M2 Pyruvate Research Articles

Drug pair of Cornus officinalis and Radix achyranthis bidentatae improves renal injury of hypertension by regulating metabolic reprogramming mediated by eNOS

ObjectiveTo explore the effects and possible mechanisms of the drug pair Cornus officinalis and Radix achyranthis bidentatae (SYR-NX) on improving hypertensive kidney damage. MethodSYR-NX, a formulation of Cornus officinalis and Radix Achyranthis Bidentatae with a dose ratio 1:2.5, was used in this experiment. We investigated the effects of SYR-NX on spontaneously hypertensive rats (SHR) fed with a high-salt diet and Human Kidney-2 (HK2) cells exposed to hypoxia. After 8 weeks of treatment with SYR-NX, blood pressure was tested, and β 2-Microglobulin(β2-MG), blood creatinine (S-cr), endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate (NADPH), M2 pyruvate kinase (PKM2), adenosine triphosphate (ATP), pyruvate, lactate, connective tissue growth factor (CTGF) and tumor necrosis factor-α (TNF-α)were measured. HK2 cells pre-treated with SYR-NX were cultured in a three-gas hypoxic incubator chamber (5 % CO2, 1 % O2, 94 % N2) for 12 h, and then eNOS, PKM2, NADPH, ATP, pyruvate, lactate, CTGF and TNF-α were assessed. ResultsSYR-NX significantly reduced SBP, DBP, β2-MG, S-cr, PKM2, pyruvate, lactate, CTGF and TNF-α, and increased eNOS, NADPH, and ATP. ConclusionSYR-NX can regulate metabolic reprogramming through eNOS and improves hypertensive kidney injury.

Icariin inhibits apoptosis in OGD-induced neurons by regulating M2 pyruvate kinase

BackgroundIschaemic stroke can lead to many complications, but treatment options are limited. Icariin is a traditional Chinese medicine with reported neuroprotective effects against ischaemic cerebral injury; however, the underlying mechanisms by which icariin ameliorates cell apoptosis require further study. PurposeThis study aimed to investigate the therapeutic potential of icariin after ischaemic stroke and the underlying molecular mechanisms. MethodsN2a neuronal cells were used to create an in vitro oxygen-glucose deprivation (OGD) model. The effects of icariin on OGD cells were assessed using the CCK-8 kit to detect the survival of cells and based on the concentration, apoptosis markers, inflammation markers, and M2 pyruvate kinase isoenzyme (PKM2) expression were detected using western blotting, RT-qPCR, and flow cytometry. To investigate the underlying molecular mechanisms, we used the PKM2 agonist TEPP-46 and detected apoptosis-related proteins. ResultsWe demonstrated that icariin alleviated OGD-induced apoptosis in vitro. The expression levels of the apoptosis marker proteins caspase-3 and Bax were upregulated and Bcl-2 was downregulated. Furthermore, icariin reduced inflammation and downregulated the expression of PKM2. Moreover, activation of the PKM2 by pretreatment with the PKM2 agonist TEPP-46 enhanced the effects on OGD induced cell apoptosis in vitro. ConclusionThis study elucidated the underlying mechanism of PKM2 in OGD-induced cell apoptosis and highlighted the potential of icariin in the treatment of ischaemic stroke.

RBMX overexpression inhibits proliferation, migration, invasion and glycolysis of human bladder cancer cells by downregulating PKM2

To investigate the role of RNA-binding motif protein X-linked (RBMX) in regulating the proliferation, migration, invasion and glycolysis in human bladder cancer cells. A lentivirus vectors system and RNA interference technique were used to construct bladder cancer 1376 and UC-3 cell models with RBMX overexpression and knockdown, respectively, and successful cell modeling was verified using RT-qPCR and Western blotting. Proliferation and colony forming ability of the cells were evaluated using EdU assay and colony-forming assay, and cell migration and invasion abilities were determined using Transwell experiment. The expressions of glycolysis-related proteins M1 pyruvate kinase (PKM1) and M2 pyruvate kinase (PKM2) were detected using Western blotting. The effects of RBMX overexpression and knockdown on glycolysis in the bladder cancer cells were assessed using glucose and lactic acid detection kits. RT-qPCR and Western blotting confirmed successful construction of 1376 and UC-3 cell models with RBMX overexpression and knockdown. RBMX overexpression significantly inhibited the proliferation, clone formation, migration and invasion of bladder cancer cells, while RBMX knockdown produced the opposite effects. Western blotting results showed that RBMX overexpression increased the expression of PKM1 and decreased the expression of PKM2, while RBMX knockdown produced the opposite effects. Glucose consumption and lactate production levels were significantly lowered in the cells with RBMX overexpression (P < 0.05) but increased significantly following RBMX knockdown (P < 0.05). RBMX overexpression inhibits bladder cancer progression and lowers glycolysis level in bladder cancer cells by downregulating PKM2 expression, suggesting the potential of RBMX as a molecular target for diagnosis and treatment of bladder cancer.

