Escherichia coli, composed of a heptapeptide with a modified adenosine monophosphate. This study was performed to evaluate the effects of Microcin C7 as a potential substrate to traditional antibiotics on growth performance, immune functions, intestinal barrier, and cecal microbiota of broilers. In the current study, 300 healthy Arbor Acres broiler chicks were randomly assigned to one of five treatments including a corn–soybean basal diet and basal diet supplemented with antibiotic or 2, 4, and 6 mg/kg Microcin C7. Results showed that Microcin C7 significantly decreased the F/G ratio of broilers; significantly increased the levels of serum cytokine IL-10, immunoglobulins IgG and IgM, and ileal sIgA secretion; significantly decreased the level of serum cytokine TNF-α. Microcin C7 significantly increased villus height and V/C ratio and significantly decreased crypt depth in small intestine of broilers. Microcin C7 significantly increased gene expression of tight junction protein Occludin and ZO-1 and significantly decreased gene expression of pro-inflammatory and chemokine TNF-α, IL-8, IFN-γ, Toll-like receptors TLR2 and TLR4, and downstream molecular MyD88 in the jejunum of broilers. Microcin C7 significantly increased the number of Lactobacillus and decreased the number of total bacteria and Escherichia coli in the cecum of broilers. Microcin C7 also significantly increased short-chain fatty acid (SCFA) and lactic acid levels in the ileum and cecum of broilers. In conclusion, diet supplemented with Microcin C7 significantly improved growth performance, strengthened immune functions, enhanced intestinal barrier, and regulated cecal microbiota of broilers. Therefore, the antimicrobial peptide Microcin C7 may have the potential to be an ideal alternative to antibiotic.Microcin C7 is an antimicrobial peptide produced by  Antibiotics are used widely in livestock production as an effective antimicrobial drug because they play important roles in disease treatment, disease prevention, and growth promotion , 2. GlobAMPs are small cationic molecules with amphipathic structures, widely distributed in living organisms , 9. AMPsEscherichia coli cells harboring a plasmid-borne mccABCDE , and the experiment is conducted in strict accordance to the animal care protocol. The basal diet  with a separate feeding trough and nipple drinkers. A 24 h constant-lighting program was used. Broilers had sal diet  was antin = 300; initial body weight (BW) = 46.67 ± 0.29 g) were obtained from Chengdu Xinjin Yunda poultry breeding cooperative. All chicks were individually weighed and randomly assigned to 1 of 5 treatments with 6 replicates of 10 broilers. Experimental treatments included a corn–soybean basal diet with no additions, a basal diet supplemented with 45 mg/kg chlortetracycline and 30 mg/kg bacitracin methylene disalicylate, and a basal diet supplemented with 2, 4, or 6 mg/kg Microcin C7. Microcin C7, which was isolated from the metabolites of Lactobacillus johnsonii by microbial fermentation, was supplied by Angeli (Chongqing) Biotechnology, Co. Ltd . The concentration of dietary Microcin C7 was determined based on findings of our previous studies (not published).One-day-old healthy Arbor Acres broiler chickens (The feeding period was divided into two stages: the starter stage (Day 1–Day 21) and the finisher stage (Day 22–Day 42). Broilers were weighed individually at the start of the trial and the end of each phase prior to the morning feeding. Feed consumption was measured at the end of each phase. All feed remaining in the feeder was weighed and subtracted from the daily allowance to determine the actual daily feed intake. The ADG, ADFI, and F/G ratio were calculated. After weighing, two broilers per pen were randomly selected, euthanized, and sampled. Blood was collected under wings, then let stand for 10 min, and centrifuged at 3,000 rpm for 15 min at 4°C, and the supernatant was collected and stored at −20°C for subsequent serum biochemical parameter measurements. After exsanguination from the jugular vein, feathers were removed, and the abdominal cavity was opened. Samples (2 cm) of the duodenum, jejunum, and ileum were excised and washed in PBS and immediately fixed in 4% (v/v) paraformaldehyde solution for characterization of small intestinal morphology. The jejunal and ileal mucosa (about 0.2 g) were scraped, and scrapings were placed in a sterile 2 ml cryopreservation tube, frozen rapidly with liquid nitrogen, and then stored at −80°C for gene expression and total sIgA analysis. Contents of jejunum, ileum, and cecum were removed and placed in a sterile 2 ml cryopreservation tube, frozen rapidly with liquid nitrogen, and then stored at −80°C for microflora measurement.Serum samples were thawed and thoroughly mixed immediately before testing. Concentrations of serum immunoglobulins A, G, and M and cytokines IFN-γ, IL-1β, IL-10, and TNF-α were measured using commercially available avian ELISA kits , according to standard procedures described by the manufacturer.Embedded tissue was deparaffinized and hydrated, cut into slices (5 μm), and then stained with hematoxylin and eosin for morphology measurements as described by Wang et al. . Villus −ΔΔCt method as described by Livak and Schmittgen  according to the instructions of the RNAiso Reagent . Purity and concentration of total mRNA in samples were evaluated using a spectrophotometer  at 260 and 280 nm, respectively. Ratios of absorption (260:280 nm) between 1.8 and 2.0 for all samples were accepted as “pure” for RNA. RNA (1 μg) was used to generate cDNA using the M5 Super plus qPCR RT kit with a gDNA remover according to the manufacturer's instructions . Primer sequences for tight junction proteins including ZO-1, Occludin, Claudin 3, Jam-2, mucoprotein Mucin 2, cytokine IFN-γ, TNF-α, IL-8, pattern recognition receptors TLR2 and TLR4, and key linker molecular MyD88 in jejunal mucosa were designed using the GenBank database from the National Center for Biotechnology Information (NCBI) and primer design software. Quantitative real-time PCR was performed with HiPer SYBR Premix EsTaq with Tli RNase H  using a StepOnePlus real-time PCR system (Applied Biosystems) on 96-well plates with 10 μl of total reaction volume of 5 μl HiPer SYBR Premix, 4 μl cDNA, 0.3 μl of forward and 0.3 μl of reverse primers (10 nmol), and 0.4 μl double-distilled water. Each reaction was run in duplicate. The PCR cycling protocol included one cycle of pre-incubation at 95°C for 30 s; 40 cycles of denaturation at 95°C for 5 s and annealing at 60°C for 30 s; one cycle of melting at 95°C for 5 s, 60°C for 60 s, and 95°C for 5 s; and one cycle of cooling at 50°C for 30 s. GAPDH was used as an internal control in this study. Average expression of the target genes relative to GAPDH was determined using the 2hmittgen . Primershmittgen .Ileal mucosa was homogenized with 0.1 M PBS, and the supernatant was centrifuged at 3,000 rpm for 15 min. BCA protein quantitative kit  was used to determine the total protein content of the ileal mucosa homogenate according to the manufacturer's instructions. Concentration of sIgA was determined by an enzyme-linked immunosorbent assay  according to the manufacturer's instructions. Data were acquired using a Microplate reader  equipped with SoftMax Pro Software. Relative abundance of sIgA in ileal mucosa was expressed as the ratio of sIgA to the total protein concentration of ileal mucosa homogenate.Lactobacillus, and Escherichia coli were quantified using real-time PCR in a StepOnePlus system. Each reaction was run in a 10 μl volume containing 5 μl HiPer SYBR Premix EsTaq , 0.3 μl of forward and 0.3 μl of reverse primers (100 nM), 1 μl template DNA, and 3.4 μl of double-distilled water for detecting total bacteria. The PCR profile was processed as follows: one cycle of pre-incubation at 94°C for 180 s; 40 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 30 s, and extension at 72°C for 60 s; one cycle of melting at 95°C for 5 s and 60°C for 60 s; and one cycle of cooling at 50°C for 30 s. To detect Lactobacillus and E. coli, each reaction was completed in a 10 μl volume containing 5 μl FastFire qPCR PreMix (Probe) , 0.25 μl of forward and 0.25 μl of reverse primers (100 nM), 0.15 μl probes, 1.5 μl template DNA, and 2.85 μl of double-distilled water. The PCR profile was determined as follows: one cycle of pre-incubation at 95°C for 600 s and 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 60 s. All samples were analyzed in duplicate. Briefly, primers and fluorescent oligonucleotide probes  according to the manufacturer's instructions and was stored at −20°C. Numbers of total bacteria, e probes  were obte probes , 27 and Lactobacillus (ATCC33323) and E. coli  were cultured anaerobically or aerobically in respective cultures including 1% glucose at 37°C from 12 to 48 h. The 16S rDNA genes of Lactobacillus and E. coli were amplified using PCR. Then, the specific PCR product was purified