Melatonin Treatment of Circadian Rhythm Sleep-Wake Disorder in Obese Children Affects the Brain-Derived Neurotrophic Factor Level.

BACKGROUND: Previous studies proved that the brain-derived neurotrophic factor (BDNF) is correlated with sleep regulation, yet how BDNF functions and reacts in the melatonin treatment of circadian rhythm sleep-wake disorder (CRSWD) among obese children remain enigmatic. Focusing on CRSWD in obese children, this study monitored their sleep efficiency and serum BDNF level changes during the treatment of melatonin. METHODS: In total, 35 obese children diagnosed with CRSWD were included in this study and administrated melatonin (3 mg/night) for 3 months. Blood samples were collected 24 hours before and after the treatment (08:00, 12:00, 16:00, 20:00, 24:00, and 04:00). Subsequently, the plasma melatonin level and serum BDNF level were measured by enzyme-linked immunosorbent assay. Sleep parameters, including sleep quality, Pittsburgh Sleep Quality Index as well as melatonin and BDNF levels before and after treatment, were recorded to profile the effectiveness and safety of melatonin treatment. RESULTS: Melatonin treatment increased plasma melatonin concentration and restored circadian rhythm. Besides, the serum BDNF level showed a significant increase, representing a strong positive correlation with melatonin concentration (p = 0.026). Patients experienced much-improved sleep efficiency (P < 0.001), with longer actual sleep time (P < 0.001), shorter sleep onset latency, and fewer awakenings after treatment (P < 0.001). Besides, melatonin was well tolerated by patients without producing severe side effects. CONCLUSION: Melatonin treatment effectively improved CRSWD among obese children with their serum BDNF levels increased, indicating that BDNF is a key regulator in CRSWD in obese children. This study may offer theoretical support for melatonin treatment of CRSWD in obese children.

Optimization of calcium pectinate gel production from high methoxyl pectin.

BACKGROUND: Calcium pectinate (CaP) gel is traditionally prepared by de-esterifying high methoxyl pectin (HMP) to low methoxyl pectin (LMP), followed by gelation with calcium. To save both time and cost in the production of CaP gel, an alternative method was developed by the addition of CaCl2 to HMP at alkaline pH. To optimize the production, response surface methodology (RSM) was used to investigate the effects of temperature (30-50 °C), time (20-40 min) and pH (8-10) on yield, calcium content of the CaP gel and the degree of esterification (DE) of pectin following decalcification of CaP (DC-pectin). RESULTS: The linear term for pH had a significant effect (P < 0.01) on all three responses, whereas interaction effects were not significant (P > 0.01), except on the calcium content (P < 0.01). The optimized process conditions (temperature, time and pH) to obtain maximum CaP-HMP gel yield (88.83%) were 50 °C, 40 min and pH 9.6, and for the highest calcium content (97.23 mg g-1 ) they were 40 °C, 30 min and pH 9.7. DC-pectin was a typical LMP with DE varying from 26.92% to 50.33%. The DE of DC-pectin could be predicted by a model that proved significant (R2  = 0.9888). CONCLUSION: The optimum conditions were established to produce CaP gels from HMP with high yield and calcium content. Also, LMP with predictable DE can be produced following a significant model. This study provides new insights into the production and application of CaP gel. © 2021 Society of Chemical Industry.

A simple preparation route for polysilicate titanium salt from spent titanium solutions.

Polysilicate titanium salt (PST) is synthesized by using spent titanium solutions and polysilicic acid (PSiA) as raw materials. PSiA could improve the aggregation ability of titanium salt flocculants and also restrain the hydrolysis of Ti4+ to stabilize titanium salts. Meanwhile, replacing titanium salt with spent titanium solutions could reduce the cost of PST and solve the problem of wastewater treatment in the titanium industry, which makes valuable waste regeneration possible. Scanning electron microscopy (SEM) results show the morphology transformation (sheet, spheroid, and sphere) of PST with different Ti/Si molar ratios. The formation process of PST is analyzed by Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). This study investigates the effect of Ti/Si molar ratios on PST flocculation performance in humic-kaolin water and actual domestic wastewater treatment. The in situ floc size change of PST is measured by laser particle size analyzer in humic-kaolin water treatment. Additionally, the performance of PST is comprehensively evaluated on flocculation and sedimentation ability, rapid sweep netting ability and stability. In short, the prepared PST in this study is suitable for treating wastewater with high turbidity and chemical oxygen demand (COD) in a wide range of pH values.

