This work described the analysis of differential protein expression of subcutaneous adipose tissue of cows that went under negative energy balance during peripartum. In particular, to the best of our knowledge, it represented an original proteomics study that was able to discriminate cows in negative energy balance up to one month before calving. We believed that our findings would open new perspectives to improve animal welfare during peripartum. To know in advance, the metabolic status of cows would permit to correct the status with appropriate measures, like diet or management.Fat mobilization in high-yielding dairy cows during early lactation occurs to overcome negative energy balance (NEB), caused by insufficient feed intake and the concomitant increased nutritional requirements. For this reason, adipose tissue represents an essential organ for healthy and performant lactation. However, only a few data are known about adipose tissue proteome and its metabolic status during peripartum. The aim of this study was to analyze the differential proteomics profiles of subcutaneous adipose tissue belonging to cows with different NEB scores (low NEB and severe NEB). Both groups were analyzed at three different time points  that were related to different levels and rates of adipose tissue mobilization. The dataset highlighted the differential expression of the same four key proteins  involved in lipid metabolism during all time points and of other 22 proteins typical of the other comparisons among remaining time points. The obtained dataset suggested that the individual variability in adipose tissue metabolism/mobilization/energy availability could be linked to the different outcomes in levels of energy balance and related physical complications among dairy cows during peripartum. After parturition, the energy intake of high-yielding dairy cows is often not sufficient to meet the elevated energy requirements for milk production. Body energy reserves, in particular, adipose tissue, are mobilized to meet this energy deficit, and this state is often referred to as negative energy balance (NEB). With the mobilization of 50 to 60 kg of adipose tissue during early lactation, lipolysis is thus the essential metabolic process to support energy supply for all physiological functions . It is wOur hypothesis took into consideration the possibility that profound metabolic changes are triggered in the cow adipose tissue at peripartum to overcome the NEB status. A massive energy amount is required to overcome the energy demand, and the mobilization of adipose tissue may play a key role. Individual variability in these mechanisms might be the cause of the different outcomes in terms of NEB status. In the present study, we provided a first proteomic survey of subcutaneous adipose tissue of dairy cows at peripartum. Proteomic profiles of cows with different scores of NEB (severe or low) at different stages of adipose tissue mobilization were comparatively evaluated. The findings of the current study contributed to the steadily increasing knowledge in the field of animal breeding management and opened new avenues in the research for the identification of novel biomarkers predictive of the animal energy status; thus, might imply the improvement of livestock breeding strategies by optimizing diet composition and management practice.All experimental protocols were approved by the ethics committee “Comité d’Ethique en Expérimentation Animale Val de Loire”  and were carried out in accordance with the guidelines of the French Council for Animal Care.n = 6) or with severe negative energy balance  according to their energy balance (EB) at 1 week postpartum . Selected animals were reared in the same environment, and calving took place in the same period. The initial weight  was 664 ± 15.5 for sNEB and 638 ± 17.9 kg for lNEB animals.From a herd of 39 animals, we selected in the present study two sub-groups of animals either with low negative energy balance . Only the morning live body weight (LBW) was used for weight analyses because the afternoon body weight was more variable. All cows were milked twice daily. At the entrance of the milking parlor, the cows were identified by an electronic collar, and the milk yield of each cow was automatically recorded . As LBW is affected by digestive contents, the estimation of empty body weight (EBW) was corrected for the digestive tract content. A change of 4.5 kg of digestive contents per kg of dry matter intake (DMI) was assumed . VariatiAdipose tissue mobilization was assessed through subcutaneous fat thickness measurements in the sacral region using an ultrasonographic examination with a linear probe . Backfat thickness was measured at one month and two weeks before calving, and at 1, 4, 8, 12, and 16 weeks postpartum. The variation of the backfat thickness relatively to week 4 before calving was calculated.®, Bayer, Leverkusen, Germany) and subcutaneous (SC) injections of 20 mg lidocaine . Subcutaneous fat was collected from the dewlap under the neck, immediately frozen in liquid nitrogen, and stored at −80 °C until use. Adipose tissue biopsies were carried out on the 6 animals with lNEB and the 7 animals with sNEB at one month before calving, one week after calving, and 16 weeks after calving. Cows had fasted for 12 h before surgery, and anesthesia was induced by intravenous (IV) injections of 12 to 14 mg of xylazine . The intra- and inter-assay coefficient of variation of plasma NEFA measures were 6% and 7.8%, respectively. Blood samples were collected from the tail vein immediately before food distribution, once per week  and twice a week . They were centrifuged at 3000× Proteomic profiles of adipose tissue samples derived from cows with a different score in NEB were analyzed. Adipose tissue from three animals with lNEB and three animals with sNEB was investigated in technical replicates over three time points for a total of 18 samples, nine belonging to the cows with the highest energetic balance, and nine belonging to the animals with the lowest energetic balance . The samw/v) CHAPS, 2% Triton, 1× protease inhibitor cocktails, 1 mM Na3VO4, and 1 mM NaF. Thereafter, samples were processed by agitation with magnets for 2 h. Afterward, protein samples were centrifuged at 14,000 rpm for 30 min to remove insoluble materials. Thus, protein extracts were precipitated in Protein Bind tubes (Eppendorf) following a procedure adapted from Wesse and Fugge [The adipose tissue samples were dissolved in lysis buffer containing 7 M urea, 2 M thiourea, 2% , and 1% w/v bromophenol blue. IPG strips were first actively rehydrated in the presence of the sample at 50 V and 20 °C for 17 h. After the rehydration step, paper wicks soaked in water were placed between cathode, anode, and gel strip for preventing high voltage to causing the burning of the strips. The voltage was gradually increased according to the following protocol: 100 V (1 h), 500 V (1 h), 1000 V (1 h), 2500 V (1 h), 5000 V until the cumulative voltage reached 50 kVh. A limitation of current up to 50 µA per gel strip was set. Following IEF, each strip was reduced for 15 min in 5 mL of solution containing 6 M urea, 2% w/v SDS, 50 mM Tris-HCl buffer, pH 8.8, and 20% v/v glycerol with 1% w/v DTT added, and then alkylated in 5 mL of the same solution with 2.5% w/v of iodoacetamide (IAA). IPG strips were then washed shortly in 1× running buffer , loaded onto 12% w/v polyacrylamide resolving gels along with the protein ladder and fixed with 0.5% w/v agarose gel. The second dimension was carried out in a Mini-Protean Tetra system . In the first step of electrophoresis, until the bromophenol blue front line entered the resolving gel, 8 mA per gel for 15 min was applied. In the second step, 16 mA per gel was applied until the bromophenol blue front line reached the bottom of the gel. Gels were then removed from the plates, washed three times for 5 min in 100 mL of deionized water, and left overnight to stain in 100 mL of preheated coomassie brilliant blue G-250 .Protein concentration in the samples was determined using Bradford assay, with BioRad protein assay stain and 2 µg/µL concentration of BSA as a standard. Optical density was measured using a spectrophotometer  at 595 nm. Proteins were separated using 2D SDS-PAGE. For isoelectric focusing (IEF) step, immobilized pH gradient (IPG) polyacrylamide gel strips  and Protean IEF Cell  were utilized. Prior to IEF, 100 µg of protein sample was dissolved in a solution containing 7 M urea, 2 M thiourea, 2% v/v ethanol and then for 2 min in 100 mL of deionized water.Gel images were acquired using a flatbed scanner  with a resolution of 600 dpi. Before scanning, gels were washed for 20 s in 70% m/z: 1046.5418, 1296.6848, 1347.7354, 1619.8223, 2093.0862, 2465.1983, 3147.4710), and to select monoisotopic peptide, MS spectra were analyzed by FlexAnalysis 3.3 software .According to Piras et al. ,15, MALDm/z: 842.509 and 2211.104) and exclusion of contaminant ions (known matrix and human keratin peaks), the peak lists were analyzed by MASCOT v.2.4.1 algorithm (www.matrixscience.com) against SwissProt database released 2019_09 restricted to Bos taurus taxonomy .After an internal calibration . For the peptide mass fingerprinting analysis (PMF), the query for database searching was set with these established parameters: carbamidomethylation of cysteines and oxidation on methionines as fixed modification and variable modification, respectively; one missed cleavage site for trypsin; maximal tolerance at 50 ppm. For protein identification assignment, only Mascot scores higher than 54 were considered significant , and centroiding. For database search analysis, the following parameters were used: carbamidomethylation of cysteines and oxidation on methionine among fixed and variable modifications, respectively; maximum of one missed cleavage; mass tolerance at 50 ppm for precursor ions and at a maximum at 0.4 Da for fragments. The database-dependent search was performed against the SwissProt database released 2019_09  restricted to other Mammalia  and Bos taurus taxonomy. The confidence interval for protein identification was set to 95% (p < 0.05), and only peptides with an individual ion score above the identity threshold were considered correctly identified.To confirm PMF identifications, the instrument was switched in LIFT mode, and MS/MS spectra were acquired with 4–8 × 10® software version 9.4. Unless differently specified, data were analyzed using the MIXED procedure for linear mixed models. A repeated effect of time  within animals was tested. The residuals from the observations generated from the mixed models were tested for normal distribution. Statistical analyses for dry matter intake, milk yield, live body weight, a variation of empty body weight, a variation of backfat thickness, energy balance, and plasma NEFA were performed with SASijkl is the dependent variable , µ is the overall mean, NEBi is the fixed effect of NEB i , Weekj is the fixed effect of week j , NEBi × Weekj is the interaction between NEBi and Weekj, and eijk is the residual error. The model used was:Least square means  estimated by the models were adjusted using the Scheffe adjustment for multiple-post ANOVA comparisons and compared. p-values, p < 0.05, were considered as significant.Differences with corresponding Variations in protein expression between lNEB cows and sNEB cows were analyzed using the Progenesis SameSpots software , Version 4.6. After evaluating the quality of the images, the module for 2D gel analysis was used to align the images, subtract background, detect, normalize, and match spots. All spots were then manually reviewed and selected for excision. p-value between different groups, and the p-values under 0.05 were