using a Gel Extraction Kit  and cloned into pEASY-Blunt vector . After verification of the sequence, the recombinant plasmid was isolated using the TIANprep Mini Plasmid Kit . The standard plasmids of Lactobacillus and E. coli were constructed successfully. The copy numbers of bacteria were calculated using the following formula: (6.0233 * 1023 copies/mol * DNA concentration (μg/μl))/(660 * 106* DNA size (bp)). Bacterial counts were performed using serial dilutions (10:1 to 10:8 dilutions) of plasmid DNA to generate the standard curve for total bacteria, Lactobacillus and E. coli. Each standard curve was constructed by linear regression of mean cycle threshold values against the logarithm of template copy numbers, ranging from 3 to 9 log10 copies. Target copy number of each sample was calculated from the standard curve . Broilers fed with antibiotic and Microcin C7 had decreased concentration of pro-inflammatory cytokine TNF-α (P < 0.05). However, all groups were similar with each other on Day 21 (P > 0.05). On Day 42, a linearly higher content of IL-10 (P < 0.05). A linearly lower content of TNF-α was observed in broilers fed Microcin C7 , IL-8 (P < 0.05), and IFN-γ (P < 0.05) was observed in broilers fed Microcin C7 , TLR2 (P = 0.03), and MyD88 (P < 0.05) was observed in broilers fed Microcin C7 . A linearly higher content of sIgA was observed in broilers fed Microcin C7 .On Day 21, broilers fed with 6 mg/kg Microcin C7 had significantly increased sIgA concentration compared with the control group  was observed in broilers fed Microcin C7  and E. coli  was observed in broilers fed Microcin C7  was observed in broilers fed Microcin C7  and 100 g/t full-tide ABP (mainly composed of cecropin) increased expression of ZO-1 and Claudin-3  and 100 g/t full-tide ABP (mainly composed of cecropin) significantly decreased the expression of pro-inflammatory cytokines IL-17A and IFN-α, which suppress intestinal inflammation , jejunum (39–72%), and ileum (24–96%) at all ages. In the ileum, the abundances of Lactobacillus are stable from 7 to 21 days of age and increased to 96.7% on Day 42 , Chongqing Rongchang Agricultural and Animal Husbandry High-tech Industry Research and Development Project (cstc2019ngzx0019), and Beijing Swine Innovation Team of Modern Agriculture Industry Technological System.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Traumatic cartilage injuries predispose articulating joints to focal cartilage defects and, eventually, posttraumatic osteoarthritis. Current clinical-standard imaging modalities such as morphologic MRI fail to reliably detect cartilage trauma and to monitor associated posttraumatic degenerative changes with oftentimes severe prognostic implications. Quantitative MRI techniques such as T2 mapping are promising in detecting and monitoring such changes yet lack sufficient validation in controlled basic research contexts. Material and Methods: 35 macroscopically intact cartilage samples obtained from total joint replacements were exposed to standardized injurious impaction with low  or high  energy levels and imaged before and immediately, 24 h, and 72 h after impaction by T2 mapping. Contrast, homogeneity, energy, and variance were quantified as features of texture on each T2 map. Unimpacted controls (n = 7) and histologic assessment served as reference. Results: As a function of impaction energy and time, absolute T2 values, contrast, and variance were significantly increased, while homogeneity and energy were significantly decreased. Conclusion: T2 mapping and texture feature analysis are sensitive diagnostic means to detect and monitor traumatic impaction injuries of cartilage and associated posttraumatic degenerative changes and may be used to assess cartilage after trauma to identify “cartilage at risk”. Associated with knee symptoms and dysfunction, focal cartilage lesions are common in the general population. Hjelle et al. reported (osteo)chondral lesions (of any type) in 61% of their patients undergoing knee arthroscopy ,3,4. TheWhile the aetiology of focal cartilage lesions is multifactorial, focal cartilage lesions are often the result of trauma ,6: MenisDue to its high soft tissue contrast and spatial resolution, non-invasiveness, and lack of radiation, Magnetic Resonance Imaging (MRI) is clearly the most powerful diagnostic tool of contemporary clinical medicine and the superordinate standard imaging modality for suspected joint and cartilage disorders ,9,10. HoInstead of merely quantifying T2 values of the superficial and deep cartilage zones in a pixel-wise manner, recent approaches have relied on more comprehensive post hoc approaches for image analysis such as texture feature analyses. During cartilage degeneration, collagen network integrity and proteoglycan content are lost . The incDespite this wealth of clinical knowledge, a basic understanding of the posttraumatic degenerative changes in cartilage and their imaging correlates is lacking. The present study’s objective was to contribute to this understanding by bringing together intact human articular cartilage, standardized injurious impaction loading with variable impaction energies, and T2 mapping and post hoc texture feature analysis. To this end,  intact cartilage tissue was subject to impaction loading using a drop-tower device as an established model for inducing posttraumatic degenerative changes ,31,32, aFollowing informed consent and Institutional Ethical Review Board approval , we obtained human articular cartilage-bone samples from 24 patients undergoing total knee replacement surgery at our institution  ,37,38,39https://www.statstodo.com, accessed 3 February 2018). Based on comparable studies [Before the study, minimum sample size had been projected using a dedicated online tool , low impact , and high impact . A custom-made drop tower device was used to standardize impaction energy levels as before  Figure b–d. BrieThese two different impaction energy levels created “mild” (LIMP) and “severe” (HIMP) structural and compositional damage in cartilage ,41. To e0) and immediately after impaction (t1) as well as 24 h (t2) and 72 h (t3) after impaction  within 6 h after sample preparation. Serial MRI measurements were performed just before  without the inflatable balloon tip was used for imaging and circumferentially enclosed the sample box. Radiofrequency pulses were applied via the scanner’s in-built body coil. Two cartilage samples were imaged at a time using morphologic and quantitative MRI  in a standard incubation unit. Culture medium was changed after each MRI series.For reproducible positioning of cartilage samples in the MRI scanner, a validated MRI-compatible device ,43 was utive MRI . BrieflyMetrics of contrast, i.e., contrast and homogeneity, metrics of orderliness, i.e., energy, and statistical metrics, i.e., variance, were calculated [Contrast assesses the extent of local variation. Cartilage areas with high contrast values display strong contrasts, i.e., pronounced differences between the highest and lowest T2 values.Homogeneity serves as a measure of uniformity by indicating similarities between pixels and their neighbours. Cartilage areas with mostly similar T2 values have high homogeneity values.Energy as computed based on the GLCM provides a measure of uniformity and orderliness. Cartilage areas with high energy values display similar T2 values and small T2 value differences in neighbouring pixels.Variance is a measure of local variation around the mean. High variance values indicate high heterogeneity and large differences in T2 values, i.e., variation from their mean.The T2 maps were generated in a pixel-wise manner using customized mono-exponential fitting routines implemented in MATLAB  as before ,39. For lculated :Contrast3, the cartilage samples and the adjacent cartilage tissue underwent standard histological work-up [Following the last MRI series at t work-up ,46. Sampadjacent cartilage tissue was assessed semi-quantitatively according to the Mankin classification of cartilage degeneration [Baseline cartilage status of the neration . Two invcartilage samples were assessed semi-quantitatively based on the Mankin classification. Posttraumatic cartilage changes such as alterations in tissue structure, i.e., clefts and other signs of surface disintegration, as well as composition, i.e., Safranin O de-staining, were noted.Following exposure to impaction or control conditions, 0, t1, t2, and t3) using Friedman’s test followed by Dunn’s post hoc test. Accordingly, relative changes for each measure (Δx) at tx were determined in reference to t0 and calculated using (Equation (3)):Statistical analysis was performed by MSH and SN using GraphPad Prism Software . Not assuming normal distributions, absolute T2 values and texture features were compared as a function of time  were used to compare relative changes Δx as well as histological changes of cartilage structure and proteoglycan content. Results are displayed