Recent Advances in Marine Algae Polysaccharides: Isolation, Structure, and Activities.

Marine algae have attracted a great deal of interest as excellent sources of nutrients. Polysaccharides are the main components in marine algae, hence a great deal of attention has been directed at isolation and characterization of marine algae polysaccharides because of their numerous health benefits. In this review, extraction and purification approaches and chemico-physical properties of marine algae polysaccharides (MAPs) are summarized. The biological activities, which include immunomodulatory, antitumor, antiviral, antioxidant, and hypolipidemic, are also discussed. Additionally, structure-function relationships are analyzed and summarized. MAPs' biological activities are closely correlated with their monosaccharide composition, molecular weights, linkage types, and chain conformation. In order to promote further exploitation and utilization of polysaccharides from marine algae for functional food and pharmaceutical areas, high efficiency, and low-cost polysaccharide extraction and purification methods, quality control, structure-function activity relationships, and specific mechanisms of MAPs activation need to be extensively investigated.

Effects of ß-cyclodextrin and methyl jasmonate on the production of vindoline, catharanthine, and ajmalicine in Catharanthus roseus cambial meristematic cell cultures.

Long-term stable cell growth and production of vindoline, catharanthine, and ajmalicine of cambial meristematic cells (CMCs) from Catharanthus roseus were observed after 2 years of culture. C. roseus CMCs were treated with ß-cyclodextrin (ß-CD) and methyl jasmonate (MeJA) individually or in combination and were cultured both in conventional Erlenmeyer flasks (100, 250, and 500 mL) and in a 5-L stirred hybrid airlift bioreactor. CMCs of C. roseus cultured in the bioreactor showed higher yields of vindoline, catharanthine, and ajmalicine than those cultured in flasks. CMCs of C. roseus cultured in the bioreactor and treated with 10 mM ß-CD and 150 µM MeJA gave the highest yields of vindoline (7.45 mg/L), catharanthine (1.76 mg/L), and ajmalicine (58.98 mg/L), concentrations that were 799, 654, and 426 % higher, respectively, than yields of CMCs cultured in 100-mL flasks without elicitors. Quantitative reverse transcription (RT)-PCR showed that ß-CD and MeJA upregulated transcription levels of genes related to the biosynthesis of terpenoid indole alkaloids (TIAs). This is the first study to report that ß-CD induced the generation of NO, which plays an important role in mediating the production of TIAs in C. roseus CMCs. These results suggest that ß-CD and MeJA can enhance the production of TIAs in CMCs of C. roseus, and thus, CMCs of C. roseus have significant potential to be an industrial platform for production of bioactive alkaloids.

Identification and classification of surface defects for digital twin models of the workpiece.

Workpiece surface defect detection is an indispensable part of intelligent production. The surface information obtained by traditional 2D image detection has some limitations due to the influence of environmental light factors and part complexity. However, the digital twin model has the characteristics of high fidelity and scalability, and the digital twin surface can be obtained by a device with a scanning accuracy of 0.02mm to achieve the representation of the real surface of the workpiece. The surface defect detection system for digital twin models is proposed based on the improved YOLOv5 model in this paper. Firstly, the digital twin model of the workpiece is reconstructed by the point cloud data obtained by the scanning device, and the surface features with defects are captured. Subsequently, the training dataset is calibrated based on the defect surface, where the defect types include Inclusion, Perforation, pitting surface and Rolled-in scale. Finally, the improved YOLOv5 model with CBAM mechanism and BiFPN module was used to identify the surface defects of the digital twin model and compare it with the original YOLOv5 model and other common models. The results show that the improved YOLOv5 model can realize the identification and classification of surface defects. Compared with the original YOLOv5 model, the mAP value of the improved YOLOv5 model has increased by 0.2%, and the model has high precision. On the basis of the same data set, the improved YOLOv5 model has higher recognition accuracy than other models, improving 11.7%, 3.4%, 6.2%, 33.5%, respectively. As a result, this study provides a practical and systematic detection method for digital twin model surface during the intelligent production process, and realizes the rapid screening of the workpiece with defects.

The main molecular mechanisms of ferroptosis and its role in chronic kidney disease.

