Gryllus bimaculatus, is registered as an edible insect by the Korean food and drug administration. The aim of this study was to investigate the effect of Gui-A-Gra on testicular damage induced by experimental left varicocele in male Sprague Dawley rats. A total of 72 rats were randomly divided into the following six groups (12 rats in each group): a normal control group (CTR), a group administrated with Gui-A-Gra 1.63 gm/kg (G1.63), a group administrated with Gui-A-Gra 6.5 gm/kg (G6.5), a varicocele (VC)-induced control group (VC), a VC-induced group administrated with Gui-A-Gra 1.63 gm/kg (VC + G1.63), and a VC-induced group administrated with Gui-A-Gra 6.5 gm/kg (VC + G6.5). Rats were administrated 1.63 or 6.5 gm/kg Gui-A-Gra once daily for 42 days. Indicators of sperm parameters, histopathology, reproductive hormones, oxidative stress, endoplasmic reticulum (ER) stress, inflammation, and mitochondrial apoptosis were analyzed to evaluate effects of Gui-A-Gra on VC-induced testicular dysfunction. Gui-A-Gra administration to VC-induced rats significantly (p < 0.05) increased sperm count and sperm motility, Johnsen score, spermatogenic cell density, serum testosterone, testicular superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, GPx4, and steroidogenic acute regulatory protein (StAR) level. Moreover, pretreatment with Gui-A-Gra significantly (p < 0.05) decreased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive cells/tubules, serum luteinizing hormone (LH), serum follicle-stimulating hormone (FSH), testicular tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), malondialdehyde (MDA), reactive oxygen species (ROS)/reactive nitrogen species (RNS) level, glucose-regulated protein-78 (Grp-78), phosphorylated c-Jun-N-terminal kinase (p-JNK), phosphorylated inositol-requiring transmembrane kinase/endoribonuclease 1α (p-IRE1α), cleaved caspase-3, and BCL2 associated X protein: B-cell lymphoma 2 (Bax: Bcl2) ratio in VC rats. These results suggest that protective effects of Gui-A-Gra on VC-induced testicular injury might be due to its antioxidant, anti-inflammatory, and androgenic activities that might be mediated via crosstalk of oxidative stress, ER stress, and mitochondrial apoptosis pathway.Gui-A-Gra, a commercial insect powder from  Varicocele (VC) is characterized by dilation and tortuosity of internal spermatic vein (pampiniform plexus), a network of veins that drains testicular tissues . VC is cOxidative stress and testicular apoptosis are well documented causes of testicular dysfunction ,3. PlasmGryllus bimarculatus powder that has recently been registered as an edible insect in Korea [Gryllus bimarculatus has pharmacological activity such as antioxidant and anti-inflammatory effects [Gryllus bimarculatus have beneficial antioxidant, hypocholesterolemic, and antimicrobial properties [G. bimaculatus include glycosaminoglycan which exhibits anti-inflammatory effects [Gryllus bimarculatus extract can prevent diabetes, liver disease, and arthritis [Gryllus bimarculatus on male reproductive functions has not been reported yet. Therefore, the objective of this study was to determine the protective effect of Gryllus bimaculatus powder for the recovery from testicular dysfunction and its associated male fertility condition in a VC-induced rat model. The roles of inflammation, oxidative stress, endoplasmic reticulum (ER) stress, and mitochondrial (intrinsic) pathway in VC testicular tissues were also examined to determine molecular mechanisms involved in the protective effect of Gryllus bimaculatus.It has been reported that antioxidant supplements can alleviate male infertility by scavenging ROS and modulating inflammatory responses and the antioxidant system . Gui-A-Gin Korea . Gryllus effects ,18. A reoperties . The con effects . Recent rthritis ,22,23. Hp < 0.05). Pretreatment with G 6.5 significantly increased sperm count in both vas deference and epididymis of VC rats (p < 0.05). Sperm motility in vas deference and epididymis in VC rats showed remarkable increases after pretreatment with G1.63 or G6.5 (p < 0.05)  . The fer < 0.05) .p < 0.05). Johnsen’s score and spermatogenic cell density in the VC group were significantly (p < 0.05) decreased compared to those in the CTR group  as compared to the CTR group. However, serum LH and FSH levels were significantly decreased in VC + G1.63 and VC + G6.5 groups. Levels of IL-6 and TNF-α were remarkably (p < 0.05) upregulated in the VC group as compared to the CTR group. Pretreatment with Gui-A-Gra significantly (p < 0.05) downregulated these parameters in the VC + G1.63 and VC + G6.5 groups (Serum testosterone level was significantly decreased in the VC group as compared to the CTR group . It was 5 groups .p < 0.05) enhanced MDA level and ROS/RNS level compared to rats in the CTR group. Treatment with G1.63 and G6.5 significantly (p < 0.05) restored these parameters in the VC group. VC-induced rats showed significant decreases of testicular levels of SOD, GPx, and catalase levels as compared to rats in the CTR group. These levels were remarkably (p < 0.05) increased after pre-treatment with G1.63 or G6.5. No significant difference in these parameters was observed between VC + G1.63 and VC + G6.5 groups.Results of lipid peroxidation parameters , ROS/RNS) and antioxidant enzymes  in testicular tissues are presented in p < 0.05) increases of GRP-78, p-IRE1α/ IRE1α, and p-JNK/JNK as compared to rats in the CTR group. In contrast, levels of GRP-78, p-IRE1α/IRE1α, and p-JNK/JNK levels in VC + G6.5 group were remarkably (p < 0.05) decreased as compared to those in the VC group. Levels of GRP 78 and p-JNK/JNK in VC + G1.63 group were lower than in the VC group, although differences between the two groups were not statistically significant. However, the p-IRE1α/IRE1α level in VC + G1.63 group was significant decreased as compared to the VC group. Levels of pro-caspase-3 were similar among all groups. VC-induced rats showed prominently increased expression of cleaved caspase-3 and Bax:Bcl2 ratio compared to rats in the CTR group, although the increase was not statistically significant compared with the CTR group. In contrast, the cleaved caspase 3 and Bax:Bcl2 ratio in the VC + G6.5 group was significantly (p < 0.05) decreased as compared to the VC group : B-cell lymphoma 2 (Bcl2) ratio, StAR, and Gpx4 were analyzed in testis tissues by western blotting. Results are shown in VC group E. The clVC-induced rats showed significantly decreased expression levels of StAR as compared to rats in the CTR group. However, treatment with Gui-A-Gra restored levels of StAR in varicocelized rats. Cell-specific expression of StAR, Grp 78, and cleaved caspase 3 proteins in testis tissues was further analyzed by immunohistochemistry staining A–C. A daThe present work used an experimental VC model to determine treatment effect of Gui-A-Gra on VC. This work extended studies on pathophysiological mechanisms associated with VC and suggested an ameliorative effect of Gui-A-Gra on VC. This study is first to report a protective effect of Gui-A-Gra on testicular dysfunction and male infertility using a VC-induced rat model. Pretreatment with Gui-A-Gra significantly improved sperm parameters, regulated levels of reproductive hormones, and promoted spermatogenesis in VC rats. Results of testicular morphological analysis, TUNEL assay, immunohistochemistry, and western blot analyses indicated that Gui-A-Gra could also decrease germ cell apoptosis.Previous studies have shown testicular hypotrophy in patients with clinical VC, but not in patients with subclinical VC . In the In our study, VC-induced rats showed degenerative changes in mammalian testis such as testicular atrophy, apoptosis of germ cell, aberration of STs, and impaired spermatogenesis. Similar findings have been reported in previous experimental VC studies ,2,3. TesLeydig cells are located in the interstitial tissue of the testis where testosterone is synthesized. They are essential for spermatogenesis . The StAIn the present study, higher concentrations of testicular MDA were detected in VC rats. The results of our study were similar to the MDA results of human testicular biopsy samples from infertile patients with VC . FurtherApoptosis is another detrimental factor involved in the impairment of sperm quality in patients with VC . IncreasGryllus bimarculatus powder material under the name of Gui-A-Gra used for the experiments was supplied from 239bio Inc. , a facility accredited for hazard analysis and critical control point (HACCP) and good manufacturing practice (GMP). Cultivated adult G. bimarculatus were subjected to 1-day of defecation before sacrifice. The G. bimarculatus were then cleansed with tap water three times, sterilized at 100 °C for 3–5 min, and dried for 6 h to have moisture less than 5%. Finally, dried G. bimaculatus were ground and homogenized. The powder was then stored at −20 °C for further use.A commercial Seventy-two 7–8 weeks old male Sprague Dawley rats were purchased from Koatech  and randomly divided into six groups (12 rats/group): (1) normal control (CTR) group; (2) Gui-A-Gra 1.63 gm/kg p.o. group (G1.63); (3) Gui-A-Gra 6.5 gm/kg p.o. group (G6.5); (4) varicocele (VC) group; (5) VC + Gui-A-Gra 1.63 gm/kg p.o. group (VC + G1.63); and (6) VC + Gui-A-Gra 6.5 gm/kg p.o. group (VC + G6.5). Rats were maintained in plastic cases (47 × 18 × 40 cm) (four rats per case) in controlled environment conditions . Rats had free access to water ad libitum and standard rat chow diet. All experiments were performed according to the Institutional Animal Care and Use Committee (IACUC) of Jeonbuk National University Hospital Laboratory Animal Center (cuh-IACUC-2017-13). After one week of acclimatization, left VC was induced for VC group, VC + G1.63 group, and VC + VC + G6.5 group of rats according to standard protocols . CTR and6 sperms/mL. Motile spermatozoa were analyzed with the formula of  × 100). Results for sperm motility are expressed as percentages.Epididymal and vas deference sperm count and sperm motility were evaluated as described previously . In brieTestis tissues were fixed in Bouin’s solution, dehydrated, embedded in paraffin wax, and sectioned into 5 μm slices. Testis sections were subjected to hematoxylin and eosin (H&E) staining and TUNEL staining  as described previously . At leasSerum levels of testosterone , luteinizing hormone (LH) , and follicle-stimulating hormone (FSH)  were measured using enzyme-linked immunosorbent assay (ELISA) per each manufacturer’s protocols.Whole tissue supernatants were prepared as described previously . TesticuMalondialdehyde (MDA) levels in testis tissues were assessed using reagents provided by Northwest Life Science Specialties LLC . Briefly, MDA reaction of a colored complex was measured at an absorbance of 532 nm using a kinetic spectrophotometer . MDA levels are expressed as μmole of MDA/mg of wet testis tissue. Testicular ROS/RNS levels were quantified using an OxiSelect in Vitro ROS/RNS Assay Kit  according to the manufacturer’s protocol. Fluorescence of dichlorofluorescein (DFC) was measured with a SpectraMax Gemini XS fluorimeter  at excitation/emission wavelengths of 480/530 nm as described previously [Paraffin-embedded consecutive testis tissue sections (5 μm in thickness) were deparaffinized with xylene, dehydrated with alcohol, and subjected to heat-mediated 1× target retrieval solution at pH 6.0 . Endogenous peroxide activity was blocked with peroxidase-blocking solution (DAKO) for 15 min and washed with 1× PBS buffer twice (5 min each). Non-specific bindings of testis section were further blocked with serum block solution for 10 min (DAKO) and incubated with primary antibodies cleaved caspase 3 , StAR , and Grp 78  at 4 °C overnight. Sections were then washed with 1× PBS twice (5 min each) and incubated with a horseradish peroxidase (HRP)-labeled micropolymer conjugated secondary antibody  at room temperature for 1 h. Prior to incubation, sections were rinsed with AEC substrate chromogens . Slides were then rinsed with deionized water for 5 min, counter-stained with hematoxylin, rinsed with distilled water, and mounted with mounting medium .Extraction of protein from testis tissue was conducted as described previously . Proteinp < 0.05. GraphPad PRISM 6.0 was used for graphical analysis .All quantitative data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using SPSS version 22 . One-way analysis of variance (ANOVA) was used to compare groups followed by Tukey’s test for post-hoc analysis. Statistical significance was considered at In summary , our res


