ROS1 gene account for up to 2% of NSCLC patients who sometimes develop brain metastasis, resulting in poor prognosis. This review discusses the tyrosine kinase inhibitor crizotinib plus updates and preliminary results with the newer generation of tyrosine kinase inhibitors, which have been specifically conceived to overcome crizotinib resistance, including brigatinib, cabozantinib, ceritinib, entrectinib, lorlatinib and repotrectinib. After introducing each agent’s properties, we provide suggestions on the best approaches to identify resistance mechanisms at an early stage, and we speculate on the most appropriate second-line therapies for patients who reported disease progression following crizotinib administration.Genetic rearrangements of the The treatment of patients affected by non-small cell lung cancer (NSCLC) has been revolutionised by the discovery of druggable mutations. ROS1 (c-ros oncogene) is one gene with druggable mutations in NSCLC. ROS1 is currently targeted by several specific tyrosine kinase inhibitors (TKIs), but only two of these, crizotinib and entrectinib, have received Food and Drug Administration (FDA) approval. Crizotinib is a low molecular weight, orally available TKI that inhibits ROS1, MET and ALK and is considered the gold standard first-line treatment with demonstrated significant activity for lung cancers harbouring ROS1 gene rearrangements. However, crizotinib resistance often occurs, making the treatment of ROS1-positive lung cancers more challenging. A great effort has been undertaken to identify a new generation or ROS1 inhibitors. In this review, we briefly introduce the biology and role of ROS1 in lung cancer and discuss the underlying acquired mechanisms of resistance to crizotinib and the promising new agents able to overcome resistance mechanisms and offer alternative efficient therapies. KRAS, BRAF, EGFR, MET and ERBB2 mutations or ALK, ROS1, RET and NTRK genomic rearrangements .,30.ROS1 xclusive . Additioth a TKI ,33,34. I(Alimta) ,36,37,38patients ,40,41,42patients . FurtherROS1-positive cancer patients are no exception. One study observed that approximately 40% of stage IV ROS1-positive lung cancer patients were diagnosed with brain metastasis . Im. ImROS1-ROS1 gene fusions, mainly due to the use of identical tests for ALK rearrangements [Fluorescence in-situ hybridisation has traditionally been considered the “gold standard” technique for the detection of ngements ,50,59. Ingements , show singements ,56. FISHngements .ROS1 rearrangement identification [ROS1 fusion alterations (and other oncogenic mutations) using either DNA or RNA as a starting material [Reverse transcriptase-polymerase chain reaction (RT-PCR) requires an RNA sample to be converted to complementary DNA (cDNA) first and then amplified, allowing specific identification of gene fusion partners. RT-PCR sensitivity and specificity are good, but it relies on RNA obtained from formalin-fixed and paraffin-embedded (FFPE) samples, which might limit its use in routine lab work . An emerfication . Howeverfication . Lastly,material . Moreovematerial . It is hmaterial . The fulROS1-rearranged lung cancers was published. A young, non-smoking male diagnosed with ALK and EGFR-negative multifocal NSCLC showed disease progression after first-line erlotinib and developed genomic alteration in ROS1. The patient was treated with 250 mg crizotinib twice per day, which led to a significant improvement in patient-reported symptoms within 7 days and dramatic shrinkage of malignant lesions at imaging within 8 weeks [Crizotinib , originally formulated as an antihepatocyte growth factor receptor (MET) agent, is a TKI against several targets including ROS1, anaplastic lymphoma kinase (ALK) and MET ,65. Criz 8 weeks .ROS1-rearranged NSCLC were included (as an expansion cohort) alongside ALK-rearranged advance NCLSC patients, all of whom were treated with crizotinib [ROS1-positive patients reported a DCR of 90%, a median PFS of 19.2 months, an ORR of 72% and an OS rate of approximately 85% at 12 months. 5 patients (10%) showed treatment-related adverse events (AEs) including vomiting, nausea, constipation, diarrhoea, fatigue, visual impairment and dizziness; the vast majority evaluated as clinically manageable with only 4% evaluated as grade 3 of 4, and no grade 5 AEs were observed [ROS1-rearranged NSCLC patients [ROS1-rearranged NSCLC. The EUROS1 Cohort study retrospectively evaluated crizotinib efficacy in 32 ROS1-positive patients and reported a disease control rate of nearly 87%, a PFS of 44% at 12 months and, remarkably, 5 patients achieved a complete response [ROS1-positive NSCLC patients included showed a DCR of 89% and an overall ORR of 54% with 43% of patients evaluated to have no-progression of disease after 12 months [In the phase I PROFILE 1001 clinical trial, 50 patients with izotinib . ROS1-popatients . Subsequpatients . Followiresponse . In the 2 months . Among 32 months . The Asi2 months . The oveROS1 gene have been identified in crizotinib-resistant forms of NSCLC  had an objective treatment response. Moreover, one patient was crizotinib-naïve and reported partial response while the other patient was crizotinib pretreated and reported stable disease. Overall, brigatinib toxicity was reported to be manageable with increased lipase (9%) as primary grade 3 or 4 AE, while serious treatment-related AEs such as dyspnoea and pneumonia occurred in 5% of patients [The ALK inhibitor brigatinib has demonstrated clinical activity against different mutations correlated with resistance to crizotinib, including ngements ,85. In tpatients . Howeverpatients ,87.ALX, KIT, MET, RET, ROS1, VEGFR2 and TIE2 [ROS1 genomic alteration who acquired resistance to crizotinib. Additionally, Sun et al. reported clinical efficacy of cabozantinib in four ROS1-positive NSCLC patients with resistance to crizotinib and ceritinib; the PFS of patients administered with cabozantinib ranged from 4.9 to 13.8 months with neutropenia, xeroderma and pulmonary embolism the primary AEs reported [The multi-kinase inhibitor cabozantinib, which has demonstrated activity against and TIE2 , and is and TIE2 ,90,91, hand TIE2 . Within reported . AlthougROS1-positive NSCLC patients administered with a daily dose of 750 mg. Among crizotinib-naïve and crizotinib pretreated patients, the overall ORR was 62% with a median PFS of 9.3 months. Among 8 patients with CNS disease, 63% reported disease control, while 25% showed an intracranial response [Ceritinib, a highly specific and robust ROS1 inhibitor, showed 20 times higher efficacy than crizotinib in a rat model , with a response . Howeverresponse . Clinicaresponse .ROS1 alteration in 2019 [ROS1 mutants [ROS1-positive NSCLC patients recruited for a phase I/II trial [Entrectinib  is an orally available, low molecular weight TKI, specifically designed to penetrate the BBB. Entrectinib received FDA approval for lung cancers harbouring  in 2019 ,47,98, h in 2019 . However mutants ,100. EntII trial ,102. AmoROS1 mutants , which confer resistance to crizotinib and ceritinib, and the ability to penetrate the BBB [ROS1-positive NSCLC patients, including both TKI-naïve and pretreated patients [Lorlatinib is a potent oral inhibitor of ALK  and ROS1 with activity against several  the BBB ,103,104.patients . A mediapatients .ALK and most ROS1 mutants  with 90 times higher efficacy than crizotinib [ROS1 rearrangements were recruited in a larger phase I study including patients with different solid malignancies and mutations  and administered with increasing dosage of repotrectinib [Repotrectinib (TPX-0005) is a low molecular weight, potent TKI with demonstrated efficacy against TRK receptors, izotinib ,107. In rectinib . While Trectinib . The abiROS1-rearranged patients [It is worth mentioning additional drugs that are thought to be potentially valuable ROS1 inhibitors. Of these, ensartinib is a low molecular weight, orally available agent currently under evaluation for paediatric patients . Fortinipatients .ROS1-rearranged malignancies [ROS1-positive lung cancers and it is currently the preferred first-line option for these patients [ROS1-rearranged cancers are relatively rare, new therapies might provide clinicians with treatment suggestions. Agents including repotrectinib and lorlatinib have demonstrated significant efficacy in the post-crizotinib setting [ALK-rearranged lung cancers can, moreover, improve understanding of ROS1-driven NSCLC cases under treatment and drive helpful clinical suggestions [ROS1-targeted therapies [ROS1 lung cancer, is essential for generation of superior compounds for combination therapies.ROS1 inhibitors have demonstrated clinical efficacy against gnancies ,104,108.patients . Unfortugestions . Of the gestions . In futuherapies . InvestiROS1-rearranged malignancies [On the clinical side, techniques for early stage identification of resistance mechanisms from tissue or blood would facilitate more precise treatment plans with a higher probability of preventing cancer progression and overcoming drug resistance. In this light, it is worth mentioning the first liquid biopsy effort for detection and monitoring of ROS1 circulating tumour DNA (ctDNA) in gnancies ,113. We ROS1 mutations. Of note, repotrectinib is currently under investigation while lorlatinib has recently been approved for ALK-rearranged NSCLC patients and is the current preferred second-line treatment in case of no evidence of G2032R mutation and/or bypass signalling [ROS1-rearranged lung cancers is essential as there are several targeted therapies available, despite the low incidence rate. The understanding of underlying molecular alterations that lead to drug resistance and disease progression, and the development of novel drugs to counteract this, is essential for the design of more precise and effective combination therapies.Its inability to penetrate the BBB renders crizotinib unsuitable for ROS1-rearranged NSCLC patients with CNS metastasis; in such cases, entrectinib, ceritinib, repotrectinib and lorlatinib are more appropriate options since these agents were specifically engineered to penetrate the BBB. With that said, although ceritinib and entrectinib are less effective towards crizotinib-resistant malignancies, they showed increased efficacy towards brain metastasis with entrectinib the current preferred and FDA-approved first-line treatment for those with metastatic brain involvement  Accordingnalling . To summ


Hemiptelea davidii (Hance) Planch is a potential valuable forest tree in arid sandy environments. Here, the complete mitochondrial genome of H. davidii was assembled using a combination of the PacBio Sequel data and the Illumina Hiseq data. The mitochondrial genome is 460,941 bp in length, including 37 protein-coding genes, 19 tRNA genes, and three rRNA genes. The GC content of the whole mitochondrial genome is 44.84%. Phylogenetic analyses indicated that H. davidii is close with Cannabis and Morus species. Hemiptelea davidii (Hance) Planch belonging to Hemiptelea of Ulmaceae is a kind of shrub or small tree with a height up to 10 m, which mainly distributed in China. It could survive well in arid sandy environment and was then recognized as a potential valuable forest tree resource in low rainfall region , China and the specimen were conserved at Shandong Provincial Center of Forest Tree Germplasm Resources (voucher number: HD2016003). The mitochondria DNA was separated from fresh leaves using an improved method according to Chen et al.  and thenH. davidii mitochondrial genome is assembled into a single circular-mapping molecule of 460,941 bp, with a GC content of 44.84%. The mitochondrial genome contains a total of 59 genes, including 37 protein-coding genes, 19 tRNA genes, and three rRNA genes. Of the protein-coding genes, nad5 is the largest gene (2016 bp) while atp9 is the smallest gene (225 bp).The cox1, cox2, cox3, matR, nad2, nad4, nad6, nad7, nad9, and rps3) were aligned with the homologous genes in other 13 species using MAFFT (Katoh and Standley H. davidii is close with Cannabis and Morus species (Sequences of 10 common protein-coding genes ( species , which i