Association of fecal calprotectin and M2 pyruvate kinase with fecal pathogenic microorganisms in gastrointestinal infection

Objective: To determine the association of Fecal Calprotectin (f-CP) and M2 Pyruvate Kinase (M2-PK) with intestinal pathogens by means of the gastrointestinal syndromic panel. Methodology: This retrospective analytic study was conducted in the laboratory department a private hospital in Jakarta from October 2021 to September 2022. The inclusion criteria comprised patients aged ≥18 years with gastrointestinal complaints who had undergone f-CP and M2-PK examinations and gastrointestinal syndromic testing. The exclusion criteria were incomplete data on laboratory examinations, follow-up examinations or treatment evaluations. We used the Mann-Whitney test with p&lt;0.05 indicating significantly different. Results: Out of 282 subjects, 69% were women and 31% men, aged 18 to 84 years (mean 50.52±15.20). In 117 subjects, single pathogen was found in 78 specimens and ≥2 pathogens in 39. Median f-CP was 77.1 µg/g (3.68-2100) and median M2-PK 3.55 U/mL (1-72.6). Mann-Whitney test found a significant association between f-CP and intestinal pathogens (p=0.048), whereas for the M2-PK, no significant association was found with intestinal pathogens a(p=0.381). Conclusion: f-CP increases in gastrointestinal infection with intestinal pathogens, but M2-PK does not increase. Therefore, f-CP can be considered a biomarker for gastrointestinal infections caused by microorganisms.

Treponema pallidum recombinant protein Tp47 activates NOD-like receptor family protein 3 inflammasomes in macrophages via glycolysis

Glycolysis is a key pathway in cellular glucose metabolism for energy supply and regulates immune cell activation. Whether glycolysis is involved in the activation of NOD-like receptor family protein 3 (NLRP3) inflammasomes during Treponema pallidum (T. pallidum) infection is unclear. In this study, the effect of T. pallidum membrane protein Tp47 on NLRP3 inflammasome activation in rabbit peritoneal macrophages was analysed and the role of glycolysis in NLRP3 inflammasome activation was explored. The results showed that Tp47 promoted NLRP3, caspase-1, and IL-1β mRNA expression in macrophages, enhanced glycolysis and glycolytic capacity of macrophage, and promoted the production of macrophage glycolytic metabolites citrate, phosphoenolpyruvate, and lactate. The M2 pyruvate kinase (PKM2) inhibitor shikonin down-regulated the Tp47-promoted NLRP3, caspase-1, and IL-1β mRNA expression in macrophages, and suppressed the Tp47-enhanced glycolysis and glycolytic capacity. Similarly, si-PKM2 significantly inhibited Tp47-promoted NLRP3, caspase-1, and IL-1β mRNA expression and the Tp47-enhanced glycolysis and glycolytic capacity in macrophages. In conclusion, Tp47 activated NLRP3 inflammasomes via PKM2-dependent glycolysis and provided a new perspective on the effect of T. pallidum infection on host macrophages, which would contribute to the understanding of the infection mechanism and host immune mechanism of T. pallidum.