Enterobacterales and its association with plasmid mediated quinolone resistance (PMQR), aminoglycoside modifying enzymes (AME) and Trimethoprim/Sulfamethoxazole (TMP-SMX) resistance is little known from North India. Therefore, the current study was aimed to investigate the frequency of Non-β-Lactam antibiotic resistance associated genes in extended spectrum β-Lactamase producing Enterobacterales. For this study, Non-Duplicate phenotypically confirmed ESBL producing Enterobacterales isolates (N = 186) were analyzed for ESBLs, PMQRs, AMEs and TMP-SMX resistance genes using polymerase chain reaction (PCR). PCR detected presence of PMQR genes in 81.29% (N = 139) of ESBL isolates (N = 171), AME genes in 60.82% and TMP-SMX resistance genes in 63.74% of the isolates. Molecular characterization of ESBL producing Enterobacterales showed 84.79% TEMbla followed by 73.68% CTX-Mbla, 43.86% SHVbla, 19.88% PER andbla 9.94% VEBbla, respectively. Analysis of PMQR genes revealed 77.7% aac(6′)-lb-cr the most commonly detected gene followed by 67.63% oqxB, 62.59% oqxA, 43.17% qnrB, 19.42% qnrD, 18.7% qnrS, 9.35% qnrA, 3.6% qepA and 2.88% qnrC, respectively. Analysis of AMEs gene profile demonstrated 81.73% aac(6′)-Ib, the most frequently encountered gene followed by 46.15% aph(3′)-Ia, 44.23% ant(3”)-Ia, respectively. A 100% prevalence of sul1, followed by dfrA (54.63%) and sul2 (15.74%) was observed. In summary, prevalence of ESBL-Producing genes (particularly TEMbla and CTX-Mbla) along with PMQR, AMEs, and TMP-SMX resistant genes may potentially aid in the transfer of antimicrobial resistance among these strains.Genetic context of extended spectrum β-Lactamase (ESBL) producing  Enterobacterales [Enterobacterales and other clinically significant Gram-negative bacteria [Enterobacterales [Enterobacterales [qnr) gene mediated (ii) quinolones modifying aminoglycoside acetyltransferase encoding genes (aac(6′)-Ib-c), (iii) plasmid-mediated quinolone efflux pumps qepA, oqxA, and oqxB [β-Lactam antibiotics are used for treating most of the human infections that are caused by Gram-negative bacteria belonging to the family cterales . Howevercterales . ESBLs abacteria ,2. Genesbacteria . Furthercterales . Chromoscterales ,5. Genercterales ,6,7,8. PEnterobacterales demonstrate resistance to TMP-SMX as well. However, little has been reported on the genetic context of various species of ESBL producing Enterobacterales and its association with, PMQR, AMEs TMP-SMX resistance exclusively from North India. Therefore, the current study was designed to analyze the distribution of non-β-lactam antibiotic resistance associated genes prevailing in ESBL producing Enterobacterales. To our knowledge, this is the first study that investigated the distribution of AME, PMQR and TMP-SMX resistance genes among ESBL producing Enterobacterales that are isolated from North India. Furthermore, this study included clinical isolates of different members of Enterobacterales , while the earlier studies limited their molecular characterization in two species (E. coli and K. pneumoniae) [Enterobacterales, an observation that has been made only a few times earlier.Literature indicates that synthesis of aminoglycoside modifying enzymes (AMEs) is an important AMR mechanism that produce high level of aminoglycoside resistance among Gram- negative bacteria ,10. Thesumoniae) ,10,13. IK. pneumoniae 16.13% (30/186), E. coli 9.67% (18/186), K. oxytoca 2.15% (4/186), P. mirabilis 1.61% (3/186) and P. vulgaris 0.53% (1/186) were resistant to all the antibiotics tested. Enterobacterales. The highest antibiotic resistance rate was noted for ampicillin 95.89% (70/73) followed by TMP-SMX 90.86% (169/186). The resistance rate of β-lactams were: cefazolin 94.08% (175/186), amoxicillin-clavulanic acid 77% (124/161), cefotaxime 82.8% (154/186), cefepime 66.67% (124/186), ceftazidime 89.78% (164/186), ceftriaxone 88.17% (164/186), cefoxitin 76.83% (126/164), cefpodoxime 86.11% (155/180), cefuroxime 86.02% (160/186), and ceftizoxime 83.3% (155/186), respectively. The resistance rate of fluoroquinolones were 75.27% (140/186), 69.89% (130/186), 76.88% (143/186), 75.27% (140/186), and 71.51% (133/186) for ciprofloxacin, levofloxacin, nalidixic acid, gatifloxacin, and moxifloxacin, respectively. Similarly, the resistance rate of aminoglycosides were 64% (119/186), 61% (114/186), 49% (91/186), and 59% (109/186) for gentamycin, tobramycin, amikacin, and kanamycin, respectively. The lowest resistance rates were associated with imipenem 43.01% (80/186) and meropenem 46.77% (87/186), respectively  obtained, ectively .Enterobacterales. Among the total phenotypically confirmed ESBL isolates (N = 186), PCR confirmed presence of ESBL genes  in 91.94% (171/186) of Enterobacterales isolates. However, genes coding for ESBL production were not detected from K. pneumoniae 2.69% (5/186), E. coli 2.69% (5/186), K. oxytoca 1.07% (2/186), P. mirabilis 1.07% (2/186), C. freundii 0.54% (1/186) and therefore, these isolates were not included for further analysis. ESBL genes were detected from pus (N = 97), respiratory tract specimens (N = 44), blood and body fluids (N = 30), respectively , E. coli 30.99% (53/171), P. mirabilis 7.6% (13/171), C. freundii 7.02% (12/171), K. oxytoca 5.2% (9/171), E. cloacae 5.2% (9/171), P. vulgaris 1.75% (3/171), and M. morganii 1.75% (3/171), respectively. Of the 91.94% (N = 171) genotypically confirmed isolates, TEMbla 84.79% (145/171) was the most common ESBL gene followed by CTX-Mbla 73.68% (126/171), SHVbla 43.86% (75/171), PERbla 19.88% (34/171) and VEBbla 9.94% (17/171), respectively.ectively . The detEnterobacterales (N = 139) as follows; K. pneumoniae 44.6% (62/139), E. coli 31.65% (44/139), P. mirabilis 8.63% (12/139), C. freundii 5.03% (7/139), K. oxytoca 5.03% (7/139), E. cloacae 2.16% (3/139), P. vulgaris 2.16% (3/139), and M. morganii 0.72% (1/139), respectively (aac(6′)-lb-cr 77.7% (108/139) followed by oqxB 67.63% (94/139), oqxA 62.59% (87/139), qnrB 43.17% (60/139), qnrD 19.42% (27/139), qnrS 18.71% (26/139), qnrA 9.35% (13/139), qepA 3.6% (5/139), and qnrC 2.88% (4/139), respectively , majority of the isolates to had co-existence with TEMbla (13/13) and CTX-Mbla (8/13) gene (qnrB gene (N = 60) demonstrated coexistence with TEMbla (46/60), CTX-Mbla (43/60), and SHVbla (27/60). In contrast, strains carrying Aac-ib-cr gene (N = 108), most of the isolates showed coexistence with TEMbla (98/108), CTX-Mbla (84/108), SHVbla (47/108), PERbla (28/108), and VEBbla (13/108) (oqxA and oqxB as well. Among the isolates that carrying oqxA (N = 87) gene, majority of the isolates were possessing TEMbla (72/87), CTX-Mbla (66/87) and SHVbla (39/87), PERbla (17/87) and VEBbla (12/87), respectively (oqxB genes was carrying TEMbla (80/94), CTX-Mbla (71/94) and SHVbla (46/94), PEblaR (15/94) and VEBbla (11/94), respectively. It was also noted that Enterobacterales isolates that having higher minimum inhibitory concentration (MIC) values for ciprofloxacin (≥16 μg/mL), nalidixic acid (≥64 μg/mL), levofloxacin (≥32 μg/mL),gatifoxacin (≥32 μg/mL) and moxifloxacin (≥32 μg/mL) mainly harboured Aac-ib-cr, qnrB, oqxA and oqxB genes. Further, the frequency of PMQR genes were high in isolates with higher quinolone MIC than their low MIC counterparts , 81.72% (152/186), 70.43% (131/186), and 90.32% (168/186), were resistant to fluoroquinolones, aminoglycosides and TMP-SMX, respectively. Out of the 91.94% (171/186) of the genotypically confirmed ESBL strains, PCR could detect 81.29% (PMQR), 60.82% (AME), 63.74% (TMP-SMX) of genes in these strains. Of the total PMQR genes (81.29%) detected, the distribution of genes in different members of ectively . PMQR geectively . Of the ectively . Among t13) gene . Similar(13/108) . This coectively . Similarterparts .Enterobacterales (N = 104) is as follows; K. pneumoniae 44.23% (46/104), E. coli 38.46% (40/104), P. mirabilis 2.89% (3/104), C. freundii 5.77% (6/104), K. oxytoca 3.84% (4/104), E. cloacae 2.89% (3/104), P. vulgaris 0.96% (1/104), and M. morganii 0.96% (1/104), respectively (aac(6′)-Ib 81.73% (85/104), followed by aph(3′)-Ia 46.15% (48/104), ant(3”)-Ia 44.23% (46/104), aac(3)-IIa 45.19% (47/104), aph(3”)-Ib 35.58%, (37/104), armA 14.42% (15/104), ant(4”)-IIa 12.5% (13/104), aac(3)-Ib 10.58%(11/104), ant(2”)-Ia 8.65% (9/104), aac(3)-Ia 6.73% (7/104), respectively (aac(2′)-Ia, aac(6′)-Ia, aac(6′)-Ic and aph(3”)-Ia were not detected in any of the strains analyzed (Out of the 60.82% (104/171) of AME genes detected, the distribution of these genes in the ectively . The disectively . Howeveranalyzed .TEMbla (43/47) and CTX-Mbla (38/47) genes. Similarly, strains carrying aac(6′)-Ib gene (N = 85) demonstrated coexistence with TEMbla (75/85), CTX-Mbla (72/85), and SHVbla (43/85). However, strains carrying ant (3”)-Ia gene (N = 46), majority of isolates showed co-existence with TEMbla (41/46), CTX-Mbla (35/46) and SHVbla (23/46). This coexistence of genes were also observed among the isolates that carrying aph(3”)-Ia and aph(3”)-Ib wherein majority of the strains were equally harbouring both TEMbla and CTX-Mbla genes (aac(6′)-Ib, aph(3”)-Ia, aac(3)-IIa, ant(3”)-Ia genes, respectively  that carried aac(3)-IIa genes (N = 47), majority of the isolate had co-existence with -M genes . Furtherectively . The ratectively .Enterobacterales isolates, the distribution of TMP-SMX resistance genes are as follows; K. pneumoniae 42.59% (46/108), E. coli 34.26% (37/108), P. mirabilis 5.56% (6/108), C. freundii 6.5% (7/108), K. oxytoca 