (45% Leu and 43% Ser) and 145 (74% Ser and 26% Pro), significant levels of polymorphism were found at the position 144  (Table S4). Pseudoviruses bearing each polymorphic residue were then generated in the background of A/Vietnam/1203/2004 and A/Anhui/1/2005, confirmed by sequencing, and then subjected to sensitivity analysis to FLD21.140 and AVFluIgG03 neutralization. Of the 12 A/Vietnam/1203/2004 envelope clones tested, six  conferred more than 10-fold resistance to FLD21.140, compared with the original WT envelope . However, replacing the leucine with serine, such as in the A/Anhui/1/2005, introduced a hydrophilic residue, which would disrupt the hydrophobic interactions around this position . By contrast, for AVFluIgG03 recognition of A/Anhui/1/2005, the S133aL mutation introduced a larger side chain, which might lead to steric clashes between HA and AVFluIgG03 . The Leu-133a and Ser-133a residues were therefore favored by FLD21.140 and AVFluIgG03 in their respective recognition. Furthermore, the residue Lys-144 on the HA globular head of A/Thailand/1(KAN-1)/2004 formed a salt bridge and a hydrogen bond with Glu-101 and Asn-102 of FLD21.140 HCDR3, respectively . The K144T mutation introduced a short and uncharged side chain, thereby disfavoring the antibody binding at this position . Conversely, although AVFluIgG03 recognized Thr-144 on the globular head of A/Anhui/1/2005, the T144K mutation would introduce a long side chain that was disfavored by AVFluIgG03 . Lys-144 and Thr-144 were therefore distinctly favored by FLD21.140 and AVFluIgG03, respectively. As for the residue at position 145, Ser-145 on the HA globular head of A/Thailand/1(KAN-1)/2004 was required for the formation of a hydrogen bond with the main chain oxygen atom of Leu-103 in FLD21.140 . Replacement of Ser-145 with Pro-145 from A/Anhui/1/2005 would disrupt the hydrogen bond and disfavor recognition by FLD21.140. By contrast, Pro-145 on the HA globular head of A/Anhui/1/2005 was required for the hydrophobic interaction with Leu-111 of AVFluIgG03 . Thus, the P145S mutation, while in favor of FLD21.140 recognition, would also disfavor AVFluIgG03 recognition. Taken together, these results highlighted the unique structural basis for the roles of the residues at positions 133a, 144, and 145 in the differential recognition by the two antibodies. Because only the residue 133a was located within the RBS, the distinct recognition between FLD21.140 and AVFluIgG03 was determined by residues both within and outside of the RBS.To investigate how the critical residues 133a, 144, and 145 determined the differences of antibody recognition, we conducted a more in-depth structural analysis of their interactions with FLD21.140 and AVFluIgG03 . In the Fig. 4in vivo and guide the future design of more effective vaccines against influenza virus infection. Over the last few years, while studying a panel of human monoclonal antibodies isolated from H5N1-infected and convalescent patients, we identified the two antibodies FLD21.140 and AVFluIgG03 that are mutually complementary in their binding and neutralizing activities against a diverse panel of H5N1 strains. In this study, we used structural and mutational studies, which uncovered that both FLD21.140 and AVFluIgG03 recognize the RBS through insertion of their HCDR3 region in a manner that directly competes with the sialic acid receptor. Such a mode of recognition is rather common, and only one exception has so far been reported, where the HCDR2 rather than the HCDR3 region mediated the recognition , in which the recognition is mediated through hydrophobic interactions in the conserved hydrophobic cavity. Other antibodies, such as H5.3, 5J8, 641 I-9, CH65, and F045-092, rely on receptor mimicry in which an aspartic acid side chain on the tip of HCDR3 forms contacts similar to those made by the carboxylic acid of the receptor . However, it should be noted that the potency