Pens were randomly assigned to 1 of 3 Zn treatments (ZNTRT): 1) control ; 2) inorganic Zn ; or 3) 120 mg supplemental Zn/kg DM as Zn-amino acid complex  for first 60 d, then a blend of ZnSO4 and Zn-AA complex  for the remainder of the trial (ZNBLD). Two dietary energy strategies (ENERGY) were formulated to reach ADG rates of 1) 1.6 kg/d (LE) or 2) 2.0 kg/d (HE) utilizing a 3 × 2 factorial arrangement (12 steers/treatment). All steers were fed LE for a 60 d growing period, then pens were randomly assigned to ENERGY treatments fed the remaining 91 d. Day 60 BW tended to be greater (P = 0.07) in steers receiving supplemental Zn vs. CON. Liver Cu was decreased in Zn supplemented steers vs. CON (P = 0.02). Liver Zn concentrations on d 56 did not differ for Zn vs. CON (P = 0.22) nor were there differences due to Zn source (P = 0.98). There were or tended to be ZNTRT × ENERGY effects for d 67–90 ADG and G:F (P ≤ 0.01), and d 122 BW and d 90–122 G:F (P ≤ 0.10) driven by improved performance for ZNBLD-HE over ZNBLD-LE, while ENERGY within CON and INZN did not differ. Day 90–122 ADG, overall ADG and overall G:F was greater (P ≤ 0.02) and d 67–90 G:F tended to be greater (P = 0.10) for HE vs. LE. No ZNTRT × ENERGY or ZNTRT effects were detected for HCW, REA, BF, KPH, MS, or YG (P ≥ 0.37) while HE increased HCW, BF, MS, and YG compared with LE (P ≤ 0.05). In the liver, ZNTRT affected d 97 MT1A expression (P = 0.03) where INZN was greater than ZNBLD or CON (P ≤ 0.02), while ZIP14 was unaffected due to ZNTRT, ENERGY, or the interaction (P ≥ 0.39). Supplying supplemental Zn as ZNBLD during the transition period appeared to improve performance measures, but no final performance advantages were noted due to increased supplemental Zn, regardless of source. Additionally, differences in liver MT1A expression may indicate differing post-absorptive metabolism between Zn sources.The objective of this study was to determine the effects of increased supplemental Zn from differing sources on growth performance of steers fed diets differing in net energy. Angus steers ( The trace mineral Zn supports a variety of metabolic processes in mammals as an important constituent in pathways such as DNA and protein synthesis . CurrentAll procedures and protocols were approved by the Iowa State University Institutional Animal Care and Use Committee (8-15-8073-B).4; INZN); or 3) blend of supplemental Zn sources (ZNBLD) where steers received 120 mg supplemental Zn/kg DM as Zn-amino acid complex  for the initial growing period of 60 d, then switched to a blend of ZnSO4 and Zn-AA complex [CON + 60 mg supplemental Zn/kg DM ZnSO4 + 60 mg supplemental Zn/kg DM as Zn-amino acid complex] for the remainder of the trial. These supplemental Zn concentrations were chosen for the current study to coincide with previous research completed by A total of 72 Angus steers (324 ± 2.1 kg) from a single source with Genemax  gain scores of 3, 4, or 5, indicating a predicted genetic value belonging in the top 60% for growth potential of tested Angus cattle , were utn = 24 steers/ZNTRT) were fed the LE diet. Beginning on d 60, 2 pens per ZNTRT (one pen from each weight block) was assigned to HE and transitioned for 7 d. Feed delivery on the first day of transition was such that steers received a similar amount of dry matter (DM) as the day before when all were receiving the LE diet. Adjustments in feed delivery over the rest of the transition period were made according to the previous day's bunk score. Following the transition, all steers were fed their respective treatment combination for a 91 d finishing period (n = 12 steers/treatment combination).The trial consisted of two phases: an initial growing period where all steers received a low-energy (LE) diet for 60 d, followed by a 91 d finishing period where half the steers transitioned to a high-energy (HE) diet while half remained on LE. The two dietary energy (ENERGY) strategies were formulated to reach targeted growth rates of 1) 1.6 kg ADG (LE) or 2) 2.0 kg ADG (HE). Diet composition and analysis are shown in During the entire trial period (158 d) all steers were fitted with unique electronic identification tags and were housed in pens equipped with GrowSafe bunks  to collect daily individual as-fed intake. Steers were fed once daily at 8:00 a.m. and provided ad libitum access to feed and water. On d 0 all steers were implanted with Component TE-IS with Tylan  and on d 90 all steers were implanted with Component TE-S with Tylan . Pre-feeding BW were recorded prior to feeding on d −1, 0, 59, 60, 66, 67, 90, 122, 157, and 158, and a 4% pencil shrink was applied to all BW measurements before calculation of ADG and gain-to-feed (G:F).Longissimus dorsi muscle. A modified Jamshidi bone marrow biopsy/aspiration needle (8 g × 10 cm needle) was inserted into the injected area to collect muscle tissue (~0.5 g wet basis). The collected tissue was rinsed with 0.1 M phosphate-buffered saline (pH 7.0) to remove any vestigial blood from the sample, placed into an acid-washed container and flash frozen in liquid nitrogen. Muscle tissue was stored at −80 °C until further analysis.Liver and muscle biopsies were collected on d 56, 97, and 153. Liver biopsies were collected using methods established by 2 EDTA Plus, and trace element K2 EDTA blood collection tubes  for analysis of plasma trace mineral (TM) and various blood metabolites.Blood was collected by jugular venipuncture on d 59, 90, 122, and 157 into serum, sodium heparin, KLongissimus dorsi muscle area (REA) 12th rib fat thickness (BF), kidney, pelvic, and heart fat (KPH), and marbling score (MS), all of which were utilized to calculate yield grade (YG) and quality grade (QG).At the end of the experiment (158 d), steers were shipped to a commercial abattoir  for slaughter followed by a 48 h chilling period. Personnel from the Hansen laboratory, blinded to treatments, were dispatched to collect carcass data, including hot carcass weight (HCW), Total mixed ration (TMR) samples of each diet were collected weekly and subsamples were taken to determine DMI. Dry matter of TMR was determined according to Inductively coupled plasma optical emission spectrometry  was used for trace mineral analysis of TMR, plasma, and liver tissue. Dried, ground, and composited TMR were acid digested prior to mineral analysis as described by −ΔΔCT).Liver and muscle samples for mRNA extraction were kept frozen with liquid nitrogen while ground with a mortar and pestle. Extraction of mRNA was conducted using the Trizol  method and the RNeasy Mini Kit  per manufacturer’s instructions. A Qubit 4 Fluorometer  was used to quantify and measure the integrity and quality of RNA. For each sample, 0.5 μg was reverse transcribed using the Superscript III kit (ThermoFisher Scientific) per the manufacturer’s instructions. Complementary DNA samples were prediluted (1:10) and 2μL were combined with 20μL of a master mix containing SYBR Green fluorescent dye , forward and reverse primers  and RNAse free water for gene expression analysis. Utilizing quantitative real-time PCR, samples were analyzed for RNA abundance of 40s Ribosomal Protein 9 . The model included the fixed effects of ZNTRT and block. Two single degrees of freedom contrasts were constructed for the growing period: 1) no supplemental Zn vs. supplemental Zn as both INZN and ZNBLD and 2) INZN vs. ZNBLD. Data collected following d 67 (finishing period) were analyzed as a 3 × 2 factorial arrangement using the Mixed procedure of SAS. The model included the fixed effects of ZNTRT, ENERGY, block, and the interaction of ZNTRT and ENERGY; steer was the experimental unit (n = 12/treatment combination) for all analyses. Finishing period plasma and IGF-1, SUN, and serum NEFA data were analyzed as repeated measures with date as the repeated effect. The compound symmetry variance structure for repeated measures was selected to achieve the lowest Akaike information criterion value for all repeated measures analysis and d 60 IGF-1 concentrations were used as a covariate for IGF-1 repeated measures. The Shapiro–Wilk test of normality was utilized and NEFA and finishing period G:F data were normally distributed by log transformation; treatment means and SEM shown are reverse transformed. Outliers were determined using Cook’s D and removed if Cook’s D ≥ 0.5. Due to health reasons unrelated to treatment one steer from ZNBLD-HI was removed from analysis following d 67. Significance was declared at P ≤ 0.05 and tendencies identified if P ≥ 0.06 and ≤0.10. Tabular values reported reflect the least square means and the PDIFF statement in SAS was utilized to determine pairwise differences.Data were analyzed as a randomized complete block design. Performance, DMI, and liver TM data for the initial 60 d growing period were analyzed using the Mixed procedures of SAS , with steer as the experimental unit (P ≥ 0.12) during this period, nor were there differences due to Zn source (P ≥ 0.20). However, d 60 BW tended to be greater in steers receiving supplemental Zn vs. CON (P = 0.07). Liver Cu was decreased in Zn supplemented steers vs. CON (P = 0.02) while no differences were detected between Zn sources (P = 0.48). Liver Mn was not different between supplemental Zn and CON (P = 0.16) but was lesser in INZN when compared with ZNBLD (P = 0.01). Day 56 liver Zn concentrations did not differ for Zn vs. CON (P = 0.22) nor were there differences due to Zn source (P = 0.98).Data for the initial 60 d growing period when all steers received the LE diet are shown in P ≥ 0.21). However, there were or tended to be ZNTRT × ENERGY effects for d 67–90 ADG and G:F (P ≤ 0.01), and d 122 BW and d 90–122 G:F (P ≤ 0.10). Collectively, these interactions are driven by improved performance during this time by ZNBLD-HE over ZNBLD-LE, while within CON and INZN, HE and LE did not differ for these measures. No ZNTRT effects were noted for interim or overall DMI, BW, ADG, or G:F during the finishing period (P ≥ 0.15). An ENERGY effect was detected where HE was greater than LE for d 90–122 ADG, overall ADG and overall G:F (P ≤ 0.02), and tended to be greater for d 67–90 G:F (P = 0.10). No ENERGY effects were detected for DMI or d 67, 90, or 122 BW, or d 122–158 ADG and d 122–158 G:F (P ≥ 0.11).Effects of ZNTRT and ENERGY strategies on steer performance during the finishing period are shown in P = 0.02) where no effect of ENERGY was detected within cattle receiving CON or ZNBLD, but within INZN, HE tended to dress greater than LE. No ZNTRT × ENERGY or ZNTRT effects were detected for HCW, REA, BF, KPH, MS, or YG (P ≥ 0.37). The HE diet increased HCW, BF, MS, and YG compared with LE (P ≤ 0.05), but ENERGY had no effect on REA or KPH (P ≥ 0.63).Carcass data are shown in P = 0.04) where INZN HE was greater than CON or ZNBLD HE and INZN LE (P ≤ 0.03) and ZNBLD LE was greater (P = 0.003) and INZN tended to be greater (P = 0.06) than CON LE. No interactions with time (P ≥ 0.24) were noted for plasma Zn concentrations; however, there was an effect of time (P = 0.0001) where concentrations were least on d 60 (1.25 mg/L) relative to d 90, 122, and 157 . No ZNTRT × ENERGY × time, ZNTRT × time, or ZNTRT effects were noted for plasma Cu (P ≥ 0.19). However, there was an ENERGY × time effect for plasma Cu (P = 0.02) where HE tended to be greater than LE on d 90 and 122  while HE and LE did not differ on d 60 and 157 . No effects of ZNTRT, ENERGY, or the interaction were observed for liver Mn or Zn (P ≥ 0.11). No ZNTRT × ENERGY or ENERGY effects were noted for liver Cu (P ≥ 0.20). Liver Cu concentrations tended to be affected by ZNTRT (P = 0.07) on d 97 where ZNBLD was lesser than CON (P = 0.04) and on d 153 where INZN was lesser (P = 0.04) and ZNBLD tended to be lesser (P = 0.06) than CON.Plasma Cu and Zn repeated measures data are shown in P ≥ 0.15; P = 0.10), where d 60 was lesser than d 90 and 157 but did not differ from d 122 while d 90, 122, and 157 did not