Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, resulting in motor deficits. The exact etiology of PD is currently unknown; however, the pathological hallmarks of PD include excessive production of reactive oxygen species, enhanced neuroinflammation, and overproduction of α-synuclein. Under normal physiological conditions, aggregated α-synuclein is degraded via the autophagy lysosomal pathway. However, impairment of the autophagy lysosomal pathway results in α-synuclein accumulation, thereby facilitating the pathogenesis of PD. Current medications only manage the symptoms, but are unable to delay, prevent, or cure the disease. Collectively, oxidative stress, inflammation, apoptosis, and autophagy play crucial roles in PD; therefore, there is an enormous interest in exploring novel bioactive agents of natural origin for their protective roles in PD. The present study evaluated the role of myrcene, a monoterpene, in preventing the loss of dopaminergic neurons in a rotenone (ROT)-induced rodent model of PD, and elucidated the underlying mechanisms. Myrcene was administered at a dose of 50 mg/kg, 30 min prior to the intraperitoneal injections of ROT (2.5 mg/kg). Administration of ROT caused a considerable loss of dopaminergic neurons, subsequent to a significant reduction in the antioxidant defense systems, increased lipid peroxidation, and activation of microglia and astrocytes, along with the production of pro-inflammatory cytokines  and matrix metalloproteinase-9. Rotenone also resulted in impairment of the autophagy lysosomal pathway, as evidenced by increased expression of LC3, p62, and beclin-1 with decreased expression in the phosphorylation of mTOR protein. Collectively, these factors result in the loss of dopaminergic neurons. However, myrcene treatment has been observed to restore antioxidant defenses and attenuate the increase in concentrations of lipid peroxidation products, pro-inflammatory cytokines, diminished microglia, and astrocyte activation. Myrcene treatment also enhanced the phosphorylation of mTOR, reinstated neuronal homeostasis, restored autophagy-lysosomal degradation, and prevented the increased expression of α-synuclein following the rescue of dopaminergic neurons. Taken together, our study clearly revealed the mitigating effect of myrcene on dopaminergic neuronal loss, attributed to its potent antioxidant, anti-inflammatory, and anti-apoptotic properties, and favorable modulation of autophagic flux. This study suggests that myrcene may be a potential candidate for therapeutic benefits in PD. Parkinson’s disease (PD) is a common aging-related neurodegenerative disease is clinically characterized by impaired motor function along with symptoms including bradykinesia, rigidity, tremor, postural changes, cognitive abnormalities, emotional, and olfactory alterations ,2. IntraOxidative stress involves the overgeneration of reactive oxygen species (ROS), which cause excessive lipid and protein oxidation, mitochondrial dysfunction, reduced kinase activity, and protein misfolding, leading to the loss of dopaminergic neurons ,8,9,10. Considering the role of oxidative and nitrative stress, inflammation, apoptosis, and autophagy in triggering dopaminergic neurodegeneration, approaches to tackle these events using bioactive agents of natural origin are gaining attention ,25,26,27To induce dopaminergic neurodegeneration, we employed a rodent model of PD induced by rotenone (ROT), a plant-extracted chemical pesticide/insecticide. It is an environmental toxin and is widely known to induce biochemical, molecular, and pathological changes, as seen in PD, which is ascribed to its mitochondrial complex-1 inhibitor properties . ROT is p < 0.001) and decrease in GSH  levels in the mid brain compared to the control group. However, myrcene treatment significantly reduced MDA levels and significantly (p < 0.05) increased GSH levels in ROT-injected rats. ROT injections also induced a significant reduction in SOD  and catalase  activities, in comparison with the control group. Similar to the improvement in GSH levels, myrcene treatment significantly (p < 0.05) improved SOD and catalase activities in ROT-injected rats.The effects of myrcene on oxidative stress markers are shown in p < 0.001), IL-1β , TNF-α , and MMP-9  levels compared to the control group. However, myrcene treatment significantly reduced the pro-inflammatory cytokines and MMP-9 levels compared to those in rats injected with ROT. In addition to pro-inflammatory cytokines, iNOS and COX-2 were also detected in midbrain samples (p < 0.001) and COX-2  expression (p < 0.001), Bcl-2  and BAX/Bcl-2 ratio)  in dopaminergic neurons was determined  and MMP-9 are shown in  samples A. ROT intermined . ROT injtermined Bp < 0.000), represented by a 50% decrease in the intensity of TH +ve striatal fibers (p < 0.000) expression in striatal fibers, as demonstrated by immunohistochemical analysis.The loss of TH-positive neurons in the SN and the subsequent reduction in striatal TH expression are characteristic pathogenic processes in PD. Therefore, we assessed the number of TH +ve dopaminergic neurons in the SN, as well as their expression in the striatum. ROT injections into the rats resulted in the loss of TH-positive neurons  and appear with large cell bodies and lesser processes along with activated astrocytes, as demonstrated by the increased expression  of GFAP-positive cells. Interestingly, myrcene treatment substantially reduced Iba-1 and GFAP expression in the striatum, compared with that in ROT-injected rats. p < 0.000), which is attributed to the preservation of dopaminergic neurons. Furthermore, ROT injections induced a substantial increase in autophagosome accumulation, as depicted by the increase in beclin-1  and LC3II/LC3I ratio . Myrcene treatment in ROT-injected rats decreased the expressions of beclin-1 and LC3II/LC3I ratio, indicating a reduction in autophagosome accumulation (p < 0.001), which connects ubiquitinated proteins with the autophagy process via beclin-1 and LC3, was examined. ROT injections resulted in a substantial increase in p62 expression, indicating inhibition of autophagic clearance by ROT. Myrcene treatment resulted in a substantial decrease in p62 expression, indicating autophagic clearance of misfolded proteins by myrcene  in α-synuclein expression in the striatum . Howevermulation . To inve myrcene . Furtherp < 0.001) at first, but gradually decreases thereafter, leading to impairment in neuronal homeostasis. ROT injections resulted in a substantial decrease in mTOR phosphorylation , according to immunoblotting analyses in the striatum  . The indTH is a rate-limiting enzyme that catalyzes the formation of L-DOPA during dopamine (DA) biosynthesis. TH deficiency in the striatum is an important characteristic of PD. DA, a neurotransmitter, is synthesized in dopaminergic neurons in the SN . Enzyme The pathological alteration of α-synuclein in ROT-induced rats is an important hallmark of PD. The exact mechanism of α-synuclein in PD is not known; however, dopaminergic neuronal loss in SN is associated with aberrant changes in α-synuclein. Various reports have shown that α-synuclein increased expression activates microglia, thus initiating the pathogenesis of PD through the production of ROS by triggering apoptosis ,36,49. CApoptosis induced by various factors, such as ROS and aberrant α-synuclein aggregation, is a key process involved in PD. Apoptosis is an evolutionary process that is detrimental to dopaminergic neurons in the SN and striatum in PD ,51,52. DAs previously stated, the build-up of α-synuclein, due to impaired autophagic processes, plays a critical role in the development of Lewy bodies and subsequent neurodegeneration in PD. Additionally, ROS are key factors in mediating neuronal metabolism and autophagy . AutophaIn addition, mTOR participates in maintaining the shape, migration, and differentiation of neuronal cells, and is thus important for the development of memory, and sustaining synaptic plasticity. Inhibition of mTOR has been shown to be associated with impairment of p70S6 kinase and 4E-BP-1, thereby promoting impaired homeostasis in neuronal growth. Hence, the expressions of mTOR, p-mTOR, and p70S6 kinase were analyzed in the present study ,60,61,62Collectively, the present study findings demonstrate that ROT injections induce oxidative stress, inflammation, and apoptosis, along with an increase in α-synuclein and altered autophagy. However, myrcene treatment mitigates the aberrant increased expression of α-synuclein, diminishes the increased levels of pro-inflammatory cytokines and inflammatory mediators, decreases oxidative stress, and favorably alters autophagic flux, and mTOR signaling eventually results in the preservation of dopaminergic neurons. Given its dietary availability, accessibility, safety, and observed efficacy, myrcene could be an important agent for its therapeutic potential in PD, and can be suggested for its nutritional and phytomedicinal uses in neurodegenerative diseases.Experiments and experimental procedures were approved and performed in accordance with the guidelines of the Animal Ethics Committee of the United Arab Emirates University (UAEU). Male Wistar albino rats weighing 280–300 g were included in the present study. The rats were maintained under standard animal house conditions at 22 ± 1 °C, 50–55% humidity, and a 12/12 h photoperiod. High-purity ROT and myrcene were obtained from Sigma-Aldrich . Radioimmunoprecipitation assay (RIPA) buffer, antibodies for inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and glial fibrillary acidic protein (GFAP) were obtained from Sigma-Aldrich . Protease and phosphatase inhibitor cocktails were purchased from Thermo Fisher Scientific . The anti-tyrosine hydrolase  antibody was purchased from Merck . LC3, p62, mTOR, phospho-mTOR, and p70S6 antibodies were obtained