and breadth of these antibodies varies despite their shared specificity and mechanism of recognition against RBS. This is largely dependent on the degree of residue variation in their recognition sites. For instance, antibodies with broader and cross-clade activity such as C05, S139/1, and F045-092 tend to recognize more conserved residues  using the Bac-to-Bac baculovirus expression system (Invitrogen). The genes encoding the globular head were cloned into the pFastBac-Dual vector (Invitrogen) with an N-terminal gp67 signal peptide for secretion and a C-terminal His6 tag for purification. Specifically, the plasmids were first introduced into DH10Bac competent cells, and the bacmids were used to transfect Sf9 insect cells using Cellfectin II reagent (Invitrogen). The recombinant baculovirus virions were harvested after 7 days. After one round of amplification, the high-titer virions were used to infect Sf9 insect cells at a density of 2 × 106 per ml. After 60 h of infection, the secreted globular head was harvested, concentrated, and buffer-exchanged to HBS buffer . The globular head was captured on nickel-nitrilotriacetic acid–agarose beads , eluted with 500 mm imidazole in HBS buffer, and then purified through gel-filtration chromatography using a Superdex 200 column . The H5N1 HA mutated globular heads of A/Vietnam/1203/2004 bearing L133aS/K144T, L133aS/S145P, K144T/S145P, and L133aS/K144T/S145P mutations, as well as of A/Anhui/1/2005 bearing S133aL/T144K, S133aL/P145S, T144K/P145S, and S133aL/T144K/P145S mutations were produced and purified in the same way as the WT globular head.The H5N1 (A/Thailand/1(KAN-1)/2004, A/Vietnam/1203/2004, A/Turkey/65596/2006, A/Anhui/1/2005, A/Beijing/01/2003) HA globular head proteins (residues Asp-55–Glu-271) were expressed in Sf9 insect cells (m H2SO4. The absorbance at 450 nm was measured using a ELISA plate reader (Bio-Rad).Reactivity of human antibodies (FLD21.140 and AVFluIgG03) against the five tested HA globular head (Asp-55–Glu-271) antigens was determined by ELISA. Briefly, 100 μl of purified HA globular heads (1 μg/ml) were used to coat 96-well ELISA plates  at 4 °C overnight. The plates were blocked with 10% bovine serum albumin or fetal bovine serum for 2h at 37 °C and then washed with PBS, 0.05% Tween 20 (PBST) three times. The tested human antibodies (diluted to 10 μg/ml) were added into the wells and incubated at 37 °C for 1 h, followed by washing with PBST. The antibodies were serially diluted 3-fold. Bound antibodies were detected with HRP-conjugated goat anti-human IgG (Promega) at 37 °C for 1 h, followed by washing. The reaction was visualized by addition of 100 μl of 3,3′,5,5′-tetramethylbenzidine substrate , and stopped by the addition of 50 μl 0.5 m sodium acetate buffer, pH 5.0. Both antibodies were cleaved to generate antigen-binding fragments (Fabs) by incubating with protease Lys-C (Sigma) at an IgG/Lys-C ratio of 4000:1 (w/w) in 10 mm EDTA, 100 mm Tris/HCl, pH 8.5, at 37 °C for 12 h. SPR experiments were run at a flow rate of 30 μl/min in HBST buffer . The surface was regenerated with 10 mm NaOH. The sensorgrams were fit with a 1:1 binding model using BIA Evaluation software .The binding kinetics and affinity of FLD21.140 and AVFluIgG03 to purified HA globular heads (Asp-55–Glu-271) were analyzed by SPR . The purified soluble globular head was covalently immobilized to a CM5 sensor chip via amine groups using the amine coupling kit (Biacore) in 10 m3–104 cells per well; obtained from ATCC) were then added to each antibody/virus mixture. The relative luciferase activity was measured after 48 h using the BrightGlo luciferase assay (Promega), according to the manufacturer's instructions. Titration curves were generated using sigmoid dose response with nonlinear fit in GraphPad Prism software version 5, and inhibitory concentration (IC50) values were determined in the region in which a 50% reduction of relative