5.56% (6/108), E. cloacae 4.63% (5/108), P. vulgaris 1.86% (2/108), and M. morganii 0.92% (1/108), respectively (sul1 100% (108/108), followed by dfrA 54.63% (59/108) and sul2 15.74% (17/108), respectively (Of the 63.74% (108/171) of TMP-SMX resistant ESBL producing ectively . These Tectively .TEMbla (95/108), CTX-Mbla (81/108) and SHVbla (51/108) genes, respectively. Similarly, strains carrying dfrA1 gene (N = 59) demonstrated coexistence with TEMbla (53/59) and CTX-Mbla (48/59)  for TMP-SMX mainly harbored sul1 and dfrA1 genes. It was also observed that the frequency of TMP-SMX genes was higher in strains with high TMP-SMX MIC values , majority of the isolates were having co-existence with  (48/59) . FurtherC values .p < 0.05) different from that found in the respiratory tract specimens  were comparable with earlier studies [TEMbla (84.79%) followed by CTX-Mbla (73.68%), SHVbla (43.86%), PEblaR (18.71%) and SHVbla (9.94%), respectively. These findings were in accordance with an earlier study wherein most prevalent ESBL gene found was TEMbla (73%) followed by CTX-Mbla (25–100%) and SHVbla (23%) [PERbla and VEBbla in the present study were comparable with the data reported by Khurana et al. [The current study investigated the frequency of non-β-lactam antibiotic resistance associated genes among ESBL producing  studies ,22. The K. pneumoniae (44.6%) was having higher number of PMQR genes detected followed by E. coli (31.65%). This observation is in accordance with an earlier study wherein, PMQR genes were more frequently encountered among E. coli, Klebsiella species, and Enterobacter species [Enterobacter cloacae in this study. This is probably due to the difference in the geographical location as the earlier study was conducted in the southern part of India (where the usage of antibiotics is different). Further, the widespread antibiotic resistance prevalent in India may be attributed to readily availability of antibiotics across the pharmacy counters. This could play a major role in increased distribution of antibiotic-resistance genes throughout the population. This study included isolates that obtained from wound, respiratory tract, body fluid and blood specimens while other studies were performed mostly using isolates that are collected from urine [aac(6′)-lb-cr (77.7%) which is in agreement with an earlier report wherein the prevalence rate was found to be 64.5% [qnrB (43.17%) was observed, which was in accordance with Yang et.al observation  [qnrD, qnrS, qnrA, and qnrC [Enterobacterales isolates were found to be oqxB (67.63%), oqxA (62.59%) and qepA (3.6%), respectively. However, this efflux pump mediated drug resistance mechanism of bacteria can be subdued using various efflux inhibitory molecules [Enterobacterales in North India. However, the oqxA and oqxB prevalence rates were comparable with earlier reports wherein the prevalence rates were found to be 88% and 30% for oqxA and oqxB genes, respectively [oqxA and oqxB genes may be attributed to the geographical distribution and type of isolates studied, as most of the studies were conducted on E. coli and K. pneumonia isolates [qepA observed (3.6%) was similar with previous data (2%) in the literature [qepA in the current study may also indicate low incidence of qepA gene among different strains of Enterobacterales across the world [TEMbla and CTX-Mbla) were more likely to carry aac(6′)-Ib-cr and qnrB genes. The genes that code for both ESBL and PMQR proteins are usually located on same plasmids and consequently that may have higher chances of transfer among the members of Enterobacterales. Therefore, it is very pertinent to comprehend the drug resistance mechanisms prevalent among the members of medically important bacteria as it may be a major concern for patient safety and in determination of therapeutic strategies.In the present study, PCRs detected presence of PMQR genes in 81.29% N = 139) of genotypically confirmed ESBL isolates (N = 171), indicating the presence of high frequency of PMQR genes among ESBL strains. Interestingly, 9 of genoeumoniae 4.6% was om urine . Of the coli 31.6%. This oand qnrC ,23. It wolecules , for insolecules . Furtherolecules . Furtherectively . Furthercoli 31.6%. This oisolates . The lowterature . This locoli 31.6%. This oEnterobacterales. This relatively higher prevalence rate of AMEs in ESBL producing strains may be due to co-existence of genes encoding ESBLs and AMEs in Gram-negative bacteria [K. pneumoniae (44.23%) and E. coli (38.46%). This was in accordance with an earlier study conducted by Haidar et al., wherein a higher prevalence of AME genes were reported among K. pneumoniae [aac(6′)-Ib (81.73%) which is in agreement with the earlier studies  [aac(6′)-Ib with other AME genes observed may be attributed to the fact that the gene coding for aac(6′)-Ib enzyme is frequently located within class I integrons. Further, it is known that the gene cassettes that carry other genes coding for AMEs can be easily incorporated into class I integrons resulting in the development of resistance to currently used aminoglycosides [aph(3′)-Ia (46.15%), followed by ant(3”)-Ia (44.23%), aac(3)-IIa (45.19%), and aph(3”)-Ib (35.58%). The higher prevalence of aph(3′)-Ia and aph(3”)-Ib is alarming as this type of resistance may usually produce high level of aminoglycoside resistance. The prevalence of other AME genes were found to be relatively low, which is comparable with earlier studies [Genes encoding AMEs are prevalent in various groups of bacteria ,30,31,32bacteria . In thiseumoniae . In the was 73%) . The preycosides . The oth-IIa 45.1%, and aponiae 44.% and E. sul1, sul2, or dfrA genes are likely to be present either on chromosome or on plasmids [Enterobacterales. Further, among the genotypically confirmed TMP-SMX resistant strains (N = 108), 42.59% of K. pneumonia isolates were carrying TMP-SMX resistance genes, followed by E. coli (34.26%). However, due to the paucity of literatures, the comparison with earlier studies could not performed. To our knowledge, this new study report, the prevalence of TMP-SMX resistance genes among various clinical strains of ESBL producing Enterobacterales. A higher prevalence of sul1 (100%) followed by dfrA (54.63%) and sul2 (15.74%), genes were noted in this study. This higher prevalence of sul1 gene may be attributed to the fact that the sul1 genes are usually located within class I integrons and this particular characteristic  might have further helped in its wide distribution [dfrA1 (33.91%) gene in the present study was in agreement with an earlier study wherein in the prevalence rate was also found to be high [Enterobacterales isolates that showed elevated MICs for TMP-SMX mainly harbored sul1 and dfrA1 genes indicating the likelihood of these genes in imparting resistance to TMP-SMX. However, no genes were detected from number of isolates that were having higher MIC values, suggesting the existence of alternative pathways of resistance in TMP-SMX resistant isolates.The TMP-SMX resistance genes such as plasmids ,34. In tplasmids . Howeverribution . The com be high . EnterobEnterobacterales. This hospital laboratory receives samples from two civil hospitals and three primary health care centers that are attached to it. Among the total samples analyzed (N = 2134), a total of 186 non-repetitive phenotypically confirmed ESBL producing Enterobacterales isolates (one organism per patient was included to avoid duplication) were obtained. All the isolates were identified by manual API® system  and the results interpreted as recommended by the manufacturer. The strains included were K. pneumoniae (N = 74), E. coli (N = 58), P. mirabilis (N = 15), C. freundii (N = 13), K. oxytoca (N = 11), E. cloacae (N = 9), P. vulgaris (N = 3) and M. morganii (N = 3). All the clinical isolates were identified by standard laboratory procedure [Between July 2018 and June 2019, a total of 2134 clinical samples  received in the Microbiology laboratory, Government Medical College and Hospital, Badaun, India were analyzed for ESBL producing strains of toca N = , E. cloaK. pneumoniae, C. freundii, K. oxytoca, E. cloacae, P. vulgaris, and M. morganii against Ampicillin (b) C. freundii, E. cloacae, and M. morganii against amoxicillin- clavulanic acid (c) C. freundii, and E. cloacae against cefoxitin (d) P. vulgaris, and M. morganii against cefpodoxime, respectively [E. coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853).The antibiotic susceptibility testing was conducted by modified Kirby Bauer disc diffusion method as recommended by the Clinical Laboratory Standards Institute (CLSI) . The antectively . QualityThe MIC of fluoroquinolones , aminoglycosides  and TMP-SMX were determined by broth micro dilution method and the results were interpreted in accordance with the CLSI guidelines . The MICE. coli, K. pneumoniae, and K oxytoca). However, zone size with ≤22 mm cefpodoxime (10 μg), ≤22 mm with ceftazidime (30 μg), ≤27 mm with cefotaxime, indicated ESBL production for P. mirabilis [ESBL activity was initially screened using disk diffusion method as recommended by CLSI . Isolateirabilis .The double-disk synergy diffusion test (phenotypic confirmatory test for ESBL) was carried out as per CLSI recommendations. Briefly, ceftazidime (30 μg) and cefotaxime (30 μg) alone and in combination with clavulanic acid  were used. The ESBL production is confirmed when there is an increase of zone diameter (≥5 mm) around disk with antibiotic-clavulanic acid combination .E. cloacae, C. freundii, P. vulgaris, and M. morganii strains. This test was performed as disc diffusion assay on Mueller-Hinton agar (MHA). Briefly, antibiotic discs containing aztreonam (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), and cefotaxime (30 μg) were kept 30 mm apart (center to center) around amoxicillin-clavulanic acid (20 μg + 10 μg) disc on MHA plate inoculated with the organism to be tested. The MHA plates were incubated at 37 °C for 24 h. An increased zone of inhibition of any of the test antibiotic towards amoxicillin-clavulanic acid was considered as ESBL production [E. coli ATCC 25922 (non-beta-lactamases producer) and K. pneumoniae ATCC 700603 (ESBL producer), respectively.The disc approximation test was used to confirm the ESBL production in Enterobacterales using QIAamp DNA Mini Kit . The DNA samples obtained by this procedure were segregated into two aliquots; first aliquot was used as template for the subsequent PCR reactions and the second aliquot was stored at −80 °C , Ghaziabad, India) for future use.Bacterial DNA was obtained from the phenotypically confirmed ESBL strains of Enterobacterales isolates were subjected to molecular characterization of the relevant encoding genes such as SHV, blaCTX-M, blaTEM, blaPERbla, and VEBbla by PCR using primers and PCR conditions shown in All the phenotypically confirmed ESBL producing Enterobacterales isolates that resistant to fluoroquinolones were subjected to PCRs for detection of (i) qnr proteins coding genes such as qnrA, qnrB, qnrC, qnrD, and qnrS (ii) quinolones modifying aminoglycoside acetyltransferase coding genes (acc(6′)-Ib-cr) and (iii) plasmid-mediated quinolone efflux pump protein encoding genes  using primers and PCR conditions depicted in ESBL producing Enterobacterales were screened for AMEs coding genes aac(2′)-Ia, aac(3)-Ia, aac(3)-Ib, aac(3)-IIa, aac(6′)-Ia, aac(6′)-Ib,aac(6′)-Ic,ant(2”)-Ia, ant(3”)-Ia, ant(4”)-IIa, aph(3′)-Ia, aph(3”)-Ia, aph(3”)-Ib, armA using primers described in ESBL producing aminoglycosides resistant strains of sul1, sul2 and dfrA genes using primers and PCR conditions shown in 4)2SO4, 3 mM MgCl2, 0.2% Tween 20), 200 mM of each dNTPs, 0.5 µM of each primer, and 2.0 units of AURA Taq DNA polymerase. Amplification was carried out as follows: initial denaturation at 95 °C for 5 min; 30 cycles of denaturation at 95 °C for 30 s, annealing at 50–62 °C for 30 s, extension/elongation at 72 °C for 45 s; and a final elongation step at 72 °C for 5 min. PCR-generated products were detected by electrophoresis of 7 μL of each amplification mixture in 2% agarose gels in 1% Tris Borate-EDTA buffer and 0.5 µg/mL ethidium bromide.All isolates phenotypically resistant to TMP-SMX were subjected to PCR for detection of To identify the ESBL, PMQR, AME, and TMP-SMX resistance genes detected in the PCR assays, automated DNA sequencing of the amplicons were conducted. More specifically, multiplex PCR-generated products were separated in 2% low melting agarose gel with 1% Tris-acetate-EDTA buffer. The PCR products were excised from the agarose gel and purified using the QIAquick PCR purification kit  as recommended by the manufacturer. The nucleotide sequencing of amplicons was conducted using an ABI 3730xl DNA Analyzer . Basic Local Alignment Search Tool (BLAST) program was used to compare each ESBL, PMQR, AME, and TMP-SMX resistance gene sequences against those available in gene bank at the National Center of Biotechnology Information database.p < 0.05 were considered as significant.Chi-squared test was used to compare the association between the origin of strain and type of resistance genes detected. The null hypothesis will be accepted if the presence of genes in all the groups  were similar. Dunn’s multiple comparisons test was performed to compare the differences in the number of resistance genes obtained between two categories of samples. All statistical analyses were performed using Graph pad Prism . The statistical difference values showing Enterobacterales and the association of these genetic determinants with PMQR, AME, and TMP-SMX resistance genes in the north India. The current study demonstrated widespread occurrence of PMQR, AME, and TMP-SMX drug resistant genetic determinants in the ESBL producing Enterobacterales strains. Screening of PMQR genetic elements in ESBL producing Enterobacterales strains revealed high prevalence of both aac(6′)-Ib-cr and qnrB. However, molecular analysis of AMEs producing Enterobacterales strains showed high prevalence of aac(6′)-Ib followed by aph (3′)-Ia. On examination of TMP-SMX resistant strains, sul1 was found to be the most frequently encountered gene followed by dfrA. The association of ESBL-producing genes with the PMQR, AMEs, and TMP-SMX resistance genes may potentially aid in transfer of drug resistance determinants among these strains. Therefore, a complete understanding of PMQRs, AMEs, and other drug resistance mechanisms will help in determining rationale of treatment and infection control measures in hospital settings.In summary, this is the first study that investigated the occurrence of genetic determinants prevailing in ESBL producing


Introduction: Intensive oncological treatment integrated with resection of metastases raised the clinical outcome of metastatic colorectal cancer (MCRC). In clinical practice, complex evaluation of clinical , and biological  parameters addresses tailored, personalized multidisciplinary treatment strategies. Patients with MCRC unsuitable for first-line intensive medical treatments are prevalent and showed worse clinical outcome. After progression to oxaliplatin-based chemotherapy, aflibercept/FOLFIRI significantly improved clinical outcome, even if no survival benefit was reported in adjuvant fast relapsers by aflibercept addition. The case reported a young-elderly (yE) patient with KRAS mutant colorectal cancer rapidly progressing to adjuvant chemotherapy, unfit owing to comorbidities, with multiple pharmacogenomic alterations, who gained long-term survival in clinical practice by multidisciplinary treatment strategy consisting of first-line and re-introduction of aflibercept-containing chemotherapy and two-stage lung metastasectomies.Case presentation: A 71-years-old yE patient, unfit for intensive oncological treatments owing to Cumulative Illness Rating Scale (CIRS) stage secondary, affected by KRAS c.35 G>T mutant colorectal cancer, rapidly progressing with lung metastases after adjuvant XelOx chemotherapy, reached long-term survival 66 months with no evidence of disease after first-line and re-introduction of tailored, modulated aflibercept (4 mg/kg) d1,15-irinotecan (120 mg/m2) d1,15-5-fluorouracil (750 mg/m2/day) dd1–4, 15–18; and secondary radical bilateral two-stage lung metastasectomies. Safety profile was characterized by limiting toxicity syndrome at multiple sites (LTS-ms), requiring 5-fluorouracil discontinuation and aflibercept reduction (2 mg/kg), because of G2 hand-foot syndrome (HFS) for >2 weeks, and G3 hypertension. Pharmacogenomic analyses revealed multiple alterations of fluoropyrimidine and irinotecan metabolism: severe deficiency of fluorouracil degradation rate (FUDR), single nucleotide polymorphisms of UGT1A1*28 variable number of tandem repeats (VNTR) 7R/7R homozygote, ABCB1 c.C3435T, c.C1236T, MTHFR c.C667T homozygote, DPYD c.A166G, TSER 28bp VNTR 2R/3R heterozygote.Conclusions: In clinical practice, a complex management evaluating clinical parameters and RAS/BRAF genotype characterizing an individual patient with MCRC, particularly elderly and/or unfit owing to comorbidities, is required to properly address tailored, multidisciplinary medical and surgical treatment strategies, integrated with careful monitoring of superimposing toxicity syndromes, also related to pharmacogenomic alterations, to gain optimal activity, and long-term efficacy. KRAS/NRAS/BRAF genotype . We previously demonstrated that first-line intensive FIr-B/FOx triplet chemotherapy plus bevacizumab reached an objective response rate (ORR) of 82%, median progression-free survival (PFS) of 12 months, and overall survival (OS) of 28 months . Integragenotype . In non-tastases . In patitastases . Patienttastases , 8.KRAS/NRAS/BRAF genotype) parameters addresses tailored, multidisciplinary treatment strategies  and biological  d1, capecitabine (825 mg/m2 bid) d1–14, cycles repeated every 21 days, for six cycles. Safety profile was characterized by LTS-ms, specifically G2 HFS associated with G2 anemia  d1,15-irinotecan (120 mg/m2) d1,15-5-fluorouracil (750 mg/m2/day) dd1–4, 15–18, cycles repeated every 28 days  of secondary lung metastases with intra-lesion necrosis  mutant MCRC rapidly progressing to adjuvant chemotherapy with bilateral lung metastases, unfit for intensive medical treatment owing to comorbidities, who was treated by first-line and re-introduction of aflibercept-containing chemotherapy followed by two-stage lung metastasectomies and gained in clinical practice by multidisciplinary treatment strategy long-term OS 66 months of metastatic disease with no evidence of disease at PFI 40 months.The case reported a yE patient with KRAS c.35 G>T (G12V) mutation  mutation  and shownd death , with sind death ; the poond death , 19. KRA cancers .KRAS c.35 G>A mutant status was significantly associated with worse clinical outcomes of patients with MCRC treated with FIr-B/FOx compared with KRAS/BRAF wild-type and other KRAS mutant patients  , consistidedness . Adjuvanidedness , 8; in Sidedness , 30 and with PFS , 32. Aflwith PFS . Afliberwith PFS . Afliberwith PFS . At progS mutant . TripletS mutant .Then, because of long-term control of bilateral lung metastastes during first-line aflibercept/irinotecan and re-challenge, bilateral resections of lung metastases were performed. The diagnosis of mucinous lung metastases may justify such a long OS 66 months of metastatic disease without evidence of disease at PFS 50 months and PFI 40 months from second stage lung metastasectomies, thus realizing the effectiveness of the integrated lung metastasectomies. In a retrospective Spanish real-life analysis of 32 patients who underwent surgical resection after aflibercept/FOLFIRI , PR was Reported yE, unfit patient underwent first-line MCRC treatment with reduced doses of aflibercept, irinotecan, and fluorouracil. Nevertheless, safety profile was characterized by LTS-ms, particularly G2 HFS lasting >2 weeks with different other G2–G1 toxicities. Because of limiting HFS and multiple pharmacogenomic alterations, 5-fluorouracil was discontinued; then, LTS-ms was observed, characterized by G3 hypertension, and re-introduction was planned with further aflibercept dose reduction. Reported prevalent toxicities with FOLFIRI/aflibercept were diarrhea (19.3%), mucositis 13.7%), asthenia (16.9%), HFS 2.8%), hypertension (2.9%), arterial (1.8%) and venous (7.9%) thromboembolic events, neutropenia (36.7%), and thrombocytopenia (3.3%) %, hypert. In Span.7%, asthUGT1A1*28 homozygote 7 repeats, ABCB1 c.C3435T and C1236T heterozygote, MTHFR—c.C667T homozygote, DPYD c.A166G heterozygote, and TSER 28bp heterozygote VNTR 2R/3R. Dihydropyrimidine dehydrogenase gene (DPYD) and UGT1A1 SNPs variably influence fluoropyrimidines and irinotecan tolerability (DPYD c.496A > G (P = 0.022) and c.1896 T > C (P = 0.027), trendly with UGT1A1*28 SNPs (P = 0.054) , ABCB1 (71%), CYP3A4 (14%), and DYPD (15%), in the reported range , thus predicting occurrence of LTS-ms in patients at risk of gastrointestinal LT  . UGT1A1*tropenia –49. 5-FUtropenia , 51. To ed range , 49. MosRAS/BRAF genotype of patients with MCRC, particularly the elderly and/or those who are unfit because of comorbidities, is required to properly tailor multidisciplinary medical and surgical treatment strategies to gain optimal activity and long-term efficacy, integrated with careful monitoring of toxicity syndromes, potentially related to pharmacogenomic alterations.In clinical practice, a complex management evaluating patient-related clinical parameters and Written informed consent was obtained from the patient for the publication of any potentially identifiable images or data included in this article.GB and ER were the medical oncologists responsible for clinical and bioclinical management of patient. AD'A, SG, and EAR were abdominal and thoracic surgeons responsible for surgical management of patient. MS performed pharmacogenomic analyses. All authors read and approved the final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AB declared a past co-authorship with one of the authors MS to the handling editor.