Point-of-care isoenzyme type M2 pyruvate kinase quick stool test for the detection of gastric cancer in Rwanda: A pilot study.

e18530 Background: Isoenzyme type M2 pyruvate kinase (M2-PK) can only be detected in gastrointestinal (GI) tumors. Detection of M2-PK on a small, portable platform with minimal training required, could offer a streamlined approach for the diagnosis of GI cancers especially in low-income countries (LIC's). We evaluated the feasibility of M2-PK stool test to detect gastric cancer in Rwanda. Methods: Participants with GI symptoms were screened and scheduled for upper endoscopy during the 5th Annual Rwanda Endoscopy Week. Participants who underwent gastric biopsy and provided a stool sample were enrolled. Three Rwandan medical centers participated. ScheBo M2-PK Quick test (Biotech AG, Giessen, Germany) was used to test fecal tumor M2-PK. Study approval was obtained from Dartmouth Health, UConn Health, and University of Rwanda IRB's. Descriptive statistics were estimated. Study is currently enrolling patients to reach the desired sample size (N = 110). Results: A total of 35 patients were consented; 26 patients did not have a biopsy, 1 did not provide a stool sample, 1 was lost to follow-up, and 7 were analyzable. 57.1% were male (n = 4) and 42.9% females (n = 3) with ages ranging from 50 to 71 years old. Males accounted for 66.6% of the cases of gastric cancer (n = 2). The most predominant symptoms reported were dyspepsia (66.7%), and weight loss (33.3%). Among those with hemoglobin levels available the mean was 8.63 g/dL (SD ± 0.71). Only one patient completed staging imaging with CT scans. Two patients with positive M2-PK stool tests had a proven diagnosis of gastric cancer (100%). One of them had a diagnosis of GIST. There was only 1 confirmed gastric adenocarcinoma in 5 patients with a negative M2-PK stool test, yet two patients were told to repeat the endoscopy procedure. Conclusions: A point-of-care (POC) quick stool test to screen gastric cancer is feasible in Rwanda. Study participation interest was high among Rwandan patients. Due to a lack of flexible endoscopes in LIC's, screening is restricted to patients living near regional or urban endoscopy centers, which contributes to the growing prevalence of gastric cancer and health disparities. Cancer control programs in LIC's can be implemented and maintained iteratively with realistic expectations and interventions tailored to the local population, its cultural values, and economic infrastructure. Thus, an emerging POC M2-PK stool test can triage patients to early medical attention when suspected gastric cancer.

Abstract #1404234: Hypoglycemia & Lactic Acidosis Due to Warburg Effect

Warburg effect (Aerobic Glycolysis) is a form of cellular adaptation by cancer cells to support its biosynthetic requirement, when in the presence of abundant oxygen and mitochondria, cell utilizes anaerobic pathway to convert glucose to lactate producing only 2 ATP’s. This phenomenon has been studied extensively and clinically can results in high glucose uptake by the tumor cells causing hypoglycemia and type B lactic acidosis. 41-year-old male with no PMHx admitted with weakness, instability, vision changes and unintentional 30 lbs weight loss x 3 months. In the ER he was found to be have BP 90/60, HR 113 and afebrile with Glucose of 20, lab showed AKI with lactic acidosis (Cr. 2.17 and lactic acid >10) and CT Abd/Plv showed multiple liver metastatic disease and entrocolitis. Started on IV fluids with dextrose, Pressor support, Antibiotics and admitted to ICU for care. Workup showed HIV positive and antibiotics coverage was broadened. Kidney function continued to worsen and started on CRRT. Patient’s hypoglycemia persisted despite dextrose infusion and endocrine team was consulted. Work up showed appropriately low insulin & c-peptide with negative sulfonylurea screen. Biopsy of liver confirmed Large B-Cell Lymphoma, but not a candidate for therapy due to acute illness. Patient lactic acidosis persisted >10 despite therapy suggestive of Type B lactic acidosis. Family changed goals of care & patient passed away within 48 hours of comfort care measures. Presence of hypoglycemia and lactic acidosis is a medical emergency suggesting hyper metabolic state with poor tissue oxygenation, mostly caused by severe inflammatory resulting in Type A Lactic Acidosis. Rarely this is seen in normoxic condition called Type B Lactic acidosis and one of the potential causes is Warburg effects, in patients with solid and hematological malignancy. This is triggered by oncogenic lesions such as expression of hypoxia-inducible factor-1α (HIF1α) causing up regulation of major glycolytic enzymes like hexokinase and Tumor M2 Pyruvate Kinase. This provides cancer cell with metabolic intermediates required for synthesis of cellular components for cell proliferation and also produces less amounts of free radicals. Proliferating cancer cells also outpace the vascular supply and warburg effect ensures survival in the hypoxic environment. Patients with type B lactic acidosis and hypoglycemia poses grim prognosis with mortality around 75% (7 out of 29 patient survivals, in chart review of cases). Therapeutic measure includes: tumor directed therapies, IV Bicarb Infusion, IV Dextrose, Renal Replacement Therapy and Thiamine replacement. Purpose of this case presentation is to further enhance clinical evidence of Warburg effect.