as mean ± standard deviation. Due to this study’s exploratory character and the large number of comparisons involved, the level of significance was set to p ≤ 0.01 to reduce the number of statistically significant, yet clinically and scientifically (most likely) insignificant findings.The different impaction energy levels and control conditions at tAll 35 samples underwent full MRI and histologic reference evaluation.Macroscopic evaluation revealed no fracturing of the subchondral bone lamella in any sample. However, considerable dents corresponding to the metallic tip’s geometry were observed at the impaction site following HIMP exposure (11/14 samples) a, while adjacent cartilage tissue was grossly intact as indicated by mean MSSs of 1.8 ± 0.8 . Baseline cartilage status was dominated by slight signs of histologic degeneration such as surface fibrillation, focal hypercellularity or slight Safranin-O de-staining, translating to MSSs of 1 or 2 in most tissue specimens.Histologic assessment revealed that Cartilage samples harvested after impaction and/or standard incubation revealed impaction energy level-associated tissue damage. Even though not significant, surface disintegration was higher with higher impaction energy levels  to 35.1 ± 7.4 ms (t3) , from 37.0 ± 6.4 ms (t0) to 39.0 ± 8.8 ms (t3) , and from 29.4 ± 5.1 ms (t0) to 31.1 ± 6.6 ms (t3) . After LIMP exposure, T2 values were significantly increased in all ROIs, from 32.0 ± 2.4 ms (t0) to 40.3 ± 5.2 ms (t3) , from 38.2 ± 4.3 ms (t0) to 47.5 ± 6.1 ms (t3) , and from 26.1 ± 2.8 ms (t0) to 33.9 ± 6.9 ms (t3) . Post hoc analysis revealed these differences to be significant between t0 and t2 and between t0 and t3 (0) to 55.9 ± 8.3 ms (t3) , from 42.9 ± 5.8 ms (t0) to 67.2 ± 19.7 ms (t3) , and from 27.7 ± 6.0 ms (t0) to 44.4 ± 11.4 ms (t3) . Significant post hoc differences were found between t0 and t1 (except for deep layer), t0 and t2, and t0 and t3  to 0.28 ± 0.11 (t3) (p = 0.270). After HIMP exposure, however, contrast values were significantly increased from 0.24 ± 0.09 (t0) to 0.40 ± 0.17 (t3) (p < 0.001). Opposite observations were made for homogeneity that remained relatively constant in controls (p = 0.615) and tended to decrease in LIMP-exposed samples (p = 0.166) but decreased significantly from 0.88 ± 0.05 (t0) to 0.82 ± 0.06 (t3) (p < 0.001) in HIMP-exposed samples. Energy as a metric of orderliness was characterized by distinct changes, too. While energy was undulating in controls (p = 0.510), it decreased considerably from 0.43 ± 0.20 (t0) to 0.30 ± 0.07 (t3) (p = 0.016) and from 0.35 ± 0.13 (t0) to 0.24 ± 0.16 (t3) (p = 0.006) after LIMP and HIMP exposure, respectively, thereby tending towards (LIMP) or reaching statistical significance (HIMP). Variance as a statistical metric was undulating, too, in controls, while it underwent moderate, yet non-significant increases from 111.0 ± 94.9 (t0) to 143.2 ± 69.4 (t3) (p = 0.015) following LIMP exposure and strong and significant increases from 118.3 ± 79.9 (t0) to 311.4 ± 264.8 (t3) (p = 0.005) following HIMP exposure. Group-wise comparisons at individual time points revealed no significant differences at t0, indicating largely similar baseline texture feature and sample homogeneity prior to exposure. For contrast, homogeneity, and energy, significant group-wise differences were found at t2 and t3, while for variance, significant differences were only observed at t3.Radiomic texture features were significantly different only after HIMP exposure, and not in controls or after LIMP exposure . MetricsImpaction-induced quantitative changes as outlined above were reflected by corresponding qualitative changes in the T2 maps . ControlThe most important finding of this study is that advanced MRI acquisition and postprocessing techniques, i.e., quantitative T2 mapping and texture feature analysis, may be used to (i) differentiate the severity of supraphysiological impact injuries of cartilage and (ii) monitor post-traumatic degenerative changes.lack of basic research available on the association of T2 mapping and traumatic cartilage injury. Even though a solid body of clinical evidence is available to support the potential of T2 mapping in evaluating posttraumatic cartilage changes [Prior to its initiation, this study had been motivated by the  changes , the cha changes . After A changes , while o changes  and thre changes . In cont changes  as well  changes . This, oinjurious cartilage impaction is associated with increasing T2 values as a function of impaction energy level and time. Based on the sensitivity profile of T2 versus structural and compositional cartilage properties, these increases reflect numerous single-impact-associated posttraumatic changes in cartilage [This study clearly demonstrates that artilage ,49,50,51artilage , and proartilage ,41,48. Nincreases in T2 may be secondary to numerous posttraumatic changes that are excellently reviewed in [On the tissue level, the impaction-induced iewed in . For onciewed in . For anoiewed in , as welliewed in . Yet, evradiomic texture features, too, as refined imaging biomarkers of cartilage trauma. Again, changes were clear and significant after HIMP exposure, while they were moderate and only tended towards significance after LIMP exposure. Across the spectrum of texture features assessed, variance and metrics of contrast and orderliness were significantly increased  and decreased  as a function of impaction energy. Additionally, these features were different between the various time points, indicating lower textural uniformity and growing structural disorder. These changes may be considered a sign of cartilage damage, too, as described before [Beyond mean T2 values, this study focused on d before ,26,44.changes over time. These aspects may be explained by the concurrence of posttraumatic changes in cartilage that are induced either immediately through the impaction itself or delayed through cell death and the induction of matrix-degrading enzymes. These processes reduce biosynthetic capacity or bring about progressive degradation that may explain the gradual alterations in T2 characteristics. Most likely, these processes are at the root of the hyperintense bands that traversed the cartilage sample in the transitional zone parallel to the subchondral lamella. Observed after both low- and high-energy exposure, similar histologic changes have been reported before [Notably, changes in T2, i.e., absolute values and texture features, were subject to gradual and drastic d before ,41. Folld before . The banzonal changes of the superficial and deep tissue layers were roughly similar in terms of relative changes in T2. Considering the direct impaction of the cartilage surface, intuitively, one would expect larger changes of superficial than deeper zones. Yet, the viscoelastic nature of cartilage, its unique compressive properties and tight attachment to the underlying subchondral bone provide efficient mechanisms for absorption of physiologic and supraphysiologic loads throughout the entire tissue depth [Notably, ue depth . Once thue depth .limitations. First, the experimental in vitro design necessitated excision and preparation of cartilage and its prolonged incubation, thereby limiting the clinical translatability of our findings. Prolonged incubation in media may artificially increase tissue hydration, thereby increasing T2 values. As this was observed for otherwise unaffected control samples, these gradual increases in T2 provide the background against which the impaction-induced changes must be considered. Additionally, scanning was performed at room temperature, again affecting T2 values. As the MRI measurements were performed in a standardized manner, this bias may be considered systematic. Additional studies using in situ human whole-knee joint configurations and more physiologic impaction methods are thus required to confirm our findings. As an additional caveat, however, resultant intra-tissue changes secondary to impaction are largely dependent on the experimental framework conditions. Standardized impaction of cartilage samples induces more severe changes than similar impaction of intact joints [0 after injurious impaction, particularly in the deep tissue layer [This study has numerous t joints . Second,t joints  and may ue layer  Althoughue layer ,62,63,64ue layer . Yet, thue layer ,67,68,69ue layer ,71,72,73This study demonstrates that advanced MRI acquisition and postprocessing techniques, i.e., quantitative T2 mapping and texture feature analysis, are sensitive diagnostic means to detect and monitor traumatic impaction injuries of cartilage and associated posttraumatic degenerative changes. If corroborated by additional in situ and in vivo studies, the close association of changes in T2 and texture features, impaction energy level, and time render this technique diagnostically promising to assess cartilage tissue after trauma and to detect cartilage at risk.


