M2H has been cloned, the in vivo function of its product is unknown. Here, we generated stable T2 homozygous transgenic rice plants in which expression of endogenous M2H was suppressed (RNAi lines). However, we failed to generate M2H overexpression transgenic rice due to failure of somatic embryogenesis. The M2H transcript level showed a diurnal rhythm with a peak at night concomitantly with the peak concentration of 2OHM. RNAi rice showed a reduced M2H mRNA level and 2OHM and melatonin concentrations. The unexpected decrease in the melatonin concentration was caused by redirection of melatonin into cyclic 3-hydroxymelatonin via a detour catabolic pathway. Thus, the decrease in the melatonin concentration in M2H RNAi rice led to slowed seedling growth and delayed germination. By contrast, the transient increase in the melatonin concentration was of greater magnitude in the M2H RNAi than the wild-type rice upon cadmium treatment due to possible suppression of melatonin degradation. Due to its higher concentration of melatonin, the M2H RNAi rice displayed tolerance to senescence, salt, and tunicamycin stresses. Therefore, the increase in the melatonin concentration caused by suppression of melatonin degradation or by overexpression of melatonin biosynthetic genes enhances stress tolerance in rice.Melatonin 2-hydroxylase (M2H) catalyzes the conversion of melatonin into 2-hydroxymelatonin (2OHM), which is present in plants at a higher concentration than melatonin. Although  The emergence of melatonin on Earth is estimated to have occurred several billion years ago, and to be closely synchronized with the appearance of non-purple sulfur bacteria and prokaryotic cyanobacteria . Due to 1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), and N2-acetyl-5-methoxykynuramine (AMK), suggestive of the presence of a melatonin metabolism pathway [In addition to the successful cloning of all genes involved in melatonin biosynthesis from tryptophan in plants , the cat pathway . In mark pathway ,29 via n pathway . Of note pathway , suggest pathway , however pathway . Thus, iArabidopsis [M2H gene to determine whether over- or under-production of 2OHM plays a role in plant growth, development, and response to abiotic stresses.We previously reported that 2OHM induces mitogen-activated protein (MAP) kinase activation comparable to that by melatonin in bidopsis . In suppbidopsis ,35. HoweM2H (GenBank accession number AK119413), full-length M2H was introduced into the pIPKb002 binary vector as described previously [M2H cDNA was first amplified by polymerase chain reaction (PCR) using a primer set  and a cDNA provided by the National Institute of Agrobiological Sciences [attB adaptor primer set . The resulting rice M2H PCR product was gel-purified and cloned into the pDONR221 Gateway vector  via the BP (attB × attP) recombination reaction. Next, the pDONR221-M2H entry vector was recombined with the pIPKb002 destination vector via LR (attL × attR) recombination to yield pIPKb002-M2H, which was transformed into Agrobacterium tumefaciens strain LBA4404. To suppress M2H expression, the pTCK303 binary vector was used as previously described [M2H cDNA fragment was amplified by PCR using the following primer set: M2H-F 5’-ACTAGT ATG CCC GCCGTG GCC-3’ (SpeI site underlined) and M2H-R 5’-GAGCTC GTG TCG TACCTG-3’ (SacI site underlined) and the M2H cDNA. The M2H PCR product was first cloned into the T&A cloning vector  for further cloning experiments. From the T&A:M2H plasmid, the antisense M2H insert was digested by SacI and SpeI, while the sense M2H insert was digested by KpnI and BamHI in the T&A vector. The antisense M2H fragment (SacI and SpeI insert) was first ligated into the pTCK303 vector. Thereafter, the sense fragment of the M2H insert was sequentially ligated into the pTCK303 vector plasmid harboring the corresponding M2H antisense fragment. The resulting pTCK303:M2H RNAi binary vector was transformed into A. tumefaciens LBA4404, and into rice (Oryza sativa Dongjin) as previously described [To over-express rice Sciences . The PCRescribed . Brieflyescribed .2/s1 for seven days. In addition, rice seeds were soaked in distilled water for three days at 28 °C, and the germinated seeds were transplanted into soil under the conditions described above.The dehusked rice seeds were surface-sterilized and grown on half-strength Murashige and Skoog (MS) medium  in vertically oriented polystyrene square dishes . The rice seedlings were grown at 28 °C under a 12/12 h light/dark cycle at a photosynthetic photon flux density of 100 µmol/mATPase a, HKT1, P5CR, rbcL, and sodCp [M2H , Actin1 , M3H , BIP1 , BIP4 , CNX , and PDIL1-1 . GenBank accession numbers were M2H (AK119413), M3H (AK067086), Actin1 (Os03g50885), UBQ5 (AK061988), ATPase a (AK121120), rbcL (KM088016), HKT1 (AB061311), sodCp (AB026724), P5CR (AK070184), BIP1 (AK119653), BIP4 (AK106696), CNK (AK069118), and PDIL1-1 (AK068268).The RNeasy Plant Mini Kit  was used to extract total RNA from the rice samples, which was subjected to DNase I (Qiagen) treatment to remove any contaminating DNA. Aliquots (1 µg) of total RNA were reverse-transcribed using RevertAid reverse transcriptase  with 500 ng of oligo(dT)18 primer at 42 °C for 1 h. Aliquots (0.2 µL) of cDNA were used as templates for PCR amplification with the appropriate primer sets using rice UBQ5 as an equal loading control as previously described . Real-tind sodCp . Other pg for 15 min, boiled at 95 °C for 30 min, and placed on ice for 5 min. The MDA concentration was determined at wavelengths of 440, 532, and 600 nm using a spectrophotometer (Optizen Pop-Bio). Malondialdehyde (MDA) was quantified using a molar extinction coefficient of 156/nmol/L/cm as previously described [2O or 3 mM MES (pH 5.8) buffer and the conductivity of the solution was measured using a conductivity meter . To measure the hydrogen peroxide concentration, frozen leaves (0.05 g) were ground to a powder under liquid nitrogen and homogenized with 1 mL homogenization buffer (50 mM Na-P (pH 6.5), 1 mM hydroxylamine). The extract was centrifuged at 12,500× g for 15 min at room temperature. The supernatant was collected and mixed with 0.5 mL reaction buffer . The extract was clarified by centrifugation at 12,500× g for 10 min. The H2O2 concentration was determined at 410 nm using a spectrophotometer . H2O2 was quantified using a molar extinction coefficient of 0.28/μmol/L/cm.To measure malondialdehyde (MDA) concentrations, frozen rice tissues (50 mg) were ground to a powder in liquid nitrogen using a TissueLyser II (Qiagen) and extracted with 1.5 mL homogenization buffer containing 0.5% thiobarbituric acid (TBA) and 20% trichloroacetic acid (TCA). The supernatant was centrifuged at 13,000× escribed . To measMelatonin and 3-hydroxymelatonin concentrations were quantified by high-performance liquid chromatography (HPLC) as previously described . Pulveri2 and incubated for the indicated times under a 12/12 h light/dark cycle. For tunicamycin (Tm) treatment, 8-day-old rice seedlings grown in half-strength MS medium were challenged with 1 µg/mL Tm for two days under a 12/12 h light/dark cycle at 25 °C. Next, Tm-treated seedlings were transferred to conical tubes without Tm for two days for recovery. Salt treatment was carried out using 2-week-old rice plants grown in soil pots immersed in 0.2 M NaCl for three days under a 12/12 h light/dark cycle at 25 °C. All rice tissues were immediately frozen in liquid nitrogen and stored at −80 °C for further analyses.Senescence treatments were performed in two different ways. First, 10 detached leaves of rice plants grown for six weeks in soil pots were incubated in 50 mL polypropylene conical tubes containing water and subsequently at 25 °C for seven days under a 12/12 h light/dark cycle. Second, rice leaves were cut into 1 cm diameter disks and floated on 3 mM MES (pH 5.8) buffer at 25 °C under dark conditions for five days. For cadmium treatment, a group of 10 segments (detached leaves from 6-week-old plants grown in a soil pot) were transferred to 50 mL polypropylene conical tubes containing 25 mL water with or without 0.2 mM CdCl4OH (containing 80% acetone). Chlorophyll concentrations were determined at wavelengths of 647, 644, and 750 nm using a spectrophotometer  as previously described [Rice samples stored at −80 °C were pulverized to powder using a TissueLyser II instrument . The powder (100 mg) was extracted with 1 mL 0.1 M NHescribed .For germination tests, healthy and uniform seeds were surface-sterilized with 100% ethanol for 30 s followed by 2% sodium hypochlorite for 5 min. The resulting seeds were rinsed three times in deionized water for 5 min. Twenty-five seeds were placed in 3 mL distilled water in 6-well plates, which were wrapped in transparent plastic to prevent water evaporation. The seeds were germinated at 28 °C under LD (14 h light/10 h dark) conditions. The germination percentage was calculated after 36 h. Seeds were considered to have germinated if the length of the emerging shoot was >1 mm. Each treatment comprised 3 replicates, each of 25 seeds.p < 0.05 according to Tukey’s post hoc honestly significant difference (HSD) test. Data are presented as means ± standard deviations of triplicate determinations.Data were subjected to analysis of variance using IBM SPSS Statistics 23 software . Means with different letters or asterisks are significantly different at M2H expression in rice peaks at night during the entire growth stage; in contrast, M3H expression peaks at night during late vegetative growth peaks around eight weeks after seeding [M2H expression, we quantified the M2H mRNA level in 14-week-old rice leaves growing in the field by qRT-PCR. As shown in M2H mRNA level was minimal during the day and increased > 25-fold at night, suggestive of a diurnal rhythm. Similarly, the 2OHM level also peaked at night  or darkness (DD). Diurnal M2H expression was not observed under the LL or DD condition, indicative of a dark-induced diurnal rhythm rather than a free running internal circadian rhythm. Notably, M2H expression significantly increased at the late stage of the LL condition, indicating that M2H responds to continuous light stress as does DWARF4, a key brassinosteroid biosynthesis gene [M3H expression was increased significantly under the DD condition, despite its similar diurnal rhythm to M2H [M2H and M3H have different functions in rice plants.According to the Rice XPro Database , M2H exp seeding . To confsis gene . Conversm to M2H . This fiAgrobacterium harboring the binary vector for three days, during which T-DNA was incorporated into the rice genome. Thereafter, calli were screened on N6 selection medium containing 2 mg/L 2,4-D and 50 mg/L hygromycin for 30 days in the dark to generate transgenic calli expressing the transgene. All three transgenes were successfully transformed into the rice genome, as indicated by generation of healthy hygromycin-resistant calli. M3H, which is responsible for cyclic 3-hydroxymelatonin (c3OHM) synthesis, was employed as a transformation control because it is readily transformed and regenerated via somatic embryogenesis [M3H overexpression and M2H downregulation calli (RNAi) grew rapidly and generated shoots , we generated T2 homozygous rice plants in which M2H was downregulated by selfing and screening T2 seeds. Consequently, three independent T2 RNAi lines were chosen for examination of the phenotypic, biochemical, and