The 2022 global human monkeypox outbreak emphasizes the importance of maintaining poxvirus research, including enriching a basic understanding of animal models for developing and advancing therapeutics and vaccines. Intravenous administration of monkeypox virus in macaques is arguably one of the best animal models for evaluating the efficacy of medical countermeasures. Here we addressed one criticism of the model, a requirement for a high-titer administration of virus, as well as improving our understanding of monkeypox virus pathogenesis. To do so, we infected macaques with a challenge dose containing a characterized inoculum enriched for the extracellular form of monkeypox virus. Although there were some differences between diseases caused by the enriched preparation compared with a relatively similar unpurified preparation, we were unable to reduce the viral input with the enriched preparation and maintain severe disease. We found that inherent factors contained within the serum of nonhuman primate blood affect the stability of the monkeypox extracellular virions. As a first step to study a role of the extracellular form in transmission, we also showed the presence of this form in the oropharyngeal swabs from nonhuman primates exposed to monkeypox virus. Although many have long viewed monkeypox as an emerging threat, monkeypox models have had the main initiative of providing evidence of efficacy for licensing medical countermeasures for smallpox. With the exception of Tembexia™, therapeutics have utilized the intravenous monkeypox virus (MPXV) nonhuman primate (NHP) model to help support licensure. In light of the global 2022 monkeypox outbreak, the screening and subsequent approval of medical countermeasures using the NHP model have proven to be of great benefit. That is, evidence from NHP studies supporting the licensure of smallpox countermeasures can concomitantly provide credence for the treatment of human monkeypox . Unlike There are two major morphogenic forms of poxviruses, the mature virion (MV) and the enveloped virion (EV) . EVs havThe in vivo role of EV in the pathogenesis of monkeypox is poorly defined, but there are multiple biological properties described for EV that likely contribute to its in vivo function. These properties include antibody and complement resistance, and relatively faster binding and entry kinetics than MV ,11,12,13The exact role of EV in the pathogenesis and transmission of orthopoxviruses is still unknown. By analyzing plasma samples generated from immunocompromised rabbits infected with vaccinia virus, strain IHDJ, Payne proposed a role of EV in dissemination . It is iThere has also been evidence that EV could be important in transmission. Payne and Kristensson provided evidence for the presence of EV as the major form of virus in the oropharyngeal region of mice infected via intranasal instillation with cowpox virus . DespiteHere, we described a characterized preparation of EV enriched monkeypox virus by its morphogenic composition. We compared the EV enriched preparation to a common preparation of monkeypox virus Zaire (unpurified) given at lethal and semi-lethal doses. We found that the new preparation may have had little, if any impact on disease, and did not affect lethality. We also found that the EVs within our preparation were not stable when administered intravenously. As a primer towards transmission research, we also present data for EV being present in the oral swabs of MPXV-exposed NHPs by electron microscopy.Monkeypox virus strain Zaire was propagated from scab material on chorioallantoic membranes, followed by one passage in LLC-MK2 cells, two passages in BSC-40 cells, and two passages in Vero E6 cells. This material was received from the Centers for Disease Control and Prevention (CDC) and passed in MA104 cells. This virus will be referred to as the master stock and was provided by John Huggins (USAMRIID).The master stock of virus was thawed, sonicated, and vortexed four times. MA104 cells were inoculated at a multiplicity of infection (MOI) of 0.5 (5 plaque forming units [pfu] for every 10 cells). The flasks were incubated in a 37 °C incubator and rocked every 15 min for 1 h. DMEM supplemented with 5% fetal bovine serum was added to each flask, after which the flasks were returned to the incubator. After 3 days, the cells were monitored for cytopathic effect (CPE) daily. One day after CPE was observed in 100% of the monolayer, the supernatant was removed, cells were scraped and pooled, and three freeze thaw cycles were performed. The material was then subjected to three sonication/vortex cycles and centrifuged to remove cells and larger cell debris. The supernatant was collected and aliquots were made into pre-labeled tubes. This material was the “crude” or “unpurified” working stock. A portion of this material was purified via a sucrose cushion, followed by a sucrose gradient  and was 6 pfu/mL, (approximately 1× LD50 [7 pfu/mL (or greater) would be less useful as it is a dose typically used to evaluate fulminant disease in the lesional MPXV NHP models. Therefore, multiple conditions were utilized to maximize the titer of the enriched virus stock.Before exposing the animals to a fresh preparation of monkeypox virus, the conditions were defined. The dose response for the intravenous model is quite steep and relatively small differences in titer could equate to more severe disease ,27. The  1× LD50 ) so that7 cells/flask) with crude working stock MPXV. The virus was adsorbed for 1 h, monolayers washed 4× with warmed EMEM with 2% heat-inactivated serum and provided a liquid overlay of the same media (10 mL). The supernatant was collected and clarified  by centrifugation (1000× g for 10 min at 4 °C). For all preparations, with the exception of the EV characterization experiments, an MOI of 30 and a harvest time of 24 h were used. So that only freshly prepared virus was used, this process was repeated for each experiment.Fresh MPXV for the enriched EV preparations was produced by infecting Vero E6 cells  for 1 h in a 37 °C incubator, with gentle rocking every 15 min. Two milliliters of EMEM containing 2% heat-inactivated FBS was added to each well and allowed to incubate for 4 days at 37 °C. The wells were then stained with a gentian violet solution (0.4% gentian violet) for at least 30 min, after which the stain was removed and the monolayers washed with distilled water. Plaques were subsequently enumerated.Freshly prepared stocks would have to be used to avoid issues with EV integrity. We first had to define a methodology to both understand the morphogenic makeup and also the repeatability of our virus preparation prior to animal inoculation. A fresh virus was prepared 24 h prior to intravenous exposure implementing the aforementioned conditions. Since the route of exposure was intravenous, we tested whether or not the susceptibility of the virions to MV-neutralizing antibody would change after the mechanical stress of expunging the material through a syringe . For the fresh preparation (EV) inoculums, the supernatant was tested by plaque assay by pipetting or by expunging through a 1 mL syringe, in the presence or absence  of a 1:50 dilution of MAb-7D11 (anti-MV antibody).Because the EV inoculum would have to be prepared from a fresh propagation on the day of challenge, initial experiments were conducted to ascertain the quantity of total virus, repeatability, and presence and relative quantity of EV. MPXV EV was propagated as stated above (viruses and cells) at an MOI of 3 and 30, and harvested at two time points, 24 and 48 h. The collected material was treated with either PBS or anti-MV antibody, MAb-7D11 and incubated for 30 min at 37 °C and plaque titrated.Although it has been reported that EVs are relatively resistant to the effects of serum that is homologous with the species from which the virions were propagated, we sought to confirm that intact EV could be responsible for potential differences observed by examining the susceptibility of monkeypox virus EV to NHP serum. To do so, MV neutralization assays and comet inhibition assays  were conMonkeypox virus was adsorbed to Vero E6 cells for 2 h before washing four times with warmed (37 °C) PBS. The wells were subsequently overlayed with EMEM containing 10% of either HI FBS, cynomolgus macaque serum, HI cynomolgus macaque serum, AGM serum, or HI AGM serum. Before addition to the well, 7D11 (1:500) or an equal volume of PBS was added to each. As a no comet control, 1.5% methylcellulose  was also used. Plates were placed in a 37 °C incubator for 5 days at an approximate angle of 10 degrees. Plates were removed, stained with crystal violet, and washed with PBS. Pictures were acquired and qualitatively evaluated.2O, followed by at least a 1 h exposure to 1% osmium tetroxide fumes before removal from Biosafety Level 3. Grids were then rinsed with distilled water five times and examined using a JEOLJEM-1011 transmission electron microscope.We questioned whether EV could potentially play a role in respiratory transmission by confirming its existence in oral/throat samples by IEM. To do so, stocks and oropharyngeal swabs were diluted 1:1 with PBS, added to grids, absorbed for 20 min, and washed three times with PBS. The grids were subsequently blocked for 20 min using 4% PBS-buffered normal goat serum. A 1:100 dilution of either MAb-5D8 (anti-MV) or MAb-10F10 (anti-EV), primary antibodies , was added, incubated for 45–60 min, and washed three times with PBS. This procedure was repeated for the application of the 1:40 secondary antibody (gold-conjugated). The grids were then fixed in 2% glutaraldehyde for 20 min and washed three times using dHMacaca fascicularis) were screened for neutralizing antibodies to monkeypox virus prior to infection as described below. We opted to use cynomolgus macaques since they tend to exhibit more severe disease than rhesus macaques and, as such, are more sensitive to the virus [Adult, male Mauritius cynomolgus macaques  were collected. Blood samples were collected via the femoral vein for all bleeds. Aliquots of EDTA blood were placed in a −80 °C freezer. Serum tubes were allowed to clot, centrifuged and serum removed according to manufacturer’s instructions. Serum was immediately utilized for blood chemistries. All exposures were performed by intravenous infusion via the saphenous vein.g for 10 min, and supernatants removed before processing for IEM. All samples were freshly prepared. Postmortem examinations were performed by a board-certified veterinary pathologist in USAMRIID’s Veterinary Pathology Division.An extra set of oropharyngeal swabs were collected from animals (unvaccinated) reported in Golden et al. , placed To confirm animals were not previously exposed to orthopox viruses, neutralization assays were performed. Serum from animals was heat-inactivated (56 °C for 30 min) and serially diluted in MEM alpha with 2% heat-inactivated FBS and HEPES. A target of 100 pfu/100 uL was added to each dilution and allowed to incubate at 4 °C overnight. Both positive (vaccinia