Signal transducer and activator of transcription 3 (STAT3) is a transcriptional regulator of a number of biological processes including cell differentiation, proliferation, survival, and angiogenesis, while cyclin-dependent kinases (CDKs) are a critical regulator of cell cycle progression. These proteins appear to play central roles in angiogenesis and cell survival and are widely implicated in tumor progression. In this study, we used the well-characterized US National Cancer Institute 60 (NCI60) human tumor cell lines to screen the in vitro anti-cancer activities of our novel small molecule derivatives (NSC765690 and NSC765599) of salicylanilide. Furthermore, we used the DTP-COMPARE algorithm and in silico drug target prediction to identify the potential molecular targets, and finally, we used molecular docking to assess the interaction between the compounds and prominent potential targets. We found that NSC765690 and NSC765599 exhibited an anti-proliferative effect against the 60 panels of NCI human cancer cell lines, and dose-dependent cytotoxic preference for NSCLC, melanoma, renal, and breast cancer cell lines. Protein–ligand interactions studies revealed that NSC765690 and NSC765599 were favored ligands for STAT3/CDK2/4/6. Moreover, cyclization of the salicylanilide core scaffold of NSC765690 mediated its higher anti-cancer activities and had greater potential to interact with STAT3/CDK2/4/6 than did NSC765599 with an open-ring structure. NSC765690 and NSC765599 met the required safety and criteria of a good drug candidate, and are thus worthy of further in-vitro and in-vivo investigations in tumor-bearing mice to assess their full therapeutic efficacy. The eansition . Under pansition ,11. CDKsansition . Thus, tansition .A number of key oncogenic abnormalities, including amplification of cyclin D; inactivation of CDKN2A p16); and deletions/mutations upstream of cyclin D, such as activating mutations of phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)/the B-raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatase and tensin homolog (PTEN) deletion ,14, were; and delSTAT3 is a cytoplasmic transcription factor involved in a number of biological processes including cell differentiation, proliferation, survival, and angiogenesis . OverexpThe translational value of knowledge of cancer biology into developing effective prognostic/diagnostic and therapeutic strategies for clinical practice remains disappointing . HoweverNSC765599 and NSC765690 are small molecule derivatives of salicylanilides , which were previously synthesized and evaluated for biological activities in our Lab . We had 2 for 48 h. Cells were fixed with a sulforhodamine B (SRB) solution [50 (concentration needed to kill 50% of cells by cytotoxic activity) for each cell line tested, and GI50 (concentration needed to inhibit 50% of cancer cell growth) [NSC765690 and NSC765599  were subsolution  followed growth) . Results50, TGI and LC50 were set as the endpoints.NSC765599 and NSC765690 were screened for potential drug target using SwissTarget prediction algorithm, which predicts potential drug target based on the principle of similarity . In addiThe three-dimensional 3D) structure of palbociclib (CID: 5330286) and SH-4-54 (CID: 72188643) were retrieved in SDF file format from the PubChem database, while the 3D structures of NSC765690 and NSC765599 were drawn out in sybyl mol2 format using the Avogadro molecular builder and visualization tool vers. 1.XX  and wereD structu50) in rats and environmental toxicity were predicted using GUSAR software [The drug-likeness, medicinal chemistry, and pharmacokinetics, including the adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of NSC765690 and NSC765599 were analyzed using SwissADME software developed by the Swiss Institute of Bioinformatics . The drusoftware .Spearman’s rank correlation was used to assess the correlations of NSC765599 and NSC765690 fingerprints with NCI synthetic compounds, standard agents, and molecular targets. The COMPARE correlation threshold was set to ≥45 common cell lines, ≥0.1 correlation coefficient, and ≥0.05 standard deviation. The growth inhibition by NSC765690 and NSC765599 in single-dose assay was obtained by subtracting the positive value on the plot from 100, i.e., a value of 40 would mean 60% growth inhibition.Both NSC765690 and NSC765599 exhibited anti-proliferative effect against all the 60 panels of NCI human cancer cell lines . Further50 values ranged from 0.14~2.79 μM in NSC76569-treated cell lines and 0.219~5.55 μM in NSC765599-treated cell lines. Consistent with the activities demonstrated in single-dose treatments, the NSCLC, melanoma, renal, and breast cancer cell lines were most sensitive to NSC765690 treatment, while leukemia, CNS, ovarian, and colon cancer cell lines were the least sensitive to NSC765690 , NSCLC , CNS , renal , leukemia , and breast  cancer cell lines . The GI5SC765690 . Analysit active .p values in the range of 0.44~0.62. In addition, NSC765690  and NSC765599  shared anticancer mechanism with a number of standardized drugs in the NCI database (p = 0.1~0.3) with the expression of multiple genes, most of which are proteins involved in cell cycle progressions , with database . The molressions .Using NSC765690 and NSC765599 as query molecules on the SwissTargetPrediction algorithm, a computer-based drug target prediction tool that identifies the most probable macromolecular targets of a small molecule, on the basis of similarity with a known actives compound in the library , we idenUsing molecular docking studies, we found that both NSC765690 and NSC765599 exhibited strong interactions with the crystal structure of apo CDK2 , CDK4/cyclin D3 , CDK6/cyclin , and STAT3 . The observed interactions and binding affinities indicated that CDK2 is the most favored receptor for both compounds , while NOur careful analysis of the interactions between NSC765690 and the receptors revealed a higher number of conventional H-bonding, pi interactions, and Van der Waal forces created on the ligand backbone with a higher number of amino acids residues than does NSC765599 and palbociclib, in the CDK2/4/6 binding cavities . NSC765650) according to OECD classification  = 0~6) and lipophilicity (XlogP3 = −0.7~5) having slight outlying values of −5.71 and 5.47 respectively. Both compounds demonstrated good synthetic accessibilities, highly probable GIA absorption, and bioavailability, but poor BBB permeation , Table 7Despite advances in treatment modalities, cancer survival ratios are still disappointing; thus, developing a novel chemotherapeutic strategy may improve the prognosis of the cancer patient . At presp = 0.15~0.36) antitumor mechanistic fingerprints with known inhibitors of growth factor receptor and cell cycle transition, and DNA damage inducer. However, the modest correlation with NCI-standard agents suggests that NSC765599 may have a unique mechanism of action not common to standard agent database. The strong antitumor pattern correlation (p = 0.44~0.6) between NSC765690 and NSC765599 fingerprint with NCI synthetic compounds suggests a mechanism of action similar to that of the seed compounds. Unfortunately, the top-ranked correlated compounds antineoplastic-643812 (p = 0.61) and N-(3-chloro-2-methylphenyl)-2-hydroxy-3-nitrobenzamide (p = 0.62) are yet to be mechanistically investigated, while combretastatin A-4 with p-value 0.58 (NSC765690) and p-value 0.53 (NSC765599) have been reported for the mechanism of action, indicating cell cycle arrest and inhibition of tubulin [p = 0.1~0.3) were low, indicating the possibility of a new mechanism not captured by COMPARE.The NCI60 cell lines have been well-characterized for genetic and protein expression patterns, and, a computational tool, the DTP-COMPARE algorithm, has been developed to identify the molecular targets and mechanism(s) of action for the unknown compound from known drugs as well as the known molecular targets in the NCI databases . Interes tubulin , hence pThe COMPARE prediction, together with in-silico SwissTarget and PASS prediction collectively identified STAT3/CDK2/4/6 as the most probable target for NSC765690 and NSC765599, and thus were further studied for ligand–receptor interactions. Interestingly, we found that NSC765690 and NSC765599 docked well into the receptor cavities of STAT3, CDK2, CDK4, and CDK6, with CDK2 being the most favored receptor while CDK4 appeared to be the least favored. Comparatively, the higher binding affinities and closer proximity of NSC765690 to the receptors suggested that NSC765690 is a better ligand for CDK2, CDK4, and CDK6 than is NSC765599.NSC765690 and NSC765599 are both derivatives of NDMC101. A close look at the structure-related activities of NSC765690 and NSC765599 revealed that cyclization of salicylanilide core scaffold  was the The unique stability of NSC765690 in the binding sites of CDK2, CDK4, and CDK6 could be attributed to the larger number of H-bond and pi interactions including pi–cation, pi–anion, pi–alkyl, pi–pi stack, and pi–pi T-shaped interactions between NSC765690 and the receptors. The large numbers of pi interactions, which mostly involve charge transfer, help in intercalating the NSC765690 in the receptors’ binding cavities. The higher affinity of NSC765690 was also associated with the presence of a larger number of Van der Waal forces created on its backbone with respective amino acids Phe82, Leu83, Asp145, Ala144, Asn132, Gln131, Thr14, Thr158, Glu12, Gly13, Gly11, Asp86, Gln85 and His84 of CDK2; Leu64, Arg60, Leu113, Ala63, Leu31, Phe163, Phe160, Leu161, Glu76 and Arg114 of CDK4; and Phe71, Ser296, Leu295, Phe127, and His73 of CDK6, which undoubtedly created a strong cohesive environment, thereby stabilizing the complex formed . Althoug50), an adverse effect that follows a single dose exposure to a substance, is an important task in drug design and risk assessment of chemicals [50 values predicted for oral, intravenous, intraperitoneal, and subcutaneous administration indicated that both NSC765690 and NSC765599 were non-toxic and will be well-tolerated by the experimental animals. In addition, NSC765690 and NSC765599 met the criteria of a good drug candidate (The concept of drug-likeness gives useful guidelines for identifying potential drug candidates during the early stage of drug discovery and development . Estimathemicals ,61,62. Tandidate , suggestIn conclusion, this study demonstrated the anti-proliferative effect of NSC765690 and NSC765599 against the 60 panels of NCI human cancer cell lines, and dose-dependent cytotoxic preference for NSCLC, melanoma, renal, and breast cancer cell lines. STAT3/CDK2/4/6 signatures appear to be potential druggable candidates for the small molecules and interacted with high binding affinity, indicating the potential of NSC765690 and NSC765599 to dysregulate expressions of STAT3/CDK2/4/6 signature and, consequently, compromised cell survival. Further in-vitro and in-vivo confirmation studies are ongoing in our lab.