considered statistically significant. Statistical analysis was performed using the Progenesis Stats module on the log-normalized volumes for all spots. Stats module performed automatically a one-way ANOVA on each spot to evaluate the The functional classification of the identified proteins was performed using the PANTHER classification system . The GenHigh confidence (score 0.700) protein-protein interactions and relations between proteins were investigated via the STRING database of protein-protein interactions  using thAdipose tissue samples were collected from cows in lNEB and cows in sNEB. The classification of samples based on energy balance was available only at 1 and 16 weeks postpartum when the EB level could be measured according to milk yield . Therefop < 0.0001), but there were no differences among EB groups from calving to week 16 postpartum. However, significant differences among least-square means of sNEB and lNEB groups were found in the samples collected at week 4 before calving and the samples collected at week 16 after calving. They consisted of the variation of empty body weight  protein, together with rates of fat tissue mobilization, as important players for the different response to negative energy balance among experimental groups. Interestingly, the FABP family is the master switch of the PPAR signaling pathway , and itsAmong all differentially expressed proteins, six were found to be overrepresented in the comparisons over all the time points. One of them (fatty acid-binding protein) was highly abundant in sNEB in the time point lapse ranging from one month before calving up to 16 weeks after calving. Apolipoprotein A-I was strongly overrepresented in the period after calving (one week to 16 weeks). It is the major component of high-density lipoprotein(HDL), and its major role is related to the transport of fat molecules from the cells. Its overrepresentation in sNEB cows after calving follows the trend of FABP4, suggesting both proteins as active players in resuming the impaired function of adipose tissue of sNEB cows. According to previously published experimental evidence on a mouse model , ApoA-I The non-esterified fatty acid can be again converted into triacylglycerol (TAG) from the liver and stored in hepatocytes. This is mainly the reason why up to 40% of dairy cows develop fatty liver disease and could explain as well such an increased amount of ApoA-1 in adipose tissue. Based on the evidences described by Karavia and colleagues  on a mouFatty acid-binding protein’s main function is related to lipid transport and has the capability to bind with high-affinity hydrophobic ligands as saturated and unsaturated long-chain fatty acids and eicosanoids (leukotrienes and prostaglandins) . Its strFatty acid-binding protein 4 (FABP4) is mainly expressed in adipocytes and macrophages. It is responsible for the development of insulin resistance and inflammation , it can Most of the underrepresented proteins were assigned to a variety of metabolic processes. Expanding the section of the lipid metabolic process, it was possible to observe that the underrepresented proteins involved in this biological process were annexin A1 and annexin A2. Relevant experimental evidence documents the key role of these proteins in the regulation of fat tissue metabolism. One of its major roles is linked to fat accumulation in cows . We founInsulin resistance could represent another important feature to take into account. The differential protein expression of adipose tissue in cows has been evaluated in relation to insulin resistance . We evalThe generation of precursor metabolites and energy highlighted in the panther pie chart b were coActin-related protein 10 was another protein that was found to be less abundant among all the time points in sNEB. Its role is mainly related to intracellular trafficking and microtubule-based movement. It was again in agreement with the previously mentioned theory about an sNEB adipose tissue more oriented to lipid transport in other organs and then to energy storage.The results obtained showed several differences in the protein profile involved in biological processes and pathways, as glycolysis and lipid metabolism and transport. The most important differences highlighted that glycolysis was dramatically underrepresented in adipocytes of sNEB cows, and, on the contrary, lipid transport out of adipocytes was highly increased B. This oThe major requirement of energy is due to the necessity of lipids necessary for milk production. The adipose tissue of sNEB cows seems to be more efficient in lipid transport and mobilization and less efficient in the glycolytic pathway. This may produce a faster metabolism of adipose tissue of sNEB cows that are as well faster in depleting the fat deposit . This di


The aim of this study was to improve the physicochemical properties of cocoa extract (CE) using hot-melt extrusion (HME) for pharmaceutical proposes. A mixture design was applied using three distinct hydrophilic polymeric matrices . Systems obtained by HME were evaluated using morphologic, chromatographic, thermic, spectroscopic, and diffractometric assays. The flow, wettability, and dissolution rate of HME powders were also assessed. Both CE and its marker theobromine proved to be stable under heating according to thermal analysis and Arrhenius plot under isothermal conditions. Physicochemical analysis confirmed the stability of CE HME preparations and provided evidence of drug–polymer interactions. Improvements in the functional characteristics of CE were observed after the extrusion process, particularly in dissolution and flow properties. In addition, the use of a mixture design allowed the identification of synergic effects by excipient combination. The optimized combination of polymers obtained considering four different aspects showed that a mixture of the Soluplus, Plasdone S630, and Eudragit E in equal proportions produced the best results , therefore making the pharmaceutical use of CE more feasible. Theobroma cacao L.), one of the main Brazilian agricultural commodities, is largely composed of the flavonoid theobromine (TB) . Th. Th14]. The optimized response was calculated from the all responses obtained, considering the formulation conditions for maximum dissolution and flowability with an importance score value of 3 since those are assays directly accessed by the pharmaceutical industry, and minimizing the dispersibility and contact angle with an importance score value of 1 since they are complementary assays.w/w). The proportion was chosen based on previous tests. The PMs were used to manually feed a co-rotating conical twin-screw extruder . Material characteristics were exploited to set the extrusion conditions, which were adjusted to allow the appropriate extrusion of each material with continuous flow. The temperature and rotation used are described in Physical mixtures (PMs) of CE and the selected polymers  were prepared with a mortar and pestle, maintaining the CE–polymer proportion of 3:7  as mobile phase; and flow rate of 1.2 mL min−1. The method was validated following the International Conference on Harmonization parameters and proved to be selective against the polymers and CE interferents and linear (r = 0.9997). The limits of quantification and detection were 0.15 μg mL−1 and 0.05 μg mL−1, respectively.Quantification of TB within CE, in extrudates, and in dissolution studies was performed through a reversed-phase chromatographic method with UV detection at 274 nm using the HPLC model LC-20AT . The method was adapted from a previous report . The opeThe morphology of TB and CE, as well as of CE-polymer mixtures before and after HME processing, were assessed by optical microscopy using a stereoscope  coupled to a video camera and with the aid of a scanning electronic microscope .TGA were carried out using a DTG-60H . Kinetic analyses using isothermal conditions were performed for TB and CE. Samples were heated at selected temperatures  and maintained at constant temperature until 5% of mass loss had occurred. An Arrhenius plot was built based on the experimental data, and the activation energy of each sample was calculated from the linear regression .−1 at a heating rate of 5 °C min−1 from 30 to 400 °C.Linear heating experiments were performed in TB and CE, as well as in CE-polymer mixtures before and after HME processing using platinum pans and under a nitrogen atmosphere or synthetic air with a flow rate of 50 mL min−1, and the step size was 0.02 degrees. The diffraction patterns were obtained at angles between 5 and 60° (θ–2θ).XRPD spectra of TB and CE as well as CE-polymer mixtures before and after HME processing were collected using a D8 FOCUS XRPD . The scan speed was 2 degrees min−1 at an optical resolution of 4 cm−1.FTIR analyses of TB and CE as well as CE-polymer mixtures before and after HME processing were performed on a Varian 640 FTIR spectrometer using an ATR imaging accessory . Spectra were recorded between 4000 and 600 cmMultiple flow measurements were assessed using a powder characteristic tester, PT-N . From these acquired data, powder flowability was determined according to Carr based on the angle of repose, angle of spatula, compressibility, and uniformity tests .Dispersibility was calculated by dropping 10 g of the powder into a cylindrical tube up to a watch glass of 100 mm diameter positioned on the bottom. The amount of powder recovered from the watch glass was compared with the initial powder to calculate the dispersibility in percentages. All measurements were performed in triplicate, and the flowability index and dispersibility were calculated using the Hosokawa software.The contact angle was measured by CAM-PLUS contact angle meter . Tablets of each system were prepared using a hydraulic press with a compression force of 4.28 kgf for 5 s. Purified water was dropped onto the surface of the tablets, for which the contact angles were immediately measured. Results are represented as the mean of 10 replicates.