differ from each other. Plasma NEFA concentrations  differed over time (P = 0.0001), where d 60 was greater than all other time points. No ZNTRT × ENERGY × time effect was noted for plasma IGF-1 concentrations (P = 0.30). A ZNTRT × ENERGY effect was noted for plasma IGF-1 concentrations (P = 0.04) where CON LE was greater than CON HE and INZN HE (P ≤ 0.02) and ZNBLD HE was greater than CON HE  where on d 122 LE was greater than HE (P = 0.0039) while on d 90 and 157 ENERGY did not differ (P ≥ 0.20). A tendency for a positive correlation was noted between plasma IGF-1 concentrations and liver MT1A mRNA expression on d 90 , which can be interpreted as when plasma IGF-1 concentrations increase MT1A expression decreases, but no correlation was detected on d 153 .No ZNTRT, ENERGY, ZNTRT × ENERGY, or interactions with time were noted for SUN or plasma NEFA concentrations (P ≥ 0.32). In d 97 liver, ZNTRT affected MT1A expression (P = 0.03) where INZN was greater than ZNBLD or CON (P ≤ 0.03) while ZNBLD and CON did not differ  on liver MT1A on d 153  also increased Zn retention. Overall, if animals are not Zn deficient plasma Zn has limited use as a Zn biomarker as it does not appear to be related to whole animal Zn retention.Plasma Zn was adequate in all treatments , and onlSteers receiving supplemental Zn exhibited limited advantages in a final performance in the present study. This is in accordance with previous studies where supplementing similar concentrations of Zn as Zn oxide to finishing heifers did not improve final BW or ADG , 2019. VIt is well recognized that increasing dietary energy increases cattle growth rate , 1995. IPrevious research in ruminants and humans has shown that IGF-1 concentrations will change depending on nutritional status . Followi4 and Zn-amino acid complex and future work is needed to better understand this discrepancy between sources.Increasing dietary energy improved performance measures in finishing cattle regardless of Zn supplementation strategy; however, targeted improvements in growth rate of HE over LE were not achieved. Collectively, increasing supplemental Zn during the growing period improved performance, but limited final performance advantages were noted due to increased Zn supplementation concentrations. Differences in liver gene expression of the Zn storage protein metallothionein suggest post-absorption metabolism may differ between ZnSO


Staphylococcus aureus generates and releases extracellular vesicles (EVs) that package cytosolic, cell-wall associated, and membrane proteins, as well as glycopolymers and exoproteins, including alpha hemolysin, leukocidins, phenol-soluble modulins, superantigens, and enzymes. S. aureus EVs, but not EVs from pore-forming toxin-deficient strains, were cytolytic for a variety of mammalian cell types, but EV internalization was not essential for cytotoxicity. Because S. aureus is subject to various environmental stresses during its encounters with the host during infection, we assessed how these exposures affected EV production in vitro. Staphylococci grown at 37 °C or 40 °C did not differ in EV production, but cultures incubated at 30 °C yielded more EVs when grown to the same optical density. S. aureus cultivated in the presence of oxidative stress, in iron-limited media, or with subinhibitory concentrations of ethanol, showed greater EV production as determined by protein yield and quantitative immunoblots. In contrast, hyperosmotic stress or subinhibitory concentrations of erythromycin reduced S. aureus EV yield. EVs represent a novel S. aureus secretory system that is affected by a variety of stress responses and allows the delivery of biologically active pore-forming toxins and other virulence determinants to host cells. Staphylococcus aureus is a pathogenic bacterium that causes a wide spectrum of human diseases, ranging from mild skin lesions and surgical wound infections, to invasive and life-threatening infections, such as pneumonia, osteomyelitis, endocarditis, and bacteremia [S. aureus isolates are resistant to commonly used antibiotics, and efforts to develop a vaccine for the prevention of staphylococcal infections have eluded success [S. aureus infections is attributed to a wide array of virulence determinants that are associated with the cell surface, such as protein adhesins [cteremia . Many S. success . The patadhesins  and glycadhesins , or secradhesins , superanadhesins , and proadhesins .S. aureus toxins are mainly expressed and secreted during the post-exponential phase of bacterial growth. These exoproteins enhance bacterial virulence by directly lysing host cells, orchestrating intracellular signaling events, or activating T cells, thereby playing important roles in the pathogenesis of staphylococcal disease [S. aureus toxins can be divided into three categories according to their effects on host cells: (1) PFTs lyse host cells by forming pores in the plasma membrane in a receptor-dependent manner [S. aureus strains in nonimmune hosts may lead to toxic shock syndrome, a life-threatening disease that is characterized by rash, hypotension, fever, and multiorgan dysfunction. Human consumption of food containing preformed enterotoxins may result in acute food poisoning [ disease ,9. S. aut manner . (2) Phet manner . (3) Pyroisoning .S. aureus also produces a variety of extracellular proteases, which can promote bacterial invasion and dissemination or dampen host innate immunity by directly degrading host proteins [S. aureus exoproteins are secreted through the general secretory (Sec) pathway [In addition to toxins, proteins ,13,14. Sproteins ,15. Exceproteins , most S. pathway . Toxins S. aureus strains, including antibiotic-resistant isolates, has been characterized during the past decade [S. aureus EVs package a diverse array of bacterial components, including cytosolic, surface, and membrane proteins, as well as surface adhesins, lipoproteins, and toxins [S. aureus EVs is a complex process. We demonstrated that alpha-type phenol-soluble modulins promote EV biogenesis by disrupting the bacterial cytoplasmic membrane, whereas peptidoglycan crosslinking and autolysin activity modulate EV production by altering the permeability of the cell wall [S. aureus isolates exhibit dose-dependent cellular toxicity [S. aureus toxins do not include their signal sequence, consistent with the observation that the toxins are packaged as biologically active molecules. Most S. aureus exoproteins are synthesized in the cytoplasm as preproteins with an N-terminal signal peptide. Recognition of the signal sequence by the Sec machinery leads to translocation of the preprotein across the cell membrane in an unfolded state. Following cleavage of the signal peptide by a type I signal peptidase [S. aureus EVs are generated by a budding process from the cell membrane under turgor pressure [Extracellular vesicles (EVs) are nano-sized, spherical, bi-layered membrane vesicles that are secreted by eukaryotes, archaea, and bacteria . The gent decade ,22,23,24d toxins ,20,21,24toxicity ,26 sincetoxicity ,27,28,29eptidase , the proeptidase . Becausepressure , secreteS. aureus has also evolved adaptive mechanisms to survive stresses encountered in the environment and within the host during infection, such as iron limitation, oxidative stress, and exposure to antimicrobial agents [Production of outer-membrane vesicles (OMVs) has been demonstrated to be a stress response characteristic of Gram-negative bacteria ,33,34,35l agents ,42,43,44S. aureus EVs, which represent a novel secretory system to deliver bacterial virulence determinants to human cells. We provide experimental evidence to show that the generation of S. aureus EVs is influenced by various environmental stresses, and those encountered in vivo, such as iron limitation and oxidative stress, enhance EV production and may contribute to the pathogenesis of staphylococcal infections.In this study, we describe the toxin content and the biological activities of S. aureus JE2 [We purified EVs from the culture supernatants of reus JE2 , a USA30S. aureus isolates [A proteomic analysis of purified JE2 EVs revealed a cargo comprised of 180 proteins , includiisolates ,25,28,29isolates  reportedS. aureus strains [S. aureus.Alpha-hemolysin (Hla), the best-characterized PFT component of EVs ,25,27,29 strains ,21,23,28 strains . By affeS. aureus EVs [S. aureus EV production by disrupting the integrity of the bacterial cytoplasmic membrane [PSMα, PSMβ, and delta hemolysin, detected in most reus EVs ,24,27,28reus EVs . In addiS. aureus strains [S. aureus JE2 produces SEK, SEQ, and SElX, but only SElX was detected in purified EVs [S. aureus MSSA476 EVs carry SEA, SEK, and SEQ, but not SEC or SEH (encoded by the genome) [More than 20 staphylococcal enterotoxin (SEs) and staphylococcal enterotoxin-like (SEls) antigens have been identified from different  strains , and manfied EVs . Likewisfied EVs , such asfied EVs . S. aure genome) . Mastiti genome) .S. aureus isolates produce exfoliative toxins (ETs) with serine protease activity, and expression of these toxins in the superficial layers of the skin results in staphylococcal scalded skin syndrome [S. aureus strains JE2 and M060 produce ETA, but it was identified only in M060 EVs [S. aureus strains M060, 03ST17, and 06ST1048 [Various syndrome . S. aureM060 EVs . As show06ST1048 .S. aureus isolates may produce up to 10 proteolytic enzymes, including serine proteases, serine protease-like (Spl) proteins, cysteine proteases, and one metalloprotease, and these enzymes modulate the stability of S. aureus proteins and target host molecules for degradation [S. aureus JE2 EVs, but other proteases, including aureolysin, SplA, SplC, SplD, and SplE, are produced by JE2 but were not detected in EVs [S. aureus strains, including MSSA476, ATCC 14458, and M060 [radation . SplB and in EVs . Proteasand M060 ,21,23  operon [agr was assessed by incubating EVs in vitro with different cell types. JE2 EVs were toxic for A549 human epithelial cells at doses as low as 1 µg/mL, whereas EVs from JE2∆agr exhibited negligible toxicity  and PSMs. As predicted, JE2 EVs, but not EVs from JE2∆agr, were cytolytic for HL60 cells (agr∆sae showed negligible cytotoxicity (The expression of many of the ) operon . The reltoxicity A. Consisemolysis B. HL60 n60 cells C. Similatoxicity D.psmα1–4), or a mutant lacking all five leukocidins and Hla (denoted as ΔΔΔΔΔ). As shown in agr mutant and the ΔΔΔΔΔ mutant exhibited negligible cytotoxicity. In contrast, EVs purified from the LAC∆psmα1–4 mutant showed only a modest decrease in cytolytic activity compared to that of WT LAC EVs  release. Blockage of EV internalization by dynasore did not reduce cytotoxicity in comparison to the DMSO control . Becausereus EVs  were ablS. aureus has developed adaptive mechanisms to respond to environmental changes and stressors encountered during infection [S. aureus EVs under different physiological stresses.To survive in a hostile host environment, nfection ,55,56. AS. aureus may encounter shifts in temperature as it transits from the anterior nares, the skin, or from inanimate objects to deeper tissues during the establishment of infection. Similarly, higher temperatures associated with fever may be encountered by the bacterium during an acute infection. To determine whether these temperature shifts affect EV production, S. aureus JE2 was cultivated to an OD of 1 at either 30 °C, 37 °C, or 40 °C. As shown in S. aureus doubling time during growth at 30 °C was longer than that of cultures grown at 37 °C or 40 °C. A quantitative analysis of EV protein yield showed that significantly more EVs were recovered from the 30 °C cultures than from cultures grown at the higher temperatures  and hydrogen peroxide (H2O2) [S. aureus may also encounter oxidative stress as a result of interactions with host phagocytes that produce NADPH oxidase and generate O−2 during the