molecular features. The M2H mRNA level was significantly downregulated in seven-day-old seedlings of all of the RNAi lines (M2H mRNA level was four-fold lower in the RNAi lines than the wild type (M2H RNAi rice than in the wild type. The wild-type shoots produced 2OHM at 20 ng/g fresh weight (FW) during the day compared to 6 ng/g FW for the RNAi lines on average. During the night, the 2OHM level in shoot was 30 ng/g FW in the wild type, which was 1.5-fold higher than that of the wild type during the day , which encodes the penultimate step enzyme in melatonin biosynthesis [M2H RNAi rice. First, M2H RNAi seeds were seeded in soil pots and cultured in a growth chamber for seven days under a 12/12 h light/dark cycle at 28 °C. The M2H RNAi seedlings were shorter than the wild type  synthesis [SNAT1 and SNAT2 suppression [M2H suppression inhibited only shoot growth, indicative of a different physiological role for M2H compared to SNAT1 and SNAT2.Exogenous melatonin application promotes germination and seedling growth in cucumber and wheat ,43. Howeynthesis ,19,40, hild type a,b, indisoil pot d,e wheresoil pot f. UnlikeAi lines , the M2Hynthesis ,44. In cpression , M2H supM2H RNAi rice was attributable solely to 2OHM deficiency, we measured the melatonin concentration. The M2H RNAi rice had a lower melatonin concentration than the wild type (N-acetylserotonin O-methyltransferase (ASMT). The transcript level of TDC, T5H and ASMT was elevated in the M2H RNAi rice compared to the wild type, indicating a reduced melatonin concentration in the M2H RNAi rice based on the feedback regulation of genes. To determine whether the reduced seedling growth in the M2H RNAi rice is associated with reactive oxygen species (ROS), we measured the level of H2O2. The H2O2 level did not differ between the wild-type and RNAi rice, suggesting that ROS are not involved in the reduced seedling growth of the latter.To examine whether the reduced seedling growth in the ild type a. This sM2H suppression, rather than melatonin, affects the synthesis of c3OHM, we quantified the c3OHM concentration in seven-day-old seedlings treated with 100 µM melatonin rhizospherically for 24 h. As shown in M2H RNAi lines compared to the wild type  and rbcL (photosynthesis) in the M2H RNAi rice were higher than those in the wild type  and superoxide dismutase  was enhanced in the M2H RNAi rice. In particular, the expression of the potassium transporter gene HKT1, which inhibits the transport of Na+ to the shoot, was increased in the M2H RNAi rice  tolerance, as indicated by lower MDA and H2O2 concentrations than the wild type (Arabidopsis [M2H RNAi rice did not show a significant increase in the expression of chaperone genes such as BIP1 (binding protein), CNX , and PDIL1–1 , suggesting involvement of chaperone genes in melatonin-mediated ER stress tolerance. In sum, the increased melatonin concentration due to inhibition of 2OHM conversion plays an important role in the tolerance of rice to various stresses.The ild type . Tm indubidopsis . The M2Hse-like; ) whereasSNAT, a pivotal enzyme in melatonin synthesis, are present from cyanobacteria to land plants whereas orthologs of M2H are absent in cyanobacteria and aquatic plants but present in land plants [M3H orthologs are only present in land plants. These data suggest that only land plants are capable of synthesizing 2OHM and c3OHM, whereas all organisms can produce melatonin. The emergence of M2H genes of the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily matches the diversification of this superfamily during the evolution of land plants [We assessed the phylogenetic lineages of genes related to melatonin, 2OHM, and c3OHM synthesis using the non-redundant protein sequence database ,42. Orthd plants . The M3Hd plants . This imd plants . By contd plants .M2H suppression does not explain the reduced seedling growth and germination rate in the M2H RNAi rice -knockout plants, in which a decreased melatonin concentration in chloroplasts resulted in premature leaf senescence [The increased melatonin concentration in ild type . M2H is ild type . Thus, snescence . This phnescence ,18. Thernescence . Given tnescence ,55.Arabidopsis [Arabidopsis [Compared to other melatonin metabolites, such as 6-hydroxymelatonin, c3OHM, and 4-hydroxymelatonin, 2OHM does not possess potent antioxidant activity because its reactions with peroxy radicals are endergonic . Thus, ibidopsis  because bidopsis ,58. Thusbidopsis . This sibidopsis . By contArabidopsis [M2H plays an important role in regulating the melatonin concentration during the daily light/dark cycle. Therefore, suppression of M2H plays an important role in orchestrating melatonin levels by upregulating the expression of key melatonin biosynthetic genes such as TDC, T5H, and ASMT  which is more inert than melatonin [The melatonin concentration also shows a diurnal rhythm in bidopsis  possiblybidopsis , unfoldebidopsis  and BR bbidopsis . The daybidopsis ,62. Thusand ASMT . Surpriselatonin . Thus, iM2H RNAi rice seedlings produced lower 2OHM as well as melatonin in normal growth condition. Melatonin decrease in the M2H RNAi rice was ascribed to the increased synthesis of 3OHM via a detour catabolic pathway. Thus, the M2H RNAi rice exhibited a melatonin-deficiency phenotype such as a reduced germination rate and retarded growth. By contrast, this catabolic flux regulation was abolished upon challenge with cadmium stress. The M2H RNAi rice produced more melatonin than the wild type, leading to tolerance of cadmium, salt, senescence, and tunicamycin stresses. Therefore, M2H controls the melatonin level in plants under normal and stressed conditions. 2OHM rather than c3OHM is functionally linked to the physiological role of melatonin in plants by regulating its synthesis during normal plant growth and in the presence of stresses. The function of the diurnal rhythm in 2OHM concentration in plants warrants further study.In conclusion, we investigated for the first time the physiological roles of endogenous 2OHM in rice plants by using the molecular genetic strategy. The


RPL22 is either deleted or mutated in 36% of CRC and provide new insights into its mechanism of action. Indeed, Rpl22 inactivation causes the induction of its highly homologous paralog, RPL22L1, which serves as a driver of cell proliferation and anchorage-independent growth in CRC cells. Moreover, RPL22L1 protein is highly expressed in patient CRC samples and correlates with poor survival. Interestingly, the association of high RPL22L1 expression with poor prognosis appears to be linked to resistance to 5-Fluorouracil, which is a core component of most CRC therapeutic regimens. Indeed, in an avatar trial, we found that human CRC samples that were unresponsive to 5-Fluorouracil in patient-derived xenografts exhibited elevated expression levels of RPL22L1. This link between RPL22L1 induction and 5-Fluorouracil resistance appears to be causal, because ectopic expression or knockdown of RPL22L1 in cell lines increases and decreases 5-Fluorouracil resistance, respectively, and this is associated with changes in expression of the DNA-repair genes, MGMT and MLH1. In summary, our data suggest that RPL22L1 might be a prognostic marker in CRC and predict 5-FU responsiveness.We have previously demonstrated that loss of the tumor suppressive activity of ribosomal protein (RP) RPL22 predisposes to development of leukemia in mouse models and aggressive disease in human patients; however, the role of RPL22 in solid tumors, specifically colorectal cancer (CRC), had not been explored. We report here that  Rpl22, which does not affect normal T cell development, markedly accelerates the development of thymic lymphoma driven by a MyrAkt2 transgene . The. TheRPL2pression . The incne ModeK  and the ne ModeK , using tne ModeK , repressne ModeK . Because samples . Similar samples . Thus, RRpl22 has been lost or inactivated. The effect of Rpl22L1 on anchorage independent growth is not restricted to colon cancer cell lines. Overexpression of RPL22L1 in RPL22-expressing wild type, immortalized MEF increased their proliferation relative to control transfected cells (We previously determined that RPL22 loss increased both cell proliferation and anchorage-independent growth [ed cells . Moreoveed cells .http://bit.ly/2qJPf7B) using CBioportal revealed a gain in Rpl22L1 copy number in 18% of patient samples  on a multi-tumor and colon cancer tissue microarrays (TMA). Because no commercial antibodies against RPL22 or RPL22L1 gave reliable IHC results in patient tumor samples, we developed an anti-human RPL22L1 polyclonal antibody and verified its specificity and its function in immunoblotting, immunofluorescent staining, and immunohistochemistry . IHC anaGiven the association of elevated RPL22L1 expression and poor outcomes, we wished to explore the mechanistic link. In doing so, we focused on chemoresistance, since 5-FU-based chemotherapy remains a mainstay in the treatment of CRC and 2 of the 4 deceased RPL22L1 high patients in our cohort failed 5-FU based therapy. To assess a potential role for RPL22L1 in resistance to 5-FU based therapy, we performed an avatar trial in mice using CRC patient-derived-xenograft (PDX) samples  Tables. in vivo  on RPL22L1 expression by ModeK and HCT116 cells. No increase in RPL22L1 expression was observed , raisingin vivo .We next wished to gain insight into the mechanism by which changes in RPL22L1 expression influence 5-FU sensitivity. 5-FU resistance in CRC has long been linked to inactivation of mismatch repair (MMR) genes, such as MLH1 –23. LikeRPL22L1 amplification strengthened its prognostic value. Indeed, patients with either RPL22L1 copy number gain or MGMT induction (upregulation & amplification) exhibited significantly reduced overall survival (OS) relative to patients with MGMT induction alone (MGMT or RPL22L1 (RPL22L1 copy number gain or MLH1 loss (mutation & downregulation) also exhibited reduced OS , we wished to determine if linking changes in those genes to RPL22 loss also occurs in solid cancers, including CRC, where the RPL22 locus is deleted in ~36% or CRC patients. RPL22 loss results in the induction of its paralog, RPL22L1, because RPL22 normally functions to repress RPL22L1 expression [We have previously reported that RPL22 functions as a tumor suppressor in T-acute lymphoblastic leukemia . We now pression . The indpression , 22.RPL22L1 induction and poor outcomes for CRC patients. Patients with RPL22L1 copy number gain trended toward reduced survival; however, the reduction in survival was not statistically significant when considered in isolation. We think this is likely because RPL22L1 expression is primarily controlled post-transcriptionally and so the prognostic value of RPL22L1 itself is best assessed by measuring RPL22L1 protein levels or in combination with RPL22 loss, which induces RPL22L1 protein expression. In agreement, patients with RPL22L1 copy number gain combined with RPL22 loss display significantly reduced disease-free survival. Moreover, when RPL22L1 induction is considered together with alterations in DNA repair enzymes (MGMT and MLH1) modulated by RPL22L1, its prognostic value becomes even clearer, as CRC patients exhibiting these alterations exhibit significantly reduced OS. We think this is likely because the modulation of these proteins (MGMT and MLH1) by RPL22L1 alters the repair of DNA damage induced by 