−1 as medium, following the FDA recommendations for immediate-release solid oral dosage forms. The temperature was maintained at 37 °C, and paddle speed was adjusted to 75 rpm. Samples containing the equivalent of 250 mg of TB, a possible therapeutic dose, were added to the dissolution vessel [Dissolution profiles of CE and HME extrudates were determined in a dissolution system, Ethik model 299 , using 250 mL of HCl 0.1 mol Ln vessel . Aliquotn vessel ,22.p) was fixed at 0.05. Statistical analysis was performed using GraphPad Prism 6 and IBM SPSS Statistics 22. Normality was previously tested for all data. Results with parametric behavior were compared using one-way ANOVA followed by Tukey post-test. Results with no parametric behavior were evaluated through Kruskal-Wallis test followed by Dunn’s post-test. Significance level (Tpeak of first derivative = 305 °C), involving a complete sample weight loss. Decomposition of CE, meanwhile, occurred in a similar range of temperature (210–330 °C) as a well-defined decomposition first step with 43% weight loss (Tpeak of first derivative = 279 °C), likely corresponding to the decomposition of TB and other compounds found in CE such as polyphenols and organic matter [2) or oxidant atmosphere (synthetic air) showed the same profile, suggesting that oxygen did not affect the decomposition kinetics under the conditions studied.Thermogravimetric tests were performed in CE samples as well as in the main marker TB in order to evaluate thermal stability. TGA analysis of TB using a constant heating rate revealed a monophasic drug decomposition in the range of 230–330 °C (−1 (r = 0.967) and 122.0 kJ mol−1 (r = 0.985), respectively, which are fairly high values when compared with those found for other drugs, which are usually within the range of 40–100 kJ mol−1 [−1 [The Arrhenius kinetic approach was applied based on TGA isothermal studies in an inert atmosphere. The activation energy values of TB and CE were 173.1 kJ molkJ mol−1 . Indeed,ol−1 [−1 . AccordiExtrusion conditions were settled to obtain continuous extrusion, and processing temperatures were established at the range of 150–185 °C, above at least 40 °C of the glass transition of polymers for single matrix systems based on previous studies with the same polymers [The morphological aspects of samples observed with optical microscopy and SEM  showed tThe thermal profile of mixtures was determined before (PM) and after the extrusion process (HME). TGA results showed no significant difference in thermal stability after processing. In fact, the extrudates presented a thermal profile equal to the sum of their individual compounds  without The crystallinity of the systems could not be measured by thermal techniques once the TB melting event occurred after its thermal degradation . TherefoXRPD of HME samples  indicate−1, the band related to C–N at 1222 cm−1, and the bands associated with the two carbonyl group vibrations in the meta position at 1664 and 1544 cm−1 , proving its poor flow capacity . MeanwhiThe thermal shear caused by HME is not only capable of greatly increasing the degree of interaction between the drug and polymer but also gives rise to a dense and uniform matrix producing granules with excellent flow characteristics, as previously described ,11.Together with the flowability determination, the dispersibility (%), which measures the tendency of a powder to scatter in the air, was also assessed. Once again, there was a great difference in performance between CE (37.8%) and HME extrudates (below 11%), as described in Although considerable improvements in flowability and dispersibility have been reached through HME processing, differences in these properties can be observed according to the composition of the polymer matrix, as shown in the response surfaces in p < 0.001) with r2 = 0.963  reveals that Sol presents the lowest coefficient of the predictive equation, therefore leading to lower contact angles. Moreover, the combination of polymers favors the reduction of the contact angle, especially in the regions of the contour plot that combine PVP and EuE . The amphiphilic properties of Sol and the wettability enhancing activity attributed to PVP and EuE as described in the literature corroborate our results [Although all formulations showed good wettability, marked differences could be observed between them, which indicate that the polymer composition plays an important role in this property. In fact, the response surface B built f results .Dissolution profiles of CE as supplied, compared with those of CE extrudates, are shown in p = 0.011; r2 = 0.597) shows that regions composed of mixtures containing predominantly Sol and EuE led to DE30 results above 85%. In fact, the coefficient for the Sol-EuE interaction presented a high value, strongly contributing to the predictive equation  was found with a polymeric mixture composed of 23.1% Sol, 23.8% PVP, and 23.1% EuE, which practically corresponds to the central region of the contour plot. The expected responses for this formulation were DE30 of 84%, flowability of 87%, dispersibility of 8.4%, and contact angle of 48°, which are nearly the same as the results determined experimentally for the formulation HME Sol-PVP-EuE.Moreover, a large central region, whose composition leads to desirability greater than 0.7, is observed in the contour plot (gray area of contour diagram). The possibilities with the best performance include a formulation containing 45.5% Sol, 11.9% PVP, and 12.6% EuE and a formulation containing 4.9% Sol, 32.2% PVP, and 33.6% EuE .Solid dispersions of CE were effectively produced using a single-step HME process. Natural extract stability was preserved while dissolution and flow properties were improved. Despite the high stability of the TB crystal lattice, there was physicochemical evidence of drug-polymer interactions. The use of a mixture design allowed the identification of synergistic effects with excipient combination. In fact, the optimized formulation obtained considering four different responses showed that a mixture of Sol, PVP, and EuE in equal proportions produced the best results, making the pharmaceutical use of CE more feasible.


Although primary duodenal adenocarcinoma (DA) is a rare malignancy representing ~ 0.5% of all gastrointestinal cancers, the incidence of DA is more frequent in Lynch syndrome. Because of its rarity, treatment strategies or optimal chemotherapeutic regimens have not been clearly defined for advanced DA.A 72-year-old woman with Lynch syndrome visited our hospital with a right upper abdominal pain. Computed tomography (CT) showed wall thickness with enhancement in the second portion of the duodenum and adjacent abdominal wall, which suggested direct tumor invasion to the abdominal wall. Upper gastrointestinal endoscopy (UGE) showed a large ulcerative tumor in the second portion of the duodenum, and histological analysis revealed a poorly differentiated adenocarcinoma. A cT4N0M0, cStage IIB  DA was diagnosed. After three courses of chemotherapy with S-1 and oxaliplatin (SOX), follow-up CT and UGE showed shrinkage of the duodenal tumor. Therefore, the patient underwent pancreaticoduodenectomy with lymph node dissection with curative intent. Histological examination showed a pathological complete response to SOX therapy. The postoperative course was uneventful, and the patient was discharged on postoperative day 29. The patient received no adjuvant chemotherapy, and there has been no evidence of recurrence 6 months after the operation.SOX therapy provided a remarkable response and can be an optimal chemotherapeutic regimen for advanced DA in Lynch syndrome. Primary duodenal adenocarcinoma (DA) is a rare malignancy, representing ~ 0.5% of all gastrointestinal cancers, and is often diagnosed at an advanced stage –3. Surgi3 and a decreased hemoglobin level of 10.8 g/dL. Serum levels of the tumor markers carcinoembryonic antigen and carbohydrate antigen 19–9 were within normal limits. She had a history of four resections of different parts of the colon because of colon cancer associated with Lynch syndrome. At the age of 36, she was diagnosed with transverse colon cancer, and a partial resection of the transverse colon was performed. At the age of 44, she was diagnosed with cecal cancer for which ileocecal resection was performed. At the age of 45, she was diagnosed with sigmoid colon cancer, and a sigmoidectomy was performed. At the age of 72, she was diagnosed with descending colon cancer, and a partial resection of the descending colon was performed. Pathological evaluation revealed a pT2N0M0 pStage I tumor based on the seventh edition of the Union for International Cancer Control TNM staging. Her family history fulfilled the Amsterdam II and revised Bethesda criteria. Her father died of colon cancer in his 40s, one of her brothers had colon cancer at the age of 39 years, one of her cousins died of colon cancer in his 30s, and her son had ascending colon cancer at the age of 35 years; these observations suggested Lynch syndrome. After genetic counseling, a written informed consent was obtained from the patient, and we examined her for microsatellite instability (MSI). The five microsatellite markers BAT25, BAT26, NR21, NR24, and MONO27 exhibited replication errors in the descending colon cancer resected in 2017. Therefore, the patient’s colon cancer was considered to be a high-frequency MSI (MSI-high) tumor. Further genetic testing was performed using DNA from the patient’s peripheral blood. The analyses revealed one missense mutation [c.676C > T (p.Arg226)] in the MLH1 gene, thus confirming Lynch syndrome.A 72-year-old woman complained of right upper abdominal pain at the time of a routine check-up for colon cancer. A physical examination revealed a hard, palpable mass with pain in the middle part of the upper abdomen approximately 5 cm in diameter. Laboratory data showed an elevated leukocyte count of 10,100 cells/mmContrast-enhanced computed tomography (CT) showed wall thickness with enhancement in the second portion of the duodenum and adjacent abdominal wall, suggesting direct tumor invasion to the abdominal wall Fig. . There w2 S-1 orally on days 1–14 and 100 mg/m2 oxaliplatin intravenously on day 1 of a 21-day cycle. Grade 1 adverse effects based on the National Cancer Institute Common Toxicity Criteria  were neutropenia, fatigue, appetite loss, and stomatitis, all of which improved with conservative treatment.The patient was scheduled for combination chemotherapy with SOX: 80 mg/mAfter three courses of chemotherapy with SOX, follow-up abdominal contrast-enhanced CT revealed reduced wall thickness of the second portion of the duodenum and the adjacent abdominal wall Fig. . There wThe patient underwent pancreaticoduodenectomy with combined resection of the adjacent abdominal wall and regional lymph node dissection with curative intent 3 weeks after the last administration of chemotherapy. During the operation, no peritoneal dissemination or lymph node swelling was observed. Gross examination of the surgically resected specimen showed an ulcerative lesion measuring ~ 2.0 cm Fig. a. PatholThe postoperative course was uneventful, and the patient was discharged on postoperative day 29. The patient received no adjuvant chemotherapy, and there has been no evidence of recurrence 6 months after the operation. Postoperative surveillance is being planned according to the Japanese Society for Cancer of the Colon and Rectum Guidelines 2016 for the Clinical Practice of Hereditary Colorectal Cancer .Primary DA is one of the rare malignancies representing ~ 0.5% of all gastrointestinal cancers, although it accounts for > 50% of small bowel adenocarcinomas (SBAs) , 2, 8. BDA is included in SBAs, and the outcomes for all SBAs are grouped together in many studies. Owing to the rarity of SBAs, prospective clinical trials are limited, and treatment guidelines or optimal chemotherapeutic regimens have not been clearly defined. Initially, SBAs were treated with chemotherapy based on the regimen used for gastric cancer. In 1984, Jigyasu et al. reported in a retrospective study that a 5-fluorouracil (5-FU)-based regimen for advanced SBAs achieved a response rate (RR) of 7.1% and a median OS time of 9 months . In a prThe role of preoperative therapy for patients with locally advanced, clinically unresectable DAs has not been well documented. A retrospective study involving unresectable or recurrent DA who was treated with preoperative chemotherapy or chemoradiation found that 9 of 10 patients showed the conversion to resectable disease after the therapy, suggesting prolonged survival after conversion to resectable disease . AnotherLynch syndrome is a known risk factor for SBA, as are familial adenomatous polyposis, Crohn’s disease, Peutz–Jeghers syndrome, and celiac disease. In Lynch syndrome, the risk of developing SBA within a lifetime is reported to be ~ 4%, almost the same as the lifetime risk of CRC in the general population , althougWe report that chemotherapy for a locally advanced DA made the surgical procedure possible and achieved pCR in Lynch syndrome. To the best of our knowledge, this is the first case of a DA patient with Lynch syndrome achieving a pCR. This case indicates that SOX therapy can be a good regimen for advanced DA even in Lynch syndrome. Since DA is a rare malignancy but occurs relatively frequently in Lynch syndrome, further clinical reports will be needed to establish the most appropriate chemotherapy regimen.