The term ferroptosis, coined in 2012, has been widely applied in various disease research fields. Ferroptosis is a newly regulated form of cell death distinct from apoptosis, necrosis, and autophagy, the mechanisms of which have been extensively studied. Chronic kidney disease, characterized by renal dysfunction, is a common disease severely affecting human health, with its occurrence and development influenced by multiple factors and leading to dysfunction in multiple systems. It often lacks obvious clinical symptoms in the early stages, and thus, diagnosis is typically made in the later stages, complicating treatment. While research on ferroptosis and acute kidney injury has made continuous progress, studies on the association between ferroptosis and chronic kidney disease remain limited. This review aims to summarize chronic kidney disease, investigate the mechanism and regulation of ferroptosis, and attempt to elucidate the role of ferroptosis in the occurrence and development of chronic kidney disease.

An atypical protein kinase C (PKC zeta) plays a critical role in lipopolysaccharide-activated NF-kappa B in human peripheral blood monocytes and macrophages.

We have reported that the bacterial LPS induces the activation of NF-kappaB and inflammatory cytokine gene expression and that this requires the activity of small GTPase, RhoA. In this study, we show that an atypical protein kinase C isozyme, PKCzeta, associates functionally with RhoA and that PKCzeta acts as a signaling component downstream of RhoA. Stimulation of monocytes and macrophages with LPS resulted in PKCzeta activation and that inhibition of PKCzeta activity blocks both LPS-stimulated activation of NF-kappaB and IL-1beta gene expression. Our results also indicate that transforming growth factor beta-activated kinase 1 acts as a signaling component downstream of PKCzeta in cytokine gene transcription stimulated by LPS in human peripheral blood monocytes and macrophages. The specificity of this response suggests an important role for the Rho GTPase/PKCzeta/transforming growth factor beta-activated kinase 1/NF-kappaB pathway in host defense and in proinflammatory cytokine synthesis induced by bacterial LPS.

[Comparative protein analysis of K562 cell apoptosis induced by 6-gingerol].

OBJECTIVE: To establish two-dimensional electrophoresis (2-DE) pattern for proteome analysis on K562 cells before and after treatment with 6-gingerol, and mass-spectrometry was applied to identify and analyze the differentially expressed proteins. METHODS: K562 cells were treated with 6-gingerol. Cell proliferation was analyzed by CCK-8 assay. Total protein of K562 cells was extracted by 2D-DIGE and then imaged by SDS gel scanning. The differentially expressed proteins were identified using Imagine Master 2D Platinum 6.0 software and functionally classified by MALDI and MALDI-TOF MS. RESULTS: 6-gingerol could significantly inhibit K562 cells proliferation and the efficacy was concentration and dose-dependent. After being treated for 24, 48, 72 h, IC50 was 22.86, 15.75, 11.18 microg/mL, respectively. 42 differentially expressed proteins were identified, including 19 up-regulated expressed proteins and 10 down-regulated expressed proteins. CONCLUSION: Proteomic technique can be used to screen multiple proteins associate with the anti-leukemia effect of 6-gingerol, involving some important proteins related to oxidative stress, cell cycle regulation, apoptosis signal transduction, biosynthesis and glycometabolism.

[Reactive oxygen species and mitochondrial membrane potential changes in leukemia cells during 6-gingerol induced apoptosis].

OBJECTIVE: To observe the effects of 6-gingerol on reactive oxygen species (ROS) and mitochondrial membrane potential(deltapsim) of chronic myeloid leukemia K562 cells and human acute T lymphoblastic leukemia MOLT4 cells, to investigate the role of mitochondrial pathway in the signal transduction of leukemia cell. METHODS: With different concentrations of 6-gingerol treatment, using 2,7-dichloro fluoresceinciactate (DCFH-DA) as ROS probe, rhodamine-123 as deltapsim probe, the levels of ROS and deltapsim of K562 cells and MOLT4 cells were tested by flow cytomentry. RESULTS: After treated with 6-gingerol, the ROS levels of K562 cells were significantly higher than control group (P < 0.01), while the deltapsim were significantly lower than control group (P < 0.01), and the ROS levels of MOLT4 cells were significantly higher than control group (P < 0.05). CONCLUSIONS: 6-gingerol can significantly increase ROS levels of K562 cells and MOLT4 cells, decrease deltapsim of K562 cells,induce apoptosis of leukemia cells by mitochondrial pathway.

Cyanogenesis in Macadamia and Direct Analysis of Hydrogen Cyanide in Macadamia Flowers, Leaves, Husks, and Nuts Using Selected Ion Flow Tube-Mass Spectrometry.

