Ganoderma mastoporum collected in Vietnam was purified to afford eight compounds, including three triterpenoids and five steroids. The purified compounds were examined for their inhibitory effects against superoxide anion generation and elastase release. Among the tested compounds, ergosta-4,6,8(14),22-tetraen-3-one (3) exhibited the most significant inhibition towards superoxide anion generation and elastase release with IC50 values of 2.30 ± 0.38 and 1.94 ± 0.50 µg/mL, respectively.The methanol extracts of the fruiting bodies of  Ganoderma (Polyporaceae) are extensively used as a Chinese remedy for the treatment of tumors [G. lucidum attract much attention because it is a well-known source of various therapeutic agents [Ganoderma genus afforded a series of triterpenoids and steroids [G. mastoporum is a fungus widely distributed in Southern China, Northern Vietnam, Malaysia, Philippines, and reported to contain sesquiterpenoids [G. mastoporum and their bioactivities are merited. In our continuous program aimed at the discovery of anti-inflammatory lead drugs from the natural sources, the chemical composition of the fruiting bodies of G. mastoporum collected in Vietnam was investigated to search for the bioactive constituents. In the present study, we wished to report the identification of eight compounds, as well as their inhibitory effects on superoxide anion generation and elastase release.The genus f tumors ,2, hepatf tumors , enhancef tumors . Speciesc agents ,6. Previsteroids ,11,12,13steroids ,15. G. mrpenoids . TherefoGanoderma mastoporum were extracted with methanol, and the combined extracts were concentrated under reduced pressure to give a deep brown syrup. The crude extract was suspended in water and partitioned with ethyl acetate to afford ethyl acetate and water soluble fractions, respectively. Purification of the ethyl acetate fraction by a conventional combination of column chromatographies yielded three triterpenes: Δ1-lupenone (1) [5), lucidumol B (6) [β-ol (2) [3), ergosterol peroxide (4) [7) [β,5α-dihydroxy--ergosta-7,22-dien-6-one (8) [Air-dried and powdered fruiting bodies of none (1) , ganoderol B (6) , and fivβ-ol (2) , ergostaxide (4) , ergosta (4) [7) , and 3β,-one (8) , respectN-formyl-L-methionyl-leucyl-phenylalanine/cytochalasin B (FMLP/CB) [2 and 8 did not display significant inhibition of superoxide anion generation and elastase release. Other tested compounds 1 and 3–7 demonstrated inhibitory effects towards superoxide anion generation and elastase release in a concentration-dependent manner (data not shown). The IC50 values were in the range between 2.30 ± 0.38 and 6.36 ± 0.36 μg/mL against superoxide anion generation, and between 1.94 ± 0.50 and 5.01 ± 0.82 µg/mL against elastase release, respectively, compared with the reference compound LY294002 (IC50 of 0.40 ± 0.02 and 1.53 ± 0.25 µg/mL towards superoxide anion generation and elastase release). Among the tested compounds, ergosta-4,6,8(14),22-tetraen-3-one (3) exhibited the most significant inhibition towards superoxide anion generation and elastase release with IC50 of 2.30 ± 0.38 and 1.94 ± 0.50 µg/mL. From the above results, the following structure-bioactivity relationships were deduced: triterpenoids or steroids possessing 3-hydroxy rather than 3-ketone functionalities, such as 2, 4, 6, and 8, displayed weaker superoxide anion generation inhibitory effects. Comparison of the bioactivity of 2 and 7 also exhibited this tendency. The 24,25-dihydroxy substitutions in 5 and 6, and the 5-hydroxy group in 8 decreased the inhibition of superoxide anion generation. There is similar tendency in the inhibition of elastase release. However, lucidumol B (6) displayed more significant inhibition of elastase release than 5 and 7.The purified compounds were subjected to the examination for inhibition of superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB) , and the1–8 were also subjected to assays of DPPH free radical scavenging activities and ferrous ion chelating capability [1 and 3–7 is mediated by modulating cellular signaling pathways [1-lupenone (1) and ergosterol peroxide (4) were reported to exhibit the inhibitory effects on LPS-induced iNOS-dependent NO production in RAW 264.7 cells [β-ol (2), ergosterol peroxide (4), and ergosta-7,22-dien-3-one (7) also inhibited superoxide anion generation release by rat neutrophils [The formation of superoxide anion in neutrophils can be inhibited by modulating cellular signaling pathways, but also by direct radical scavenging. Therefore the purified constituents pability . None ofpathways . In the .7 cells ,26,27. Mtrophils . In the 1H- and 13C-NMR, COSY, NOESY, HMQC, and HMBC spectra were obtained on the Bruker Avance III-400 NMR spectrometer , with tetramethylsilane (TMS) as internal standard and the chemical shifts were reported in δ values (ppm). The electrospray ionization (ESI) mass spectrum was determined using an Agilent 1200 LC-MSD Trap spectrometer . Column chromatography (CC) was performed on silica gel . Thin layer chromatography (TLC) was conducted on precoated Kieselgel 60 F 254 plates (Merck) and the compounds were visualized by spraying with 10% (v/v) H2SO4 followed by heating at 110 °C for 10 min.Melting points were determined using Yanagimoto MP-S3 apparatus . Optical rotations were measured using a JASCO DIP-370 polarimeter . The UV spectra were obtained on a Hitachi UV-3210 spectrophotometer , and IR spectra were recorded on a Shimadzu FTIR-8501 spectrophotometer . Ganoderma mastoporum (Mont) Pat.) was collected at the Puhuong National Park of Nghean Province, Vietnam, in November 2010 and identified by Dr. Ngo Anh, Department of Biology, Hue University. A voucher specimen (Vinh-TSWu 20100805) was deposited at the herbarium of the Department of Chemistry, Vinh University.The basidiomycete (Ganoderma mastoporum (4.3 Kg) were air-dried, powdered, and extracted with methanol (10 L × 3) at ambient temperature, and the combined extracts were concentrated under reduced pressure to give a deep brown syrup (125 g). The crude extract was suspended in water and partitioned with ethyl acetate to afford ethyl acetate (30 g) and water soluble (95 g) fractions, respectively.The fruiting bodies of n-hexane and acetone gradients (15:1 to 1:1) to afford five fractions. Fractions 1–2 and 4–5 did not display significant triterpenoid and steroid spots monitored by TLC so that they were not further purified. Fraction 3 was a triterpene-rich fraction visualized by spraying with 10% (v/v) H2SO4 followed by heating at 110 °C for 10 min. It was subjected to silica gel column chromatography eluted with chloroform and methanol gradients (100:1 to 1:1) to afford three subfractions. Subfraction 3-1 was purified by SiO2 CC (eluted with chloroform/ethyl acetate 20:1) and further recrystallization of the resulted minor fractions with chloroform/methanol to yield Δ1-lupenone  and ergosta-7,22-dien-3β-ol , respectively. Subfraction 3-2 was repeatedly purified by SiO2 CC and preparative TLC (pTLC) (eluted with benzene/ethyl acetate 20:1) to afford ergosta-4,6,8(14),22-tetraen-3-one , ergosterol peroxide , and ganodermanondiol , respectively. Subfraction 3-3 was purified by SiO2 CC (eluted with benzene/acetone 50:1) to yield three minor fractions (3-3-1~3-3-3). Fractions 3-3-1 and 3-3-2 were repeatedly purified by SiO2 CC and pTLC (eluted with benzene/ethyl acetate 20:1) to afford lucidumol B , and ergosta-7,22-dien-3-one , respectively. Fractions 3-3-3 was further recrystallized with chloroform /methanol to result in 3β,5α-dihydroxy--ergosta-7,22-dien-6-one .The ethyl acetate soluble extracts were subjected to silica gel column chromatography (CC) with Neutrophils were isolated with a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes. Blood was drawn from healthy human donors (20–30 years old) by venipuncture into heparin-coated vacutainer tubes, using a protocol approved by the institutional review board at Chang Gung Memorial Hospital .The assay of the generation of superoxide anion was based on the SOD-inhibitable reduction of ferricytochrome c . CalculaDegranulation of azurophilic granules was determined by elastase release as described previously . The resThe DPPH free radical scavenging assay was executed according to the reported method . The DPPThe capability of chelating ferrous ions was determined by the reported method with some modifications . The rel50) was computer-assisted (PHARM/PCS v.4.2). Statistical comparisons were made between groups using Student’s t test. Values of p less than 0.05 were considered to be statistically significant.Results were expressed as mean ± S.E.M. Computation of 50% inhibitory concentration (ICG. mastoporum collected in Vietnam were characterized and their inhibition of superoxide anion generation and elastase release by human neutrophils in response to FMLP/cytochalasin B was examined. The results provide evidence for the use of the fruiting bodies of G. mastoporum as herbal medicines in the treatment of inflammatory diseases, and these purified principles may be potentially useful in developing new anti-inflammatory therapeutic agents.In summary, three triterpenoids and five steroids from the fruiting bodies of


Sexual behavior is the open manifestation of a complex interplay between psychophysiological mechanisms that either facilitate or inhibit sexual thoughts, desires, and associated behaviors. Whereas sexual excitation has been widely studied, less is known about the impact of inhibitory control mechanisms that enable individuals to refrain from sexual cognition and behavior. The present study examined: (1) the relationship between general and sexual inhibitory mechanisms (as measured through self-reports and computer-based tasks), (2) the relation between sexual inhibitory processes at cognitive and motor-motivational levels and with sexual inhibition as an individual trait, and (3) the predictive value of these parameters on sexual thoughts (cognition) and behavior. We demonstrate that general inhibitory control  and the specific inhibition of sexual responses represent distinct processes that require at least partly different control mechanisms. Similarly, the ability to inhibit sexual visual input and the ability to suppress sexually driven responses seem to be two independent processes. The different inhibitory processes distinctively predicted the frequency of sexual thoughts and sexual behavior. We propose that these different inhibitory mechanisms are at play during different phases of sexual regulation  and that a specific deficit in one of these processes may underlie the distinctive symptomatology and comorbidity of sexual disorders. Human sexuality is an essential part of our existence and comprises a broad range of aspects, including cognitive, emotional, behavioral, and physiological processes. These processes are modulated by a complex interplay between inhibitory  and excitatory  mechanisms, shaping the manifestation of sexual thoughts (cognition), desire , and behavior. The successful inhibition of sexual expressions is a natural and adaptive reaction to contextual factors  task to measure the control over a motivationally driven motor response, thus comprising a peripheral inhibitory component. We formulated the following propositions:Sexual motor inhibition (AA) relates to general motor response inhibition (measured by the Go/No-go task) as both processes require withholding a dominant motor response.The second factor of Sexual Inhibition (SIS2) trait relates to general self-control (BSCS) as both traits comprise the ability to withhold actions that lead to pleasurable consequences by means of higher-order processing such as preventing negative consequences or pursuing long-term goals.We further aimed to test whether different sexual inhibition processes relate to each other and to sexual inhibition as trait, or whether they comprise distinct constructs. As we proposed