Key to this strategy is the identification of the cells to produce such cartilage intermediates efficiently. We applied a cell selection strategy based on published skeletal stem cell markers using mouse embryonic limb cartilage as cell source and analysed their potential to form bone in an in vivo ectopic assay. FGF2 supplementation to the culture media for expansion blocked dedifferentiation of the embryonic cartilage cells in culture and enriched for stem cells and progenitors as quantified using the recently published CD marker set. However, when the stem cells and progenitors were fractionated from expanded embryonic cartilage cells and assessed in the ectopic assay, a major loss of bone forming potential was observed. We conclude that cell expansion appears to affect the association between cell identity based on CD markers and in vivo bone forming capacity.The healing of large bone defects remains a major unmet medical need. Our developmental engineering approach consists of the  Several tissue engineering strategies have been developed to overcome these challenges and one of them is based on bone developmental engineering. This approach involves the in vitro manufacturing of a living cartilage tissue construct that upon implantation forms bone in vivo by recapitulating endochondral ossification taking place during embryonic development.Large bone defects can be caused by major trauma, infection, prosthetic revision, bone tumour resection or non-healing fractures and in clinical practice, their healing remains a therapeutic challenge. Current treatments such as iliac crest autografts or cadaver allografts require multiple and repetitive interventions and are associated with various risks resulting in a high socio-economic burden+ chondrocytes. These chondrocytes proliferate, organize in columns and enter hypertrophy under the control of an Ihh/PTHrP loop. After cell maturation into Runx2+ hypertrophic chondrocytes, a shift in matrix synthesis occurs from collagen type II to type X. This matrix calcifies and is replaced by bone by invading osteoblasts and transdifferentiating non-apoptotic hypertrophic chondrocytes, both characterized by Osterix expression and secretion of osteoid matrix4.Briefly, during that process, Prrx1 expressing limb mesenchymal cells condense and differentiate into Sox95. Lineage tracing experiments in mice have shown that during bone repair, osteoblasts and osteoclasts originated from the bone marrow, endosteum and periosteum, but that callus chondrocytes were primarily derived from the periosteum6. More recently, it has been shown that human periosteal cells can be primed in vitro, by using conditioned medium and cell aggregation, to a cartilaginous intermediate tissue able to develop into bone ectopically and facilitate healing in an orthotopic long bone defect7. However, these cells still generate excessive fibrous tissue. Enrichment for the osteochondrogenic precursors is expected to result in an enhanced bone forming potential and improved purity of cell based treatments.The cell sources to engineer cartilage intermediates can be diverse with the periosteum currently considered an excellent cell source12. In another study, “rainbow” mice displayed a high frequency of clonal regions in the growth plate of adult mice, characterized by Alpha V Integrin (CD51) expression but negative for CD45 and TER11913. This population was subsequently divided into eight subpopulations based on differential expression of CD105, CD90.2, CD200 and 6C3 cell surface markers. By combining this strategy with in vivo and in vitro approaches, they mapped bone, cartilage and stromal development from a postnatal mouse skeletal stem cell to its downstream progenitors in a hierarchical program similar to hematopoiesis13.Several studies in mice focused on the identification and contribution of skeletal stem cells and osteochondroprogenitors in bone development, homeostasis and fracture healing. These cells are found either in the zone underneath the growth plate, blood vessel niches or the periosteum. Several molecular markers have been associated with these cells such as Nestin, Gremlin1, Leptin Receptor, Gli1 and Periostinin vivo. To select the desired cell subsets, we used previously described cell surface markers13 and studied them separately in an established in vivo ectopic bone formation assay in nude mice.In the current study, we have optimized the prospective isolation of stem and progenitor cell populations from the mouse embryonic hind limb cartilage 14.5 dpc and studied their potential for cartilage and bone formation in vivo ectopic bone forming assay. Cell tracking experiments revealed the contribution of donor cells to the osseous tissue. We then purified from the embryonic cartilage cells two cell populations, namely the mouse skeletal stem cell (mSSC) and a Pre-progenitor (PreP), a direct descendent of the mSSC, and demonstrated their bone forming potential in the ectopic assay. We showed however that their potential is heavily influenced by the hydrogel encapsulating the cells. Next, when expanding the embryonic cartilage cells in vitro in the presence of FGF2, a standard ligand used in stem cell expansion protocols, an enrichment for stem cells and progenitors as quantified using the CD marker set was observed. However, a major loss of in vivo bone formation was observed, suggesting the lack of predictive value of the markers for in vivo bone forming potential, when in vitro expansion is performed.We show that primary mouse embryonic cartilage cells (ECC) continue their developmental program and form a bone organoid in an in vivo, we focused on the total cell preparations from femur anlagen of 14.5 dpc mouse embryos. They were dissected and the embryonic cartilage cells were released using collagenase. In the in vivo bone formation assay, we used two different hydrogel encapsulation protocols, collagen type I and alginate. The latter allows for the ECC to form bone in an attachment-free environment. The cells were encapsulated in respective gels and implanted subcutaneously behind the shoulders of nude mice  which were encapsulated in collagen hydrogel and implanted subcutaneously  and CD51+CD45−TER119−CD105−CD90.2−CD200+ skeletal stem cells (mSSC)  Fig. . The mSSSC) Fig. . FollowiTwo sets of genes were analysed: those relevant for endochondral ossification Fig.  and to pIn conclusion, and as anticipated, the expression of early chondrogenic markers  in mSSC was reduced, whereas the expression of early mesodermal markers  was increased. We could see a significant differential expression of Hoxa10 in PreP and mSSC, one of the genes associated with limb positional information and proximal distal outgrowth and with activation of the pre-osteoblast marker Runx2.in vivo, in collagen I or alginate hydrogel. The mSSC were encapsulated at a concentration of 32,000 cells and the PreP at 200,000 or 32,000 cells per implant. The hydrogels were implanted into nude mice and retrieved after three weeks in vivo  led to bone formation through the endochondral pathway both ectopically and in a large bone defect. This effect was mediated primarily by the increased expression of bone morphogenic protein 2 (BMP2). Thus, we investigated the addition of FGF2 to the culture media to prevent the dedifferentiation of the ECC in vitro. The ECC were expanded for two passages on a gelatin-coated surface and in standard growth medium or in the same medium supplemented with FGF2  induced dedifferentiation of the ECC towards a spindle shaped appearance. Expression of genes involved in endochondral ossification decreased rapidly and was undetectable already after one passage. Prior workGF2 Fig. . Gene exGF2 Fig. . Interesin vitro for mSSC and PreP as defined by the CD markers.We compared the amount of mSSC and PreP in the ECC, expanded in standard Fig.  or FGF2 in vivo assay. Thus, ECC were expanded on gelatine in standard or FGF2 supplemented medium for two passages. PreP and mSSC from both culture conditions were sorted by FACS, encapsulated in collagen hydrogel and implanted ectopically in nude mice . For the collagen hydrogel, the shape of the implant can be adjusted depending on the mould in which the gel is allowed to set, increasing its versatility to use in different defect sizes or shapes. Alginate, a natural anionic polysaccharide polymer obtained from brown seaweed, is been studied extensively in biomedical engineering due to its biocompatibility, low toxicity, relative low cost and mild gelation methods17. Alginate has several advantages over non-injectable 3D scaffolds when used for bone regeneration. It can be introduced into the body in a minimally invasive manner; it can fill irregular shaped defects and can be used as delivery system for slow release of bone inducing factors. The disadvantages are insufficient mechanical properties to allow load bearing in the initial stages of regeneration and inability to degrade under physiological conditions. Nonetheless, alginate is frequently used for cartilage generation and anchorage independent chondrocyte cultures. Chondrogenic potential of stem cells seems linked to the morphology of the encapsulated cells (preferably preservation of the round cell shape) and alginate promotes this rounded shape and thus the cellular differentiation and maturation process19. We have shown that the embryonic cartilage cells form cartilage and bone after three weeks in vivo when encapsulated in collagen I hydrogel. Thus, the cells were able to maintain their developmental fidelity, suggesting that the bone formation is a cell intrinsic process. This has been demonstrated by previous work in our group20. However, when the cells were encapsulated in alginate, bone formation was very limited. Thus, a scaffold that provides a niche to allow initial cell attachment has a great influence on the cells potential and in which direction their differentiation goes. Furthermore, the alginate might keep the cells in a more chondrogenic state, thereby hindering further development into bone. Additionally, it must be noted that with alginate the invasion of blood vessels and recruitment of osteoblasts is limited. Nonetheless, we detected small areas of osteoid positive matrix in the hypertrophic cartilage tissue islands in the alginate gel. In the cell-tracing studies, we detected eGFP+Osterix+ donor cells beyond the cartilage-bone perimeter, in the osseous tissue. These results combined may suggest differentiation of donor cells to osteoblasts. Contamination of the cell preparation with osteoblastic precursors could not be detected by qPCR, but cannot be excluded. Therefore, donor cells contributing to the osseous tissue could be osteoblastic precursors, or hypertrophic chondrocytes undergoing transdifferentiation. Recently, Yang, et al.21 was able to tag Col10a1 expressing hypertrophic chondrocytes during mouse development and adulthood and track their fate. They showed survival of these cells during the cartilage-to-bone transition and differentiation into osteoblasts in foetal and postnatal endochondral bone, which could persevere into