oxidative burst. O−2 can undergo dismutation to H2O2 that can be used by the myeloperoxidase complex to produce the bactericidal compound hypochlorite. Ciprofloxacin and other bactericidal antibiotics have also been shown to increase oxidative stress in bacterial cells by increasing the production of reactive oxygen species [e (H2O2) . S. aure species .2O2 was added to log-phase cultures at concentrations ranging from 0 to 1 mM. To counteract peroxide degradation, fresh H2O2 was added at four intervals until the culture reached an OD of 1.0. S. aureus growth rates were minimally affected by the subinhibitory H2O2 concentrations that were used in our studies  of erythromycin  [Iron is an essential nutrient for both human and microbes and serves as a cofactor in many biological processes. The host tightly regulates iron distribution within the body, and this serves as an innate immune mechanism against invading microbes. Iron in humans is sequestered intracellularly, complexed within hemoglobin inside erythrocytes. Free iron is tightly bound by transferrin, lactoferrin, and haptoglobin ,61. Bactrculosis ,63 was erculosis . To inveyl (DIP) , at concS. aureus EV yield was enhanced in a dose-dependent fashion with increased concentrations of DIP in the medium , and LAC ΔΔΔΔΔ [S. aureus strains were cultivated with shaking in TSB to an OD of 1.2. The culture supernatants were filtered and concentrated 25-fold with a 100-kDa tangential flow filtration system . EVs were pelleted from the retentate by ultracentrifugation at 150,000× g for 3 h at 4 °C. To remove non-membranous proteins, protein aggregates, and other contaminants, EV samples were overlaid by gradient layers of Optiprep medium ranging from 40% to 15%. After centrifugation for 16 h, aliquots of 1 mL fractions were subjected to SDS-PAGE and silver stained. Fractions enriched for EVs were pooled and concentrated by diafiltration with phosphate buffered saline (PBS). Purified EVs were filtered again and stored at −80 °C. Proteomic analyses of EVs by liquid chromatography–tandem mass spectrometry (LC–MS/MS) were reported previously [EVs were purified from AC ΔΔΔΔΔ  as descrAC ΔΔΔΔΔ . Brieflyeviously ,27.S. aureus EVs were visualized by TEM as described [g. The pelleted cells were fixed in 4% paraformaldehyde for 2 h at room temperature, and then placed in PBS containing 0.2 M glycine to quench free aldehyde groups. The fixed cell pellets were cryoprotected by incubating with PBS containing 2.3 M sucrose, frozen in liquid nitrogen, and sectioned on an ultramicrotome at −120 °C. The sections were transferred to formvar/carbon coated copper grids and stained with antibodies to S. aureus EVs, followed by a protein A-gold (5 nm) conjugate. The grids were counterstained and embedded by incubation with 0.3% uranyl acetate in 2% methyl cellulose. The samples were imaged on a JEOL1200EX electron microscope  equipped with an AMT 2k CCD camera .Purified escribed . For immTHP-1 cells  were maintained and differentiated as described . The hum545 nm of the supernatant with an ELISA reader. Differentiated THP-1 macrophages grown in 48-well plates were incubated with EVs or lysis buffer for 4 h at 37 °C. Cytotoxicity was assessed by measuring the LDH level in culture supernatants using an LDH cytotoxicity assay kit.The relative cytotoxicity of EVs (1 to 20 µg/mL) toward different cell types was determined. A549 lung epithelial cells were incubated overnight in a 96-well plate at 37 °C with EVs. Cytotoxicity was assessed by measuring the LDH in culture supernatants with an LDH cytotoxicity assay kit . Differentiated HL60 cells or human neutrophils were seeded in 96-well plates and treated with EVs, 1% Triton X-100 lysis buffer, or LukSF-PVL  for 4 h at 37 °C. Cytotoxicity was measured with a CellTiter kit . A 2% rabbit erythrocyte suspension was mixed with EVs or the positive control Hla  in a 96-well plate for 1 h at 37 °C. The erythrocytes were pelleted by centrifugation, and cytotoxicity was determined by measuring the ODS. aureus JE2 was used to inoculate 100 mL of prewarmed  TSB medium. The cultures were cultivated with shaking (200 rpm), and bacterial growth curves were generated by recording optical density (OD650 nm) readings until the post exponential phase of growth (OD650 1.2). Culture supernatants were collected by centrifugation  at 4 °C and filter sterilized with a 0.45 µm filter. After the supernatant was concentrated 4-fold with a 100-kDa tangential flow filtration system , the retentate was filtered again and centrifuged at 150,000× g for 3 h at 4 °C to pellet the vesicles. The EVs were resuspended in 250 µl sterile PBS and sterilized by passage through a 0.45 µm filter. EV protein concentrations, determined with a BioRad protein assay, were expressed relative to EVs harvested from 37 °C cultures. Immunoblot analyses on each sample were performed as described below.To assess the effects of growth temperature on EV production, 100 µl of an overnight culture of 650 of 1.2. Bacterial growth curves were generated with each culture to assess relative growth rates of treated and untreated samples. Culture supernatants were collected, concentrated by tangential flow filtration, and EVs were isolated as described above.To measure the effects of a variety of stressors on EV production, we inoculated 100 mL TSB supplemented with or without 1% ethanol, 1 or 2% NaCl, 0.2, 0.3, or 0.4 mM 2,2-dipyridyl, 1, 2.5, or 5 µg/mL ciprofloxacin, or 0.05 µg/mL erythromycin as described above. The cultures were incubated with shaking (200 rpm) at 37 °C to an ODS. aureus cultures treated with H2O2 were inoculated as described above and cultivated at 37 °C until early log phase (OD650 ~0.2). Bacterial cells were pelleted  at 4 °C and resuspended in an equal volume of fresh 37 °C TSB medium supplemented with final concentrations  of H2O2. Fresh H2O2 was added to each culture at OD readings of 0.4, 0.6, 0.8 to counteract H2O2 degradation. Cultures were grown to an OD650 of 1 before collecting the culture supernatants for EV isolation and analyses.S. aureus EVs. Alternative the membranes were incubated with 1 µg/mL of Hla mAb 6C12, rabbit anti-LukF-PV, or LTA mAb . After washes with PBST, the membranes were incubated at RT for 1 h with either HRP-conjugated goat anti-rabbit antibody or HRP-conjugated goat anti-mouse antibody . The membranes were washed with PBST, developed with a chemiluminescent HRP substrate , and imaged with an iBright FL1500 instrument . Signal intensity of dot blot mages  were quantified by Image J . The chemiluminescence of treated samples was compared relative to untreated samples for each stress condition at the optimal EV dilution.EVs from stressed culture conditions were isolated as described above; 100 µL of serial twofold dilutions of the EV samples were applied to nitrocellulose membranes using a 96-well Bio-dot apparatus . To block the staphylococcal IgG binding proteins Spa and Sbi, the membranes were blocked for 1 h at room temperature with either rabbit or guinea pig IgG (2 µg/mL) in PBS + 5% skim milk. The membranes were washed with PBST (PBS + 0.05% Tween-20) and incubated overnight at 4 °C with one of four primary antibodies. These included sera (diluted 1:1000 in PBS + 0.1% Tween-20 and 5% skim milk) collected in a previous study  wherein t-test. A p value of ≤0.05 was considered significant.Statistical analyses were performed using Prism 7.0 . Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparison test. Samples treated with or without ethanol or Em were analyzed by an unpaired Student


Chronic kidney disease (CKD) is the known cause of morbidity and mortality among diabetes mellitus (DM) patients. Targeted screening of renal impairment based on estimated glomerular filtration rate (eGFR) among DM patients has potential benefits in early identification and treatment of CKD. Hence, this study was aimed to estimate the magnitude of renal impairment using eGFR among type 2 DM patients.An institution-based cross-sectional study was conducted from February-1 to April 30/2020 among 422 type 2 DM patients in Northeast Ethiopia. Data were collected by the semi-structured questioner and serum creatinine measurement. The collected data were edited into Epi-data manager version 4.4.1.0, and the analysis was performed by SPSS-25. The Simplified Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology (CKD-EPI), and Cockcroft-Gault (C-G) equations were used to calculate eGFR.2, according to the MDRD, CKD-EPI, and C-G equations, respectively. Female sex, , duration ≥ 10 years , age ˃60 years , hypertension , poor glycemic control , and body mass index  were significantly associated with renal impairment.Of all study participants, 82(19.4%), 92(21.8%), and 103(24.4%) had eGFR < 60 ml/min/1.73 mRenal impairment was prevalent among type 2 DM patients. Older age, female sex, duration, hypertension, poor glycemic control, and BMI were significantly associated with renal impairment. Chronic kidney disease (CKD) is a known public health problem worldwide with an estimated prevalence of 8–16% [The prevalence of CKD is growing rapidly , 2 princDue to the global epidemic of type 2 DM with disproportionate effects in the developing countries, the rate of diabetic kidney disease is growing rapidly in these countries , 2, 10. In Ethiopia, the prevalence of DM is growing alarmingly. In parallel, diabetes-related complications are rising. Additionally, in Ethiopia, renal complications of diabetes may go undetected due to limited diagnostic resources and late presentation of cases , 19.Since diabetic kidney disease constitutes a major cause of chronic kidney disease, routine screening of renal impairment based on glomerular filtration rate among DM patients is therefore extremely important, as detecting chronic kidney disease during its initial stages provides the opportunity for early therapeutic interventions to slow its progression and to improve outcomes , 21.Using serum creatinine alone is unreliable because its level is dependent on a number of factors that have nothing to do with a patient’s renal function. Instead an estimated glomerular filtration rate (eGFR) derived from prediction equations has been suggested as an important measure for assessing renal function , 23.Increasing evidence suggests that reduced eGFR is a strong predictor of renal morbidity and mortality in DM patients , 25. FurThus, the finding of this study is helpful to establish the clinical relevance of providing eGFR for routine screening of renal impairment. The findings will also help the clinicians to appreciate the magnitude of renal impairment using eGFR among type 2 DM patients. It is also hoped that the study will provide an impetus for further research on renal complications in type 2 DM patients.Hence, this study was designed to determine the magnitude of renal impairment among type 2 DM patients attending at public hospital in Northeast Ethiopia, based on eGFR derived from the Modification of Diet in Renal Disease (MDRD), Cockcroft-Gault (C-G), and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations.A Hospital-based cross-sectional study was conducted among type 2 DM patients from February-1 to April 30/2020. The study was undertaken among 422 type 2 DM patients who were registered at the follow-up clinic in Debre Berhan referral hospital, Northeast of Ethiopia.All adults type 2 DM patients (≥ 18 years) who have a follow-up in the diabetic clinic in the study period were considered in the study, whereas patients diagnosed as having renal disease before a diagnosis of DM and diagnosis of a recent attack of acute kidney disease by medical experts, critically ill patients, dialysis patients, and under medications affecting kidney function were excluded from the study.a/2= 1.96), and 5% margin of error. Then the minimum sample size obtained was 384. After adding a 10% non-response rate, a total of 422 type 2 DM patients