5-FU based chemotherapeutic treatments, rendering those patients’ CRC more resistant. Studies are ongoing to determine whether RPL22L1 can function as a driver in CRC beyond the effects we have observed in vitro. It is important to re-emphasize that RPL22L1 is primarily regulated post-transcriptionally and so the relationship between its induction and patient outcomes is best assessed by measuring protein levels. Consistent with this perspective, our study of RPL22L1 protein levels and outcomes revealed an association with poor outcomes in CRC. Nevertheless, these analyses are based on a relatively small sample size and so further validation on a large, independent CRC cohort will be required to fully assess the prognostic potential of RPL22L1 in CRC.Our data reveal a link between poor colorectal cancer prognosis and RPL22L1 overexpression. This is of great importance because CRC incidence in increasing worldwide, and so prognostic biomarkers that predict outcomes and can be used to guide therapy are needed. This is particularly true in later stage CRC, where surgery is not applicable and chemotherapy is ineffective –14. WhilAn important remaining question is whether, in addition to serving as a prognostic marker for aggressive CRC, RPL22L1 induction might also play a role in influencing CRC disease course. Our in vitro data suggests that RPL22L1 induction might influence disease course in two ways. First, RPL22L1 induction might promote the aggressiveness of CRC, since ectopic expression of RPL22L1 promotes both proliferation and anchorage independent growth of immortalized colon epithelial cells and CRC cell lines. Likewise, RPL22L1 induction could also affect response to treatment. Indeed, 5-FU based chemotherapy is a mainstay of CRC treatment , 28–30 aTaken together, these results reveal that the RP, RPL22L1, is capable of promoting CRC growth and anchorage independence, as well as therapeutic resistance. Consequently, gaining insight into its mechanism of action and the cellular pathways it regulates may enable the identification of therapeutic vulnerabilities in CRC patients with RPL22 loss and/or RPL22L1 induction.S1 FigRPL22 shallow deletion occurs in up to 36% of CRC samples. Data were collected from TCGA colorectal adenocarcinoma studies n = 223. B. Representative qRT-PCR analysis of Rpl22 and Rpl22l1 mRNA performed in triplicate is plotted graphically as the mean ± SD. Rpl22 and Rpl22l1 mRNA expression was normalized to Gapdh mRNA levels in MEF cells isolated from Rpl22+/+, Rpl22+/- and Rpl22-/- mice. C. Immunoblotting of extracts of tissues collected from Rpl22+/+ and Rpl22-/- mice reveals that RPL22L1 expression is markedly induced by loss of Rpl22. Elevated expression of RPL22L1 was observed in normal colon. D. Immunoblotting of the human CRC cell line, HCT116, reveals that is expresses higher levels of RPL22L1 than the normal colon epithelial cell line CCD841 (Control). E. Immunoblotting of normal colon epithelial cell lines and human CRC cell lines reveals that colon cancer cells express higher levels of RPL22L1 and lower levels of RPL22 relative to normal colon epithelial cell lines. GAPDH served as loading control. Quantification of band intensity by ImageJ is listed on the bottom.A. (TIF)Click here for additional data file.S2 FigA-B. Overexpression of RPL22L1 (RPL22L1-HA) in SW480 colon cancer cells promotes cell proliferation (A) and knockdown of Rpl22L1 in HT29 colon cancer cells inhibits cell proliferation (B). Data is depicted as average of cell number ± SD from triplicate measurements every two days. Student’s t-test was used for biostatistical analysis between groups . C. Knockdown of RPL22L1 with two different shRNA (left) inhibits cell proliferation (right) of HCT116 cells. D. Overexpression of Rpl22L1 in immortalized or Ras-transformed MEF cells promotes cell proliferation as determined by cell numbers (left) and colony formation (right). Student’s t-test was used for statistical analysis between groups. **, p<0.005.(TIF)Click here for additional data file.S3 FigA. Immunoblot analysis reveals that knockdown of RPL22L1 by two different shRNA hairpins in HEK293 cells eliminates immunoreactivity. GAPDH served as loading control. B. Immunofluorescent staining with anti-RPL22L1 followed by FITC anti-rabbit secondary antibody, reveals that RPL22L1 knockdown abrogates staining. C. Knockdown of RPL22L1 in HEK293 cells attenuates staining with anti-RPL22L1 in IHC. D. IHC staining with anti-RPL22L1 of spleens from WT, Rpl22l1-/-, and Rpl22-/- mice. Images at 20x magnification are shown, which validate the specificity of the antibody. E. IHC with anti-RPL22L1 in human normal colon or colon adenocarcinoma samples. IgG in normal colon tissue is used as the negative control. F. Representative image of strong (left) and weak (right) staining of human colon cancer by anti-RPL22L1 in IHC. The staining was quantified by VECTRA automated quantitative image system and presented with H-Score.(TIF)Click here for additional data file.S4 Fig Multi-tumor TMA  and colon cancer TMA  with map information including sample ID were stained with IgG control or anti-RPL22L1. N and EN denote normal tissue samples, while T1 and ET1 denote tumor samples.(TIF)Click here for additional data file.S5 FigData are plotted as percentage of lymph node involvement (A) or tumor size (B) in RPL22L1 low and RPL22L1 high colon adenocarcinoma patient samples. There is a significant correlation between elevated RPL22L1 expression and increased lymph node involvement (A) but not with tumor size (B).(TIF)Click here for additional data file.S6 FigA. Representative images illustrate increased RPL22L1 IHC staining of colon cancer PDX samples (bottom) and in normal colon (top). Staining intensity was quantified using a VECTRA automated quantitative image system (right): green indicates stroma (top right) and brown indicates tumor staining (bottom right). A map of the tumor samples and patient information are found in Table S1 and S2. B. Immunoblot analysis revealed that chronic 5-FU treatment (3mo) of HCT116 or ModeK cells to induce resistance did not result in RPL22L1 induction. The RPL22L1 band is quantified by Image J with background correction and normalized to loading control, Actin.(TIF)Click here for additional data file.S7 FigWe found 6 of 7 of the RPL22L1 high PDX samples exhibited expression of MSH6 and PMS2. Representative images illustrate RPL22L1 high and RPL22L1 low staining. The top two RPL22L1 high samples are clearly MSS because of the strong nuclear staining of MSH2 and PMS2, while the bottom RPL22L1 low sample appears to be microsatellite unstable (MSI), since it lacks PMS2 staining.(TIF)Click here for additional data file.S1 Tablen.a.- not analyzed; met–metachonous metastasis; syn–synchronous metastasis.(TIF)Click here for additional data file.S2 Tablen.a.–not analyzed; T/C = tumor volume (treated/control); Scores according to T/C value: 50–100% = -; 35–50% = +; 21–35% = ++; 6–20% = +++; 0–5% = ++++. RTV—Relative tumor volument.(TIF)Click here for additional data file.S3 TableCytoplasmic staining, p = 0.038; Nuclear staining- p = 0.003. RPL22L1 high staining is defined as the top quartile of H scores.(TIF)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.


CYP2C9, CYP2D6 and CYPOR genes are responsible for some varied drug responses observed across human populations. There is limited data available regarding the pharmacogenetic polymorphisms and frequency distributions of major allele variants in the Pakistani population. The present in silico mutagenesis study conducted on genotype pharmacogenetic variants and comparative analysis with a global population aims to extend the currently limited pharmacogenetic available evidence for the indigenous Pakistani population. Extracted genomic DNA from 244 healthy individuals’ venous blood samples were amplified for distinct variant loci in the CYP2C9, CYP2D6 and CYPOR genes. Two-way sequencing results were compared with standard PubMed data and sequence variant loci confirmed by Chromas. This study revealed significant variations in CYP2C9 , CYP2D6 (rs16947 and rs1135840), and CYPOR  variants in intraethnic and interethnic frequency distributions. In silico mutagenesis and three-dimensional protein structural alignment analysis approaches clearly exposed the possible varied impact of rare CYPOR (rs781919285 and rs562750402) single nucleotide polymorphisms (SNPs) and confirmed that the influences of CYP2C9 and CYP2D6 variants are consistent with what was found in earlier studies. This investigation highlighted the need to study pharmacogenetic relevance loci and documentation since evidence could be utilized to elucidate genetic backgrounds of drug metabolism, and provide a basis for future pharmacogenomic studies and adequate dose adjustments in Pakistani and global populations.Diverse distributions of pharmacogenetically relevant variants of highly polymorphic  Genetic constitution determines the varied interethnic metabolizing capacities of various drug metabolizing enzymes . The recCYP2C9, CYP2D6, and CYPOR genes. The basic phenotype or genotype findings have greatly contributed to the determination of an individual’s metabolic capacity that acts as an important tool for safe and rational drug administration. In the future, this practice will be beneficial to decrease the prevalence of adverse drug reactions and treatment failures particularly for narrow therapeutic index drugs (NTI-drugs) [Principally, the oxidative metabolism of xenobiotics, including most clinically used drugs, is mediated by the phase-I heme-containing members of the cytochrome P450 (CYPs) enzyme superfamily. Cytochrome P450 oxidoreductase (CYPOR) is indispensable in the metabolic activities mediated by all microsomal CYPs . The CYPCYP2C9 exists in different polymorphic forms and 60 alleles have been identified so far, most of which alter the metabolic activity of the enzyme [CYP2C9*2 (rs1799853-R144C), CYP2C9*3 (rs1057910-I359L), and CYP2C9*14 (rs72558189-R125H) [CYP2C9*1). The CYP2C9*2 allele is more moderately defective while CYP2C9*14 was found to be highly defective in in vitro studies [CYP2C9, one of the most abundant CYP enzymes, constitutes 50% of the CYP2C subfamily and is involved in the oxidative metabolism and clearance of up to 15–20% of clinically important drugs, including phenytoin and warfarin . CYP2C9 e enzyme . CYP2C9**2 rs179953-R144C,9-R125H) . These a studies .CYP2D6, “the only functional gene in the CYP2D human subfamily”, is one of the extensively investigated members in the genetic polymorphism context [CYP2D6 represents only 2% of total human hepatic CYPs and is non-inducible by environmental factors. This little amount is enough to metabolize more than 25% of currently marketed drugs. CYP2D6 substrate drugs including antidepressants, selective serotonin-reuptake inhibitors, antiemetics, beta-blockers, and opioids are mostly lipophilic in nature [CYP2D6 alleles and sub-variants are available at the human CYP allele nomenclature website [CYP2D6 reported alleles, some are fully functional, less functional and non-functional or null alleles [CYP2D6 polymorphisms [ context . CYP2D6 n nature ,14,15. T website , with th alleles . A populorphisms .CYPOR gene is highly polymorphic and more than sixty SNPs have been identified relating to variations in CYPOR protein contents [CYPOR mutations and polymorphisms is available at [CYPOR