that sexual inhibition does not compromise a unitary process and that different mechanisms withhold the inhibition at different phases of the cascade of sexual arousal responding, we used two distinct paradigms to assess sexual inhibition. In addition to the AA task, which measures sexual motor inhibition, we selected the Negative Affective Priming (NAP) task. This task allowed us to study sexual inhibition at a central cognitive level, as subjects are required to ignore an affective distractor while attending a non-affective target; in a post-trial, the subjects are required to respond to a stimulus with the same content type than the one that was previous inhibited, which causes a response delay compared to non-priming trials. We expected:The two sexual inhibitory processes are not related, as one comprises cognitive processing (NAP) and the second (AA) comprises a rather “hot” component, as individuals are required to either approach or avoid sexual stimuli.The second factor of Sexual Inhibition relates to sexual motor inhibition as process (AA) since both constructs imply the control over sexually rewarding actions and thus comprise a strong motivational and peripheral component.Finally, since we hypothesized that different inhibitory mechanisms play a distinct role at different levels of the sexual arousal unfolding, and because sexual responses and related behaviors result from the balance between excitatory and inhibitory mechanisms, we tested whether different general and sexual inhibition traits and processes along with sexual excitation  predicts the frequency of sexual thoughts as sexual cognitive inhibition reveals the control over central processes through attention.The sexual motor inhibition (AA) and general inhibition processes (Go/No-go) relate directly to sexual behavior since they reflect the motor control over preponderant responses.Sexual inhibition (second factor) and general inhibition (self-control) as traits have predictive value regarding sexual behavior, as both constructs measure the ability to control pleasurable actions at the foresight of long-term consequences.Sexual excitability is a significant predictor of sexual thoughts and sexual behavior.As women and men respond differentially during sexual cognitive and inhibitory processes Dewitte,  and becaM age = 24.46; SD = 4.32) were recruited through advertising in Maastricht University halls and in a Facebook group created to recruit participants for research at Maastricht University. All of the participants were financially reimbursed for their participation with 10 euros in vouchers. The study was approved by the local ethical committee. A total of 90.3% of participants were Bachelor or Master students; from the rest, three participants had completed university and two had completed high school. Approximately half of the participants were single (53.8%); the others had been in a relationship for an average period of 2.7 years (SD = 2.85). Single and coupled men did not differ in age nor in individual traits  measured by self-reports.A total of 52 self-declared heterosexual male participants  and one non-sexual (Go/No-go task). The order in which the two sexual tasks were presented was counterbalanced. The Go/No-go task was always presented between the sexual tasks to prevent habituation to sexual stimuli. The questionnaires were completed at the end of the session. To increase the confidence of participants on the anonymity of their answers and increase their comfort, their identities were registered as number codes and they were left alone in the room while answering.Our version of the task , the content of the distractor picture in the prime trial matched the content type of the target in the probe trial, whereas in the control trial sequences (No Priming) the content of these two pictures differed. The target in the probe trial could be sexual or non-sexual. This way, there were four different conditions: Sex Priming , Sex No Priming , Non-Sex Priming , and Non-Sex No Priming  Fig. a. The prAs the inhibitory effect is implied from the response delay in priming conditions, we calculated a sexual priming index by subtracting the reaction times in Sex No Priming trials to those of Sex Priming trials. Because we were specifically interested in the sexual priming effect, we subtracted a non-sexual priming index to the sexual priming index ([Sex Priming–Sex No Priming]–[Non-Sex Priming–Non-Sex No Priming]) to exclude the non-sexual inhibitory component. This index was the main score used for this task. A higher index indicated a stronger sexual priming effect and thus a stronger sexual inhibition.This task consisted of four blocks with 48 stimuli each, half of them of sexual content and the rest with non-sexual content  were displayed on gray background . Participants could choose from a 1 to 5-point Likert scale ranging from This 45-item scale measures the propensity for sexual inhibition and excitation. It contains one factor quantifying sexual excitation  and two factors quantifying sexual inhibition: (1) SIS1–Inhibition derived from threat of sexual performance failure, distraction or lack of physical stimulation  and (2) SIS2–Inhibition due to the threat of performance consequences, such as risk of being caught or sexually transmitted diseases . Response options were on a 4-point Likert scale , participants answered according to their experience during the last 4 weeks how often they: (1) thought about sex, (2) watched pornography, (3) masturbated, and (4) had sex (intercourse with penetration). Participants could answer on a 6-point Likert scale, ranging from Mean and SD for the self-reported measures and sexual behaviors are given in Table F = 15.52, p = .001; Priming: M = 837, SD = 143, No Priming: M = 811, SD = 145, and an interaction between stimuli and condition, F = 8.15, p = .006; Table F = 1.11, p = .29; Sex: M = 820, SD = 147 ms; Non-Sex stimuli: M = 829, SD = 145 , showing that the omission of visual sexual stimuli requires stronger inhibition.A 2 (Stimuli: Sex vs. Non-Sex) × 2 (Condition: Priming vs. No Priming) ANOVA revealed a main effect of priming condition, 145 Fig. , the priF = 13.93, p = .001; Sex trials: M = 1024, SD = 198 ms, Control trials: M = 1055, SD = 183 ms, which is in concordance with previous evidence  = .28, p = .59; Approach trials: M = 1037, SD = 188 ms, Avoid trials: M = 1041, SD = 195 ms, and there was a significant interaction between stimuli and condition, F = 8.07, p = .006, indicating a faster response approaching sexual stimuli over the other conditions (Table A 2 (Stimuli: Sex vs. Control) × 2 (Condition: Approach vs. Avoidance) ANOVA revealed that participants were faster responding to sexual than to control stimuli, Dewitte, . ReactioM = 242, SD = 36 ms, Go trials: M = 358, SD = 34 ms; t = − 15.49, p = .001). For further analyses, false alarms were log-transformed to correct skewness. To examine whether relationship status would influence performance on the implicit measures, we conducted a series of tests, which revealed that single and partnered men did not differ significantly in their performance on none of the three tasks.Participants committed on average 19.51 SD = 14.62) false alarms and 7.86 (SD = 8.91) misses , self-control (BSCS), sexual cognitive inhibition (NAP), sexual motor inhibition (AA), and sexual inhibition as a trait (SIS2) were significant Table .Table 4CTo investigate whether sexual motor inhibition, sexual cognitive inhibition, and sexual inhibition as a trait were related, we performed correlation analyses Table . There wTo investigate whether sexual inhibition, general inhibition, self-control, and sexual excitation were related to the frequency of different sexual manifestations, we performed correlation and multiple regression analyses. In Table To predict the frequency of sexual thoughts, we entered the Sexual Cognitive Inhibition (NAP) index and Sexual Excitation (SES) scores to the model. To predict the frequency of pornography watching, masturbation, and intercourse, we entered the sexual motor inhibition (AA) and general inhibition  indices, along with the self-control (BSCS), sexual inhibition (SIS2), and sexual excitation (SES) trait scores in separate models. To exclude the fact that sexual cognitive inhibition (NAP) would predict the frequency of sexual behavior or that sexual motor inhibition (AA) and sexual inhibition as a trait (SIS2) would predict the frequency of sexual thoughts, we repeated the same analyses including both sexual inhibition processes and sexual inhibition as trait into the models. These variables were included stepwise into the model as we did not have a priori predictions about which variables would be more relevant in predicting the different sexual manifestations.R2 = .21; p = .005; Table R2 = .16; p = .016; Table R2 = .08; p = .04; Table The regression analysis showed that NAP and sexual excitability predicted the frequency of sexual thoughts  along with sexual excitation as a trait distinctively predicted the frequency of two spheres of sexuality: thoughts and behavior.Regarding the question of whether sexual inhibition and general inhibition as traits and processes would constitute different or related constructs, our results showed no significant relationship between sexual and general inhibition as processes. Contrary to our prediction in Hypothesis 1, the ability to inhibit a sexually driven response (AA) seems independent from the general ability to suppress a preponderant response (Go/No-go). Similarly, we did not find support for Hypothesis 2 which predicted that the general ability to control behavior regarded as trait (self-control) would relate to the degree to which individuals inhibit their sexual arousal due to the risk of undesired consequences (SIS2). Altogether, these findings do not support that sexual inhibition derives from a general inhibitory domain and instead requires specific cognitive mechanisms. Although general inhibition deficits can impact on sexual behavior as seen in frontal brain damage patients  involves the capacity to inhibit sexual visual information. With this regard, it had been debated whether the negative priming effect reflected retrieval or inhibition processes  did not relate to sexual inhibition as a trait (SIS2). This may indicate that the Approach–Avoidance paradigm is not sensitive enough to reflect the inhibition of sexual arousal in quotidian circumstances. It is also possible that what is measured by this factor of the SIS  is inherently different from the motor response to approach or avoid a sexual stimulus; whereas the latter constitutes an intentional action, the former one refers to the automatic body response as a product of fear of the sexual performance consequences .Finally, we examined whether the different inhibitory processes and traits, on the one hand, and sexual excitability, on the other hand, would distinctively predict specific sexual expressions. We expected that the sexual cognitive inhibition would predict the frequency of sexual thoughts (Hypothesis 5). Our results supported this hypothesis, which is in line with the process model of emotion regulation Gross, , statingTo inhibit sexual behavior, other inhibitory mechanisms are needed. Hypothesis 6 predicted that sexual motor inhibition and general inhibition as processes would directly relate to sexual behavior. The ability to inhibit sexually driven motor responses  predicted the frequency of watching pornography. This finding is in line with previous research showing that this task correlates with sexual behavior at the implicit level (viewing time of erotic stimuli) but not at the explicit level   did not predict the frequency of any sexual manifestation. This was unexpected, as in a previous study sexually compulsive individuals committed more false alarms and misses in the same task  would also predict sexual behavior. Self-control was also a