from Cell Signaling Technology . The apoptotic polyclonal markers, Bax and Bcl-2, were purchased from Abcam . The monoclonal mouse anti-α-synuclein antibody was purchased from BD Biosciences . Anti-ionized calcium-binding adapter molecule 1 (Iba-1) antibody was procured from Wako Chemicals . Alexa Fluor 488, a fluorescent secondary antibody, was obtained from Thermo Fisher Scientific . Biotinylated goat anti-rabbit secondary antibody was procured from Jackson Immune Research Laboratories . Biochemical assays were performed using kits available commercially. All other analytical grade chemicals used in these experiments were procured from local distributors. n = 15). Group I (control): Rats received olive oil, DMSO in mygliol alone in an amount equal to myrcene, and ROT-alone treatment; Group II: Rats were treated with ROT  along with olive oil (similar amount used for dissolving myrcene); Group III: Rats were orally treated with myrcene (50 mg/kg) dissolved in olive oil and DMSO in mygliol; Group IV: Rats were pretreated with myrcene (50 mg/kg), followed by ROT . In all groups, treatments were carried out 5 days a week for a period of 28 days.PD was induced in experimental rats by administering ROT injections , as standardized in our laboratory and as described previously ,14,15. AFollowing anesthesia with pentobarbital (40 mg/kg body weight), the animals underwent intracardiac perfusion with phosphate buffered saline (PBS) (0.01 M). For immunofluorescence analysis, the rats were perfused with PBS (0.01 M), followed by 4% paraformaldehyde. The brain was aseptically removed, and the midbrain and striatum were extracted for further experiments. Whole brains were incubated in 4% paraformaldehyde for 48 h, followed by sucrose treatment for 3 consecutive days. The midbrain was homogenized in KCl buffer containing 10 mM Tris-HCl, 140 mM NaCl, 300 mM KCl, 1 mM ethylenediaminetetraacetic acid, and 0.5% Triton-X100 at pH 7.5, following the addition of protease and phosphatase inhibitors. Tissues were centrifuged at 13,000 rpm for 30 min at 4 °C. The supernatants were analyzed using ELISA and biochemical assays. Striatum tissues were processed using RIPA buffer for Western blot analysis.MDA assay was performed to evaluate lipid peroxidation using the North-West Life Science MDA detection kit , following the manufacturer’s instructions. Sample or calibrators (100 μL), thiobarbituric acid (100 μL), BHT (5 μL), and acid reagent (100 μL) were dispensed in an Eppendorf tube. The samples were incubated at 60 °C for 1 h, followed by centrifugation for 2–3 min at 2000 rpm. The reaction mixture was transferred to a 96-well ELISA plate, and the absorbance was read at 532 nm. The absorbance data were processed, and the results were calculated and presented as micromolar MDA.GSH content was analyzed using a commercially available GSH assay kit  in all experimental groups. Briefly, the samples were deproteinized using 5% 5-sulfosalicylic acid, and 10 μL of sample or standard was dispensed in a 96-well plate, followed by 150 μL of a working mixture consisting of assay buffer, 5,5′-dithiobis (2-nitrobenzoic acid), and GSH reductase. NADPH (50 µL) was added to the reaction mixture for optimal color development, and kinetic readings were recorded for 6 min at 412 nm. GSH content was calculated and expressed as micromolar GSH. 2O2) and incubating for 20 min. Next, 30 μL of KOH was added to terminate the reaction, and 30 μL of catalase purpald was added for optimal color development to complete the reaction. Catalase potassium periodate (10 μL) was then added to stop the reaction. Absorbance was recorded at 540 nm and catalase activity was calculated in nM/mL/min. For SOD estimation, 10 μL of sample or standard was added to a 96-well microplate, followed by 20 μL of xanthine oxidase to start the reaction. The microplate was incubated for 30 min at room temperature. Thereafter, absorbance was read at 450 nm and SOD activity was recorded in U/mL.SOD and catalase activities were estimated in brain samples using commercial kits procured from Cayman Chemicals Company , following the manufacturer’s protocols. For the catalase assay, 20 μL of the sample or standard, 30 μL of methanol, and 100 μL of assay buffer were dispensed in a 96-well plate. The reaction was initiated by adding 20 μL of hydrogen peroxide  and MMP-9 were measured in midbrain tissues using commercially available kits . The plates were coated with primary antibody in PBS  and incubated overnight. Next, the plates were washed thrice, blocked with 300 μL of 1% bovine serum albumin (BSA), and incubated for 1 h. The plates were washed thrice, and 100 μL of the pre-prepared standard and samples were added to the plates, and incubated for 2 h. After incubation, the plates were washed thrice, and 100 μL of secondary antibody was added, followed by 2 h of incubation. Subsequently, the plates were washed thrice, and 100 μL of HRP was added, again washed thrice, and 100 μL of TMB was added to develop the color. The colorimetric reaction was stopped by adding 50 μL of 2N sulfuric acid. Absorbance was recorded at 450 nm, and the concentrations of pro-inflammatory cytokines and MMP-9 were calculated as pg/mL. The striatal sections of all experimental groups were used to stain astrocytes (GFAP) and microglia (Iba-1). Sections with 30 µm thickness were washed twice in a 24-well tissue plate using the floating section method. Briefly, striatum tissue sections were blocked with a blocking reagent consisting of 10% normal goat serum in PBS, 0.3% Triton-X100, and 1% BSA for 1 h. The sections were incubated overnight with primary antibodies GFAP (1:1000) and Iba-1 (1:1000) at 4 °C representing the expression of astrocytes and microglia, respectively. The sections were washed twice with PBS and incubated with the corresponding fluorescent secondary antibody (Alexa 488) (anti-rabbit or anti-mouse) specific to the primary antibody. The sections were incubated for 1 h and washed twice with 1x PBS, and mounted on slides. The sections on slides were mounted using fluorescent mounting media and Vecta stained with DAPI. Photomicrographs were visualized under a fluorescence microscope (Nikon Eclipse Ni). From each brain, a minimum of three coronal sections at a similar level of the striatum were used to analyze the number of activated microglial cells and astrocytes. After random selection of three different fields of equal area, the images were analyzed using the ImageJ software . An outline was drawn around the region of interest, and the area, circularity, and mean fluorescence were determined considering numerous adjacent background readings. Total corrected cellular fluorescence (TCCF) was calculated using the formula: TCCF = integrated density - area of selected cell × mean fluorescence of background readings). All readings were determined by an observer blinded to the experimental protocols to avoid any bias in the calculation. Data are presented as percentages of the control.The striatal tissues were used for Western blotting to determine the expression of specific proteins. Western blotting was performed as described previously . Striatal tissues were homogenized in RIPA buffer supplemented with phosphatase and protease inhibitors. Proteins (35 µg) from each sample were loaded and separated using SDS-PAGE. Proteins from the gels were electro-transferred onto a polyvinylidene fluoride membrane using the trans-turbo Bio-Rad method. The membrane was blocked with 5% nonfat dry milk in PBS with 0.1% Tween 20 (PBST) for 1 h. After blocking, the membranes were washed with PBST and incubated overnight at 4 °C with the following primary antibodies: α-synuclein (1:750), COX-2 (1:2000), iNOS (1:1000), Bax (1:2000), Bcl-2 (1:500), mTOR (1:1500), phospho-mTOR (1:900), p62 (1:900), LC3 (1:800), and p70S6K (1:900). The membranes were washed with PBST, and secondary antibodies specific to the primary antibodies were added to the membrane and incubated at room temperature for 1 h. The membranes were developed for protein bands using a Chemiluminescence Pico Kit Thermo Fisher Scientific, . The membranes were stripped and re-probed for β-actin to determine the total protein content. The observed bands were quantified using the ImageJ software.2O2. The samples were again washed with PBS twice and blocked using blocking buffer  for 1 h. After blocking, sections were washed with 1x PBS, and primary antibody was added  overnight at 4 °C. The sections were washed twice with 1% PBS, and biotinylated secondary antibody (1:1000) was dispensed and incubated for 1 h. The sections were again washed twice and developed with an avidin–biotin peroxidase complex system  followed by 3,3′ diaminobenzidine (DAB) to visualize and examine TH immunoreactivity. Sections were then mounted on slides with DPX media and cover slips, examined under a light microscope, and representative images were analyzed. Cryoprotected rat brains were serially sectioned (25 µm) using a cryostat . Serial sections of the SN were washed twice using PBS. The sections were inactivated for peroxidase using 1% HThe loss of nerve fibers in the SN area was analyzed. The stained sections were examined for loss of TH-immunoreactive (TH-ir) neurons. Briefly, sections at three to five different levels of the medial terminal nucleus region were counted. The counts are represented as a percentage of control to naive cells ,14,15. SThe protein content in each sample was quantified using the Pierce BCA protein assay kit Thermo Fisher Scientific,  following the manufacturer’s protocol.p < 0.05 was considered statistically significant.The data were analyzed using one-way analysis of variance followed by Tukey’s test. The criterion of statistical significance between different experimental groups was established by employing the SPSS12 software. Values are expressed as mean ± standard error of the mean (SEM).