allelic variants are CYP enzyme and substrate dependent [CYPOR is essential for all reactions catalyzed by CYPs for drugs, steroid hormones and xenobiotics . All miccontents . An onlilable at . The funependent ,24.CYP2C9, CYP2D6 and CYPOR genes in all Pakistani geographical populations is available. In the present work, we investigated the genotype and allele frequencies in CYP2C9 , CYP2D6 (rs16947 and rs1135840) and CYPOR  genes, and compared our findings with 11 corresponding world populations reported in the 1000 Genomes Project phase-III database [CYPOR missense variations and to explain the influences of CYP2C9 and CYP2D6 SNPs corresponding to Pakistani people.By now, no investigation indicating the genotype and allele frequencies distributions of CYP2C9, CYP2D6 and CYPOR, major allele variants from global populations and a comparative analysis technique was used. We compared our study results with world populations from all continents, as previously listed. The frequency distribution data of eleven world populations was retrieved from the 1000 Genomes Project phase-III database [In total, 244  healthy Pakistani volunteers from all provinces and administrative territories, aged 18–63 years, were randomly selected. The individuals were subdivided into smaller ethnic groups  based on reported ethnic classification  and the database ,27.CYP2C9, CYP2D6, and CYPOR genes. The targeted SNP sites were amplified using PCR, and Primer3 [https://blast.ncbi.nlm.nih.gov/Blast.cgi) and SNP BLAST (https://www.ncbi.nlm.nih.gov/snp) were used. Each 50 µL PCR reaction mix consisted of 1 µL of DNA (50 ng/µL), 5 µL 10× PCR buffer, 2 µL of each primer (1000 µM), 4 µL deoxynucleotide triphosphate (2.5 mM), and 0.325 µL r-Taq polymerase (5 U/µL). Amplification was performed using an Eppendorf  thermal cycler, and the PCR profile included an initial denaturation of 2 min at 94 °C followed by 32–34 cycles of 30 S at 94 °C, 20 S at 60 °C, and 30 S at 72 °C; a final extension step at 72 °C for 10 min; and being held at 4 °C. Amplicons were confirmed for band sufficiency and specificity on a transilluminator following agarose gel electrophoresis, and they were purified before sequence analysis. Purified amplicons were sequenced using the same forward and reverse primers by Sangon Biotech Co., Ltd. . For the detection of polymorphisms in CYP2C9, CYP2D6 and CYPOR genes, all SNPs were identified followed by human reference genome assembly (GRCh38). The full length standard reference sequences for human CYP2C9 (NC_000010.11), CYP2D6 (NC_000022.11) and CYPOR (NC_000007.14) genes were obtained from the GenBank, NCBI [http://evs.gs.washington.edu/EVS/). For accuracy, every DNA fragment was sequenced in both the forward and reverse directions, and sequence reads were observed for base pair changes to identify the specific variations using Chromas Lite software,  [Individual gDNA was screened out by sequencing after PCR amplification for clinically relevant polymorphisms in  Primer3  was usednk, NCBI  and usedp < 0.05 level was considered as statistically significant. Intraethnic allele frequency comparison between different Pakistani regions was carried out using one-sided binomial proportion test. For each test, calculated p-values were adjusted for the multiple testing using q-value  of NCBI. In silico mutagenesis of identified point substations were created in the 3D protein structures of wild-type CYP2C9, CYP2D6 and CYPOR after deleting water molecules. For the evaluation of the structural/functional effect of every SNP, each amino acid replacement corresponding to a reported missense variation was made individually. The process was performed by keeping the replaced residues free, fixing the rest of structure, taking into account the secondary structure protein features, and independent ultimate structural aligning to the corresponding wild-type. The modeling procedures were performed using SYBYL 6.0 software  and the molecular visualizations were performed on PyMOL Molecular Graphics System, Version 1.6  [The X-ray structures of CYP2C9 (PDB ID: 1OG2), CYP2D6 (PDB ID: 2F9Q), and CYPOR (PDB ID: 3QE2) were retrieved from the worldwide available archive for structural data of biological macromolecules, the Protein Data Bank (PDB) . They weer, LLC) ,38,39.CYP2C9, CYP2D6, and CYPOR compared to others. All DNA samples extracted from healthy volunteers’ venous blood were analyzed for thirty-six SNPs in coding and related noncoding regions (p < 0.05)  . The dis < 0.05) .CYP2C9, one of the most abundant members of the human CYP2 isoform, has been extensively studied for polymorphisms in different ethnicities including Caucasian, African-American, and Asian populations [www.fda.gov/default.htm). The CYP2C9 gene was successfully genotyped for rs1799853 (CYP2C9*2/R144C), rs1799853 (CYP2C9*2/R144C), and rs72558189 (CYP2C9*14/R125H) polymorphisms, and allele frequencies were calculated for them. The frequencies of rs1799853, rs1057910 and rs72558189 variant alleles were calculated as 1.04, 9.26, and 4.12%, respectively. Genotype frequencies of rs1799853 and rs1057910 did not deviate significantly from Hardy–Weinberg equilibrium, while the p-value for rs72558189 was calculated as <0.05 , as shown in CYP2C9 polymorphisms rs1799853 (CYP2C9*2), rs1057910 (CYP2C9*3), and rs72558189 (CYP2C9*14) are variants associated with reduced enzymatic activity leading to poor metabolizing phenotype [CYP2C9*14 defective allele in the Pakistani population and will provide a clear indication for genetic test benefits in narrow therapeutic range substrate dosing, such as for warfarin and phenytoin  percentages in individuals from the Azad Kashmir (AZK) region. Polymorphism rs1135840 related variant allele G was calculated as having a 35.21% frequency and observed to be significantly different from Hardy–Weinberg equilibrium with a p-value of <0.05, as shown in p < 0.05), shown in CYP2D6 gene products with the ultrarapid metabolizer phenotype [CYP2D6 gene, suggested to be a protective factor for cancer risk, however, was found less frequently (30%) in the Pakistani population. The homozygote (GG) and G carriers (AG) of rs16947 polymorphism in the CYP2D6 gene were reported to be negatively associated with cancer incidences and considered to be protective against cancer risks [The highly polymorphic proteins ,47. Polyhenotype ,49. A rehenotype . The rs1er risks . The cany higher .78% percer risks .CYPOR gene product is an important electron donating flavoprotein and essentially required for all reactions catalyzed by CYPs. The CYPOR gene was successfully genotyped for three nonsynonymous , four synonymous  substitutions, and 11 related noncoding variants. The CYPOR rs781919285 and rs562750402 human variations were reported only in 0.001652 and 0.002480%, respectively, in dbSNP, and no information was provided in the Human Cytochrome P450 (CYP) Allele Nomenclature Database [CYPOR gene, the non-synonymous rs1057868 polymorphism was statistically significant in African and Japanese ethnicities, while rs1057870 and rs2228104 synonymous variations were found significantly different from African, East Asian, and Admixed American population groups. The rs1135612 polymorphism identified in the CYPOR gene was observed statistically significant in genotype and allele frequency distributions in the studied population, when compared with previously reported populations (p < 0.05) shown in CYPOR rs781919285 and rs562750402 human variations were not reported in any of the compared populations. However, in the Pakistani population, though there were relatively diverse intraethnic frequencies, CYPOR rs1057868 was found to be almost uniformly distributed  wild type and two site mutants generated by in silico mutagenesis found in Pakistani people . The ami alleles . The sub alleles . The oth channel . The strvariants  and is avariants .The identified missense variations in the Pakistani population are shown in a typical three-dimensional structural of human CYPOR protein molecule in CYPOR SNPs (rs781919285 and rs562750402) following relatively diverse intraethnic, interethnic and distribution patterns and the detection of defective CYP2C9 (rs72558189) and CYP2D6 (rs1135840) polymorphisms with the highest frequencies in the healthy Pakistani population. This study also supports how allele frequency distributions of human biotransformation genes follow geographic and/or diverse ethnic specific patterns rather than random distributions.To the best of our knowledge, this study is the first to reveal the identification of rare


KMT5B (OMIM: 610881), may fit into a category of epigenetic modifier genes that are critical for typical neurodevelopment, such as EHMT1 and ARID1B, which are associated with Kleefstra syndrome (OMIM: 610253) and Coffin-Siris syndrome (OMIM: 135900), respectively. Based on our current knowledge of the H4K20 methyl modification, we discuss emerging themes and interesting questions on how this histone modification, and particularly KMT5B expression, might impact neurodevelopment along with current challenges and potential avenues for future research.Neurogenesis is an elegantly coordinated developmental process that must maintain a careful balance of proliferation and differentiation programs to be compatible with life. Due to the fine-tuning required for these processes, epigenetic mechanisms  are employed, in addition to changes in mRNA transcription, to regulate gene expression. The purpose of this review is to highlight what we currently know about histone 4 lysine 20 (H4K20) methylation and its role in the developing brain. Utilizing publicly-available RNA-Sequencing data and published literature, we highlight the versatility of H4K20 methyl modifications in mediating diverse cellular events from gene silencing/chromatin compaction to DNA double-stranded break repair. From large-scale human DNA sequencing studies, we further propose that the lysine methyltransferase gene,  While several residues on H3  are subjected to methylation (reviewed elsewhere . In thisDrosophila melanogaster proteins (Suppressor of variegation 3-9 (Su(var)3-9), Enhancer of zeste (E(z)), and the homeobox gene regulator Trithorax (Trx)) [KMT enzymes responsible for establishing H4K20 methylation marks are part of a large family of proteins that contain a SET domain: Initially named after three x (Trx)) . Enzymesx (Trx)) ,16. Skp2 and degraded by CRL4cdt2 [cdh1-dependent ubiquitination which permits entry back into G1 [CDT2-mediated degradation of KMT5A and in the DNA damage repair process [KMT5A  is exclusively a H4K20 mono-methyl transferase ,18 with CRL4cdt2 ,25 prior into G1 . While K into G1  at K248  into G1 , an inte process . Other i process  and Numb process . Protein process . Recent  process . The enzymes KMT5B  and KMT5C  are highly homologous and have been investigated together in many studies ,32. All KMT5B/C is enriched in peri-centric heterochromatic regions and is typically a mark of silenced heterochromatin . InvestiNSD1 (OMIM: 606681) are associated with Sotos syndrome, characterized by childhood overgrowth, developmental delay, intellectual disability, muscle hypotonia, and seizures [NSD2 (OMIM: 602952) is associated with Wolf-Hirschhorn syndrome, characterized by facial dysmorphisms, developmental delay, intellectual disability, muscle hypotonia, and seizures [The NSD (nuclear receptor SET domain-containing) family of histone