luciferase activity was identified.The pseudovirus-based neutralization assay was conducted as described previously , 64. BriThe expression plasmids encoding the genes of WT and mutant HAs were used to transiently transfect HEK293T cells. Approximately 40 h later, the transfected cells were harvested, trypsinized, fixed with 4% paraformaldehyde, stained with FLD21.140 and AVFluIgG03 antibodies at a concentration of 10 μg/ml, and detected using an anti-human IgG-Alexa Fluor 488 secondary antibody (Santa Cruz Biotechnology) using a FACSCalibur flow cytometer (BD Biosciences).m (NH4)2SO4 and 0.1 m BisTris, pH 5.5. All crystals were cryoprotected by soaking in reservoir solution with 20% v/v glycerol for several seconds and then frozen in liquid nitrogen before data collection. Diffraction data were collected on the BL17U beamline at the Shanghai Synchrotron Research Facility (SSRF)  at 37 °C for 12 h, and the digested Fab and Fc fragments were separated by loading the mixture onto a protein A column. The Fab fragment was collected in the flow-through, and the Fc fragment was captured on the protein A beads. The flow-through was further purified by gel filtration . The purified globular heads (A/Thailand/1(KAN-1)/2004) and FLD21.140 Fab were mixed at a molar ratio of 1:1, incubated at 4 °C for 1 h, and then purified by gel-filtration chromatography on a Superdex 200 column . For crystallization, the globular head–FLD21.140 Fab complex was concentrated to 20 mg/ml in HBS buffer. Crystals were successfully grown at 18 °C using the vapor diffusion method in sitting drops, which consisted of equal volumes of protein and reservoir solution containing 2.0 y (SSRF)  and procy (SSRF) . All dat5DUT) and structures of the variable and constant domains of heavy and light chains available in the Protein Data Bank with the highest sequence identities. Iterative refinements using PHENIX and model building with COOT were performed to complete the structural refinement  were loaded onto protein A sensors and incubated with varying concentrations of WT and mutant globular heads in the same buffer. All the assays were carried out with agitation set to 1000 rpm at 25 °C. The experiments included six steps: 1) baseline (60 s); 2) IgG loading onto the protein A sensors for 120–200 s; the threshold of capture levels was set to 1 or 3 nm; 3) second baseline (120 s); 4) association of the globular head for the measurement of kon (60–300 s); 5) dissociation of the globular head for the measurement of koff (60–300 s); 6) regeneration and neutralization. The protein A sensors were regenerated for 5 s in regeneration buffer  and then neutralized for 5 s in kinetics buffer. This was repeated three times. Five concentrations of heads were used, including the highest and lowest concentrations. Data processing and analysis were performed using Octet analysis software version 9.0 . Experimental data were fitted using a 1:1 binding model for all assays. All sensorgrams used for fitting of kon and koff as well as the steady-state analysis curves are shown in Fig. S3.The binding kinetics of FLD21.140 and AVFluIgG03 IgGs to the WT and mutant globular heads were measured via bio-layer interferometry using an Octet Red96 instrument . The FLD21.140 and AVFluIgG03 IgGs diluted at 10–20 μg/ml in kinetics buffer (10 mY. Z., P. W., T. Z., X. W., and L. Z. conceptualization; Y. Z., P. W., S. G., and X. W. data curation; Y. Z., P. W., and S. G. software; Y. Z., P. W., S. G., and L. Z. formal analysis; Y. Z., P. W., X. W., and L. Z. validation; Y. Z., P. W., J. S., and G. W. investigation; Y. Z., P. W., X. W., and L. Z. visualization; Y. Z., P. W., J. S., S. G., G. W., and S. F. methodology; Y. Z., P. W., X. W., and L. Z. writing-original draft; Y. Z., P. W., X. W., and L. Z. writing-review and editing; P. Z. and M. L. resources; X. S. project administration; X. W. and L. Z. supervision; X. W. and L. Z. funding acquisition.