In human medicine, the progression from early neoplasia development to either complete resolution of tumorigenesis and associated inflammation or chronicity and fatal outcomes remain difficult to predict. Resolution of inflammation is an active process that stimulates the termination of the inflammatory response and promotes return to homeostasis, while failure in resolution contributes to the development of a number of diseases. To understand how resolution pathways contribute to tumorigenesis, we defined and employed a cumulative score based on the expression level of genes involved in synthesis, signaling, and metabolism of the D-series resolvin (RvD). This score was used for comparative analyses of clinical, cellular, and molecular features of tumors, based on RNA-sequencing (RNA-seq) datasets collected within The Cancer Genome Atlas (TCGA) program. Our results indicate that higher RvD scores are associated with better clinical outcome of patients with head and neck squamous cell carcinoma (HNSC), and with molecular and cellular signatures indicative of enhanced anti-tumor immunity and better response to immune-checkpoint inhibitors (ICI), also in human papilloma virus (HPV) negative HNSC subtypes. Thus, higher activity of the RvD pathway identifies patients with improved resolution and a more efficient immune reaction against cancer. Acute inflammation is a beneficial immune response aimed to restore tissue integrity after pathological injuries. However, if not properly regulated, a chronic inflammatory state may ensue and drive a number of diseases including cancer . Indeed,Resolution represents the ideal outcome of acute inflammation and is an active process that encompasses the removal of the causative agent, a temporal shift in in the composition of the cellular infiltrate, i.e., from polymorphonuclear neutrophils (PMN) to monocytes-macrophages that, in turn, initiate the non-phlogistic phagocytosis of dangerous agents and apoptotic PMN . These hALOX15 gene), 12-LOX (ALOX12), or 5-LOX (ALOX5) on arachidonic acid (LX) or docosahexaenoic acid  (reviewed in [FPR2) and DRV/GPR32 (GPR32). DRV/GPR32 was also reported as a receptor for RvD5, while RvD2 conveys pro-resolving functions through DRV2/GPR18 (GPR18) (reviewed in [HPDG) and the eicosanoid oxidoreductase Prostaglandin Reductase 1 (PTGR1) [SPM are mainly produced by the actions of 15-LOX (iewed in  via the iewed in ). FinallWe and others recently reported pre-clinical evidence that the stimulation of resolution is beneficial to counteract primary and metastatic neoplasia ,11,12,13Here, we show that higher RvD scores identify HNSC patients with improved clinical outcome, and with a molecular signature indicative of enhanced anti-tumor immunity and response to immune-checkpoint inhibitors (ICI). Thus, higher activity of the RvD pathway is associated with better immune reaction, improved resolution, and beneficial response to cancer.ALOX15 and ALOX5 that work together and dependently (multiplication), with further dependence (multiplication) on receptors , and inverse dependence (division) with inactivating enzymes (HPGD or PTGR1 addition due to their independent actions)  A. The fiTumor-specific HNSC RNA-seq datasets were then integrated with clinical features of matched patients, retrieved through cBioPortal  and straALOX15, ALOX15, FPR2, GPR18, GPR32, HPGD and PTGR1 were leveraged from 516 bulk tumor HNSC RNA-seq data, profiled by TCGA and plotted as a heatmap of individual tumor samples , another HPV+ tumor, as well as in different malignancies such as urothelial bladder carcinoma (BLCA), mesothelioma (MESO) and skin cutaneous melanoma (SKCM), further corroborating its tumor-protective role [GPR32, another RvD receptor, proved to be a worse prognosis index in other cancers such as kidney chromophobe (KICH), cervical kidney renal papillary cell carcinoma (KIRP) and in low grade glioma (TCGA-LGG). Similarly, other RvD receptors are associated with both protective and dangerous effects, depending on the cancer type. Conversely, the expression of the inactivating enzyme PTGR1 is associated with an increased risk of death, in particular in HPV− HNSC, while it is protective in kidney renal clear cell carcinoma (KIRC). Therefore, genes involved in the RvD pathway affect malignant progression in a cancer- dependent manner, thus underlying the importance of lipid mediators during tumorigenesis. The expression levels of the distinct genes of the RvD pathway  samples A. To eva samples B. As shoive role . On the ALOX5 + ALOX12) × (ALOX15 + ALOX5) × (FPR2 + GPR32)/(HPGD + PTGR1), applying the same principles of the RvD score, fails to predict HNSC survival  and clinical outcome , tumor necrosis factor α (TNF) and interferon γ (INFG)  in the RvD high group A,B. In aγ (INFG) C. In acc+ and CD4+, T cells, NK cells, monocytes/macrophages, dendritic cells, mast cells, eosinophils, neutrophils). CIBERSORT effectively determines the overall immune content in RNA-seq datasets when compared against other experimental methods [+, CD8+ T lymphocytes , Cytotoxic T-Lymphocyte Antigen 4 (CTLA4), Lymphocyte Activation Gene 3 (LAG3), Programmed Cell Death Protein 1 (PDCD1) may be used as biomarkers to identify putative ICI responsive patients [CD274, CTLA4, LAG3 and PDCD1 bulk RNA-seq HNSC were retrieved and compared amongst RvD high and RvD low score patients (ICI-targeting programmed cell death protein-1 (PD-1) and its ligands, programmed death ligand-1 (PD-L1)/2, rejuvenate antitumor immunity and produce clinical responses in subsets of HNSC, even if the majority of patients display primary resistance and do not benefit from these agents ,29. Apprpatients ,31,32. Tpatients C. This apatients A,B, indiGZMA and GZMB, respectively), perforin (PRF1) and the cytolytic score [+ T cell tumor cytotoxicity. RvD high tumors proved increased gene expression levels of anti-tumor effectors, including GZMA, GZMB, PRF1, and cytolytic score  involved in CD8+ T cell function in patients with high RvD score tumors  with LM10 as the signature matrix  [To validate these findings, we determined whether RvD could prompt the anti-tumor activity of human leukocytes against HPV-negative cancer. To this end, blood borne human leukocytes were treated with vehicle or RvD1 (as a prototype of RvD with an increased score in the biobank atlas) and added on top of UM-SCC-23 cells. The growth curve of cancer cells was monitored with a real-time impedance assay in a small-scale in vitro system, as shown in . As show4 cells) . Of inteIn this study we defined and exploited a RvD score to stratify HNSC patients accordingly to their resolution pathway activity, using a bioinformatic approach to evidence clinical, cellular, and molecular differences among patient cohorts . + T cells, and increased expression of immunotherapy targets, predicting a better response to ICI treatment  that proved comparable to immunohistochemistry , we dete in HNSC ,40, but  in HNSC . Therefo in HNSC ,43,44,45+ T cells as the main affected cellular effector in high RvD tumors. Indeed, both bulk DEG  but also of other genes encoding inhibitory receptors on lymphocytes   that stimulate immune responses. Unleashing the potential of CD8+ T cells in high RvD HNSC by ICI may therefore activate a potent cytotoxic reaction against cancer cells. Stratification accordingly to their RvD score may thus identify HNSC patients with a potential better response to ICI, thus allowing a specific tailoring of therapies only to responsive patients, with a considerable advantage in terms of the limitation of side effects and social costs. CD8herapies . ICI  C,D. Furt+ lymphocytes, our results revealed a crucial impact of the RvD pathway activity in modulating PMN functions. Robust PMN infiltration in cancer tissues has been frequently associated with worse clinical outcome, although PMN can act as both pro-tumoral and anti-tumoral effectors depending on their phenotype [In addition to CD8henotype . PMN infhenotype . Collectively, these results suggest that the stimulation of the RvD pathway  could be beneficial to orchestrate the anti-tumoral activity of immune cells. Along these lines, our previous studies proved that RvD modulates the macrophage phenotype and controls the production of cytokines/chemokines/growth factors with pro-tumor properties ,43,44. Ffirebrowse.org, accessed on 1 April 2022). Clinical data of Head and Neck Squamous Cell