were included in the study. A systematic random sampling technique based on participants’ order of flow (every third patient) was used for selecting the study participants. The required sample size of the study was estimated using a single population proportion formula, by considering: P = 50% , confidence interval (CI) of 95% , and blood pressure were measured by nurses working in the clinic.2) as (kg/m2) and classified as: Normal weight (18–24.9 kg/m2), overweight (25–29.9 kg/m2), and obesity (≥ 30 kg/m2) [After calibrating the equipment’s and with patients being bare-footed and wearing light clothes, height and weight were measured using a stadiometer, and an electronic weight scale, respectively. Body mass index (BMI) was computed as weight (kg) divided by the square of the height (m0 kg/m2) . WC was 0 kg/m2) , 28. AlBlood pressure was measured using an aneroid sphygmomanometer with an adult cuff size after 5 minutes of rest in sitting position in the right upper arm while placing the hand at the level of the heart. An average of three measurements with five minutes intervals between measurements was recorded. Participants were considered to have hypertension if systolic blood pressure (SBP) ≥ 130 mmHg and/or diastolic blood pressure (DBP) ≥ 80 or if they were taking any antihypertensive drugs .Current smoker was defined as smoking one or more cigarettes per day in the last 3 months; former smoker: a subject who had stopped smoking before 3 months; nonsmoker: those had no habit of smoking .Concerning glycemic control, four consecutive fasting blood sugar (FBS) measurements  were taken from the patient’s medical records for calculating the mean FBS level. Then participants were classified as good glycemic control (average of four consecutive FBS were 80–130 mg/dl) and poor glycemic control (average of four consecutive FBS > 130 mg/dl mg/dl or < 80 mg/dl mg/dl ) .Three milliliters of venous blood samples were collected with an aseptic venipuncture technique for serum creatinine analysis. After separating the serum, serum creatinine analysis was automated on ECHO XPC automatic chemistry analyzer . All the participants were advised to have the other two serum creatinine checked-up within the next two months after the first check-up with keeping a 1-month interval with each check-up. Then three measurements with a 1-month interval were taken and the mean values of three measurements were recorded for analysis. Normal serum creatinine values were ≤ 1.5 mg/dl for males and ≤ 1.3 mg/dl for females .−1.154 × age(years)−0.203 × 0.742  × 1.210 (as our population are Africans), C-G equation as: eGFR=(140 - age) x body weight (kg) / 72 x SCr x 0.85 for female [α x max −1.209 x 0.993age x 1.018  x 1.159 (as our participants black), where Scr is serum creatinine in mg/dL, κ is 0.7 for females and 0.9 for males, α is − 0.329 for females and − 0.411 for males, min indicates the minimum of Scr/κ or 1, and max indicates the maximum of Scr/κ or 1 [2), mild, moderate, and severe renal impairment if eGFR was 60–89.9, 30–59.9, and, 15–29.9 ml/min/1.73 m2, respectively. For this study, renal impairment was considered as eGFR < 60 ml/min/1.73 m2.The estimated Glomerular Filtration Rate (eGFR) was estimated using the MDRD equation  as: eGFRr female , and Chrr/κ or 1 . Renal sBefore entry, data were checked, cleaned, coded, and sorted, for any lost values. Then data were edited into Epi-data manager version 4.4.1.0 and exported to statistical software for social sciences (SPSS) version 25 software. The data were also checked for its distribution and outliers before analysis. Then the data were processed by using descriptive analysis, including frequency tables, cross-tabulation, and summary measures. Categorical variables were stated as number (percentage) whereas the continuous data as means ± standard deviation (SD).To determine the independently associated variables, associations were investigated using binary logistic regression analysis. All independent variables with p-value < 0.25 in the unadjusted model of logistic regression analysis were included in the multivariate model (backward stepwise) to control the effect of confounding variables and to identify independently associated factors with renal impairment in the final model. The degree of associations was expressed by using the odds ratio (ORs) with 95% CI. P-value with < 0.05 was considered statistically significant.A total of 422 type 2 DM patients were invited and all of them were volunteered to participate in the study making the response rate of 100%. From the total of the study participants, 229 (54.3%) were female, and 260 (61.6%) were from urban areas. Their ages were ranged from 36 to 80 years old, with a mean (± SD) of 54.16 (± 10.61) years, with 302 (71.6%) of them aged below 60 years old. Nearly three fourth (73.7%) of the respondents were married, 206 (48.8%) of respondents attained high school and above, and 141 (33.4%) of respondents were government employees (Table 2 with 288 (68.2%) were classified as normal/underweight. About 91 (21.6%) of the study participants also had higher waist circumference for the gender-specific normal range.The mean (± SD) of DM duration was 6.2 (± 4.6) years with a minimum and maximum of 1 year to 24 years, respectively. Regarding anthropometric measurements, the mean (± SD) of BMI of the participants was 23.28 (± 3.64) Kg/mThe majority (82.9%) of the participants had no family history of kidney disease (FH-KD), 371 (87.9%) never smoked, and 396 (93.8%) had no history of cardiovascular disease. Current alcohol consumption habit was observed in 72 (17.1%) of participants. Regarding the treatment modality of the participants, nearly three fourth (73.9%) were taking oral hypoglycemic agent(s) only, while 110 (26.1%) were on a combination of insulin and oral hypoglycemic agents.The mean systolic and diastolic blood pressures of participants were 133.69 (± 14.48) and 82.9 (± 11.10) mmHg, respectively. Nearly two fifths (39.1%) of participants were hypertensive with 140 (84.8%) of them were on antihypertensive treatments. More than three fifths (66.6%) of patients were presented with the level of poor glycemic control. Mean SCr (± SD) of the participants was 0.96 (± 0.32) mg/dl, and the majority of participants (88.9%) were with normal serum creatinine levels (Table 2) values of the participants estimated according to the MDRD, CKD-EPI, and C-G equations were 99.66 (± 34.24), 88.78 (± 22.32), and 84.87 (± 29.18) ml/min/1.73 m2, respectively.The mean eGFR , 21.8% (95%CI = 17.9% − 25.7%), and 24.4% (95%CI = 20.3% − 28.5%), using the MDRD, CKD-EPI, and C-G equations, respectively. From those with renal impairment, 43.9%, 51.1%, and 57.3% of participants had normal serum creatinine level for MDRD, CKD-EPI, and C-G equation, respectively.In this study, the prevalence of renal impairment, defined as the presence of GFR < 60 ml/min/1.73 m2), while 113 (26.8%), 114 (27.0%), and 124 (29.4%) had eGFR of 60–89.9 mL/min/1.73 m2 in MDRD, CKD-EPI, and C-G equation, respectively. None of the participants had eGFR < 30 mL/min/1.73 m2 in all the three equations , 216  of the participants had eGFR of  when the MDRD formula was used to assess renal function. When CKD-EPI equation was used age ˃60 years , female sex , ≥ 10 years duration of DM , presence of hypertension , and increased BMI  were significantly associated with the presence of renal impairment.In multivariable analysis, older age , female sex , long duration of DM , poor glycemic control , hypertension  and increased BMI  were significantly associated with the presence renal impairment , female sex , ≥ 10 years duration of DM , poor glycemic control , and hypertension  were significantly associated with the presence of renal impairment  was comparable with the study conducted in Australia (23.1%) , and UniWhen the renal function was assessed using the C-G equation, the magnitude of renal impairment (24.3%) was in line with the study in Tanzania (24.7%) , Thailan2. Therefore, without measuring eGFR the clinician may not be informed of the existence of renal impairment and be wrongly reassured that renal function is well.Indeed, the SCr measurement is the most common method for assessing kidney function, use of creatinine alone can show the function of the kidney in many diabetic patients falsely normal , 46. In In this study, we found that using the C-G equation estimate a higher magnitude of renal impairment (24.3%) than the MDRD and CKD-EPI equations, which estimate19.4% and 21.8%, respectively. C-G equation does not incorporate race, which may influence creatinine excretion  might beIn this study, gender difference has a significant association with renal impairments when renal function was assessed by the three equations in which the female gender was reported to be a non-avoidable risk factor for renal impairment. This is in agreement with the findings in Sweden   and UK  and activation of their receptor, thereby generation of reactive oxygen species (ROS) and a chronic subacute inflammatory process which play a pivotal role in the development of chronic kidney disease. Also, activation of AGEs receptors’ induces glomerular matrix production and increases oxidative stress, which promotes epithelial-mesenchymal transdifferentiation of renal tubular cells, thus contributing to interstitial fibrosis and renal histological change .It was also likely that the difference in the prevalence of renal impairments might result from differences in the duration of DM using the MDRD, CKD-EPI, or C-G equation. Compared to respondents with a short duration of DM, respondents with a long duration had greater odds for the occurrence of renal impairments. This corresponds with the findings of several studies that reported that the possibility of developing renal impairment was greater among patients with longer duration of DM , 30.In this study, BMI has a significant association with renal impairments when renal function was assessed by both MDRD and CKD-EPI equation. The results of this study were in agreement with various studies , 38 whicAnatomical and physiological changes related to obesity including an increase in glomerular size, increased mesangial matrix and mesangial cell proliferation, hypertrophy and effacement of podocytes, and increase growth factors and adipokine alterations which may lead to fibrosis were also another possible mechanism for renal impairments in obese individuals .Unlike a study conducted in Sweden  in whichThe strength of this study is that it is one of the few studies with including the three equations for estimating GFR from serum creatinine level.The study has its limitations that need to be considered. The uses of eGFR rather than direct measured GFR for assessing renal impairment, use of FBS instead of HbA1c for assessing the level of glycemic status, and due to selection bias during selecting participants there may be reduction in the external validity. Due to limited resources, the non-diabetic renal diseases of course may also coexist the study participants were not excluded and the equation used for estimating GFR was not validated among adult populations of Ethiopian origin were also the limitation of the study.The prevalence of renal impairment was 19.4%, 21.8%, and 24.3% when using MDRD, CKD-EPI, and C-G equations, respectively but only 11.1% would have been classified as having renal impairment if SCr used alone for assessing kidney status. Older age, female sex, longer duration of DM, hypertensive, increased BMI, and poor glycemic control were significantly associated with renal impairment. Physicians should not depend on serum creatinine only as a measure of kidney function instead use the serum creatinine to estimate eGFR and assessing of renal impairment. Thus, early screening based on eGFR would allow more aggressive measures to be taken to prevent further progression and adverse outcomes.