TRPM7 and its homologue TRPM6 have been implicated in a variety of cellular functions and is critically associated with intracellular signaling, including receptor tyrosine kinase (RTK)-mediated pathways. Emerging evidence indicates that growth factors, such as EGF and VEGF, signal through their RTKs, which regulate activity of TRPM6 and TRPM7. TRPM6 is primarily an epithelial-associated channel, while TRPM7 is more ubiquitous. In this review we focus on TRPM7 and its association with growth factors, RTKs, and downstream kinase signaling. We also highlight how interplay between TRPM7, Mg2+ and signaling kinases influences cell function in physiological and pathological conditions, such as cancer and preeclampsia.The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed chanzyme that possesses an ion channel permeable to the divalent cations Mg T,5. T2+, nditions ,11,12,13lar Mg2+ . HoweverTRPM6 and TRPM7 have been linked to many signaling pathways. Emerging evidence indicates an important role for these systems in growth factor signaling through receptor tyrosine kinases (RTK). The C-terminal α-kinase domain induces phosphorylation of many downstream targets involved in RTK signaling including annexin A1, calpain II, myosin II, PLCγ2, Src, and SMAD2 , and reg2+ transport across cell membranes, TRPM7, together with other Ca2+ channels, regulates Ca2+ influx in many cell types i. TRPM7 is regulated by vasoactive factors, such as AngII, aldosterone, endothelin-1 and bradykinin [2+]i and attenuated Ang II-mediated VSMC growth i conditions, another Mg2+ binds to α and γ-phosphates and to the asparagine amide nitrogen within the catalytic loop i is low, PLC activation is associated with TRPM7 inhibition, whereas in normal [Mg2+]i conditions, activation of PLC increases TRPM7 currents [2+-dependent activation of ERK, since ERK inhibition reduced TRPM7 expression [2+ homeostasis and cell migration and invasion [RTKs induce phosphorylation of downstream kinases that influence TRPM7 activity . Howevercurrents . In prospression . Ang II,pression . Bradykiinvasion . The infinvasion . In HEK2invasion . While kinases regulate TRPM7, these kinases themselves may be targets of TRPM7. MAP kinases are regulated by TRPM7. In human endothelial cells, downregulation of TRPM7 using a siRNA approach was associated with increased ERK1/2 phosphorylation with no effects on p38MAPK or JNK activation . On the A regulatory role of TRPM7 in MAPK signaling has also been observed using pharmacological approaches in cancer tissue and cell lines. In glioblastoma cells, naltriben, a pharmacological activator of TRPM7 channel, enhanced ERK1/2 phosphorylation and proliferation, with no effect on the PI3K/AKT pathway (150). Carvacrol, a non-specific inhibitor of TRPM7, suppressed phosphorylation of ERK1/2 and AKT, reducing cell proliferation ,134,135.2+-induced increase in mRNA expression of chemotaxis-related genes was attenuated by TRPM7 siRNA and PI3K inhibition, suggesting a link between TRPM7 and PI3K activation mediated by Mg2+.The PI3K/AKT pathway is also affected by TRPM7. In mouse chondrocytes, overexpression of TRPM7 stimulated the AKT pathway, while TRPM7 silencing inhibited AKT signaling . In humaSTAT3 is also modulated by TRPM7. TRPM7 siRNA reduced activation of STAT3 (Tyr705) by affecting phosphorylation of the upstream protein JAK2, a non-receptor tyrosine kinase . Reduced2+ has versatile biological functions, contributing to all vital cellular processes, including stability of tertiary structures of DNA and RNA, energy metabolism, enzyme activity, signaling, cell cycle progression and cell differentiation [2+ and Mg2+, an interaction between TRPM7 and RTK downstream effectors, including PI3K, AKT and ERK1/2, have been implicated [TRPM7-regulated Mgntiation ,141. Thentiation . The regntiation . TRPM7 wntiation ,145,146.plicated ,148,149.2+ supplementation partially attenuated the phenotype [Developmental experiments using zebrafish models of TRPM7 mutants showed abnormal pancreas development, which was associated with increased expression of Suppressor of cytokine signaling (SOCS)-3a, the zebrafish homologue to the human SOCS3, which is part of a classical