Carcinoma  were retrieved from cBioPortal  [The HNSC RNA-seq data  were downloaded from FireBrowse .timer.cistrome.org, accessed on 1 April 2022) was used to evaluate the association between lipid gene expression and clinical outcome, adjusted for age, gender, stage and purity.Cox proportional hazard model in the Gene_Surv module of TIMER2.0 . Similarly to [ALOX5 and ALOX15), RvD receptors  and enzymes involved in RvD degradation (HPDG or LTB4DH). The final score was calculated as follows (genes involved in production x sum of gene receptors / sum of degradation genes):The HNSC RNA-seq data were used to filter out 11 genes currently identified as key in D-series resolvin pathway  and low RvD (the lower 100 samples). Differential gene expression and statistical significance  among the two groups were calculated using the mRNA module of cBioPortal. mRNAs with a q-value < 0.05 and with a log2 fold-change (−log2 FC) ± 1 (corresponding to 2-fold change) were defined as differentially expressed. The functional association of genes and pathways selectively regulated in the high RvD group were identified by querying the list of differentially expressed genes in The Database for Annotation, Visualization and Integrated Discovery (DAVID) . A falseTo quantify intratumoral infiltration levels of immune cell fractions, bulk RNA-seq gene expression data from high and low RvD groups were analyzed with the impute cell fraction module of CIBERSORT , using L+ T cell, CD4+ T cell, NK cell, monocyte, dendritic cell, mast cell, eosinophil and neutrophil gene expression profiles.To attribute cell-specific gene expression profiles, bulk RNA-seq gene expression data from high and low RvD groups were analyzed with the impute cell expression module, group mode of CIBERSORT . For theDifferential gene expression between RvD high versus RvD low samples was calculated for each gene in paired leukocyte subsets and visualized as a heatmap using the BioVinci software. Genes with a −log2 FC > ± 2 in each population were considered differentially expressed and were analyzed with IPA to identify the pathways and biological functions significantly affected . Function and pathway analyses were retrieved by IPA filtering experimentally observed relations in immune cells with stringent filters.+ T cells-specific gene expression of GZMA, PRF1, CTLA4, LAG3 and PDCD1 from bulk tissues at sample-level, a high-resolution module of CIBERSORT was used. Differential expression of selected genes in CD8+ T cells were then calculated with GraphPad Prism version 8.0.To impute CD8AKT1, AKT2, AKT3, B2M, BCL2L1, CD247, CD274, CD28, CD80, CDK2, CSK, CSNK2A1, CTLA4, FOXP3, GSK3B, HLA-DRB1, IFNG, IFNGR1, IFNGR2, IL12B, IL2RA, IL2RG, JAK1, JAK2, JAK3, LAG3, LCK, MR1, NGFR, PDCD1, PDCD1LG2, PIK3C3, PIK3CA, PIK3CB, PIK3CD, PIK3CG, PIK3R4, PRKCQ, PTPN11, RNF2, TGFB1, TGFB2, TGFB3, TNF, TNFRSF1A, TNFRSF1B and TYK2) reported as mRNA expression z-scores relative to diploid samples.The gene expression levels of four key targets of immunotherapies  CD274  were used as markers of anti-tumor CD8+ T lymphocyte immunity as in [PRF1 and GZMA RSEM normalized values as in [Expression levels of genes encoding effector molecules released by cytotoxic CD8ty as in . RNA-seqes as in . Tumor samples were also filtered according to their positivity to HPV mRNA and analyzed as described above to identify immunotherapy targets in HPV positive tumors.4 cell/well on iCelligence E-plate L8 (ACEA Biosciences) for 2 h before the addition of effectors. Buffy-coats from healthy donors were obtained from the ASL2 Lanciano-Vasto-Chieti  transfusion center after informed consent. Human leukocytes (effector cells) were isolated after erythrocyte sedimentation in dextran (Merck KGaA) for 15 min at room temperature, washed, treated for 15 min at 37 °C with vehicle (0.05% Ethanol) or RvD1  (10 nM) and added on top of UM-SCC-23 at an effector: target ratio of 10:1. Cell growth was monitored continuously up to 48 h with the real-time impedance assay (ACEA Bioscience) as in [For cell-growth studies, the University of Michigan Squamous Cell Carcinoma-23   were maie) as in . UM-SCC-t tests were used to compare two samples normally distributed. Data were expressed as mean ± SEM of the number of biological replicates indicated in each figure legend. Values of p < 0.05 were considered statistically significant.Statistical significance was evaluated with Graphpad Prism version 8.00. Multiple samples were compared with ANOVA. Paired or unpaired Student + T cells, and transcriptional profiles predictive of a better response to ICI treatment. In summary, here we defined a RvD score to estimate D-series resolvin pathway activity and function in tumor specimen from the TCGA consortium. Our results demonstrate that HNSC patients, both positive and negative for HPV presence, with high a RvD score presented longer disease-free survival, enhanced anti-tumor immunity mainly driven by CD8


N = 5959) sampled from December 2019 to September 2020 and “2021 genomes”  sampled from 15 January to 15 March 2021. All genomes were aligned to the SARS-CoV-2 reference genome and amino acid polymorphisms were called with quality filtering. Thereafter, 74 codons (loci) in 14 genes including orf1ab polygene (N = 9), orf3a, orf8, nucleocapsid (N), matrix (M), and spike (S) met the 0.01 minimum allele frequency criteria and were selected to construct multilocus genotypes (MLGs) for the genomes. At these loci, 137 mutant/variant amino acids  were detected with eight VOC-defining variant alleles, including N KR203&204, orf1ab , orf3a H57, orf8 S84, and S G614, being predominant globally with > 35% prevalence. Their persistence and selection were associated with peaks in the viral transmission and COVID-19 incidence between 2020 and 2021. Epidemiologically, older patients (≥20 years) compared to younger patients (<20 years) had a higher risk of being infected with these variants, but this association was dependent on the continent of origin. In the global population, the discriminant analysis of principal components (DAPC) showed contrasting patterns of genetic clustering with three  and two (North and South America) continental clusters being observed for the 2020 and 2021 global populations, respectively. Within each continent, the MLG repertoires (range 40–199) sampled in 2020 and 2021 were genetically differentiated, with ≤4 MLGs per repertoire accounting for the majority of genomes sampled. These data suggested that the majority of SARS-CoV-2 infections in 2020 and 2021 were caused by genetically distinct variants that likely adapted to local populations. Indeed, four GISAID clade-defined VOCs - GRY (Alpha), GH (Beta), GR (Gamma), and G/GK (Delta variant) were differentiated by their MLG signatures, demonstrating the versatility of the MLG tool for variant identification. Results from this proof-of-concept multilocus genotyping demonstrates its utility for SARS-CoV-2 genomic surveillance and for monitoring its spatiotemporal epidemiology and evolution, particularly in response to control interventions including COVID-19 vaccines and chemotherapies.Since its emergence in 2019, SARS-CoV-2 has spread and evolved globally, with newly emerged variants of concern (VOCs) accounting for more than 500 million COVID-19 cases and 6 million deaths. Continuous surveillance utilizing simple genetic tools is needed to measure the viral epidemiological diversity, risk of infection, and distribution among different demographics in different geographical regions. To help address this need, we developed a proof-of-concept multilocus genotyping tool and demonstrated its utility to monitor viral populations sampled in 2020 and 2021 across six continents. We sampled globally 22,164 SARS-CoV-2 genomes from GISAID . They comprised two study populations, “2020 genomes” ( SARS-CoV-2, the causative virus of the COVID-19 pandemic, emerged in December 2019 and has since infected more than 500 million and killed over 6 million people globally. Public health interventions including testing and isolation of infected individuals, wearing of face masks, and the rollout of vaccines in 2021 in many affected countries have contributed to significant declines in the transmission and incidence of SARS-CoV-2 infections ,4,5. Othorf1ab polygene, which encodes 16 non-structural proteins (nsp1–16) associated with viral replication. The remaining one-third encodes