adulthood. Studies are now being undertaken to further investigate the molecular pathways underlying this transdifferentiation phenomenon23.We encapsulated the embryonic cartilage cells in either collagen or alginate hydrogel for implantation et al.13, and later the publication of the detailed protocol24 enabled studies of the skeletal stem cell hierarchy, whether in long bone fracture healing studies25, disease models such as diabetes mellitus26 or for tissue engineering purposes for healing large bone defects (our study).Recent lineage tracing studies in mice investigated the existence of a mouse skeletal stem cell which contributes to the skeletal tissues during development, homeostasis and regeneration. However in these studies, the focus was on genes and proteins expressed intracellularly, making it difficult to study these cell populations outside of their natural environment and in a tissue engineering context. The identification of the mouse skeletal stem cell and its downstream progenitors by Chan, 13. The most likely reason for this discrepancy is that the previous report used young adult (P6) mice, thus much older than our material. These findings support the general notion that the amount of stem cells decreases with age of the individual. It would be interesting to investigate whether there is a higher percentage of stem cells earlier in development, at the onset of limb development at 10.5dpc, when the cells already express Gremlin, a proposed stem cell marker9.Embryonic cartilage cells obtained from femur anlagen of 14,5dpc mouse embryos contain about 1% of mSSC. This is significantly more than 0.1% reported in previous studies27.Besides endochondral relevant genes, we measured the expression of Meis1 and Hox genes, more specifically the analogs 9–11. These genes show a proximal-distal expression pattern, in which Meis1 is expressed most proximally, Hoxa9 and Hoxa10 is expressed in stylopod and Hoxa11 in the zeugopod. We observed an increased expression of Hoxa10 in mSSC and PreP. Loss of Hox10 paralogous genes results in severe stylopod mispatterning. Our result that Hoxa10 is slightly upregulated in mSSC isolated from stylopod limb cartilage is therefore an interesting finding. However, the expression of these genes is not uniform and dependent on the tissue type. Additional, in literature, an involvement of Hoxa10 in bone formation has been reported. The gene responds to BMP2 stimulation, and thereby contributes to osteogenic lineage specification through activation of Runx2 and directly regulates osteoblastic genes such as alkaline phosphatase, osteocalcin and bone sialoprotein genesin vivo bone tissue engineering assay.In our study, we designed a protocol to investigate the potential of the mSSC and the PreP to form bone when encapsulated in a hydrogel in our established 24 making a comparison between these two procedures possible  to isolate sufficient number of stem cells and progenitors, we sought to develop an in vitro expansion protocol for these embryonic cartilage cells. One of the ligands typically used in such applications is FGF230. We were able to expand the ECC almost 10-fold over two passages, leading to sufficient cell numbers for isolation of mSSC and PreP. The application of FGF2 did help in the maintenance of BMP2 expression and the inhibition of ACTA2, a fibroblast marker as compared to standard growth medium. Subsequent flow cytometry analysis showed an enrichment for mSSC and PreP after two passages of expanding ECC in the presence of FGF2. However, when introducing the fractioned cells in the ectopic assay, the in vivo bone formation was significantly reduced and large amounts of fibrotic tissues was found instead. Additionally, no significant difference in bone tissue was found between progenitors isolated from ECC expanded in FGF2 or GM. Subsequent gene expression analysis of P2-mSSC and P2-PreP showed a significant reduction in the expression levels of genes driving osteochondral ossification compared to P0-mSSC and P0-PreP. Even though the FGF2 supplementation led to an increase in the cell number of the investigated progenitor populations as defined by the CD marker set, it did not preserve their capability to form bone tissue according to the endochondral ossification pathway. Therefore, we concluded that in our expansion protocol, the reported stem cell markers may not be useful for prospective cell expansion and selection, and more importantly for successful in vivo bone formation. It must be noted that this study is performed on embryonic cells, and that young/adult cells could give different results. It is known that the chromatin of embryonic cells is less compact and more permissive to change than cells that are more adult. In embryonic cells, promoters of many developmental regulator genes share an unusual bivalent chromatin pattern, which keep these genes in a poised conformation, ready for expression or silencing in response to cues31. Adult stem cells dissected from their specific niches had more time to fix their epigenetic signature determining their cellular identity. Similar experiments using adult cells could give more insights into these aspects.One of the most severe limitations when working with primary cells is their availability. Therefore, a cell expansion step is typically used to bypass this limitation and increase the number of cells for downstream applications. A challenge when expanding primary cells is to find the conditions to preserve their identity and, most importantly, their All animal experiments were conducted according to the European and Belgian legislation and with approval of the Animal Ethics Committee of the faculty of Biomedical Sciences of the KU Leuven (project license 221/2015). SWISS, NMRI nu/nu mice (Janvier labs) and B6.Cg-Tg(ACTb-eGFP) mice were used in these studies. For timed matings, male and female SWISS mice were placed together and left overnight. The next day (0.5 dpc) the success of the mating was assessed by the presence of a vaginal plug. At 14.5 dpc pregnant females were sacrificed by cervical dislocation.in vitro by seeding them on pre-coated gelatin surface  at 20,000 cells/cm2 and cultured in a humidified incubator at 37 °C with 5% CO2 in standard medium with or without supplementation with 10 ng/mL FGF2 (Peprotech). Upon reaching 90% confluence, the cells were passaged twice using the same media conditions.Embryos were harvested, staged, and the femurs were dissected and digested with collagenase II (Thermo Scientific) for 40 min at a concentration of 24 mg/ml in standard growth medium  under continuous rotation. After digestion, the embryonic cartilage cells were passed through a 70 µm cell strainer to obtain a single cell suspension. Cells were expanded Single cells suspended in 2% FBS in PBS (Lonza) were stained with fluorochrome-conjugated antibodies against CD45, TER119, CD51, CD200, CD105 and CD90.2 (Ebioscience – Thermo Scientific). All antibodies were used at their optimal concentrations as determined by titration. Three controls were used: unstained cells, single stained compensation controls (UltraComp eBeads – Thermo Scientific) and fluorescence-minus-one (FMO) controls. An FMO control contains all the tested antibodies in the panel, minus one of them. This gives a strong negative control to work with since it allows to take into account how the other stains in the panel influence the left out parameter. These FMOs were used as gating controls to accurately discriminate positive vs negative signals. The step-by-step gating strategy is described in Fig. 2 (Sigma). Either of the gels were subcutaneously implanted behind the shoulders of NMRI nu/nu mice, anaesthetized with ketamine-xylazine (80 mg/kg b.w.t. ketamine and 5 mg/kg b.w.t xylazine - IP). After implantation, the mice received analgesics . After one, two or three weeks, gels were retrieved for ex vivo nano computed tomography (nCT) and histological analysis.Single cells  were encapsulated in two different hydrogels: collagen or alginate, at a volume of 150 uL or 50 uL. Single cells were pelleted down, suspended in 5 mg/mL collagen gel I (Corning) prepared according to the manufacturer’s protocol and allowed to gelate in moulds for 30–40 min at 37 °C. Similarly, pelleted cells were suspended in 1,5% alginate (Sigma) and chemically crosslinked with 102 mM CaClFor cell tracing experiments, ECC were derived from ACTb-eGFP mice and encapsulated in collagen I gel as described above. Implants were retrieved after one, two or three weeks. Retrieved explants were fixed in 4% paraformaldehyde overnight at 4 °C, washed with PBS and decalcified in 0.5 M EDTA/PBS solution for 14 changes (of 24 h) prior to 30% sucrose cryoprotection and embedding in OCT . Samples were sectioned on a cryostat (Microm HM560) at 6 µm and stored at −80 °C until staining. Dual immunofluorescence staining for eGFP and Sox9, Runx2 or Osterix (Txf) was performed by combining an anti-eGFP (Abcam: ab139700) and anti-Txf  (1/1000 in blocking buffer: 5% goat serum in TNB) primary antibody. After 1.5 h, secondary antibodies Dylight 488 (Abcam: ab96951) and Alexa Fluor 546 (Life Technologies) (1/200 and 1/500 in blocking buffer respectively) were incubated for 1 h. The samples were counterstained with Hoechst 33342 (1/200 in PBS) (stock solution 10 mg/mL - Sigma) for 5 min and mounted with Fluoroshield mounting medium (Sigma). The mounted samples were imaged using a fluorescence microscope (Olympus BX63).Retrieved explants were fixed in 4% paraformaldehyde overnight at 4 °C, washed with PBS and stained with 20% Hexabrix in PBS overnight at room temperature and imaged using a Phoenix NanoTom M (GE Measurement and Control Solutions). To generate the X-rays, a diamond-coated tungsten target was applied. The system was operated at a voltage of 60 kV and a current of 190 µA, and a 0,1 mm aluminium filter was used to reduce beam hardening during the acquisition. The exposure time was 500 ms and 2400 images were acquired over 360° using the fast scan mode (frame averaging = 1 and image skip = 0). The explants were examined at 3.02 µm isotropic voxel size. During reconstruction , a beam hardening correction of 8 was applied. Region of interest selection, thresholding and bone volume over tissue volume  calculations were determined using CTan software (Bruker).Explants were decalcified in 0.5 M EDTA/PBS solution for 14 changes (of 24 h) prior to dehydration, embedding in paraffin wax and sectioning at 5 µm. Slides were stained with Haematoxylin & Eosin, Safranin-O and Fast Green, Mason’s Trichrome and Picrosirius Red. Images were taken with an Olympus DP73 microscope. Polarized light microscopy to visualize collagen fibers after Picrosirius Red staining was performed with a Leica DMR microscope and SPOT imaging software.2O2. For Ki67, an antigen retrieval step  was performed. Primary antibodies targeting Ki67 (Abcam ab16667) or ColX (Cosmo Bio LS-LB-0092) were incubated overnight in blocking buffer (TBS with 5% goat serum). The next day, the biotynilated secondary antibody (Abcam ab6720) was incubated for 30 min. An ABC kit (Vectastain PK4000) was added to amplify the biotin-streptavidin reaction and provide the detection by HRP and the DAB substrate (DAKO K346711). Slides were counterstained with Haematoxylin and mounted with Pertex.Proliferation and hypertrophy were detected with Ki67 and ColX immunohistochemistry staining respectively. Briefly, slides were dewaxed, dehydrated and hydrated before blocking cellular peroxidase activity with H−ΔCt method and normalized for the expression of the reference gene β-actin. Specific forward and reverse oligonucleotide primers were used, and are listed in Supplementary Table RNA from primary, cultured or sorted cells was extracted using the RNeasy Mini Kit (Qiagen) or RNeasy Micro Kit (Qiagen) and converted to cDNA with the Revert Aid H Minus First strand cDNA synthesis kit (Thermo Scientific) according to the manufacturer’s protocols. Quantification of gene expression was carried out using SYBR Select (Master mix - Applied Biosystems) based qPCR reaction on the StepOne Plus System (Applied Biosystems). Expression levels were analysed using the 2Biological independent replicates for each experiment are indicated in the respective figure legends. Data was analysed in SPSS . Details of the statistical tests performed for each experiment can be found in the representative figure legends. Graphs show raw data as individual data points, overlaid with a bar indicating the mean. We present a typical histological section from each of the experiments.Supplementary information