Macadamia has increasing commercial importance in the food, cosmetics, and pharmaceutical industries. However, the toxic compound hydrogen cyanide (HCN) released from the hydrolysis of cyanogenic compounds in Macadamia causes a safety risk. In this study, optimum conditions for the maximum release of HCN from Macadamia were evaluated. Direct headspace analysis of HCN above Macadamia plant parts (flower, leaves, nuts, and husks) was carried out using selected ion flow tube-mass spectrometry (SIFT-MS). The cyanogenic glycoside dhurrin and total cyanide in the extracts were analyzed using HPLC-MS and UV-vis spectrophotometer, respectively. HCN released in the headspace was at a maximum when Macadamia samples were treated with pH 7 buffer solution and heated at 50 °C for 60 min. Correspondingly, treatment of Macadamia samples under these conditions resulted in 93%-100% removal of dhurrin and 81%-91% removal of total cyanide in the sample extracts. Hydrolysis of cyanogenic glucosides followed a first-order reaction with respect to HCN production where cyanogenesis is principally induced by pH changes initiating enzymatic hydrolysis rather than thermally induced reactions. The effective processing of different Macadamia plant parts is important and beneficial for the safe production and utilization of Macadamia-based products.

Alliin alters gut microbiota and gene expression of colonic epithelial tissues.

Alliin is a natural organosulfur-containing phytochemical in garlic. It is possible that alliin can regulate the gut microbiota for its strong antimicrobial activity against many pathogens. Here, we assessed whether alliin impacts the distal small intestinal bacteria, hence the cecal microbiota, thus altering the gene expression of colonic epithelial tissues (CETs). Eighty mg/kg alliin was orally administered to rats for 14 days, and the 16S rDNA from small intestinal and cecal microbiota as well as mRNA from CETs were sequenced and analyzed. The results showed that alliin consumption affected microbiota composition in both the small intestine and cecum, although there was only one specific genus, Allobaculum that was significantly altered in the rat cecum. The altered composition of microbiota indirectly impacted 174 genes in the CETs. Specifically, five genes, including RT1-Ba, RT1-Bb, Cd80, Madcam1, and Aicda, indicated this consumption related to the intestinal immune network for IgA production. PRACTICAL APPLICATIONS: We firstly reported alliin consumption in vivo potentially affected the intestinal immunity of healthy rats by slightly alteration of microbiota composition in small intestine and cecum. The alteration subsequently amplified, resulting in the change of the colonic epithelial expression of several genes related to the intestinal immune network for IgA production. Hence, we suggested the alliin consumption may potentially affect the immune system of healthy individuals by alteration of gut microbiota and epithelial gene expression.

Analysis of anti-platelet aggregation components of Rhizoma Zingiberis using chicken thrombocyte extract and high performance liquid chromatography.

BACKGROUND: The conventional procedure for screening bioactive components from traditional Chinese medicine is time-consuming, expensive and low efficient. Therefore, some alternative strategies are needed urgently. A novel method for screening anti-platelet aggregation components from oleoresins was developed using chicken thrombocyte extract and high performance liquid chromatography. METHODS: The anti-platelet aggregation components of oleoresins were combined with receptors, channels and enzymes of chicken thrombocytes under physiological environment. Unbound substances were washed away and bound compounds were eluted using specific phosphate buffered solution (PBS). Compounds released from target sites were collected and analyzed by high performance liquid chromatography and LC-MS. The activity of three compounds which were screened from this model was confirmed using platelet aggregation pharmacology in vivo. RESULTS: There were four typical compounds that bound to the thrombocytes: 6-gingerol, 8-gingerol, 6-shogaol and 10-gingerol, and all had shown anti-platelet aggregation activities. Eight-gingerol displayed the best anti-platelet aggregation effect. CONCLUSIONS: Chicken thrombocyte extract can be used to isolate chemicals that are ligands of the receptor or other bio-targets on the platelet. This may therefore be a simple and efficient method to screen for anti-platelet aggregation compounds from traditional Chinese medicine.

A subset of esophageal squamous cell carcinoma patient-derived xenografts respond to cetuximab, which is predicted by high EGFR expression and amplification.