structural proteins such as the spike glycoprotein (S), matrix (M), envelope (E), and nucleocapsid (N), and accessory proteins including ORF3a and ORF8 [Phylogenomic analysis of SARS-CoV-2 whole genomes has been used to differentiate variants when source-tracking infections and inform public health control efforts. However, the former analysis can be computationally challenging when dealing with thousands of genomes . Additioand ORF8 .orf1ab P4715L, orf3a G251V, orf8 L84S, N R203K, and N G204R have been implicated in high infectivity and replication [VOCs such as the Indian Delta variant have been associated with the presence of “evolutionary-relevant” or “adaptive” mutations, including L452R, T478K, N501Y, and D614G in the S protein, a target of cell- and vaccine-mediated immunity . In the lication ,14,15 anlication . The epilication , making lication ,15,18,19Multilocus genotyping of amino acid polymorphisms at putatively adaptive loci in SARS-CoV-2 could be a useful genetic tool to monitor the viral epidemiology, transmission dynamics, and evolution in different populations and regions worldwide. To reduce the complexity in the SARS-CoV-2 genomic space, multilocus genotyping using SNPs and/or amino acid polymorphisms at ~20 loci in the SARS-CoV-2 genome was used to differentiate closely-related variants that caused local  and globSince the pandemic begun, thousands of SARS-CoV-2 genomes have been made publicly available in the Global Initiative on Sharing Avian Influenza Data (GISAID) database . In thisAll the data used for this study were obtained from GISAID . At the Individuals from whom the infections were isolated and sequenced were referred to as “patients” for the purpose of this study. Based on the available metadata associated with the 2020 genomes, patient age was stratified into four categories: 0–19, 20–39, 40–59, and ≥ 60 years, as described elsewhere . Clinica614 and mutant allele as S G614. Contiguous alleles at the loci investigated in a gene were considered as the genotype and those across ≥ 2 genes were considered the multilocus genotype (MLG) for a SARS-CoV-2 genome. Only loci with ≥ 2 alleles, each with a minor allele frequency of 0.01, were used for the MLG construction. These criteria were implemented to ensure unbiased construction of MLGs [All genomes were aligned against the SARS-CoV-2 Wuhan reference strain (NC_045512.2) using minimap version 2.17 . Amino a of MLGs .eH  were estimated using poppr V2.8.5 [vegan [Within a continent, the number of unique MLGs, expected number of unique MLGs or eMLGs , and expected heterozygosity or r V2.8.5 . To asse5 [vegan . The evepoppr. The p-value < 0.05.To determine which loci in the SARS-CoV-2 genome were co-evolving, i.e., whether infections carried multiple mutations at different loci that were non-randomly associated, we used all the MLGs sampled in a continent to estimate a standardised index of association  to 1 . GST values ranging from 0 to 0.09, 0.1 to 0.19, and ≥0.2 indicate little, moderate, and great genetic differentiation, respectively [adegenet [ggplot2 [Phyloviz V2 was used to construct networks of minimum spanning trees [To determine whether SARS-CoV-2 infections from different continents could be genetically differentiated based on the MLGs carried by the SARS-CoV-2 genomes, we estimated the Nei G in mmod . The GSTectively . We alsoectively . Brieflyadegenet  to identadegenet . By addi[ggplot2 . This prng trees .p-value was <0.05.Statistical analysis was performed in R v3.5.2  and STATp-value = 0.002). The majority of SARS-CoV-2 genomes reportedly originated from Asia (26.5%) and <10% originated from Africa, North America, and South America each , orf3a, orf8, M, N, and S genes (Orf1ab 2796 (in NSP3), N 203, S 320, S 477, and orf3a 251 were the most polymorphic with five alleles each . This suggests that the majority of mutations in the orf1ab polygene were not under strong selection compared to those in the structural proteins, including the S protein [The “2020 genomes” comprised seven GISAID clades: G (22%), GH (23.3%), GR (25.3%), L (4.3%), O (12.1%), S (10%), and V (3%). Seventy-four loci with SNPs that resulted in amino acid changes met the inclusion criteria for genotype and MLG construction. They were in 14 protein coding genes:  S genes . Loci inles each . Of the s ~21 kb , but the protein ,36,37.orf1ab mutant alleles were detected infrequently with prevalences below 25% during the early phase of the pandemic, i.e., Feb–May 2020, and thereafter declined sharply as transmission peaked in all the six continents , F3606 , and L4715 . The remaining five included two in the accessory proteins, orf3a H57 and orf8 S84, and three in two structural genes, S G614 and N KR203&204 (this genotype defines variants in the GR clade). These mutant alleles have been associated with infectivity, disease severity, and immune dysregulation [orf1ab L4715, S G614, and N KR203&204 [501 mutant allele, which was associated with high infectivity among UK variants that caused nationwide outbreaks in September 2020 [The majority of the ntinents . In contntinents . They ingulation ,38 and hgulation ,40. In pR203&204 , rose shR203&204 , suggestR203&204 . Interesber 2020 . Our datber 2020 .orf1ab , orf3a 57, orf8 84, N (203 and 204), and S 614, considered informative for clade/variant designation [orf8 84 (North America), N 203 and 204 (Asian), and orf3a 57 (Europe) , orf1ab 3606 (Africa and Asia), orf3a 57 (Africa and South America), and orf8 84 (Africa and North America), they were less likely to harbour infections that carried mutant alleles at S 614  . Particuat S 614 . NonsynoWe constructed a multilocus genotype (MLG) for each of the 22,164 genomes included in this study to evaluate the utility of the 74 polymorphic loci as a genetic tool for differentiating SARS-CoV-2 variants and for monitoring their evolution in different geographical regions. Among the 5959 genomes in the 2020 study population, 472 unique MLGs were detected in the global population, with MLG repertoire sizes ranging from 40 for South America to 185 for Asia . The meaeH ≥ 0.26) and lowest in South America   . This pa = 0.15) A. This op-value < 0.001)  in Africa, Europe, and South America  , which s< 0.001) . Followi America , which i America . Indeed,p-value < 0.001) between NSP12 and ORF8, NSP12 and S, ORF8 and S, and ORF3a and NSP2  among their loci. Co-selection of mutant alleles across multiple genes may indicate potential recombination signatures and and/or epistatic interaction between specific gene pairs to enhance SARS-CoV-2 fitness [Interestingly, although several of the genomes carried multiple mutations across ≥ 2 genes, pairwise LD estimates showed that the multilocus LD was driven by key mutant alleles that likely co-evolved . That isand NSP2 B, which and NSP2 . A potenvariants . This ex fitness ,49.ST to visualise relationships, identify genetic clusters, and to estimate the genetic differentiation among MLG repertoires from the six continents. The network analysis indicated that 11 major clonal complexes  existed in the “2020 genomes”  except between Oceania and Asia, Oceania and Europe, and Europe and South America (ST ≥ 0.111) within continents  to each continent , which i America F. In addntinents , demonstWe then tested the versatility of our multilocus genotyping tool to differentiate SARS-CoV-2 genomes with respect to their GISAID clade designation . The 16,ST ≥ 0.333) from their respective 2021 viral populations: Africa , Asia , Europe , North America , and South America   . Oceaniaorf1ab polygene in comparison to the structural genes including N and S suggested that the majority of orf1ab mutations were not under strong selection in the 2020 viral population. In contrast, the predominance and spatiotemporal persistence of the eight mutant alleles, N KR203&204, orf1ab , orf3a H57, orf8 S84, and S G614, indicate their selective advantage. As demonstrated by the detection of significant multilocus LD, SARS-CoV-2 variants including the VOCs may have acquired multiple co-evolving mutations to enhance their transmissibility. Epidemiologically, the risk for variant-specific infections among vulnerable populations may vary across different demographics and geographical regions, intimating that future control interventions need to target all age groups.Genomic tools are needed to differentiate SARS-CoV-2 variants causing COVID-19 and to monitor their