Drosophila melanogaster and Caenorhabditis elegans and the mammalian Class I TRIM9 regulate axon branching and guidance in response to the guidance cue netrin, whereas mammalian TRIM46 establishes the axon initial segment. In humans, mutations in TRIM1 and TRIM18 are implicated in Opitz Syndrome, characterized by midline defects and often intellectual disability. We find that although TRIM67 is the least studied vertebrate Class I TRIM, it is the most evolutionarily conserved. Here we show that mammalian TRIM67 interacts with both its closest paralog TRIM9 and the netrin receptor DCC and is differentially enriched in specific brain regions during development and adulthood. We describe the anatomical and behavioral consequences of deletion of murine Trim67. While viable, mice lacking Trim67 exhibit abnormal anatomy of specific brain regions, including hypotrophy of the hippocampus, striatum, amygdala, and thalamus, and thinning of forebrain commissures. Additionally, −/−Trim67 mice display impairments in spatial memory, cognitive flexibility, social novelty preference, muscle function, and sensorimotor gating, whereas several other behaviors remain intact. This study demonstrates the necessity for TRIM67 in appropriate brain development and behavior.Class I members of the tripartite motif (TRIM) family of E3 ubiquitin ligases evolutionarily appeared just prior to the advent of neuronal like cells and have been implicated in neuronal development from invertebrates to mammals. The single Class I TRIM in  Trim67 results in malformations of a subset of subcortical brain regions and of cortical and subcortical myelinated fiber tracts, as well as deficits in spatial memory, motor function, sociability and sensorimotor gating. Interactions between TRIM67 and both the netrin receptor DCC and TRIM9, and abnormalities in netrin-sensitive brain regions are noted. We conclude that TRIM67 is critical for appropriate brain development and behavior.Class I tripartite motif (TRIM) E3 ubiquitin ligases play important roles in neuronal development and function, some of which occur downstream of netrin. TRIM67 is the most evolutionarily conserved of this class and is developmentally regulated and brain enriched. Deletion of murine E3 ubiquitin ligases mediate covalent attachment of ubiquitin to specific substrates. This posttranslational modification targets substrates for degradation or affects substrate localization, interactions, or function . In deveTrim9 deletion is also associated with aberrant migration, morphogenesis, and synapse organization of adult-born neurons in the murine dentate gyrus, and severe deficits in spatial learning and memory , thalamus, and amygdala. Additionally, there were decreases in the size of the internal capsule and the dorsal commissures, including the corpus callosum and hippocampal commissure. The consequences of Trim67 deletion on brain anatomy and behavior indicate that TRIM67 plays critical, yet undefined roles in brain development and function.Here, we show that TRIM67 interacts with its paralog TRIM9 and the netrin receptor DCC. We found that deletion of murine Trim9, which encodes the RING and two BBox domains, was flanked by LoxP sites , and Dr. Eva Anton (University of North Carolina at Chapel Hill), respectively.Mouse lines were on a C57BL/7 background and were housed and bred at the University of North Carolina with approval from the Institutional Animal Care and Use Committee. Timed pregnant females were obtained by placing male and female mice together overnight; the following day was designated as E0.5 if the female had a vaginal plug. We previously generated mice in which the first exon of xP sites . Exon 1 Antibodies: TRIM67 rabbit polyclonal generated using murine TRIM67 recombinant protein aa45-73 ; TRIM9 rabbit polyclonal antibody  was described previously . The plain vitro in modified RIPA buffer . Protein from embryonic and adult tissues was obtained by dissection at E15.5 and postnatal day (P) 78, respectively, followed by lysis in modified RIPA buffer. SDS-PAGE and immunoblot analysis were performed using standard procedures with far-red-conjugated secondary antibodies (LI-COR Biosciences) imaged with an Odyssey Imager (LI-COR Biosciences).Embryonic day (E) 15.5 dissociated cortical neuron cultures were prepared as previously described , and lysCoimmunoprecipitation assays were conducted according to standard procedures. Briefly, HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen) by manufacturer protocol. Protein A/G beads (SCBT) coupled with a mouse anti-myc (9E10) antibody were incubated with lysates overnight at 4°C. Beads were washed three times with lysis buffer and bound proteins were resolved by standard SDS-PAGE and immunoblot analysis.Mice were given one 5-min trial on the plus maze, which had two walled arms  and two open arms. The maze was elevated 50 cm from the floor, and the arms were 30 cm long. Animals were placed on the center section (8 × 8 cm) and allowed to freely explore the maze. Measures were taken of time in, and number of entries into, the open and closed arms.Mice were tested in a Plexiglas cage located in a sound-attenuating chamber with ceiling light and fan. The cage contained 5 cm of corncob bedding, with 20 black glass marbles (14 mm in diameter) arranged in an equidistant 5 × 4 grid on top of the bedding. Subjects were given access to the marbles for 30 min. Measures were taken of the number of buried marbles (two thirds of the marble covered by the bedding).Several days before the olfactory test, an unfamiliar food  was placed overnight in the home cages of the mice. Observations of consumption were taken to ensure that the novel food was palatable. Sixteen to twenty hours before the test, all food was removed from the home cage. On the day of the test, each mouse was placed in a large, clean tub cage (46 cm long × 23.5 cm wide × 20 cm high), containing paper chip bedding (3 cm deep), and allowed to explore for 5 min. The animal was removed from the cage, and one Froot Loop was buried in the cage bedding. The animal was then returned to the cage and given 15 min to locate the buried food. Measures were taken of latency to find the food reward.Individual mice were placed in a tall plastic cylinder located on a hotplate, with a surface heated to 55°C . Reactions to the heated surface, including hindpaw lick, vocalization, or jumping, led to immediate removal from the hotplate. Measures were taken of latency to respond, with a maximum test length of 30 s.Mice were given a 1-h trial in an open field chamber (41 × 41 × 30 cm) crossed by a grid of photobeams . Counts were taken of the number of photobeams broken during the trial in 5-min intervals, with separate measures for locomotion  and rearing movements. Time spent in the center region of the open field was measured as an index of anxiety-like behavior.Subjects were given two acoustic startle tests , one at age 10–11 weeks and another at age 17–19 weeks. Mice were placed in a small Plexiglas cylinder within a larger, sound-attenuating chamber. The cylinder was seated on a piezoelectric transducer, which allowed vibrations to be quantified and displayed on a computer. The chamber included a ceiling light, fan, and a loudspeaker for the acoustic stimuli. Background sound levels (70 dB) and calibration of the acoustic stimuli were confirmed with a digital sound level meter (San Diego Instruments).Each session consisted of 42 trials that began with a 5-min habituation period. There were seven different types of trials: the no-stimulus trials, trials with the acoustic startle stimulus  alone, and trials in which a prepulse stimulus  occurred 100 ms before the onset of the startle stimulus. Measures were taken of the startle amplitude for each trial across a 65-ms sampling window, and an overall analysis was performed for each subject’s data for levels of prepulse inhibition at each prepulse sound level  × 100]).In the first test session, mice were given three trials on an accelerating rotarod  with 45 s between each trial. Two additional trials were given 48 h later. Revolutions per minute was set at an initial value of three, with a progressive increase to a maximum of 30 across 5 min . Measures were taken for latency to fall from the top of the rotating barrel.A track of Whatman filter paper (Sigma-Aldrich) was placed in a 7.5-cm-wide, 40-cm-long Plexiglas corridor closed at one end and open to a 10 × 10-cm Plexiglas chamber at the other end. The chamber was covered with an opaque cloth and filled with several pellets of dry food. Mouse forepaws and hindpaws were painted with two colors of Crayola Washable Kids’ Paint  before the mouse was placed at the closed end of the Plexiglas corridor. Once mice had walked the length of the corridor, they were given 45 s in the recovery chamber before being returned to their home cage.Mice were held near a circular rubber gasket, suspended from a pulley, such that the mouse grasped the loop . Mice weThis procedure consisted of three 10-min phases: a habituation period, a test for sociability, and a test for social novelty preference. For the sociability assay, mice were given a choice between being in the proximity of an unfamiliar, sex-matched C57BL/6J adult mouse (stranger one) versus being alone. In the social novelty phase, mice were given a choice between the already-investigated stranger one, versus a new unfamiliar mouse (stranger two). The social testing apparatus was a rectangular, three chambered box fabricated from clear Plexiglas. Dividing walls had doorways allowing access into each chamber. An automated image tracking system (Noldus