Chrysomallon squamiferum) is highly adapted to deep-sea hydrothermal vents and has drawn much interest since its discovery. However, the limited information on its genome has impeded further related research and understanding of its adaptation to deep-sea hydrothermal vents.The scaly-foot snail (Gigantopelta aegis), which inhabits similar environments. Using Oxford Nanopore Technology, 10X Genomics, and Hi-C technologies, we obtained a chromosome-level genome of C. squamiferum with an N50 size of 20.71 Mb. By constructing a phylogenetic tree, we found that these 2 deep-sea snails evolved independently of other snails. Their divergence from each other occurred ∼66.3 million years ago. Comparative genomic analysis showed that different snails have diverse genome sizes and repeat contents. Deep-sea snails have more DNA transposons and long terminal repeats but fewer long interspersed nuclear elements than other snails. Gene family analysis revealed that deep-sea snails experienced stronger selective pressures than freshwater snails, and gene families related to the nervous system, immune system, metabolism, DNA stability, antioxidation, and biomineralization were significantly expanded in scaly-foot snails. We also found 251 H-2 Class II histocompatibility antigen, A-U α chain-like  genes, which exist uniquely in the Gigantopelta aegis genome. This finding is important for investigating the evolution of major histocompatibility complex (MHC) genes.Here, we report the whole-genome sequencing and assembly of the scaly-foot snail and another snail (Our study provides new insights into deep-sea snail genomes and valuable resources for further studies. Bathymodiolus platifrons) showed that, while most of the genes present in a related shallow-water mussel (Modiolus philippinarum) have been retained, many gene families have expanded in the B. platifrons genome. These families include those that are associated with stabilizing protein structures, removing toxic substances from cells, and the immune response to symbionts , Octopus bimaculoides [GCF_0 011 94135.1], Biomphalaria glabrata [GCF_000457365.1], Crassostrea gigas [GCF_000297895.1], Lottia gigantea [GCF_000 327385.1], Pomacea canaliculata [GCF_003073045.1], Pinctada fucata [GCA_0 022 16045.1], and Helobdella robusta [GCF_000 326865.1] from the NCBI database and C. squamiferum and G. aegis from this research) were compared using blastp with the E-value threshold set as 1e−7. Then, alignment segments of each protein pair were concatenated using the in-house software Solar v0.9.6 [The TreeFam tool   was usedr v0.9.6 . H-scorer v0.9.6 .RRID:SCR_011812) [A. californica—C. gigas (∼516.3–558.3 MYA), A. californica—P. canaliculata (∼310–496 MYA), A. californica—O. bimaculoides (∼551–628 MYA), C. gigas—H. robusta (∼585–790 MYA), and C. gigas—P. fucata (394  MYA) [We obtained 406 one-to-one single-copy orthology gene families based on gene family classification. Then, these gene families were extracted and aligned using guidance from amino acid alignments created using the default parameters of the MUSCLE   programm_011812) , 74 was 94  MYA)  were useC. squamiferum and G. aegis using BWA-MEM (v0.7.12-r1039)  [RRID:SCR_002105) [RRID:SCR_001876) [Approximately 50× clean WGS reads were mapped to genomes of _010910)  with def_002105)  and “Sor_001876)  with def_001876) .We used CAFE v2.1  to analyCNP0000854. The raw sequencing reads were also uploaded to the SRA database under accession No. CNP0000854. All supporting data are available in the GigaScience GigaDB database [The genome assemblies of these 2 genomes have been deposited in GenBank under accession No. database .Supplementary Figure S1.17-mer frequency distribution for C. squamiferum and G. aegis genomes.Supplementary Figure S2.Construction of Phylogenetic trees for ten representative molluscs using coding sequences of 407 single-copy orthologs.Supplementary Figure S3.Box plot of Ka and Ks values of 1,324 single copy orthologous genes from two deep-sea snails, one shallow-water snail, and two fresh-water snails.Supplementary Figure S4.Expansion pattern of HTR4 genes in two deep-sea snails.Supplementary Figure S5.KEGG enrichment analysis of unique gene families of G. aegis.Supplementary Table S1.Statistics of raw sequencing data of Chrysomallon squamiferum.Supplementary Table S2.Statistics of raw sequencing data of Gigantopelta aegis.Supplementary Table S3.Summary from the genome assembly of Chrysomallon squamiferum without using Hi-C data.Supplementary Table S4.Lengths of the 16 chromosomes assembled for Chrysomallon squamiferum.Supplementary Table S5.BUSCO assessment of the assembled genome of Chrysomallon squamiferum using metazoa_odb9 database.Supplementary Table S6.Summary of the genome assembly for Gigantopelta aegis.Supplementary Table S7.BUSCO assessment of the assembled genome for Gigantopelta aegis using the metazoa_odb9 database.Supplementary Table S8.General statistics of predicted protein-coding genes of Chrysomallon squamiferum.Supplementary Table S9.General Statistics of Predicted Protein-coding Genes of Gigantopelta aegis.Supplementary Table S10.Summary of predicted gene functions of the Chrysomallon squamiferum gene set.Supplementary Table S11.Summary of predicted gene functions of the Gigantopelta aegis gene set.Supplementary Table S12.Summary of repeat contents in four selected species.Supplementary Table S13.Gene family clusters in selected species.Supplementary Table S14.Estimation of mutation rates of two deep-sea snails.Supplementary Table S15.Ka and Ks values of 1,324 single copy orthologous genes from five snails.Supplementary Table S16.KEGG enrichment of expanded gene families of C. squamiferum.Supplementary Table S17.KEGG enrichment of unique gene families in G. aegis genome.bp: base pairs; BTBD6: BTB/POZ domain-containing protein 6; BUSCO: Benchmarking Universal Single-Copy Orthologs; BWA: Burrows-Wheeler Aligner; CAFE: Computational Analysis of gene Family Evolution; CAT: catalase; CR1: chicken repeat 1; DLD: dihydrolipoyl dehydrogenase; DLST: dihydrolipoyl succinyltransferase; DMBT1: deleted in malignant brain tumours 1; DNB: DNA nanoball; GAFT: glutamine-fructose-6-phosphate transaminase; GATK: Genome Analysis Toolkit; Gb: gigabase pairs; GP340: glycoprotein-340; Hi-C: high-throughput chromosome conformation capture; HIF-1α: hypoxia-inducible factor-1α; HIV: human immunodeficiency virus; Hsp90: heat shock protein 90; HTR4: 5-hydroxytryptamine receptor 4; IDH: isocitrate dehydrogenase; IUCN: International Union for Conservation of Nature; kb: kilobase pairs; KEGG: Kyoto Encyclopedia of Genes and Genomes; LINE: long interspersed nuclear element; LTR: long terminal repeat; Mb: megabase pairs; MHC: major histocompatibility complex; ML: maximum likelihood; MYA: million years ago; NCBI: National Center for Biotechnology Information; OGDC: α-ketoglutarate dehydrogenase complex; OGDH: oxoglutarate dehydrogenase; ONT: Oxford Nanopore Technologies; ORF: open reading frame; PAML: Phylogenetic Analysis by Maximum Likelihood; PSMC: pairwise sequential Markovian coalescent; SAG: salivary agglutinin; SNP: single-nucleotide polymorphism; SRA: Sequence Read Archive; SRCR: scavenger receptor cysteine-rich; TCA: tricarboxylic acid; TE: transposable element; TLR13: Toll-like receptor 13; TNF: tumour necrosis factor; Txn1: Thioredoxin 1; WGS: whole-genome sequencing.The authors declare that they have no competing interests.This work was supported by the National Key R&D Programme of China (No. 2018YFC0310702).Zongze Shao, S.L., G.F., and X.L. conceived and managed this project and amended the manuscript. X.Z., Y.Z., L.M., and I.S. performed the evolutionary analysis and wrote the manuscript. L.M., J.C., and Y.S. performed genome assembly and annotation. J.B., S.L., X.F., C.W., Zenghua Shao, H.L., N.L., and L.W. were responsible for sample collection, DNA extraction, and library construction.giaa139_GIGA-D-20-00187_Original_SubmissionClick here for additional data file.giaa139_GIGA-D-20-00187_Revision_1Click here for additional data file.giaa139_Response_to_Reviewer_Comments_Original_SubmissionClick here for additional data file.giaa139_Reviewer_1_Report_Original_SubmissionTakeshi Takeuchi, Ph.D -- 7/31/2020 ReviewedClick here for additional data file.giaa139_Reviewer_1_Report_Revision_1Takeshi Takeuchi, Ph.D -- 10/9/2020 ReviewedClick here for additional data file.giaa139_Reviewer_2_Report_Original_SubmissionMathew J. Jenny -- 8/5/2020 ReviewedClick here for additional data file.giaa139_Reviewer_3_Report_Original_SubmissionReuben William Nowell, Ph.D. -- 8/10/2020 ReviewedClick here for additional data file.giaa139_Supplemental_Tables_and_FiguresClick here for additional data file.