Pyrazolines are five-membered heterocycles possessing two adjacent nitrogens. They have attracted significant attention from organic and medicinal chemists due to their potent biological activities and the numerous possibilities for structural diversification. In the last decade, they have been intensively studied as targets for potential anticancer therapeutics, producing a steady yearly rise in the number of published research articles. Many pyrazoline derivatives have shown remarkable cytotoxic activities in the form of heterocyclic or non-heterocyclic based hybrids, such as with coumarins, triazoles, and steroids. The enormous amount of related literature in the last 5 years prompted us to collect all these published data from screening against cancer cell lines, or protein targets like EGFR and structure activity relationship studies. Therefore, in the present review, a comprehensive account of the compounds containing the pyrazoline nucleus will be provided. The chemical groups and the structural modifications responsible for the activity will be highlighted. Moreover, emphasis will be given on recent examples from the literature and on the work of research groups that have played a key role in the development of this field. Cancer is one of the leading causes of death worldwide. According to the World Health Organization reports, about one in six deaths globally is attributed to cancer with more than 9 million deaths in 2018  phenyl}-1,3,5-triazin-2-amine, which is currently in clinical development for the treatment of Acute Myeloid Leukemia (AML) [Out of the three possible triazine isomers, only the 1,3,5-triazine and the 1,2,4-triazine rings have been incorporated in a pyrazoline hybrid structure so far. The former, also referred to as ia (AML) . Howeveria (AML) .70  and breast HS-578 (0.644 μM). For comparison purposes, since we have mentioned the activities of many compounds against MCF-7, MDA-MB-231, and HCT-116 the corresponding values found were 1.26, 1.45, and 1.28 μM, respectively, for compound 72, 1.42, 1.71, and 1.59 μM, respectively, for compound 73 and, finally, 1.27, 1.63, and 1.55 μM for compound 74.More recently, Insuasty et al. reported the synthesis and evaluation of a series of chalcone derived 1,3,5-triazine containing 2-pyrazolines. Seventeen of them were selected and screened by NCI, where compounds 72–74  were pro2–74 -one, used as control, was the least active. As an example, the ratio Vt/Vo for the tumor growth was found to be 1.15, 1.75, 2.37, and 4.31 after 2, 8, 14, 20 days, respectively, for compound 75 and 1.38, 9.88, 27.66, and 40.21 after 2, 8, 14, 20 days, respectively, for the starting triazine.A series of ten 3-thioxo-1,2,4-triazines bearing pyrazolines among other heterocycles (oxazoles and pyridines), was prepared and evaluated against various cancer cell lines in vitro and in vivo . The autcular 75  which fo76  while being inactive against normal green monkey kidney cell line Vero B (IC50 > 50 μg/mL). Özdemir et al. synthesized a series of 12 pyrazoline hybrids with various heterocycles  attached via a thioethanone linker group to the nitrogen-1 of the pyrazoline ring [77  or selective androgen receptor modulators (SARMs) . Antican78 was identified as one of the most active ones . The other members of this library involve various substituents  of the steroidal framework or formation of a new pyrazoline fifth ring fused on the D-ring. Compounds derived from pregnenolone were the first approach to this family of hybrid molecules. Banday et al. performed in vitro screening of the synthesized molecules and compound ts R—see  like flu17,20-lyase, Schneider and co-workers reported the synthesis of pregnenolone-pyrazoline conjugates as potential anticancer agents [79, which is one of the most potent, showed growth inhibition equal to 50.3% and 69.3% against MCF-7 and A2780 at 10 μM whereas the S-isomer displayed 25.8% and 52.3%, respectively.After having studied various steroid hybrids with heterocycles, including pyrazoles  as inhibr agents . Interes2 and MDA-MB-230 cell lines [2 (IC50 = 28.97 μM) and no cytotoxity against MDA-MB-230. Among the 58 pyrazoline derivatives tested , compound 80  while having low cytotoxicity against SGC-7901, A549, and HeLa. In general, all pyrazolines demonstrated lower activities than those of the pyrazole analogues, indicating that the pyrazole fragment may play an important role in the cytotoxic activities of these conjugates [83  followed by 2-chloride and 2-hydroxyl [Kankala et al. performed the synthesis and anticancer evaluation of a series of steroidal pyrazoline conjugates derived from a steroidal glycoside extract of pound 84 , having hydroxyl .50 > 50 μM against PBMC) [85 (50 = 10.6 μM) and one of the most active against HeLa (IC50 = 22.5 μM). Following these findings, they performed a second screening on three more derivatives [86 .Shamsuzzaman et al. investigated the anticancer potential of B-ring fused steroidal pyrazoline conjugates. Derived from cholest-5-en-7-one, these derivatives were found, in principle, non-toxic to normal cells (ICst PBMC) . CompounBMC) [85  was idenivatives , concludives [86  was the 2 cancer cell lines revealed lower activities than the parent molecule [50 values of 6.3, 16.4, and 21.3 μg/mL against HepG2, A549, and SKOV3, respectively). From the pyrazoline derivatives, only compound 87 (50 = 18.9 μg/mL). Interestingly, the chalcone precursors were more potent, but none of them reached higher activity than ursulic acid against HepG2 cells.Finally, anticancer evaluation of pyrazoline and chalcone derivatives of ursulic acid against A549, SKOV3, and HepGmolecule . Ursulicpound 87  showed s88 . In addition, in silico studies pointed out that 89 demonstrates good binding interactions with the human IKK-β protein.Meanwhile, in a very recent publication, Chaudhary et al. reported the synthesis and biological evaluation of curcumin-pyrazoline condensates as anticancer agents against human cervical cancer HeLa . CompounThe idea of designing hybrid molecules consisting of two or more therapeutic units in a single molecule is generally new. The strategy of molecular hybridization has been employed by medicinal chemists to develop novel bioactive compounds that aim towards multiple targets, being at the same time less vulnerable to drug resistance. Pyrazolines are versatile molecules with potent anticancer activities, thus they have been selected by many researchers for the development of such structures.In this review, we presented a variety of literature reports on anticancer pyrazoline hybrids containing an additional heterocyclic or non-heterocyclic framework. Overall, the structures described were more potent than their parent compounds.Alongside, we tried to shed light onto structure activity relationships wherever it was possible and for the compounds having at least moderate activities. 1-Pyrazolines and 3-pyrazolines are certainly underrepresented and have not been investigated adequately. However, even for 2-pyrazolines, since this field comprises of relatively recent works, there are not many comprehensive structure activity relationship studies for many cancer cell lines, protein targets, toxicology, and in vivo tests.p-methoxyphenyl groups at C-3 and C-5 of the ring usually enhance cytotoxicity, as does, in many cases, the trimethoxyphenyl unit inspired from combretastatin A4. In parallel, in other studies, electron withdrawing groups, mostly fluoride or chloride increased the activity. Coumarin, quinoline, indole, and thiazole components have been found as very potent. Overall, there is not a generalized SAR rule for enhancing the cytotoxicity and it is heavily dependent on the structure configuration or functionalization of the conjugate molecule.Among the hundreds of structures, one can identify some common modalities; groups, atoms, and structural conformations that favor the anticancer activities. As a general trend, To conclude, pyrazoline is an important five membered nitrogen heterocycle. The ring is quite stable and has inspired chemists to carry out various structural modifications to enhance activity, selectivity, and/or pharmacological properties. This has propelled the development of distinct pyrazolines towards the area of molecular hybridization to achieve maximum multimodal activity, as it has been highlighted by the content of this review. Since this topic is relatively new, it is anticipated that a lot of frontier research will be performed, and we believe that scientists working on such derivatives will find valuable information to further advance the structural diversity and anticancer applications of novel pyrazoline-based hybrid drugs.