Ethovision) provided measures of time spent within 5 cm of the Plexiglas cages (the cage proximity zone), and entries into each side of the social test box.At the start of the test, the mouse was placed in the middle chamber and allowed to explore for 10 min, with the doorways into the two side chambers open. After the habituation period, the test mouse was enclosed in the center compartment of the social test box, and stranger one was placed in one of the side chambers. The stranger mouse was enclosed in a small Plexiglas cage drilled with holes, which allowed nose contact. An identical empty Plexiglas cage was placed in the opposite side chamber. Following the placement of the stranger and the empty cage, the doors were reopened, and the subject was allowed to explore the social test box for a 10-min session. At the end of the sociability phase, stranger two was placed in the empty Plexiglas container, and the test mouse was given an additional 10 min to explore the social test box.The water maze consisted of a large circular pool (diameter = 122 cm) partially filled with water , located in a room with numerous visual cues. The procedure involved three different phases: a visible platform test, acquisition in the hidden platform task, and reversal learning. In the visible platform test, each mouse was given four trials per day, across 2 d, to swim to an escape platform cued by a patterned cylinder extending above the surface of the water. For each trial, the mouse was placed in the pool at one of four possible locations (randomly ordered), and then given 60 s to find the visible platform. If the mouse found the platform, the trial ended, and the animal was allowed to remain 10 s on the platform before the next trial began. If the platform was not found, the mouse was placed on the platform for 10 s, and then given the next trial. Measures were taken of latency to find the platform and swimming speed via an automated tracking system (Noldus Ethovision).For the hidden platform task, the platform (diameter = 12 cm) was submerged and the water made opaque using white Crayola Washable Kids’ Paint . Each animal was given four trials per day, with 1 min per trial, to swim to the hidden platform. Criterion for learning was an average group latency of 15 s or less to locate the platform. Mice were tested until the group reached criterion, with a maximum of 9 d of testing. When criterion was reached, mice were given a one min probe trial in the pool with the platform removed. Selective quadrant search was evaluated by measuring percentage time spent in the target quadrant and number of crossings over the location where the platform (the target) had been placed during training, versus the corresponding area in the opposite quadrant. During the subsequent reversal learning phase, the escape platform was placed in a different quadrant, and mice were tested until the group reached the learning criterion, using the same procedure as with initial acquisition. A second one min probe trial was conducted at the end of the reversal learning phase.All adult mice used for neuroanatomical studies were anesthetized with an intraperitoneal injection of 1.2% avertin and intracardially perfused with 1× PBS followed by 4% paraformaldehyde (PFA). Brains were removed and fixed in 4% PFA for an additional 48 h, rinsed with 1× PBS, and washed in 70% EtOH for at least 24 h before vibratome sectioning at 100-µm thickness. Embryonic brains were removed at E15.5 and drop-fixed in 4% PFA for 72 h, rinsed with 1× PBS, and embedded in 1% agarose before cryoprotection with 30% sucrose and cryostat sectioning at 20-µm thickness.Sections were blocked in 0.2% Triton X-100 and 10% BSA in 1× PBS for 2 h on a rocker at room temperature. Solution was replaced with primary antibodies in blocking solution overnight at 4°C. Sections were washed three times for 20 min each in 1× PBS, and then incubated with secondary antibodies in blocking solution for two h at room temperature. Sections were then incubated with 300 nM DAPI in 1× PBS for 30 min and then washed three times for 20 min each with 1× PBS. Sections were mounted in mounting media  and sealed with nail polish. Fluorescently labeled sections were imaged on a Zeiss LSM 780 confocal microscope using a 20× Plan-Apochromat objective. Embryonic cortical neurons cultured on nitric-acid-washed coverslips and fixed with 4% PFA were stained by the same protocol and imaged on an Olympus IX81-ZDC2 inverted microscope using a 100 × 1.49 NA UAPON DIC/TIRF objective.Five-week-old brains from an equal number of male and female mice from both genotypes were sectioned and stained with Black Gold II  per manufacturer protocol, mounted in DPX mounting media , and imaged on a Leica WILD M420 macroscope using an APOZOOM lens. Sections were aligned to bregma using a reference atlas, and individual regions were outlined as described below and measured using ImageJ. White matter in the striatum was defined using the Trainable Weka Segmentation v3.2.2 plugin for ImageJ  trained All structures present in each hemisphere  were reported as the average size of the structure in one hemisphere. Fiber tracts were demarcated by myelin staining. The thicknesses of the corpus callosum, hippocampal commissure and anterior commissure were measured at the midline perpendicular to the direction of the tract. The cortical thickness was determined by measuring the area of a cortical region divided by the average of the lengths of the cingulum and external surfaces to produce an average thickness along the length of the cortex. Hippocampal area was defined as the area bounded by the fimbria, thalamus, habenula, third ventricle, and corpus callosum, which included both the dentate gyrus and Ammon’s horn. To measure the area of the lateral and basolateral amygdala, which are bounded by the external and amygdalar capsules, boundaries along these capsules were drawn on black gold II myelin stained sections and were connected with a straight perpendicular line at the medial inferior ends. The striatum was bounded by the lateral ventricles, and black gold stained corpus callosum, external capsule, and anterior commissure in anterior sections, and the lateral ventricles, corpus callosum, external capsule, globus pallidus, and internal capsule in posterior sections. The thalamus was delineated by the habenulae, hippocampus, fimbriae, internal capsules, and a straight line drawn between the inferior tip of the internal capsules. Anatomic regions were compared to reference atlases to confirm boundaries , NCBI GenPept , and EnsemblMetazoa (accession number CapteT155810) databases. Sequences were compared using the ClustalX2 multiple sequence alignment, with default parameters and three full alignment iterations . PhylogeF value was determined. Within-group comparisons were conducted to determine side preference in the three-chamber test for social approach and for quadrant preference in the Morris water maze.Behavioral data were analyzed using one-way or repeated measures ANOVA, with factor genotype. Separate ANOVAs for each sex were conducted for measures of body weight. Fisher’s protected least-significant difference (PLSD) tests were used for comparing group means only when a significant fl/flTrim67 strain corpus callosum comparisons, Wilcoxon signed-rank tests were used. For unpaired single measurements, Mann–Whitney pairwise comparisons were made. A χ2 test was used to compare the heterozygote birth rate to the predicted Mendelian distribution. For all comparisons, α = 0.05.For all brain regions measured in multiple sections, comparisons were performed by two-way ANOVA. Sections and genotypes were treated as groups with each brain a subject, with genotype as the tested factor. For the paired brain weight and Nex-Cre:D. melanogaster trim9 suggested that human TRIM9 was the most evolutionarily conserved . Mice carrying the flTrim67 allele were crossed with CMV-Cre mice to delete Trim67 in the germline and generate −/−Trim67 mice, confirmed by multiplexed PCR genotyping . We developed specific polyclonal antibodies to TRIM9 and TRIM67 using unique regions within their respective N termini  and regulate netrin-dependent axonal responses  and 4 adult −/−Trim67  littermate-paired brains, sectioned and stained for myelin  but did decrease the thickness of the corpus callosum  was also reduced when compared to +/+NexCre/Trim67/Tau-lox-STOP-lox-GFP littermates . The intensity of GFP in the hippocampal commissure of these mice was insufficient to permit quantification of the thickness of this tract.All these findings prompted us to investigate netrin-dependent fiber tracts in mmissure . We measr myelin A. Deleticallosum B. Additi−/− mice B, inset.promoter  and Tau-GFP mice , resultiTrim67 deletion on fiber tracts, we noted that −/−Trim67 brains appeared smaller than +/+Trim67 counterparts; postfixation weight of −/−Trim67 brains was indeed ∼10% less than littermate counterparts  and 13 −/−Trim67 mice  aged six weeks at the start of behavioral testing. Over the course of the behavioral testing, there was no significant effect of loss of Trim67 on body weight or overall growth in either male or female mice , a measure of anxiety. Normal behaviors in these tests indicate loss of Trim67 does not detectably impact sensory abilities, general locomotion, or anxiety behaviors.With the neuroanatomical defects in  regimen  has been regimen . For theale mice A. There ale mice B. In an ovements C or in t−/−Trim67 brains, have been associated with spatial learning and memory deficits . −/−Trim67 mice took significantly longer to find the hidden platform, and never reached the criterion for learning . In the test for sociability  and thirteen −/−Trim67  mice were assayed for gait and muscle function. Assessment of footprints on a linear track , when normalized to CPu area, white matter was increased in −/−Trim67 brains , indicating a reduction in gray matter in −/−Trim67 brains. Despite equal white matter in the striatum, the cross-sectional area of the internal capsule, where these fibers fasciculate, was smaller in −/−Trim67 brains (In addition to circuits in the hippocampus and cortex, striatal circuits are implicated in spatial learning and memory and motor activity, particularly in the CPu . Since ltermates A to dete− brains B, which − brains C. Althou− brains D.