methyl transferases consists of three enzymes: NSD1, NSD2 , and NSD3 , all of which have been implicated in neurodevelopment and cancer syndromes . Mutatioseizures . NSD2 , recruited via the interaction of its S102 residue and MDC1, is responsible for this local increase . Other eThe dynamic regulation of H4K20 methylation includes not only the methyl transferases but also demethylases, or “erasers.” These enzymes contain flavin-dependent amineoxidase and α-ketoglutarate dependent Jumonji-C (JmjC) domains for demethylating activity . Lysine demethylase 4A , consisting of tandem hybrid tudor domains, is a histone demethylase that has dual specificity toward H4K20me3 and H3K4me3 . KDM4A aPHF8 are associated with Siderius X-linked mental retardation with cleft lip/palate [PHF8 ortholog in zebrafish (phf8) increases H4K20me1 levels, causes craniofacial abnormalities and apoptosis in the brain and neural tube, and impairs jaw development [A class of demethylases in the JmjC family is characterized by an N-terminal PHD (plant homeodomain zinc finger) domain and a catalytic JmjC domain that recognize methylated lysines . In humap/palate . Morpholelopment . Mice deelopment . In anotelopment . Data suelopment , a key pLysine-specific histone demethylase 1A  is primarily described as a H3K4me1 and me2 demethylase implicated in phenotypes of cognitive impairment that resemble Kabuki syndrome (OMIM: 147920) ,56,57. HThe mono, di, and tri-methyl marks are dynamically read by specialized reader/effector proteins that bind methyl-lysine residues via motifs such as chromo, tudor, MBT, WD40, BAH, ADD, ankyrin, PHD, and zn-CW .Malignant brain tumor D1 (MBTD1) binds H4K20me1 and plays a role in mouse oocyte maturation, TP53BP1-mediated DSB repair, checkpoint activation , and chromosome alignment during mitosis . Lethal The cell cycle checkpoint protein, p53-binding protein 1 (TP53BP1), is an important reader of H4K20me states as a co-activator of p53, a tumor suppressor that is rapidly activated in response to DNA damage and cellular stress , as descFanconi Anemia Complementation Group D2 , recently identified as a H4K20me2 reader, is associated with the FA/BRCA DNA repair pathway, which promotes HR over NHEJ . FANCD2 H4K20 methylation has been pathologically implicated in neurodevelopmental disorders (NDDs); however, this has come primarily from demethylase enzymes and the NSD family of methyltransferases, which target multiple histone lysine residues . While mKMT5B (SUV420H1) has been most implicated in human phenotypes through multiple independent sequencing studies. While initially highlighted using computational modeling in 2015 [KMT5B harbors more potentially pathogenic de novo mutations in individuals with NDDs than would be expected by chance under multiple statistical models [KMT5C and no de novo mutations in KMT5A [PHF8) with an X-linked inheritance pattern [KMT5B as an NDD-associated gene was a targeted sequencing study that did not include any of the other H4K20-associated genes [Among the KMT H4K20 methyl writer family,  in 2015 , two larl models ,74. Exoml models ) found tin KMT5A ,74. It iin KMT5A . De novoin KMT5A , suggest pattern ,76 are oKMT gene family (SNVs and copy number variants (CNVs); KMT5A; 11 CNVs, 24 unique missense/frameshift/stop-gained/coding-complex SNVs  representing 60,706 unrelated individuals sequenced as part of various disease-specific and population genetic studies [Variant class scores (z and pLI) taken from the ExAC Browser , autism spectrum disorder (ASD), or developmental delay (DD)  [Most of the 43 individuals carrying lay (DD) . Based oificity) ,77,80, pBrain-specific functional details and expression patterns of H4K20 methylation and the KMT enzymes  are currently not well understood, but available expression datasets and model systems give us some leads.http://help.brain-map.org/display/devhumanbrain/Documentation). These gene expression data show that KMT5B and KMT5C transcripts are most highly expressed prenatally, with a decrease to steady state levels after birth  in uniqse brain . Highestal layer . InteresD. melanogaster homolog for KMT5A) in D. melanogaster larval brains causes disorganization of highly proliferative regions of the optic lobes. Based on the severe chromosomal condensation defects seen in mutant flies [Kmt5a homozygous-null mice can only be recovered at 2.5 days post-conception (cleavage stage) [Loss of PR-Set7  . Embryone stage) . These dSETD8a and SETD8b (Kmt5a), suv420h1 (Kmt5b), and suv420h2 (Kmt5c) are ubiquitously expressed before the onset of zygote gene expression, with higher expression in the head region [KMT5B/C orthologs in Xenopus embryos results in defective eye and melanocyte differentiation, reduced cell proliferation, and increased apoptosis during development, likely resulting from impairing transition from a pluripotent state to a differentiating state, highlighting a role for KMT5B/C enzymes in neural fate determination [Kmt5b-null mice (deletion of the entire SET domain) show perinatal lethality, while Kmt5c null mice have no apparent phenotype [Kmt5b and Kmt5c recapitulates the perinatal lethality seen in Kmt5b-null mice, suggesting that there is a profound need for Kmt5b during development [Clues to the roles of KMT5B and KMT5C in the developing brain come from zebrafish, amphibian, rodent, and primate model systems. In situ hybridization in zebrafish shows that zebrafish orthologs d region ,91. Morpmination . Consistmination —is enricmination . Conditimination , yet incmination . The reahenotype . Deletioelopment .While many of these studies are circumstantial at implicating H4K20 methylation in various neuropathological states, they give credence to the ability of H4K20me1 and me2 to modulate juvenile-to-adult plasticity states in the brain and the role of H4K20me3 locking in neural phenotypes. KMT5B that decrease activity or potential gain-of-function missense mutations may have more profound consequences during brain development, when there is a need for rapid cell proliferation and differentiation  in mice. Actively proliferating myoblasts in primary limb bud mesenchymal cultures and undifferentiated C2C12 muscle cell lines show high H4K20me1 levels , which g cancers ,104,105  cancers .Our understanding of the role of epigenetic regulation in neurodevelopment is just beginning. The main support for our argument that KMT5B is involved in neurogenesis through maintenance of the neural stem cell pool comes primarily from the analysis of available human and mouse RNA expression datasets ,83. HoweEither through the process of gene expression regulation or DSB repair, it is clear that the KMT enzymes are crucial for regulating cell cycle dynamics, proliferation, differentiation, cell fate determination, and specification of lineage programs ,107. Lac


One of the potent compounds, OTS193320, was further analyzed in in vitro studies. The compound decreased global histone H3 lysine 9 tri-methylation levels in breast cancer cells and triggered apoptotic cell death. Combination of OTS193320 with doxorubicin (DOX) resulted in reduction of γ-H2AX levels as well as cancer cell viability compared to a single agent OTS193320 or DOX. Further optimization of inhibitors and their in vivo analysis identified a compound, OTS186935, which revealed significant inhibition of tumor growth in mouse xenograft models using MDA-MB-231 breast cancer cells and A549 lung cancer cells without any detectable toxicity. Our results suggest that the SUV39H2 inhibitors sensitize cancer cells to DOX by reduction of γ-H2AX levels in cancer cells, and collectively demonstrate that SUV39H2 inhibition warrants further investigation as a novel anti-cancer therapy.Protein methyltransferase SUV39H2 was reported to methylate histone H2AX at lysine 134 and enhance the formation of phosphorylated H2AX (γ-H2AX), which causes chemoresistance of cancer cells. We found that a series of imidazo[1,2- MLL-rearranged leukemia in a rat xenograft model pyridine compounds that could inhibit SUV39H2 methyltransferase activity. We have characterized one of the newly synthesized compounds, OTS193320 pyridine derivative compounds to have weak SUV39H2 methyltransferase inhibitory activity with IC50  values of <10 μM. Through intensive structure-activity relationship-based drug development, we obtained novel compounds that shared a common chemical structure as shown in Figure 50 of 22.2 nM) and a growth suppressive effect of SUV39H2-positive A549 lung cancer cells (IC50 of 0.38 μM).Through screening of a commercially available compound panel, we identified some imidazo-  (Perkin Elmer: NET155H001MC) in the assay buffer . N-terminal GST fused SUV39H2  (in-house) was added to start reaction and incubated at room temperature for 3 hours. The reaction was stopped by adding 2.5 mg/mL of Streptavidin SPA beads in bead buffer . The radioactive signal was measured with Trilux-Microbeta counter (Perkin Elmer). IC50 was calculated with XLfit.The compounds OTS193320 or OTS186935 were serially diluted (10 doses) and mixed with biotin conjugated histone H3 peptide (1-21)  (Millipore: 12-403) and adenosyl-L-Methionine, S-[Methyl-Cells were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer at 4°C for 1 h, then permeabilized in 0.1% Triton X-100 (Sigma-Aldrich) for 3 min at room temperature, and blocked with 3% BSA for 1 h at room temperature. Fixed cells were incubated with primary antibodies overnight at 4°C, followed by incubation with Alexa Fluor-conjugated secondary antibodies  , and obs7 cells) were injected subcutaneously in the left flank of female NOD.CB17-Prkdcscid/J mice (Charles River Laboratory). A549 cells (1 × 107 cells) were injected subcutaneously in the left flank of female BALB/cAJcl-nu/nu mice . When MDA-MB-231 and A549 xenografts reached approximately 150 to 250 mm3, animals were randomized into groups. Number of mice in a group was 6 for the study with MDA-MB-231 and A549 in combination with DOX, and 3 for the study with A549 and single treatment of OTS186935. For intravenous administration, OTS186935 and doxorubicin hydrochloride (Kyowa Hakko Kirin Co. Ltd.) was formulated in a 5% glucose solution and 0.9% sodium chloride injection  and injected via tail vein. An administration volume of 10 mL/kg of body weight was used. The doses administered were indicated in the main text and figures. Tumor sizes were measured every other day or twice weekly by using a caliper. The results were converted to tumor volumes (mm3) by the formula length × width2 × 1/2. Tumor growth inhibition (TGI) was determined by the formula {1 – (T – T0) / (C – C0)} · 100, where T and T0 are the mean tumor volumes on day 14 and day 0, respectively, for treatment groups, and C and C0 are those for the vehicle control group. Tumor mass was dissected from mice on day 14 and tumor weight was determined and then fixed with 4% paraformaldehyde.The animal experiments were performed at OncoTherapy Science, Inc. in accordance with their Institutional Guidelines for the Care and Use of Laboratory Animals. MDA-MB-231 cells  in a steamer for 20 minutes. Anti-Ki67 antibody  was applied on tissue sections for one hour incubation at room temperature in a humidity chamber. Following TBS wash, the antigen-antibody binding was detected with Envision+ system  and DAB+ chromogen . Tissue sections were briefly immersed in hematoxylin for counterstaining and were covered with cover glasses.


Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome. Steroid hormones and bile acids are potent regulators of hepatic carbohydrate and lipid metabolism. Steroid 5β-reductase (AKR1D1) is highly expressed in human liver where it inactivates steroid hormones and catalyzes a fundamental step in bile acid synthesis.Human liver biopsies were obtained from 34 obese patients and AKR1D1 mRNA expression levels were measured using qPCR. Genetic manipulation of AKR1D1 was performed in human HepG2 and Huh7 liver cell lines. Metabolic assessments were made using transcriptome analysis, western blotting, mass spectrometry, clinical biochemistry, and enzyme immunoassays.AKR1D1 expression decreased with advancing steatosis, fibrosis and inflammation. Expression was decreased in patients with type 2 diabetes. In human liver cell lines, AKR1D1 knockdown decreased primary bile acid biosynthesis and steroid hormone clearance. RNA-sequencing identified disruption of key metabolic pathways, including insulin action and fatty acid metabolism. AKR1D1 knockdown increased hepatocyte triglyceride accumulation, insulin sensitivity, and glycogen synthesis, through increased de novo lipogenesis and decreased β-oxidation, fueling hepatocyte inflammation. Pharmacological manipulation of bile acid receptor activation prevented the induction of lipogenic and carbohydrate genes, suggesting that the observed metabolic phenotype is driven through bile acid rather than steroid hormone availability.In human liver biopsies, AKR1D1 mRNA is down-regulated with advancing NAFLD suggests that it may have a crucial role in the pathogenesis and progression of the disease.Genetic manipulation of AKR1D1 regulates the metabolic phenotype of human hepatoma cell lines, driving steatosis and inflammation. Taken together, the observation that  •AKR1D1 expression is downregulated in patients with non-alcoholic fatty liver disease•AKR1D1 regulates bile acid availability in human hepatocytes•AKR1D1 silencing drives insulin sensitivity, lipid accumulation and fuels hepatocyte inflammation•Alterations in FXR and LXR activation underpin some, but not all, of the metabolic effects of AKR1D1 silencing NAFLD is common, with an unselected population prevalence of 30%, rising to over 80% in obese T2DM patients. It is a spectrum of disease ranging from simple steatosis through to inflammation  and eventual scarring, fibrosis and cirrhosis with a significant risk of development of hepatocellular carcinoma (HCC) alongside increased cardiovascular mortality . NAFLD ii.e. androgens, glucocorticoids, and bile acids -palmitate. [2H]-palmitate enrichments were determined by gas chromatography–mass spectrometry using a 5890 GC coupled to a 5973 N MSD . Ions with mass-to-charge ratios (m/z) of 270 (M + 0) and 271 (M + 1) were determined by selected ion monitoring  using the 1/40 dilution from a standard curve generated from a pool of all cDNAs as the calibrator for all samples. The relative expression ratio was calculated using the following: Ratio = ΔCt[target]/ΔCt[ref] and expression values were normalised to 18SrRNA, unless stated otherwise  and reductions in the expression of enzymes regulating lipid export [ATP-binding cassette subfamily A1 (ABCA1)] and oxidation [peroxisome proliferator-activated receptor α (PPARα)] (Metabolic assessments were made in HepG2 cells following ecreased a. Consisncreased b. Howevencreased c. In add(PPARα)] a. The paAKR1D1 knockdown HepG2 cells , whilst fatty acid oxidation decreased, as measured by reduced 3-hydroxybutyrate concentrations in the cell culture media (via a number of different pathways. De novo lipogenesis (DNL) is the synthesis of fatty acids, namely palmitate (16:0), from non-lipid precursors and this was measured by incorporation of deuterated water into cellular TAG and phospholipids. AKR1D1 knockdown increased DNL both in total cellular TAG and in the phospholipid fraction .Consistent with the mRNA expression data, there were increased phosphorylated and total protein levels of ACC in G2 cells d. Total re media e and f. fraction g. CompleAKR1D1. However, no differences in the expression of genes involved in carbohydrate and lipid metabolism or intracellular TAG accumulation were observed .Parallel metabolic assessments were made in HepG2 cells over-expressing 3.6AKR1D1 knockdown significantly increased the expression of CYP7A1, FASN, ACC1, SREBF1, SCD1, AKT1, GLUT1 and GLUT9 in human hepatoma cells. Cortisol treatment  of HepG2 cells failed to regulate the mRNA expression of these genes, suggesting that the phenotype we observed in our AKR1D1 knockdown experiments was unlikely to be due to changes in glucocorticoid availability . Additional treatments with CA  and CDCA  were performed. Whilst CA had no effect on gene expression, CDCA treatment significantly prevented the induction of CYP7A1, FASN, SCD1, SREBF1, AKT1 and GLUT1 mRNA expression caused by AKR1D1 knockdown (nockdown a and b.FS)-Hydroxycholesterol (22-S HC)  decreased CYP7A1, GLUT1, FASN and SCD1 expression, whilst the LXRα antagonist selectively decreased SREBF1 expression . Treatment with GW4064 normalised the expression of all target genes to levels seen in scrambled controls  resulted in partial rescue of the expression levels of FASN, ACC1 and SREBF1, but had no impact on the induction of expression of the other genes caused by AKR1D1 knockdown (We therefore tested the hypothesis that the observed phenotype could arise from decreased FXR activation (due to decreased bile acid synthesis and availability) or increased LXR activation secondary to increased accumulation of oxysterols and bile acid precursors. We proposed that FXR agonism and/or LXR antagonism would have the potential to rescue the phenotype in our cells. The FXR agonist GW4064  and the LXRα antagonist 22, which is induced in response to an inflammatory insult and has been implicated in the pathogenesis of NAFLD [AKR1D1 knockdown increased the mRNA expression of iNOS and the expression of the pro-inflammatory cytokines IL-1β and IL-6 in Huh7 cells  or vehicle, and pro-inflammatory cytokine mRNA expression were explored. In Huh7 cells, GW4064 restored the expression levels of IL-1β only, but had no impact on the induction of expression of IL-6, IL-8 or iNOS caused by AKR1D1 knockdown. Similarly, GW4064 failed to restore cytokine mRNA expression in HepG2 cells, suggesting, in total, that the inflammatory response caused by AKR1D1 knockdown is not entirely FXR mediated . Therefore, potential differences in the regulation of SREBF1 by FXR activation between rodents and humans cannot be excluded. In parallel, FXR activation also increases fatty acid oxidation within hepatocytes through induction of PPARα [At a cellular level, FXR activation supresses SREBP-1c expression in mice , an actiof PPARα . FXR hasof PPARα ,53, whilof PPARα ,55.AKR1D1 knockdown was the only mechanism responsible for our observations, some of our data would conflict with the published literature. Obeticholic acid  treatment of human liver slices increased the expression of both SLC51A and B [AKR1D1 knockdown experiments. An explanation for these findings may lie in analysis of the composition of the bile acid pool following AKR1D1 knockdown. The differing affinities of both primary and secondary bile acids to activate (or antagonize) bile acid receptors are well described [If limiting FXR activation by 1A and B , and thiescribed  and, theescribed  althoughSREBF1, FASN, ACC1 and SCD1, has been well described [AKR1D1 knockdown on lipogenic gene expression, although to a lesser extent when compared to FXR agonism. The role of LXRβ in hepatic lipogenesis has been conflicting. A study from Repa et al. [AKR1D1 knockdown on lipogenic gene expression, highlighting, in total, the additive role of LXRs in the metabolic phenotype observed following AKR1D1 knockdown.The role of LXR receptor activation to regulate lipogenic gene expression, including escribed . In our a et al.  revealeda et al. . In our via 5αR activity) and low levels of CDCA , which can then drive liver injury.Inflammation and liver injury are hallmarks in NAFLD/NASH; lipid accumulation within hepatocytes has also been proposed to trigger the activation of inflammatory pathways . Furtherin vitro and in vivo, by reducing the expression of pro-inflammatory cytokines [AKR1D1 knockdown, suggests that AKR1D1 regulates inflammatory response in a both NF-κB-dependent and NF-κB-independent way of action. .There is increasing evidence on the role of FXR as a regulator of cellular inflammation and immune response; activation of the receptor induces the expression of anti-inflammatory genes  as well ytokines ,70. In o5AKR1D1 is down-regulated across the spectrum of NALFD, and that this drives a detrimental impact on hepatocyte lipid accumulation and inflammation. Further studies are clearly warranted to explore both the mechanisms by which this occurs as well as the potential to manipulate activity to alter NAFLD progression.In conclusion, we have shown that Study development, N.N., L.L.G. and J.W.T.; Methodology, N.N., C.G., N.J.D., L.H.; Investigation, N.N., L.M., S.A., C.G., M.v.d.B., C.M., B.S., R.S.G., A.A., B.A.H.; Writing - Original draft, N.N., S.A., J.W.T.; Writing - Review & Editing, N.N., W.A., H.U.M., T.P., J.R., L.H., J.W.T.; Supervision, W.A., L.H., J.W.T.; Funding Acquisition, J.W.T.Medical Research Council ; NIHR Oxford Biomedical Research Centre ; British Heart Foundation (senior fellowship to LH ref. FS/15/56/31645); National Institute of Environmental Health Sciences (P30-ES013508 awarded to TMP); NIHR Birmingham Biomedical Research Centre . The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health or the National Institute of Environmental Health.This work was supported by the Nothing to declare. TMP is a consultant for Research Institute for Fragrance Materials, is a recipient of a sponsored research agreement from Forendo, and is founding director of Penzymes, LLC.


RNA recognition motif (RRM) is the most common RNA binding domain across eukaryotic proteins. It is therefore of great value to engineer its specificity to target RNAs of arbitrary sequence. This was recently achieved for the RRM in Rbfox protein, where four mutations R118D, E147R, N151S, and E152T were designed to target the precursor to the oncogenic miRNA 21. Here, we used a variety of molecular dynamics-based approaches to predict specific interactions at the binding interface. Overall, we have run approximately 50 microseconds of enhanced sampling and plain molecular dynamics simulations on the engineered complex as well as on the wild-type Rbfox·pre-miRNA 20b from which the mutated systems were designed. Comparison with the available NMR data on the wild type molecules  served to establish the accuracy of the calculations.The Free energy calculations suggest that further improvements in affinity and selectivity are achieved by the S151T replacement. RNA is an outstanding target for oncological intervention. Engineering the most common RNA binding motif in human proteins  so as to bind to a specific RNA has an enormous pharmacological potential. Yet, it is highly non trivial to design RRM-bearing protein variants with RNA selectivity and affinity sufficiently high for clinical applications. Here we present an extensive molecular simulation study which shed light on the exquisite molecular recognition of the empirically-engineered complex between the RRM-bearing protein Rbfox and its RNA target pre-miR21. The simulations allow predicting a variant, the S151T, which may lead to further enhancement of selectivity and affinity for pre-miR21. RNA recognition motif (RRM) is the largest family of eukaryotic RNA-binding proteins  following ref. [The conformational entropy has been estimated by calculating the chain’s conformational entropy from the distribution of the backbone  and side-chains rotameric angles, [χing ref. . The calhttp://www.shiftx2.ca/) [D software (https://brooks.chem.lsa.umich.edu/) [http://www.shiftx2.ca/performance.html). We run SHIFTX2 and LARMORD on each frame extracted from the un-restrained simulations every 10 ps of the free pre-miR20b and of the Rbfox•pre-miR20b complex, for which experimental CS are available. The chemical shifts predictions for these 48,000 pre-miR20b and 48,000 Rbfox•pre-miR20b conformers were then linearly averaged to make a final prediction for the protein’ 13Ca, 13C’, 13Cb, 15N and for the RNA’s 13C and 1H CS.Protein‘ chemical shifts (CS) were predicted using SHIFTX2 v. 1.07 (tx2.ca/) , 38. LARch.edu/)  was usedrNOE, the simulated NOE’s 〈ri,j〉 were calculated according to:ri,j is the interatomic distance between atoms i and i, and the sum runs over the Nf trajectories frames. The average distance violation was defined as:K-means clustering algorithm was used to cluster this group of frames based on the RMSD of the complex. The representative structures of the 20 clusters obtained constitute the “MD-adapted structure ensemble”: sets of atomic coordinates (deposited as PDB files at https://doi.org/10.5281/zenodo.1297931) that capture the flexibility and the conformers suggested by MD simulations while still retaining the highest possible level of agreement with the primary NMR data.For the set of experimental upper bound distance restraint ol as in . In partS1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S1 FigHistograms from PCs analysis in Cartesian space calculated from the trajectories with independent projection of the PCs on the separate trajectories of the pre-miR20b Click here for additional data file.S2 Figcalculated over heavy atoms with respect to the initial structure of the Rbfox protein in simulations of (A) the free state , sim.1 a(PDF)Click here for additional data file.S3 FigDetails of the calculations are reported in the Materials and Methods section.