Leaf senescence is an essential physiological process that is accompanied by the remobilization of nutrients from senescent leaves to young leaves or other developing organs. Although leaf senescence is a genetically programmed process, it can be induced by a wide variety of biotic and abiotic factors. Accumulating studies demonstrate that senescence-associated transcription factors (Sen-TFs) play key regulatory roles in controlling the initiation and progression of leaf senescence process. Interestingly, recent functional studies also reveal that a number of Sen-TFs function as positive or negative regulators of plant immunity. Moreover, the plant hormone salicylic acid (SA) and reactive oxygen species (ROS) have been demonstrated to be key signaling molecules in regulating leaf senescence and plant immunity, suggesting that these two processes share similar or common regulatory networks. However, the interactions between leaf senescence and plant immunity did not attract sufficient attention to plant scientists. Here, we review the regulatory roles of SA and ROS in biotic and abiotic stresses, as well as the cross-talks between SA/ROS and other hormones in leaf senescence and plant immunity, summarize the transcriptional controls of Sen-TFs on SA and ROS signal pathways, and analyze the cross-regulation between senescence and immunity through a broad literature survey. In-depth understandings of the cross-regulatory mechanisms between leaf senescence and plant immunity will facilitate the cultivation of high-yield and disease-resistant crops through a molecular breeding strategy. Arabidopsis, leaf senescence occupies nearly half of the leaf life history from the onset of senescence to completely dead , and ATbidopsis , also pl cinerea ,43,44,45with PCD . Similarroot cap . Furtherll death  and it bth [HIN1 . Howeverreatment . ANAC032r stress ,45. Addi2O2-induced NAC TF and functions as a negative regulator of longevity in Arabidopsis [JUB1 causes the accumulation of DELLA proteins and antagonizes SA pathway, resulting in impaired resistance to Pst DC3000, but increases resistance against necrotrophic fungus A. brassicicola [2O2 accumulation is observed in jub1 mutants compared to Col-0 plants, whereas overexpressing JUB1 could decrease the cellular H2O2 level, suggesting that JUB1 is involved in the regulation of biosynthesis and/or metabolism of H2O2 [nac017, nac082, or nac090 accelerates cell death and leaf senescence, while overexpressing lines show the opposite effect, indicative of their negative regulatory roles in leaf senescence [ICS1 and EDS5. Different from NAC090, ANAC017 predominantly suppresses ROS levels.JUNGBRUNNEN1  is an Hbidopsis . The ovesicicola . Additio of H2O2 . Recentlnescence . NAC090 anac019 anac055 anac072 triple mutant shows increased resistance to P. syringae pv. maculicola (Psm) ES4326 [anac019 anac055 double mutant exhibits enhanced resistance to necrotrophic fungus B. cinerea, while the overexpression of ANAC019 or ANAC055 has the opposite effects [ICS1 [NAC019, NAC055, and NAC072 (RD26) positively regulate age-dependent leaf senescence . However) ES4326 . In cont effects . ANAC019ts [ICS1 , suggestts [ICS1 .ORS1 overexpression lines reveals that several potential targets of ORS1, such as WRKY40 [WRKY75 [FMO1 [ALD1 [In addition to the above-mentioned transcription factors, several NAC TFs, such as NAC059 (ORS1)  and NTL4s WRKY40 , WRKY75 75 [FMO1 ,88, and O1 [ALD1 , play crO1 [ALD1 ,64, whicArabidopsis [Oryza sativa WRKY6 (OsWRKY6), and Chimonanthus praecox WRKY71 (CpWRKY71) are positive regulators of leaf senescence [ICS1 to activate its transcription. Moreover, WRKY75 suppresses the transcription of CAT2 to repress H2O2 scavenging, which results in an accumulation of ROS level. OsWRKY6 directly binds and positively regulates OsICS1 to enhance SA accumulation, leading to constitutive activation of several PR genes [WRKY TFs are one of the largest families of plant-specific TFs, with more than 70 members in bidopsis . Althougnescence ,69,70,91nescence ,65,69. Onescence , and it nescence . WRKY75 WRKY53 promoter and suppresses its expression [wrky18 wrky40 and wrky18 wrky60, as well as the triple mutant wrky18 wrky40 wrky60 display enhanced resistance to P. syringae, but are more susceptible to B. cinerea than Col-0 plants. Moreover, the double mutant wrky18 wrky40 is more resistant to the biotrophic powdery mildew fungus Golovinomyces orontii [wrky18 wrky40 wrky60 exhibits a higher level of SA-responsive gene PR1, implying that they negatively regulate SA signaling.As a negative regulator of leaf senescence, WRKY18 directly binds the W-boxes of pression . WRKY18  orontii ,74. The Arabidopsis [WRKY70 results in impaired resistance to necrotroph A. brassicicola, whereas enhances the resistance against biotroph Erysiphe cichoracearum [WRKY70 and WRKY54 act redundantly to negatively regulate leaf senescence in bidopsis , functiobidopsis . WRKY70 bidopsis . The eleracearum , suggestCAT2 to suppress its transcription, thus resulting in the decreased H2O2-scavenging activity [gbf1 mutant shows a decreased H2O2 level and delayed senescence phenotypes [GBF1 overexpressing plants are more resistant, whereas gbf1 is susceptible, to Pst pathogens when compared to Col-0 [lux-2 shows earlier yellowing as compared with Col-0 [lux mutants display impaired disease resistance against P. syringae and SA- and JA-mediated defense signaling [tcp19 or tcp20 single mutant shows slightly early senescence phenotype in the dark, whereas tcp19 tcp20 double mutant displays significantly accelerated senescence, suggesting that TCP19 and TCP20 redundantly regulate leaf senescence [tcp19 tcp20 has yet to be studied, the TCP19 was found to bind the promoters of ICS1, PBS3 (avrPphB SUSCEPTIBLE 3), PAD4, and EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1), indicative of their possible involvement in plant immunity [In addition to NAC and WRKY TF families, other TF family members have also been reported to regulate leaf senescence and plant immunity . GBF1, aactivity . Accordienotypes . The GBFto Col-0 . The cirto Col-0 ,96. One th Col-0 , and luxignaling . TCP TFsignaling . The tcpnescence . Althougimmunity .SAG101, a gene encoding an acyl hydrolase, plays a positive role in leaf senescence [Arabidopsis HLS1 (HOOKLESS1) encodes a putative histone acetyltransferase and negatively regulates dark-, pathogen-, and ABA-induced leaf senescence [hls1 mutant. The loss of HLS1 function leads to an increased susceptibility to B. cinerea with larger disease lesions, enhanced necrosis, and chlorosis when compared to controls [hls1 mutants also display enhanced disease symptoms after inoculation with Pst DC3000 (AvrRpm1), but they show a level of bacterial growth under Pst DC3000 inoculation, suggesting that HLS1 manipulates defense responses associated with ETI [Arabidopsis. The expression of two positive regulators of leaf senescence, WRKY53 and SAG201, is repressed by JMJ16 in an age-dependent manner through reducing H3K4me3 levels at these loci [jmj16 mutants display an early senescence phenotype, suggesting that JMJ16 is an important epigenetic regulator of leaf senescence via demethylating H3K4 at SAGs in an age-dependent manner. RNA_seq and ChIP_seq reveal that lots of potential target genes of JMJ16, such as WRKY33, CBP60G, EDS5, and PR5, are associated with plant immunity. However, the disease resistance phenotype of jmj16 is not reported so far.In addition to TFs, other types of genes are also involved in the regulation of leaf senescence and disease resistance . SAG101,nescence . SAG101 nescence . Arabidonescence . A highecontrols . Howeverwith ETI . JMJ16 iese loci . The jmjArabidopsis MAPKs, MPK3 and MPK6, play crucial roles in response to biotic and abiotic stresses. MPK3/6 are involved in PTI and ETI [mpk6 mutants show delayed senescence phenotypes [mpk6 mutants, the MKP2-suppressed line (MPK2_RNAi) displays early senescence phenotypes [B. cinerea, suggesting that MKP2 and PP2A regulate disease resistance against necrotrophs in different mechanisms [Two  and ETI , and mpkenotypes . MAPK phenotypes . Contrarenotypes ,83. Anotchanisms .Arabidopsis FtSH4, a mitochondrial AAA-protease, mediates autophagy and senescence [ftsh4 mutant shows a significantly increased SA level and accelerated leaf senescence and cell death. Although the function of FtSH4 in disease resistance has not been studied, several SA biosynthesis and signaling genes, such as SID2, NDR1, and NPR1, are upregulated in the ftsh4 mutant. Furthermore, the loss of SID2, NDR1, or NPR1 functions in the ftsh4 mutant restores the early senescence phenotypes. In addition, several WRKY genes, including WRKY40, WRKY46, WRKY51, WRKY60, WRKY63, and WRKY75, display elevated expression levels in ftsh4 mutant. Together, the above data imply that FtSH4 plays a negative role in plant immunity.nescence , and theAge- and pathogen-induced changes in SA and ROS can be regulated by a large number of Sen-TFs, as mentioned above. Although Sen-TFs are involved in these two processes, there are still significant differences between them. The spatial and temporal expression of Sen-TFs is strictly regulated, and their transcript levels are gradually upregulated during leaf senescence. Consistently, the contents of SA and ROS gradually increase as the leaf ages. By contrast, pathogen-evoked acute induction of Sen-TFs gene expression as well as the contents of SA/ROS would usually return to basal levels after a certain period of time.Arabidopsis during leaf senescence. In addition to age, the functional differences of the common regulatory components of SA and ROS are likely dependent on the tissue specificity of gene expression. For instance, NTL9 functions in guard cells for flg22-triggered induction of SA synthesis- related genes and is required for stomatal immunity [SAGs in other cell types, such as mesophyll cells. Multi-omics analysis of specific cell types (single-cell sequencing) would contribute to the understanding of the functional differences of Sen-TFs on leaf senescence and plant immunity.The physiological state of plants, such as age, has complex and delicate regulation in gene expression. For example, the “NAC troika” precisely governs the time-dependent regulatory shift for NAC-TFs in immunity . HoweverOther phytohormones generally regulate senescence and immune responses by antagonistically or synergistically interacting with the SA/ROS signaling pathway because SA and ROS act as central players in leaf senescence and plant immunity . Cross-tPst pathogen secretes a mimic of JA-Ile virulence factor coronatine (COR) to suppress SA-dependent defenses by activating MYC2 and NAC019/055/072, which repress SA synthesis and/or promote SA metabolism [coi1 mutant [coi1 or jar1-1 mutants [SID2 and negatively regulate the accumulation of SA. The ein3 eil1 double mutant thus constitutively accumulates SA, and ROS production is diminished in ethylene-insensitive mutants under Flg22 treatment [OsrbohA/OsrbohB to activate their transcription [ERF1 in response to necrotrophic pathogens [MYC TFs in response to the herbivorous insect [PLETHORAs (PLTs), which are responsible for stem cell niche maintenance [ET synergistically interacts with JA in plants in response to stresses and generally antagonizes SA signaling . Howevertabolism . Consist1 mutant . JA indu mutants . ET is p mutants  and the  mutants . EIN3/EIreatment . In ricecription . ET syneathogens . JA proms insect ,135. As s insect . Auxin es insect . Howeverntenance . JA antantenance .ICS1 transcriptional level is down-regulated in ABA receptor mutant pyl. In rice, ABA significantly suppresses the expression levels of OsWRKY45 and OsNPR1 [TE module is a regulatory hub that is involved in the cross–talks between GA and ABA [Tiller Enhancer (TE), encoding an activator of the APC/CTE E3 ubiquitin ligase complex, interacts with and degrades ABA receptor OsPYLs. SnRK2 phosphorylates TE to interrupt