significant predictor of the frequency of watching pornography. In quotidian life, one should overcome pleasant temptations in order to achieve mid- and long-term goals. In this regard, self-control is relevant to regulate a behavior that can be highly rewarding, and unlike intercourse, risk-free and easily accessible.The frequency of intercourse was predicted by sexual inhibition as a trait (SIS2). This construct emphasizes the decrease in sexual arousal and erectile response under the risk of future consequences. Therefore, the inhibition of sexual physiological responses occurring through higher-order processing  seems crucial in the regulation of dyadic sex. This is relevant since the failure of this inhibitory mechanism would presumably lead to harmful consequences for the individual and for others. In fact, this factor has been previously related to risky sexual behavior . For ethical reasons, the participants were aware that the study would include erotic material which could also have resulted in a biased self-selected sample.In addition, as occurs with small samples, the current study entails the risk of false negative results. However, the alpha values of the non-significant results were substantially far beyond the threshold of significance. Another problem of small samples is the potential inaccuracy in the magnitude of the correlation values . Future studies can include additional physiological indicators of sexual arousability  to have a more integrative perspective.Although we tried to include ecologically valid measurements for sexual behavior , we assessed those only via self-reports. To study sexual inhibition and sexual behavior, future studies can also consider to include behavioral non-computer-based measurements (for examples, see Gailliot & Baumeister, This work aimed to study the relationship between different inhibitory mechanisms and their specific role in regulating sexuality at the cognitive and at the behavioral level. The presented evidence supports the notion that sexual inhibition is distinct from one’s general ability of inhibitory control. Along with clinical observations in brain damage patients showing that sexual disinhibition is not always accompanied by a general inhibition deficit (Baird et al.,


Zymomonas mobilis under anaerobic, aerobic, and N2-fixing conditions. Our phosphoproteome analysis revealed 125 unique phosphorylated proteins, belonging to major pathways such as glycolysis, TCA cycle, electron transport, nitrogen metabolism, and protein synthesis. Quantitative analysis revealed significant and widespread changes in protein phosphorylation across growth conditions. For example, we observed increased phosphorylation of nearly all glycolytic enzymes and a large fraction of ribosomal proteins during aerobic and N2-fixing conditions. We also observed substantial changes in the phosphorylation status of enzymes and regulatory proteins involved in nitrogen fixation and ammonia assimilation during N2-fixing conditions, including nitrogenase, the Rnf electron transport complex, the transcription factor NifA, GS-GOGAT cycle enzymes, and the PII regulatory protein. This suggested that protein phosphorylation may play an important role at regulating all aspects of nitrogen metabolism in Z. mobilis. This study provides new knowledge regarding the specific pathways and cellular processes that may be regulated by protein phosphorylation in this important industrial organism and provides a useful road map for future experiments that investigate the physiological role of specific phosphorylation events in Z. mobilis.Protein phosphorylation is a post-translational modification with widespread regulatory roles in both eukaryotes and prokaryotes. Using mass spectrometry, we performed a genome wide investigation of protein phosphorylation in the non-model organism and biofuel producer  Zymomonas mobilis, a facultatively anaerobic alphaproteobacterium, possesses several desirable characteristics for industrial biofuel production. These include fast glucose catabolism, high ethanol yield (up to 96% of consumed glucose), low biomass production, resilience to inhibitors present in lignocellulosic hydrolysates, and tolerance to high ethanol and sugar concentrations (Z. mobilis possesses nitrogenase and can efficiently fix atmospheric nitrogen (N2) without affecting ethanol yield, which adds potential economic and environmental benefits to ethanol production  and isoprenoid fuels derived from the methylerythritol phosphate (MEP) pathway  strain was first grown on a rich medium plate anaerobically at 30°C for 3 days. This was followed by inoculation of rich medium with a single colony from the plate. A 2 mL ZM4 minimal medium was inoculated with liquid rich medium overnight culture and grown for 16 h. This culture was then used to inoculate a 25 mL minimal medium culture to a starting optical density of 0.05 (measured at 600 nm). The cells were grown to O.D600 of 0.5 before being collected by centrifugation for 15 min at 4500 rpm   ; cell peLysis and digestion were performed as previously described . BrieflyPhosphopeptide enrichment was performed using a previously described titanium (IV) immobilized metal affinity chromatography (Ti-MAC) method . In briem/z, fragmentation by higher-energy collisional dissociation (HCD) with normalized collision energy (NCE) = 30, maximum injection time = 118 ms, AGC = 2 × 105 and fragment ions were analyzed by the Orbitrap with R (resolution) = 60,000. Each sample was analyzed with and without the advanced precursor determination (APD) toggled . These p-values were converted to q-values using permutation-based FDR correction. For normalization to protein abundance, phosphorylation log2 signals were mean normalized across each sample for each site independently, and the process was repeated for protein log2 label-free quantitation signals. A protein normalized signal was then calculated for each sample by subtracting the mean normalized protein value from the corresponding mean normalized phosphorylation site value, followed by calculating the fold changes and q-values in Perseus software platform.Raw files were analyzed with MaxQuant software program; default settings were applied except for match between runs and label free quantitation, which were toggled on . The intZ. mobilis across three different growth conditions: anaerobic, aerobic, and N2-fixing conditions. Cells were grown in defined minimal media using glucose as the single carbon source and ammonia as the nitrogen source, except for N2-fixing conditions in which no ammonia was added. For each condition, five biological replicates were generated. Samples for phosphoproteome analyses were taken during mid-exponential growth  (Z. mobilis proteins (125/1892 proteins). This fraction is similar to the fraction of phosphorylated proteins previously reported in other bacteria such as E. coli (9.1%) B. subtilis (4.2%), and K. pneumoniae (5.4%)  and S. coelicolor (STY: 46.8/48/5.2) , B. subtilis (6.71%), and K. pneumoniae (4.64%). We also observed that nearly all (>95%) proteins phosphorylated on Ser2 and Thr2 had their first methionine removed, as it has been observed in other bacteria (We performed a genome wide investigation of protein phosphorylation in 00 ∼0.5) . Tryptic00 ∼0.5) . Enriche00 ∼0.5) , of whic00 ∼0.5) . In para00 ∼0.5) . The nume (5.4%) . We catee (5.4%) . The sere (5.4%) . This waeumoniae , but som/48/5.2) ; Table 1 or Thr2 . Interesbacteria .2-fixing growth conditions that affected several major pathways and cellular processes  pathway  and alcohol dehydrogenase  ( pathway  instead revisiae . The ED ZMO1596) .2, Tyr339), glyceraldehyde phosphate dehydrogenase  had three phosphorylation sites , and pyruvate kinase  had four phosphorylation sites . Interestingly, we observed a general trend for increased phosphorylation in nearly all glycolytic enzymes during aerobic and N2-fixing growth  was phosphorylated at Ser12 under aerobic and N2-fixing conditions but not under anaerobic growth. The high prevalence of phosphorylation sites in Z. mobilis glycolytic enzymes bears similarity to previous phosphoproteome analyses of other bacteria  showing that most of their glycolytic enzymes are phosphorylated , all other enzymes in the ED glycolytic pathway were phosphorylated under at least one growth condition . We ideng growth . Some phorylated .2 and Ser311) and ADH 2 . PDC displayed significantly increased phosphorylation on both Ser2 and Ser311 under aerobic and N2-fixing conditions. In contrast, ADH 2 was specifically phosphorylated at Ser126 only under anaerobic growth and at Ser99 only under N2-fixing conditions  was phosphorylated at Ser257 and Ser262, and putative malate dehydrogenase  was phosphorylated at Ser2 and Thr540 (2-fixing conditions (540 increased significantly under both aerobic and N2-fixing conditions while phosphorylation of PEPC (Tyr317 and Thr323) decreased significantly under N2-fixing conditions  was phosphorylated at Tyrd Thr540 . Unlike nditions . Only MDnditions .Z. mobilis, the nitrogenase complex is encoded by the NifHDK genes. NifHDK expression is controlled by the transcription factor NifA, a master regulator that also controls expression of many other genes involved in nitrogen fixation  that includes the NifA gene, the nitrogenase operon (nifHDKENX-fdxB-nifQ), two operons involved in nitrogenase maturation (nifB-fdxN and iscN-nifUSVW-modD), and the Rnf operon (rnfABCDGEH) encoding the Rnf electron transport complex  in bacteria and archaea is carried out by the highly conserved nitrogenase complex. In most nitrogen-fixing bacteria, including fixation . In Z. m complex .2-fixing growth. NifH was phosphorylated at Ser90 and NifD was phosphorylated at Ser60, Ser53, Thr42, and Ser286 exclusively under N2-fixing conditions (2 was observed in both anaerobic and N2-fixing conditions but displayed a significant increase in phosphorylation under N2-fixing conditions (111) only under N2-fixing conditions  and NifD  during Nnditions . NifD phnditions . NifQ  was phosphorylated at Serc growth . In γ-prn status . Z. mobi178) and RnfG (Thr205) proteins in N2-fixing conditions. In contrast, RnfC was phosphorylated (Ser5) exclusively under N2-fixing conditions  form a transmembrane complex that catalyzes reverse electron flow from NADH to reduce ferredoxin/flavodoxin, which acts as the electron donor to nitrogenase  Figure . We obsenditions . Althoug4+) assimilation into glutamate takes places via the glutamine synthetase/glutamate synthase cycle . GS catalyzes the ATP-dependent production of glutamine from NH4+ and glutamate. GOGAT then takes this glutamine and α-ketoglutarate to produce two molecules of glutamate , but this enzyme appears to be missing in Z. mobilis. GS activity is extensively regulated in response to intracellular levels of glutamine, α-ketoglutarate, and other metabolites. Post-translationally, GS is regulated via adenylyation/deadenylylation by the bifunctional adenylyl transferase (AT). AT activity is directly modulated by glutamine, which favors GS adenylyation . AT activity is also regulated by regulatory protein PII: uridylyated PII interacts with AT to promote GS deadenylylation  while non-uridylyated PII favors adenylyation. Uridylylation of PII by uridylyl transferase  is in turn favored by low glutamine and high α-ketoglutarate levels  to be differentially phosphorylated at Tyr51 and Ser26 under different growth conditions  on EF-Tu that were located on domains II and III. These phosphorylation events could potentially modulate EF-Tu interaction with aa-tRNAs  to the ribosome . EF-Tu f binding . Here wenditions , suggestaa-tRNAs . These tnditions . Interesnditions .84 of L7/L12 is a highly conserved surface residue that is thought to be critical for its interaction with EF-Tu; its mutation to alanine has been reported to significantly decrease binding efficiency of the ternary complex to the ribosome  exclusively under aerobic and N2-fixing conditions while L3 (Thr214 and Tyr218) phosphorylation increased under these two growth conditions  in the phototrophic bacterium R. palustris , adenylylation  or nitration (anabaena) (49 under N2–fixing conditions or in response to high AKG concentrations has been observed in cyanobacteria (S. elongatus and Synechocystis sp.)  . In additis sp.) . In Z. mnditions . Phosphoactivity  It wouldZ. mobilis (In vitro experiments have shown that phosphorylation of ribosomal proteins can alter ribosomal activity (2-fixing growth may play a role at regulating translation in Z. mobilis under these conditions. For example, changes in phosphorylation of L7/L12, L3, and L13 could potentially affect ribosomal activity by modulating interactions with elongation factors EF-Tu and EF-G. Similarly, differential phosphorylation of EF-Tu across growth conditions may also serve as a regulatory mechanism of protein synthesis (Paralleling observations in other bacteria , we foun mobilis . In vitractivity , 2011. Tynthesis .Z. mobilis increases under what may be considered “non-optimal” growth conditions, i.e., aerobic and N2-fixing conditions vs. anaerobic growth with abundant ammonia. This suggests that phosphorylation may be particularly important for regulating metabolism during adverse growth conditions.Our results show that overall protein phosphorylation in Z. mobilis that we have presented here provides new knowledge regarding the specific proteins, pathways, and cellular processes that may be regulated by phosphorylation in this important industrial organism. The results of this study provide a useful road map and a unique hypothesis-generating resource to establish future genetic and biochemical experiments that investigate the physiological role of specific phosphorylation events in Z. mobilis.The genome-wide phosphoproteome analysis of http://www.ebi.ac.uk/pride/archive/projects/PXD014065.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014065 and can be accessed via this link: MT, AH, JC, and DA-N designed the study. MT prepared the samples for the analysis. AH performed the mass spectrometry experiments. MT, AH, and DA-N analyzed the data. MT and DA-N wrote the manuscript. All authors read and approved the final version of the manuscript.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.


Magallana gigas) harvested from a sewage-impacted production site and then subjected to site-specific management procedures. These procedures consisted of relocation of oysters to an alternative production area during the norovirus high-risk winter periods (November to March) followed by an extended depuration (self-purification) under controlled temperature conditions. Significant differences in norovirus RNA concentrations were demonstrated at each point in the management process. Thirty-one percent of oyster samples from the main harvest area (Site 1) contained norovirus concentrations > 500 genome copies/g and 29% contained norovirus concentrations < 100 genome copies/g. By contrast, no oyster sample from the alternative harvest area (Site 2) or following depuration contained norovirus concentrations > 500 genome copies/g. In addition, 60 and 88% of oysters samples contained norovirus concentrations < 100 genome copies/g in oysters sampled from Site 2 and following depuration, respectively. These data demonstrate that site-specific management processes, supported by norovirus monitoring, can be an effective strategy to reduce, but not eliminate, consumer exposure to norovirus genome copies.Oysters contaminated with norovirus present a significant public health risk when consumed raw. In this study, norovirus genome copy concentrations were determined in Pacific oysters ( Norovirus infections are the most common cause of non-bacterial gastroenteritis worldwide. In Europe, the peak of illness cases occurs in the winter months, with most patients experiencing relatively mild symptoms. During these epidemic winter periods, the norovirus distribution in the human population, and therefore in sewage, is ubiquitous  is located in an estuary that has a number of potential sources of sewage contamination. The most significant of them is a secondary wastewater treatment plant (WWTP), serving a population equivalent of 1200 with the discharge outlet located approximately 1.5 km away from Site 1. Further significant contamination sources (such as septic tanks or other wastewater treatment plants) may also impact on the production site but these are more distant. Throughout the study period, this production area was classified as a category A site, based on Magallana gigas) were generally harvested from Site 1 during the months of April to September inclusive. Over the winter months , production switched to Site 2, an alternative category A site situated at the estuary mouth and approximately 4.5 km away from the WWTP discharge outlet , and 2 (winter), all oysters were depurated before placing on the market. During the summer, oysters were depurated for 2–3 days at ambient temperatures. During the winter months, oysters were depurated for between 2 and 9 days at elevated temperatures.3/h providing effective aeration and adequate dissolved oxygen levels in the tanks to allow active functioning of the oysters. Each tank was fitted with 4 UV sterilisation lamps  and a set of three 300 W aquarium water heaters, which were used when the seawater temperature dropped below 12 °C. During the study, the average seawater temperature measured during depuration in winter (October to March) was 13.3 °C (min = 8.1 °C and max = 19.7 °C). Prior to depuration, oysters were washed briefly and placed in shallow, open mesh plastic trays (8–10 kg of oysters per tray). Trays were stacked up to four layers high, with a maximum of 800 kg of oysters placed in each of the tanks for between 2 and 9 days.Depuration undertaken by the shellfish producer was routinely performed in recirculating tanks of seawater using ultraviolet (UV) disinfection. In total, seven depuration tanks were used during the study period. Tank dimensions were approximately 6 by 0.7 by 1.3 m. The maximum volume of each tank was 6000 L of seawater pumped from a nearby pit on the estuary shore that flooded during high water. This seawater was filtered and UV treated prior to entering the depuration system. The seawater flow rate was approximately 10 mBetween January 2015 and April 2016, samples consisting of 10 live oysters were collected at the three production points on a weekly basis. Sampling schedules varied throughout the study period depending on season and are outlined in Table All oyster samples collected during the study period were transported to the laboratory under chilled conditions (< 15 °C) and received within 48 h of harvesting. Analysis began within 24 h of receipt to the laboratory.Between January and March 2015, a sample was collected from the unused Site 1 and duplicate samples were collected from Site 2 each week. In total, five samples were collected weekly from the depuration tanks during the winter months. This consisted of triplicate samples of oysters purified for 2–3 days (short-term depuration) and duplicate samples of oysters depurated for 7–9 days (long-term depuration).Between November 2015 and March 2016, duplicate samples were collected weekly from Site 1 and 2 as well as following short-term and long-term depuration.In total, 55 samples were taken from Site 1 and 65 samples from Site 2 during the winter months. In the same period, 74 and 50 samples were collected following short-term  and long-term  depuration, respectively.Sampling frequency was reduced during the summer months in line with the expected reduction in norovirus detection rates. Between April and October 2015, a single sample was collected weekly from the unused Site 2, duplicate samples were harvested from Site 1 and following short-term depuration.In total, 50 samples were collected from Site 1 and 36 samples from Site 2 during the summer months. In the same period, 89 samples were collected following short-term depuration.Oysters were prepared in accordance with ISO 15216-1:2017 ( 0) for evaluation of virus extraction efficiency similar to that described by Costafreda et al. (−1). Samples were incubated at 37 °C for 60 min with shaking at 150 rpm followed by 15 min at 60 °C. Finally, after centrifugation at 3000×g for 5 min, supernatants were retained for RNA extraction.Briefly, oysters were cleaned under running potable water. Ten oysters per sample were shucked and the digestive tissues (DT) dissected out. The dissected DT was finely chopped using a sterile razor blade and mixed well. Two grams of DT was then spiked with 10 µl of the internal process control (IPC) virus  and the NucliSENS® MiniMAG® extraction platform and eluted into 100 µl of elution buffer. RNA extracts were stored at − 80 °C until the RT-qPCR analysis was conducted. RNA was also extracted from 10 µl of the IPC sample for evaluation of extraction efficiency. A single negative extraction control (water only) was processed alongside the oyster samples.Oysters were analysed for the norovirus concentrations using standardised quantitative real-time reverse transcription PCR (RT-qPCR) (“ISO 15216-1:2017” 5 copies/µl) were included in duplicate on each RT-qPCR run. The number of RNA copies in norovirus-positive samples was determined by comparing the CT value to the standard curves. The final concentration was then adjusted to reflect the volume of sample analysed and expressed as the number of detectable virus genome copies per gram of DT.RT-qPCR analysis was carried out using the Applied Biosystems AB7500 instrument  and the RNA Ultrasense one-step qRT-PCR system (Invitrogen). The reaction was prepared by combining 5 µl of the extracted RNA sample and 20 µl of the reaction mix containing 500 nM forward primer, 900 nM reverse primer, 250 nM sequence specific probe, 1x ROX reference dye and 1.25 µl of enzyme mix. Previously described primers QNIF4 . The threshold cycle (CT) value obtained for samples spiked with the ECRNA was compared to the results obtained in the absence of the sample (5 µl of water used instead) and used to estimate RT-PCR inhibition expressed as a percentage. In accordance with ISO 15216-1:2017, oyster samples with RT-PCR inhibition below the 75% were accepted for inclusion in this study.The presence of inhibitors was checked by spiking an additional 5 µl of each sample RNA with 1 µl of either norovirus GI or norovirus GII external control RNA  and negative extraction controls (blank sample carried through the RNA extraction step) were included in each RT-PCR analysis in order to control for cross-contamination.The limit of quantification (LOQ) for both, norovirus GI and norovirus GII assays was 100 genome copies/g. Results were presented as total norovirus concentration calculated as a sum of GI and GII results. To facilitate the statistical analysis of the results, any result demonstrating a norovirus concentration < LOQ was assigned a value of half of the LOQ value (50 genome copies/g) for each genogroup.t test was performed in Microsoft Excel to compare the norovirus concentration results obtained from the three sampling points. Results with values p < 0.05 were deemed statistically significant.An unpaired, two-tailed Norovirus concentrations in oysters followed a clear seasonal trend at both Sites 1 and 2 . The maximum and mean norovirus RNA concentrations detected in oysters from Site 1 were 1191 and 353 genome copies/g, respectively. This is compared with maximum of 467 and mean of 117 genome copies/g at Site 2. Following depuration, these figures were reduced to 341 and 58 gc/g (< LOQ), respectively.However, there was a significant gradation in the norovirus RNA concentrations detected in oysters sampled at Site 1 and 2, and following depuration during the same period . This confirms finding in