MAPK mutations favor HNSCC survival, revealing the broad clinical utility of MAPK pathway mutations in prognosis and potentially in precision immunotherapy. TP53 mutations (median ∼14 yr). We explored underlying outcome-favoring mechanisms with omics followed by preclinical models. Strikingly, multiple hotspot and non-hotspot MAPK mutations  all abrogated ErbB3 activation, a well-established HNSCC progression signal. Inhibitor studies functionally defined ERK activity negatively regulating phospho-ErbB3 in MAPK-mutants. Furthermore, pan-pathway immunoprofiling investigations identified MAPK-mutant tumors as the only “CD8+ T-cell–inflamed” tumors inherently bearing high-immunoreactive, constitutive cytolytic tumor microenvironments. Immunocompetent MAPK-mutant HNSCC models displayed active cell death and massive CD8+ T-cell recruitment in situ. Consistent with CD8+ T-inflamed phenotypes, MAPK-mutant HNSCC patients, independent of tumor-mutational burden, survived 3.3–4 times longer than WT patients with anti-PD1/PD-L1 immunotherapies. Similar prognosticity was noted in pan-cancers. We uncovered clinical, signaling, and immunological uniqueness of MAPK-mutant HNSCC with potential biomarker utilities predicting favorable patient survival.MAPK pathway mutations affect one-fifth of head and neck squamous cell carcinoma (HNSCC). Unexpectedly, MAPK pathway aberrations are associated with remarkably long patient survival, even among patients with  The incidence of head and neck squamous cell carcinoma (HNSCC) is approaching 0.7 million/year globally . DespiteTP53 wild-type (WT) status which allows proper execution of drug-induced apoptosis by functional p53  responses in HNSCC, with proven biology demonstrated in PI3K-mutant, PI3K-activated preclinical models of HNSCC and retrospective patient cohorts  positivity in patients’ tumors can be potentially used as a favorable prognostic biomarker (mainly for cancer from the oropharynx subsite), which can clinically stratify patients for treatment de-intensification purposes . The bioonal p53 . Similar cohorts . These sTP53 mutations (median ∼14 yr), much longer than that of HPV-positive HNSCC (median ∼5.5 yr). The favorable prognosticity of MAPK pathway mutations in HNSCC was found to be independent of HPV. Subsequent molecular dissections revealed two plausible underlying mechanisms operative by MAPK mutations in patient tumors, followed by preclinical HNSCC models. First, multiple hotspot and non-hotspot MAPK mutations  all abrogated ErbB3 activation, an established signal for HNSCC progression. Inhibitor studies functionally defined ERK activity negatively regulating p-ErbB3 in MAPK-mutants. Second, comprehensive pan-pathway immune landscape investigations identified MAPK-mutant tumors as the only “CD8+ T-cell–inflamed” tumors with inherently immunoreactive tumor microenvironments with constitutive cytolysis, subsequently validated in immunocompetent HNSCC models. As low tumoral phospho-ErbB3 levels and elevated CD8+ T-cell infiltrations are recently established events driving good patient survivals in HNSCC , were able to identify subsets of HNSCC patients benefiting significantly from anti-PD1/PD-L1 immunotherapy clinically. Similar prognosticity of MAPK pathway mutations were noted in pan-cancer immunotherapy settings. Our study uncovers novel clinical, biological, and immunological uniqueness of MAPK-mutant HNSCC and may indicate important clinical utilities of MAPK pathway mutations in this cancer.Here, we first reported that MAPK pathway mutations in HNSCC predict remarkably long patient survival, even among patients bearing in HNSCC , our stuAs of today, the clinical significance of somatic genomic aberrations in HNSCC remains underexplored. Here, using a comprehensive pan-pathway approach, we examined the prognostic impact of mutational events of seven major cancer-related signaling pathways on HNSCC patient survivals with The Cancer Genome Atlas (TCGA) dataset . These include three major mitogenic pathways, the PI3K, MAPK(ERK), and JAK/STAT pathways; two differentiation-related pathways, the Notch and TGF-β/Smad pathways; the inflammation-related NF-κB pathway; and the stem cell–related WNT pathway, representing a total of 81% of cases . PathwayNOTCH1 mutations predict recurrences with poor outcomes .Among all seven pathways examined, only mutations in the MAPK pathway  and the Notch pathway  are associated with patient outcomes . Mutatiooutcomes . Unexpec p.E322K ; Fig S2)TP53-mutated patients  (TP53 double mutant” patients (N = 363) have an extreme long median OS of 169.25 mo (∼14 yr), which is 4.77 times longer than that of the “MAPK-WT/TP53-mutated” counterparts . The double mutant patients also have a 55.26% reduction in chances of death (OR = 0.4474) versus MAPK-WT counterparts .Most strikingly, MAPK pathway mutations are also prognostic for markedly improved survival even among iveness) . In factP = 0.01003, 0.03372, respectively, Table S1). Importantly, unlike HPV-positive HNSCC with favorable outcomes, MAPK pathway mutations span multiple head and neck anatomic subsites, including the oral cavity sites, larynx, oropharynx, and others (Table S2). More than 87% (83/95 cases) of MAPK pathway-mutated tumors are HPV-negative. Upon HPV stratification, MAPK pathway mutations are still found to be prognostic for HPV-negative HNSCC , but potentially associated with lower alcohol intake per TCGA alcohol history, and a higher occurrence in females .Table S1 Subsite distribution of MAPK pathway mutations in HNSCC tumors (upper panel) and subsite distributions of HPV positivity in TCGA HNSCC cohort (lower panel).Table S2 Pan-cancer analyses results for associations between MAPK proteome components  in patient tumors and overall survival in 33 cancer types (TCPA database).Table S3 H/N/K-RAS, A/B-RAF, RAF-1, MAP2K1/2, and MAPK1) were also prognostic of favorable HNSCC patient outcome  expression versus control  expressions in HNSCC cell models  and the hotspot ARAF p.S214F mutations inhibited p-ErbB3(Y1289) expressions. In addition to these mutants, overexpression of either MAP2K1-WT or BRAF-WT was also sufficient to inhibit p-ErbB3(Y1289) expressions by ∼60% and 50%, respectively.ErbB3 activation contributes to HNSCC proliferation  and is ag/tcpa/, , 2017a) in HNSCC , we thusl models  and S4A.Top 19 proteins significantly associated with patient OS in the HNSCC TCPA dataset.Table S4 MAPK1 p.R135K and HRAS p.G12S mutations and the HSC-6 cell line harboring MAPK1 p.E322K, >100–200% increases of p-ErbB3 expression were observed, whereas such an increase was not observed in MAPK-WT cells (HSC-4). The data suggested MAPK (ERK) activity negatively regulates p-ErbB3 in a mutant-specific manner in HNSCC. Such a mutant-specific phenomenon was further supported by proteomic findings in MAPK-mutant HNSCC cell lines  and 0.4333  levels. Therefore, we further tested this non-canonical MAPK/ErbB3 relationship using the pharmacological GDC-0994 MAPK1/2-RSK inhibitor in HNSCC cells harboring endogenous MAPK pathway mutations. As shown in a [CCLE] ) and in Response to therapy is an important factor determining cancer patient survival. Yet, there was no clear association between MAPK mutations and responses to cisplatin, docetaxel, 5-flurouracil, methotrexate, and cetuximab in CCLE HNSCC drug-sensitivity database . SimilarSupplemental Data 1, there were 1,793 significant differentially expressed genes (DEGs) in MAPK-mutated versus WT HNSCC tumors with P-values < 0.05 and false discovery rate (FDR) < 0.05, among which 130 protein-coding DEGs showed a >1.41-fold difference (log2 fold-change > 0.5) in gene expression with FDR < 0.05 (Perforin (PRF1), and four granzymes, GZMA (Cytotoxic T-Lymphocyte-Associated Serine Esterase-3), GZMB (Cytotoxic T-Lymphocyte-Associated Serine Esterase-1), GZMH (Cytotoxic Serine Protease C), and GZMK (Tryptase II) were all significantly up-regulated in MAPK-mutated tumors  differentially expressed protein-coding genes (DEGs) in MAPK-mutated tumors (versus WT) with all immune-related genes highlighted in red.A list of 130 significantly up-regulated .A complete list of 1,793 significant differentially expressed genes (DEGs) in MAPK-mutated tumors versus WT tumors in TCGA HNSCC provisional cohort . Most importantly, MAPK-mutant tumors have the most remarkable “CD8+ T-cell–inflamed status” along with most significant and concurrent increases in all three major immunoreactive signature scores, including high cytolytic process (CYT)  and neutrophil infiltration levels in MAPK-mutant tumors  and neutrophils, which were discrete from signatures for B cell, CD4+ T-cell, and macrophage infiltrations  analysis , 2017 ress (CYT) , T-effecss (CYT) , and antss (CYT) , all eviss (CYT) . Whereas in situ . We alsotrations . Overall as well  and S8B.+ T-cells (accompanied by dendritic cells and neutrophil infiltrations) by immunohistochemistry, consistent with the TIMER-predicted CD8+ T-cell–inflamed, immunoactive microenvironments borne by MAPK-mutated HNSCC tumors in TCGA , whereas such infiltrates were not detected in WT tumors .Increases of CD8+ T-cell infiltration in HNSCC tumor microenvironment alone are known to independently predict favorable patient survival  Hartman. Our in- in TCGA  and S10.+ T-cell infiltration in HNSCC dictate patient outcomes , and their abilities to induce intratumoral CD8+ T-cell infiltration in vivo were compared. As shown in HRAS p.G12V (mHRAS p.G12V), mMAPK1 p.D319N , and mMAPK1 p.E320K mutations , were all potent recruiters for CD8+ T-cell infiltration in situ. Furthermore, consistent with our findings that MAPK-mutated human HNSCC tumors have inherently high active cytolytic signatures  were all survival-favoring features among HNSCC patients . Importantly, MAPK pathway-mutated HNSCC patients bearing high IFN-γ scores, high CYT scores, and high T-eff scores have long-term survivals . At the most relaxed median cutoff, both the IFN-γ and T-eff scores could still separate patient survival (P < 0.05), but not CYT score (P = 0.2) (In addition to high CD8P = 0.2) –S13.Notably, among MAPK-mutated HNSCC patients, we found that those with high IFN-γ, CYT, or T-eff scores (both at 20% and 50% cutoffs) do not significantly overlap with patients of low p-ErbB3  and oropharyngeal cancer (N = 32) were two major cancers with sufficient case numbers for survival analysis. Strikingly, among advanced oral cancer patients treated with PD1/PD-L1 inhibitors, MAPK pathway mutations appeared to predict a 3.3 times longer median OS versus WT   . This wact test) .Notably, as high as 49% of HNSCC tumors (53/110) sequenced by + T-cell–inflamed/cytolytically active HNSCC patients who may potentially benefit from ICIs. Our study uncovers novel clinical, biological, and immunological uniqueness of MAPK-mutant HNSCC and may indicate wide clinical utilities of MAPK pathway mutations in this cancer.Overall, MAPK pathway mutations may represent novel and important HNSCC biomarkers for prognosis in HNSCC. Based on our transcriptome findings, validated in immunocompetent models, MAPK mutations likely identify highly CD8TP53-mutated HNSCC patients (median OS >14 yr). Thus, MAPK mutations may represent novel prognostic or even de-intensification biomarkers for HNSCC with broad applicability in terms of HNSCC subsites.We reported for the first time that MAPK pathway mutations have significant survival-favoring clinical, signaling, and immunological impacts in HNSCC. Among seven major cancer-signaling pathways examined, MAPK (ERK) pathway mutations, predominated by many activating mutations, were prognostic for remarkably long patient survivals. This is contradictory to the known role of MAPK-mitogenic signaling in HNSCC tumorigenesis and progression . This fiHRAS and MAPK1 hotspot mutations and potentially others, have remarkable tumoral ErbB3-suppression . Mechanistically, our findings identified a previously undescribed mechanism of p-ErbB3 regulation by MAPK pathway-mutants. Such a negative regulation of p-ErbB3 by ERK activity is uniquely found in MAPK-mutant, but not in MAPK-WT HNSCC. Most importantly, MAPK-mutant HNSCC tumors are the only tumors having significant “CD8+ T-cell–inflamed” and inherently immunoactive tumor microenvironment (versus six other pathway-mutant tumors), with constitutive cytolysis. The ability of MAPK mutations to drive a CD8+ T-cell–inflamed status in vivo with marked apoptosis is proven in