the interaction between TE and OsPYLs. GA reduces SnRK2s levels and inhibits APC/CTE-mediated degradation of OsPYLs. ABI4 positively regulates ARR5 (Arabidopsis RESPONSE REGULATOR 5) expression, a negative regulator of CK signaling, but it suppresses the transcript level of PIN-FORMED 1 (PIN1), indicating that ABI4 mediates the cross-talks among ABA, CK, and auxin [Recent studies have greatly expanded our understandings of the cross-talks between ABA and other hormones. ABA antagonizes plant immune responses by repressing SA responses. The d OsNPR1 . Meanwhid OsNPR1 . MYC2 isd OsNPR1  and the d OsNPR1 . Moreoved OsNPR1 . The antd OsNPR1 . One of d OsNPR1 . The SnR and ABA . Rice Tind auxin . SnRK2.3nd auxin . The crond auxin ,151.PR1 gene, suggesting that CKs can also function in the SA signaling pathway [2O2 levels through restraining H2O2 generation to be implicated in the regulation of stomatal opening. BRs are a unique class of plant steroid hormones that mediate growth and development. BRs signals are perceived and transduced by the receptor-like kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED KINASE1 (BAK1). Pathogen Pythium graminicola manipulates plant BR pathway to antagonize SA- and GA-mediated defenses to promote its infection [2O2 levels in apoplast [GAs, CKs, auxins, and BRs also play important roles in plant development and immune system. GAs regulate these two processes by mediating the degradation of DELLA proteins . DELLAs  pathway . CKs rec pathway . Auxins  pathway . Biotrop pathway . Auxins nfection . Meanwhiapoplast .PDF1.2 and PR1, respectively. In addition, a high concentration of JA is required for the induction of H2O2 accumulation, thus JA might promote leaf senescence by partially interacting with ROS [Phytohormones SA, ET, JA, ABA, and BRs play positive roles in regulating leaf senescence, whereas CK, auxins, and GA delay this process . The conwith ROS .SA and ROS play important roles in the regulation of plant development as well as biotic and abiotic stress responses .2+ influx into the cells, activate MAPK cascades, and regulate the redox of TFs, thus activate the expression of defense-related genes [Biotic stresses (focused on pathogen attacks) cause changes of SA and ROS contents by multiple layers of transcriptional regulation . SA is ped genes ,160. Raped genes ,10.Drought, salt, cold, and heat are the most common adverse abiotic stresses that seriously influence plant growth and development. Water deficit increases the levels of SA/ROS, and drought stress induces SA-responsive PR genes , suggestsid2 seeds is hypersensitive to salt stress. The endogenous H2O2 is elevated in wild type and sid2 seeds under high salinity. However, the level of H2O2 is reduced after treatment with SA, suggesting that SA plays an essential role in seed germination under salt stress by balancing ROS [SA is also involved in salt tolerance. The germination of cing ROS . The loscing ROS .Arabidopsis, NTL6 has been demonstrated to be an important regulator in linking cold signals with pathogenesis [PR1, PR2, and PR5. However, NTL6-mediated PR genes expression is independent of SA [Cold stress also increases the levels of SA/ROS , indicatogenesis . Cold cont of SA . Recentlnt of SA . Howevernt of SA .cpr5 (constitutive expressor of PR genes), a SA constitutive accumulation mutant, displays a high thermotolerance, while SA-deficient NahG transgenic plants exhibit an opposite phenotype. The overexpression of WRKY39 enhances thermotolerance by upregulating SA-related genes [Benzothiadiazole (BTH), an SA analogue, can induce thermosensitive genes, indicating that SA is involved in heat stress . Applicaed genes . ROS acced genes .SAGs are also involved in regulating plant immunity, and vice versa. Thus, in-depth understandings of the cross-regulatory mechanisms between leaf senescence and plant immunity will facilitate the cultivation of high-yield and disease-resistant crops through molecular breeding strategy.Numerous signaling components are involved in both leaf senescence and plant immunity, which suggests that these two events are not independent biological processes and share partial regulatory networks. It is apparent that SA and ROS play important and overlapping roles in leaf senescence and disease resistance. The co-players promote leaf senescence and immune response by converging on the SA and ROS pathways, which explains the phenomenon that the early-senescing mutants with increased SA and/or ROS levels are more resistant to biotrophic pathogens, but more susceptible to necrotrophic pathogens. The relationships between these two processes have not been systematically studied, despite outstanding progresses have been made over the past few decades in the individual field of leaf senescence and plant immunity. Based on our above-mentioned gene network analysis, it is reasonable to speculate that a large number of


Flavivirus are distributed globally and cause significant human disease and mortality annually. Flavivirus infections present a spectrum of clinical manifestations, ranging from asymptomatic to severe manifestations, including hemorrhage, encephalitis and death. Herein, we describe 3 case reports of cerebrovascular involvement in patients infected by dengue and Zika viruses in Sao Jose do Rio Preto, São Paulo State, Brazil, a hyperendemic area for arbovirus circulation, including dengue, yellow fever, chikungunya and Saint Louis encephalitis viruses. Our findings highlight the potential threat that unusual clinical manifestations may pose to arbovirus disease management and recovery.Arthropod-borne viruses (arboviruses) of the genus  Flavivirus includes several arboviruses that are considered significant threats to global public health [The genus c health , includic health . Conversc health  and its c health . While mc health ,14,15,16c health , describc health ,19, Guilc health ,20,21,22c health , transvec health ,25, encec health ,27,28, ac health ,33,34,35Studies suggest that brain bleeding and strokes are associated to virus-induced and/or immune-mediated endothelium injury and platelet dysfunction ,36,37. TThese case series were submitted and approved by the Ethical Review Board  of the School of Medicine of São José do Rio Preto (FAMERP), São Paulo, Brazil. Confidentiality was ensured by de-identifying of all questionnaires and samples before data entry and analysis.Through an arbovirus surveillance system already stablished in the city, all dengue-suspected cases with warning signs (DwWS) or severe disease (SD) were monitored by our team from admission to discharge. Between November 2018 and June 2019, 31,534 cases were laboratory-confirmed as dengue in the city, of which 551  cases were classified as DwWS or SD. Among them, 28 cases  were defined as SD according to the 2009 World Health Organization (WHO) dengue classification criteria , and 20 The samples were subjected to molecular and serological analyses, including Real Time Multiplex PCR (RT-PCR), Enzyme-Linked Immunosorbent Assay (ELISA) and Rapid Immunochromatographic assay (ICA), according to the sample collection time and onset of symptoms, following Brazilian and WHO guideline recommendations ,58. Deta® Viral RNA  following the manufacturer’s recommendations.Serum and CSF samples were used for viral RNA extraction and Real Time Multiplex PCR (RT-PCR). Briefly, virus RNA (vRNA) was extracted from 140 µL of sample using the Kit QIAmp® Kit by Promega. In fourplex reaction mixtures, 50 pmol (each) of DENV-1- and DENV-3-specific primers, 25 pmol each of DENV-2- and DENV-4-specific primers, and 9 pmol of each probe were combined in a 50-µL volume total reaction mixture. Real-time PCR was performed on a 96-well plate using the QuantStudio™ Dx instrument. Cycle threshold (Ct) values of less than 38 were interpreted as positive. Primer and probe sequences are available from the authors upon request [One-Step Real time multiplex PCR assays were performed using the GoTaq request .® Dengue NS1 ELISA , human anti-DENV IgM ELISA  and the EUROIMMUN human IgM anti-ZIKV ELISA . CSF samples were screened using the NovaTech human anti-DENV IgM and anti-ZIKV IgM ELISA kits . All assays were performed according to the manufacturer’s recommendations using the corresponding positive and negative controls for quality control. ELISA plates were read using a Spectramax Plus ELISA reader at 450 nm . Results were expressed in Standard Units (PU) and interpreted as <0.8 negative, >0.8 and <1.1 equivocal and >1.1 positive.Serum samples were screened for exposure to dengue and Zika infection using the PanBio®  rapid immunochromatographic test kit was used for the detection of NS1 antigen and IgM and IgG DENV antibodies. Briefly, for NS1 antigen detection 100 µL of sample (serum or CSF) was applied to the corresponding well and allowed to incubate for 20 min. The presence or absence of antigen was determined by the absence or presence of the corresponding band. For the detection of IgM and IgG antibodies, 10 µL of sample (serum or CSF) was applied to the corresponding well, and 30 µL of diluent was added and allowed to incubate for 15–20 min. An NS1 antigen test was interpreted as positive by the presence of 2 color lines (T band and C band) within the 15–20 min incubation period regardless of which color line appears first and negative by the presence of 1 color line within the 15–20 min incubation period. An IgM or IgG antibody test is interpreted as positive when the control (C) line and the IgM (M) or IgG (G) line are visible and negative when only the control (C) line is visible.The SD BIOLINE Dengue DUO3. No acute mass or vascular lesions were observed by computed tomography . A new serum sample obtained upon her admission to ICU was tested positive for DENV by IgM and IgG ELISA and ICA, respectively, and to DENV-2 by RT-PCR . Her hemmography A; howevemography B–H. Rheu3 and platelets count 83,000/mm3). One day later, he developed mental confusion and decreased consciousness level. Immediate computed tomography showed evidence of hypodense images in right parieto-occipital (0.5 cm) and left front-temporal (1.6 cm) areas, middle-line deviation (1.5 cm) and left lateral ventricular and cerebellar cisterns compression, compatible as subarachnoid hemorrhage . Seven days later, the patient exhibited sudden reduced mental awareness, cardiac arrest and death. The last laboratory exams showed hematocrit levels of 22% and platelet count of 35,000 mm3.A 73-year-old male with diabetes and prostatic neoplasia with bone metastasis was admitted to our hospital with symptoms of myalgia, headache, nausea, vomiting, arthralgia and exanthema that appeared 2 days prior to his admission. He was conscious, communicating and with stable vital signs , glucose 48 mg/dL, protein 43 mg/dL, bacteria culture negative). IgM capture ELISA on serum and CSF samples were both negative for DENV and positive for ZIKV. The patient was subjected to cerebral drainage, received supportive care treatment and 53-days-later was discharged, totally dependent on others for daily functions, such as hygiene care, food and mobility.A 65-year-old male presented to our hospital with symptoms of headache, altered consciousness level (confusion), drowsiness, dark urine and malaise that occurred a week prior to his admission. Upon admission, examination showed jaundice, disorientation, absence of neck rigidity or pupil alterations, a blood pressure of 158/90 mmHg and a heart rate of 78 bpm. Interview of his relatives indicated an absence of symptoms, including fever or dengue-like symptoms, up to the point of hospital admission and no recent travel history. NMR showed posterior meningium thickening (data not shown) and a reduced cerebral activity  was observed in the electroencephalogram cerebral. A subsequent brain NMR performed 48 h later showed the presence of subarachnoid hemorrhage in the cerebellar tent, cerebellar sickle and right hippocampal groove A,B. CereIn