negative feedback system that influences cytokine signal transduction by reducing activation of JAK-STAT3. Mghenotype . Using ghenotype .The effects of TRPM7 in pathologic cell differentiation has been intensively investigated in cancer cells, including pancreas, ovary, breast and adenocarcinoma of lungs and prostate . Express2+ influx and phosphorylation of signaling kinases, Smad1/5 and p38 MAPK, whereas TRPM7 knockdown decreases MSC proliferation and viability, and induces cell death [2+ supplementation promoted osteogenic differentiation through the activation of Notch signaling in MSCs, which was decreased by 2-APB [Cancer stem cells are cells found within tumors and hematological cancers that have features of normal stem cells, particularly the ability to self-renew and differentiate into multiple cell types . They arll death . Additioby 2-APB ,156,157.by 2-APB . Togetheby 2-APB .2+, have also been implicated in preeclampsia. In a study examining placentas from women with preeclampsia compared to placentas from normotensive women, pre-eclamptic placentas had reduced expression of TRPM6 and TRPM7 [2+ homeostasis. These phenomena were associated with decreased placental VEGF expression in preeclampsia versus normotensive pregnancies, effects normalized by treatment with MgSO4 [2+ in preeclampsia. This is clinically important because it provides a mechanistic basis for the therapeutic use of MgSO4 as the drug of choice for preeclampsia [Interactions between RTKs, such as VEGFR and EGFR, TRPM6/7 and Mgnd TRPM7 , which wth MgSO4 . Togetheclampsia .2+ homeostasis are regulated by EGF signaling through EGFR. Inhibition of EGFR signaling impairs TRPM6 function leading to renal Mg2+ wasting and consequent hypomagnesemia in cancer patients treated with EGFR inhibitors [2+ and Mg2+ mobilization. Changes in TRPM7 activity and altered Mg2+ homeostasis have a significant effect on tyrosine kinase signaling. Hence, RTKs influence TRPM7, which in turn may influence tyrosine kinase signaling. Most studies have focused on EGFR signaling, but growing evidence indicates that VEGFR and PDGFR also signal through TRPM7. The putative cross-talk between TRPM7 and RTKs has clinical relevance, because inhibitors of RTKs, including VEGFR and EGFR, are increasingly being used to treat cancer and inflammatory diseases. Hence, knowing mechanisms whereby these drugs cause hypomagnesemia is important so that unwanted side effects can be managed in a mechanism-specific manner. The field of TRPM6/7 and RTKs is still immature and further studies are required to unravel the complex interplay between these systems. TRPM7 and TRPM6 are unique proteins characterized by their channel and kinase domains, which differentially regulate cell signaling and function. TRPM6 is mostly expressed in epithelial cells of the kidney and gastrointestinal system, whereas TRPM7 is ubiquitously expressed. In the kidney, TRPM6 and cellular Mghibitors . It is s


The expression and significance of CD276 in non-small cell lung cancer (NSCLC) was explored.The BioGPS database was used to analyze the expression level of CD276 in normal tissues. Studies on the expression of CD276 in NSCLC patients using the Oncomine database. The prognostic roles of CD276 in NSCLC was studied using the Kaplan-Meier plotter database.The BioGPS database showed CD276 expression in all the human normal tissues. Compared with normal lung tissue, CD276 gene highly expressed in NSCLC tissue at mRNA level (P<0.05). The expression level of CD276 gene was negatively correlated with overall survival (OS) of NSCLC patients. Subgroup analysis showed that CD276 expression level had a significant effect on OS of patients with lung adenocarcinoma, while in squamous cell carcinoma its expression level had no significant effect on OS.According to the information mined from the tumor gene database, CD276 mRNA was found highly expressed in NSCLC tissue and the expression of CD276 has a significant impact on survival of NSCLC patients, which provides an important theoretical basis for further study of the role of CD276 in the occurrence and development of NSCLC. Its incidence and mortality are increasing year by year . Non-sma+ and CD8 + cells. As a co-stimulatory molecule, CD276 signal induces cellular immunity. CD276 enhances the induction of cytotoxic T cells and selectively stimulates interferon gamma production (IFNγ) [CD276 is a type I transmembrane protein and its extracellular domain consists of two immunoglobulin constant (IgC) and variable (IgV) domains . CD276 in (IFNγ) . Howevern (IFNγ) , therebyThere are few studies on whether CD276 plays a role in NSCLC and its function in the development of NSCLC. This study used a database to analyze the expression of CD276 gene in NSCLC and its impact on prognosis.22.1Analysis of CD276 expression in normal human tissues was made using BioGPS database [2.2Oncomine database is currently the largest number of oncogene arrays integrated data mining platform in the world . In this2.3Online survival analysis was performed using the lung cancer dataset of the Kaplan-Meier Plotter (KM Plotter) . The scr2.4The database is a search tool for analyzing biological gene or protein interactions, including biological databases and network resources for proven and predictable protein-protein interactions . In this2.5t test. The relationship between CD276 gene expression and prognosis was analyzed by Kaplan-Meier model. The survival rate between the two groups was compared by Log-Rank test. The test level was α=0.05. The data used were analyzed by online statistical analysis. P<0.05 was considered statistically significant.The difference in CD276 gene expression between NSCLC and normal lung tissue was analyzed by 33.1The BioGPS database showed that the CD276 gene was expressed in all normal tissues of the human body, with higher levels in bronchial epithelial cells and the uterus .3.2A total of 436 different types of microarray studies were collected in the Oncomine database, and 29 of them had statistically significant differences in CD276 expression, including 22 studies of increased CD276 expression and three studies of decreased CD276 expression .3.3Proc Natl Acad Sci [PLoS One [Cancer Res [Genome Res [A search in the Oncomine database found that 6 studies had focused on the expression of CD276 in NSCLC and normal lung tissue since 2003, including a total of 591 samples . The artAcad Sci , PLoS OnPLoS One , Cancer ncer Res , Genome nome Res . A meta-3.4Data mining of Oncomine showed that CD276 was highly expressed in different types of NSCLC research arrays compared with normal lung tissue, and the difference was statistically significant . In the Hou et al, the expression of CD276 mRNA was 1.13 times higher in lung adenocarcinoma (45 cases) than in normal lung tissues (65 cases) (P = 6.14E-4, 3.5To further clarify the relationship between CD276 expression level and the prognosis of patients with NSCLC, KM Plotter database analysis showed a survival curve of 1145 patients with high CD276 expression and low expression group, and found that CD276 expression level has a significant impact on overall survival of patients. Compared with 589 patients with low-expression NSCLC, 556 patients with high CD276 expression had significantly lower OS (HR=1.56, logrank P=1.4e-07, 3.6The protein network interacting with CD276 was obtained by String database analysis and the number of nodes was 11 . Among t4At present, the incidence and mortality of lung cancer in the world are the highest among all tumors. In recent years, the application of chemotherapy, radiotherapy, targeted drugs and immunotherapy in lung cancer has significantly improved the 5-year survival rate of NSCLC . Due to Studies have found that CD276 is expressed at both transcriptional and translational levels in six different non-small cell lung cancer cell lines . StudiesThe BioGPS database is an online gene annotation database that contains information on public resources and data generated by Novartis Research . The resIn summary, through the deep mining of NSCLC-related gene information in the database, it was found that CD276 is highly expressed in NSCLC tissues and is associated with the prognosis of NSCLC. The results of these researches provide clues and evidence for further experimental research, and provide theoretical support for further research on molecular mechanisms.