BACKGROUND: Paediatric Huntington disease with highly expanded mutations (HE-PHD; >80 CAG repeats) presents atypically, compared to adult-onset Huntington disease (AOHD), with neurodevelopmental delay, epilepsy, abnormal brain glucose metabolism, early striatal damage, and reduced lifespan. Since genetic GLUT-1 deficiency syndrome shows a symptom spectrum similar to HE-PHD, we investigated the potential role of the two main glucose transporters, GLUT-1 and GLUT-3, in HE-PHD.
METHODS: We compared GLUT-1 and GLUT-3 protein expression in HE-PHD, juvenile-onset (JOHD), and AOHD brains (n = 2; n = 3; n = 6) and periphery (n = 3; n = 2; n = 2) versus healthy adult controls (n = 6; n = 6). We also investigated mitochondrial complexes and hexokinase-II protein expression.
RESULTS: GLUT-1 and GLUT-3 expression were significantly lower in HE-PHD frontal cortex (p = 0.009, 95% [CI 13.4, 14.7]; p = 0.017, 95% [CI 14.2, 14.5]) versus controls. In fibroblasts, GLUT-1 and GLUT-3 expression were lower compared to controls (p < 0.0001, 95% [CI 0.91, 1.09]; p = 0.046, 95% [CI 0.93, 1.07]). In the frontal cortex, this occurred without evidence of extensive neuronal degeneration. Patients with HE-PHD had deregulated mitochondrial complex expression, particularly complexes II-III, levels of which were lower in frontal cortex versus controls (p = 0.027, 95% [CI 17.1, 17.6]; p = 0.002, 95% CI [16.6, 16.9]) and patients with AOHD (p = 0.052, 95% [CI 17.0, 17.6]; p = 0.002, 95% [CI 16.6, 16.7]). Hexokinase-II expression was also lower in HE-PHD frontal cortex and striatum versus controls (p = 0.010, 95% [CI 17.8, 18.2]; p = 0.045, 95% [CI 18.6, 18.7]) and in frontal cortex versus patients with AOHD (p = 0.013, 95% [CI 17.7, 18.1]). Expression JOHD levels were consistently different to those of HE-PHD but similar to those of AOHD.
CONCLUSIONS: Our data suggest a dysfunctional hypometabolic state occurring specifically in paediatric Huntington disease brains.
BACKGROUND: \'5 × 1000\' Personal Income Tax donation to LIRH Foundation; Italian Ministry of HealthRC2301MH04 and RF-2016-02364123 to CSS.

OBJECTIVE: Limited research exists on translation of in-vitro glucose measurement interfering compounds to the in-vivo situation. We investigated whether Point-of-Care glucose measurements by Accu Chek Inform II (ACI II) were accurate to monitor glucose concentrations during surgery with general anesthesia by comparing with the reference laboratory hexokinase plasma glucose test.
METHODS: Patients undergoing surgery with general anesthesia were included. Anesthesia was maintained with either Sevoflurane or Total intravenous anesthesia (TIVA). Prior to and after induction, blood glucose was measured with ACI II and the hexokinase test. Bland-Altman analysis was performed to assess method agreement. Subgroup analyses on glucose measurement differences per type of maintenance anesthesia were performed.
RESULTS: Thirty-nine patients were included, and 78 measurements were performed. All paired measurements had clinically acceptable agreement with a percentage error of 10.0% (95% CI 8.0 to 11.9). The mean difference (95% limits of agreement) between ACI II and hexokinase for all measurements was 0.0 mmol/L (-0.7 to 0.7 mmol/L). Before induction (n = 39), mean difference was -0.1 mmol/L (-0.6 to 0.4 mmol/L), and after induction (n = 39), mean difference was 0.1 mmol/L (-0.8 to 0.9 mmol/L). Further investigation showed the difference varied per test for patients receiving Sevoflurane compared to patients receiving TIVA (-0.2 ± 0.4 mmol/L vs. 0.4 ± 0.3 mmol/L, p < 0.001). Before and after induction, the difference between ACI II and hexokinase measurements increased for patients receiving Sevoflurane compared to patients receiving TIVA (0.4 ± 0.4 mmol/L vs. -0.4 ± 0.3 mmol/L, p < 0.001).
CONCLUSIONS: The agreement between glucose measurements using ACI II and the reference laboratory hexokinase test was clinically acceptable with a percentage error of 10.0% (95% CI 8.0 to 11.9). The use of TIVA may negatively affect the measurement performance of the ACI II.