Escherichia coli, many cell division proteins interact with FtsZ and modulate Z-ring assembly, while others direct cell wall insertion and peptidoglycan remodeling. Here, we show that ZapE, an ATPase that accumulates during late constriction, directly interacts with FtsZ and phospholipids in vitro. In the presence of adenosine triphosphate (ATP), ZapE induces bundling of GTP-induced FtsZ polymers; however, ZapE also binds FtsZ in the absence of GTP. The ZapE mutant protein ZapE(K84A), which is defective for ATP hydrolysis, also interacts with FtsZ and induces FtsZ filament bundling. In vivo, cultures of zapE deletion cells contain a low percentage of filamentous cells, suggesting that they have a modest division defect; however, they are able to grow when exposed to stress, such as high temperature and limited oxygen. When combined with the chromosomal deletion of minC, which encodes an FtsZ disassembly factor, ΔzapE ΔminC cells experience growth delays that slow proliferation at high temperature and prevent recovery. This synthetic slow growth phenotype after exposure to stress suggests that ZapE may function to ensure proliferation during and after stress, and this is exacerbated when cells are also deleted for minC. Expression of either ZapE or ZapE(K84A) complements the aberrant growth phenotypes in vivo suggesting that the division-associated role of ZapE does not require ZapE ATP hydrolysis. These results support that ZapE is a stress-regulated cell division protein that interacts directly with FtsZ and phospholipids, promoting growth and division after exposure to environmental stress.During pathogenic infections, bacterial cells experience environmental stress conditions, including low oxygen and thermal stress. Bacterial cells proliferate during infection and divide by a mechanism characterized by the assembly of a large cytoskeletal structure at the division site called the Z-ring. The major protein constituting the Z-ring is FtsZ, a tubulin homolog and GTPase that utilizes the nucleotide to assemble into dynamic polymers. In  Escherichia coli and most bacteria, cell division requires the highly conserved tubulin-like FtsZ protein  and under anaerobic conditions 85 and Walker B 141(CFDEF)145 nucleotide-binding sites . ZapA, -B, -C, and -D localize to the Z-ring early in the cell division pathway and regulate the development of the Z-ring . ZapE wang sites . The reqin vitro by destabilizing FtsZ polymers in an adenosine triphosphate (ATP)-dependent manner  vesicles. ZapE bundles FtsZ polymers in the presence of ATP, which is in contrast to disassembly activity that was previously reported  back into the native locus. MinC is an FtsZ polymerization inhibitor that prevents division near the cell poles , or kanamycin (50 μg ml–1), where indicated. Gene deletions were constructed by Lambda red recombination as described (zapE or zapE(K84A) were constructed as described  expression plasmids were constructed by restriction cloning  and site-directed mutagenesis into pET-28a expression vector with an N-terminal 6 × -histidine tag.escribed , and chrescribed . CulturezapE and zapE(K84A) with an N-terminal 6 × -histidine tag were transformed and overexpressed in E. coli BL21 (λde3). Cell cultures were grown to an OD600 of 0.8 and induced with IPTG (0.5 mM) for 4 h at 30°C. Cultures were centrifuged, and pellets were lysed by French press in 25 mM Tris (pH 7.5) buffer containing 0.1 mM TCEP, 10 mM MgCl2, 150 mM KCl, and 10% glycerol. Soluble cell extracts were collected by centrifugation at 35,000 × g for 30 min, applied to a cobalt column, and eluted with an imidazole gradient (0 mM to 150 mM). Collected protein was fractionated on a sephadex G25 column to remove imidazole and small contaminants. Native FtsZ was expressed in E. coli BL21 (λde3) and purified as described (E. coli BL21 (λde3) and purified as described  for 5 min and then passed through a Sephacryl S-200 (25 ml) column with 25 mM Tris (pH 7.5) buffer containing 0.1 mM TCEP, 10mM MgCl2, 150 mM KCl, and 10% glycerol, and, where indicated, equilibrated with ATP (0.5 mM). Eluting fractions (250 μl) were collected (0.5 ml min–1) and analyzed by the Bradford protein assay and the proteins were visualized by SDS-PAGE and Coomassie staining.Vectors containing escribed . ZapE coescribed . Where i2 in the absence or presence of SUVs (250 μg ml–1), where indicated, was monitored by measuring the release of inorganic phosphate with time using the Biomol Green phosphate detection reagent (Enzo Life Sciences) at 23°C as described  in the absence and presence of ZapE or ZapE(K84A) (8 μM) with or without ATP or ATPγS (4 mM), where indicated, at 23°C for 10 min and collected by centrifugation for 20 min at 250,000 × g, Supernatants and pellets were resuspended in equivalent volumes of LDS sample buffer (Life Technologies) and analyzed by SDS-PAGE and Coomassie staining.FtsZ polymers were assembled by incubating FtsZ (6 μM) in 50 mM HEPES buffer, pH 7.5, containing 150 mM KCl, 20 mM MgCl2. Where indicated, reaction mixtures (80 μl) containing ZapE (0 to 8 μM) or ZapE(K84A) (4 μM), with or without FtsZ (8 μM), were monitored at 23°C using an Agilent Eclipse fluorescence spectrophotometer with both excitation and emission wavelengths set to 450 nm with 5 nm slit widths, as described (Light scattering (90° angle) was performed in 50 mM MES (pH 6.5) buffer containing 100 mM KCl and 10 mM MgClescribed . Protein2 at 23°C for 1 h to allow binding. After washing with TBS containing Tween-20 (0.01%), the membrane was probed with rabbit polyclonal antisera (anti-ZapE) and then goat anti-rabbit antibody coupled to horseradish peroxidase. The membrane was developed by chemiluminescence, and spot density was analyzed by NIH ImageJ. Anti-ZapE antiserum was generated in rabbits using purified H6-ZapE (ThermoFisher).Binding of ZapE to FtsZ was assayed by dot blot. Briefly, bovine serum albumin (BSA) (10 μM), ZapE (10 μM), and FtsZ (10 μM) alone or with GDP (2 mM) or GTP (2 mM) were spotted (3 μl) onto a nitrocellulose membrane. The membrane was blocked with BSA (2%) in tris-buffered saline (TBS) then incubated with ZapE (8 μM) and ATP (4 mM) in 50 mM HEPES (pH 7.5) buffer containing 150 mM KCl and 20 mM MgClin vitro. ZapE (5 μM), ZapE(K84A) (5 μM), and/or Gfp-FtsZ (10 μM) reaction mixtures (40 μl), in 50 mM MES (pH 6.5) buffer containing 100 mM KCl and 10 mM MgCl2, were incubated with ATP (4 mM) and, where indicated, GTP (2 mM) at 23°C for 10 min. Reactions were filtered through 100 kDa Nanosep polyethersulfone filters by centrifugation at 16,900 × g for 20 min. Protein present in filtrate and retentate were analyzed by SDS-PAGE and Coomassie staining, and densitometry was performed using NIH ImageJ.Filter retention assays were performed to detect interactions between fluorescent Gfp-FtsZ and ZapE –1) for 5 min at 23°C in the presence or absence of ATP (4 mM), where indicated in 50 mM HEPES (pH 7.5) reaction buffer containing 150 mM KCl and 20 mM MgCl2. Phospholipid recruitment assays with ZapE and Gfp-FtsZ (3 μM) were performed by incubating Gfp-FtsZ with GTP (4 mM), where indicated, in reaction buffer for 2 min and then added to a reaction containing ZapE (1 μM) pre-assembled with SUVs (500 μg ml–1) and ATP (4 mM) and then incubated for an additional 2 min at room temperature. Phospholipid vesicles and associated proteins were collected by low-speed centrifugation at 21,000 × g for 15 min. Supernatants and pellets were analyzed by SDS-PAGE and Coomassie staining. Percent of protein in the pellet fraction was determined by densitometry using NIH ImageJ.Phospholipid recruitment assays with ZapE were performed by incubating ZapE (1 μM) or ZapE K84A (1 μM) together with small unilamellar vesicles (SUVs)  strains were grown overnight in LB (Lennox) media at 30°C. The next day, cultures were diluted into fresh LB (Lennox) medium standardized to OD600 of 0.05, grown for 3 h at 35°C, and then the final OD600 was measured. Cells were then harvested by centrifugation at 3,000 × g for 5 min, resuspended in 100 μl of 1 × PBS with EDTA (1 mM), and fixed to glass slides with poly-L-lysine-coated coverslips or, where indicated, applied to glass slides with agarose gel pads  containing M9 minimal medium supplemented with 0.4% glucose. Cells were imaged by DIC microscopy using a Zeiss AxioCam HRc high-resolution camera, processed using Adobe photoshop CS6, and measured using ImageJ software.MG1655, MG1655 Δ2), ZapE (8 μM), ZapE (K84A) (8 μM), FtsZ (8 μM), and ATP (4 mM), where indicated, were incubated for 5 min at 23°C, then GTP (1.5 mM), was added to induce complex formation, where indicated, and reactions were incubated for an additional 15 min at room temperature. Samples were applied to a 300-mesh carbon/formvar coated grid, fixed with glutaraldehyde (2.5%), and negatively stained with uranyl acetate (2%). Samples were visualized at × 10,000— × 25,000 direct magnification by transmission