recent years, flavivirus-associated neurological manifestations have often been reported. However, their incidence rate remains unclear for both Zika and dengue ,61, espeThe current dengue clinical classification, proposed by WHO in 2009, includes central nervous system (CNS) involvement in the definition of SD requiring intensive care and supportive management . DENV inSeveral factors may contribute to neurological manifestations of dengue, ranging from direct viral invasion of CNS to vascular and metabolic abnormalities , includiSimilar to DENV, ZIKV neurovirulence and tropism have been well-established with a myriad of syndromes, such as CZS, GBS and others ,82,83,84The NS1 glycoprotein is involved in viral replication, immune evasion, and vascular leakage during flavivirus infection . In addiβ, IL-1β and IL-12) and endothelial cell monolayer permeability via toll-like receptor (TLR) 4 activation [Rhodobacter sphaeroides lipopolysaccharide (LPS-RS) inhibits vascular leakage [A recent in vivo and in vitro study demonstrated that flavivirus secreted NS1 alters endothelial cell permeability and induces disruption of critical membrane components. While ZIKV NS1 has caused increased vascular permeability in both umbilical vein and developing brain endothelial cells, DENV NS1 has induced hyperpermeability in more tissues, such as the lung, skin, liver, brain and placenta . Thus, Ntivation  and effetivation . Further leakage . Since NWhile the clinical association between flavivirus and cerebrovascular events is challenging, obtaining a differential diagnosis is even more challenging. According to national and international guidelines, the presence of NS1 antigen, DENV RNA and/or anti-dengue IgM is enough for diagnosis of acute infection ,58. As pFlavivirus [β among other cytokine by monocytes [β upregulation was observed in human brain microvascular endothelial cells after ZIKV infection, followed by decreased proinflammatory cytokine levels when IL-1 receptor antagonist was added to model [Cerebrovascular events in ZIKV-infected patients are even less frequent than in dengue infected patients . Consideavivirus , and theavivirus , it is ravivirus . The endonocytes . The IL-to model . Once thto model . Indeed,to model ,111,112,Because of ZIKV short viremia, serum RNA-negative results do not exclude recent ZIKV infection. For this reason, since February 2016, when Zika emergence became a public health concern , the CenBoth DENV and ZIKV may infect the central nervous system through a hematogenous route by crossing the BBB facilitated by the inflammatory response and permeability increase . Althoug


Type 2 diabetes mellitus (T2DM) and osteoarthritis (OA) are common chronic diseases that frequently co-exist. The link between OA and T2DM is attributed to common risk factors, including age and obesity. Several reports suggest that hyperglycemia and accumulated advanced glycosylation end-products might regulate cartilage homeostasis and contribute to the development and progression of OA. Metformin is used widely as the first-line treatment for T2DM. The drug acts by regulating glucose levels and improving insulin sensitivity. The anti-diabetic effects of metformin are mediated mainly via activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), which is an energy sensing enzyme activated directly by an increase in the AMP/ATP ratio under conditions of metabolic stress. Dysregulation of AMPK is strongly associated with development of T2DM and metabolic syndrome. In this review, we discuss common risk factors, the association between OA and T2DM, and the role of AMPK. We also address the adaptive use of metformin, a known AMPK activator, as a new drug for treatment of patients with OA and T2DM. Osteoarthritis (OA) and type 2 diabetes mellitus (T2DM) are common chronic diseases worldwide. About 237 million people, or 3.3% of the world’s population, suffer from OA, a condition that is much more common in older individuals ,2. AboutOA is the most common degenerative joint disease, affecting an estimated 12%–15% of the global population aged 25–74 years . Over 70T2DM is a form of diabetes characterized by high blood sugar, insulin resistance, and a relative lack  of insulin. Hyperglycemia leads to development of a metabolic syndrome (MetS) that initiates chronic inflammation in patients ,19,20. TCo-existence of OA and T2DM in older adults is common. Although the reason for increased prevalence of arthritis in patients with T2DM is not clear, shared risk factors such as aging, obesity, and chronic inflammation may be one explanation .Aging is one of the most common and dominant risk factors for development of OA and T2DM. In both diseases, cell function declines with age . In OA, 2) or overweight (30 kg/m2 ≥ BMI ≥ 25 kg/m2) [Obesity is a well-known risk factor for initiation and progression of knee OA and T2DM ,35. Up t5 kg/m2) . Excess 5 kg/m2) ,46. Most5 kg/m2) . InsulinWhereas excess weight might explain the increased risk of OA in weightbearing joints such as the knee and hip, it is difficult to explain onset of OA in non-weight-bearing joints such as those in the hand . FurtherOA and T2DM are recognized as low grade inflammatory conditions, and the pathogenic roles for inflammatory mediators have been elucidated. IL-1, a well-studied cytokine in the context of OA, plays a prominent role in inducing expression of MMPs and other catabolic genes ,54. IL-1IL-1β induces production of IL-6, IL-8, and leukemia-inducing factor, all of which have additive or synergistic effects on the chondrocyte catabolic cascade . ConsequOncostatin M acts synergistically with IL-1, IL-1β, and TNF-α to suppress expression of a number of genes associated with differentiated chondrocyte phenotypes; such genes include those encoding ACAN and COL2A1. ADAMTS-5, an aggrecanase belonging to the ADAMTS family  of extracellular proteinases, is considered to be the major aggrecan-degrading enzyme involved in cartilage degradation in OA. Several studies show marked regulation of ADAMTS-4 and ADAMTS-5 after stimulation with TNF-α and oncostatin M, as well as IL-1-induced expression of ADANTS-4, by both human and mouse chondrocytes ,61. Both mechanical stress and inflammatory mediators induce NF-κB (p65/p50)- and activate mitogen-induced protein kinase (MAPK)-mediated downstream signaling pathways, which are abnormally activated in osteoarthritic chondrocytes . The canIn addition to NF-κB pathways, mechanical and inflammatory stimuli in articular chondrocytes MAPK pathways through the ERK, c-Jun N-terminal kinase (JNK), and p38 kinase cascades. Activated transcription factors, including those of the ETS, AP-1, and C/EBP families, regulate expression of genes related to catabolic and inflammatory responses. JNK-driven activation of AP-1, MEK/ERK-induced phosphorylation of ETS factors, and p38-mediated activation of C/EBPβ and RUNX2 participate in induction of MMPs and in regulation of catabolic and inflammatory responses ,66.Under obese conditions, pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β activate the JNK and IKKβ/NF-κB pathways in adipocytes, hepatocytes, and associated macrophages; moreover, MCP-1 and other chemokines play essential roles in recruiting macrophages to adipose tissue . ObesityAs obesity progresses, adipocytes release adipokines such as leptin, adiponectin, resistin, and visfatin from adipose tissue, which is considered to be a metabolic endocrine organ . These aLeptin levels in OA cartilage are higher than those in normal articular cartilage, and expression levels of adipokines increase upon inflammatory stimulation . In addiAnother adipokine expressed in adipose tissue, adiponectin, plays a role in cartilage hemostasis by increasing expression of tissue inhibitor of metalloprotease-2 (TIMP-2), and by decreasing IL-1β-induced expression of MMP-3, in chondrocytes . In addiElevated leptin levels are associated with insulin resistance and with development of T2DM, obesity, and hypertension. MetS is more common in T2DM patients with increased leptin levels ,83,84,85T2DM and OA are linked by the chronic systemic inflammation related to MetS ,87. UndeAnother pathogenic role of hyperglycemia is induction of AGEs; age-related accumulation of AGEs in articular cartilage results in a pathogenic environment and, ultimately, induces symptoms of OA such as stiffness and cartilage degradation ,93. HighHuman chondrocytes express functional insulin receptors that respond to physiologic insulin concentrations. Expression and activity of insulin receptors in OA chondrocytes is lower than that in normal chondrocytes . TreatmeIn conclusion, low grade inflammation, oxidative stress, and dysregulation of cell function mediated by aging, obesity, and hyperglycemia are common risk factors for OA and T2DM, suggesting a strong relationship between the two diseases ,98.This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.Recent studies demonstrate that the risk for OA in obese patients with MetS is higher than that for obese patients without MetS . In addiDrug repositioning is a promising field that identifies new therapeutic opportunities for existing drugs . Drug reThe link between OA and T2DM means drug repositioning can be used to identify new interventions. In terms of drug repositioning, well-known oral anti-diabetic drugs such as thiazolidinedione and lipid-lowering medications such as statins have been investigated as supplements to symptomalleviating treatments for OA in OA patients with T2DM ,103. FroAMPK plays an important role in insulin signaling, whole-body energy balance, and metabolism of glucose and fats . It is aMechanistically, AMPK activation is associated with inhibition of TNF-α-mediated NF-κB signaling pathways, which are critical for pro-inflammatory effects. AMPK also downregulates the JAK/STAT signaling pathway, a crucial driver of cytokine signaling, cell growth, and apoptosis ,115,116.Since diabetic patients are at higher risk of bone degradation, anti-diabetic mediations may have beneficial effects against bone disorders ,118,119.T2DM patients are more likely to have hand or knee OA than non-diabetic subjects. Conversely, subjects with OA have an higher risk of developing T2DM than age- and sex-matched non-OA counterparts. Based on the biological effects mediated by targeting the pathogenic mechanisms of OA, metformin (in addition to weight loss) might be considered as a potential disease-modifying agent for the obese phenotype of knee OA. A recent prospective cohort study reported a relationship between metformin use and reduced progression of knee OA in obese individuals; indeed, compared with non-users, those using metformin showed a decreased rate of medial cartilage volume loss over 4 years, and a trend toward a decreased risk of undergoing total knee replacement over 6 years . AnotherIn this review, we discussed common risk factors for OA and T2DM, which include aging, obesity, and cytokine- and adipokine-mediated inflammation. These shared pathogenic factors link development of two chronic diseases: OA and T2DM. Accumulated evidence suggests that T2DM could be an independent risk factor for OA development. These findings raise the possibility that medications used to treat T2DM can also be used to treat OA . Among t