other studies conducted in Europe EFSA  and highFollowing the outbreak associated with oysters harvested from Site 1, the producer introduced additional management controls which included the use of an alternative harvest site (Site 2) during the winter (November to March) and depuration for extended periods under controlled temperature conditions. Nevertheless, the additional controls resulted in a limited reduction in the frequency of norovirus detection in marketed oysters during winter with norovirus detected in 91, 89 and 81% of oysters from the main production site (Site 1), alternative harvest site (Site 2) and following depuration, respectively. However, a reduction in norovirus concentration was observed in the oysters following implementation of the management procedures. It was notable that the majority (84%) of oyster samples following depuration contained norovirus RNA concentrations below the LOQ. Therefore, while management procedures employed during this study did not significantly lower the frequency of norovirus detection in marketed oysters, they reduced the concentration of norovirus copies, to which consumers were exposed.50) to be between 18 and 1015 genome equivalents for norovirus GI.I  genome equivalents for susceptible individuals  and its value as an added public health control is questionable. The limited value of depuration periods extended beyond the 3 days as observed here is consistent with laboratory-based studies reported elsewhere for other shellfish species , only a slight drop in the number of oyster samples containing norovirus was observed. However, there was a notable drop in the average concentration of norovirus in oysters before and after depuration with most (90%) of oysters following depuration for 7–9 days containing < 100 genome copies/g. This is compared with just 55% of oysters containing norovirus RNA concentrations < 100 genome copies/g prior to depuration. A possible explanation for the reduction in norovirus RNA concentrations observed in this study is that norovirus was sequestered into tissues outside of the digestive tissue. Given that the ISO standard method used in this study examines the digestive tissue only, we cannot determine from this study whether this is the case but we can find no evidence in the literature of such sequestration into alternative tissues. On average oysters purified for 7–9 days contained slightly lower concentrations of norovirus than oysters depurated for 2–3 days indicating that depuration for extended periods may further reduce norovirus RNA concentrations. However, the additional reduction in norovirus RNA concentrations in oysters depurated beyond the 3 days was not significantly different (In summary, in the absence of current regulatory standards, we believe that a site-specific management approach, such as described here and supported by norovirus monitoring, can reduce consumer exposure to norovirus genome copies. This may provide additional, if not complete, consumer protection. However, despite the anecdotal evidence presented here, i.e. lack of illness reports, it is not possible to determine the public health benefits of this approach. Therefore, there remains a clear requirement for further work to better characterise the relationship between norovirus RNA concentrations in oysters as judged by RT-qPCR and illness outcomes.


Metacommunity theory highlights the potential of β-diversity as a useful link to empirical research, especially in diverse systems where species exhibit a range of stage-dependent dispersal characteristics. To investigate the importance of different components and scales of β-diversity in community assembly, we conducted a large-scale disturbance experiment and compared relative recovery across multiple sites and among plots within sites on the rocky shore. Six sites were spread along 80 km of coastline and, at each site, five plots were established, matching disturbed and undisturbed quadrats. Recovery was not complete at any of the sites after 1 year for either epibenthos  or infauna. Significant differences in recovery among sites were observed for epibenthos but not for infauna, suggesting that different community assembly processes were operating. This was supported by epibenthos in the recovering plots having higher species turnover than in undisturbed sediment, and recovery well predicted by local diversity, while infaunal recovery was strongly influenced by the epibenthic community's habitat complexity. However, infaunal community recovery did not simply track formation of habitat by recovering epibenthos, but appeared to be overlain by within-site and among-site aspects of infaunal β-diversity. These results suggest that documenting changes in the large plants and animals alone will be a poor surrogate for rocky shore community assembly processes. No role for ecological connectivity (negative effect of among-site β-diversity) in driving recovery was observed, suggesting a low risk of effects from multiple disturbances propagating along the coast, but a limited resilience at the site scale to large-scale disturbances such as landslides or oil spills. This γ) diversity. β-diversity can be decomposed to reflect the importance of different processes affecting community assembly: species replacement , richness difference (one community has more uniquely held species than another), and nestedness (the species present at a site with low numbers of species are a subset of the species present at richer sites) , where n is the total number of line segments, d is the distance between the start and endpoints of the line, and L is the cumulative length of all line segments , where d was set at the highest level of resolution on the 1 : 5000 map of the Ligurian coastline. Sinuosity (GIS) was defined as: S = l/lsf; where l is the cumulative length of all line segments, and lsf is the distance between the start and endpoints of the transect. Sinuosity (GIS) is calculated in the same way as sinuosity (chain) measurements, but at a larger scale: the sinuosity (chain) taking into account the site-scale roughness of the substrate.(i) Habitat complexity in terms of sinuosity of the shoreline was measured at different scales using the following indices: (a) sinuosity (chain), within site, by way of carefully laying a chain  along the coast following minor indentations at the same tidal height as our disturbed plots (five measurements per site); (b) two different measures of fractal dimension were used to provide measurements of habitat rugosity at the larger scale of the whole site extent (150 m); both these indices were calculated using ARCGIS. Fractal (GIS) was defined as: (ii) Given the different complexity of the dominant macroalgal assemblages, an algal habitat complexity index (AHCI), indicating three-dimensionality and shelter provision, was calculated (see ), based −2, www.comuni-italiani.it) by the shortest distance (m) to the site.(iii) An urbanization index for each site was calculated by dividing the population density of the closest urban centre (ind km3 sec−1) of the closest river divided by the distance (m) from the sampling site. This simple index is a good surrogate for river effects because of the weak near shore currents and the dominance of weather conditions on water movement.(iv) A river index was calculated as the river flow (dm(v) An exposure index was calculated as the aspect of each site relative to the main wave direction (225°).To characterize differences within and between sites, the following indices were calculated.2.4.(i) Relative recovery of epibenthos and infauna for each plot was estimated as the difference between paired disturbed and undisturbed samples 1 year after disturbance, based on Bray-Curtis dissimilarities of raw count data as percentages (i.e. community recovery = 100 - % Bray-Curtis dissimilarity).(ii) We calculated β-diversity, based on presence–absence data, at two scales, within and among sites, based on undisturbed data only . Within-(iii) We decomposed the undisturbed β-diversity into replacement, richness difference and nestedness, based on the Jaccard group (presence/absence data) and using the Podani family of calculations ,13:Rrepl) = 2 × min / (a + b + c),relativized species replacement (Rrd) = |b − c|/(a + b + c),relativized richness difference (Rnest) = (a + |b − c|)/(a + b + c), if a > 0 and 0 if a = 0, which equates to 1–Rrepl,relativized nestedness  On disturbed plot data we calculated the replacement, nested and richness difference at the among-site scale only.2.5.2.5.1.An initial PERMANOVA analysis was performed on epibenthic and infaunal composition data separately, to test for treatment and site effects .2.5.2.t-tests for each component separately. This was done for the among-site scale only (n = 15).Differences in the relative contribution of different components of β-diversity between undisturbed and experimentally disturbed communities were tested by paired 2.5.3.n of 30 for the regression analyses. Parsimonious models were produced by backwards selection based on p-values, with terms only removed if doing so did not result in a significant increase in deviance [p statistic. None of the final models retained variables with a variance inflation factor greater than 5, nor did they include polynomials.Multiple regression was used to develop the best predictive model for each assemblage type. All environmental and β-diversity variables described above were used in the modelling, with nonlinearities incorporated by using log transformations, polynomials and interactions. We expected differences in relative recovery to be influenced by both within-site and between-site processes. While some variables were only available at the larger scale (among sites), other variables  differed at the plot level within sites, resulting in an deviance –33. Over3.3.1.2 plots, recovery was not complete for either infauna or epibenthos, with the average recovery per site ranging from 30 to 60 for infauna and 15 to 59 for epibenthos). For the infauna there was no significant site*treatment interaction, but a significant treatment effect (p = 0.007 and p = 0.008), while FRA exhibited the fastest recovery (p = 0.029). The other three sites also displayed a significant treatment effect .One year after clearing the 1 mt effect . Epibent3.2.t-test, n = 15, p = 0.0004; figure 1a), prediction 1, and conversely relativized nestedness and richness difference were higher in undisturbed plots than in disturbed ones . Results were not so clear for the infaunal β-diversity components, with significant effects only observed for relativized richness difference . Overall, the sites ranked based on nestedness were different for infauna and epibenthos (figure 1a). A significant effect of infauna and epibenthos was not detected on the relativized richness difference proportion of β-diversity for either undisturbed or disturbed plots . Epibenthic and infaunal communities from both the undisturbed and the disturbed plots demonstrated higher proportions of relativized nestedness than replacement .Across sites, epibenthic relativized replacement was higher for disturbed plots than undisturbed ones  was only observed for the epibenthic communities. Our models showed no indications that sites with higher connectivity to the regional species pool recovered faster for epibenthos or infauna (prediction 2). Our third prediction (that relationships between recovery and β-diversity would differ for epibenthos and infauna) was the most strongly supported of our three predictions.Environmental factors were not important either for epibenthic or infaunal recovery. Instead, our results emphasize the importance of local biotic features in affecting the relative recovery rates of rocky shore communities. Epibenthic  recovery was faster at homogeneous sites (i.e. sites characterized by low within-site β-diversity) and where high numbers of taxa were