MiR-133b Promotes Esophageal Squamous Cell Carcinoma Metastasis.

Evaluation of biological changes at the molecular level has important clinical implications for improving the survival rate of esophageal squamous cell carcinoma (ESCC). Therefore, we plan to analyze and elucidate the expression of microRNA-133b (miR-133b), M2 pyruvate kinase (PKM2), and signal transducer and activator of transcription 3 (STAT3) in ESCC and their associated clinicopathological significance. The 72 patients with ESCC were selected as the experimental study group. Normal adjacent tissues (NAT) were matched as the control group. In this study, in situ hybridization was used to detect the expression of miR-133b in ESCC, and tissue expressions of PKM2 and STAT3 were detected by immunohistochemistry, and literature review was conducted. Studies had shown that the positive expression of miR-133b in NAT was significantly higher than that in ESCC (χ2 = 9.007, P = .003). PKM2 and STAT3 in ESCC had a significantly higher positive expression levels than those of NAT (χ2 = 56.523, P = .000; χ2 = 72.939, P = .000). From correlation analysis, there was a negative correlation between miR-133b and PKM2(r = -0.515, P < .001), a negative correlation between miR-133b and STAT3(r = -0.314, P = .007), and a positive correlation between PKM2 and STAT3(r = 0.771, P < .001). In ESCC, our study demonstrated that downregulation of miR-133b and upregulation of PKM2 and STAT3. We predict that miR-133b may inhibit the STAT3 pathway by downregulating PKM2.

ASO-Based PKM Splice-Switching Therapy Inhibits Hepatocellular Carcinoma Growth.

Antisense oligonucleotides are used to induce a change in PKM isoform usage in hepatocellular carcinoma, reversing the Warburg effect and inhibiting tumorigenesis.

Norisoboldine Attenuates Sepsis-Induced Acute Lung Injury by Modulating Macrophage Polarization via PKM2/HIF-1α/PGC-1α Pathway.

This study aimed to investigate the effect of norisopoldine (NOR) on acute lung injury in septic mice. Lipopolysaccharide (LPS) was used to establish sepsis induced acute lung injury (ALI) in mice. The dry and wet weight of mice lung was detected, and the pathological changes of lung were observed by hematoxylin and eosin (H&E) staining. Bronchoalveolar lavage fluid (BALF) was detected. Inflammatory factors in BALF were detected by enzyme-linked immunosorbent assay (ELISA). The polarization of macrophages in lung tissue was detected by flow cytometry. The markers of M1 and M2 macrophages were detected by RT-PCR. LPS induced RAW264.7 cells were treated with NOR. Inflammatory response, macrophage polarization, glycolysis, and M2 pyruvate kinase (PKM2)/hypoxia inducible factor-1α (HIF-1α)/peroxisome proliferator activated receptor-γ co-activator 1-α (PGC-1α) signaling pathway were detected. NOR could effectively alleviate sepsis induced ALI, and reduce the number of total cells, total protein concentration, neutrophils, macrophages in BALF. NOR decreased the level of inflammatory factors and promoted macrophages from M1 to M2 type in vivo and vitro. Moreover, NOR could activated PKM2, and inhibited PKM2 from cytoplasm to nuclear, attenuated HIF-1α expression, and increased PGC-1α and peroxisome proliferator-activated receptor (PPAR)-γ expression. In addition, NOR inhibited glycolysis and promoted oxidative phosphorylation in RAW264.7 cells. Furthermore, PKM2 inhibitors could reverse the effect of NOR on PKM2/HIF-1α/PGC-1α signaling pathway in RAW264.7 cells. NOR alleviated sepsis induced AIL in mice, inhibited the inflammatory response, promote M2 polarization of macrophages through regulating PKM2/HIF-1α/PGC-1α signaling pathway.

Biomarkers in chronic inflammatory bowel disease - present and future

Inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohn's disease (CD) are complex diseases that require a complex approach in the diagnosis, treatment and monitoring of the effect of the applied therapy. To achieve these goals, in addition to classical invasive еndoscopies, various non-invasive biomarkers are applied. A review of the used classical (ESR, C-reactive protein, lactoferrin, fecal calprotectin, M2 pyruvate kinase, myeloperoxidase) and new biomarkers (human beta-defensin-2, matrix metalloproteinases, human nucleic acid (DNA and miRNA)) was done, highlighting their advantages and disadvantages.