Inula britannica contains various phenolic compounds with prophylactic properties. This study aimed to determine the anti-adipogenic effect of an I. britannica flower aqueous extract (IAE) and its underlying mechanisms in the 3T3-L1 preadipocytes and to identify the phenolic compounds in the extract. Treatment with IAE inhibited the adipogenesis of 3T3-L1 preadipocytes by showing a dose-dependently suppressed intracellular lipid accumulation and significantly mitigated expression levels of lipogenesis- and adipogenesis-associated biomarkers including transcription factors. IAE exerted an anti-adipogenic effect through the modulation of the early phases of adipogenesis including mitotic clonal expansion (MCE). Treatment with IAE inhibited MCE by arresting the cell cycle at the G0/G1 phase and suppressing the activation of MCE-related transcription factors. Furthermore, IAE inhibited adipogenesis by regulating the extracellular signal-regulated kinase 1/2 and Akt signaling pathways. Protocatechuic acid, chlorogenic acid, kaempferol-3-O-glucoside, and 6-methoxyluteolin, which are reported to exhibit anti-adipogenic properties, were detected in IAE. Therefore, modulation of early phases of adipogenesis, especially MCE, is a key mechanism underlying the anti-adipogenic activity of IAE. In summary, the anti-obesity effects of IAE can be attributed to its phenolic compounds, and hence, IAE can be used for the development of anti-obesity products.The flower of  Adipose tissue is crucially involved in various biological functions such as energy homeostasis, hormonal regulation, and metabolism by secreting the hormones, growth factors, and adipokines as well as function as an energy reservoir ,2. HowevAdipogenesis, which is a multi-step process of adipocyte formation, involves commitment of pluripotent mesenchymal stem cells into preadipocytes and the differentiation of preadipocytes into mature adipocytes. The 3T3-L1 cell line, which is derived from the murine preadipocyte, is a well-established model for studying adipogenesis and can be differentiated into mature adipocytes under experimental conditions ,9. AdipoAdipogenesis is modulated by multiple transcription factors such as signal transducer and activator of transcription-3 (STAT3), CCAAT/enhancer-binding proteins (C/EBPs), and peroxisome proliferator-activator receptor (PPAR)-γ . During Inula britannica is a flowering wild plant distributed in eastern Asia and is used for medicinal purposes based on traditional applications. The flower of I. britannica exhibits neuroprotective, antimicrobial, anti-tumor, and anti-inflammatory properties  = 315.25656), which was consistent with the results of previous studies on the major phenolic compounds of I. britannica flower [O-glucoside (54.842 ± 0.191 μg/mg), followed by chlorogenic acid (16.540 ± 0.094 μg/mg), 6-methoxyluteolin (6.669 ± 0.286 μg/mg), and protocatechuic acid (3.998 ± 0.027 μg/mg). However, the levels of gallic acid were lower than the limit of quantification. These results suggest that IAE suppresses lipid accumulation by inhibiting the development of adipogenesis, which may be mediated by identified anti-obesogenic phenolic compounds in the extract.The phenolic compounds of IAE were examined using UPLC-ESI-Q/Orbitrap tandem mass spectrometry analysis and quantified using HPLC analysis . IAE coma flower ,27,28. Aa flower . The pheI. britannica by demonstrating the anti-adipogenic effect of IAE and its underlying cellular mechanisms in the 3T3-L1 preadipocytes. Additionally, phenolic constituents of IAE were identified.Obesity is reported to be associated with increased risk of onset of various degenerative diseases including metabolic syndrome morbidities. The aberrant growth of adipose tissue, which is a characteristic feature of obesity, is dependent on the hypertrophy and/or hyperplasia of adipocytes . AdipocySrebp1c (SREBP-1c), Fasn (FAS), Fabp4 (FABP4), Plin1 (perilipin), and Scd1 (SCD-1), thereby establishing an adipocyte phenotype [Edgeworthia gardineri flower, Aster spathulifolius, and Viburnum opulus fruit extracts have been reported to inhibit development of adipogenesis by regulating the expression of C/EBP-α and PPAR-γ, which results in decreased accumulation of intracellular lipids [The differentiation of adipocytes accompanies upregulated de novo lipogenesis, which is modulated by various transcription factors such as C/EBPs and PPARs and results in the accumulation of intracellular lipids . In parthenotype ,22. Numehenotype . Similarhenotype . Therefor lipids ,40,43. OKIP1, a CDK inhibitor protein. However, hormonal stimulation induces the degradation of CDK inhibitor proteins and consequently promotes cell cycle progression by sequential activation and assembly of cyclin D1/CDK4/CDK6, cyclin E/CDK2, cyclin A1/CDK2, and cyclin B1/CDK1 (cdc2) complexes. In particular, cyclin E/CDK2 and cyclin A1/CDK2 complexes play crucial roles in cell cycle progression by mediating the G1/S phase transition and S phase progression. Furthermore, C/EBP-β and STAT3 are activated by acquiring DNA-binding capacity during the S phase [KIP1 contributes to the inhibition of MCE and adipocyte differentiation, whereas the degradation of p27KIP1 promotes adipogenesis and increases fat mass, indicating that p27KIP1 potentially inhibits hyperplasia [KIP1. Additionally, IAE inhibited the expression of C/EBP-β and the activation of STAT3. Consistent with preceding results, previous studies have reported that plant extracts and their phytochemicals inhibit adipogenesis and lipid accumulation through suppression of MCE, which was dependent on the regulation of cell cycle progression and/or suppression of transcription factors [Gleditsia sinensis fruit extract suppressed MCE and adipogenesis through the inhibition of p27KIP1 degradation and the activation of STAT3 [During the early phases of adipogenesis, quiescent preadipocytes are induced to undergo MCE upon hormonal stimulation. Hence, growth-arrested preadipocytes synchronously re-enter the cell cycle and consequently undergo one or two rounds of mitosis, which increases the numbers of adipocytes and results in adipocyte hyperplasia ,22. Desp S phase ,12,22,46 S phase . Severalerplasia ,49. Ther factors ,51,52. FKIP1 [Clitoria ternatea flower and coffee extracts inhibited MDI-induced phosphorylation of ERK 1/2 and Akt, which resulted in the inhibition of adipogenesis development [The ERK 1/2 and Akt cellular signaling pathways are involved in MCE and adipogenesis, depending on the modulation of proliferation, differentiation, and lipid metabolism of adipocytes ,33,34,53KIP1 ,22,33. TKIP1 ,49. Our elopment ,50.O-glucoside, and 6-methoxyluteolin. Dietary flavonoids such as kaempferol, quercetin, and curcumins inhibited the adipogenesis of 3T3-L1 preadipocytes by suppressing MCE and regulating adipogenic transcriptional cascades [p-coumaric acid suppressed intracellular lipid accumulation by inhibiting the expression of lipogenic enzymes, which results from the downregulated expression of C/EBP-α and PPAR-γ [Phenolic compounds including phenolic acids and flavonoids have been demonstrated to exhibit anti-obesogenic properties such as the suppression of adipogenesis and the degradation of intracellular lipids ,38,39. Tcascades ,49. For cascades . Similard PPAR-γ ,39. Mored PPAR-γ ,55. AddiThis study revealed the anti-obesity potential of IAE by demonstrating the anti-adipogenic effects of IAE and elucidating the underlying mechanisms during the differentiation of 3T3-L1 preadipocytes. The inhibitory effect of IAE on MCE during the early phases of adipogenesis primarily contributed to the suppressed expression of adipogenesis-associated biomarkers, which resulted in the inhibition of adipogenesis and lipogenesis. Additionally, IAE exerted an anti-adipogenic effect through the regulation of the ERK 1/2 and Akt cellular signaling pathways. Furthermore, IAE comprised anti-obesogenic phenolic compounds. Although further in vivo studies are needed to confirm the anti-obesity properties of IAE, the findings of this study indicate that IAE can potentially prevent obesity by regulating the hyperplasia and hypertrophy of adipocytes. Thus, IAE might be considered as a potential therapeutic agent for obesity. Overall, IAE could be potentially used for the development of prophylactic and therapeutic products for obesity.


Bothrops jaracaca induce rapid hemostatic disturbances in victims, leading to systemic bleedings, thrombocytopenia and consumption coagulopathy. Although circulating levels of coagulation factors recover rapidly after administration of specific antivenom therapy, it is unclear if B. jararaca venom (BjV) upregulates the mRNA synthesis of hepatic hemostasis-related factors, or if the recovery occurs under basal conditions after the neutralization of venom components by antivenom. Thus, we aimed to investigate if BjV regulates gene expression of important hemostasis-related factors synthetized by the liver. On that account, Swiss mice were injected with saline or BjV , and after 3, 6 and 24 h blood samples and liver fragments were collected to analyze mRNA expression by real-time qPCR. Increased gene expression of fibrinogen chains, haptoglobin and STAT3 was observed during envenomation, particularly at 3 and 6 h. At 24h, mRNA levels of F10 were raised, while those of Serpinc1, Proc and Adamts13 were diminished. Surprisingly, F3 mRNA levels were steadily decreased at 3 h. Gene expression of Thpo, F7, F5 Tfpi, Mug1 was unaltered. mRNA levels of Vwf, P4hb, F8, F2, Plg, and Serpinf2 were minimally altered, but showed important associations with Nfkb1 gene expression. In conclusion, snakebite envenomation upregulates hepatic mRNA synthesis particularly of fibrinogen chains, and acute-phase markers. This response explains the fast recovery of fibrinogen levels after antivenom administration to patients bitten by B. jararaca snakes.Few studies have addressed gene expression of hemostasis-related factors during acute thrombo-hemorrhagic diseases. Bites by the lanced-headed viper  Bothrops jararaca snakebites induce hemostatic disturbances in man and animals, but no information is available about how the liver, the main organ involved in the synthesis of hemostasis-related factors, copes with this condition. In an effort to understand how these factors recover after B. jararaca snakebites, we evaluated the hepatic gene expression of hemostatic-related factors after experimental envenomation in mice. We noticed that a marked increase in gene expression of fibrinogen chains occurred 3 and 6 h after subcutaneous administration of B. jararaca venom, explaining thereby the rapid recovery of the coagulation disturbance after administration of specific antivenom to patients. Interestingly, gene expression of other coagulation factors consumed during envenomation, e.g. factors V, VIII, and prothrombin, was scarcely affected over time, while the expression of tissue factor mRNA was promptly decreased at 3 h. Moreover, gene expression of acute-phase proteins was modified by envenomation, as well as of the transcription factor STAT3. Gene expression of NF-κB1 was not altered significantly over time, but it was associated with the regulation of mRNA levels of various hemostasis-related genes. Our results showed that B. jararaca envenomation induces a rapid change in gene expression of some coagulation factors and acute phase proteins. In 1.8–2.7 million patients bitten by various venomous snakes worldwide annually, hemostatic disorders are commonly observed , leadingBothrops genus–popularly known as jararacas in Brazil–inhabit most regions in Central and South America, where they are an important public health problem [Bothrops jararaca (BjV), a snake species found in the southeastern region of Brazil, contains various enzymatic and non-enzymatic proteins–e.g., phospholipases A2, snake venom serine proteinases (SVSP), snake venom metalloproteinases (SVMP), L-amino acid oxidases, disintegrins and C-type lectins, among others [Snakes from  problem , 9, 10. g others . These pg others –14, leadg others , 16. Besg others . This exg others , 19.B. jararaca snakebites include direct proteolytic activity of snake venom enzymes on fibrinogen, prothrombin, and factors X, VIII and V, in a mechanistic process that is distinct from the extrinsic and intrinsic coagulation pathways, and by a feedback mechanism of these activated factors, especially meizothrombin or thrombin, which ultimately leads to activation and consumption of coagulation factors in circulation, as well as exacerbated secondary fibrinolysis [B. jararaca envenomation [Hemostatic abnormalities evoked