immunoglobulin) and negative (media only) controls were concomitantly prepared. Titration of the samples was performed in duplicate wells by plaque assay. Results were reported as a percent reduction of the negative control.Extractions and QPCR were performed as described previously . BrieflyHematological data were generated on an ACT 10 Beckmann Coulter using whole EDTA blood. Abaxis Piccolos were used to evaluate clinical chemistries using Abaxis Chem12 or Chem13 reagent disks using serum samples.A postmortem examination  was performed on all animals, either as soon as death occurred or after euthanasia of terminally ill or moribund animals. All tissues were immersion-fixed in 10% neutral buffered formalin for a minimum of 21 days, according to Institute protocol.Formalin-fixed tissues for histologic examination were trimmed, processed, and embedded in paraffin according to established protocols . HistoloThe act of freezing/thawing and/or sonication of fresh preparations of vaccinia virus will destroy the integrity of the outer envelope of EV . To test5 versus 2.1 × 104 pfu/mL, in 24 h with limited CPE (≤1) and a tighter coefficient of variation (CV) (5 pfu/mL), whereas the 48 h incubation produced similar virus in the supernatant (6.6 × 105 pfu/mL) with more CPE (≥2) and a greater % CV  to evaluate total virus output A. An MOIion (CV) A. Using ion (CV) B. Again,ter % CV B. To proter % CV . From th5 pfu/mL and 2.4 × 105 pfu/mL, respectively). The syringe and pipet samples containing MV-neutralizing antibody were also quite similar, 2.6 × 104 pfu/mL and 2.3 × 104 pfu/mL, respectively, with a total neutralization of 90.2% and 90.7%, (5 pfu/mL), alluding to a maximum EV content of approximately 6.6% (4 pfu/mL of antibody resistant EV.Samples were prepared from the supernatant using a syringe or pipet and treated with either PBS or 7D11 (1:50). Both the syringe and pipet PBS titers for the inoculum were similar  and partially lethal (5 × 106 pfu/mL) dose are included for comparison. More details of this research will be published elsewhere [Data from animals intravenously inoculated with crude preparation of MPXV at a uniformly lethal . In all three NHPs, an attenuated disease course, as compared to the 5 × 106 pfu/mL or 5 × 107 pfu/mL IV model, was noted  survived to the study endpoint and were humanely euthanized on day 30 post-exposure (approximately 12 days after disease resolution). The third animal  was humanely euthanized on day 18 post-exposure due to a decline in clinical condition and was not considered moribund due to poxvirus infection. Because this animal’s disease course was not typical, it will be discussed separately.Gross, histopathologic and immunohistochemical findings in Animals #1 and #2 were reflective of a mild infection, with virus dissemination post-exposure and extensive viral clearance as evidenced by little to no poxviral antigen staining in target tissues at day 30 post-exposure. No MPXV-related macroscopic observations were noted in the lungs from either of the two surviving monkeys. Microscopic pulmonary findings typical of MPXV infection were noted in Animal #2, characterized by subpleural necrotizing and lesions that were immunopositive for MPXV antigen. Cutaneous macroscopic observations consisted of desquamated scars on the face, arms, legs, axillary and inguinal regions, and/or abdomen. Microscopically, cutaneous lesions were characterized by histiocytic inflammation and epithelial hyperplasia. Enlarged axillary and/or inguinal lymph nodes were observed macroscopically in both surviving monkeys. Microscopically, lymphoid hyperplasia was noted in multiple lymphoid organs; this was considered to be a nonspecific finding reflecting antigenic stimulation. All remaining tissues from the two monkeys euthanized on day 30 post-exposure were immunonegative for MPXV antigen.Animal #3 was humanely euthanized on day 18 post-exposure due to necrosis and ulceration in the hind limb attributed to extravasation of the challenge inoculum. Cutaneous lesions consistent with monkeypox infection were widely disseminated in inguinal and axillary areas, lips, chest, abdomen, arms, legs, and particularly striking over the affected right hind limb. Microscopically, cutaneous lesions were characterized by histiocytic inflammation, necrosis, and epithelial degeneration with intracytoplasmic inclusion bodies typical of poxviral inclusions. A solitary 3–4 mm pale nodule was observed on the pleural surface of the lungs. Microscopically, the nodule was characterized by a focal necrotizing and proliferative lesion, typical of MPXV infection. Additional microscopic findings attributed to EV infection consisted of necrotizing and proliferative steatitis surrounding the mesenteric lymph node. Immunohistochemistry revealed extensive immunoreactivity of macrophages and fibroblasts in the lung lesion, skin and underlying skeletal muscle of the affected hind limb, and perinodal mesenteric adipose tissue. Minimal immunoreactivity was detected in macrophages in lymphoid tissues. Circulating leukocytes were not immunoreactive.Since no discernible change in virulence was noted in the animal experiments, we questioned whether this was because the EV had no additional role during intravenous exposure, or if the route of exposure influenced the morphogenic state of the virus. Based on the literature, where EVs were protected from the effect of host-homologous complement, our initial hypothesis  was that the effects of serum would minimally impact the integrity or infectivity of the extracellular virus . Prelimip > 0.05). The crude preparation did not differ greatly from the freeze/thawed material (p > 0.05) and ranged from 98% to 99% (To show that the effect was specific to the fresh preparation (EV), crude stock virus was also tested concomitantly . All sam% to 99% D. Since % to 99% C,E. It iTo further illustrate this point, comet inhibition assays were performed . ExtraceNext, we questioned whether EV could play a role in transmission by addressing whether EVs were present in oral samples. Oral swabs were collected from two nonhuman primates three days after intravenous exposure to the MV form of monkeypox virus. Samples were collected and examined by IEM to detect the EV forms of monkeypox virus. In one animal, particles were present that labeled with anti-EV antibody, 10F10, to varying degrees A. From tOne of the major drawbacks to the monkeypox intravenous macaque model is the unnatural quantity of virus required to produce severe disease. Although other routes of exposure can reduce the input of virus required for lethality, ≥5 logs of virus are still needed and skin lesions are reduced . TherefoThe envelope of the extracellular virus is thought to be more fragile than that of the intracellular mature virus and purification, freeze/thaw cycles, and sonication are known to disrupt the membrane . AdditioHere, we characterized the morphogenic makeup of a fresh preparation of monkeypox virus propagated for 24 h in the Vero E6 cell line at a high MOI. Experiments at 24 and 48 h using MOIs of 3, 10, and 30 were also conducted and, with the exception of virus propagated for 24 h at an MOI of 3 96%), neutralization was between 88% and 91%, similar to those used for further experimentation and animal exposure. Sonication and freeze/thaw cycles liberated the enclosed MV-like particle increasing the neutralization to 97%–99%, but never 100%. Immunoelectron microscopy confirmed the presence of EV in the fresh preparation and the relative absence of EV from our historical stock. These data indicate that approximately 7%–9% of viruses in the inocula were EV with intact outer membranes. Based on the results of our comet assay, where comets were reduced in the presence of MV-neutralizing antibody, it is likely that more EV may be present, but lack outer membrane integrity. This would explain the initial high neutralization of the fresh preparations (approximately 90%), where the extracellular form of the virus should be prevalent, as exhibited by our comet assays . Ma et a6%, neutr6 pfu/mL for our inoculum, a dose that could cause a severe but not uniformly lethal disease in NHPs. We chose to continue the experiment and rationalized that the properties of EV could still make a significant difference while only comprising 7%–9% of the inoculum; in essence, we theorized that the small proportion of EV in the inoculum could disproportionately affect infection and disease. In addition to resistance to complement, EV have also been shown to have faster binding and entry kinetics [5 pfu/mL, of MV [We were unable to obtain our target titer of approximately 5 × 10kinetics ,42,43 ankinetics ,38,43. TL, of MV . This isL, of MV  and reviL, of MV ).We next tested whether or not expunging the supernatant through a syringe would damage the EV outer envelope. We found that the conditions used to produce the inoculum for exposure were repeatable and the mechanical stress induced by mimicking an intravenous exposure did not affect the integrity of the EV membrane, as determined by the MV-neutralizing assay.7 pfu intravenous model. With that being said, the onset and magnitude of viral DNAemia was strikingly similar to the 5 × 107 pfu model. This is despite two of the animals that received the entire inoculum intravenously  having qPCR levels far below the limit of detection. The magnitude of the viremia was greater (2 log10) and peaked later than reported results using traditionally prepared virus at a similar dose. Interestingly, no viable virus was detected from whole blood samples during the course of infection. It is possible that virus contained within the blood samples at early time points were not detectable due to the inherently high limit of detection when assaying blood samples. As for subsequent samples, it has been shown that animals exposed to similar doses of crude virus develop neutralizing antibody as early as day 5 [Exposure of cynomolgus macaques with similar material produced a mild smallpox-like disease. All animals survived the challenge, although Animal #3 was humanely euthanized prior to study termination for other reasons. Of the remaining animals, cutaneous lesions were few (64 and 203 lesions) and appeared concomitantly with lymphadenopathy on day 6. Although increases in temperature were noted, and interestingly biphasic in one case, a febrile state, as defined by a 2 °C increase from baseline, was not achieved in either animal. These findings are comparable to cynomolgus macaques exposed to crude preparations of monkeypox virus at a similar dose , but mucas day 5 . InteresWe examined whether EV could have been responsible for the small changes exhibited in vivo. From our MV specific neutralization assays, we found that freshly prepared EV became susceptible to MAb-7D11 in the presence of 50% serum, but not heat-inactivated serum, obtained from two nonhuman primate species. Furthermore, it required freeze/thaw fracturing to allow the MV-neutralizing antibody to affect the infectivity of the remaining (roughly 8%) EV. These