immunocompetent HNSCC models. As low tumoral phospho-ErbB3 levels and elevated CD8+ T-cell infiltrations are recently established events indicative of good patient survivals in HNSCC , MAPK1/3(ERK2/1), RPS6KA1, SHC1/2/3/4, GRB2, and Erk1/2-specific DUSP3/5/6/7/9. The PI3K pathway was defined as AKT1/2/3, PIK3CA/B/D/G/2A/2B/2G, PIK3AP1, PIK3IP1, PDK1, MTOR, TSC1, TSC2, PTEN, RICTOR, RPTOR, RHEB, and PIK3R1/2/3/4/5/6. The NF-κB pathway was defined as TAB1/2/3, MAP3K7/14, CHUK, IKBKB, IKBKG, NFKBIA, NFKBIE, REL, RELA/B, NFKB1/2, LTBR, TNF, TNFAIP3, TNFSF11/13B, TNFRSF1A/8/11A/13C, BTRC, CYLD, NLRC5, TRADD, CD40, CD40LG, LTA, TRAF2/3/5/6, IL1B, and IL1R1. The JAK/STAT pathway was defined as JAK1/2/3, STAT1/2/3/4/5A/5B/6, PTPN11, IL6, IL6R, IL6ST, and SOCS3. The Notch pathway was defined as DLL1/3/4, JAG1/2, NOTCH1/2/3/4, NUMB, DTX1/3L, NEDD4, MAML1, RBPJ, POFUT1, HES1/5, and HEY1/2/L. The WNT pathway was defined as WNT1/3A/5A/5B/7A, CTNNB1, HNF1A, FZD1/2/3/7/8/9/10, AXIN1, LEF1, LOXL2, DVL2/3, NKD1/2, TAB1/2, GSK3B, CSNK1A1, NLK, and LRP5/6. The TGF-β/Smad pathway was defined as SMAD1/2/3/4/5/6/7/9, TGFB1/2/3, TGFBR1/2, INHBA/B/C/E, NODAL, ACVR1/1B/1C/2A/2B, BMP2/4/7, BMPR1A/B/2, AMHR2, LTBP1, BAMBI, ZFYVE9, SMURF1/2, and LIMK1. All TCGA whole-exome sequencing data and clinical data of TCGA are downloaded from the www.cbioportal.org  generated from Kaplan–Meier curves as in P-value < 0.05 (significant) would we consider them having favorable/unfavorable OS. CCLE-proteomic database was downloaded from DepMap portal (http://www.depmap.org) as published by the CCLE study . The Platinum-A (PLAT-A) retrovirus packaging cell line was purchased from Cell Biolabs. The HSC-6 cell line was a generous gift from Dr J Inazawa , and SCC VII mouse HNSCC cell line was kindly provided by Dr Sven Branduau . Pt-25 primary cultures were prepared from a female recurrent HNSCC patient. For GDC-0994 treatment, the cells were plated at ∼30% confluency for overnight and then subjected to either vehicle or 0.5 μM of GDC-0994 for 30 min. The cells were then washed with 1× PBS, and protein lysates were prepared for Western blot analyses.2. Retroviruses were removed from the infection medium at postinfection, and cells were then cultured in their respective culture media. Gene expression were then validated by Western blotting. For mouse HNSCC tumor cell inoculation  was used for ectopic expression of genes and mutants into HNSCC cells. In brief, the desired genes/mutants cloned into the pMXs-puro retroviral expression vector backbone were transfected into PLAT-A cells using Lipofectamine 3000 (Thermo Fisher Scientific) for the generation of retroviruses. Retroviruses were filtered through a 0.45-μm mixed cellulose ester membrane filter to remove cell debris and subsequently used for infection of FaDu cells or SCC VII cells for 48–72 h at 37°C, 5% COckground ), 2–3 miCells were washed with cold 1× PBS and lysed with the NP40 lysis buffer . Cell lysates were centrifuged, and the supernatant was quantified with Protein Assay Dye Reagent (Bio-Rad). 50 µg of protein lysates were mixed with a 4× protein loading dye and then separated by SDS–PAGE. Separated proteins were transferred to nitrocellulose membrane, which was then blocked with 5% nonfat dry milk  and probed with primary antibody at 4°C overnight. Primary antibodies include AKT (#9272), pi-AKT (#9271), ARAF(#4432), pi-ARAF(S299) (#4431), BRAF (#2696), pi-BRAF(S445) (#2696), pi-ErbB3 (#2842), ErbB3 (#12708), MAPK (#9102), pi-MAPK (#9101), pi-MEK1/2 (#9154), and RSK1 (#8408), all from Cell Signaling Technology, USA. Anti-β-actin (sc-69879) antibody was from Santa Cruz. Anti-MEK1/2 (YT2714) and anti-N/H/K-RAS (YT2960) antibodies were from ImmunoWay. The probed membrane was then washed three times with 1× TBST, followed by a 1–2-h incubation with the respective secondary antibody , followed by 3× washings with 1× TBST. ECL detection solution was then applied onto the membrane for the development of chemiluminescence, which was captured by autoradiography.+ T-cell, CD4+ T-cell, macrophage, neutrophil, and dendritic cell) in 512 TCGA-HNSCC head and neck squamous carcinoma tumor samples are extracted from the TIMER Web site (https://cistrome.shinyapps.io/timer/_w_20aca96c/immuneEstimation.txt) and grouped according to the mutational information downloaded from cBioPortal . The Wilcoxon rank sum test was performed to calculate the statistical significance between two groups in the TIMER plots.We adopted the methodology of TIMER analysis for immune infiltration level estimation , 2017b. https://portal.gdc.cancer.gov/repository?facetTab=files&filters=%7B%22op%22%3A%22and%22%2C%22content%22%3A%5B%7B%22op%22%3A%22in%22%2C%22content%22%3A%7B%22field%22%3A%22cases.project.project_id%22%2C%22value%22%3A%5B%22TCGA-HNSC%22%5D%7D%7D%2C%7B%22op%22%3A%22in%22%2C%22content%22%3A%7B%22field%22%3A%22files.data_category%22%2C%22value%22%3A%5B%22Transcriptome%20Profiling%22%5D%7D%7D%5D%7D&searchTableTab=files)   using GDGZMA and PRF1 as previous described by GZMA, GZMB, PRF1, IFN-γ, EOMES, and CD8A (offset 0.01)  was calculated by the geometric mean of the TPM of et 0.01) . AntitumMAP2K1 and MAPK1. It also captured selection regions of ARAF, BRAF, HRAS, KRAS, and MAP2K2 to cover all well-known hotspot sites. The sequenced locations for these partially sequenced genes are listed in the following table, and variants were called by the Ion Reporter Software (Thermo Fisher Scientific).Tumor tissues and blood samples were collected from patients under written informed consents according to clinical research approvals by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong East Cluster Research Ethics Committee , the Joint Chinese University of Hong Kong–New Territories East Cluster Clinical Research Ethics Committee , Hong Kong SAR, and the Research Ethics Committee, Kowloon West Cluster , Hong Kong SAR. Genomic DNA from samples were extracted with the DNeasy Blood & Tissue Kit (QIAGEN), followed by quantification and targeted sequencing by next-generation sequencing using the IonS5 platform (Thermo Fisher Scientific). All samples were sequenced with a mean depth of >500× using a custom-designed gene panel on major MAPK pathway genes. The custom gene panel consisted of amplicons that covered all exons of Patient tumors were freshly fixed with 10% formalin and dehydrated in a serial manner in ethanol. The formalin fixed and paraffin embedded tumor samples were then sectioned and dewaxed and rehydrated in xylene, 100% ethanol, 70% ethanol, and running tap water. Antigen retrieval was performed at 95°C for 20 min in citrate buffer . The VECTASTAIN Elite ABC Universal PLUS Kit Peroxidase (Horse Anti-Mouse/Rabbit IgG) (Cat. no. PK-8200) was used for immunohistochemical staining. Endogenous peroxidase activity was quenched by BLOXALL Blocking Solution, followed by blocking in 2.5% Normal Horse Serum for 20 min at room temperature. CD8 mouse antihuman antibody , CD11c rabbit antihuman antibody , and neutrophil elastase rabbit antihuman antibody  and phospho-HER3/ErbB3(Tyr1289) (21D3) rabbit antihuman antibody  were used as primary antibodies to stain patient tumors for overnight incubation at 4°C. CD8 rabbit antimouse antibody  and cytokeratin mouse antibody  were used. The secondary antibody (prediluted biotinylated horse antimouse/rabbit IgG secondary antibody) was added for 1-h incubation at room temperature. For signal amplification and detection, the slides were incubated with VECTASTAIN Elite ABC reagent for 30 min and ImmPACT DAB EqV solutions for 30 s. After counterstaining with hematoxylin for 1 min, clearing, and mounting, pictures were taken under a light microscope. The Roche In Situ Cell Death Detection Kit, Fluorescein (Cat. no. 11684795910) was applied for TUNEL assay. Proteinase K solution  was used to pretreat the rehydrated sections. Then the TUNEL reaction mixture was incubated on the section in dark at 37°C for 1 h in dark. The section was mounted with VECTASHIELD vibrance antifade mounting medium (Cat. no. H-1800).


The structures were determined based on spectroscopic evidence including 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry (HRESIMS) data, along with comparisons to reported data. The leaves were used to extract compounds with different solvents. The extracts were tested for antioxidant activity with a variety of in vitro tests including 2,2-diphenyl-1-picrylhydrazyl (DPPH•), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate (ABTS•+), ferric reducing antioxidant power assay (FRAP), superoxide, and nitric oxide radical scavenging assays. The dichloromethane fraction was most active, displaying significant free radical scavenging activity. The n-butanol fraction also showed notable activity in all assays. Therefore, these findings support the potential use of A. vacillans leaves as an antioxidant medication due to the presence of polyphenolic compounds.A new dihydroisocoumarin glucoside, vacillanoside ( Aloe spp. are members of the bitter or Asphodelaceae family (previously known as Liliaceae). This family is represented by more than 600 species endemic to tropical and southern Africa, Madagascar, Jordan, the Arabian Peninsula, East Asian countries, and various islands in the Indian Ocean + , m/z 529.1320 [M+Na]+ , and m/z 545.1069 [M+K]+  with 162 amu more than that of 2, suggesting the presence of a monosaccharide moiety. A significant fragment using the high-resolution electron impact mode (HR-EIMS) appeared at 327.0868 corresponding to C18H15O6 (M+-glucose). A positive Molisch’s test reflected the glycosidic nature − at m/z 579.1509  with 17 degrees of unsaturation. Thirty carbon resonances were clear in the 13C-NMR and were sorted in a DEPT experiment into two oxymethylenes , 16 methines, and 12 quaternary carbons. The HR-EI-MS for both compounds showed strong peaks at 256.0735 with a relative abundance of 100% calculated for C15H12O4 and assigned to the aloe-emodin anthrone aglycone  in 10 and  in 11. These data matched data reported for caffeic acid + , m/z 507.1500 [M+H]+ , m/z 529.1320 [M+Na]+ , m/z 545.1069 [M+K]+ .White amorphous powder (1 mg); [α]23D + 16.8° ; UV λmax MeOH (log ε) nm: 209 (4.63), 244 (4.42), 300 (3.92), 330 (3.61); IR (KBr) vmax 3400, 1720, 1640, 1606, 1511, 1240 cm−1; 1H and 13C NMR .Red amorphous powder (6 mg); [α] NMR see ; HR-ESI-23D − 4.7° ; UV λmax MeOH (log ε) nm: 209 (4.63), 244 (4.42), 300 (3.92), 330 (3.61); IR (KBr) vmax 3400, 1720, 1640, 1606, 1511, 1240 cm−1; 1H and 13C NMR , m/z 581.1651 [M+H]+ , m/z 603.1469 [M+Na]+ .Red amorphous powder (8 mg); [α] NMR see ; HR-ESI-t-test. Data were expressed as mean ± SD, and the difference was considered significant at p < 0.05 compared to the control. All statistical calculations used OriginLab software  and Microsoft Excel.Analysis of variance (ANOVA) was used to evaluate significance differences, followed by the Student’s 3), 3,4 dihydro-6 glucopyranozyl-8-hydroxy-3--methyl-1H-[6), and two new anthraquinone derivatives, vacillantins A and B (10 and 11) were isolated from the leaves of A. vacillans (Asphodelaceae) together with nine known compounds . The structures of these compounds were elucidated through extensive spectroscopic analyses. The total alcohol extract and different fractions were tested for their antioxidant activities in five spectrophotometric assays. The dichloromethane fraction exhibited promising free radical scavenging activity in most of the assays. Our findings add new information to the literature on the structural diversity and pharmacological activities of Aloe species. Our results suggest A. vacillans as a potential source of secondary metabolites with pharmacological and industrial importance. Moreover, these results advocate further investigation of the remaining fractions with the aim of isolating bioactive compounds exhibiting interesting biological capacities.In summary, a new dihydroisocoumarin derivative, vacillanoside (