electron microscopy using a JEM-2100 80 Kev instrument.For electron microscopy imaging, reactions containing buffer  strains were grown overnight in LB (Lennox) medium at 30°C. The next day, cultures were diluted into fresh LB  or LB (Lennox) NaCl 5 g/L medium, where indicated, standardized to an OD600 of 0.05 and grown for 3 h under mild heat shock conditions in static, closed culture at 42°C, or aerobically at 37°C and shaking. After the allotted time, growth was measured by OD600, and cultures were diluted (2-log) into LB  medium. Dilutions were spot plated (5 μl) onto Lennox agar LB  agar, and plates were incubated at room temperature. Images of colony growth were collected, and biomass was quantified in ImageJ software to determine spot density in the zone of growth.MG1655, MG1655 ΔzapE, MG1655 ΔminC, MG1655 ΔminCΔzapE, MG1655 ΔminCΔzapE:zapE, and MG1655 ΔminCΔzapE:zapE(K84A) strains were grown overnight in LB (Lennox) media at 30°C. The next day, cultures were diluted into fresh LB  to an OD600 of 0.05 and grown for 3 h under mild heat shock conditions in static, closed cultures at 42°C, or aerobically at 37°C with shaking. Cells were harvested, normalized to total number of cells for SDS-PAGE, and then cell extracts were analyzed by Western blotting. Membranes were blocked with BSA (2%) in TBS containing Tween-20 (0.005%), and ZapE was detected with rabbit polyclonal antisera raised against ZapE followed by goat anti-rabbit antibody coupled to horseradish peroxidase. Membranes were developed using chemiluminescence.MG1655, MG1655 ΔEscherichia coli ZapE was previously reported to engage FtsZ in vitro and localize to the Z-ring in vivo (max). The reaction velocity plateaued near ∼1 pmol Pi min–1 pmol–1. Curve fitting to the Michaelis–Menten equation calculated a Vmax of 1.1 ± 0.09 pmol Pi min–1 pmol–1 with a Km of 1.7 ± 0.36 mM, under the conditions tested (–1 pmol–1) than ZapE with the N-terminal histidine tag, suggesting that the addition of a tag sequence to the C-terminus of ZapE may interfere with ATPase activity  as an N-terminal histidine fusion protein to quantitatively measure ATP hydrolysis. We observed no phosphate released in reactions containing ZapE(K84A) (6 μM) and ATP under the conditions tested, in contrast to wild-type ZapE  with ZapE (0 to –8 μM) in the presence of ATP (4 mM) for 5 min, added GTP (1.5 mM) to induce polymerization, and monitored the change in light scatter with time. We observed that as ZapE concentration increases, the light scattered and maximum intensity recorded also increased, with a greater than twofold increase in maximum light scatter comparing ZapE (0 μM) and ZapE (8 μM)  . The incE (8 μM) . In a coE (8 μM) . We alsoE (8 μM) .E. coli proteins reported to bundle FtsZ polymers , ZapE (10 μM), FtsZ (10 μM), FtsZ (10 μM) with GDP (2 mM), and FtsZ (10 μM) with GTP (2 mM) were applied as a spot to a nitrocellulose membrane and then blocked with BSA. A solution of ZapE (8 μM) with ATP (4 mM) was incubated with the membrane to allow for potential binding. Binding was then probed with a ZapE antibody. Our results show that ZapE binds FtsZ similarly under all conditions tested and that ZapE does not bind to the control spot containing BSA . These r in vivo . In the  in vivo . The amo in vivo . Finally in vivo , suggestE. coli PL directly in a phospholipid recruitment assay (–1) in the absence and presence of ATP (4 mM), and then supernatant and PL-pellet fractions were collected by low-speed centrifugation and analyzed by SDS-PAGE  ; however, cultures of MG1655 ΔzapE had a mean cell length of 3.86 ± 6.15 μm (n = 200), which is 38.8% longer than wild-type cells . We performed microscopy on cells in log phase and determined that both strains, zapE + (restored) and zapE(K84A), had a cell length distribution similar to wild-type MG1655 cells, and no filamentous cells were observed (zapE:zapE (restored) cells were observed to have a mean length of 2.26 ± 0.55 μm (n = 200), and MG1655 ΔzapE:zapE(K84A) cells were observed to have a mean length of 2.31 ± 0.60 μm (n = 200)  restores the moderate filamentation defect, similar to zapE.ZapE was reported to be an ATPase and also a cell division protein in e (42°C) . First, pe cells , with a  at 35°C . Notablyperature . Next, tobserved . Addition = 200) . These rzapE cells on solid LB medium without O2 by dilution spot-plating; however, we observed no major differences in the extent of growth of the two strains after 24 h  and 2.32 ± 0.12 μm (n = 100), respectively], with occasional cell filaments observed under both conditions   .zapE in combination with chromosomal deletion of another FtsZ assembly regulator, such as minC, further altered cell growth or morphology, we constructed a strain deleted for both minC and zapE by Lambda Red recombination. It was previously reported that deletion of either slmA or clpX caused a synthetic phenotype in combination with deletion of minC  at the chromosomal zapE locus restored the ability to recover after exposure to stress  and non-stressed conditions . We observed that ZapE was present in wild-type cells under both stressed and non-stressed conditions and not present in ΔzapE or ΔzapEΔminC deletion strains  is defective for ATP hydrolysis; however, ZapE(K84A) binds to and bundles FtsZ polymers. Many AAA + ATPases  form hexameric rings, and may be stabilized when bound to nucleotide , deletion of zapD leads to filamentation and a decrease in viability, suggesting the role of ZapD as a regulator of FtsZ   or clpX (synthetic filamentous)  . Our stumentous) . MinC spll poles . In cellThe original contributions presented in the study are included in the article/ED, RD, CT, and JC contributed to conception and design of the study. ED, RD, CT, and CF contributed to assay development and performed experiments. JM performed electron microscopy. ED wrote the first draft of the manuscript. CT, CF, JM, and JC wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Aedes albopictus is a competent vector of several viral arboviruses including yellow fever, dengue fever, and chikungunya. Several vital mosquito behaviors  are primarily dependent on the olfactory system for semiochemicals detection and discrimination. However, the limited number of studies hampers our understanding of the relationships between the Ae. albopictus olfactory system and the complex chemical world.The Asian tiger mosquito Ae. albopictus mosquitoes of different sexes, ages and physiological states, and found odorant receptor 11  enriched in non-blood-fed female mosquitoes. Then, we examined the odorant preference with a panel of physiologically and behaviorally relevant odorants in Xenopus oocytes.We performed RT-qPCR assay on antennae of The results indicated that AalbOr11 could be activated by ten aromatics, seven terpenes, six heterocyclics, and three alcohols. Furthermore, using post-RNA interference (RNAi) hand-in-cage assay, we found that reducing the transcript level of AalbOr11 affected the repellency activity mediated by (+)-fenchone at a lower concentration (0.01% v/v).Anopheles gambiae Or11. In addition, RNAi and bioassay suggested that AablOr11 might be one of the receptors mediating (+)-fenchone repellency activity. Our study attempted to link odor-induced behaviors to odorant reception and may lay the foundation for identifying active semiochemicals for monitoring or controlling mosquito populations.Using in vitro functional characterization, we found that AalbOr11 was a broadly tuned receptor. Moreover, we found that AalbOr11 shared a conserved odorant reception profile with homologous The online version contains supplementary material available at 10.1186/s13071-022-05158-1. Aedes albopictus is a vector for viral pathogens causing human diseases including yellow fever, dengue fever, and chikungunya. Due to strong ecological plasticity and a wide range of biting hosts, it is implicated in outbreaks of these diseases in areas where their primary vector, Aedes aegypti, is absent or outnumbered by Ae. albopictus [Ae. albopictus are increasingly becoming a global health concern [Ae. albopictus olfactory system and odorants might provide important information for developing active semiochemicals for monitoring or controlling populations.Female bopictus , 35, 41. concern , 29. Man concern , 31, 32.The olfactory system is essential for mosquitoes, since vital behaviors such as finding carbohydrate sources, hosts for blood meals, and oviposition sites and avoiding predators are dependent primarily on its detection of blends of volatile molecules from the complex chemical world . As two Anopheles darlingi) to 180 (Culex quinquefasciatus) [Anopheles gambiae have been identified and the majority has been deorphanized, which means that corresponding ligands of Ors have been found [An. gambiae, Ae. albopictus has extremely different behaviors, such as daytime biting and oviposition sites in containers [Aedes have been defined; for