evolution in different geographical regions. Here, we identified 74 polymorphic loci located in 14 genes in 22,164 SARS-CoV-2 whole genomes isolated from clinical infections that occurred in 2020 and 2021. The low prevalence of mutant alleles in the NSPs and the low genetic diversity in the Although extensive sampling of symptomatic infections is encouraged during surveillance activities, it is paramount that asymptomatic infections, which constitute the majority of COVID-19 cases, are included during surveillance in order to detect novel variants. Among the 2020 and 2021 variants that caused COVID-19, our multilocus genotyping tool identified continental clusters, which suggested geographical structure in the global viral population. Furthermore, the DAPC results suggested that the majority of infections in 2021 and 2021 were caused by genetically distinct variants. In particular, in the 2021 viral population, we observed considerable genetic differentiation among variants in the GISAID clades including GRY (Alpha), GH (Beta), GR (Gamma), and G/GK (Delta variant), which were largely responsible for successive waves of transmission and COVID-19 outbreaks in multiple countries. These findings are consistent with previous reports using phylogenomic analysis, and thus demonstrate the utility of the 74 polymorphic loci as a proof-of-concept multilocus genotype tool for monitoring the viral evolution. However, the limited genetic differentiation of the admixture populations suggest that more polymorphic loci may be needed to accurately differentiate closely related variants.The samples used in this study reflect both health policy decision-making and sampling strategies implemented in the countries at the time. The data presented in this study, though they are based on publicly available data and are thus relevant, need further investigations to draw definite conclusions on the associations between age, gender, and geographical region and the SARS-CoV-2 variant causing COVID-19. Nearly all the genomes sampled lacked metadata on country of infection, instead showing the country of detection, making it difficult to infer a source of infection based solely on the MLG data. Furthermore, the LD structures detected in this study population may change as the virus continues to evolve, particularly in response to control interventions such as vaccines. Thus, continuous genomic surveillance is warranted. We look forward to future studies aiming to improve the MLG genotyping tool. Such studies would need to factor in polymorphisms in the most recently emerged variants, including SARS-CoV-2 Omicron variants. This study did not include the Omicron genomes since, at the time of the study from January 2020 to March 2021, they were not publicly available. The Omicron variants emerged in November 2021.


Ferula is the third largest genus of the Apiaceae family, its species are utilized as a remedy for diverse ailments all over the world. F. sinkiangensis K. M. Shen  is mainly found in Xin-jiang Uygur Autonomous Region, China. Traditionally, it is utilized for treating various illnesses such as digestive disorders, rheumatoid arthritis, wound infection, baldness, bronchitis, ovarian cysts, intestinal worms, diarrhea, malaria, abdominal mass, cold, measles, and bronchitis. It can produce different classes of metabolites such as sesquiterpene coumarins, steroidal esters, lignans, phenylpropanoids, sesquiterpenes, monoterpenes, coumarins, organic acid glycosides, and sulfur-containing compounds with prominent bioactivities. The objective of this work is to point out the reported data on F. sinkiangensis, including traditional uses, phytoconstituents, biosynthesis, and bioactivities. In the current work, 194 metabolites were reported from F. sinkiangensis in the period from 1987 to the end of 2022. Nevertheless, future work should be directed to conduct in vivo, mechanistic, and clinical assessments of this plant`s metabolites to confirm its safe usage. People have utilized plants since ancient times for different reasons: food, clothing, shelter, decoration, and construction . Their uFerula is the third largest genus of Apiaceae family that comprises about 180 species. Its species commonly exist in Asian and Mediterranean regions e.g., Iran, Turkey, Algeria, Afghanistan, Saudi Arabia, Pakistan, China, and India D and Xray analyses. They had UV absorbance at 320–330 nm and a common fragment at m/z 185 in MS D analyses. Their inhibitory potential on NO production induced by LPS using nitrite and MTT assays in BV2 cells. Compounds 3, 5, 8, 25, 28–31, 38, 40, 41, 46, and 56 possessed noticeable inhibition of NO production in over-activated BV2 cells, (IC50s 1.2–93.8 μM) compared to minocycline (IC50 65.5 μM), while 27, 23, 52, and 57–59 had weak inhibitory capacity (IC50 > 00 μM). It was noted that 28 (IC50 1.2 μM) revealed the potent in vitro anti-neuroinflammatory that was confirmed by docking to TLR4/MD-2. Structure-activity relation showed that chain sesquiterpene coumarins with C-10` acetoxy group  had powerful activity than the ones with C-10`-OH, an unsaturated fatty chain or 4-decylenic acid ester . A-ring substitution pattern affected the potential of monocyclic derivatives with opened B-ring. The α,β-unsaturated ketone  increased the effect, while the seven-membered ring-A resulted from C-4′-O–C–3` linkage  remarkably repressed NO producing relative to six-membered ring-A derivatives. In the compounds with opened A-ring , increase the length of oxyacyl side chain at C-3` weakened the anti-neuroinflammatory activity decane-type sesquiterpene skeleton was purified from the 95% EtOH extract of the seeds. Its 2′S/3′R/5′R/7′R/10′R configuration was determined based on NMR and CD analyses. It had moderate and weak anticancer activity against AGS (IC50 12.7 μM) and Hela (IC50 82.9 μM) cells, respectively compared to taxol  [A novel metabolite (ctively) .52) possessed dose-dependent and time-dependent apoptosis in CLL (chronic lymphocytic leukemia) that was more sensitive to 52 than PBMCs. It is noteworthy that IL-4 could not decline 52-caused apoptosis in CLL  against AGS cells with less anticancer to GES-1 . It boosted AGS cells apoptosis with elevated Bax/Bcl-2 ratios, ROS generation, lessened mitochondrial-membrane potential, and PARP and caspase 3 activation resulting in mitochondrial apoptosis pathway activation. It also arrested the G0/G1 phase of the cell cycle, increased P27, P53, and P16, expression, and diminished cyclin E, cyclin D, Cdk2, and Cdk4 expression in cancer cells. Therefore, it could be developed into anti-cancer therapy [52 also demonstrated anticancer capacity against BGC-823 and AGS, with less toxic influence on the normal GES-1 gastric cells. It was proven to prevent gastric cancer cell invasion, growth, and migration by disconcerting the Wnt signaling pathway. Additionally, it exhibited no harm in the in vivo BGC-823 xenograft model as evidenced by no observed abnormality in daily diet, body weight, liver function, and histological features of the spleen, liver, lung, kidney, and heart tissue. This further supported the previous evidence of its promising potential for treating gastric cancer [Umbelliprenin -62 had marked proliferation inhibition capacity on PANC-1 cells  and moderate activity against CFPAC-1 cells  compared to taxol . It was noted that the anticancer potential of (+)-61 was more powerful than (-)-62 [63–65 had anticancer activity (EC50s 22.78–65.38 μM) against IOZCAS-Spex-II, where 65 was the most potent (EC50 22.78 μM) relative to camptothecin (EC50 51.27 μM) [The anticancer investigation against SW1990, CFPAC-1, Capan-2, and PANC-1 revealed that (+)-n (-)-62 . Also, 61.27 μM) .69 and 70 against AGS, MGC-803, and GES-1 using MTT assay. Only 70 possessed the notable anticancer potential against MGC-803 and AGS  with less toxicity against GES-1 (IC50 36.73 μM) comparing with taxol , whereas the other metabolites had no notable potential. The mechanistic study revealed that 70 was found to elevate Bax/Bcl-2 ratios, as well as RB, P16, and P27 expression and decrease cyclins (D1 and E), Cdk4, P53, and Cdk2, resulting in apoptosis and G0/G1 cell cycle arrest in AGS cells. This compound could be a potential candidate for gastric cancer therapy [Wang et al., examined the activity of  therapy .F. sinkiangensis seeds afforded 77–80 that displayed weak to moderate anticancer activity on AGS and Hela cells in the MTT assay [77, 80, and 81 had a weak anticancer capacity against AGS, HeLa, A549, and PC3 cell lines in the MTT assay (IC50s 54.4–167.3 µM) [TT assay   .84 and 86 were found to significantly prohibit the migration and invasion of TNBC) cell lines. On the other hand, 88 and 89 promoted the HUVECs) proliferation which was more remarkable than bFGF  in the wound-healing assay [In the CCK-8 assay, ng assay .179) and D (180), along with 158, 163, 164, 166–170, 174, and 181 were separated from the resin 95% EtOH extract and specified by spectral and CD analyses. In the MTT assay against AGS, MGC-803, and GES-1, they had no or weak potential (IC50s 18.89 to 182.46 μM) [New phenylpropanoid derivative; sinkiangenones C (2.46 μM) .