BACKGROUND: Epidermal growth factor receptor (EGFR) is reportedly overexpressed in most esophageal tumors, but most targeted therapies showed no efficacy in non-selected patients. This study aims at investigating the adaptive cetuximab subset in a cohort of esophageal squamous cell carcinoma (ESCC) patient-derived xenografts (PDXs). METHODS: A large panel of ESCC PDXs has been established. The copy number, mRNA expression and immunohistochemistry (IHC) of key EGFR pathways have been examined along with cetuximab response. A preclinical trial on a randomly selected cohort of 16 ESCC PDXs was conducted, and the genomic annotations of these models were compared against the efficacy readout of the mouse trial. RESULTS: The trial identified that 7 of 16 (43.8%) responded to cetuximab (ΔT/ΔC <0 as responders). The gene amplification and expression analysis indicated that EGFR copy number ≥5 (P=0.035), high EGFR mRNA expression (P=0.001) and IHC score of 2-3 (P=0.034) are associated with tumor growth inhibition by cetuximab, suggesting EGFR may function as a single predictive biomarker for cetuximab response in ESCC. CONCLUSIONS: Overall, our results suggest that an ESCC subtype with EGFR amplification and overexpression benefits from cetuximab treatment, which warrants further clinical confirmation.

Protection against oxidative stress in diabetic rats by wheat bran feruloyl oligosaccharides.

Diabetes is one of the most costly of the chronic diseases and is increasing in epidemic proportions in developing countries. It has been found that some antioxidants play a role in protection against oxidative stress, which is associated with diabetes. In this study, enzyme-released feruloyl oligosaccharides from wheat bran were given intragastrically (ig) to test their effect on antioxidant capacity, body weight restoring capacity, and serum glucose level in alloxan-induced diabetic Sprague-Dawley (SD) rats, using sodium ferulate and vitamin C as positive control groups. The levels of blood glucose, total antioxidant capacity (TAOC), and malondiadehyde (MDA) and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and xanthine oxidase (XOD) were determined in rat serum, liver, and testes. Feruloyl oligosaccharides significantly increased TAOC level, GSH-Px, and SOD activities, but decreased blood glucose and MDA levels and XOD activity in serum, liver, and testes of diabetic rats compared to diabetic controls. Feruloyl oligosaccharides were, overall, more efficient in mitigating oxidative damage in diabetic rats than sodium ferulate and vitamin C. In this feruloyl oligosaccharide feeding study, the antioxidant restoring capacity varied across the tissues observed, and also the activity change of the various antioxidant enzymes varied within a single tissue. Feruloyl oligosaccharides showed greater antioxidant capacity in vivo than in vitro when compared with vitamin C.

Thermolysis kinetics and thermal degradation compounds of alliin.

To investigate thermolysis kinetics and identify degradation compounds, alliin solutions were heated at 60, 80, and 89°C. The degradation compounds of alliin were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS) and ultra-pressure liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). The results showed that the thermal degradation kinetic of alliin could be described by a first-order reaction and k=4.38×1017exp (-142494/RT), where k is the reaction rate constant, min-1; R is gas constant; T is the absolute temperature, K. Degraded compounds, including S-allyl-l-cysteine and ethers, such as allyl alanine disulfide, allyl alanine trisulfide, allyl alanine tetrasulfide, dialanine disulfide (cysteine), dialanine trisulfide and dialanine tetrasulfide, were identified by HPLC-MS, MS/MS and UPLC-HRMS. Allyl alanine tetrasulfide was identified for the first time in alliin. The results show that alliin is unstable and significant numbers of organosulfur compounds are generated under high temperature treatment.

Immunomodulatory Activity of Polysaccharide-Protein Complex from the Mushroom Sclerotia of Polyporus rhinocerus in Murine Macrophages.

A novel water-soluble polysaccharide-protein complex (PRW1) isolated from the sclerotia of an edible mushroom Polyporus rhinocerus which was purified by membrane ultrafiltration could significantly activate murine macrophages RAW264.7 in vitro. PRW1 had a molecular weight of less than 50 kDa and was found to be a highly branched heteropolysaccharide-protein complex composed of 45.7 ± 0.97% polysaccharide and 44.2 ± 0.41% protein. Based on the results of total acid hydrolysis, methylation analysis, and Fourier transform infrared spectroscopy, the carbohydrate moiety of PRW1 was found to be a ß-d-mannoglucan with its backbone containing →1)-d-Glcp-(4→, →1)-d-Glcp-(6→, and →1)-d-Manp-(2→ residues (molar ratio of 5:4:6) and having terminal d-Glcp as side chain (degree of branching of 0.62). In vitro studies showed that PRW1 significantly induced NO production and enhanced the release of a variety of cytokines including G-CSF, GM-CSF, IL-6, IL12p40/70, MCP-1, MCP-5, MIP-1-α, MIP-2, RANTES, sTNFRI, and TNF-α. Mechanistically, PRW1 treatment triggered ERK phosphorylation to activate macrophages within 15 min and significantly increased the expression level of inducible NOS after 6 h. In summary, this study indicates that PRW1 derived from the sclerotia of P. rhinocerus is a potential immunomodulatory agent for cancer immunotherapy.