results were confirmed by comet assay where secondary plaque formation (EV spread) was drastically reduced in the presence of 10% nonhuman primate serum. The addition of MAb 7D11 further reduced the presence of comets, as well. By itself, the comet assay suggests that serum and MAb 7D11 might work by the same mechanism, most likely taking advantage of ruptured EV, since MAb 7D11 also dramatically reduced comet formation. Some caution must be taken when examining mechanism of EV neutralization, as CEV and EEV could be different . The datAlthough the experiments presented in our report cannot completely differentiate the mechanism or define the exact membrane status of the viral particle, they provide evidence that few, if any, intact EVs were effectively administered to the cynomolgus macaques. Our results also question whether the morphological constituents of monkeypox viremia could be of the extracellular form or if we may be utilizing the wrong cell lines/methodology. Vero cells have been shown to have the regulators of complement activation (RCA) thought to provide resistance . We did Similar to other reports with vaccinia virus, MAb-7D11 neutralized monkeypox virus ,43,44. IIt is unclear what morphogenic form of the virus is naturally transmitted from host-to-host, but poxvirus canon dictates that EVs are too fragile to manage this task. One must consider that if transmission of MPXV and VARV is by direct contact (via respiratory droplets), it would logically follow that robust attributes are not required for the nominal period outside of the host. Moreover, the viral membrane may be stabilized by components in the extracellular environment. Additionally, it should be asked which particle is more capable of infection. Relative to MV, EV infection is kinetically more favorable ,38,42, rMore studies should be conducted to inquire if the EV phenomena (aggregated EV bundles) present in the oral samples have some role in transmission or are just an artifact. If real, the aggregation of monkeypox virus may be the reason for inefficient respiratory transmission and/or be a mechanism for environmental stability, possibly similar in function to A-type inclusion bodies produced by some poxviruses ,51,52. I


Subsequently,we compared the statistical models correlating the outcome of thereaction in different solvents with molecular descriptors producedfor each of these proposed configurations. The models allude to theimportance of different interactions in controlling selectivity ineach of the studied solvents. As a proof-of-concept for the practicalityof our approach, the models in chloroform ultimately led to loweringthe ketone loading to only two equivalents while retaining excellentyield and enantio- and diastereo-selectivity.The critical influence of solventeffects on proline-catalyzedaldol reactions has been extensively described. Herein, we apply multivariateregression strategies to probe the influence of different solventson an aldol reaction catalyzed by proline modified at its secondarysphere with boronic acids. In this system, both  Notably, in an enzyme, both the substrate and the activesite have an ideal microenvironment to interact and react selectively.2 Conversely, in a reaction flask, the interactionbetween a substrate and molecular catalyst is controlled by the interplaybetween their steric and electronic characteristics, which in turninfluences the way they assemble into a reactive intermediate. Furtherconfounding the situation, this interplay is highly influenced bythe surrounding solvent molecules.3 Thisstark difference is exemplified by the observation that many reactionsthat occur at room temperature enzymatically, oftentimes need lowtemperatures and long reaction times in vitro toafford desirable levels of selectivity.4 In organocatalysis, wherein selectivity is often governed by noncovalentinteractions,5 solvents significantly impactthe structural organization and degrees of freedom of intermediatesand transition states.13 This pronounced sensitivity of reaction outcomes to solvent changeshas led to unusual solvent combinations.The pursuit of reactionsthat deliver levels of selectivity similarto those provided by enzymes is at the core of asymmetric catalysis.6 Subsequently, List demonstratedthat the addition of chloroform (CHCl3) to a dimethyl sulfoxide(DMSO)/acetone solvent system, could speed up the reaction, minimizeelimination and increase er.7 Similar phenomenawere observed for this reaction when using more structurally complexcatalysts. Xiao and co-workers demonstrated that catalyst II in CHCl3 afforded the aldol adduct with 75:25 er, whileincreased er was obtained in acetone or a 1:1 mixture of acetone anddichloromethane (DCM). An even more drastic response to solvent variationswas identified by Shirai and co-workers (III), who reportedthat DCM and acetone delivered opposite enantiomers.9 Furthermore, changing the solvent to DMSO modulated theer to 88:12. The impact of solvent on cyclic ketones as aldol donorsis also marked  as aresponse to solvent variation , and methanol (MeOH). With the aim of obtaininga broad spread of results suitable for modeling, boronic acids withdifferent steric, electronic, and substitution patterns were representedin the data set .21 The parameters used in this process were intended to captureboth the electronic and steric variation due to different substitutionpatterns. The electronic nature of the structures is represented byNBO charges, and their steric nature is depicted by bond lengths,distances, and dihedral angles as well as Sterimol parameters. Thedirectional components of the dipole moment and specific bond stretchingfrequencies are stereoelectronic hybrids affected by the location,size, mass, and electronic nature of the substituents. The componentsof the dipole moment for all of the structures were taken with respectto the center of the boronic acid aromatic ring. The models with thehighest goodness-of-fit Q2 leave-one-outcross-validation values were identified for each proposed structurein each solvent system . We decided to usethis Q2 value as the selection criterionrather than the R2 because it not onlyreflects the goodness-of-fit but also the predictive robustness ofeach model, thus minimizing the likelihood of overfitted models beingselected.Molecular descriptors were extracted for each library,correlatedagainst all of the experimental data and the best performing modelwas identified .Under neat reaction conditions, wherein 18 equiv ofcyclopentanonewere used as both reactant and solvent, the model with the best fitwas based on the boronic acid–aldehyde π interactionstructure 4.f. The tructure 4.g. The tructure 4.h. The  solvent 3. Nevert solvent 4.i. The 5 width on the boronic acid aryl ring, andthe component of the dipole moment on the plane of the aryl ring inthe direction of the ortho and meta positions. Based on the assumption that both stereocenters are formedin the same step, we were expecting to identify models stemming fromthe same structural configuration for both the enantio- and diastereo-selectivity.Nevertheless, these types of selectivity are controlled by the facefrom which and with which the aldehydes approach.20 Therefore, it is not unlikely that they may be decoupledbecause of the fine balance between possible spatial arrangementsof the reaction components. Moreover, since the diastereoselectivityis fairly low in hexane showing a narrow distribution in responseto changes in the structure of boronic acid, it could indicate anaveraging across different configurations. Thus, the fairly good fitof the model with boronic acid alone could in fact also representan averaging across different structures or interactions in whichboronic acid takes part. Enantioselectivity, on the other hand, seemsto be more significantly impacted by different boronic acids and isperhaps more stringently controlled by a specific interaction or intermediaterepresented by the structure of boronic acid bound to proline.In all of the solvents besides MeOH and hexane the er valueswereexcellent for all boronic acids tested. In hexane, as opposed to MeOH,the er varied significantly as a result of the boronic acid structure.This variation enabled correlating the libraries of descriptors toenantioselectivity in hexane and the best fit was based on the boundboronic acid–proline structure 5. The pa3 or ACN as solvents with only6.8 equiv of cyclopentanone already surpassed our previously optimizedneat conditions.19 Given these results,we wondered whether the models could provide an indication as to whichof these systems could withstand further reduction in the amount ofketone without an erosion of selectivity. For the reaction in CHCl3 the bound boronic acid–proline structure providedthe best fit . We assumethat this disparity could indicate that both types of interactionsor species come into play in both cases, yet in CHCl3 acovalently bound proline–boronic acid species may play a moreimportant role.As stated above, we set out to study solvent effects withthe intentionof uncovering mechanistic aspects of our secondary-sphere guided aldolreaction, but in the process were also hoping to identify conditionswith lower aldol donor loading. Gratifyingly, the excellent experimentalresults obtained using CHClbest fit 4.h, wherbest fit 4.g. We s3 model was the total dipole moment (μtot)on the boronic acid moiety . Carbonyl charges aretightly correlated with pKa and Hammetvalues;22 therefore, we interpreted thisparameter to reflect binding strength between the proline and boronicacids. This assumption led us to presume that binding in CHCl3 is strong enough to have less of an impact on selectivitybecause, in contrast to ACN, the most important parameter in the CHCl3 model did not signify binding. Taken together, these hypothesesled us to select the reaction in CHCl3 as a more appropriatecandidate for lowering ketone loading. Indeed, when we lower the ketoneloading enantio- and diastereoselectivity are preserved; however,the yields in 1 h reactions are diminished. When we moved to slightlyhigher reaction times we were able to lower the ketone loading to2 equiv with excellent er and dr values .Because the bound boronic acid–prolinestructure 4.d proviiety see 4.h, wheriety see S11. Carb19 where both NMRand mass spectrometry suggested that the active catalyst in this systemconsisted of one proline unit bound to boronic acid(s). To addressthis discrepancy, we decided to measure mass spectra of boronic acidin the presence of proline and cyclopentanone in all of the testedsolvents. Due to low solubility, this analysis was not possible inhexane; however, in ACN, CHCl3, and MeOH we were able tonot only identify masses that can be attributed to boronic acid–prolineadducts but also to their enamines . This result supports our hypothesis that theactive catalyst could contain a boronic acid–proline adductbecause such adducts seem to form a putative reactive intermediatein the catalytic cycle. To try to settle this result with a possibleπ interaction between the boronic acid and aldehydes, we