Inhibition of the ketolytic acetyl CoA supply to tumors could be their “Achilles heel”

Inhibition of the ketolytic acetyl <scp>CoA</scp> supply to tumors could be their “Achilles heel”

Diagnostic Value of Pyruvate Kinase Isoenzyme Type M2 in Colon Cancer Proven with Colonoscopy.

Background:Colonoscopy is the gold standard for colon cancer screening; it is also associated with a high cost and complication. Proliferating cells, in particular tumor cells, express a dimeric isoenzyme of pyruvate kinase, termed M2 pyruvate kinase (M2-PK). The aim of this study was to determine the diagnostic accuracy of fecal M2-PK for colon cancer.Materials and Methods:Forty-nine patients with colon cancers and 49 healthy controls were selected consecutively among individuals undergoing screening colonoscopy for various indications. The diagnosis was confirmed by histology. M2-PK measurements were done by enzyme-linked immunosorbent assay of fecal occult blood test (FOBT) and immunological FOBT (IFOBT) according to the manufacturer's instructions.Results:M2-PK > 9 (U/mL) was the best cutoff point in the detection of colon cancers. In this cutoff point, sensitivity and specificity were 87.8% and 91.8%, respectively, and accuracy was 89.8%. The sensitivity and specificity of IFOBT were 93.9% and 100%, respectively, and accuracy was 96.9%. The sensitivity and specificity of FOBT were 65.3% and 100%, respectively, and accuracy was 82.6%.Conclusion:IFOBT with high sensitivity and specificity and accuracy and low cost is the best fecal screening test. The current study suggests that fecal M2-PK can be used for high-risk colon cancer patients and negative IFOBT that refused colonoscopy as a precolonoscopy screening test.

The Use of Spectral Ellipsometry and Raman Spectroscopy in Screening Diagnostics of Colorectal Cancer

Diagnostic pilot monitoring of the groups of healthy patients and patients with colorectal cancer (CRC) at different stages of the disease was carried out using Raman spectroscopy (RS) and measuring the state of polarized light near the conditions of observation of surface plasmon resonance (SPR). The reaction of the blood serum antigens with antibodies to serum M2 pyruvate kinase (M2-PK) nears the conditions of observation of SPR was used for diagnosing; for this, the Ellipse-SPEC spectral ellipsometric complex created at the Rzhanov Institute of Semiconductor Physics (Siberian Branch, Russian Academy of Sciences) and having a high sensitivity, accuracy, and nondestructive nature of the effect on the studied sample, was used in the work. The peak intensities at 1005, 1157, and 1520 cm–1 in Raman spectra in CRC patients as compared with healthy individuals (as well as the intensity of surface plasmon resonance) were significantly lower, correlating with the stage of the disease and the presence of metastases; this allows to consider these optical methods for studying the blood serum as promising diagnostic approaches in diagnosis CRC, including in the early stages of the disease.

Differential Expression Patterns of Glycolytic Enzymes and Mitochondria-Dependent Apoptosis in PCOS Patients with Endometrial Hyperplasia, an Early Hallmark of Endometrial Cancer, In Vivo and the Impact of Metformin In Vitro.