by inolysis , 20–23. nomation , 22, 24,nomation , 25. Granomation . In healnomation , 27.B. jararaca envenomation [ca. 90 h and is synthetized at a rate of ca. 34 mg/kg/day [B. jararaca [Due to the effectiveness of specific serotherapy to treat coagulopathy and thrombocytopenia evoked by nomation , restoranomation , based onomation , 30, sugg/kg/day . Thus, ig/kg/day , not expjararaca . We hypojararaca , a modelB. jararaca snakes was obtained from the Laboratory of Herpetology, Butantan Institute . Bothrops antivenom was kindly donated by Butantan Institute (lot: 1305077). All reagents were of analytical grade or better.Lyophilized venom from adult specimens of Male Swiss mice (n = 30), weighing 30–35 g, were obtained from the Animal Facility of Butantan Institute, and were maintained with free access to food and water. The experimental procedures involving mice were in accordance with National Guidelines, and were approved by the Institutional Animal Care and Use Committee from Butantan Institute (CEUAIB 4388061115) and from the School of Medicine, University of São Paulo (protocol 188/15). All procedures involving animals were in accordance with the National Guidelines from Conselho Nacional de Controle de Experimentação Animal (CONCEA) . Blood aMice were randomly assigned to two experimental groups . Mice from the saline control group were injected subcutaneously (s.c.) with sterile saline alone (vehicle), and mice from the BjV group were injected with freshly diluted BjV . The dose of BjV was based on a previous report , and it 2EDTA for complete blood cell counts. Blood was also added to plastic flasks containing the anticoagulant CTAD , and plasma was obtained by centrifugation at 2500 g for 15 min at 4°C. Bothrops antivenom (1 vol. to 100 vol. of blood) was added to all flasks to halt the in vitro activity of BjV after blood collection. Liver fragments from the central region of the left lateral lobe (50–100 mg) were collected, immersed in 500 μL of RNAlater  and stored at -80°C until used.After 3, 6 or 24 h, mice were anesthetized with isoflurane (induction and maintenance at 2.5%) for the collection of blood and tissues. Blood was collected from the caudal vena cava into plastic syringes, and added to plastic flasks containing 269 mM NaPlatelet counts were obtained using an automated cell counter  and plasma fibrinogen levels were measured with a colorimetric assay .Liver fragments were removed from RNALater, immersed in 1 mL of TRIzol reagent, incubated for 5 min at room temperature, and disrupted using FastPrep-24  for 60 s at 6.5 m/s. Total RNA extraction and purification were performed with TRIzol Plus RNA Purification Kit , following the manufacturer’s instructions. RNA concentration was quantified with Qubit 2.0 Fluorometer and Qubit RNA Assay Kit, Broad Range , following the manufacturer’s instructions. RNA quality was determined by micro-fluidic capillary electrophoresis in the Agilent 2100 Bioanalyzer  using the RNA Nano6000 kit, and all RNA samples showed a RNA Integrity Number (RIN) ≥ 7.9 . A pool Total RNA (1 μg) was reversed transcribed using the iScript cDNA Syntesis Kit , according to the manufacturer’s instructions.https://www.ncbi.nlm.nih.gov/tools/primer-blast/) or obtained from PrimerBank (https://pga.mgh.harvard.edu/primerbank/) [Genes and primers are listed in erbank/) . Primer Vwf, Adamts13, F3, F7, F8, F5, F10, Tfpi, and Thpo; 10 ng template per reaction for the genes Rplp0, P4hb, F2, Serpinc1, Proc, Plg, Serpinf2, Fga, Fgg 2, Mug1, Nfkb1 and Stat3; 1 ng template per reaction for the genes Fgb, Fgg 1 and Hp. The reaction mixture contained cDNA templates (in 1–2 μL), 5 μL of 2X Fast SYBR Green Master Mix , and 1 μL of gene-specific primers , in a final volume of 10 μL. PCR reactions were carried out in triplicate in 96-well format plates  using a StepOnePlus Real-Time PCR System , in a total of 40 cycles, following manufacture instructions.For qPCR reactions, cDNA samples were diluted to obtain 1–100 ng template per reaction, depending on the analyzed gene: 100 ng template per reaction for the genes Gapdh, Actb, Hprt, Rps29, B2m and Rplp0), and analyzed with geNorm [Rplp0 showed the best stability expression , and was chosen as the reference gene for the normalization of qPCR data. The relative gene expression in mice from saline control and BjV groups was analyzed by the 2-ΔΔCT method, using the pool of mRNA from six normal mice, loaded in each experiment, as the reference sample [-ΔΔCT values. For statistical analyses, ΔΔCT values were used to compare experimental groups using ANOVA and correlation tests (see underneath).For the normalization of data, amplification efficiencies of six reference genes were tested .Sample size, i.e., the number of mice allocated in each experimental group, was based on previous data of experimental envenomation that resulted in falls in platelet counts and fibrinogen levels around 50–75% at 3 h after injection . Normal Scatterplots and Pearson’s correlation matrices were created to examine the correlation coefficients and linear regression between variables, taking into account data from control and experimental groups together; whenever statistically significant correlation coefficients were observed (p< 0.05 and correlation coefficients> 0.700), the data were split into control and experimental groups and re-analyzed independently to check if the significance was due to false correlations between both groups. Confidence intervals (95%) for correlation values were estimated using a percentile bootstrap, using 1000 replicates, and reported inside square brackets. Correlation coefficients, linear regressions and ANOVA analyses were performed in SPSS (version 22).As expected, platelet counts  and fibrFga (Fgb (Fgg 1 (Fgg 2 (Hp), which showed a 4 to 6-fold increase at all time intervals , Fgg1  and Fgg2 . In contrast, regarding the BjV group, Fga expression only predicted 3–30% of the variance of Fgb and Fgg, unveiled by weak correlation coefficients  among these genes. These findings indicate that the regulatory mechanisms of Fga transcription may differ from those of other fibrinogen chains during envenomation.Fibrinogen synthesis seemed a coordinated process, once mRNA levels of fibrinogen chains correlated significantly among each other   [F3 gene expression was noticed in the liver. In fact, F3 (tor (TF) , 46, 47,fact, F3  was the F10 mRNA levels   was shown to be involved in the dimerization of VWF, a key step for VWF regulation [Vwf and P4hb  and plasminogen (gene Plg)–was significantly correlated  as index to predict this variation, these data suggest that Nfkb1 accounted for 60–80% of the expression variance of important hemostatic genes, indicating that Nfkb1 could be a relevant gene involved in the regulation of hemostasis-related genes.NF-κB1 is a transcription factor that plays an important role in immunity and inflammation . NF-κB1 .g., Vwf , P4hb  , 52, 55.ntivenom . The incke venom . The cooke venom –60, and ke venom , 61. Durxpressed , and FDPFurthermore, we showed that not only was that the gene expression of fibrinogen chains upregulated by envenomation, but also that of haptoglobin. Indeed, the stimulation of fibrinogen and haptoglobin genes is a well-known positive acute-phase response (APR) . APR is B. jararaca envenomation, the contribution of the inflammatory reaction or raised FDP levels to the upregulation of gene expression of fibrinogen chains and other APP in liver is difficult to separate. Infusion of the C. rhodostoma thrombin-like enzyme induced a rapid increase in Fga, Fgb and Fgg, peaking at 12–16 h [Hp around 24 h post-stimuli. Our findings showed that the relative mRNA levels of Fga, Fgb, Fgg1, Fgg2, and Hp were already elevated at 3 h, and tended to peak at 6 h. Thus, both the inflammatory reaction and the rapid rise in FDP levels tended to accelerate the synthesis of these genes during B. jararaca envenomation. Moreover, secondary infections, which occur in some patients bitten by B. jararaca at later periods [During  12–16 h . On the  12–16 h  or turpe 12–16 h , induced periods , might uStat3 expression was indeed increased in the acute-phase of B. jararaca envenomation. IL-6 signaling not only activates STAT3, but also induces Stat3 expression, via IL-6 responsive element in the gene promoter, explaining thereby the Stat3 mRNA increase in hepatocytes during envenomation and the generation of an APR [Reasoning about important transcription factors in hepatocytes, which could be linked to APR, we evaluated gene expression of STAT3 and NF-κB1. Hepatic STAT3 functions as a mechanism for the control of systemic inflammation, and upon stimuli IL-6 activates STAT3, which in turn induces the transcription of APPs genes , 70. In f an APR , 72.Fga mRNA expression nor plasma fibrinogen levels were strongly associated with the gene expression levels of other fibrinogen chains after venom injection. In rats, gene expression of Fga differs from that of Fgb or Fgg, since the response of Fga to IL-6 stimuli is not mediated by STAT3 [Our results showed that neither by STAT3 , 73. Eveby STAT3 , 70, butBothrops envenomation, the hemostasis-related genes Vwf, P4hb, Thpo, F7, F8, F5, F2, Tfpi, Plg, Serpinf2 and Mug1 showed no quantitative difference in gene expression, but their expression was associated among each other and with Nfkb1. Interaction between transcription factors STAT3 and NF-κB is important for the transcriptional regulation of several genes related to modulation of inflammatory responses [Nfkb1 may also exert suppressor activity on inflammatory pathways [Nfkb1 may also be associated to the regulation of hemostasis-related genes. Moreover, our findings indicate that the restoration of prothrombin and factors V, VIII and X, which are decreased during envenomation [Despite the coagulopathy observed following esponses , 51. Howpathways  and our nomation , 21, seeB. jararaca envenomation [F7 mRNA levels are neither altered. However, we showed for the first time a decrease in hepatic F3 mRNA, which occurred as soon as 3 h after BjV administration, returning to control levels only at 24 h. TF expression is well known to differ depending on the tissue being analyzed, and to be regulated by several transcription factors, pathways and stimuli [Circulating levels of factor VII were not altered during nomation , and her stimuli . Interes stimuli , 76. FurF3 gene expression during envenomation, gene expression of Adamts13, Serpinc1 and Proc decreased, whilst that of F10 increased at a later period of envenomation (24 h). Similar results were reported in an experimental model of direct liver hyperplasia and were associated to liver regeneration [Proc and Serpinc1, and Plg and Serpinf2 were already reported to be associated with liver function and liver regeneration [Differently from the early pattern of neration . This maneration , 79.Although we could not evaluate all proteins whose genes have been studied due to limitations in the blood volume obtained from mice, it is important to emphasize that few studies have addressed how gene expression of various hemostatic and inflammatory factors synthetized by the liver occurs under steady state conditions and under acute systemic injuries. Understanding these mechanisms, using models that disturb both systems simultaneously, is important to increase our knowledge about how coagulation and inflammation are regulated under protein and gene levels.In conclusion, our results demonstrate that envenomation by BjV induces different expression patterns of hemostasis-related genes. The association of gene expression of hemostasis-related factors, inflammatory proteins and transcription factors reaffirms the complex interactions that occur during envenomation. Moreover, the rapid induction of mRNA synthesis of fibrinogen chains explains the fast recovery of fibrinogen levels observed in bitten patients after antivenom therapy.S1 Table(PDF)Click here for additional data file.