Escherichia coli, followed by purification. Both proteins exhibited effective anti-vesicular stomatitis virus (VSV) activity in Vero, F81 (feline kidney cell), Madin–Darby bovine kidney (MDBK), Madin–Darby canine kidney (MDCK), and porcine kidney (PK-15) cells, showing broader cross-species antiviral activity than the INTERCAT IFN antiviral drug. Furthermore, the recombinant feIFN-ωa and feIFN-ωb proteins demonstrated antiviral activity against VSV, feline coronavirus (FCoV), canine parvovirus (CPV), bovine viral diarrhea virus (BVDV), and porcine epidemic diarrhea virus (PEDV), indicating better broad-spectrum antiviral activity than the INTERCAT IFN. The two novel feIFN-ω proteins (feIFN-ωa and feIFN-ωb) described in this study show promising potential to serve as effective therapeutic agents for treating viral infections in pet cats.Cats are becoming more popular as household companions and pets, forming close relationships with humans. Although feline viral diseases can pose serious health hazards to pet cats, commercialized preventative vaccines are lacking. Interferons (IFNs), especially type I IFNs ), have been explored as effective therapeutic drugs against viral diseases in cats. Nevertheless, there is limited knowledge regarding feline IFN-ω (feIFN-ω), compared to IFN-α and IFN-β. In this study, we cloned the genes encoding feIFN-ωa and feIFN-ωb from cat spleen lymphocytes. Homology and phylogenetic tree analysis revealed that these two genes belonged to new subtypes of feIFN-ω. The recombinant feIFN-ωa and feIFN-ωb proteins were expressed in their soluble forms in  As cats continue to become more popular as household companions and pets , a varieInterferon omega (IFN-ω), which is produced primarily by leukocytes, displays similar structure and physicochemical characteristics to type I IFNs, and researchers have focused on developing therapeutic drugs based on feline IFN-ω (feIFN-ω) to treat viral infections in cats ,12. FeLVEscherichia coli. The molecular characteristics of feIFN-ωa and feIFN-ωb were analyzed, and the species-specific and broad-spectrum antiviral activities of both were evaluated in vitro. Our results suggest that the feIFN-ωa and feIFN-ωb proteins described in this study show promising potential to serve as effective therapeutic agents against feline viral infections.In this study, two novel genes encoding feIFN-ω, called feIFN-ωa and feIFN-ωb, were cloned from spleen lymphocytes of cats stimulated by VSV infection combined with poly(I:C) treatment. These genes were then expressed in their soluble forms in 2. Feline coronavirus (FCoV) was propagated in F81 cells, porcine epidemic diarrhea virus (PEDV) was propagated in Vero cells, VSV was propagated in Vero cells, bovine viral diarrhea virus (BVDV) was propagated in MDBK cells, and canine parvovirus (CPV) was propagated in MDCK cells.The plasmid pMD19-T (simple) and the prokaryotic soluble expression plasmid pCold-TF were used in this study. African green monkey kidney (Vero) cells (ATCC CCL-81), Madin–Darby bovine kidney (MDBK) cells, porcine kidney (PK-15) cells, Madin–Darby canine kidney (MDCK) cells, and feline kidney (F81) cells were kept in our laboratory and maintained in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum  at 37 °C with 5% COn = 5) were purchased from a pet market in Harbin, China. Animal experiments were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol (2018NEAU052x) was approved by the Ethical Committee for Animal Experiments at Northeast Agricultural University, Harbin, China (20 July 2018).Healthy Dragon Li cats  until the cytopathic effect (CPE) was observed to exceed 90%, and then a freeze–thaw was performed. The supernatants were harvested and stored at −80 °C until required. The cats were intramuscularly injected daily with 2 mL of 100 TCID50 VSV combined with 500 μL of 1.0 mg/mL poly(I:C) for three consecutive days. On day 15 post-infection, the cats were euthanized, and the splenic lymphocytes were isolated aseptically and used for total RNA extraction with the TRIzol Total RNA Isolation kit  according to the manufacturer’s instructions. Subsequently, first-strand cDNA was reverse transcribed using a QuantiTect Reverse Transcription kit  according to the manufacturer’s instructions. The genes encoding feline IFN-ω were obtained by PCR amplification with the degenerate primer pair of F: 5′-GGTACCATGGCCCTCCTGCTCCCTCTRCT-3′ (containing a Kpn I restriction site) and R: 5′-GGATCCTCAYGATKACGCSSGRTCTCCAT-3′ (containing a BamH I restriction site), which were designed according to the gene sequences of known feIFN-ω published in the GenBank . The genes were then subcloned into the pMD19-T (simple) plasmid and further subjected to sequencing and bioinformatics analyses.Confluent monolayers of F81 cells were infected with VSV at an MOI of 0.1 for 1 h at 37 °C in 5% COhttp://www.cbs.dtu.dk/services/SignalP-3.0/; phosphorylation sites were analyzed by the NetPhos3.1 server at http://www.cbs.dtu.dk/services/NetPhos/; N-glycosylation sites were analyzed by the NetNGlyc1.0 server at http://www.cbs.dtu.dk/services/NetNGlyc/; O-glycosylation sites were analyzed by the YinOYang1.2 server at http://www.cbs.dtu.dk/services/YinOYang/; subcellular localization was analyzed by the TargetP 1.1 server at http://www.cbs.dtu.dk/services/TargetP; transmembrane regions were analyzed by the TMHMM 2.0 server at http://www.cbs.dtu.dk/services/TMHMM/; antigen epitopes and hydrophobicity were analyzed by the BepiPred 1.0 server at http://www.cbs.dtu.dk/services/BepiPred-1.0/; and secondary and three-dimensional structures were predicted by SOPMA at https://npsa-prabi.ibcp.fr/cgi-bin/npsa.Homology and phylogenetic tree analysis of the feline IFN-ω genes isolated in this study were analyzed using DNASTAR and MEGA7 software. In addition, the characteristics of the feIFN-ω genes and proteins were analyzed by several online bioinformatics software programs: signal peptide cleavage sites were analyzed by the SignalP 3.0 server at E. coli BL21 (DE3) competent cells and validated by PCR and sequencing analyses, thus generating the recombinant E. coli strain pCold-feIFN-ω/BL21. We then used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis to determine the optimal conditions for expression of the recombinant feIFN-ω in pCold-feIFN-ω/BL21 cells via induction by isopropyl β-D-thiogalactoside . Briefly, for optimization of the IPTG concentration, the recombinant E. coli strain was grown in Luria–Bertani broth  supplemented with 100 μg/mL ampicillin at 37 °C until the optical density at 600 nm was approximately 0.5. Then, IPTG was added at final concentrations of 0.4 mmol/L, 0.6 mmol/L, 0.8 mmol/L, 1.0 mmol/L, or 1.2 mmol/L, and the cultures were continually cultivated for another 6 h. After centrifugation at 12,000× g for 10 min, the cell pellets were lysed and analyzed by 12% SDS-PAGE. To determine the optimal induction time, the recombinant strain was induced by the optimized final concentration of IPTG for 4 h, 6 h, 8 h, 10 h, and 12 h. Following centrifugation and cells lysis, the proteins were again analyzed by 12% SDS-PAGE.In this study, the genes encoding feIFN-ω that were isolated by RT-PCR were subcloned as a KpnI and BamHI-generated  gene fragment into the prokaryotic soluble expression plasmid pCold-TF, giving rise to recombinant pCold-feIFN-ω. After that, the recombinant plasmid was transformed into 2+ affinity chromatography columns according to the manufacturer’s instructions, followed by confirmation analysis using SDS-PAGE. The purified feIFN-ω protein was stored at −80 °C until use. VSV was used as a virus model to evaluate the antiviral activity of the recombinant feIFN-ω via the in vitro microdose cytopathic effect inhibition assay (MCIA) according to the method described previously [2 for 18 h. F81 cells without IFN treatment were used as a control group. After incubation, VSV (MOI = 1) was prepared as described above, added into the 96-well plate, and incubated for 8-12 h until the CPE of the cells in the viral control group reached 100%. Next, the culture medium was removed, and the cells were stained with 0.2% crystal violet in 20% ethanol at 37 °C for 30 min. The cells were then destained with 0.1% acetic acid in 50% ethanol at 37 °C for 5 min before determining the absorbance of each well at 595 nm. Each sample was performed with eight biological replicates and three technical replicates. The antiviral activity of the IFN was calculated as the method previously described, which is expressed as units per milligram according to the ratio of IFN titer and protein concentration [The fusion protein expressed by the pCold-feIFN-ω/BL21 bacteria cells was subjected to cleavage by the 3C protease . The target recombinant feIFN-ω protein was then purified using Nieviously ,26 with ntration . In paran = 3) for three independent experiments. Statistical analyses were performed using GraphPad Prism V5.0 software. Tukey’s multiple comparison tests and one-way analysis of variance (ANOVA) tests were used to analyze the significance of the differences between groups: * p < 0.05; ** p < 0.01; and *** p < 0.001.The results are shown as mean ± SEM (We first collected splenic lymphocytes from cats infected with VSV and treated with poly(I:C) simultaneously. We then performed RT-PCR assays using a pair of degenerate primers to identify two genes encoding feIFN-ω, referred to as feIFN-ωa and feIFN-ωb a. These We constructed a phylogenetic tree comparing the feIFN-ωa/ωb genes identified in this study with other IFN gene sequences published in GenBank from different animals using the software DNASTAR  and MEGA7. The results showed that the feIFN-ωa/ωb genes belonged in the type I IFN family, but that their evolutionary relationship to the known feline IFN-ω genes already published in the GenBank  was distThe characteristics of the novel feline IFN-ω proteins (feIFN-ωa and feIFN-ωb) were analyzed using several online bioinformatics software programs, including the identification of potential signal peptide cleavage sites, N-glycosylation sites, O-glycosylation sites, phosphorylation sites, subcellular localization, and transmembrane regions. The results from these detailed analyses are displayed in E. coli BL21 competent cells, thus generating the recombinant protein expressing E. coli strains pCold-feIFN-ωa/BL21 and pCold-feIFN-ωb/BL21. After inducing their expression with IPTG, the rfeIFN-ωa  and VSV as the viral model that was propagated on F81 cells. As shown in We then tested whether the antiviral activities of rfeIFN-ωa and rfeIFN-ωb were species-specific. To address this question, we performed MCIAs with VSV propagated in several different cell lines from different animal species, including F81 cells (cat), Vero cells (monkey), MDCK cells (dog), MDBK cells (cattle), and PK-15 cells (pig). As shown in Next, we wanted to investigate whether the antiviral activities of the recombinant feIFN-ωa and feIFN-ωb proteins were also broad-spectrum. We performed MCIAs using a wide range of different viruses, including VSV, FCoV, CPV, BVDV, and PEDV. As shown in Currently, cats are continuing to become more and more common as household pets. We know of a wide variety of viral infections that can seriously endanger the health of pet cats, such as FeLV, FIV, FIPV, FCoV, and feline calicivirus (FCV). Interferon represents a promising potential therapeutic agent that can effectively treat pet cats infected with these viruses. In this study, we identified two new genes encoding feline IFN-ω (feIFN-ωa and feIFN-ωb) in the spleen lymphocytes of cats. Following sequence homology analysis, we found that feIFN-ωa and feIFN-ωb shared maximum nucleotide sequence homologies of 91.88% and 90.20%, and maximum amino acid homologies of 91.6% and 89.4% with the 13 previously published subtypes of feline IFN-ω, respectively. In addition, phylogenetic tree analysis of IFNs in cats and other species constructed using neighbor-joining analysis revealed that feIFN-ωa and feIFN-ωb do belong to the type I IFN family, but their evolutionary relationship to known feline IFN-ω genes was distant, indicating that feIFN-ωa and feIFN-ωb were indeed new subtypes of feline IFN-ω. We deposited these two genes into the GenBank with the accession numbers MK682680 (feIFN-ωa) and MK682681 (feIFN-ωb), thus enriching the IFN-ω data submitted to the GenBank .In this study, the characteristics of the feIFN-ωa and feIFN-ωb proteins, such as signal peptide sequences, signal peptide cleavage sites, phosphorylation sites, glycosylation sites, antigen epitopes, hydrophobicity, and transmembrane regions were analyzed using bioinformatics to provide better theoretical guidance for the functional study of these proteins. The mature proteins, with normal biological activity, were formed only after the signal peptide sequence was removed from the precursor protein, thus allowing them to be