PARP1 is a novel independent prognostic factor for the poor prognosis of chordoma.

OBJECTIVE: To evaluate the expression of PARP1 in chordoma and analyzed its association with clinical factors and patients' prognosis. METHODS: The expression of Poly (ADP-ribose) polymerase 1 (PARP1) in chordoma specimens from 74 chordoma patients (50 primary and 24 recurrent tumors of 50 patients)and 20 distant normal tissue specimens was measured by immunohistochemical staining. The association of PARP1with the clinical factors and patients' prognosis was also analyzed. RESULTS: Of all the chordoma samples, 78% showed high expression of PARP1, whereas, only 10% of distant normal tissues expressed a high level of PARP1 (p< 0.01). Chi-square analysis revealed that high expression of PARP1 was significantly correlated with tumor recurrence (p< 0.01) and invasion into surrounding muscle (p< 0.01), while the data did not indicate any association with patients' gender, age, tumor location and size (p> 0.05). Kaplan-Meier survival curve and log-rank test showed that continuous disease free survival time (CDFS) was significantly shorter in the PARP1-positive group than in the PARP1-negative group (P= 0.019). CONCLUSION: High expression of PARP1 is significantly associated with chordoma invasion and recurrence. PARP1 may become a potential biomarker for chordoma in predicting its recurrence and patients' prognosis.

Cetuximab response in CRC patient-derived xenografts seems predicted by an expression based RAS pathway signature.

Cetuximab is an approved treatment for metastatic colorectal carcinoma (mCRC) with codon 12/13-KRAS mutations, recently questioned for its validity, and alternative mutation-based biomarkers were proposed. We set out to investigate whether an expression signature can also predict response by utilizing a cetuximab mouse clinical trial (MCT) dataset on a cohort of 25 randomly selected EGFR+ CRC patient-derived xenografts (PDXs). While we found that the expression of EGFR and its ligands are not predictive of the cetuximab response, we tested a published RAS pathway signature, a 147-gene expression signature proposed to describe RAS pathway activity, against this MCT dataset. Interestingly, our study showed that the observed cetuximab activity has a strong correlation with the RAS pathway signature score, which was also demonstrated to have a certain degree of correlation with a historic clinical dataset. Altogether, the independent validations in unrelated datasets from independent cohort of CRCs strongly suggest that RAS pathway signature may be a relevant expression signature predictive of CRC response to cetuximab. Our data seem to suggest that an mRNA expressing signature may also be developed as a predictive biomarker for drug response, similarly to genetic mutations.

A set of defined oncogenic mutation alleles seems to better predict the response to cetuximab in CRC patient-derived xenograft than KRAS 12/13 mutations.

Cetuximab is a standard of care for treating EGFR-expressing metastatic colorectal carcinoma (mCRC) exclusive of those with KRAS mutations at codons 12/13. However, retrospective analysis has recently suggested that KRAS-G13D patients can still benefit, while only a fraction of KRAS wild-type patients can benefit, from the treatment. We set out to test this contradicting issue experimentally in an independent cohort of patient derived xenograft (PDX) diseases. We conducted a mouse clinical trial (MCT) enrolling a random cohort of 27 transcriptome sequenced CRC-PDXs to evaluate cetuximab activity. The treatment responses were analyzed against the KRAS 12/13 mutation alleles, as well as several other well-known oncogenic alleles. If the response is defined by >80% tumor growth inhibition, 8/27 PDXs (~30%) are responders versus 19/27 non-/partial responders (~70%). We found that indeed there are no significantly fewer KRAS-12/13-allele responders (4/8 or 50%) than non-/partial responders (7/19, or 37%). In particular, there are actually no fewer G13D responders (4/8, or 50%) than in non-/partial responders (2/19 or 10.5%) statistically. Furthermore, majority of the non-/partial responders tend to have certain activating oncogenic alleles (one or more of the following common ones: K/N-RAS-G12V/D, -A146T, -Q61H/R, BRAF-V600E, AKT1-L52R and PIK3CA-E545G/K). Our data on an independent cohort support the recent clinical observation, but against the current practiced patient stratification in the cetuximab CRC treatment. Meanwhile, our data seem to suggest that a set of the six-oncogenic alleles may be of better predictive value than the current practiced stratification, justifying a new prospective clinical investigation on an independent cohort for confirmation.