The underlying mechanisms of polycystic ovarian syndrome (PCOS)-induced endometrial dysfunction are not fully understood, and although accumulating evidence shows that the use of metformin has beneficial effects in PCOS patients, the precise regulatory mechanisms of metformin on endometrial function under PCOS conditions have only been partially explored. To address these clinical challenges, this study aimed to assess the protein expression patterns of glycolytic enzymes, estrogen receptor (ER), and androgen receptor (AR) along with differences in mitochondria-dependent apoptosis in PCOS patients with and without endometrial hyperplasia in vivo and to investigate the effects of metformin in PCOS patients with endometrial hyperplasia in vitro. Here, we showed that compared to non-PCOS patients and PCOS patients without hyperplasia, the endometria from PCOS patients with hyperplasia had a distinct protein expression pattern of glycolytic enzymes, including pyruvate kinase isozyme M2 isoform (PKM2) and pyruvate dehydrogenase (PDH), and mitochondrial transcription factor A (TFAM). In PCOS patients with endometrial hyperplasia, increased glandular epithelial cell secretion and infiltrated stromal cells in the glands were associated with decreased PDH immunoreactivity in the epithelial cells. Using endometrial tissues from PCOS patients with hyperplasia, we found that in response to metformin treatment in vitro, hexokinase 2 (HK2) expression was decreased, whereas phosphofructokinase (PFK), PKM2, and lactate dehydrogenase A (LDHA) expression was increased compared to controls. Although there was no change in PDH expression, metformin treatment increased the expression of TFAM and cleaved caspase-3. Moreover, our in vivo study showed that while endometrial ERβ expression was no different between non-PCOS and PCOS patients regardless of whether or not hyperplasia was present, ERα and AR protein expression was gradually increased in women with PCOS following the onset of endometrial hyperplasia. Our in vitro study showed that treatment with metformin inhibited ERα expression without affecting ERβ expression. Our findings suggest that decreased glycolysis and increased mitochondrial activity might contribute to the onset of ERα-dependent endometrial hyperplasia and that metformin might directly reverse impaired glycolysis and normalize mitochondrial function in PCOS patients with endometrial hyperplasia.

An allostatic mechanism for M2 pyruvate kinase as an amino-acid sensor.

We have tested the effect of all 20 proteinogenic amino acids on the activity of the M2 isoenzyme of pyruvate kinase (M2PYK) and show that, within physiologically relevant concentrations, phenylalanine, alanine, tryptophan, methionine, valine, and proline act as inhibitors, while histidine and serine act as activators. Size exclusion chromatography has been used to show that all amino acids, whether activators or inhibitors, stabilise the tetrameric form of M2PYK. In the absence of amino-acid ligands an apparent tetramer–monomer dissociation Kd is estimated to be ∼0.9 µM with a slow dissociation rate (t1/2 ∼ 15 min). X-ray structures of M2PYK complexes with alanine, phenylalanine, and tryptophan show the M2PYK locked in an inactive T-state conformation, while activators lock the M2PYK tetramer in the active R-state conformation. Amino-acid binding in the allosteric pocket triggers rigid body rotations (11°) stabilising either T or R states. The opposing inhibitory and activating effects of the non-essential amino acids serine and alanine suggest that M2PYK could act as a rapid-response nutrient sensor to rebalance cellular metabolism. This competition at a single allosteric site between activators and inhibitors provides a novel regulatory mechanism by which M2PYK activity is finely tuned by the relative (but not absolute) concentrations of activator and inhibitor amino acids. Such ‘allostatic’ regulation may be important in metabolic reprogramming and influencing cell fate.

Native Mass Spectrometry Gives Insight into the Allosteric Binding Mechanism of M2 Pyruvate Kinase to Fructose-1,6-Bisphosphate.

The various oligomeric states of the M2 isoform of pyruvate kinase (PKM2) were distinguished using native mass spectrometry. The effect of PKM2 concentration on its dimer-tetramer equilibrium was monitored, and a value for the dissociation constant ( Kd) of the two species was estimated to be 0.95 μM. Results of binding of fructose-1,6-bisphosphate (FBP) to PKM2 are shown and provide insight into the allosteric mechanism and changes in the oligomerization status of PKM2. The average Kd for binding of FBP to the PKM2 tetramer was estimated to be 7.5 μM. It is concluded that four molecules of FBP bind to the active PKM2 tetramer whereas binding of FBP to the PKM2 dimer was not observed. It is suggested that either FBP potentiates rapid tetramer formation after binding to apo PKM2 dimers or FBP binds to PKM2 apo tetramers, thus driving the dimer-tetramer equilibrium in the direction of fully FBP-bound tetramer. The binding occurs in a highly positively cooperative manner with a Hill coefficient ( n) of 3.