–C (182–184) possessed anticancer activity on the AGS cell line , whereas 184 was the most active in comparison to taxol (IC50 4.69 μM) and induced AGS cell apoptosis in the MTT assay. Unfortunately, they (IC50 ˃100 μM) had no effect against HeLa and K562 cells [192 exhibited anticancer activity on AGS cells (IC50 36.9 μM) [Sinkiangensis A62 cells . Whilst 36.9 μM) .F. sinkiangensis resin anticancer activity against Caco-2, HC-T116, MFC, and HepG2 cells in the SRB assay. EtOAc fraction was found to have the potent anti-proliferative and apoptotic effects against all tested cell lines. This was correlated to its content of sesquiterpene coumarins [The petroleum ether, EtOAc, n-BuOH, and MeOH fraction possessed of oumarins .45 prohibited (IC50 4.0 μM) BJ09/H1N1 (influenza A/Beijing/7/2009 H1N1) infection in MDCK cells [63–65 and the coumarins: 66 and 67 displayed insecticidal potential  against S. exigua 3rd instar larvae  compared to camptothecin  [120) and guaiol (122)  possessed toxic potential on the S. exigua insect 3rd instar larvae [120 exhibited antifungal effect against plant pathogens: inhibitory effects on Pyricularia grisea, Alternaria alternata, and Botrytis cinereal  compared to carbendazim  [Besides, CK cells . The monrtality) . From thDrug addiction is a prime health concern that influences a growing number of persons and gives rise to severe economic and social burdens to economy society ,57. Desp133 and 135 was obtained from the F. sinkiangensis crude essential oil. A mixture of 133 and 135  significantly repressed the morphine abstinence syndrome and physiological addiction in rats and mice [50 values of its iv and ip injections were 1.42 g/kg and 1.66 g/kg, respectively [A mixture of In 2006, Wang reported in his patent that the resin extract in the form of capsules, powder, injection, drop pills, granules, tablets, or oral liquid) ameliorated the influences of serious and moderate long-time drug addictions in addicted patients, indicating its potential for treating subjects addicted to morphine, opioid, diamorphine, and marijuana ,38. It iF. sinkiangensis roots water-soluble polysaccharides revealed in vitro PTP1B (protein-tyrosine phosphatase 1B) competitive inhibition  [FSPs-a (acidic polysaccharides) fraction of  91.23%) . In anot 91.23%) .F. sinkiangensis resin extracts possessed antiulcer capacity, whereas CHCl3 extract (%inhibition 48.43) had comparatively better antiulcer potential than the n-BuOH and EtOAC  extracts comparing to famotidine (%inhibition 45.37) [F. sinkiangensis resin n-BuOH, EtOAc, and MeOH fractions significantly scavenged DPPH, whereas the EtOAc fraction was the most effective and the petroleum ether fraction was weakly active in the DPPH assay [In the in vivo antiulcer assay, different n 45.37) . In the PH assay .Lateolabrax japonicus  with F. sinkiangensis was found to notably promote L. japonicus foraging and better digestive enzyme activity and fish growth performance, where 10 g/kg was appropriate in the fish diet. Thus, it had an efficient role in L. japonicus farming and could have potential in the aquaculture industry as aquafeed formulation [In 2020, Xu et al., reported that feeding mulation .Medicinal plants are fundamental to humans and utilized for thousands of years in various cultures to treat or prevent diseases or promote health and well-being . Many coF. sinkiangensis is among the most valuable species of the genus Ferula that possess various traditional applications in treating various disorders such as bronchitis, diarrhea, malaria, gastric disorders, and rheumatoid arthritis. In this work, 194 metabolites have been characterized from various parts of this plant, including aerial parts, seed, roots, gum resin, oleo-gum resin, and resins (Herbal medicines have been utilized for thousands of years as principal therapeutic agents for treating various human illnesses in many countries. Recently, a growing number of studies have been carried out to prove the efficacy of these medicines against the assigned disorders. d resins .It was obvious that the majority of metabolites have been distinguished from resin extract. These metabolites belong to various chemical classes. Sesquiterpene coumarins with their structural diversity and contents represent the main and substantial metabolites of this plant .F. sinkiangensis. F. sinkiangensis had metabolites with marked antifungal and insecticidal capacity that can be valuable in agriculture for insect and plant pathogens control, however, additional field assessment is requested. Its metabolites; 61, 62, 65, 70, and 120 had notable anticancer potential against different cancer cell lines. These findings would provide evidence for the application of this and its fractions in treating cancers. Compound 12 had marked anti-inflammatory and anti-neuroinflammatory potential, revealing its potential as a lead metabolite for therapeutic intervention in various illnesses such as ischemic stroke, Alzheimer`s disease, and cerebral ischemia. Many studies proved the anticancer and apoptotic potential of 52 against different cancer cell lines particularly the gastric cancer cells with no toxic effect on the normal cells and no observed abnormality in daily diet, body weight, liver function, and histological features of the spleen, liver, lung, kidney, and heart tissue. This further supported its promising potential for treating gastric cancer as foods or folk medicine, however, additional clinical investigations are needed. To find out more metabolites with unique structures and bioactivity, more phytochemical investigations are demanded and indispensable. Also, new technologies such as metabolomics, transcriptomics, genomics, and proteomics can be applied for discovering more metabolites from this valuable medicinal plant. The plant`s mechanism in treating gastric disorders and rheumatoid arthritis is insufficiently explored. Additionally, in-depth in-vivo and in vitro studies of the other bio-activities mechanisms are required. Meanwhile, toxicological, pharmacokinetic, preclinical, quality control, and clinical studies are insistent to estimate the safety and rationale usage of this plant. Finally, the integration of traditional knowledge into ecology-based research for the endangered medicinal plant protection must be promoted.Many studies surmised that these compounds may have a substantial contribution in many of the reported activities of