designeda proline catalyst that cannot form an ester derivative with boronicacid. Thus, proline methyl ester was prepared and submitted to ourreaction conditions in ACN with three different boronic acids thatspread over the dr range  and a stirringbar, was placed under argon . To this flask, 25 mgof activated molecular sieves (3 Å) and a boroxine derivative(0.03 mmol) were also added. Next, 0.8 mL of a stock solution of internalstandard 1,3,5-trimethoxybenzene  and water (11.3μL of water per 1 mL of solvent) in 37.5% V of cyclopentanoneand 62.5% V of a given solvent was also added to the solids in thevial. The reaction was then allowed to stir for 1 h and after thatquenched with a saturated solution of ammonium chloride. The organicphase was then collected, and the water phase was extracted once againwith chloroform. The organic phases were then combined and dried overmagnesium sulfate. Lastly, solvent was removed under reduced pressure,and the reaction crude was directly analyzed via 1H NMR for yieldand diastereoselectivity determination and via chiral-HPLC for determinationof enantioselectivity.For the subsetsin chloroform and hexane, due to low water solubility in this solvents,water was added to each individual reaction instead of being usedas a component in the solvent stock solutions. A 5 mL vial, containing5.8 g of proline (0.05 mmol) and a stirring bar, was placed underargon . To this flask was added 25 mg of activatedmolecular sieves (3 Å), and a boroxine derivative (0.03 mmol)were also added. Next, 0.8 mL of a stock solution of internal standard1,3,5-trimethoxybenzene  in 37.5% V of cyclopentanoneand 62.5% V of a given solvent was also added to the solids in thevial. To this mixture was added 9 μL of water (0.5 mmol). Thereaction was then allowed to stir for 1 h and after that quenchedwith a saturated solution of ammonium chloride. The organic phasewas then collected, and the water phase was extracted once again withchloroform. The organic phases were then combined and dried over magnesiumsulfate. Lastly, solvent was removed under reduced pressure, and thereaction crude was directly analyzed via 3. The organic layer was dried over sodium sulfate and concentratedto give a crude residue which was then purified via column chromatographyover silica gel using hexane–ethyl acetate or hexane–DCMas an eluent to afford pure product. Diastereoselectivity and yieldwere determined by 1H NMR analysis of the crude aldol product.The ee of the aldol product was determined by chiral-phase HPLC analysis.Unlikethe reaction indifferent solvents, the reactions for studying the stoichiometry ofketone, those with methyl prolinate, and blank reactions were notperformed in batch. These reactions were performed using the followingprocedure: proline , boroxine derivative (0.03mmol), 1,3,5-trimethoxybenzene , 25 mg of molecularsieves 3 Å, 0.8 mL of cyclopentanone, and 9 μL of waterwere placed in a screw-capped vial under argon . Themixture was stirred for 15 min at ambient temperature followed byaddition of aldehyde (0.5 mmol). After completion of the reaction,the reaction mixture was treated with saturated aqueous ammonium chloridesolution, and the whole mixture was extracted 3 times with CHCl


Our results showed that body weight of mice was significantly decreased in HBM and HCM groups from 1 to 4 weeks compared with the HFD group. The mRNA expression of ACAA2, ACACB, and SLC27A5 genes involved in the lipid metabolism in liver tissue were significantly elevated in HCM group, relatively to HFD and HBM group. In addition, the adipocyte number, size and lipid accumulation in the liver were significantly decreased in HCM group compared with the HFD group by H&E staining and oil red O staining, but was not change in HBM group. The mRNA levels of TNF-α and IL-1β inflammatory genes were significantly increased in HBM group, relatively to HFD and HCM group, which is consistent with results from inflammatory cell infiltration and tissue disruption by colon tissue sections. In conclusion, dietary supplementation of cow milk has beneficial effects on loss of weight and lipid metabolism in obese mice.The aim of this study was to evaluate the effects of buffalo milk and cow milk on lipid metabolism in obese mice. Milk composition analysis showed fat, protein, and total solid content in buffalo milk was higher than cow milk, while the lactose content of buffalo milk was lower than cow milk. After milk metabolite extraction and LC-MS/MS analysis, differential metabolites were mainly enriched in “linoleic acid metabolism pathways,” “pentose and glucuronate interconversion pathways,” and “metabolism of xenobiotics by cytochrome P450 pathways.” We fed three groups of C57BL/6J mice ( The complexity of obesity as a disease increases the susceptibility of obese individuals to develop other metabolic diseases, such as diabetes, metabolic syndrome, and cardiovascular diseases. A high fat diet will cause lipid metabolism disorder and fat accumulation in the mouse body, leading to causing obesity . The livcis-9 and trans-11 isoform of conjugated linoleic acid , n = 6 animals per high-fat diet + 0.4 mL buffalo milk + 0.4 mL normal saline as HBM group, n = 6 animals per high-fat diet + 0.8 mL cow milk as HCM group). For equal protein and energy in HBM group and HCM group was diluted with normal saline. The HFD in this study contained 60% fat . All the mice were housed under standard laboratory conditions  and at least on their diets for 5 weeks. The body weight of each mouse was recorded weekly. After 5 weeks, the serum and tissue samples were collected and stored for further studies.The animal experiment was approved by the Institutional Animal Care and Use Committee of Yangzhou University, and the study was conducted at Yangzhou University . Eighteen 8-week-old male C57BL/6J mice were obtained from the Comparative Medicine Animal Center of Yangzhou University. Following a week-long acclimation period, principle of near body weight was randomly divided into three groups . Each sample was placed in tubes with a preservative  and submitted to Dairy One Cooperative Inc.  for analysis of milk related indicators.The metabolite in buffalo and cow milk were extracted as described previously , 20. In At week 5 of intervention, mice were briefly food deprived (between 3 and 8 h starting at 06:00) prior to tissue and blood collection as described in detail . Serum g–ΔΔCT.Total RNA was extracted and purified from colon and liver tissues using the Trizol tissue kit according to the manufacturer instructions . Reverse transcription (RT) was performed using an RT Kit . RT reaction mixtures contained 1 μg total RNA and 1× PrimeScript RT Master Mix in a final volume of 20 μL, and were conducted for 15 min at 37°C. Reverse transcriptase was inactivated by heating to 85°C for 5 s. Quantitative RT-PCR (qRT-PCR) assays were performed using SYBR Premix Ex Taq TM II Kit . The qRT-PCR reaction mixture contained 1 × SYBR Premix Ex Taq TM II, 0.4 μM each forward and reverse primers, and 2 μL cDNA templates in a final volume of 20 μL. Reactions were performed as follows: initial denaturation at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. The primers used are listed in Small pieces of colon tissue were fixed in 10% phosphate-buffered saline (PBS) formalin and embedded in paraffin wax for hematoxylin and eosin (H & E) staining. After dehydration, the slices were sealed with neutral gum. The histology slides were performed using an optical microscope , with images being captured at a final magnification of 200×. The adipocyte area was measured using image analysis software .Oil red O staining was performed after small pieces of liver tissue was immersed in 60% isopropanol. Finally, these slides were sealed with glycerin gelatin. The histology slides were performed using an optical microscope , with images being captured at a final magnification of 200×. The fatty droplets area in hepatocytes was observed by oil red O staining and image analysis software .P < 0.05), while the same letters represent no significant differences (P > 0.05).MetaboAnalyst 5.0  was usedP < 0.01). However, the lactose content of buffalo milk was lower than that of cow milk . In addition, abdominal fat and liver tissue were analyzed. The index of abdominal fat and liver tissue from mice treated with buffalo and cow milk had no profound alter compared to the HFD group , but was not change by buffalo milk intervention.To explore the effect of buffalo and cow milk on hepatic lipid accumulation in mice induced by high fat diet, we performed H&E and Oil red O staining on the liver tissues . As showP < 0.05; To explore the effect of buffalo and cow milk on morphology of colon tissues in mice with high fat diet treatment, the colon H&E staining was performed. The HCM group had no significant change in the morphology of colon compared to HFD group, but morphology of colon was damaged by buffalo milk . ConsistDietary active substance may exert a key role in relieving the comorbidities of obesity. The protein and fat of milk are the most important nutrients, and are important indicators to measure milk quality . The conin vivo studies (In our study, mice body weight was significantly decreased in HBM and HCM compared to HFD group. Studies have demonstrated a relationship between milk intake and body weight in children and adolescents , 24, 25. studies , 33. Pre studies . Our datAt present, there are many animal studies on obesity. Lee’s index, as an index reflecting the degree of obesity, was first considered to be suitable for the evaluation of mouse obesity model . On the Previous studies have shown that dairy products reduce the risk of metabolic syndrome by reducing obesity and blood lipids, and also indirectly suggest that some components from dairy products improve liver fat disease and damage . Lipid mThe liver is an important organ which maintains the balance of lipid metabolism in the body, and the disorder of liver lipid metabolism will cause excessive fat accumulation in liver tissues, resulting in systemic inflammation and fatty liver. Studies have found that prolonged consumption of HFD causes lipid accumulation in liver and adipose tissue, and obesity is often characterized by an increase in number and size of fat cells . High faObesity is characterized by chronic low-level activation of the innate immune system and macrophage-induced metabolic inflammation. The interaction between adipocytes and macrophages plays an important role in the regulation of immune responses. Studies have suggested that obesity is associated with chronic inflammation of many tissues, including the liver, adipose tissue, and intestines , 48. TheIn this study, mice fed by high fat diet were perfused with cow milk and buffalo milk, and body weight gain of mice was significantly decreased by cow milk and buffalo milk perfusion. The cow milk can enhance the mRNA expression of ACAA2, ACACB, and SLC27A5 genes involved in the lipid metabolism in liver tissue. Moreover, area of lipid accumulation in liver was significantly decreased by cow milk intervention. These findings provide a better understanding of the role of cow milk in fatty liver, and thus dietary supplementation of cow milk may have beneficial effects on lipid metabolism in the context of obesity.