found in the nearby undisturbed plots. Similarly, infaunal recovery increased with increased recovery of the epibenthos (prediction 3), negatively mediated by local habitat structure . However, for infaunal recovery, effects of local habitat were overlain by aspects of β-diversity at both the local and among-site scale.For macroalgae, limited macroalgal propagule dispersal and/or colonization through vegetative propagating thalli contribute to the domination of local over regional processes in recovery. Vegetative propagating thalli appear to be less vulnerable to a variety of physical and biological factors  than are sexual propagules, typical of canopy-forming species . Thus, vThe model for the infauna emphasizes the importance of recovering macroalgae influencing differences in recovery. Interestingly, despite the importance of macroalgae in habitat creation for infauna, the recovery of these communities did not simply track habitat formation (per cent explained less than 45%), with other processes also indicated as important . In particular recovery was increased when both average site replacement and among-site β-diversity were high. While previous surveys of macroalgae and associated infauna have emphasized the importance of site α-diversity and the habitat architecture created by different algal morphologies , our resUsing β-diversity as an indicator of ecological connectivity in this large-scale experiment indicated no reliance on the regional species pool for epibenthic or infaunal recovery, thus decreasing the risk of cumulative impacts propagating along the coast. Conversely, this implies limited resilience at the site scale (10–100 m) to large-scale disturbance associated with impacts such as landslides, oil spills or shoreline modification.Our results can be scaled up to the regional scale because we employed an unusually large scale of disturbance encompassing much of the entire tidal range in the Mediterranean and we replicated our experiment across a range of sites that encompassed major rocky shore habitat variation in the region. We have much to learn about resilience, connectivity and thresholds of recovery from unfavourable states. Mixing experimental studies of disturbance and recovery with information on different scales of heterogeneity in biodiversity allows us to gather information on a range of scales and ecological processes. In this system, we found that local ecological features associated with β-diversity and biogenic habitat formation influenced community assembly through habitat filtering and species replacement (turnover) in disturbed plots. An important implication of this is our ability to interpret survey data when the disturbance history is unknown ,37. NatuConnectivity is the consequence of interactions between natural history, transport vectors and the quantity, quality and spatial arrangement of habitat patches, across landscapes. As such, integrative measures or surrogates for connectivity are likely to be imperfect. Nevertheless, as disturbance to coastal habitats increases and biodiversity continues to be eroded, connectivity and the current state of ecological communities will play an important role in understanding the consequences of change in different ecosystems. Increasing the disturbance regime ultimately leads to the homogenization of habitats and communities. These effects are likely to occur faster as the spatial scale of disturbance increases . Our fin


Neuronal growth cones are essential for nerve growth and regeneration, as well as for the formation and rearrangement of the neural network. To elucidate phosphorylation-dependent signaling pathways and establish useful molecular markers for axon growth and regeneration, we performed a phosphoproteomics study of mammalian growth cones, which identified >30,000 phosphopeptides of ∼1,200 proteins. The phosphorylation sites were highly proline directed and primarily MAPK dependent, owing to the activation of JNK, suggesting that proteins that undergo proline-directed phosphorylation mediate nerve growth in the mammalian brain. Bioinformatics analysis revealed that phosphoproteins were enriched in microtubules and the cortical cytoskeleton. The most frequently phosphorylated site was S96 of GAP-43 (growth-associated protein 43-kDa), a vertebrate-specific protein involved in axon growth. This previously uncharacterized phosphorylation site was JNK dependent. S96 phosphorylation was specifically detected in growing and regenerating axons as the most frequent target of JNK signaling; thus it represents a promising new molecular marker for mammalian axonal growth and regeneration.  •Phosphoproteomics of mammalian growth cone membranes reveals activation of MAPK•JNK is the activated MAPK in growth cones and phosphorylates S96 of GAP-43•pS96 of GAP-43, the most frequent site, is observed in growing axons•pS96 is biochemically detected in the regenerating axons of the peripheral nerves Neuroscience; Developmental Neuroscience; Bioinformatics; Proteomics The growth cone, a specialized, highly motile structure formed at the tips of extending axons of developing neurons , is crucin vitro phosphorylation systems that do not necessarily represent the in vivo situation. Phosphoproteomics is an important, novel, and powerful technique for comprehensive and quantitative identification of in vivo phosphorylation sites , a vertebrate neuron-specific protein involved in nerve growth  rat forebrain identified more than 30,000 phosphopeptides at greater than 95% confidence see . The conWe classified the kinase substrates in GCMs into various categories based on the number of phosphorylation sites A and theIceLogo web server , p38, and JNK, among which JNK appeared to be the most likely kinase candidate for mammalian GCM phosphorylation. First, several recent reports showed that JNK is involved in multiple steps of mammalian brain development . Second, elegans . TogetheTo test this hypothesis, we produced eight phospho-specific Abs against the high-frequency SP/TP phosphorylation sites  axons in mammals, including humans, can regenerate . In lighin vitro phosphorylation studies  The high frequency of P-directed phosphorylation in GCMs , as reveC. elegans (Our results suggest that mammalian nerve growth requires activation of JNK. Surprisingly, this conclusion is essentially consistent with the requirement for JNK signaling in axon regeneration, consistent with the results of a large number of mutant screens in  elegans . We conc elegans , this wa elegans ; however elegans  during m elegans . Althoug elegans , its funOur results show that pS96 of GAP-43 is associated with normal mammalian axon growth  and 5 anIt is important to note that JNK is also a negative regulator of axon growth and can induce axon degeneration . AccordiAll methods can be found in the accompanying


This study explored changes in hoof shape related to the application of horseshoes. Past research has indicated that horse hooves change shape in response to gallop exercise, so this study sought to separate the changes due to exercise from changes due to wearing horseshoes. Amongst the most important findings were that proximal (coronary band) hoof circumference decreased more when horses were shod compared to barefoot, and hoof angle decreased when horses were shod but increased when horses were barefoot over a seven-week interval. A better understanding of the relationship between hoof shape and shoeing could help improve horse management practices. n = 11) wearing shoes versus barefoot. An ANOVA appropriate to a crossover design was used to assess the differences in the change in hoof shape over the seven-week period and significance was set at p < 0.05. Results are displayed as the mean difference for horses when shod versus barefoot ± the SEM for the left (L) and right (R) front hooves. Proximal hoof circumference (PHC) decreased when horses were shod and barefoot, but this decrease was greater when horses were shod . Hoof angle increased slightly when horses were barefoot and decreased when they were shod . Sole length decreased more when horses were barefoot, but this was only significant for the right fore . Solar circumference increased when horses were barefoot but decreased when shod . This is the first study to show a significantly lower PHC when horses were shod compared to barefoot. The study suggests that shod horses may benefit from a shorter shoeing interval to help mitigate the changes in hoof angle.This crossover study tested the hypothesis that hoof shape would differ after a seven-week period of horses ( There is little doubt amongst equine industry stakeholders that hoof health is integral to the soundness of a horse. The shape (or conformation) of the hoof may have serious implications for the soundness of the animal and hoof problems are a major cause of lameness in the horse ,2,3. SpeThe domestic horse hoof is a continuously growing biological material that is frequently subjected to human intervention. Both hoof trimming and the individual farrier performing the trim have significant effects on the shape of the hoof capsule ,7. AlthoPrevious research has found that during a period of race training the hoof decreased in hoof angle  and decrn = 5). Seven weeks later, during data collection 2, the shoes were carefully removed from shod horses, and all the horses had measurements taken (Post 1). The horse’s hooves were then trimmed and measurements were taken (Pre 2). Shoes were then applied to Group B (n = 6). After a second seven-week period the shoes were carefully removed and the last set of measurements (Post 2) were collected. Eleven adult Quarter Horse mares  were used in this study. Horses underwent both treatments, shod and barefoot, in a crossover design. Horses were randomly assigned to their first treatment. Shod horses had the left and right front hooves shod, and the rear hooves were left bare. At the beginning of the study, in data collection 1, the horse’s feet were all trimmed by the same farrier and the first set of measurements were collected (Pre 1). Group A horses were shod , one centred on the dorsal hoof wall , and one on the medial hoof wall  . At eachHorses were unshod and maintained on a seven-week trimming interval  prior to the beginning of the study. The horses were housed at the same facility, managed under the same conditions, fed the same diet, and underwent no forced exercise during the trial (June to September). Horses were group-housed in paddocks with a mostly sand base. No horses received hoof supplements or hoof dressings. Three of the mares were lactating and pregnant; five mares were lactating and not pregnant; two mares were pregnant but not lactating; and one mare was neither pregnant nor lactating. Horses wore the same type of standard steel shoe in a suitable size. Hoof measurements were taken before the application of the shoe and after careful removal of the shoe by the qualified farrier. The farrier used the same standard trimming protocol that had previously been used on these horses and trimmed the hooves so that they were balanced. Horses received the same basic trim regardless of which group they would be in. None of the horses had a history of any serious lameness (including laminitis or navicular), they remained free of lameness throughout the study, and none of the horses had obviously hoof abnormalities  The horses did have a varied exercise history, with some horses having competed extensively and others having no performance record. However, all the horses had been retired for at least 12 months prior to the beginning of the study. All procedures were approved by the University of Melbourne Animal Ethics Committee (1011571.1).p < 0.05. The left and right front hooves were analysed separately for ease of interpretation.An analysis of variance (ANOVA) appropriate to a crossover design was used to assess the effect of treatment on the