−/−Trim67 mice (−/−Trim67 mice (+/+Trim67 brains (−/−Trim67 brains (Trim67 results in decreases in the size of some, but not all, brain regions and fiber tracts in the adult.Deficits in sensorimotor gating have been associated with abnormalities in the sensory nuclei of the thalamus . In ligh−/− mice  and TRIM−/− mice C, we mea−/− mice A. Thalam−/− mice B. Howeve+ brains C, indica+ brains D,E is pr+ brains , we comp− brains D. These Trim67 deletion results in a complex set of behavioral deficits and malformation of several brain regions and axonal fiber tracts in adult mice. In humans, the TRIM67 gene, which thus far has not been linked to any human disorders, is within the q42.2 band of chromosome 1, a region highly associated with heritable neurologic conditions (TRIM67 to other genes linked to Alzheimer’s disease and schizophrenia (DISC1; SIPA1L2; DISC2, C1orf131, GALNT2; Trim67 are intriguing, potentially suggesting possible involvement of TRIM67 in human disorders or behaviors. Indeed, a small GWAS of patients with neuroticism, a personality trait that often occurs with major depression and anxiety disorders, identified variations in several genes, including TRIM67 (Here, we generated a novel knockout mouse and specific antibody to investigate the largely unstudied, yet evolutionarily conserved E3 ubiquitin ligase, TRIM67. We show that TRIM67 is enriched in the cerebellum and, to a lesser extent, other brain regions in the adult mouse. Furthermore, in the embryo, TRIM67 is enriched in the developing cortex, diencephalon, and midbrain. We found that nditions . The proa DISC1; , Parkinsg TRIM67 . HoweverTrim67 being deleted globally, sensorimotor gating deficits in prepulse inhibition of acoustic startle were not present at 10–11 weeks but appeared by 17–19 weeks. This suggests that TRIM67 may also be necessary for maintenance of brain function in mature animals. The spatial and motor learning impairments also suggest a role for TRIM67 in remodeling of the hippocampus, classically associated with the Morris water maze task (The change in TRIM67 protein levels from embryonic to adult brains suggests that TRIM67 could function both in developing neurons, for example the embryonic cortex, as well as mature neurons, such as cells of the adult cerebellum. Indeed, despite aze task . This staze task .−/−Trim67 brains which, based on black-gold staining, is due to decreased non-myelinated tissue. Given the normal size of the cortex and some portions of the affected nuclei, this may be due to abnormal maturation and development of specific types of neurons, as opposed to overall proliferation. This distinction will require additional studies to determine which neuron types express TRIM67, and at what points during development or maturity TRIM67 is expressed. The pattern of TRIM67 expression in the embryonic brain however supports a role for TRIM67 in neuronal development after proliferation, as TRIM67 protein levels are highest in regions of the developing cortex and hippocampus containing migrating and maturing, postmitotic neurons. This may offer an explanation for the reduction in adult gray matter, as a large portion of this tissue consists of neurites as opposed to cell bodies. A previous study on TRIM67 suggested a role in the formation and elongation of neurites (−/−Trim67 mice alternatively may originate from decreases in branching of axons in the affected brain regions, whereas the cell bodies of the affected neurons reside in distant nuclei. As TRIM67 interacts with the axon guidance receptor DCC, which can promote axon branching (−/−Trim67 mice, including the callosal and hippocampal commissures, and the stria terminalis and stria medullaris.There is apparent hypotrophy of the hippocampus, striatum, thalamus, and amygdala in neurites , and theneurites , both ofranching , this isTrim67 deletion, the changes in cellular structure that lead to fiber tract malformation, the time course of and underlying cause of behavioral abnormalities in −/−Trim67 mice, and the contribution of TRIM67 variations to human neurologic disorders. Furthermore, although TRIM67 is the most evolutionarily conserved vertebrate Class I TRIM, unlike the invertebrate TRIM ortholog, loss of Trim67 does not fully phenocopy the axon tract defects seen in mice lacking Dcc. Future studies will need to examine whether this is due to compensation from other Class I TRIM proteins such as TRIM9, which interacts with both DCC and TRIM67, or TRIM1 and TRIM18, whose mutation in humans is associated with midline birth defects.These experiments have laid a foundation for investigating the role of TRIM67 in development and function of the brain. The role of TRIM67 ubiquitin ligase activity, its substrates, and the consequences of their ubiquitination remain to be identified. Further studies will be required to elucidate not only this molecular function of TRIM67 in developing and adult neurons, but also whether TRIM67 plays a role in DCC-dependent responses, the underlying cause of brain hypotrophy resulting from


L/D = 100) and long carbon fibers . It was observed that SCFs and LCFs built efficient network structures during the foaming process. Furthermore, the foaming process significantly increased the specific electromagnetic interference shielding effectiveness from 10 to 60 dB. In addition, cellulose/carbon fiber composite foams possessed good mechanical properties and low thermal conductivity of 0.021–0.046 W/(m·K).Lightweight electromagnetic interference shielding cellulose foam/carbon fiber composites were prepared by blending cellulose foam solution with carbon fibers and then freeze drying. Two kinds of carbon fiber (diameter of 7 μm) with different lengths were used, short carbon fibers (SCF,  With the development of science and technology, mankind has developed a variety of electronic technologies, such as broadcasting, television, and microwave technology, which have greatly improved human living conditions. However, these technologies are accompanied by electromagnetic pollution ,2,3,4, w4 solution to reduce graphite oxide, and deposited the reduced graphite on the surface of carbon fibers by electrophoretic deposition. Its electromagnetic shielding effectiveness reached up to 37.8 dB (8.2–12.4 GHz). Al-Saleh  (mL·g−1) = 3.85 × 10−2 (wM)0.76 [The cellulose was provided by Hubei Chemical Fiber Group Ltd.  in the form of cotton linter pulp, in which the α-cellulose content was more than 95%. The cellulose needed to be pretreated before using—washed in distilled water and then oven dried for 24 h. The viscosity-average molecular weight ((Mw)0.76 . Carbon Cellulose solution was prepared according to a previous method . FourteeV) and the mass (m), and then the density (ρ) was calculated through the equation: ρ = m/V.Scanning electron micrographs (SEM) were taken on a Hitachi S4800 scanning electron microscope  with 3 kV accelerating voltage, at magnification of 50 and 5000 respectively. The foams in their wet state were treated in liquid nitrogen, immediately snapped and then freeze dried. The samples were sputtered with gold, then were observed and photographed. Pore size was read from the SEM images. FT-IR spectra were recorded on a Nicolet FTIR spectrometer  by KBr pellet method. The dried cellulose foams were cut into cubes to measure the volume . The electrical conductivity of composites was measured using the four point method on a resistivity/Hall measurement system . The contact points of the samples were coated with silver paste to reduce the contact resistance. Voltage and current data was recorded after the display became stable following full electrical connections between the probes and composites. At least five measurements were made for each set of conditions.bσ) and compression properties of the foams were measured on a universal testing machine  according to ISO 527-2, 1993 (E) at a speed of 5 mm·min−1, respectively. The bσ values recorded were the average of five measurements.The tensile strength (L/D rates of 100 (SCF) and 300 (LCF) respectively, were added into cellulose foam to create electromagnetic shielding and insulation foam. Carbon fibers were treated with 98 wt % H2SO4 for several hours to remove hydrophobic reagents on the surface, and oxidize the carbon fiber to make it more hydrophilic. These changes were measured by FTIR, which is shown in COOH appears near 1720 cm−1, and there is a characteristic peak of the carboxylate (COO–) group near 1590 cm−1, indicating that the surface of the carbon fibers was attached after the sulfuric acid treatment. The peak intensity at 1650 cm−1 is derived from the hydrogen bond in the acid [−1, which possibly due to carboxyl coupling vibration and hydroxyl deformation vibration. The characteristic peak absorption of the hydroxyl group near 3440 cm−1 is also greatly enhanced, indicating that the number of hydroxyl groups is greatly increased. A main peak splitting into several small peaks near 1160 to 1030 cm−1 may be the stretching vibration absorption peak of C–O in different groups such as carboxyl group, lactone group, and phenol group, and the small absorption peak at 1160 cm−1 is the stretching vibration absorption peak of the C–O bond in the carboxyl group. The small absorption peak at 1060 cm−1 is the absorption peak of the C–O group in the lactone group, and the peak appearing at 1030 cm−1 may be the stretching vibration absorption peak of the CO group in the phenol group. It is indicated that after electrochemical oxidation treatment, the reactive functional groups attached to the surface of the carbon fibers are carboxyl groups, hydroxyl groups, and lactone groups, which will be compatible with cellulose.In this research, two kinds of carbon fiber, the acid . A weak After acid treatment, the carbon fibers were added to cellulose solution to prepare composite foams. Electrical conductivity is critical for EMI shielding efficiency, because it is an intrinsic ability of a material for absorbing electromagnetic radiation . As showIn general, the foaming process has two effects on the electrical conductivity of polymer composites . One effThe malfunction of electronics can be hazardous, as the electronics can be associated with strategic systems such as aircraft, nuclear reactors, transformers, control systems, communication systems, etc. . Figure According to the results, SCFs were oriented in cellulose lamellae and separated by bubbles, which made the network not so good. LCFs connected with each other directly to make the conduct network good even at low content. Good network means larger electron conductivity, leading to better EMI properties. Furthermore, it was proposed that the spherical air bubbles in the foam structure enhanced the attenuation of incident electromagnetic microwaves by multiple reflection and decay between the cell wall and fillers . As indiFoams always have good thermal insulation properties, which is quite useful in the area of building materials. The thermal conductivity of the foams is shown in In summary, we have developed an easy and fast approach for scalable fabrication of lightweight cellulose/carbon fiber composite foam, based on a cellulose dissolution and regeneration process. Acid treated short carbon fibers (SCF) and long carbon fibers (LCF) were added into the cellulose solution to produce EMI composite foams. Carbon fibers built conductive networks, where SCFs were most likely oriented in the bubble cell wall and LCFs penetrated through the bubbles. LCF/cellulose foams showed better electrical conductivity and higher EMI shielding property (60 dB of LCF20). Furthermore, cellulose/carbon fiber composite foams exhibited well-defined thermal insulation and tensile properties. The comprehensive study of cellulose composite foams based on other powerful absorbers will be required in the future, in order to develop useful materials for EMI shielding in high-tech fields.