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University.MJ and GZ designed the whole experiment and verified the validity of experiment and checked the results. KZ, QY, and XM performed the experiment, including chemical analysis, and statistical analysis. MJ, TY, and ZM worked on the manuscript. ZC, XG, YH, and ZM participated in the experiment design and gave valuable advice. All authors have read and approved the final version of this 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Funneliformis mosseae) under low P (no added P) or optimal P (200 mg kg−1) for 53 days. Low P stress significantly inhibited shoot and root growth, photosynthesis, tissue P content, and root P concentration in both genotypes. Shengrui 999 was more tolerant to P stress with less reduction of these traits compared to Zhongke 11. Shengrui 999 had a higher AMF infection rate than Zhongke 11 at both P levels. Under P deficit, inoculation with AMF significantly promoted plant growth and P uptake in both genotypes with more profound effects seen in Zhongke 11, whilst Shengrui 999 was more dependent on AMF under optimal P. Low P stress inhibited the growth and physiological attributes of both genotypes. The small-rooted Shengrui 999 was more tolerant to low P than Zhongke 11. Inoculation with AMF alleviates low P stress in both genotypes with a more profound effect on Zhongke 11 at low P and on Shengrui 999 at high P conditions.Soil available phosphorus (P) is one of the main factors limiting plant growth and yield. This study aimed to determine the role of arbuscular mycorrhizal fungi (AMF) in P-use efficiency in two maize genotypes with contrasting root systems in response to low P stress. Maize genotypes small-rooted Shengrui 999 and large-rooted Zhongke 11 were grown in rhizoboxes that were inoculated with or without AMF ( Zea mays L.), as an important grain crop, feed crop, industrial material, and energy resource. It is one of the most widely planted crops in the world with the highest total production [Phosphorus (P) is one of the key nutrients that is necessary for plant growth and development, and it is mainly absorbed and utilized by plant roots via the phosphate transporter protein carriers. However, most of the P in the soil exists in the form of insoluble organic P, which is also characterized by high adsorption and low mobility, leading to the available P being scarce in soil . The scaoduction ,5. Howevoduction ,7,8.Large amounts of P fertilizer have been widely applied to meet the plants requirement for P ensuring plant growth and to achieve a high yield in maize production. However, the excessive use of chemical fertilizer not only inhibits soil beneficial microorganisms but also causes environmental pollution ,10. PlanThe aim of this study was to investigate how mycorrhizal inoculation and root morphological traits respond to low P stress in two maize genotypes with contrasting root systems elected from recent studies using a semi-hydroponic root phenotyping system . The stup < 0.01), but had no significant effect on root–shoot dry mass ratio of both maize genotypes regardless of inoculation   . p > 0.05)  . Without AMF inoculation, the two genotypes exhibited significant differences in response to low P stress with a greater inhibition on shoot and root growth in Zhongke 11 than in Shengrui 999 . Under op < 0.01), and the total root length, root surface area, and shoot P content , but significantly decreased root diameter (p < 0.01) and PUE (p < 0.05) of both genotypes  , had a nUnder low P conditions, Zhongke 11 was more responsive to AMF than Shengrui 999. Inoculation with AMF significantly increased the values of seven traits  for both genotypes . ZhongkeVisual root growth through the glass plates showed increased differences among the four treatments with time in both genotypes . Low P tUnder optimal P, AMF inoculation diminished the correlation between the total root length and root dry weight a, and thUnder optimal P, AMF inoculation decreased the correlation between photosynthesis and the shoot dry weight a and shoThe plants root system architecture (RSA) traits include three main categories: topological properties, geometric properties, and physiological properties . RSA is It is well accepted that inoculation with AMF improves P acquisition, especially when the availability of soil P is low. The extending hyphae of AMF can greatly increase the absorption area and can help solubilize P in the soil, thus enhancing P uptake and promoting plant growth and yield, particularly for species with poor root development ,43,44,45The formation of AM can alter RSA to tolerate low P stress, and the effect of AM on RSA is usually attributed to the improvement of P absorption. In addition, plants themselves can resist low P stress by reducing the diameter of the root system, making the roots thinner, and producing more branches. This study showed that among the morphological traits, average root diameter was significantly reduced after inoculation of AMF. This result may indicate possible complementarity between root morphology and mycorrhiza in P uptake under low P stress. Ma et al., (2018) suggested that plants evolved thinner roots to reduce their dependence on AMF for nutrient uptake  Figure . The resAMF inoculation also significantly increased photosynthesis of both maize genotypes. The improvement in photosynthesis that was associated with AMF is mainly due to the improvements in the availability of P and other nutrients that supply the photosystem . The carThese results indicate that AMF inoculation can alleviate low P stress through the improvement of RSA, plant growth, photosynthesis, and P uptake . Most AMBrassica napus), in which the P-efficient genotype developed more dominant root morphological traits, intensive rhizosphere acidification, greater exudation of carboxylates, P uptake, and shoot and root dry weight than a P-inefficient genotype [The two genotypes showed significant genotypic variation in RSA including total root length, total surface area, and root volume, confirming our previous observations from phenotyping studies . The largenotype . It has genotype ,71,72  than in Zhongke 11 (large-rooted) . TawarayZea mays L.), two P treatments, and two arbuscular mycorrhizal (AM) inoculation levels. Genotype Shengrui 999 had a smaller root system while Zhongke 11 had a larger root system and were selected from the recent phenotyping study for root traits [−1) and optimal P . The inoculum that was used in this study was AM fungus Funneliformis mosseae BGC NM02A, propagated with Trifolium repens using the pot culture method [−1 (+AM) of the inoculum and the non-inoculation control was given the same dosage of sterilized inoculum with bacterial filter liquor (−AM). There were four replicates in each treatment.The experiment was conducted in rhizoboxes in a temperature—controlled glasshouse with day/night temperatures of 23 (±5) °C/16 (±3) °C and an average relative humidity of 65–75% at Northwest A&F University, Yangling  from November to January 2020. This study used a randomized complete block design that comprised of two genotypes of maize  were constructed from polyvinyl chloride (PVC). One side of the rhizobox was made of transparent plexiglass plate, which can be used to observe the dynamic growth of roots and covered with a black plastic sheet to avoid light exposure to the roots during the experiment. Rhizoboxes were placed on steel stands at an angle of 30° and spaced 0.05 m apart from each another. A total of 32 rhizoboxes (2 × 2 × 2 × 4) were used.−1, total nitrogen 253 mg kg−1, available nitrogen 19.6 mg kg−1, available P 2.54 mg kg−1, available potassium 131 mg kg−1, and pH 7.5. For the optimal P treatment (200 mg kg−1), P was supplied in the form of Ca(H2PO4)2 and mixed thoroughly with the soil together with the base fertilizer: nitrogen (100 mg kg−1) and potassium (120 mg kg−1). For the low P treatment, only the base fertilizer was added. The soil that was used in the experiment was taken from Yangling farmland (0–20 cm soil layer). The soil was air-dried, sieved (2 mm), and mixed with an equal weight of air-dried washed fine river sand. The physical and chemical properties of the mixed soil were as follows: organic matter 3.90 g kgEach rhizobox was filled with 3.3 kg of the above soil mixture amended with base fertilizer with or without P addition. Prior to the soil filling, the bottom of each rhizobox was covered with a layer (2 cm) of small rocks to allow free draining. Each rhizobox was filled with the soil mixture with the top 10–20 cm layer filled with the soil amended with 100 g of AM inoculum for the AM inoculation treatment. For rhizoboxes without AMF inoculation, 5 mL of the fungicide filtrate of the same amount of AMF inoculum was added to the soil. Finally, the top 2 cm of the rhizoboxes was topped up with the untreated soil mixture. 2O2 for 10 min. After rinsing with water, the seeds were soaked in saturated CaSO4 solution for 8 h. A total of four homogenous pre-germinated seeds of the same genotype were sown in two holes (two seeds per hole) at a depth of 2 cm in each rhizobox, ensuring that the seeds were in contact with the glass wall. After emergence, two plants from different holes were retained in each rhizobox and were considered as one replication. Uniform seeds were surface sterilized in 10% HThe plants were watered manually from the top as required to maintain the soil water content close to field capacity and the rhizoboxes were randomly repositioned weekly to minimize environmental effects. The root growth was traced once a week commencing on the 10th day after transplanting (DAT). Visible new roots through the glass panels were traced on the transparent sheets using a marker pen. Immediately afterwards, the visible new roots were also marked on the glass panels to facilitate the next observation of the newly grown roots. Root tracing was done 4 times: DATs 10, 17, 24, 31) when some roots had reached the bottom of the rhizoboxes. The traces were scanned at 300 dpi using a portable scanner  and root images were analyzed for root length using WinRhizo Pro software .All the plants were harvested 53 DAT for assessments. Before harvest, the shoot height and leaf number were measured manually and photosynthesis was measured using a portable photosynthesizer at 9:00–11:30 am on a sunny day. The latest fully expanded leaf of each plant was used for measuring photosynthesis. On the day of harvest, plants and root systems in each rhizobox were photographed before and after the glass wall was removed. The two plants in each rhizobox were harvested by cutting the shoots from the roots along the soil layer. The shoots were dried in an air-forced oven at 75 °C for 72 h to a constant weight to determine the shoot dry weights. The root