example, AaegOr4 is considered to be the key receptor for distinguishing humans from other animals [Aedes are still "orphan receptors" (no corresponding ligands have been found), which hampers our understanding of its olfactory system.Mosquitoes possess various numbers of the Or family, varying from 18 (sciatus) , 18, 23.en found . In compntainers , 27, 36, animals , and Aal animals , 39. DueAnopheles, Culex, and Aedes  data, we found that non-blood-fed (NBF) female mosquitoes possessed higher transcript levels of AalbOr11 than males (unpublished). In addition, the alignment of amino acid sequences indicated that Or11 was conserved among three major disease-transmitting vectors: pellents , 28, 42.Ae. albopictus used in this study was kindly provided by Zhejiang Provincial Center for Disease Control and Prevention, China. The colony was originated from a population collected from Sichuan, China, and has been maintained in insectary for 15 years without exposure to any insecticides. Mosquitoes were maintained at 27 ± 1 °C, 70 ± 10% relative humidity (RH), with a photoperiod of 14:10 (light/dark).The colony of To assess the relative transcript abundance of AalbOr11 in different sexes and ages, gene expression assays were conducted using the antennae of male and non blood-fed female mosquitoes at 1, 3, and 5 days post-eclosion (dpe). In addition, the mosquitoes were collected at 1 h, 48 h, and 96 h after blood-feeding to identify the effects of the blood-feeding behavior on relative transcript abundance of AalbOr11. After the mosquitoes were cold-anesthetized, their antennae were cut off with dissecting scissors and immediately placed in RNAlater-ICE  on ice prior to RNA extraction. The total RNA was extracted using TRIzol reagent  and isolated according to the manufacturer’s instructions. Then, 1 μg of total RNA was used as template for complementary DNA (cDNA) synthesis by reverse transcription using a PrimeScript™ RT Reagent Kit with gDNA Eraser  following the manufacturer’s instructions.−ΔΔCt method.The expression profile of AalbOr11 was determined using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). β-actin was used as the housekeeping gene , and qPCR was performed using a TB Green Premix Ex Taq II Kit . Each 20 μL qPCR reaction mixture consisted of 10 μL 2× TB Green Premix Ex Taq II mix (Tli RNase H Plus), 0.8 μL of each primer (10 μM), 2 μL diluted cDNA template, 0.4 μL ROX Reference Dye II (50X) , and 6 μXenopus laevis frog ovarian lobes using standard procedures as described previously [Gene-specific primers were designed based on coding sequences of putative AalbOr11 (XM_029861619.1) and identified AalbOrco (AALF000221/XM_029877254.1). PCR was performed using the following gene-specific primers containing Kozak motif (GCCACC): AalbOr11 F: GCCACCATGCAGCTCAAAGACGAATGGAT; AalbOr11R: TTAGCCGGCAGCTTGCTTCAGGA; AalbOrco F: GCCACCATGAACGTCCAGCCGACAAAGTA; AalbOrco R: TTATTTCAACTGCACCAACACCA. Subsequently, PCR products through sequencing validation were subcloned into pT7TS vector with the In-Fusion HD cloning kit . The capped RNA (cRNA) was prepared from linearized vectors and purified by mMESSAGE mMACHINE T7 kit  according to the manufacturer’s instructions. Mature healthy oocytes (stage V–VII) were isolated from female eviously . Each oo–1 M with DMSO. Odorant-induced current at a holding potential of –80 mV was recorded from the injected Xenopus oocytes using a two-electrode voltage-clamp setup . Data acquisition and analysis were carried out with an Axon Digidata 1550B and pCLAMP 10 software . Information regarding odorants used in electrophysiological recordings is provided in Additional file Odorant stock solutions were prepared at 10TAATACGACTCACTATAGGGACGACGTTTACGACAATCCG, AalbOr11-R: TAATACGACTCACTATAGGGATCCCAGAAAATCGCCTTCT; EGFP-F: TAATACGACTCACTATAGGGCCACAAGTTCAGCGTGTCCG, EGFP-R: TAATACG.ACTCACTATAGGGAAGTTCACCTTGATGCCGTTC. PCR products were amplified with Phusion High-Fidelity DNA Polymerase , and the PCR procedure was conducted with the following settings: 98 °C for 30 s, 35 cycles of 98 °C for 15 s, 60 °C for 30 s, and 72 °C for 10 s, followed by a final 10 min extension step at 72 °C. The purified and sequenced PCR fragments were used as the template for synthesizing dsRNA of AalbO11 and EGFP. The dsRNA molecules were synthesized using the TranscriptAid T7 High Yield Transcription Kit  according to the manufacturer’s protocol.The genes of interest (GOI) were amplified using gene-specific primers that included T7 promoter sequences (underlined), AalbOr11-F: 2 pad to be anesthetized. The anesthetized mosquitoes were lined up on the side for injection, and 1000 ng of dsRNAs in 0.5 μL volume was injected into one side of the thorax with a PLI-100 injector (World Precision Instruments). Post-injection, mosquitoes were put in new cages and supplied with sugar water (10% wt/vol). Males (ratio 1:1) were released into the cage for mating.Three-day-old female mosquitoes were collected and placed on a clean COThe hand-in-cage assay method used was identical to that described previously . Forty 4Ae. albopictus to identify the transcript level of AalbOr11 in mosquitoes of different sexes, ages, and physiological states. The transcription level of AalbOr11 in females was significantly higher than that in males among 1 dpe (P = 0.0021), 3 dpe (P = 0.0000001), and 5 dpe (P = 0.00005) mosquitoes , was used as the preliminary screening concentration. A total of 26 odorants elicited currents on AalbOr11/AalbOrco, of which the currents induced by seven odorants were greater than 300 nA. The seven strong ligands comprised two terpenes (+)-fenchone and (−)-fenchone, and five aromatics 3-methylindole, 2-ethyltoluene, indole, acetophenone, and 2-ethylphenol -fenchone, elicited robust mean current (~ up to 1500 nA) at a dose of 10–3 M, whereas at a dose of 5 × 10–6 M, (+)-fenchone only induced current to 10 nA, which activated AalbOr11/AalbOrco at half-maximal effective concentration (EC50) of 139.5 μM. Another compound, 2-ethyltoluene, which belongs to the aromatics, elicited lower responses at a dose of 10–3 M; however, its EC50 was 62.19 μM, thus representing the most sensitive ligand , and the knockdown effect of AalbOr11 was maintained for at least 5 days    -fenchone, as the subject to investigate odorant-induced behavior. Our hand-in-cage assay showed that (+)-fenchone elicited repellency in ion Fig. b. We conays Fig. a. Then 273) Fig. b. Moreov00) Fig. b. By conAe. aegypti, Anopheles coluzzii, and An. gambiae [Ae. aegypti antennae might contribute to human preference, and the differentially expressed Or4 responded to sulcatone, a human odorant, to discriminate human and non-human animals [Age-, sex-, and feeding state-dependent transcript level changes in odorant receptors have been found in other mosquitoes, such as  gambiae , 37, 40, gambiae . Our res animals . Our eleAnopheles, Culex, and Aedes. AalbOr11 shared 58.39%, 58.49%, and 69.5% overall sequence identity with Or11 of An. gambiae, An. coluzzii, and Cx. quinquefasciatus, respectively. Or11 of Ae. aegypti, another mosquito species belonging to the subfamily Aedes, was up to 93.57% identical to AalbOr11 -fenchone (0.01% v/v). In addition, reduced protection was observed with knockdown mosquitoes at a higher concentration (0.1%), although it was not statistically significant. On one hand, (+)-fenchone-elicited repellency might involve multiple Ors of Ae. albopictus. On the other hand, RNAi treatment reduced transcript levels by only c. 80%, and the remaining AalbOr11 still played a significant role in (+)-fenchone-mediated repellent activity. The possible link between reception and behavior has also been found in Cx. quinquefasciatus [The strong ligand of AalbOr11, (+)-fenchone, has been found to confer repellency to several insects, including  aegypti , Drosoph suzukii , and Proruncates . Similarasciatus . The sigasciatus . RNAi anWe found that AalbOr11 was highly conserved among three major disease-transmitting vectors and was enriched in non blood-fed female mosquitoes. According to in vitro functional characterization, AalbOr11 was a broadly tuned receptor. In addition, RNAi and bioassay suggested that AablOr11 may be one of the receptors mediating (+)-fenchone repellent activity. Our study provides further information regarding the mechanisms of olfactory-mediated mosquito behavior , and also provides new insight into active semiochemical identification for monitoring or controlling mosquito populations.Additional file 1: Figure S1. Aligned amino acid sequence from Aedes, Culex, and Anopheles. AablOr11 refers as Aedes albopictus Or11, AaegOr11 refers to Aedes aegypti Or11, CquiOr11 refers to Culex quinquefasciatus Or11, CpipOr11 refers to Culex pipiens pallens Or11, AgamOr11 refers to Anopheles gambiae Or11, AcolOr11 refers to Anopheles coluzzii Or11.Additional file 2: Table S1. Compounds used in both electrophysiological recordings and behavioral assays.