(PDF)Click here for additional data file.S4 Fig27GGCAUG33 (left) and G28GCAUG33 (right) in complex with Rbfox versus time in the six MD simulations performed ((A) RNA backbone dihedral angles calculated over the aggregated simulations of the pre-miR20b Rbfox complex ((PDF)Click here for additional data file.S5 Fig20-C40, U21-A39, A22-U38, G23-C37, calculated over the aggregated simulations ((A) Bp and (B) bps parameters for base pairs G(PDF)Click here for additional data file.S6 FigεRMSD of the pre-miR20b loop (r-U27GGCAUG33) in the three χOL3 -CP-OPC MD simulations (OL3 -CP-OPC)). C. RNA backbone dihedral angle histograms calculated over the aggregated simulations. The green dots indicate the values of the angles in the lowest energy structure of the NMR ensemble 2n7x from which the simulations were started.(A) 2D representation of the free pre-miR20b loop structure. (B) Time development of the (PDF)Click here for additional data file.S7 FigThe loop conformation in the initial structure is shown as the grey overlay.(PDF)Click here for additional data file.S8 Fig20-C40, U21-A39, A22-U38, G23-C37, U24-U36, U25-C35, U26-A34, calculated over the entire MD (dark blue) and NMR (light blue) ensembles.(A) bp and (B) bps parameters for base pairs G(PDF)Click here for additional data file.S9 FigAverage Na+distribution for the (A) free pre-miR20b ((PDF)Click here for additional data file.S10 Fig< 0.5 nm and θ <30°. The distributions are calculated for G29-R184, G29-F126, U32-H120 and G33-F160 pairs in the individual unrestrained trajectories (Stacking geometries are described by the center of mass distance d and the angle θ between the planes of the bases and amino acid side chain. Amino acid and nucleobase are considered stacked if d(PDF)Click here for additional data file.S11 FigD [Calculated and experimentally measured  chemicalD  for the (PDF)Click here for additional data file.S12 FigThe grey square represents the standard deviation. Additional details on the calculations are reported in the Materials and Methods section.(PDF)Click here for additional data file.S13 FigThe blue dot represents the calculated average value; the red one corresponds to the experimental value. A similar behaviour is observed in the other simulations performed on the system.(PDF)Click here for additional data file.S14 Fig13C and (B) 1H atoms of pre-miR20b in the free state  (PDF)Click here for additional data file.S15 FigRepresentation as in (PDF)Click here for additional data file.S16 Fig29-R184, G29-F126, U32-H120 and C33-F160 pairs in replica exchange Click here for additional data file.S17 Fig(PDF)Click here for additional data file.S18 Fig29 with F126 and R184.(A) Top view of the structure in the simulation , sim. 23(PDF)Click here for additional data file.S19 Fig33, R147 and D118.(A) Top view of the structure in the simulation , sim. 24(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


As for NS5B, RASs at positions 159 and 316 was observed only in GT 1b strains. This study highlighted that SVR rate in clinical routine in Brazil was similar to randomized clinical trials (89–98%). Our research provided genetic data about the circulation of resistant variants in Brazil. Despite its presence, most of identified baseline mutations did not negatively impact treatment outcome. Genetic diversity of circulating strains suggested that most of the Brazilian HCV chronic carriers are susceptible to new therapeutic regimens including recently approved DAAs.The selection of viral strains with resistance-associated substitutions at hepatitis C virus (HCV) NS5A and NS5B genes is considered one of the limiting factors for achieving sustained virologic response (SVR) to combination of direct-acting antivirals daclatasvir (DCV) and sofosbuvir (SOF). Since 2015, this interferon-free regimen has been available in Brazilian clinical routine for treating mono- and HCV/HIV-coinfected patients chronically infected with genotypes 1 and 3. Our aim was to assess SVR rate for Brazilian patients chronically infected with genotypes 1 and 3 after DCV/SOF therapy and the frequency of baseline RASs in HCV NS5A and NS5B genes. Serum samples were collected from 107 monoinfected patients and 25 HCV/HIV co-infected patients before antiviral therapy with DCV/SOF. Genetic diversity of NS5A and NS5B genes was assessed by direct nucleotide sequencing. Overall, SVR rate was 95.4% (126/132), and treatment failure occurred in five monoinfected and one HCV/HIV co-infected patient. NS5A RASs frequency was higher for HCV/HIV patients (28%) than monoinfected patients (16.8%). No difference was evidenced between mono- and HCV/HIV-coinfected groups (15% vs. 16%) regarding NS5B gene. Genotype (GT) 1b strains had significantly more baseline substitutions in NS5A (31.6%) than GT 1a and 3a. At least one primary NS5A RAS described in literature at  It is estimated that 71 million people worldwide are chronically infected with hepatitis C virus (HCV) and approximately 2.3 million individuals have HCV/HIV-coinfection [The availability of all-oral direct-acting antivirals (DAAs) has further increased sustained virological response (SVR) rates, the primary objective for a successful therapy, usually evaluated 12 weeks after treatment conclusion . These dDespite the promising results, the selection of viral strains with resistance-associated substitutions (RASs) at HCV NS5A and NS5B genes can be considered one of the limiting factors for failures to DAA combinations. Viral resistance is characterized by positive selection of viral variants that carry amino acid substitutions responsible to reduce susceptibility to certain drug . Each faConsidering drug-specific RASs, mutations with high fold change seem to have increased clinical relevance in inducing treatment failure when associated with drug- and host-related factors (presence of cirrhosis and prior HCV treatment) . RASs inFew studies in Brazil have evaluated the effectiveness of DAA therapeutic regimens in Brazilian patients  and the role of RASs in a flaw treatment outcome . ResearcBased on inclusion criteria indicated in 2015 clinical guidelines for using therapeutic regimen with DCV and SOF, this study enrolled 132 NS5A and NS5B inhibitors-naïve individuals chronically infected with HCV GTs 1 or 3a with advanced fibrosis (METAVIR score F3) or cirrhosis (F4), which attended the National Institute of Infectology Evandro Chagas (INI) and Gaffrée & Guinle University Hospital (UNIRIO). Between 2015 and 2017, serum samples were collected from 107 HCV monoinfected patients  and 25 from HCV/HIV co-infected patients  before antiviral therapy with SOF/DCV with or without RBV (12 or 24 weeks). As determined in 2015 Brazilian Clinical Guideline, RBV addition may be performed especially in patients with cirrhosis, with no response to prior therapy and patients with HCV/HIV co-infection regardless of the degree of fibrosis. Regarding therapy duration, a 12-week regimen was standardized for treatment-naïve GT 1 patients while a 24-week regimen was indicated for GT 1 mono- and HCV/HIV-coinfected patients which had already been experimented with DAAs and/or individuals with cirrhosis Child-Pugh B and C. All GT 3 patients received a 12-week therapeutic regimen. All mono- and HCV/HIV-coinfected patients have completed therapy with SOF/DCV with or without RBV. Fourteen of the monoinfected patients and one HCV/HIV coinfected patient had already been experienced with first-generation PIs boceprevir or telaprevir. Serum samples from six non-responder patients with detectable viral load after the end of therapy period were included in our analyses. SVR rates were accessed 12 weeks after treatment conclusion. HCV viral loads was measured by Abbott Real Time HCV assay  with a limit of detection of HCV RNA > 12 IU/mL or > 1.08 Log IU/mL.Samples were collected after obtaining written informed consent from each patient. This study was approved by the ethics committee from Oswaldo Cruz Foundation (CAAE 68116417.2.0000.5248) and by the ethics committee of Gaffrée & Guinle University Hospital (Number 204.445).Taq DNA Polymerase High Fidelity (Thermo Fisher Scientific).Viral RNA was extracted from serum samples 200 μL) using High Pure Viral Nucleic Acid Kit  following manufacturer’s recommendations. HCV NS5A ~1600 bp) and NS5B (~1500 bp) genes were amplified by one-step reverse-transcription (RT) with polymerase chain reaction (PCR) followed by a second round of PCR (nested-PCR) using specific primers designed for each subtype  and 53 °C, 53 °C and 62 °C (second PCR) for GTs 1a, 1b and 3a, respectively. PCR products were submitted to 1.5% agarose gel electrophoresis, stained with ethidium bromide and visualized under UV light.NS5A and NS5B products were purified using High Pure PCR Product Purification Kit (Roche Life Science) and concentration was estimated with Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific) for each sample. Purified products were subjected to nucleotide sequencing reactions in both directions using Big Dye Terminator v3.1 Cycle Sequencing kit  according to the manufacturer’s instructions and analyzed on ABI 3730 DNA automated sequencer (Applied Biosystems). After assembly of overlapping contigs, HCV NS5A and NS5B nucleotide sequences were submitted to GenBank database under accession numbers MK135170-MK135301 (NS5A gene) and MK135302-MK135433 (NS5B gene).Nucleotide sequences were aligned in MEGA version 7.0  togetherUnivariate analyses were used to associate the presence of baseline NS5A RASs between HCV GTs. Fisher’s exact test and Pearson chi-square were selected to test the significance level of associations, which was assessed at the 0.05 probability level. Statistical analyses were performed using software Epi Info version 7.1 .Clinical, virological and therapeutically baseline data of 132 patients studied are provided in Overall, SVR rate in both groups was 95.4% (126/132) after 12 or 24-weeks of treatment. The majority of patients had non-specific symptoms during therapy, such as headache, anemia, nausea and fatigue. Therapeutic failure with SOF/DCV occurred in six (4.6%) individuals, five monoinfected and one HCV/HIV co-infected patient. Clinical and resistance data of non-responders are described in SVR rates in each HCV GTs for both groups were 92.7% (51/55) for GT 1a, 100% (57/57) for GT 1b and 90% (18/20) for GT 3a. For patients with HCV monoinfection, GT 1b had a higher response rate  followed by GT 3a  and GT 1a . Regarding co-infected individuals, GT 3a had a response rate of 75% (3/4), while for GTs 1a and 1b SVR was 100%.p = 0.0040). Similarly, resistance analyses for HCV NS5B gene indicated a frequency of amino acid substitutions in GT 1b strains of 35% (20/57) whereas for GTs 1a and 3a no mutations were identified for both groups.In the present study, NS5A RASs frequency was higher in HCV/HIV patients  than in monoinfected patients . Regarding NS5B RASs, similar frequency was found for both groups (16% vs 15%). Filtering the presence of NS5A RASs by HCV GTs, GT 1b strains  had significantly more resistance mutations when compared to GTs 1a  and 3a  together (p = 0.0076). Similarly, it was observed a statistically significant association for the presence of baseline NS5A RASs between GT 1b and 1a (p = 0.0164) and for GTs 1b, 1a and 3a (p = 0.0171).Considering monoinfected group, GT 1b strains had significantly more substitutions than other GTs  [et al. (2016) [In 2015, DAAs SOF and DCV were included in Brazilian clinical guidelines for the treatment of HCV chronic infection. This combined therapy had been the most used by hepatologists in clinical routine, due to its effectiveness to different patient profiles: treatment-naive, non-responders to previous therapies and HCV/HIV co-infected individuals. Until November 2015, treatment options available in Brazil were limited to a protocol with "first-wave" PIs directed to patients infected with HCV GT 1. Those who experienced previous failure did not have other