samples were rinsed with tap water, and a small part of roots was randomly cut from multiple directions and stored in 10 mL centrifuge tubes with 70% ethanol solution as mycorrhizal microscopy samples, the rest of the root samples were stored in plastic bags in a 4 °C refrigerator for later root scanning. The roots were scanned in greyscale at 300 dpi using a desktop scanner and were analyzed in WinRHIZO as mentioned above to obtain root morphology data including root length, average root diameter, root surface area, and root volume. After scanning, the roots were oven-dried to determine root dry weights. The root–shoot mass ratio (RSM) was calculated as the root dry weight divided by the shoot dry weight.The determination of P concentration: the dried root and shoot parts were ground into a powder, then 0.1 g of sample was put into the digestion tube, and 5 mL of concentrated sulfuric acid was added for digestion at 360 °C. The P concentrations of the root and shoot parts were measured by molybdate-blue colori-metric method , and P cThe determination of AMF infection rate: the root samples were cut into 1-cm long pieces, followed by bleaching with 5% KOH , acidifying in 2% HCl , and staining in 0.01% acid fuchsin . The extent of root length colonization was estimated using the gridline intersect method :AMF infep < 0.05, LSD). Pearson’s correlation coefficient was used to analyze the relationship between morphological and physiological traits. The graphs were drawn in Origin 2018 .The observed data were calculated and sorted by Microsoft Excel. Values for 16 traits were analyzed by three-way ANOVA using SPSS 20.0  to test the significance of different factors  and their interactions (vice versa when the soil P was optimal. Given that plant available P is generally deficient in soils, the non-renewable nature of P fertilizers, and the high cost and negative environmental impact of heavy fertilizer application, it is crucial to efficiently manage and P applications by planting P-efficient genotypes with AMF inoculation. Future studies with more genotypes and long-term field trials are required to determine the roles of root system architecture, rhizosphere physio-chemistry, and beneficial microbes for maize under low P conditions.The study demonstrated that low P stress significantly inhibited shoot and root growth of both maize genotypes, reduced leaf photosynthesis rate, plant P content, and PAE. Genotypic variation in response to low P stress was observed with a more profound inhibitory effect on the large-rooted genotype Zhongke 11 compared to the small-rooted Shengrui 999. Zhongke 11 was more sensitive to low P stress than Shengrui 999. AMF inoculation significantly promoted plant growth, root–shoot dry mass ratio, photosynthesis rate, P uptake, and PAE under both P conditions. Zhongke11 was more dependent on AMF than Shengrui 999 under low P conditions and


In engineering structures that are subject to cyclic loading, monitoring and assessing fatigue crack growth (FCG) plays a crucial role in ensuring reliability. In this study, the acoustic emission (AE) technique was used to monitor the FCG behavior of 2.25Cr1Mo0.25V steel in real-time. Specifically, an AE multi-parameter analysis was conducted to qualitatively assess the crack growth condition and quantitatively correlate the crack growth rate with AE. Various AE parameters were extracted from AE signals, and the performances of different AE parameters were analyzed and discussed. The results demonstrated that four stages of FCG, which correspond to macrocrack initiation, stable crack growth with low crack growth rate, stable crack growth with high crack growth rate, and unstable crack growth, are distinctly identified by several AE time domain parameters. The sudden and continuous occurrence of many AE signals with high count (>100) and high energy (>40 mV·ms) can provide early and effective warning signs for accelerated crack growth before final failure occurs. Moreover, linear correlations between crack growth rate and different AE parameters are established for quantifying crack growth. Based on the AE multi-parameter analysis, it was found that the count, energy, and kurtosis are superior AE parameters for both qualitatively and quantitatively characterizing the FCG in 2.25Cr1Mo0.25V steel. Results from this research provide an AE strategy based on multi-parameter analysis for effective monitoring and assessment of FCG in engineering materials. As a valuable structural health monitoring technique, the acoustic emission (AE) technique has been widely used for the real-time damage monitoring and integrity assessment of materials and structures in a variety of situations. Acoustic emissions are essentially the transient stress waves that are produced as a result of the rapid release of energy from localized sources within material under stress . The pridC/dN) as a function of stress intensity factor range (ΔK) in commercial-purity zirconium and divided the AE into three stages, which corresponded to the three common stages of the curve of fatigue crack growth rate  methods such as infrared thermography (IRT)  and digis et al.  presentes et al.  recommens et al. ,10,11. FK for predicting the fatigue crack length [m and C are material constants. Roberts et al. [K of S275 JR steel plate during FCG, which can be represented by a formula similar to the Paris–Erdogan law. This formula can be expressed by:p and B are material constants and can be determined by experiments. By eliminating logΔK from Equations (1) and (2), the relationship between Note that the above investigations mainly focus on the qualitative characterization of FCG with the aid of AE. Moreover, the effort has also been made to quantitatively correlate multiple AE parameters with k length ,16,17,18k length :(1)log of the CrMoV steel was 0.15 C, 0.10 Si, 2.30 Cr, 0.98 Mo, 0.30 V, 0.54 Mn, 0.05 Al and balance Fe. The compact tension (CT) specimen was machined from a CrMoV steel plate to perform the FCG test at room temperature. The design of the specimen size see  is basedThe AE signals generated during the FCG process were collected by using an advanced AE monitoring system  including a high-sensitivity AE sensor, a preamplifier, and a data acquisition system. The AE sensor, R15α, is a resonant piezoelectric sensor that has a resonant frequency of 150 kHz and an operating frequency range of 50–400 kHz. The sensor was fixed on the specimen surface using vacuum silicone grease as couplant and the position of the sensor is shown in To perform the AE multi-parameter analysis, eight time domain parameters including amplitude, count, energy, information entropy, rise angle (RA), root mean square (RMS), kurtosis and crest factor were first extracted from each AE signal recorded during the fatigue process for characterizing the FCG behavior. Furthermore, the Fast Fourier Transform (FFT) was performed to provide frequency information about AE signals because the frequency is generally regarded as an effective measure in discriminating different failure types ,25. ThusN of two specimens. The final crack sizes are close to 20 mm and the fatigue lives reach approximately 190,000 cycles. The crack growth rate is calculated based on the secant method, which can be expressed as follows:ith fatigue crack size, and K in double logarithmic coordinates for both specimens. The K and the data points can be fitted using the classical Paris–Erdogan crack growth law. The Paris–Erdogan law constants and the fitting coefficient R2 can be found in In stage A, noticeably high crack growth rates are observed. This is because after the onset of fatigue loading, the specimen undergoes significant plastic deformation and the crack starts to initiate from the notch. At the same time, a number of AE signals emerge in this stage. A large proportion of signals show low amplitudes of less than 60 dB and counts of less than 50. However, some AE signals with higher amplitude and energy can also be sporadically observed. This is more evident in the plots of entropy and RA value because many data points with high numerical values can be easily seen, indicating the AE source is active in this stage.When the crack propagates into stage B, however, the crack growth rate slightly decreases and remains low. The crack grows almost linearly and the crack size at the end of stage B is less than 2.7 mm, indicating this stage is related to stable crack growth with a low During stage C, the crack length increases by approximately 5 mm, and the crack growth rate exhibits an obvious increase and significant fluctuations compared with that in stage B. This evidence suggests stage C corresponds to the stable crack growth with a higher When the crack further grows into stage D, which is close to the final failure of the specimen, a substantial rise in crack growth rate along with dramatic fluctuations can be evidently observed. The crack size increases by more than 12 mm and the crack growth rate increases about six times. These observations indicate stage D is associated with rapid or unstable crack growth before failure. Similar to the AE characteristics in stage C, the numerical values of all AE parameters in stage D exhibit a substantial increase. For instance, the maximum values of count and kurtosis are almost double those in stage C. Such distinct AE results are in good agreement with the rapid increase of To further compare the AE characteristics during different stages of FCG, the AE count and energy of signals generated during each stage of two specimens were statistically analyzed and the results are illustrated in boxplots. As can be seen in To further quantify the performance of FCG characterization by different AE time domain parameters, the coefficient of variance (CV) of each parameter was calculated and compared. A CV measures relative dispersion of a dataset and is calculated by dividing the standard deviation by the mean . A higheIn short, based on the combined analyses of AE multi-parameters and crack growth behavior, four stages of FCG can be distinctly identified and differentiated. Ostash has claimed that the fatigue fracture of materials generally includes three stages, that is, macrocrack initiation, macrocrack growth and spontaneous fracture . The perα and β are constants and can be determined by experiments, and X indicates the growth rate of AE data within a certain number of fatigue cycles N. Specifically, in this study, X can represent the AE amplitude rate, count rate, energy rate, entropy rate, RA rate, RMS rate, kurtosis rate and crest factor rate. The Equation (5) obtained in this work is also consistent with the previous results that there is an empirical linear relationship between crack growth rate and AE growth rate in long fatigue crack growth of metallic materials [aterials ,15,16,18Note that the accuracy of crack growth rate predictions depends on the use of the AE parameter in the quantitative relationship. If an improper AE parameter is used for constructing the linear correlation, a large error in the predicted crack growth rate may be obtained. To determine the superior AE parameters in describing Equation (5), linear least squares regressions are conducted. As shown in The tested specimen shows a typical brittle fracture with a flat fracture surface. To further understand the microcosmic mechanism of FCG, the fracture surface morphology of 2.25Cr1Mo0.25V steel was investigated with the aid of SEM. (1)Based on the combined analyses of AE time domain parameters and crack growth rate, four stages of FCG  of 2.25Cr1Mo0.25V steel can be distinguished. The four stages correspond to crack initiation, stable crack growth with low crack growth rate, stable crack growth with high crack growth rate, and unstable crack growth, respectively. The continuous emergence of a large number of AE signals with high count (>100) and high energy (>40 mV·ms) in stages C and D can help to provide early and effective warning signs for accelerated crack growth.