CFAP57) loss-of-function mutations  recessively segregating with male infertility. A mouse model mimicking the mutation (c.2872C>T) was generated and recapitulated the typical MMAF phenotype of CFAP57-mutated individuals. Both CFAP57 mutations caused loss of the long transcript-encoded CFAP57 protein in spermatozoa from MMAF-affected individuals or from the Cfap57-mutant mouse model while the short transcript was not affected. Subsequent examinations of the spermatozoa from Cfap57-mutant mice revealed that CFAP57 deficiency disrupted the inner dynein arm (IDA) assembly in sperm flagella and that single-headed IDAs were more likely to be affected. Thus, our study identified 2 pathogenic mutations in CFAP57 in MMAF-affected individuals and reported a conserved and pivotal role for the long transcript-encoded CFAP57 in IDAs’ assembly and male fertility.Multiple morphological abnormalities of the sperm flagella (MMAF) are the most severe form of asthenozoospermia due to impaired axoneme structure in sperm flagella. Dynein arms are necessary components of the sperm flagellar axoneme. In this study, we recruited 3 unrelated consanguineous Pakistani families with multiple MMAF-affected individuals, who had no overt ciliary symptoms. Whole-exome sequencing and Sanger sequencing identified 2 cilia and flagella associated protein 57 ( CFAP43), CFAP44, CFAP47, CFAP61, CFAP65, CFAP69, CFAP70, CFAP91, CFAP251, and dynein axonemal heavy chain 17 (DNAH17) after being mutated  surrounding a pair of singlets , with radial spokes extending from each of the peripheral doublets toward the central pair , 26. Innd DNAH17 , 27–29. d DNAH17 –34. Defed DNAH17 , 35. HowCFAP57 stop-gain mutations  cosegregating with the MMAF phenotype in all 3 families. We generated Cfap57-mutant mice and found that mutant male mice were infertile due to MMAF. Both CFAP57 mutations caused loss of the long transcript-encoded CFAP57 protein in spermatozoa from MMAF-affected individuals. Further studies showed that the defect in Cfap57-mutant sperm flagella was caused by abnormal assembly of IDA proteins, of which the single-headed IDA protein was the most severely affected. Collectively, these findings demonstrate that loss-of-function mutations in CFAP57 caused MMAF and male infertility in mice and humans, providing evidence for an essential and specific role for long transcript-encoded CFAP57 in male reproduction.In this study, we recruited 3 unrelated consanguineous Pakistani families with 7 infertile men, 4 of whom were diagnosed with asthenozoospermia. WES and Sanger sequencing identified 2 homozygous https://doi.org/10.1172/jci.insight.166869DS1). The scanning electron microscopy (SEM) analysis results of spermatozoa from infertile cases further demonstrated the presence of MMAF , P2 , and P3 ; 2 infertile brothers in family 2, P4  and P5 ; and 2 infertile brothers in family 3, P6  and P7  . Despite of MMAF . In addiCFAP57  as candidate MMAF-causing mutations. Both CFAP57 mutations were located in the regions of homozygosity (RoHs) of affected individuals , encoding 2 CFAP57 proteins with 1,250 aa and 698 aa, respectively. Reverse transcription polymerase chain reaction (PCR) revealed that both transcripts of CFAP57 existed in human testes and nasal epithelial cells (CFAP57 (NM_001195831.2)  .CFAP57 mutations were predicted to introduce a premature stop codon, which may result in mRNA degradation or truncated proteins  mice  to imitate the M1 mutation identified in family 1  did not detect the WT and truncated CFAP57 proteins in M/MCfap57 mouse testes  and ODA markers  along with a microtubule marker (α-tubulin) in sperm from  pattern , A–D, wh+/M mice . Surpris+/M mice . IDAs ar+/M mice . The morse sperm . Overallse sperm .M/MCfap57 mouse testes, these IDA proteins were expressed at levels comparable to those in +/+Cfap57 and +/MCfap57 mice  that were recessively cosegregated with male infertility in 3 Pakistani families. Semen analysis of affected men revealed typical MMAF phenotypes. Both CFAP57 mutations have been verified to cause loss of long transcript-encoded CFAP57 protein in spermatozoa from MMAF-affected individuals or from a Cfap57-mutant mouse model. Extensive analysis using M/MCfap57 mice revealed that loss of the long transcript of Cfap57 disrupted IDA assembly in sperm flagella and single-headed IDAs were more likely to be affected. Our study provides direct evidence for the pathogenicity of CFAP57 loss-of-function mutations in MMAF-affected individuals and determines an essential role for the long transcript-encoded CFAP57 in spermiogenesis in humans and mice.In this study, we identified 2 stop-gain mutations in DNAH1 and DNAH6  results, the abnormalities of double-headed IDAs and ODAs may be the consequence of the disorganized ultrastructure in sperm flagella from M/MCfap57 mice, resulting from the IDA assembly failure in M/MCfap57 mice. Therefore, it is supposed that the integrity of the axoneme structure in mutant mouse sperm is more susceptible to IDA assembly failure than in humans.It is noteworthy that pathogenic mutations in corresponding IDAs or IDA assembly genes cause not only the loss of IDAs but also severely disorganized axonemal structures in sperm flagella in humans and mice , 28, 42.flagella . In addiCFAP57  was identified in a PCD-affected man, but his reproductive status was unknown (CFAP57 mutations claimed no PCD symptoms except asthenozoospermia. It should be noted that both mutations (M1 and M2) identified in our study only affected the long transcript of CFAP57 (NM_001195831.2), while the previously reported mutation affected both transcripts (NM_001195831.2 and NM_152498.3)  and the man harboring homozygous CFAP57 mutation (c.1762C>T) displayed a severe PCD-like phenotype. Together, these results suggest that the long and short transcripts of CFAP57 have distinct functional roles, the long transcript is essential for the development of sperm flagella, and the short one is critical for the development of cilia, which is worth exploring further.Previously, a pathogenic mutation in  unknown . In our 52498.3) . Since t52498.3) , 44, it 52498.3) , B–D. BeOur study provides a potentially novel direction for investigating the basis of structural heterogeneity between cilia and flagella. Although they have a high similarity in ultrastructure, the key IDA assembly factors, such as CFAP57, play essential roles in cilia and flagella through distinct transcripts from a single gene. Our study also suggests that different transcripts from one gene can be involved in different biological processes. When evaluating the specific effects of such gene mutations in human disorders, it is necessary to comprehensively consider the effects of mutations on all transcripts of the gene.CFAP57 cause MMAF and male infertility in mice and humans due to the disruption of IDA assembly. Our findings highlight the essential and conserved role of CFAP57 isoforms in cilia and flagella and improve our understanding of sperm flagellar anomaly etiology and pathophysiology. CFAP57 can be used as a genetic screening marker in genetic counseling and diagnosis of MMAF and male infertility.In conclusion, our study based on MMAF-affected individuals and the corresponding mutant mouse models demonstrates that homozygous loss-of-function mutations in In the present study, 3 Pakistani consanguineous families were recruited, with families 1, 2, and 3 having 3 , 2 , and 2  infertile brothers, respectively. Semen parameters, including semen volume, sperm concentration, and sperm motility, were analyzed at least twice for P2, P3, P6, and P7 in accordance with guidelines established by WHO , family 2 (II:2 and II:4), and family 3  using the FlexiGene DNA Kit (QIAGEN) and fragmented using a Covaris Focused-ultrasonicator. Whole-exome capture and sequencing were performed using AIExome Enrichment Kit V1 (iGeneTech) and HiSeq 2000 platform (Illumina) following standard procedures. The reads were aligned to the human genome reference assembly (hg19) using BWA-MEM, and Picard software was used to remove PCR duplicates. DNA sequence variants were called using the Genome Analysis Toolkit HaplotypeCaller. Variants were annotated using ANNOVAR , and canA CFAP57 polyclonal antibody was generated in rabbits using aa 701–900 of human CFAP57 (UniProt accession Q96MR6) as antigens by Dia-an Biotechnology.HEK293T (ATCC) cells were cultured as we previously described  and tranImmunoblotting was performed as we previously described , 49, 53.The preparation of human semen smears and immunofluorescence staining were conducted as we described . The smeM/MCfap57 mice were generated by using CRISPR/Cas9 technology  containing 4% glutaraldehyde, 4% PFA, and 0.2% picric acid at 4°C for at least 8 hours. SEM and TEM assays were conducted as we previously described , 56.t test for independent data. P < 0.05 was considered significant.Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software). Values in tables and graphs are expressed as mean ± SD. Two groups of mice were compared with Student’s 2-tailed The USTC Ethical Committee approved this study. An informed consent form was obtained from each participant before the study began.AM, BZ, and HM designed the experiments; AM, JC, XH, and BZ performed the experiments; AM wrote the manuscript; HA, TA, IA, ZM, SD, WS, BS, GM, FI, MAK, and AK recruited the families, performed semen analysis, and collected samples; QS, ZM, HZ, HM, LW, BX, and QL modified the manuscript; JZ, DZ, YZ, and HZ performed the WES sequencing and WES data analysis; and QS and HZ conceived and supervised the study and article drafting. All authors contributed to the report.