change in hoof shape over a seven-week period. The model included the effects of treatment (shod and barefoot), sequence , and period (one and two). Stata  was used for the analysis. Statistical significance was set at Repeatability was previously assessed in a separate study by measuring fifty horses (both forelimbs) two times each. The repeatability coefficient describes the range within which 95% of absolute differences between two readings from the same horse would be expected to lie . The repThe results are displayed in p = 0.0026; R p = 0.0002) when horses were shod, compared to barefoot. PHC decreased during both seven-week periods under both treatments. This decrease was significantly greater .Hoof angle increased slightly (became steeper) when horses were barefoot but decreased (became less steep) when they were wearing shoes. The difference between treatments (shod and barefoot) was significant .Heel width, lateral heel height, and medial heel height all showed an average increase in both hooves under both treatments, however none of the differences between treatments were significant . Sole width increased in both treatments over the seven-week period. This increase was not different between treatments (p > 0.05). Sole length decreased in both treatments over the seven-week period. Sole length decreased more when horses were barefoot. This difference was significant only for the right fore . Solar area increased more when horses were barefoot on average, but this change was not significant (p > 0.05).Solar circumference increased when horses were barefoot and decreased when horses were shod; this change was significant , but not for dorsal hoof growth (p > 0.05).Horses showed an increase in dorsal hoof growth cross mark for the right and left hooves, and a decrease in the dorsal hoof wear cross mark, under both treatments. There was a significant difference between treatments for dorsal hoof wear . Both treatment groups showed an increase in lateral hoof growth cross mark and a decrease in the lateral hoof wear cross mark, for the left and right hooves with and without shoes. The differences between shod and barefoot were nonsignificant . Both treatment groups had an increase in medial hoof growth cross mark for both front hooves and a decrease in medial hoof wear cross mark. Neither variable exhibited a significant difference between treatments . Changes in the distance from the frog to the medial wall were significant between treatments for the left hoof (L p = 0.0466), but not for the right hoof (R p = 0.5016). Changes in the distance from the frog to the toe were nonsignificant between treatments (p > 0.05).The distance from the frog to the wall of the hoof increased in all directions in both treatments. All increases were greater when the horses were barefoot, except for the distance from frog to toe on the right front hoof, which was greater when the horses were shod. Changes in the distance from the frog to the lateral wall were nonsignificant between treatments (p = 0.18) and 15.2 kg (p = 0.80) in period two. When the change in body weight was analysed by treatment the horses gained 1.4 kg more when they were barefoot compared to shod, but this difference was nonsignificant (p = 0.84).Horses showed changes in body weight during both seven-week treatment periods. The results are reported as the mean ± the standard deviation. During the first data collection the mean body weight was 417 ± 36 kg (range 340–455 kg). At the mid-point the mean body weight was 412 ± 33 kg (range 343–448 kg). At the final data collection, the mean body weight was 419 ± 28 kg (range 357–445). The mean change was −4.9 kg in period one (p = 0.18) and 15.2 kg (p = 0.80) in period two. When the change in body weight was analysed by treatment the horses gained 1.4 kg more when they were barefoot compared to shod, but this difference was nonsignificant (p = 0.84).Horses showed changes in body weight during both seven-week treatment periods. The results are reported as the mean ± the standard deviation of the mean. During the first data collection the mean body weight was 417 ± 36 kg (range 340–455 kg). At the mid-point the mean body weight was 412 ± 33 kg (range 343–448 kg). At the final data collection, the mean body weight was 419 ± 28 kg (range 357–445). The mean change was −4.9 kg in period one , sole length, and solar circumference. All horses involved in the study showed a decrease in PHC over the course of fourteen weeks. This result could be related to the season , substraHoof angle increased when horses were barefoot, becoming slightly and significantly steeper, and decreased, developing a significantly more acute angle, when they were wearing shoes. These data suggest that shod and unshod horses exhibit different changes in hoof angle during a seven-week shoeing interval. This difference is likely related to the increased wear of the unshod hoof, allowing it to maintain a steeper hoof angle. Previous research suggests that changes in hoof angle between shoeing sessions were associated with an increase in dorsal length of the hoof . MolemanThere were no significant differences between treatments for heel width or heel height. Heel width increased in both treatments over the course of the study. Heel width increased more when the horses were barefoot, but the difference was nonsignificant. Future research could explore the relationship between horseshoes and heel expansion, especially when the shoe is worn for extended periods of time.Both lateral and medial heel height increased in both treatments, but the difference was not significant. It could be expected that heel height would increase in shod horses (with the shoe protecting the heel area from wear) and decrease in barefoot horses. It should be noted that heel height is difficult to measure with a high repeatability value using the methods in this study see .Sole length decreased in both treatments over the seven-week period. The decrease was greater when the animal was barefoot, but this was only significant for the right fore. This is an interesting finding, because we would expect the sole length to increase as the hoof grew, but the decrease could be related to hoof wear at the toe. There was a slightly larger decrease in sole length when the horses were barefoot. Sole width increased in both treatments over the seven-week period. Hoof wear increased more when the horse was barefoot, but this difference was nonsignificant. This is most likely due to the increased flaring at the distal wall of the hoof in barefoot horses. The shoe could limit this deformation in the shod horse.Solar circumference was significantly different between treatments for both hooves. There was a small increase in solar circumference when the horses were barefoot, consistent with the spreading of the hoof. When horses were shod there was a small decrease in hoof circumference, indicating that the shoe may have restricted hoof spreading on the solar surface. Solar area increased when horses were barefoot. When horses were shod, the solar area increased only slightly for the left fore and decreased slightly for the right fore. The changes in solar area were not significantly different between treatments.There were no significant differences in hoof growth when the horses were shod versus barefoot. Only a few other studies have focused on the difference between hoof growth and hoof wear, by marking the horse hoof and measuring hoof growth from the coronet band to the mark and hoof wear from the mark to the ground ,25. The mean growth at the dorsal growth cross mark for barefoot horses was 0.238 mm per day for the left fore and 0.235 mm per day for the right fore. Florence and McDonnell reported a growth of 0.33–0.35 mm ± 0.02 (mean daily growth ± SE) at the dorsal midline of the hoof. They collected data from forty barefoot self-trimming ponies measured from July to September . Reilly Vermunt and Greenough suggested that hoof trimming may stimulate hoof growth in cattle . The horUnshod subjects had a mean wear of −0.17 mm for the left fore and −0.18 mm for the right fore. Florence and McDonnell reported slightly greater wear, with a mean of 0.24 ± 0.03 mm to 0.52 ± 0.04 mm . Our subFor this study, more wear was predicted when the hoof was not protected by a shoe. However, our results show no significant difference in wear in the unshod versus shod treatments, except at the toe. The toe was the area with the greatest amount of wear in unshod horses, and the significant difference in wear between treatments at this location demonstrates the protective nature of the shoe.The distance from the frog to the hoof wall increased in all three directions , indicating that the solar area of the hoof was growing or spreading. There were no significant differences between shod and barefoot horses except the distance from the frog to the medial hoof wall showed significantly less spreading in the shod horses. The fact that only the left hooves showed a significant difference could be due to numerous factors, including limb preference or handedness (horses were not assessed for limb preference in this study). Several studies have identified asymmetries between the left and right limbs. Some level of asymmetry is likely to be biologically normal and associated with horse-level laterality . The lack of significant differences between treatments for many of the solar values could be due to the fact that horses had wear of the distal edges of the hoof wall in both treatments. There was a significant difference between treatments for dorsal wear, therefore it would be expected that the frog to toe variables would also differ between treatments. There was no significant difference, perhaps because the heel became more underrun as the toe grew out, and hence the position of the frog in relation to the toe did not change. It is also possible that changes to the frog and sole happened simultaneously, and thus changes in the position of the frog were not detected or that a small change was undetected by our methods.p = 0.84). The horses were measured with a commercially available body weight tape and this is a limitation of the study. Several studies have evaluated the accuracy of different commercially available weight tapes and found that there is a large variability in weight estimated and low correlation with body weight measured on a scale [During the first seven-week period the horses had a mean decrease in body weight and in the second seven-week period they showed a mean increase in body weight. When the change in body weight was analysed by treatment, the horses gained 1.4 kg more when they were barefoot compared to shod but this difference was nonsignificant  used was also relatively small. We were able to distinguish statistical differences with this small group, but more horses would have been advantageous. Measurements that were taken while the foot was loaded, such as PHC, may have been influenced by changes in body weight. There were no significant differences in body weight from period 1 to period 2, but this should still be considered. A separate study done by this lab indicated little, to no differences in PHC when horses gained a considerable amount of weight over a 60-day period, however that data has not been published.This study demonstrates a significant effect of shoeing on hoof shape. Horses that were shod displayed a larger decrease in proximal hoof circumference, a decrease in hoof angle, a reduction in solar circumference, and less wear at the toe than occurred when they were barefoot. This study is the first to describe morphometric changes in hoof shape between horses when they are shod versus barefoot. It is the first study to demonstrate a change in PHC related to shoeing. A better understanding of the factors that influence hoof shape may lead to better hoof management practices that minimise the risk of injury to the horse.