secreted outside the cell membrane ,29. We uE. coli expression systems are still the most widely used prokaryotic expression system for protein production with their characteristics of simple procedures, large-scale production, and low cost [E. coli expression systems in an insoluble inclusion body form that has not been modified or folded correctly, resulting in the loss of its biological activity [E. coli strains and induction by IPTG, the rfeIFN-ωa/ωb proteins were expressed in their soluble forms and analyzed via SDS-PAGE. We optimized expression conditions for the rfeIFN-ωa and rfeIFN-ωb proteins and purified them using His-tag Ni2+ affinity column chromatography. Both proteins showed antiviral activity against VSV in microdose cytopathic effect inhibition assays using F81 cells. Our results provide a basis for further studies into the development of rfeIFN-ωa and rfeIFN-ωb as therapeutic agents for viral infections. Meanwhile, our data provide evidence that the pCold-TF expression system can be used to produce IFN with full bioactivity.Insect/baculovirus expression systems are one of the most effective eukaryotic expression systems for preparing feline IFNs. These expression systems are capable of making post-translational modifications, such as glycosylation, which allows proteins to fold correctly, thus producing highly active and stable IFNs ,34,35,36low cost ,39,40. Gactivity ,42. To oactivity ,44. FollRecombinant feline interferon-ω (rfeIFN-ω) was the first licensed immunomodulator for use in treating viral infections in cats, especially FIV and FeLV infections ,18,45. FRecently, combination antiviral therapy has become a common practice in treating feline viral infections due to pharmacokinetics and the short half-life of IFN alone ,49. TherE. coli. Our data showed that purified recombinant feIFN-ωa and feIFN-ωb had broad-spectrum antiviral activity in homologous and heterologous animal cells, suggesting they are candidates for the development of effective therapeutic agents to be used against viral infections in pet cats. And, our research is underway to systematically evaluate the effectiveness of the two novel feIFNs as therapeutics agent for cat viral infections in vivo. In addition, the reported feIFN-ω sequences will enrich the IFN data submitted to GenBank.In this study, two new subtypes of feline IFN-ω (ωa and ωb) were identified and characterized. They shared a maximum nucleotide sequence homology of 91.88% and 90.20% and a maximum amino acid homology of 91.6% and 89.4% with the 13 previously known subtypes of feIFN-ω, respectively. We analyzed the characteristics of feIFN-ωa and feIFN-ωb in detail using bioinformatics followed by soluble expression and optimization of induction conditions in


Correction to: BMC Genet (2016) 17:144https://doi.org/10.1186/s12863-016-0455-5Following publication of the original article , it has This has led to a misinterpretation of the percentage of the genome that was sequenced. However, none of the remaining analyses were affected.Please find below the corrections to the text (organized per article PDF page number). The authors have also added a column with the 1C values in Additional file Page 1, Abstract. Where it reads: “A partial genome assembly, representing 24% of the total size...”, it should read: “A partial genome assembly, representing 48% of the total size...”Page 5 - Where it reads: “The homoploid genome size  was assessed through the formula”, it should read: “The holoploid genome size  was assessed through the formula”.Page 5 - Where it reads: “The obtained values were expressed in picograms (pg) and in giga base pairs (Gb), using the formula by [72] (1 pg = 0.978 Gb)”, it should read: “The obtained values were expressed in picograms (pg) and in giga base pairs (Gb), using the formula by [72] (1 pg = 0.978 Gb). The monoploid genome size (1Cx) was obtained by dividing the holoploid genome size by the ploidy level, in this case, diploid, and was also expressed in picograms and giga base pairs.”Philaenus spumarius and P. maghresignus estimates of genome size were 5.27 ± 0.25 pg (5.15 Gb) and 8.90 ± 0.20 pg (8.90 Gb), respectively. In P. spumarius, males and females differed significantly in genome size , with males presenting on average a lower genome size  than females  .”, it should read: “Philaenus spumarius and P. maghresignus estimates of monoploid genome size were 2.63 ± 0.13 pg (2.58 Gb) and 4.45 ± 0.10 pg (4.35 Gb), respectively. In P. spumarius, males and females differed significantly in monoploid genome size , with males presenting on average a lower genome size  than females  .”Page 9 - Where it reads: “P. spumarius genome.”, it should read: “In total, 1,218,749,078 bp were assembled which based on the total estimated monoploid genome size of 2.58 Gb, correspond to approximately 48% of the P. spumarius genome.”Page 10 - Where it reads: “In total, 1,218,749,078 bp were assembled which based on the total estimated genome size of 5.3 Gb, correspond to approximately 24% of the Page 12 - Where it reads: “... an insect species with a very large genome (5.3 Gb)...”, it should read: “... an insect species with a very large genome (2.58 Gb)...”Moreover, the authors would like to inform the readers that the raw reads of the RAD libraries used for association analyses are available in NCBI under the accession PRJNA572593.The authors apologize to the editor and readers for any inconvenience caused by this error.Furthermore, they would like to thank Roberto Biello (John Innes Centre) and Saskia Hogenhout (John Innes Centre) for having brought this matter to their attention.Additional file 2: Table S1-S13. Lists of colour-associated SNPs obtained for each pairwise comparison and association analyses; genic and genotypic differentiation tests; pairwise Fst estimates among dorsal colour phenotypes; SNP correlation value (r2) in linkage disequilibrium analyses; Genome size estimates; Assembly statistics for genome and transcriptome; and lists of blast results.


Brassica napus was revealed by transcriptomic and metabolomic analyses.The molecular mechanism underlying white petal color in Brassica napus L.) is one of the most important oilseed crops worldwide, but the mechanisms underlying flower color in this crop are known less. Here, we performed metabolomic and transcriptomic analyses of the yellow-flowered rapeseed cultivar ‘Zhongshuang 11’ (ZS11) and the white-flowered inbred line ‘White Petal’ (WP). The total carotenoid contents were 1.778-fold and 1.969-fold higher in ZS11 vs. WP petals at stages S2 and S4, respectively. Our findings suggest that white petal color in WP flowers is primarily due to decreased lutein and zeaxanthin contents. Transcriptome analysis revealed 10,116 differentially expressed genes with a fourfold or greater change in expression  in WP vs. ZS11 petals, including 1,209 genes that were differentially expressed at four different stages and 20 genes in the carotenoid metabolism pathway. BnNCED4b, encoding a protein involved in carotenoid degradation, was expressed at abnormally high levels in WP petals, suggesting it might play a key role in white petal formation. The results of qRT-PCR were consistent with the transcriptome data. The results of this study provide important insights into the molecular mechanisms of the carotenoid metabolic pathway in rapeseed petals, and the candidate genes identified in this study provide a resource for the creation of new B. napus germplasms with different petal colors.Rapeseed (The online version contains supplementary material available at 10.1007/s00425-020-03536-6. Carotenoids, among the most important pigments in plants, are composed of various combinations of isoprene units, including several oxygen-free carotenes and oxygen-containing xanthophylls      (Cunningham and Gantt The carotenoid biosynthetic pathway has been well characterized in many plant species (Cunningham and Gantt MYB305 by RNAi resulted in reduced β-carotene levels (Liu and Thornburg RCP1 (R2R3-MYB) led to the downregulation of all carotenoid biosynthetic genes and reduced carotenoid contents in RNAi Mimulus lewisii flowers  RNAi tomato fruits  produced light-green unripened fruits and yellowing ripened fruits with reduced levels of chlorophyll and carotenoids, respectively, in their pericarps  individuals. Furthermore, total carotenoid contents in rapeseed sprouts are affected by light treatment, and differences in flower color were ascribed to higher carotenoid contents in yellow-flowered cultivars compared to white-flowered cultivars  and S4  in WP and in petals at the corresponding stages in ZS11. We identified the components of the carotenoid biosynthetic pathway in the four pools of petals via multiple reaction monitoring (MRM) using the absolute quantification method. We also analyzed the transcriptomes of petals at the B5 (5 mm long buds), B7 (7 mm long buds), S1 , and S3  stages in WP and the B5, B8 (8 mm long buds), S1, and S3 stages in ZS11 and measured the expression of the carotenoid biosynthetic genes by transcriptomic analysis and quantitative reverse-transcription PCR (qRT-PCR). Finally, we systematically analyzed the regulatory genes of the carotenoid biosynthetic pathway. The results of this study reveal comprehensive changes in the metabolomic and transcriptomic networks underlying the formation of white and yellow petals in rapeseed at different developmental stages, providing important insights into the molecular mechanisms of the carotenoid biosynthetic pathway. Moreover, the candidate genes identified in this study will provide a resource for the creation of new flower colors germplasms in B. alboglabra, and was selected from progenies of the advanced backcross population with the white-flowered individual as the donor parent and the normal yellow-flowered rapeseed as the recurrent parent. The common yellow-flowered rapeseed cultivar ‘ZS11’ and the white-flowered inbred line ‘WP’ were cultivated in the experimental station at the Rapeseed Engineering Research Center of Southwest University in Beibei , Chongqing city, China. White and yellow petals at different stages were collected for metabolomics analysis in March 2018, and samples for RNA-sequencing (RNA-Seq) and qRT-PCR validation were collected in March 2019. Petals from flowers at the bud phase and the flowering phase were sampled from both lines, including the following: petals from 5 mm long WP buds (WB5P); petals from 5 mm long ZS11 buds (YB5P); petals from 7 mm long (the maximum length before flowering) WP buds (WB7P); petals from 8 mm long (the maximum length before flowering) ZS11 buds (YB8P); petals from open WP buds before the petals spread (WS1P); petals from ZS11 open buds before the petals spread (YS1P); petals from WP flowers when the petals were half spread (WS2P); petals from ZS11 flowers when the petals were half spread (YS2P); petals from WP flowers when the petals had just fully spread (WS3P); petals from ZS11 flowers when the petals had just fully spread (YS3P); petals from WP flowers two days after the petals had fully spread (WS4P); and petals from ZS11 flowers two days after the petals had fully spread (YS4P). The ZS11 petals at stages S1-S4 are shown in Fig. ‘WP’ is a stable white-flowered inbred line derived from a distant hybridization between the normal yellow-flowered rapeseed and the white-flowered WS2P, WS4P, YS2P, and YS4P petal samples were freeze-dried and ground into a fine powder using a mixer mill  with zirconia beads for 1 min at 30 Hz. Each sample (100 mg) was weighed and extracted with n-hexane:acetone:ethanol  with 0.01% butylated hydroxytoluene (BHT) (g/mL) at room temperature. The sample was vortexed for 30 s, subjected to ultrasound-assisted extraction for 20 min at room temperature, and centrifuged for 5 min at 10,000 g at room temperature. After repeating these steps, the two supernatants were collected and evaporated to dryness under a nitrogen gas stream. The residue was dissolved in 200 μL mixing solution , vortexed for 30 s, subjected to ultrasound-assisted extraction for 2 min, and centrifuged for 5 min at 10,000 g. The supernatants were absorbed and filtered (0.22 µm pore size) prior to LC–MS analysis.www.shimadzu.com.cn/) equipped with a C30 column . HPLC analysis was performed using a binary solvent system comprising acetonitrile:methanol  (0.01% BHT) as mobile phase A and methyl tert-butyl ether (0.01% BHT) as mobile phase B. The A:B (v/v) gradient program was 85:15 at 0 min, 75:25 at 2.0 min, 40:60 at 2.5 min, 5:95 at 3.0 min, 5:95 at 4.0 min, 85:15 at 4.1 min, 85:15 at 6.0 min. The flow rate was kept at 0.8 mL/min, and the column temperature was maintained at 28 °C. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (Q TRAP)-MS system. Linear ion trap (LIT) and triple quadrupole (QQQ) scans were acquired on an API 6500 Q TRAP LC/MS/MS system equipped with an APCI Turbo Ion-Spray interface operating in negative ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The APCI source operation parameters were as follows: ion source, turbo spray; source temperature, 350 °C; ion spray voltage (IS) voltage, 3.5 kV; the ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively; and the collision gas (CAD) was set at medium. DP and CE for individual MRM transitions were performed with further DP and CE optimization. A specific set of MRM transitions was monitored for each period according to the plant hormones eluted within this period. The MRM for each line was performed in triplicate. Three spears were used for each repeat.Each 5 μL supernatant sample was injected into a HPLC system  listed on the abscissa and peak areas listed on the ordinate. The peak areas of absorbance (carotenoids) in all samples were calculated based on the linear equation of the standard curve, and the absolute carotenoid contents in test samples were calculated using the formula: Carotenoid content in samples (μg/g) = B*C/1000/D, where B is the area of the integrated value of the absorbance peak in the sample substituted for the concentration from the standard curve (μg/mL); C is the volume of solution (μL); and D is the sample quantity (g). The carotenoid metabolome was analyzed by Metware Biotechnology Co., Ltd. .https://bigd.big.ac.cn/gsa.Total RNA was extracted from WB5P, YB5P, WB7P, YB8P, WS1P, YS1P, WS3P, and YS3P petal samples using an EZ-10 Total RNA Mini-Preps Kit ; three biological repeats were prepared for each sample. Sequencing library construction and transcriptome sequencing were performed by Novogene Bioinformatics Technology Co., Ltd. . The mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, and the library quality was assessed using the Agilent Bioanalyzer 2100 system. After cluster generation, the library preparations were sequenced on an Illumina HiSeq™ 4000 platform, and 150 bp paired-end reads were generated. Raw data (raw reads) in Fastq format were processed by removing reads containing adapters, reads containing poly-N, and low-quality reads, and the downstream analyses were performed on the remaining high-quality data. The raw sequencing data reported in this study have been deposited in the Genome Sequence Archive  . Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs were performed using the OmicShare tools, a free online platform for data analysis (https://www.omicshare.com/tools). GO terms and KEGG pathways with P-value < 0.05 were considered to be significantly enriched by the DEGs, and the enrichment results were plotted using online software available on OmicShare.Differential gene expression analysis of two groups  was performed using the DESeq2 R package (1.16.1). DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting BnPSYc, BnPSYg, BnPDS1a, BnPDS1b, BnPDS1c, BnPDS1d, BnZ-ISOa, BnZ-ISOb, BnCrtISOa, BnCrtISOb, BnLYCEd, BnLYCBb, BnBCH1b, BnBCH1d, BnBCH2b, BnZEPa, BnVDEb, BnNCED3c, BnNCED4b, and BnNCED4c. The cDNAs were synthesized according to the manufacturer’s protocol of PrimeScript™ RT reagent Kit with gDNA Eraser   and used as templates for qRT-PCR analysis with primers designed based on the reference B. napus gene sequences using Primer Premier 5.0. PCR was conducted using SYBR Premix Ex Taq II  (TaKaRa) in a typical 10 μL PCR mixture that included 5 μL of SYBR Premix Ex Taq II, 1 μL (100 ng) of template cDNA, 3 μL ddH2O, and 0.5 μL of each PCR primer (10 μM). Cycling conditions were 95 °C for 2 min, followed by 40 cycles of 95 °C for 3 s (denaturation), and 60 °C for 30 s . The melting curve for each PCR amplicon was obtained under the following conditions: 95 °C for 5 s and 65 °C for 5 s, followed by a constant increase in temperature from 65 °C to 95 °C at an increment of 0.5 °C/cycle. Samples were run on the Bio-Rad CFX96 Real-Time System . Relative expression of the target genes was analyzed by the 2−ΔΔCt method using BnACTIN7  as an internal control ; in addition, the yellow colorimetric values (Δb) and total chromaticity values (ΔE) of WP and ZS11 petals were significantly different . These results indicate that the major difference between WP and ZS11 petals is the variation in yellow colorimetric values during petal development.There was no significant difference in flower morphology between ZS11 and WP, except for changes in petal color during development , whereas that in ZS11 petals decreased by only 5.564% (22.432 μg/g). These results indicate that the lighter color of WP petals is mainly due to lower carotenoid contents compared to ZS11 petals at the same stage. In addition, WP petals faded from pale yellow to white from S2 to S4 primarily due to a decrease in total lutein plus zeaxanthin contents.B. napus  are summarized in Supplementary Table S1. After removing adaptor sequences, ambiguous nucleotides, and low-quality sequences, 47.1 Gb and 44.3 Gb of original data were obtained in the WP and ZS11 libraries, and the Q20 (sequencing error rate < 1%) and Q30 (sequencing error rate < 0.1%) values were ≥ 99.87% and 93.05%, respectively. The G + C percentage was 46%. After filtering by Trimmomatic, 677,351,694 and 639,227,560 reads were obtained in the WP and ZS11 libraries, respectively, with Q30 values ≥ 97.51%. Using Hisat2 to compare these reads with the reference genome of We identified 3,169, 4,573, 4,843, and 5,068 DEGs in WB5P vs. YB5P, WB7P vs. YB8P, WS1P vs. YS1P, and WS3P vs. YS3P, respectively. Compared to ZS11 petals, 1,817, 2,184, 2,521, and 2,914 genes were upregulated in WP and 1,352, 2,389, 2,322, and 2,154 genes were downregulated in WP in the comparisons WB5P vs. YB5P, WB7P vs. YB8P, WS1P vs. YS1P, and WS3P vs. YS3P, respectively  and photosystem (GO:0009521). The most highly enriched terms in the molecular function category were catalytic activity , oxidoreductase activity (GO:0016491), and transferase activity (GO:0016740). The most highly enriched terms in the biological process category were metabolic process (GO:0008152), single-organism process (GO:0044699), single-organism metabolic process (GO:0044710), and oxidation–reduction process (GO:0055114). The top 50 most significantly enriched terms are shown in Supplementary Fig. S2. KEGG analysis revealed 46 enriched pathways. The most highly enriched pathways were metabolic pathways (ko01100), biosynthesis of secondary metabolites (ko01110), plant hormone signal transduction (ko04075), and carbon metabolism (ko01200). Most importantly, the flavonoid biosynthesis (ko00941), flavone and flavonol biosynthesis (ko00944), and carotenoid biosynthesis (ko00906) pathways were significantly enriched as well .B. napus genome  in the carotenoid biosynthesis pathway were obviously expressed in WP and ZS11 petals, even though there are 13 homologous GGPS genes in the B. napus genome. However, five of six PSY genes  were clearly expressed in both WP and ZS11 petals. One PDS gene (BnPDS1d), one CrtISO gene (BnCrtISOb), and one LYCE gene (BnLYCEb) were clearly expressed in WP petals but showed almost no expression in ZS11 petals. Importantly, the FPKM values of the two VDE genes (BnVDEa and BnVDEb) were < 3, indicating that these VDE genes, which participate in the xanthophyll cycle, were expressed at very low levels in both WP and ZS11 petals. These results indicate that little violaxanthin and antheraxanthin can be transformed into zeaxanthin  in these petals. Only nine of the 30 genes in the carotenoid degradation pathway were clearly expressed in WP and ZS11 petals during various stages of petal development, including 2 CCD1 subfamily members , 2 NCED3 subfamily members , 3 NCED4 family members , and 2 NCED5 subfamily members . However, members of the BnCCD2, BnCCD7, BnCCD8, BnNCED6, and BnNCED9 subfamilies showed almost no expression in both WP and ZS11 petals, indicating that they did not function during petal development. These results provide an important reference for studying carotenoid metabolism in B. napus petals.Of the 86 genes in the carotenoid metabolic pathway, only 49 genes were obviously expressed during at least one stage in WP or ZS11 petals , with FPKM values > 5. Only two genes  at stage B7, two PDS genes (BnPDS1a and BnPDS1c) at all four stages, two Z-ISO genes (BnZ-ISOa and BnZ-ISOb) at S1 and S3, one CrtISO gene (BnCrtISOa) at all four stages, and one BCH gene (BnBCH1b) at S1 and S3. Several of these genes were highly upregulated in WP vs. ZS11 petals, including one PDS gene (BnPDS1d) at S3, one CrtISO gene (BnCrtISOb) at all four stages, one LCYE gene (BnLCYEd) at S3, one BCH gene (BnBCH1b) at stages B5 and B7, two BCH genes (BnBCH1d and BnBCH2b) at all four stages, and one ZEP gene (BnZEPa) at S3. Among genes in the carotenoid degradation pathway, two NCED DEGs (BnNCED3c and BnNCED4c) were markedly downregulated in WP petals compared to ZS11, whereas only BnNCED4b was markedly upregulated in WP petals during all four stages of development.Various genes in the carotenoid biosynthesis pathway were significantly downregulated in WP petals compared to ZS11, including the following: one B. napus cultivar ‘Darmor-bzh’. Among these, 52 BnMYB genes, 18 BnMADS genes, 55 BnNAC genes, 77 BnbHLH genes, 13 BnSBP genes, 88 BnAP2/ERF genes, 38 BnHD-ZIP genes, 14 BnNF-Y genes, and 27 BnWRKY genes were differentially expressed in WP vs. ZS11 petals at different stages of development (Supplementary Table S5).In addition to structural genes, transcription factors play vital roles in regulating the carotenoid metabolic pathway. Members of several transcription factor families, including the MYB, MADS-box, NAC, bHLH, SBP box, AP2/ERF, HD ZIP, NF-Y, and WRKY families, are the major regulators responsible for the transcriptional activation or repression of carotenoid metabolic genes (Stanley and Yuan BnaCnng29120D), one BnMADS gene (BnaC02g00490D), two BnHD-ZIP genes , one BnNFYA1 gene (BnaA03g04040D), and one BnWRKY22 gene (BnaCnng02000D) were significantly upregulated in WP vs. ZS11 petals at all four stages of development . However, only one BnMYB106 gene  were significantly downregulated in WP vs. ZS11 petals at S1, which may be related to the reduced total lutein and zeaxanthin contents in the white-flowered line. In the crtiso mutants of Chinese kale, the carotenoid isomerase gene (BoaCRTISO) was targeted and edited using the CRISPR/Cas9 system, the expression levels of CRTISO and most carotenoid and chlorophyll biosynthesis-related genes were notably lower than in the WT plants, in addition, both the total and individual carotenoid and chlorophyll concentrations were reduced, and the total levels declined by 11.89–36.33%  encodes a MIXTA-like transcription factor (NOECK) thought to negatively regulate trichome branching  led to defects in male gametogenesis and embryogenesis  regulates the expression of senescence-associated genes indirectly or through other WRKY-mediated senescence signaling pathways  W-box BnNCED4b, a gene involved in carotenoid degradation, represents a key candidate gene for the formation of white petals. The results of qRT-PCR analysis were consistent with the results of RNA-seq. Further analysis of transcription factor genes related to carotenoid accumulation suggested that BnWRKY22 might function upstream of BnNCED4b to promote the degradation of carotenoids in petals. These results provide important insights into the molecular mechanisms of the carotenoid metabolic pathway in B. napus petals. In addition, the candidate genes identified in this study provide a resource for the creation of new rapeseed materials with different flower colors.We combined metabolomic, transcriptomic, and qRT-PCR analyses to explore the mechanisms underlying the differential accumulation of carotenoids in WP and ZS11 petals. The decreased total lutein and zeaxanthin contents in WP petals cause them to fade from light yellow to white as the flowers mature. Transcriptome analysis revealed 20 DEGs in the carotenoid metabolism pathway. Of these, LDJ designed and performed all of the experiments and wrote the manuscript. XTZ, FYJ, and SSZ prepared the plant materials. MZD, CLQ, and XC performed RNA extraction. MCZ and GQM collected the phenotypic data in the field. JSW provided the WP material. CMQ and RW contributed to the experimental design. JSW and JNL approved of and modified the manuscript.Supplementary file1 (DOCX 13 KB)Supplementary file2 (DOCX 14 KB)Supplementary file3 (DOCX 14 KB)Supplementary file4 (DOCX 15 KB)Supplementary file5 (DOCX 13 KB)Supplementary file6 (TIF 31337 KB)Supplementary file7 (TIF 100077 KB)Supplementary file8 (TIF 106486 KB)Supplementary file9 (TIF 119358 KB)Supplementary file10 (TIF 118996 KB)Supplementary file11 (XLSX 12 KB)Supplementary file12 (XLSX 13 KB)Supplementary file13 (XLSX 2026 KB)Supplementary file14 (XLSX 87 KB)Supplementary file15 (XLSX 16 KB)Supplementary file16 (XLSX 11 KB)Supplementary file17 (XLSX 127 KB)Below is the link to the electronic supplementary material.