therapeutic alternatives since at that period no other DAAs options were available. In general, the real efficacy of DAAs and SVR assessment rates in non-clinical studies in Brazil could only be accessed since 2016. Consequently, the low number of national scientific studies evaluating and comparing their findings with controlled clinical trials whose SVR rates ranged from 89% to 98% depending on the infecting GT and treatment with previous drugs . In the . (2017)  and a ra. (2016)  where SVStudies indicated that previous treatment with DAAs for a specific gene may represent a negative factor for a future therapeutic success with a DAA directed to the same target likely due to drug-selective pressure , 19. Heret al. (2014) [SVR rates observed for each GT was 92.7% (51/55), 100% (57/57) and 90% (18/20) for GTs 1a, 1b and 3a, respectively. Our results are similar to that reported by Sulkowski . (2014)  in a ranIn the non-responder GT 1a monoinfected patient who failed a 12-week treatment with SOF/DCV/RBV, the primary (M28T and Q30R) and secondary (E62D) mutations observed could be associated to the treatment failure. M28T mutation confers high-level resistance to DCV (fold change > 100) and OBV (fold change > 1000), while Q30R confers a clinical impact due to the high fold-change to DCV, EBV, LDV and OBV [et al. (2017) [Here, we report the presence of RAS Y93N in a HCV NS5A sequence from a non-responder GT 1a monoinfected patient whose serum sample was isolated after treatment with SOF/DCV/RBV. A study from Wyles DL . (2017)  had repoet al. (2016) [in vivo for non-responders patients, therefore, additional studies will be needed to evaluate a possible new resistance profile associated with this mutation.Sarrazin . (2016)  have ideet al. (2017) [in vivo reports so far limited the possibility of association of this RAS with non-response. The patient also presented other negative predictive factors for non-response to treatment: (1) viral factors: high infective basal viral load (log 6.36) and HCV GT 3a infection; (2) host factors: male and chronic cirrhotic profile; and (3) therapeutic factor: reduced treatment duration (12 weeks) [In regard to NS5A sequence from HCV GT 3a, substitution A30S has previously been reported by Malta F. . (2017)  in a vir. (2017) . In the 2 weeks) . An updaet al. (2017) [et al. (2013) [p<0.05). Although its higher frequency in GT 1b, impact of the presence of RASs in treatment outcome is considered more evident in patients infected with GTs 1a and 3a [According to previous reports, baseline RASs frequency in HCV NS5A region determined by conventional and next-generation sequencing techniques presented values between 6% and 16% –27. Here. 2017)  in a stu. (2013)   [in vitro for DAAs DCV and LDV (fold change > 100). However, our analyses in vivo indicated that baseline presence of RAS L31M in HCV strains of GTs 1a (2.2%) and 1b (6.7%) did not influenced treatment outcome since all five patients achieved SVR. Additionally, low prevalence for L31M variant was also observed by Zeuzem et al. (2017) [et al. (2013) [According to Wyles . (2017)  amino ac. (2017)  (2.3%) a. (2013)  (3.1%). . (2013) . Likewiset al. (2017) [et al. (2017) [in vitro and highly associated with SOF resistance, was not observed for isolates of HCV GT 1b in our study as well as in findings from Castilho M.C. et al. (2011) [et al. (2015) [Regarding HCV NS5B RASs for monoinfected patients, the high frequency of combination L159F + C316N  found in strains of HCV GT 1b was similar to results reported by Noble C.F. . (2017)  and Pere. (2011)  and Cost. (2015) .et al. (2012) [et al. (2011) [et al. (2011) [Few Brazilian studies have analyzed NS5A and NS5B genes resistance profile for HCV/HIV co-infected patients. Further studies are needed to better understand the circulation of resistant viral subpopulations in this group. Our analyses indicated that NS5A RASs frequency for referred group was 28% (7/25) while for NS5B RASs was 16% (4/25). HCV GT 1b strains had higher frequency of NS5A  and NS5B  RASs than GTs 1a and 3a. According to Plaza Z. . (2012) , none of. (2011)  (13.3%),. (2011)  (10%) siin vivo were observed. However, most of these baseline substitutions did not seem to negatively impact treatment outcome, especially for GT 1b since all patients achieved SVR, demonstrating the importance of a combined therapy directed to different viral proteins. Our observations about circulation of resistant variants in Brazil suggested that new DAAs combination, such as OBV/DSV/PTV/r, SOF/LED and EBV/GZV, whose efficacy is not diminished by these substitutions, could be licensed and included in future clinical protocols for Brazilian chronic HCV carriers.In conclusion, this study demonstrated that SVR rate in Brazilian patients chronically infected with HCV treated with DAA regimen DCV/SOF (95.4%) was similar to results from randomized clinical trials. Our research also sought to identify the circulation of resistant variants in Brazil and if emerging RASs could have influenced or not in treatment outcome. Here, amino acid substitutions already described in literature and with limited clinical reports


Bacillus subtilis, the extracytoplasmic function σ factor σM regulates cell wall synthesis and is critical for intrinsic resistance to cell wall targeting antibiotics. The anti-σ factors YhdL and YhdK form a complex that restricts the basal activity of σM, and the absence of YhdL leads to runaway expression of the σM regulon and cell death. Here, we report that this lethality can be suppressed by gain-of-function mutations in yidC1 (spoIIIJ), which encodes the major YidC membrane protein insertase in B. subtilis. B. subtilis PY79 YidC1 (SpoIIIJ) contains a single amino acid substitution in a functionally important hydrophilic groove (Q140K), and this allele suppresses the lethality of high σM. Analysis of a library of YidC1 variants reveals that increased charge (+2 or +3) in the hydrophilic groove can compensate for high expression of the σM regulon. Derepression of the σM regulon induces secretion stress, oxidative stress and DNA damage responses, all of which can be alleviated by the YidC1Q140K substitution. We further show that the fitness defect caused by high σM activity is exacerbated in the absence of the SecDF protein translocase or σM-dependent induction of the Spx oxidative stress regulon. Conversely, cell growth is improved by mutation of specific σM-dependent promoters controlling operons encoding integral membrane proteins. Collectively, these results reveal how the σM regulon has evolved to up-regulate membrane-localized complexes involved in cell wall synthesis, and to simultaneously counter the resulting stresses imposed by regulon induction.In  Bacillus subtilis, the alternative σ factor σM is induced by cell wall antibiotics, and upregulates genes for peptidoglycan and cell envelope synthesis. However, dysregulation of the σM regulon, resulting from loss of the YhdL anti-σM protein, is lethal. We here identify charge variants of the YidC1 (SpoIIIJ) membrane protein insertase that suppress the lethal effects of high σM activity. Further analyses reveal that induction of the σM regulon leads to high level expression of membrane proteins that trigger envelope stress, and this stress is countered by specific genes in the σM regulon.Bacteria frequently produce antibiotics that inhibit the growth of competitors, and many naturally occurring antibiotics target cell wall synthesis. In  Bacillus subtilis, there are seven ECF σ factors, with the σM regulon playing a particularly important role in modulating pathways involved in peptidoglycan synthesis, cell envelope stress responses, and intrinsic resistance to antibiotics . Ov. OvyidC1Q140K protein may simply be more active or abundant in cells than the native protein. To test if increasing insertase activity is sufficient to alleviate toxicity associated with high σM, we overexpressed the wild-type 168 YidCQ140 protein using a strong IPTG inducible promoter, Pspac(hy)  and plotted against time. The slope of the linear part of the product accumulation curve was calculated using Excel and Miller Units (MU) were calculated using formula MU = Slope/OD600/V, where V is the volume of cells used for the reaction (200 μl).Bacterial cells containing the 600 ~0.4) and RNA was extracted using RNeasy Mini Kit (Qiagen). The extracted was then treated with DNase I (Invitrogen) and the quality of RNA was checked with electrophoresis. RNA was then reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green (BioRad) and the topA gene was used for reference of data normalization.Cells were grown to mid-exponential phase  Variation of yhdK colony size measurement between trials on different days with different batches of LB plates. P value was calculated using Student’s t test, and percentage changes of average colony size were shown.A) Map of SNPs from strain PY79 to 168 and distribution of SNPs contained in each congression suppressors. Genome coordinates were based on the 168 reference genome with NCBI accession number NC_000964.3. B) Transformation plates of (TIF)Click here for additional data file.S2 FigBacillus subtilis. B) Streaking of yhdK Pspac(hy)-yidC2Q148K on plates of LB or LB supplemented with 1 mM IPTG . C) Spot dilution of yhdL depletion strains with Pspac(hy) based overexpression of different YidC homologs, on LB plates supplemented with a final concentration of 1% xylose (+Xylose), 1 mM IPTG (+IPTG) or nothing (None).A) Venn diagram of function overlap and distinction of YidC1 and YidC2 of (TIF)Click here for additional data file.S3 FigM tolerance contain a charge of +2 or +3. Among the 103 samples with charge of +2 or +3, the positive charge can be located in one, two or three transmembrane segments, with the exception that no sample contains more than one positive charge in TM1 alone. B) Crystal structure of YidC from Bacillus halodurans (PDB ID 3WO6), showing the seven variable amino acid providing positive charges in the hydrophilic substrate binding chamber of the enzyme. Gly231 is not visible due to the lack of side chain of this residue. TM1-5, transmembrane region 1–5; E1, extracytoplasmic region 1; C1, cytoplasmic region 1.This figure was generated using UCSF Chimera 1.13[A) Distribution of 128 possible charge variants of YidC1 in the tested library. Theoretically, the majority of YidC1 variants in the input library have a charge of +2 to +5 in the hydrophilic groove, while experimental data from 103 samples suggests that the ones capable of providing high σmera 1.13.(TIF)Click here for additional data file.S4 FigyhdL depletion strains with YidC1 variants were streaked on LB plates with or without xylose inducer for yhdL. The positive charge of each variant was labelled next to the streaking, with a negative control “None” meaning no yidC1 variant at thrC locus , and a positive control “YidC1PY79” meaning the native YidC1 mutated into the PY79 Q140K version (HB23719). B) Spot dilution of YidC depletion strains with yidC1 variants at thrC locus. The depletion strain has its native yidC1 and yidC2 deleted, and a xylose inducible copy of PxylA-yidC2. Diluted cultures were spotted on LB without xylose (-Xyl) or with 1% final concentration of xylose (+Xyl). The negative control (None) has no yidC1 at thrC locus, while the positive control has the 168 version of yidC1 that contains a single positive charge at R73. Some YidC1 variants exhibited reduced growth ability, and the variant containing R72 K140 R144 failed to grow, although the emergence of suppressors was noted.A) (TIF)Click here for additional data file.S5 FigyhdK null, and yhdK with YidC1Q140K single amino acid substitution. B) PhtrA activity of strains in panel A during growth. C) Growth curves of WT strain 168, cssR null, cssS null, cssRS double null, and cssS null with an ectopic IPTG inducible copy under induced or uninduced conditions. D) PhtrA activity of strains in panel C during growth. The OD600 and luminescence were measured every 12 minutes. At least four biological replicates were used for each strain, and the results are shown as mean ± SEM.A) Growth curves of WT strain 168, (TIF)Click here for additional data file.S6 FigB. subtilis 168 vs. strains with the indicated mutations. B) B. subtilis yhdK null mutant vs. strains additionally mutant for the indicated gene. C) WT B. subtilis 168 vs. strains lacking either the σM-regulated promoter for spx (σPM-spx) or the sasA or secDF genes. D) B. subtilis yhdK null mutant vs. strains additionally mutant for the indicated gene. E) WT B. subtilis 168 vs. strains lacking the σM-regulated promoter (PM) for the indicated gene(s). The percentage change relative to the average colony size is shown above each Box and Whisker plot.A) WT (TIF)Click here for additional data file.S1 TableNo mutations were found in these genes.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.