G37R mice, which are a familial ALS model. AAV6/skMLCK showed the successful expression of skMLCK in the muscle tissues. Although the control did not affect the muscle force in both of the WT and SOD1G37R mice, AAV6/skMLCK enhanced the twitch force of SOD1G37R mice and the tetanic force of WT and SOD1G37R mice. These results indicate that overexpression of skMLCK can enhance the tetanic force of healthy muscle as well as rescue weakened muscle function. In conclusion, the gene transfer of skMLCK has the potential to be a new therapy for ALS as well as for other neuromuscular diseases.Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that shows progressive muscle weakness. A few treatments exist including symptomatic therapies, which can prolong survival or reduce a symptom; however, no fundamental therapies have been found. As a therapeutic strategy, enhancing muscle force is important for patients’ quality of life. In this study, we focused on skeletal muscle-specific myosin regulatory light chain kinase (skMLCK), which potentially enhances muscle contraction, as overexpression of skMLCK was thought to improve muscle function. The adeno-associated virus serotype 6 encoding skMLCK (AAV6/skMLCK) and eGFP (control) was produced and injected intramuscularly into the lower limbs of SOD1 G93A  are well recognized as an ALS mouse model because they develop motor neuron disorders similar to that of a human ALS  for 1 h at room temperature and then in primary antibody in TTBS for 1 h at room temperature followed by secondary antibody in TTBS for 30 min at room temperature. After washing with TTBS (three times for 5 min each), membranes were incubated with the ECL Western Blotting Detection Reagent . The emitted light was detected and quantified with a chemiluminescence imaging analyzer, ImageQuant LAS 4000 .12 vg/mL, 50 μL, total viral copy was 1 × 1011 vg per mice) into the right lower limb and intramuscular injections with 50 μL of saline into the left lower limb (sham) were performed. The dose of viral vector and the method of intramuscular injection were determined based on previous articles [G93A mice have shown the phenotype of muscle weakness at around 90 days after birth [At 8 weeks of age, littermate- and copy number-matched mice were randomly divided into AAV6-eGFP-T2A-FLAG-skMLCK and AAV6-eGFP treatment groups. Intramuscular injections with AAV6 vectors . cDNA synthesis was performed using Applied Biosystems TM High-Capacity cDNA Reverse Transcription Kit . Reverse transcript PCR (RT-PCR) conditions were: 25 °C for 10 min followed by 2 h at 37 °C, and 5 min at 85 °C. Droplet digital PCR (ddPCR TM) was performed on QX200 TM Droplet Digital PCR system , using the PrimePCR TM ddPCR TM Expression Probe Assay for rat. ddPCR parameters were: 10 min at 95 °C, 40 cycles of 30 s at 94 °C, 1 min at 56 °C, and 10 min at 98 °C. TATA binding protein (Tbp) was used as an endogenous control. Each probe for human MYLK2 , mouse MYK2 , and mouse Tbp  were designed and purchased from Bio-Rad custom order.Total RNA was isolated from homogenized TA muscles using NucleoSpin®  in a sample buffer dyed with bromophenol blue (BPB) containing 50 mM Tris-HCl (pH 6.8), 10% grycerol, 1% sodium dodecylsulfate (SDS), and 6% 2-mercaptoethanol (2-ME). After sonication for 3 min and centrifugation at 14,000× g for 10 min, supernatants were collected as protein solution of muscle samples.Muscle samples were homogenized using MULTI BEADS SHOCKERMuscle tissues for immunofluorescence staining were embedded in an Optimal Cutting Temperature (OCT) matrix compound , frozen with dry ice, and mounted in the cryostat. Sections were cut for 20-μm thickness.HE stain was performed by Applied Medical Research Laboratory . The slides were observed by optical microscope .N,N,N′,N′-tetramethylethylenediamine. The resolving gel was composed of 12% acrylamide, 30 μM Phos-tag acrylamide , 60 μM MnCl2, 0.1% SDS, 375 mM Tris–HCl (pH 8.8), 0.05% ammonium persulfate, and 0.25% N,N,N′,N′-tetramethylethylenediamine. Electrophoresis was performed in 0.1% SDS, 25 mM Tris, and 192 mM glycine at 150 V per gel for 80 min. After electrophoresis, the gel was soaked in EDTA (+) transfer buffer containing 10 mM EDTA, 50 mM Tris, 380 mM glycine, 0.000375% SDS, and 20% EtOH for 10 min and then in transfer buffer for 10 min. Proteins were transferred to PVDF membranes  at 15 V for 30 min in transfer buffer. Membranes were then blocked with non-fat dry milk for 1 h at room temperature, incubated with primary antibody in non-fat dry milk for 1.5 h at room temperature followed by secondary antibody in non-fat dry milk for 45 min at room temperature, and detected as with SDS–PAGE. The amount of phosphorylation was analyzed by ImageQuant TL .For analysis of RLC phosphorylation by skMLCK, samples were subjected to phosphate affinity SDS–PAGE using an acrylamide-pendant phosphate-binding tag (Phos-tag SDS–PAGE ). Phos-t2) of isolated EDL muscle was calculated using the following formula: cross-sectional area = muscle mass/(muscle length × density), the density of muscle was 1.0564 g/cm3 [2) in the present study.The surgically isolated EDL muscles from mice were mounted on the force transducer . Muscles were stimulated with two silver chloride electrodes in oxygenated Tyrode’s buffer comprised of 134 mM NaCl, 5 mM KCl, 0.34, 1 mM MgCl2, 5 mM HEPES, and glucose 2 g/L, pH 7.4 at 37 °C. First, we investigated the muscle length for maximal isometric twitch tension (Lo). After adjusting muscle length to 0.7 Lo  , musclesp < 0.05 were considered to represent a significant difference.Statistical analyses were performed using the JMP version 14 statistical software . All data are expressed as means ± standard deviation (SD). A student’s t-test was performed for the comparison between the sham and AAV injected sides. Values of G37R mice, and the skMLCK vector enhanced the twitch force of the SOD1G37R mice and the tetanic force of the WT and SOD1G37R mice. We conclude that the gene transfer of skMLCK has the potential to rescue weakened muscle function due to various diseases.The AAV6/skMLCK vector successfully showed the overexpression of skMLCK in TA and EDL muscles. The control vector did not affect the muscle force in either of the WT or SOD1


Brassica rapa (B. rapa) is an important oilseed crop in northern China, but the mechanism of its cold resistance remains unclear. Ascorbate peroxidase (APX) plays important roles in the response of this plant to abiotic stress and in scavenging free radicals. In this study, the roles of APX proteins in the cold response and superoxide metabolism pathways in rapeseed species were investigated, and a comprehensive analysis of phylogeny, chromosome distribution, motif identification, sequence structure, gene duplication, and RNA-seq expression profiles in the APX gene family was conducted. Most BrAPX genes were specifically expressed under cold stress and behaved significantly differently in cold-tolerant and weakly cold-resistant varieties. Quantitative real-time-PCR (qRT-PCR) was also used to verify the differences in expression between these two varieties under cold, freezing, drought and heat stress. The expression of five BrAPX genes was significantly upregulated in growth cones at 3 h of cold stress, while their expression was significantly lower at 24 h than at 3 h. The expression of Bra015403 and Bra003918 was significantly higher in “Longyou-7” growth cones than in other treatments. Five BrAPXs  were closely associated with abiotic stress responses in B. rapa. These candidate genes may play important roles in the response of B. rapa to low temperature stress and provide new information for the elucidation of the cold resistance mechanism in B. rapa.Winter  Brassica rapa (B. rapa)  comprises several subspecies that provide human nutrition in the form of leafy, root and stem vegetables and edible oils. It also represents the origin of the genome of Brassica “A” genome and contributes to the Brassica allopolyploid oil crops Brassica napus (B. napus) (AACC) and Brassica juncea (B. juncea) (AABB)  (AABB) . Thus, Bvailable . B. rapail crops . Overwinow 30 mm . Unfavouow 30 mm . Accordiwintered . The bre2−), hydrogen peroxide (H2O2) and hydroxyl radicals (OH−), which destroy DNA and cellular components via lipid peroxidation and protein oxidation. Most cold-tolerant plants have evolved antioxidant defense mechanisms to defend against excessive accumulation of these ROS substances, including protective substances such as ascorbic acid, superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX)  analysis showed that APX/GPX genes were significantly regulated under drought stress  play an important role as a regulator of cellular response to environmental factors in plants, including superoxide anions (Ose (APX) . APX belse (APX) . APX is sistance . The impt stress . Antioxid stress . A studyaptation . Sato etAPX genes in B. rapa and performed a comprehensive analysis of their phylogeny, chromosome distribution, motif identification, sequence structure and gene duplication. We also used RNA-seq data to identify APX genes in B. rapa and B. napus expressed in response to cold stress and used qRT-PCR data to validate the specific expression of these genes under