BOP1 or GNL3 or WDR12 or DCAF13 or IGF2BP3 or IGF2BP1 were associated with poor overall survival (OS). Conversely, overexpression of KHDRBS2/SMAD predicted high OS in these patients. ROC curve analysis showed that the eight hub genes with a better diagnostic accuracy to distinguish lung adenocarcinoma. The results provided novel insights into the pathogenesis of LUAD and the development of treatment targets and prognostic molecular markers.Lung cancer is the top cause of carcinoma-associated deaths worldwide. RNA binding proteins (RBPs) dysregulation has been reported in various malignant tumors, and that dysregulation is closely associated with tumorigenesis and tumor progression. However, little is known about the roles of RBPs in lung adenocarcinoma (LUAD). In this study, we downloaded the RNA sequencing data of LUAD from The Cancer Genome Atlas (TCGA) database and determined the differently expressed RBPs between normal and cancer tissues. We then performed an integrative analysis to explore the expression and prognostic significance of these RBPs. A total of 164 differently expressed RBPs were identified, including 40 down-regulated and 124 up-regulated RBPs. Pathway and Gene ontology (GO) analysis indicated that the differently expressed RBPs were mainly related to RNA processing, RNA metabolic process, RNA degradation, RNA transport, splicing, localization, regulation of translation, RNA binding, TGF-beta signaling pathway, mRNA surveillance pathway, and aminoacyl-tRNA biosynthesis. Survival analysis revealed that the high expression of  LIN28A cause embryonic stem cell developmental defects and a Parkinson’s disease-related phenotype (RNA-binding proteins (RBPs) are generally recognized as proteins that bind to a variety of RNAs, such as rRNAs, miRNAs, snRNAs, ncRNAs, mRNAs, snoRNAs, and tRNAs. Currently, there are more than 1,500 experimentally validated RBP coding genes in human genome, accounting for about 7.5% of all protein-coding genes . They cahenotype . ZFP36L1henotype . Besideshenotype . AlthougTo date, more and more studies have revealed that RBPs were dysregulated in tumors, which influence RNA modification and protein translation, involved in cancer development and progression . For insLung cancer is a very harmful disease with an average 5-year relative survival rate of only 18% . In recehttps://portal.gdc.cancer.gov/). The DESeq2 package (http://www.bioconductor.org/packages/release/bioc/html/DESeq2. html) was used to preprocessed raw data and excluded genes with an average count value less than 1. Besides, we also used DESeq2 package to identify the differently expressed RBPs in view of —log2 fold change (FC)— ≥1 and false discovery rate (FDR)<0.05. The GSE30219 dataset were downloaded from Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) and used as a validation cohort.The RNA-sequencing information of 524 LUAD samples and 59 normal lung tissue samples with corresponding clinical information were obtained from TCGA database   pathway analysis were performed using WEB-based Gene Set Analysis Toolkit  . Both P http://www.string-db.org/)   were usehttp://gepia.cancer-pku.cn/), which included gene expression data and corresponding survival information of 478 LUAD patients  databases and GEPIA databases (http://www.proteinatlas.org/)  . The rechttps://www.cbioportal.org/). Then, we conducted the co-expression analysis and constructed the network according to the cBioPortal’s instruction. In addition, functional enrichment analysis of hub genes alterations was evaluated by WebGestalt.The mutations and copy-number alterations information of all hub genes from were determined by utilizing segmentation analysis and GISTIC algorithm in cBioPortal — ≥1.0), including 40 down-regulated RBPs and 124 up-regulated RBPs  related to mRNA processing, regulation of translation, regulation of cellular amide metabolic processes, and metabolic processes . The uprBOP1, GNL3, WDR12, NOP2, BYSL, BRIX1, DCAF13, TFB2M, NSUN2 and DKC1. Furthermore, the MODE plugin was used to identify possible key modules from the co-expression network and the top 2 significant modules were screened , GNL3 , WDR12 , and DCAF13  were significantly related to poor OS  or IGF2BP1  was related to unfavorable prognosis of LUAD patients  or SMAD9  was negatively associated with OS  of each candidate hub gene to OS by using GEPIA. The results showed that high expression of  poor OS –3D. Conspatients –3F, whil with OS –3H. ThesGSE30219 LUAD patient cohorts , GNL3 , WDR12 , DCAF13 , IGF2BP3 , IGF2BP1 , KHDRBS2 , and SMAD9  were greater than 0.7, suggesting that the eight hub genes with a better diagnostic accuracy for LUAD (GSE30219 cohorts (In accordance with the immunohistochemistry results from the Human Protein Atlas database, we found that the expression of hub RBPs BOP1, GNL3, WDR12, DCAF13, IGF2BP3, and IGF2BP1 were obviously elevated in lung carcinoma compared with normal lung tissues, while KHDRBS2 and SMAD9 were significantly decreased in tumor tissues . Further cohorts . Moreove cohorts . In addifor LUAD . To furt cohorts .BOP1, GNL3, WDR12, NOP2, BYSL, BRIX1, DCAF13, TFB2M, NSUN2, DKC1, IGF2BP3, IGF2BP1, KHDRBS2 and SMAD9 were carried out by using cBioPortal. We found that the 14 key genes altered in 223 samples out of 507 LUAD patients (The mutations and copy-number alterations analysis of the key genes patients –9B. Thenpatients . The funpatients .Despite advances in diagnostic and treatment methods over the past few decades, the overall mortality rates of lung carcinoma remained largely unchanged. Thence, understanding the etiology and molecular mechanism of lung carcinogenesis is necessary to improve the survival rate of patients. In recent years, many studies have shown that RBPs play a pivotal role in the development and progression of various human tumors . HoweverThe function enrichment analysis demonstrated that the differently expressed RBPs were significantly related to RNA processing, RNA metabolic process, RNA binding, peptide biosynthetic process, regulation of translation, regulation of cellular amide metabolic process, catalytic activity acting on RNA, single-stranded RNA binding, poly-pyrimidine tract binding, RNA cap binding complex, ribonucleoprotein complex, mRNA 3′-UTR binding, ribonucleoprotein granule, and nucleolus. Previous studies have shown that RNA metabolism and RNA processing have been increasingly recognized in various diseases . RBPs caBOP1, GNL3, WDR12, NOP2, BYSL, BRIX1, DCAF13, TFB2M, NSUN2 and DKC1. Among these genes, NOP2, also known as p120, is found highly expressed in lung adenocarcinoma tissue and negatively associated with patients prognosis (BOP1 as Wnt/β-catenin target gene involved in induced migration, EMT, and metastasis of colorectal carcinoma (GNL3 is upregulated in colon carcinoma cell and tissue, and facilitate tumor cell epithelial-mesenchymal transition by activating the Wnt/β-catenin signaling pathway (WDR12 as a new oncogene contributes to liver cancer spread (DCAF13 is upregulated in liver cancer and significantly related to poor survival (DKC1 is deregulated in glioma and knockdown of DKC1 restrain cancer cell proliferation, invasion and migration (Furthermore, we constructed a PPI network of these differently expressed RBPs and got the top ten hub genes including rognosis . Despitearcinoma . GNL3 is pathway . WDR12 ar spread . DCAF13 survival . NSUN2 asurvival , breast survival , and ovasurvival . DKC1 isigration . By execBOP1, GNL3, WDR12, DCAF13, IGF2BP3 and IGF2BP1 were related to poor overall survival, while overexpression of KHDRBS2 and SMAD were related to better overall survival. Subsequently, we verified the expression patterns of these eight hub RBPs on translation and transcription level by using the Human Protein Atlas database and GEPIA database, the results showed that BOP1, GNL3, WDR12, DCAF13, IGF2BP3 and IGF2BP1 were upregulated, and KHDRBS2 as well as SMAD were downregulated in LUAD tissues. These findings suggested that BOP1, GNL3, WDR12, DCAF13, IGF2BP3 and IGF2BP1 may have potential carcinogenic effects, KHDRBS2 and SMAD role as tumor suppressor genes. Previous study has reported that the expression IGF2BP3 or IGF2BP1 can predict poor overall survival in lung cancer patients (DCAF13 was increased in hepatocellular carcinoma or breast cancer, and obviously associated with poor overall survival (GSE30219 cohorts.In addition, the survival analysis showed that 8 RBPs are obviously related to survival of LUAD patients. Increased expression of patients , which wsurvival . The ROCNSUN2 was the most common alterations in all copy number variations of these RBPs. A study revealed that NSUN2 gene copy number was elevated in colorectal and oral carcinoma (DCAF13 and BOP1 have high copy number gain frequency in patients with LUAD. Previous studies have shown that DCAF13 is amplified in various tumors, such as hepatocellular carcinoma (BOP1 was a frequent event in colorectal carcinoma and related to cancer occurrence (Finally, we analyzed the hub genes alterations and constructed the co-expression network by using the cBioPortal online tool for LUAD. The results showed that amplification of arcinoma .We also arcinoma , breast arcinoma , and ostarcinoma . Anothercurrence . By consBOP1, GNL3, WDR12, DCAF13, IGF2BP3, IGF2BP1, KHDRBS2 and SMAD were significantly related to prognosis of LUAD patient. These findings have the potential to provide new therapeutic targets prognostic markers for LUAD.In conclusion, the current study conducted a comprehensive bioinformatics analysis of differently expressed RBPs to identify the potential biomarkers and predict progression of LUAD. The results showed that the expression of 10.7717/peerj.8509/supp-1Table S1Click here for additional data file.