Immunoglobulin E (IgE) antibodies are well known for their role in mediating allergic reactions, and their powerful effector functions activated through binding to Fc receptors FcεRI and FcεRII/CD23. Structural studies of IgE-Fc alone, and when bound to these receptors, surprisingly revealed not only an acutely bent Fc conformation, but also subtle allosteric communication between the two distant receptor-binding sites. The ability of IgE-Fc to undergo more extreme conformational changes emerged from structures of complexes with anti-IgE antibodies, including omalizumab, in clinical use for allergic disease; flexibility is clearly critical for IgE function, but may also be exploited by allosteric interference to inhibit IgE activity for therapeutic benefit. In contrast, the power of IgE may be harnessed to target cancer. Efforts to improve the effector functions of therapeutic antibodies for cancer have almost exclusively focussed on IgG1 and IgG4 subclasses, but IgE offers an extremely high affinity for FcεRI receptors on immune effector cells known to infiltrate solid tumours. Furthermore, while tumour-resident inhibitory Fc receptors can modulate the effector functions of IgG antibodies, no inhibitory IgE Fc receptors are known to exist. The development of tumour antigen-specific IgE antibodies may therefore provide an improved immune functional profile and enhanced anti-cancer efficacy. We describe proof-of-concept studies of IgE immunotherapies against solid tumours, including a range of in vitro and in vivo evaluations of efficacy and mechanisms of action, as well as ex vivo and in vivo safety studies. The first anti-cancer IgE antibody, MOv18, the clinical translation of which we discuss herein, has now reached clinical testing, offering great potential to direct this novel therapeutic modality against many other tumour-specific antigens. This review highlights how our understanding of IgE structure and function underpins these exciting clinical developments.  Immunoglobulin E (IgE), named in 1968 ,3, was t2+-dependent) lectin-like superfamily . I. I36]. Iw motion . To date2 domains and no hinge . T. Ta ≈ 102+ binding site, although IgE binding does not require occupancy of this site . . 182]. EThese studies established that MOv18 IgE could mediate effector functions such as degranulation and tumour cell killing through cytotoxicity (ADCC) and phagocytosis (ADCP) by activating known IgE effector cells.The ability of MOv18 IgE to restrict tumour growth in vivo was studied against different rodent models including human tumour xenografts established in immunodeficient (SCID and nu/nu) mice. In immunodeficient mouse models, human effector cell populations were co-administered with MOv18 IgE because: (a) human IgE-Fc is not recognised by mouse FcεRs, and (b) in mice the high-affinity IgE receptor FcεRI is expressed only by mast cells and basophils, and is absent in key effector cells such as monocytes and eosinophils. These studies therefore took place in an in vivo system containing both target and effector cells of human origin.In an s.c. human ovarian cancer (IGROV1) xenograft grown in a SCID mouse model, animals administered with mouse/human chimeric MOv18 IgE or MOv18 IgG1, intravenously (i.v.) exhibited an initial inhibition of tumour growth up to day 19 post-tumour challenge. However, the tumours in mice administered PBMCs and MOv18 IgG1 subsequently grew to the same size as the controls. In contrast, mice administered PBMCs and MOv18 IgE exhibited reduced growth of up to 72% by day 35 post-challenge. In a range of experiments in this model, a single treatment with MOv18 IgE and PBMC significantly restricted the growth of ovarian tumours a 147]. . 147]. ISubsequently, a patient-derived xenograft (PDX) model of ovarian cancer was established from a human primary tumour sample, originating from the ascites of a moderately differentiated Grade 3, stage III ovarian serous cystadenocarcinoma. This PDX could be passaged in nude mice while retaining its human phenotype and was found to express FRα. In efficacy studies using this model, nude mice were challenged with i.p. ascites from donor human xenograft-bearing mice and were then treated with saline, human PBMCs or PBMCs plus MOv18 IgE on days 1 and 16. The mean survival time of control mice was 22 days, for those administered PBMCs alone it was 30 days, while for those administered PBMCs plus MOv18 IgE, the mean survival time was 40 days . In a stOne limitation of studies in mouse models is the need to introduce exogenous human effector cells, thus limiting the immune functions of the model and the possible duration of study as exogenous effector cells become depleted. Therefore, an immunocompetent syngeneic tumour model in Wistar Albino Glaxo (WAG) rats was designed to study efficacy as well as safety of MOv18 IgE prior to clinical translation. This model was selected based on similar expression and cellular distribution of FcεRI in rats and humans. Rat CC531 colon adenocarcinoma cells , engineeOverall, in three models of cancer including a patient-derived xenograft and an immunocompetent syngeneic model, the anti-tumour efficacy of MOv18 IgE was reliant on the presence of both an effector cell population and tumour antigen specificity. Furthermore, anti-tumour IgE was more effective than the corresponding IgG.The mechanisms by which IgE antibodies can exert their anti-tumour effects have been studied and several pieces of evidence support a role for monocytes and macrophages as key effector cells.In vitro evidence for monocyte-mediated effector functions: Monocytes mediate MOv18 IgE-dependent tumour cell killing in vitro by two pathways, ADCC and ADCP, acting through FcεRI and CD23 respectively. FcεRI-expressing primary monocytes principally exert ADCC. MOv18 IgE-potentiated ADCC by monocytes could be blocked with recombinant sFcεRIα ..191].In concordance, in previous immunodeficient mouse models of human FRα-expressing carcinoma xenografts, the administration of mouse/human chimeric MOv18 IgE or MOv18 IgG1 together with human peripheral blood lymphocytes and peripheral blood mononuclear cells did not trigger any toxic effects, despite the presence of human basophils and eosinophils, including those from allergic human donors ,179,181,Finally, dogs may be an alternative model to examine the safety and anti-tumour functions of IgE, since this species is known for susceptibility to both cancer, including spontaneous mammary carcinomas, and allergy, with strong similarities of FcεR expression and distribution on immune cells compared with humans ,208,209.Translation to clinical testing is expected to entail careful monitoring of patients and measuring functional readouts and immunological markers of type I hypersensitivity following administration of MOv18 IgE due to the potential for basophil and/or mast cell degranulation. Functional tests may monitor the propensity to trigger basophil activation and mast cell degranulation in patient blood and sera ex vivo, all measured at different points of drug administration. Monitoring would include clinical signs of type I hypersensitivity, changes in serum levels of β-tryptase, total and tumour antigen-specific IgE, circulating tumour antigen and autoantibodies to the target antigen. Specifically, serum β-tryptase elevation signifying mast cell degranulation during clinical testing may be important to help distinguish cytokine release-type infusion reactions from type I hypersensitivity ,211.Production of IgE for clinical study requires the development of GMP processes that ensure swift production of an antibody with sufficient quality, purity and stability profiles. Importantly, the product must show physiochemical and functional profiles compatible with those of the laboratory grade material. Additionally, IgE antibodies display seven glycosylation sites, six of which comprise complex N-glycans, potentially with terminal galactose, fucose and sialic acid residues, as discussed above (and illustrated for IgG in Rational design of suitable therapeutic agents should aim to take advantage of the tissue-resident immune surveillance exerted by IgE antibodies that can be directed against cancer antigens, whilst minimising the risk of the potential toxic effects of the therapeutic agent. Malignant indications could be selected according to whether tumour cells are likely to reside in tissues in which important IgE effector cells such as macrophages are also found. Indications in which tumour cells and tumour cell fragments do not circulate would be preferable, since following systemic administration of anti-tumour IgE, basophils loaded with anti-tumour IgE could encounter circulating cancer cells bearing multiple copies of the target antigen; such interactions might trigger degranulation and potential type I hypersensitivity. Important criteria for the selection of cancer antigen targets would include high expression on the tumour with minimal and restricted distribution in normal tissues away from patient circulation. Furthermore, selection of single epitopes on tumour antigens and antigens that do not shed in multimeric forms in patient circulation would be key criteria for target selection.Within the fields of Immunology, Allergy and AllergoOncology, there are many aspects of IgE biology that are yet to be explored. The most prominent unknowns in the field are: defining the dynamics of antibody trafficking to tumours, recruiting monocytes into tumour lesions and engaging local tumour-associated macrophages; pharmacokinetics in patient circulation and biodistribution in health and disease settings; the roles and anti-tumour functions of mast cells; unexplored mechanisms of action beyond the TNFα/MCP-1 cascade; the existence of modulatory mechanisms for IgE despite the lack of any known inhibitory FcεR; the impact of target antigen expression levels and distribution in tumour lesions on the anti-tumour efficacy of IgE antibodies; stratification of patients with tumours featuring immune tumour environments congruent to IgE antibody therapy; the most suitable administration route, and malignant indication to help refine treatment and maximise patient benefit.Evidence from a number of studies points to monocytes and macrophages as important effector cells that participate in the anti-tumour functions of IgE in vitro and in vivo . On the Further areas for investigation include the impact of clinically available therapies such as chemotherapies, checkpoint inhibitors, steroids, and targeted treatments on the following: effector cells and IgE therapeutic efficacy and safety; expression of IgE Fc receptors by immune cells in different cancer types and patient tumours; mechanisms by which IgE acts on the TME, including IgE receptor-expressing and non-expressing cells, and their recruitment into tumours.neu. It is to be hoped that IgE antibodies against these targets will progress along the translational pipeline towards clinical testing. The field of AllergoOncology, including the use of IgE antibodies for cancer treatment, will undoubtedly enrich our understanding of human immunity and responses in health and malignant disease, and both inform and transform the design of future immunotherapeutic agents.A number of antibodies engineered with IgE Fc regions have been shown to engender potent effector functions and restrict tumour growth in disparate model systems. These include antibodies recognizing epitopes found on clinically validated tumour targets such as HER2/