cold stress. This study provides information on the involvement of APX genes in the cold stress response in B. rapa, which will help to further elucidate the mechanism underlying strong cold resistance in B. rapa.In this study, we conducted a genome-wide identification of 118 family members of B. rapa varieties, “Longyou-7”  and “Lenox” , were selected for their wide variation in cold resistance. The overwintering rates of “Longyou-7” and “Lenox” for many years have been approximately 90 and 10%, respectively, in Shangchuan Town, Yongdeng County, Lanzhou City, Gansu Province, China , where the altitude is 2,150 m, the average annual temperature is 6.5°C, the average minimum temperature in the coldest month is approximately −14.6°C, the extreme low temperature is −26.5°C and the average annual precipitation is 175 mm  for 2 days. Three uniform seedlings of each cultivar were selected, with good growth condition and consistent growth stages, and transplanted into 15-cm diameter seedling pots filled with matrix and vermiculite . At the six-leaf stage, cold, freezing and heat stress treatments were carried out in incubators at 4°C, −4°C and 40°C, respectively. Drought stress treatment was applied by adding 200 ml of a nutrient solution containing 18% PEG6000 to the medium of each pot. Under a variety of abiotic stresses, the leaves of these two varieties with different cold resistance showed markedly different phenotypic characteristics as the treatment time progressed. Growth cones are considered to be the most direct site for sensing temperature changes and stresses, while roots (root length and root diameter) can show great variation in pots. Studies have shown that cold stress causes significant changes in phenotype and gene expression in rapeseed leaves and growth cones  file of the peroxidase domain PF00141 was downloaded from the Pfam databasebase BRAD2  following the manufacturer’s instructions and removing genomic DNA contamination. A spectrophotometer  was used to evaluate RNA concentration and mass by determining the A260/A280 and A260/A230 ratios, respectively. First-strand cDNA was synthesized with Evo M-MLV RT Premix  according to the instructions. qRT-PCR was performed on an ABI QuantStudio 5  using the SYBR® Green Premix Pro Taq HS qRT-PCR Kit  with primers provided in Supplementary Table S1. The qRT-PCR reaction conditions were as follows: 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 30 s at 60°C, followed by 65–95°C melting curve detection. The qRT-PCR efficiency of the genes was obtained by analyzing the standard curve of cDNA gradient dilution, and the gene fragment encoding B. rapa β-actin RNA was used as the internal control to normalize the amount of template cDNA. Relative expression values for each gene were computed using the comparative 2-ΔΔCT method with normalization to the internal control gene  and “Lenox” (weakly cold-resistant) under cold stress  (B. rapa cultivars “Longyou-7” (ultra-cold resistance) and “Tianyou-2” (weakly cold-resistance) under freezing stress 3. RNA-seq library was derived from leaves of B. napus cultivars “NTS-309” (strong cold resistance) and “Tianyou-2238” (weakly cold-resistant) under cold stress (25°C as a control and 4°C for 48 h) (B. napus cultivar “2016TS(G)10” (strong cold-resistance) under freezing stress   was derived from growth cones of nd 24 h) . 2. RNA-or 48 h) . 4. RNA-nd 24 h) . RNA-seqhan 0.05 . Interacp-value ≤ 0.05 was considered statistically significant. All results are represented as the mean ± standard error of the mean of at least three replications.One-way analysis of variance and Duncan’s multiple range test were used to detect significant differences among the means of the plant treatment groups using SPSS 19.0 statistical software . A APX genes in B. rapa and B. napus was completed using HMM searches with the aid of the BRAD genome database (version 1.5). These proteins were identified as having the reported peroxidase domains after sequence analysis using the SMART, CDD, Pfam and InterProScan tools. Finally, 118 and 221 candidate APX genes were obtained for B. rapa and B. napus, respectively. BrAPX amino acid residue lengths ranged from 250 aa (Bra030706) to 723 aa (Bra011683), isoelectric point (pI) values ranged from 4.40 (Bra036445) to 10.78 (Bra019132), and molecular weights ranged from 27.39 kDa (Bra017830) to 80.97 kDa (Bra011683) (APX genes in B. rapa (APX genes are more widely distributed in the B. rapa genome.The identification of a011683) . We mapp B. rapa , with moB. napus and B. rapa, a rootless maximum likelihood (ML) phylogenetic tree was constructed with APX protein sequences of B. napus and B. rapa to explore the reasons for their cold resistance   . Bra0352APX genes differentially expressed under cold stress under other abiotic stresses, we performed qRT-PCR analysis on these two varieties under heat (40°C), PEG , and freezing (−4°C) stress conditions for 3 and 24 h  were significantly expressed in cold-tolerant varieties under freezing stress and were more highly expressed than in weakly cold-resistant varieties. Bra035235 and Bra017120 were expressed in “Longyou-7” growth cones at levels 2 and 2.9 times higher than “Lenox” growth cones at −4°C under 3 h treatment, respectively. The expression of Bra003918 and Bra033040 in “Longyou-7” growth cones was 4.3, 3.4, 2.2, and 6 times higher than in ‘Lenox’ growth cones at −4°C for 3 and 24 h treatments, respectively. The expression of these genes was higher in growth cones than in leaves, suggesting that cold-tolerant varieties of growth cones have an important role in cold resistance. When leaves wilted after the cold overwintering period in northern China, these results were also shown during cold stress. After 24 h of simulated drought stress, Bra035235, Bra002269, Bra033040, Bra017120 and Bra031934 were significantly upregulated in “Longyou-7” growth cones, at levels 13.4, 6.6, 3.4, 32.9, and 10 times that of “Lenox” growth cones, respectively. The expression of Bra003918 and Bra015403 was higher in “Longyou-7” leaves than in growth cones under drought stress. Under heat stress, Bra035235, Bra003918, Bra015403, Bra033040 and Bra031934 were significantly upregulated in growth cones and leaves of both species, with higher expression in “Longyou-7” than in “Lenox”. The expression of Bra017120 in “Longyou-7” growth cones was 12.2 and 2.7 times higher than that in “Lenox” growth cones at 3 and 24 h, respectively. The results showed that B. rapa leaves were more susceptible to heat stress, and cold-tolerant varieties may additionally have had stronger heat tolerance. We performed interaction analysis on the 10 genes screened, a total of 90 pairs of interacting proteins were predicted. All nine proteins interacted with Bra011510, Bra016041, Bra017639, and Bra022425, except for Bra011683. There were 14 pairs of proteins that interacted with Bra035235 and 10 pairs of proteins that interacted with Bra017120. In addition, these genes are involved in plant peroxidase, plant methyltransferase dimerization, and phenylpropanoid biosynthesis pathways (STRING 11.5 software) (B. rapa and deserve further study.To further investigate the expression patterns of these 10 and 24 h . Five APoftware) . In summ2O2 elimination. It also plays a role in plant growth and development as well as adversity response , dehydroascorbate reductase (DHAR), and glutathione reductase (GR). The ascorbate-glutathione or Halliwell-ASADA cycle, which combines these processes, is one of the most important antioxidant systems in plants. Ascorbate and glutathione are employed as reducing substrates for scavenging of H2O2 in this cycle, and they are ultimately cycled at the expense of ATP and NAD(P)H  belongs to the peroxidase (PF00141) family, and is considered to be a key enzyme in Hresponse . The det NAD(P)H . AlthougAPX genes were identified in B. rapa, and comparisons with the B. napus APX gene family revealed that the A genome contained much less APX members than the AC genomes, indicating that APX is closely linked evolutionarily between these two species. In the B. rapa APX gene family, there have been no tandem duplication events, but there have been 62 segmental duplication events, with the majority of the BrAPX gene segmental duplications localized on chromosomes A1, A2, and A3. In B. napus, we also found greater numbers of segmental duplications on chromosomes A1, A2, and A3. This suggests that segmental duplication events have played an important role in the evolution of the BrAPX genes. The research showed that all of Brassica underwent an extra whole genome triplication (WGT) event that occurred approximately 9–15 million years ago, or even approximately 28 million years ago , whereas it was not significantly expressed in Lenox  responsive to abiotic stress were found. These genes are closely related to the abiotic stress response of B. rapa, which is worthy of further study. In addition, we have identified a number of candidate genes  that may play important roles in the response of B. rapa and B. napus to cold and freezing stresses. This work provides new ideas for studying the involvement of the APX gene family in the molecular mechanisms of cold resistance in B. rapa.In summary, we identified 118