P < 0.001 for both comparisons). Positive resistin expression was significantly associated with tumor size, grade, stage, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) status, and molecular classification; strongly positive resistin expression was associated with tumor grade, ER, PR, HER2 status, and molecular classification. Significantly positive correlations were observed between positive and strongly positive resistin expression and corresponding levels of EGFR expression. Relapse-free and overall survival was worse for patients with high levels of both proteins than for those with high levels of only one protein or normal levels of both proteins. Our evidence suggests that combined high levels of resistin and EGFR expression correlate with survival in patients with breast cancer.Elevated levels of resistin and epidermal growth factor receptor (EGFR) facilitate the development of breast cancer, although there are no reports of any correlation between these proteins. This study analyzed 392 human breast cancer tissue specimens and 42 samples of adjacent normal tissue. Rates of positive and strongly positive resistin expression were significantly higher in breast cancer tissue than in the adjacent nontumor tissue (83.2% vs. 23.8% and 20.9% vs. 0.0%, respectively;  Elevated levels of circulating resistin have been linked to a higher risk of breast cancer , 2, and Epidermal growth factor receptor (EGFR), a member of the HER family of receptor tyrosine kinases, is abnormally expressed and activated in many epithelial tumors and plays a critical role in the initiation and development of cancer via modulating downstream signaling pathways , 10. OurThis paper describes our insights derived from immunohistochemistry (IHC) analyses of resistin and EGFR expression in breast cancer and healthy normal breast tissue specimens obtained from 392 Chinese Han women. We detail the clinical prognostic significance of a positive association between resistin and EGFR expression in breast cancer.+/PR+[≥20%]/HER2–, Ki‐67 < 14%); luminal B, containing hormone receptor-positive cases that did not meet the conditions of luminal A; HER2-enriched ; or triple-negative breast cancer (TNBC) . Follow-up information was available for 239 breast cancer patients with a median follow-up time of 60 months . The Ethics Committee of the Affiliated Dongyang Hospital of Wenzhou Medical University approved this study. All study methods satisfied the relevant guidelines and regulations issued by the Affiliated Dongyang Hospital of Wenzhou Medical University.Breast cancer tissue samples were obtained from 392 untreated Chinese Han women aged 24–90 years (median 50 years) who underwent surgery in the Affiliated Dongyang Hospital of Wenzhou Medical University  between 2007and 2019. Forty-two samples of adjacent normal breast tissue were also obtained following surgical resection. Pathohistological diagnoses followed the World Health Organization breast tumor classification criteria . HistoloWe followed the methods described by Wang et al. .We followed the methods of Wang et al. . The priThe entire section was scanned and scored independently by 2 pathologists. Resistin and EGFR expression staining intensity was scored on a 4-point scale from 0 (negative) to 1 (weak), 2 (moderate), or 3 (strong) . A case We followed the methods of Wang et al. .We followed the methods of Wang et al. . MultivaP < 0.001 and P = 0.001, respectively) (P = 0.012), grade (P < 0.001), stage (P = 0.042), ER (P < 0.001) and PR (P < 0.001) status, HER2 status (P = 0.021), and molecular classification (P < 0.001). Strongly positive resistin expression was associated with higher tumor grade (P < 0.001), ER (P < 0.001) and PR (P < 0.001) status, HER2 status (P = 0.001), and molecular classification (P < 0.001).The rate of positive resistin expression in breast cancer tissue specimens was 83.2% (326/392), which included 82 (20.9%) strongly positive cases; corresponding rates in normal breast tissue specimens were 23.8% (10/42) and 0.0% (0/42), respectively. Rates of positive and strongly positive resistin expression were significantly higher in breast cancer tissue than in normal breast tissue (ctively) . As showP < 0.001) and 15.2% [10/66] (P = 0.001), respectively, r = 0.250 and P < 0.001 and r = 0.166 and P = 0.001, respectively). Similarly, as shown in P < 0.001) and 27.4% [85/310] (P < 0.001), respectively). Spearman correlation analysis revealed significantly positive correlations between strongly positive levels of resistin expression and EGFR-positive or strongly positive expression in breast cancer tissue specimens . Similarly, a positive correlation was observed between staining intensity scores of resistin and EGFR in breast cancer tissues  (Rates of positive and strongly positive EGFR expression in breast cancer tissues were 52.0% (204/392) and 32.4% (127/392), respectively. When we analyzed the relationship between resistin and EGFR expression in breast cancer tissue specimens, we found significantly higher levels of positive or strongly positive EGFR expression among resistin-positive cases  compared with resistin-negative cases (24.2% [16/66] (< 0.001) .P = 0.237, P = 0.128, P = 0.171, and P = 0.105, respectively)   had a mean RFS of 51.5 months (an estimated 5-year RFS rate of 68.9%); patients whose tumors were either resistin-positive or strongly EGFR-positive (n = 129) had a mean RFS of 54.3 months (an estimated 5-year RFS rate of 83.7%), while patients whose tumors were resistin-negative and not strongly EGFR-positive (n = 36) had a mean RFS of 54.1 months . Patients whose tumors were both resistin-positive and strongly EGFR-positive experienced worse OS compared with patients whose tumors were either resistin-positive or strongly EGFR-positive and patients whose tumors were resistin-negative and not strongly EGFR-positive (P = 0.099).As shown in Figures n = 29) had a mean RFS of 49.1 months (an estimated 5-year RFS rate of 62.1%); patients whose tumors were either strongly resistin-positive or strongly EGFR-positive (n = 73) had a mean RFS of 54.2 months (an estimated 5-year RFS rate of 79.5%), and patients whose tumors were not strongly resistin-positive and not strongly EGFR-positive (n = 137) had a mean RFS of 53.9 months . Patients whose tumors were both strongly resistin-positive and strongly EGFR-positive experienced worse OS compared with patients whose tumors were either strongly resistin-positive or strongly EGFR-positive and patients whose tumors were not strongly positive for either protein (P = 0.453). Cox proportional hazards regression analysis did not reveal any significant associations between resistin-positive and strongly EGFR-positive tumor tissue (hazard ratio (HR) = 0.736, 95%CI = 0.198–2.734, P = 0.648; 0.765, 0.110–5.323, P = 0.786), strongly resistin-positive and strongly EGFR-positive tumor tissue , and RFS or OS.As shown in Figures n = 61) had a mean RFS of 51.3 months (an estimated 5-year RFS rate of 67.2%); patients whose tumors were either resistin-positive or strongly EGFR-positive (n = 111) had a mean RFS of 55.2 months (an estimated 5-year RFS rate of 86.5%), while patients whose tumors were resistin-negative and not strongly EGFR-positive (n = 36) had a mean RFS of 54.1 months . Non-TNBC patients whose tumors were both resistin-positive and strongly EGFR-positive experienced worse OS compared with patients whose tumors were either resistin-positive or strongly EGFR-positive and also patients whose tumors were resistin-negative and not strongly EGFR-positive (P = 0.065). As shown in Figures n = 23) had a mean RFS of 48.3 months (an estimated 5-year RFS rate of 60.9%); patients whose tumors were either strongly resistin-positive or strongly EGFR-positive (n = 59) had a mean RFS of 54.5 months (an estimated 5-year RFS rate of 78.0%), and patients whose tumors were not strongly resistin-positive and not strongly EGFR-positive (n = 126) had a mean RFS of 54.6 months . Non-TNBC patients whose tumors were both strongly resistin-positive and strongly EGFR-positive experienced worse OS compared with patients whose tumors were either strongly resistin-positive or strongly EGFR-positive and patients whose tumors were not strongly positive for either protein (P = 0.135).We analyzed the effect of combined high expression of resistin and EGFR on the prognosis of non-TNBC or TNBC, ER-negative or ER-positive, HER-negative or HER2-positive tumors. As shown in Figures As shown in Figures The cytokine resistin participates in several physiological and pathological processes, including metabolism, inflammation, autoimmunity, and various cancers, including breast cancer –6, 21–24κB)/signal transducer and activator of transcription 3 (STAT3) signaling pathway-mediated induction of mesenchymal phenotypes and stemness properties [Our previous study have shown that EGFR promotes breast cancer invasion through downstream p44/p42 MAPK (ERK1/2) signaling , and resoperties . These rIn this study, both resistin and EGFR impacted adversely upon survival in breast cancer, but neither protein alone had a significant impact. Breast cancer patients whose tumors were both resistin-positive and strongly EGFR-positive, or were both strongly resistin-positive and strongly EGFR-positive, had worse RFS than all other breast cancer patients. In the non-TNBC and TNBC subgroup analyses, similar results were observed with non-TNBC, but not with TNBC. This may be due to the fact that there were too few cases for analysis. Further study should examine the effects of combined high resistin and EGFR expression on the prognosis of patients with TNBC. These results suggest that resistin and EGFR are potential clinical biomarkers of disease progression and prognosis in breast cancer and that simultaneously targeting these proteins is a potentially useful therapeutic strategy in this disease.


Candida infections  C. glabrata, in cases of azole-resistant isolates , the general and long-term therapeutic use of FLZ (or other triazoles) can result in acquisition of molecular mechanisms that enable Candida isolates to exhibit antifungal resistance  , fluconafections , 3. Howeisolates . Except sistance . It is ksistance . Fenticoisolates . Thus, aisolates  but alsosistance .C. albicans (20 isolates) and C. glabrata (30 isolates) species, respectively. In each isolate’s pair, the FLZ-resistant (derivative) isolate originated from the FLZ-susceptible or susceptible-dose-dependent  isolate following resistance development during patient infection  isolates exhibit known molecular resistance mechanisms, which consisted of upregulation of drug efflux pump-encoding genes  and/or point mutations of 14-α-lanosterol demethylase-encoding ERG11 gene . MIC values to FEZ and FLZ—both obtained as standard powders from Sigma-Aldrich —were determined using the protocol specified in the Clinical and Laboratory Standards Institute (CLSI) M27-A3 document without modifications -specific clinical breakpoints reported in the CLSI M27-S4 document (P < 0.05) differences between GM MIC values obtained for isolate groups from each species (see below), using repeated-measures analysis of variance (ANOVA) on log2 MICs followed by Bonferroni-Dunn’s multiple-comparison test  from nfection . All excications . Only foson test .in vitro, 15 C. glabrata and 1 C. albicans isolate overexpressed drug efflux pumps alone, whereas 9 C. albicans isolates combined overexpression of drug efflux pumps and ERG11 amino acid substitution(s) (Table S1). For C. albicans isolates, FEZ MIC ranges were 0.25 to 2 mg/liter among FLZ-nonresistant isolates  and 1 to 8 mg/liter among FLZ-resistant isolates . For C. glabrata isolates, FEZ MIC ranges were 0.5 to 2 mg/liter among FLZ-nonresistant isolates  and 0.5 to 4 mg/liter among FLZ-resistant isolates . C. albicans and C. glabrata, respectively. Interestingly, the GM MICs ± CIs of C. albicans FEZ MICs in FLZ-nonresistant isolates differed significantly from that in FLZ-resistant isolates . Conversely, no significant difference was seen between the GM ± CIs of C. glabrata FEZ MICs in FLZ-nonresistant isolates and that in FLZ-resistant isolates .Of 25 isolates with molecular mechanisms contributing to the FLZ resistance phenotype observed Candida species, including C. albicans and C. glabrata. Remarkably, differences were more prominent in FLZ-resistant isolates than their nonresistant counterparts but were statistically significant only for C. albicans. Our data demonstrate that FEZ exhibits higher activity than FLZ. FEZ activity was less dependent on drug efflux pump-mediated FLZ resistance in Candida species such as C. glabrata. Based on these findings, FEZ should be evaluated as a candidiasis treatment, particularly in patients with recurrent or antifungal-recalcitrant Candida infections.In conclusion, we showed that FEZ MIC values were lower than FLZ MIC values in 50 well-characterized isolates from two clinically relevant