Power of screening tests for colorectal cancer enhanced by high levels of M2-PK in addition to FOBT

Colorectal cancer (CRC) is a multistep process that involves adenoma-carcinoma sequence. CRC can be prevented by routine screening, which can detect precancerous lesions. The aim of this study is to clarify whether faecal occult blood test (i-FOBT), tumor M2 pyruvate kinase (t-M2-PK), and endocannabinoid system molecules (cannabinoid receptors type 1-CB1, type 2-CB2, and fatty acid amide hydrolase-FAAH) might represent better diagnostic tools, alone or in combination, for an early diagnosis of CRC. An immunochemical FOB test (i-FOBT) and quantitative ELISA stool test for t-M2-PK were performed in 127 consecutive patients during a 12month period. Endocannabinoid system molecules and t-M2-PK expression were detected by immunostaining in healthy tissues and normal mucosa surrounding adenomatous and cancerous colon lesions. i-FOBT and t-M2-PK combination leads to a better diagnostic accuracy for pre-neoplastic and neoplastic colon lesions. T-M2-PK quantification in stool samples and in biopsy samples (immunostaining) correlates with tumourigenesis stages. CB1 and CB2 are well expressed in healthy tissues, and their expression decreases in the presence of advanced stages of carcinogenesis and disappears in CRC. FAAH signal is well expressed in normal mucosa and low-risk adenoma, and increased in high-risk adenoma and carcinoma adjacent tissues. This study shows that high levels of t-M2-PK in addition to FOBT enhance the power of a CRC screening test. Endocannabinoid system molecule expression correlates with colon carcinogenesis stages. Developing future faecal tests for their quantification must be undertaken to obtain a more accurate early non-invasive diagnosis for CRC.

Accurate cut-offs for predicting endoscopic activity and mucosal healing in Crohn's disease with fecal calprotectin.

Fecal biomarkers, especially fecal calprotectin, are useful for predicting endoscopic activity in Crohn's disease; however, the cut-off point remains unclear. The aim of this paper was to analyze whether faecal calprotectin and M2 pyruvate kinase are good tools for generating highly accurate scores for the prediction of the state of endoscopic activity and mucosal healing. The simple endoscopic score for Crohn's disease and the Crohn's disease activity index was calculated for 71 patients diagnosed with Crohn's. Fecal calprotectin and M2-PK were measured by the enzyme-linked immunosorbent assay test. A fecal calprotectin cut-off concentration of ≥ 170 µg/g (sensitivity 77.6%, specificity 95.5% and likelihood ratio +17.06) predicts a high probability of endoscopic activity, and a fecal calprotectin cut-off of ≤ 71 µg/g (sensitivity 95.9%, specificity 52.3% and likelihood ratio -0.08) predicts a high probability of mucosal healing. Three clinical groups were identified according to the data obtained: endoscopic activity (calprotectin ≥ 170), mucosal healing (calprotectin ≤ 71) and uncertainty (71 > calprotectin < 170), with significant differences in endoscopic values (F = 26.407, p < 0.01). Clinical activity or remission modified the probabilities of presenting endoscopic activity (100% vs 89%) or mucosal healing (75% vs 87%) in the diagnostic scores generated. M2-PK was insufficiently accurate to determine scores. The highly accurate scores for fecal calprotectin provide a useful tool for interpreting the probabilities of presenting endoscopic activity or mucosal healing, and are valuable in the specific clinical context.

Profiling Proteins in the Hypothalamus and Hippocampus of a Rat Model of Premenstrual Syndrome Irritability.

Premenstrual syndrome (PMS) refers to several physical and mental symptoms (such as irritability) commonly encountered in clinical gynaecology. The incidence of PMS has been increasing, attracting greater attention from medical fields. However, PMS pathogenesis remains unclear. This study employed two-dimensional gel electrophoresis (2DE) for proteomic map analysis of the hypothalamus and hippocampus of rat models of premenstrual syndrome (PMS) irritability. Matrix-assisted laser desorption/ionisation time of flight mass spectroscopy (MALDI-TOF-MS) was used to identify proteins possibly related with PMS irritability. Baixiangdan, a traditional Chinese medicine effective against PMS irritability, was used in the rat model to study putative target proteins of this medicine. The hypothalamus and hippocampus of each group modelling PMS displayed the following features: decreased expression of Ulip2, tubulin beta chain 15, α actin, and interleukin 1 receptor accessory protein; increased expression of kappa-B motif-binding phosphoprotein; decreased expression of hydrolase at the end of ubiquitin carboxy, albumin, and aldolase protein; and increased expression of M2 pyruvate kinase, panthenol-cytochrome C reductase core protein I, and calcium-binding protein. Contrasting with previous studies, the current study identified new proteins related to PMS irritability. Our findings contribute to understanding the pathogenesis of PMS irritability and could provide a reference point for further studies.