(2)The centroid frequency of AE signals caused by FCG of 2.25Cr1Mo0.25V steel is distributed in a narrow range of 170–220 kHz. The centroid frequency may not be appropriate for assessing the crack growth condition due to low variability, however, the occurrence of such a frequency band can help to identify possible crack growth signals.(3)Linear correlations are found between crack growth rate and different AE parameters for quantifying crack growth. However, it should be noted that these quantitative correlations are only valid in current laboratory conditions. This is because AE signals are highly influenced by the sensor/source distance, specimen’s geometry and coupling quality , and con(4)The AE multi-parameter analysis is recommended for damage characterization due to its advantage of reducing errors in using individual AE parameters. In this study, based on the multi-parameter analysis, one can conclude the count, energy and kurtosis are superior parameters for both qualitatively and quantitatively characterizing the FCG of 2.25Cr1Mo0.25V steel.In the present investigation, an AE multi-parameter analysis was performed for characterizing the FCG behavior of 2.25Cr1Mo0.25V steel. In particular, a variety of time domain parameters  and frequency domain parameter , were calculated for qualitatively assessing the crack growth condition and quantitatively correlating the crack growth rate with AE data. The performance of each AE parameter was analyzed and discussed. Major conclusions can be drawn as follows:


Noninvasive prenatal testing (NIPT) is the testing of blood samples from pregnant women to screen for fetal risk of chromosomal disorders. Even though in vitro hemolysis of blood specimens is common in clinical laboratories, its influence on NIPT has not been well investigated.Peripheral blood samples were collected from 205 pregnant women and categorized according to the concentration of free hemoglobin in the plasma. After performing NIPT using massively parallel sequencing, the quality control metrics were analyzed and compared with samples that did not undergo hemolysis or samples redrawn from the same women.The specimens were divided into four groups based on the concentration of free hemoglobin: Group I , Group II , Group III , and Group IV . There was no significant difference in the quality control metrics of clinical samples with slight or moderate hemolysis (Group II and III). However, samples with severe hemolysis (Group IV) showed a significantly increased rate of duplicated reads (duplication rate) and fetal fraction, as well as decreased library concentration compared with samples without hemolysis. Moreover, the increase in fetal fraction caused by hemolysis was confirmed by redrawing blood samples in Group IV.For NIPT using massively parallel sequencing, samples with slight or moderate hemolysis (≤ 4 g/L) are acceptable. However, careful consideration should be taken regarding the use of severely hemolyzed samples (> 4 g/L), since they might increase the risk of test failure.The online version contains supplementary material available at 10.1186/s12920-022-01280-2. NIPT screens cell-free fetal DNA (cffDNA) fragments for common fetal aneuploidies, including trisomy 21 (T21), trisomy 18 (T18), and trisomy 13 (T13) –6. The fHemolysis is the destruction of red blood cells, which leads to the release of hemoglobin and other intracellular components into the blood . HemolyzIn clinical laboratories, as much as 3.3% of blood specimens show various degrees of hemolysis . The hemThe influence of hemolysis on NIPT has not been well investigated. A recent study showed that hemolysis in Cell-Free DNA Collection Tubes had no impact on the fetal fraction in NIPT . One of In this study, we used clinical specimens to comprehensively investigate the effect of hemolysis on the quality control metrics of NIPT using massively parallel sequencing, including fetal fraction, library concentration, duplication rate, mapping rate, and GC content. Our results can be used by clinical laboratories to evaluate samples on reception and determine which should be rejected.This study was conducted in two parts. In the first part, we collected specimens from clinical laboratories of the Beijing Genomics Institute (BGI) from June 2020 to December 2021. The specimens were collected from pregnant women who received a NIPT test in a clinical setting. Specimens were categorized into four groups based on their free hemoglobin concentration. The effect of hemolysis was evaluated based on five quality control metrics: library concentration, fetal fraction, GC content, mapping rate, and duplication rate. The library concentration was used to assess the quality of the library, while the other four metrics were used to assess the sequencing quality. Duplication rate was defined as the proportion of reads of duplicate copies with the same barcode and the same 5' and 3' coordinates, reflecting the complexity of libraries , , respectively. Based on a visual assessment of plasma color, Group I samples were considered non-hemolyzed, whereas those of Group II, Group III, and Group IV were recorded as mild, moderate, and severely hemolyzed samples.+1, 16+0, 15+4, and 16+2 in Group I, Group II, Group III, and Group IV, respectively. There was no significant difference in the maternal age and gestational week of individuals in Groups II, III, or IV compared with Group I  in Group IV failed to meet this threshold. These samples were processed a second time. The mean library concentration of Group I, Group II, Group III, and Group IV was 9.69 ng/μL, 11.25 ng/μL, 10.94 ng/μL, and 7.30 ng/μL, respectively. Overall, the library concentration of Group IV was significantly lower compared with Group I (p = 0.002), and the duplication rate was also significantly higher in Group IV than Group I .The other four quality control metrics, fetal fraction, GC content, mapping rate, and duplication rate, were obtained from the sequencing data. There was no significant difference between the four groups in GC content or mapping rate Table . HoweverTo further investigate the correlation between hemolysis and the quality control metrics, we calculated the Spearman's correlation coefficient Fig. . It was Of the 25 patients whose samples had severe hemolysis (Group IV), 21 agreed to come back to redraw blood. The new samples were collected within an average of eight days (3–12 days) after the first blood collection. All samples did not hemolyze at the second blood draw. We were thus able to use pairwise comparison to compare the quality control metrics of samples from the same woman with and without hemolysis.p < 0.001, Table p = 0.007). As in the between-group analysis, there was no change in the GC content or mapping rate. Interestingly, the fetal fraction was higher in the hemolyzed specimens, although they were collected an average of eight days earlier .The mean library concentration was 14.24 ng/μL for the non-hemolyzed samples, which was significantly higher than the hemolyzed specimens  during in vitro hemolysis, which would release fetal genomic DNA and thus increase the fetal fraction in maternal peripheral blood. This hypothesis was supported by a previous study, which demonstrated that most NRBCs in maternal peripheral blood before 24 weeks of gestation were of fetal origin, and after that the majority were of maternal origin . Since tThe mapping rate and GC content were not affected in hemolyzed specimens. The mapping rate is used to evaluate the proportion of uniquely mapped reads out of all input reads. When the quality of the library is low, such as following contamination by other samples, the mapping rate is low. GC content is another critical metric. The average GC content in human genomes ranges from 35 to 60%, with a mean of 41% . VariatiOne concern regarding hemolysis is the lysis of leukocytes, which releases maternal genomic DNA into the plasma. Contamination with maternal genomic DNA would introduce a great deal of background noise and interfere with NIPT analysis. To prevent the lysis of leukocytes, cfDNA collection tubes are generally used in clinical practice. cfDNA collection tubes used in this study contain preservation agents to stabilize white blood cells, inhibiting them from releasing DNA into the plasma. Thus, when hemolysis occurs, red blood cells rupture but leukocytes will not be simultaneously lysed , 38. ThiOne limitation of this study is the lack of sufficient samples with chromosomal abnormalities confirmed by amniocentesis. Such samples would facilitate the assessment of whether hemolysis affects the results of NIPT. During the period of this study, only two hemolyzed samples with confirmed prenatal chromosomal abnormalities—chromosome 22q11.2 deletion syndrome (DiGeorge syndrome)—were available. The deletion in one sample was 2.9 Mb; in the other, 1.5 Mb. The two samples had mild hemolysis, and all five quality control metrics passed the cutoffs. More importantly, NIPT successfully identified the two deletions  are acceptable for NIPT using massively parallel sequencing because no impact on the quality control metrics was found. Unnecessary rejection of samples can thus be avoided by measuring the degrees of hemolysis. However, greater attention should be paid to severely hemolyzed samples (free hemoglobin concentration > 4 g/L) because severe hemolysis significantly decreased duplication rate, increased library concentration and fetal fraction. Accepting these specimens might increase the risk of test failure and thus waste time during prenatal testing.These findings provide a scientific basis for NIPT laboratories to evaluate hemolyzed samples for high-throughput sequencing. Rejecting samples with free hemoglobin concentration > 4 g/L would reduce experimental errors which could harm patients and also lead to repeated specimen collection and analysis, increasing the cost of NIPT .Additional file 1. The CNV plot of NIPT and prenatal diagnosis of sub-chromosomal CNVs hemolyzed sample 1.Additional file 2. The CNV plot of NIPT and prenatal diagnosis of sub-chromosomal CNVs hemolyzed sample 2.


Both sebum production and composition were found to be affected by cetuximab treatment. These specific sebum lipid alterations could help to better understand the development of cetuximab-induced skin toxicity and could be useful for improving skin lesion prevention and treatments.The etiology of the skin toxicity that is a common side effect of cetuximab is still under debate. In this study, proton nuclear magnetic resonance  and squalene (SQ) was found to be induced by cetuximab administration. The reduction of these two lipid classes was also found to be associated with the severity of skin rash experienced by patients. The results of this study indicate that cetuximab-based treatment can reduce sebum gland activity, leading to an overall decrease in sebum production and the induction of specific modifications to its composition. The extent of the loss of skin barrier function may be important for determining the severity of skin toxicity development.The epidermal growth factor receptor inhibitor (EGFRIs) treatments are commonly associated with the development of adverse skin effects. This study aims to investigate the lipid composition change in sebum during cetuximab-based treatment in an attempt to identify specific metabolic signatures useful in predicting the occurrence of severe skin toxicity. Sebum from 30 metastatic colorectal cancer (mCRC) patients was collected at three time points during the targeted therapy by the application of Sebutape Compared with the standard cytotoxic effects of conventional chemotherapeutic drugs, these target EGFR therapies are characterized by low systemic side effects [Epidermal growth factor receptor (EGFR) is a tyrosine kinase transmembrane protein expressed in healthy tissue such as the gastrointestinal tract, epithelial tissues, skin, and hair follicles, and it is also involved in tumor proliferation. Given its role in cancer progression, several EGFR inhibitors (EGFRIs) have been developed in the form of monoclonal antibodies, namely cetuximab  the potential sebum lipid alterations in RAS wild-type mCRC patients undergoing cetuximab treatment [This study aims to investigate by reatment . In partIn this investigation, a group of 30 RAS wild-type mCRC patients were enrolled. The clinical characteristics of the patients, stratified according to the development of skin toxicity and chemotherapy type, are reported in 2 biweekly. Patient rash was classified during go-rounds of chemotherapy according to Common Terminology Criteria for Adverse Events (CTCAE v3.0) [Patients were subjected to different chemotherapy regimens constituted by CAPIRI, FOLFIRI, and FOLFOX, all associated with cetuximab administered at the dose of 500 mg/mAE v3.0) . The sevSebum samples were collected at different times during the progress of the chemotherapy, namely before the first administration  and after 14 and 28 days (time point “b” and “c”) from the start of the treatment, which correspond to the end of the first and second cycles of the therapy, respectively.®, Cuderm Corporation, Dallas, TX, USA) [®  tube and then stored at −80 °C. All of the procedure was performed using tweezers.Sebum samples were collected by the application of a lipid-adsorbing tape   accordin1H-NMR method. Clean, blank tapes were subjected to the same extraction procedure and were used to evaluate the presence of interfering signals resulting from the polymeric matrix of the tape.Sebum samples from a healthy volunteer were used to establish sebum analysis ® the non-adsorbing black tabs of tape were cut-off and its remainder placed into a 1.5 mL Eppendorf® tube containing 1 mL of isopropanol . Different solvents such as chloroform, ethanol, and cyclohexane were previously reported to efficiently extract lipids from Sebutape® [1H-NMR measurement. After solvent addition, the tape was vortexed for 10 s three times, bath sonicated for 20 min (cooled by ice), then vortexed again for 10 s three times. After, the solvent was transferred into a clean 1.5 mL Eppendorf® tube and centrifuged at 13,000× g rpm for 5 min to eliminate traces of skin/hairs present in the extract. A volume of 800 µL of the supernatant was placed into a clean Eppendorf® tube and evaporated under vacuum. The dried extract was then resuspended in 600 µL of deuterated chloroform  by 10 s of vortex followed by 1 min of ultrasonic bath and placed in the NMR tube.Prior to lipid extraction from the Sebutapeebutape® ,11,12,131H-NMR analysis to quantify the total amount of sebum as well as to characterize their lipid composition. Data were acquired by Bruker 400 MHz Advance spectrometer . Acquisition parameters consisted in a 20.02 ppm spectral width, 30° pulse, 4.08 s acquisition time, 1 s relaxation delay, 64,000 data points, 1600 scans. MestReNova®  software was used for processing proton free induction decay data typically by Fourier transformation, and phasing and spectra were referenced to the low levels of protonated chloroform. Only signals with relative standard deviations and relative interference intensities lower than 10% were considered. The sum of sebum peak areas was linearly correlated (R2 = 0.995) to the sebum amount measured by differential weighing of Sebutape® (before and after the application). The 1H-NMR lipid signals were assigned to sebum components using standard lipids which are representative of the main lipid classes present in sebum (Sebum lipid extracts were subjected to in sebum . The annt-test (paired or unpaired) or with non-parametric Wilcoxon or sign tests. The differences among the investigated groups were considered significant for p < 0.05. Analysis and graphing are performed with RStudio software (RStudio Team (2021), version 4.1.2 (2021-11-01), Boston, MA, USA).The data are reported as average ± standard deviation (SD), or standard error of the mean (SEM), or in the case of a non-normal distribution (established by Shapiro test), by median and interquartile ranges (IQR). Comparisons are performed by 1H-NMR sebum lipid analysis, compared with high performance liquid chromatography (HPLC), which is also applied for sebum analysis [1H-NMR allows us to obtain quantitative data simply by the integration of peak areas [analysis , does noak areas .1H-NMR sebum spectra, while signal assignments to lipid classes are reported in ® allows the efficient collection of the sebum secreted by sebaceous glands. In order to establish the optimal time required to collect the maximum amount of sebum, we quantified the sebum of a healthy volunteer obtained from tapes removed at different application times. In about 20 min the amount of sebum becomes constant  represent the main sebum lipid component (49 ± 2 w%), followed by wax esters (WE) (29 ± 2 w%) and squalene (SQ) (19 ± 1 w%), while cholesterol (COH) and cholesterol esters (CE) are minor components (3.5 ± 0.2 w%)  .1H-NMR data revealed a statistically significant decrease in sebum production after 14 (b: 0.8 ± 0.5 mg) and 28 (c: 0.9 ± 0.4 mg) days of treatment when compared with the baseline level (a: 1.0 ± 0.5 mg) , “b” and “c” .  0.5 mg) .w/w) among the three investigated time points is reported in The comparison of sebum composition expressed as weight percentage of lipids on total amount of sebum  or oxaliplatin (FOLFOX)-based chemotherapy. In order to evaluate if the severity of the rash is influenced by different kinds of chemotherapy, we stratified patients according to the above chemotherapy regimens. Performing Fisher’s exact test for non-significant differences revealed the development of severe skin rash toxicity .Sebocyte activity is affected by hormones both in terms of production and composition , suggestIn spite of these differences, male and female patients reported non-significant differences in terms of severity of rash grade. However, male patients stratified by age showed a decreasing trend in rash grade associated with increasing age but without reaching statistical significance by Fisher’s exact test . This trSebum variation associated with rash severity is of particular interest since it could be relevant for the identification of predictive biomarkers. In this context, patients were stratified according to rash grade, and alterations in sebum production and composition were evaluated, although without indicating any significant lipid pattern that can be associated with skin toxicity. Conversely, when the patients were stratified by sex, some interesting differences emerged. For male patients, a decreasing trend of SQ percentage at time point “b” was associated with the increase in rash grade , while in the female group no differences were highlighted. Given that hormones can influence sebum production and composition and differ based on menopausal status , female 1H-NMR technique to determine information on sebum production and lipid composition in a group of mCRC patients subjected to cetuximab treatment. Compared with other principal analytical techniques applied in metabolomics, such as gas and liquid chromatography coupled with mass spectrometry, 1H-NMR allows an easier sample preparation and the direct absolute quantification of metabolites with a higher reproducibility. The use of Sebutape® represents an interesting breakthrough in sebum analysis since this specific matrix device, composed by a hydrophobic, open-celled, microporous polymeric film coated with an adhesive layer [1H-NMR spectra from a single tape, without requiring the use of a cryoprobe device to improve sensitivity. The 1H-NMR analysis of the sebum extracts showed specific previously reported matrix signals corresponding to components of the adhesive layer of Sebutape® [In this study, we applied the ve layer , allows ebutape® ,14,19. H1H-NMR measurement was found in agreement with that reported in a previous study [In patients, the mean amount of sebum after 30 min of collection estimated by us study . Since tus study . The maiSebum production’s quantification is of particular interest since one of the common skin issues manifested by cetuximab-based treatments consists in itchy dry skin . In thisIn contrast with this observation, a previous study analysed the sebum variation of eight non–small-cell lung cancer patients treated with either gefitinib or erlotinib  and repoThe present study revealed a significant specific decrease in the overall sebum levels of TG and SQ after 14 and 28 days  of cetuximab treatment. Such a decrease, from the start of treatment, can be trigged by the proliferation or the activation of Cutibacterium bacteria (formerly Propionibacterium). This latter is a lipolytic microorganism which transforms TG in glycerol and produces short chain FA, such as butyric and propionic acids, which are responsible for inducing inflammation . The conIn addition to the drop in TG, Cutibacterium can also be responsible for the SQ decrease with cetuximab treatment. The latter is a COH precursor that in humans only accumulates in sebum, having both lubricant and antioxidant properties, mainly associated with its numerous double bonds that can act as scavengers of reactive oxygen species (ROS) . The SQ In sum, the sebum decrease due to the sebaceous gland distortion induced by cetuximab treatment determines the loss of skin barrier function, which could induce skin dysbiosis characterized by an over development of Cutibacterium, which influences sebum composition and triggers skin inflammation.Sebum amount and composition variations have been found to be associated with the differences in the development of cetuximab skin severity side effects. Stratifying patients according to sex revealed a decreasing trend of SQ associated with an increase in skin toxicity in male patients already after 14 days from the start of cetuximab administration. This alteration could suggest that in patients experiencing more intense rash, SQ consumption could start earlier. In the female group, stratified by age, the baseline levels of sebum TG were found to be significantly lower for G2–G3 skin rash , suggest1H-NMR for the sebum lipids analysis being appropriate, proficient, and effective, the translation into the clinical setting could represent a significant cost increase to consider [® for the monitoring of sebum production does not determine the introduction of extra costs.Despite the application of consider . Convers1H-NMR method allowed the straightforward analysis of sebum, revealing a decrease in its production and in lipid composition induced by cetuximab treatment. The two main sebum components, TG and SQ, significantly decreased, respectively, after 2 and 4 weeks of cetuximab administration. These alterations could be explained assuming an abnormal colonization of Cutibacterium, indicated as a pivotal contributor in cetuximab skin toxicity development. Gender and age resulted in important factors in determining the sebum production and composition that in turn influence skin rash severity. Further analysis concerning the quantification of single-lipid species instead of the entire class and the inclusion of fatty acids profiles could prove useful to better understand the etiology of skin rash from cetuximab toxicity. It has been demonstrated that sex and other phenotypic characteristics can influence sebum production, composition, and also treatment response. Moreover, other characteristics not considered in this study, such as diet, body mass index, and medications, may also influence the occurrence of skin toxicity. Given the limited number of patients involved in the study, a large cohort of patients is required in order to properly evaluate other contributing factors. This could help to better establish the potential of baseline sebum 1H-NMR lipid profile as a predictive biomarker of the development of skin rash associated with cetuximab treatments. Despite its limits, this exploratory study revealed a possible connection between the underlying mechanisms of the cetuximab skin rash occurrence and acne development, thus giving the opportunity to translate acne treatment in the management of skin rash induced by cetuximab treatment.The developed