The Warburg effect is an important metabolic feature of tumours, and hexokinase is the first ratelimiting enzyme of the glycolytic pathway during tumour metabolism. Among hexokinase subtypes, hexokinase 2 (HK2) is increasingly proving to be a key target for cancer treatment. This study presents the challenges and potential strategies for developing HK2 inhibitors by systematically summarising the characteristics of HK2 inhibitors reported in the literature and patents.
In this study, we analysed the HK2 active site using molecular docking and evaluated the structure, biochemical and physiological function, activity, and action mechanism of reported HK2 inhibitors using databases (Science, SCI Finder, CNKI, and WANFANG DATA).
In total, 6 natural inhibitors of HK2, 9 synthetic inhibitors of HK2, and 3 compounds with patent-pending HK2 inhibitory effects were obtained by searching 87 articles. These inhibitors have poor efficacy and specificity when used alone and have numerous side effects; therefore, there is an urgent need to develop HK2 inhibitors with improved activity and high selectivity.
HK2 has received much attention in anticancer drug development, but most previous studies have focused on elucidating the action mechanism of HK2 in carcinogenesis, whereas the development of its small-molecule inhibitors has rarely been reported. In this study, we analysed and illustrated the eutectic structure of small molecules with the catalytic structural domain of HK2 to develop highly selective and low-toxicity HK2 inhibitors.

Type 2 diabetes mellitus (T2DM) is a major global public health issue. Diet and physical exercise are modifiable factors that influence the glycaemic status of patients with T2DM. We aimed to investigate the acute effects of breakfast fruits meal sequence and postprandial exercise on the blood glucose level and dipeptidyl peptidase 4 (DPP4) activity among type 2 diabetes mellitus patients.
A randomized pilot study recruited patients with T2DM who attended two primary health care centres in Tasikmadu District, Karanganyar Regency, and Kartasura District, Sukoharjo Regency, Central Java, Indonesia, from July to October 2016. Eligible patients (4 men and 32 women) were randomly divided into four treatment groups. Venous blood samples were analyzed for fasting and one-hour postprandial blood glucose (FBG and 1 h PPG) levels and DPP4 activity. Blood glucose levels were measured using a routine hexokinase method, and serum DPP4 activity was determined spectrophotometrically after incubation with the Gly-Pro-p-nitroanilide substrate.
Fruits last meal decreased FBG level whilst fruits first meal did not significantly decrease 1 h PPG level. Both treatments had no acute effects on DPP4 activity but the addition of postprandial exercise helped lower DPP4 activity. Fruit last and first meals showed significant opposite effects on mean changes of FBG level (p < 0.05).
This preliminary report of fruits meal sequence is potentially involved in acute regulation of blood glucose levels and that it might be independent of DPP4 activity in Indonesian patients with T2DM. Moreover, postprandial exercise may be an important intervention for T2DM through the mediation of DPP4 but has no acute effects on the regulation of blood glucose levels. Further studies are required to investigate whether or not different types of fruits and longer treatment intervals can affect blood glucose levels and DPP4 activity differently. This study also gives an insight into the feasibility of conducting food order modification with or without the combination of postprandial exercise in a primary health setting for our next studies.

The highly conserved Superfamily 1 (SF1) and Superfamily 2 (SF2) nucleic acid-dependent ATPases, are ubiquitous motor proteins with central roles in DNA and RNA metabolism (Jankowsky & Fairman, 2007). These enzymes require RNA or DNA binding to stimulate ATPase activity, and the conformational changes that result from this coupled behavior are linked to a multitude of processes that range from nucleic acid unwinding to the flipping of macromolecular switches (Pyle, 2008, 2011). Knowledge about the relative affinity of nucleic acid ligands is crucial for deducing mechanism and understanding biological function of these enzymes. Because enzymatic ATPase activity is directly coupled to RNA binding in these proteins, one can utilize their ATPase activity as a simple reporter system for monitoring functional binding of RNA or DNA to an SF1 or SF2 enzyme. In this way, one can rapidly assess the relative impact of mutations in the protein or the nucleic acid and obtain parameters that are useful for setting up more quantitative direct binding assays. Here, we describe a routine method for employing NADH-coupled enzymatic ATPase activity to obtain kinetic parameters reflecting apparent ATP and RNA binding to an SF2 helicase. First, we provide a protocol for calibrating an NADH-couple ATPase assay using the well-characterized ATPase enzyme hexokinase, which a simple ATPase enzyme that is not coupled with nucleic acid binding. We then provide a protocol for obtaining kinetic parameters (KmATP, Vmax and KmRNA) for an RNA-coupled ATPase enzyme, using the double-stranded RNA binding protein RIG-I as a case-study. These approaches are designed to provide investigators with a simple, rapid method for monitoring apparent RNA association with SF2 or SF1 helicases.

Glioblastoma (GBM) is the most common malignant primary brain tumor with a universally poor prognosis. GBMs express elevated levels of hexokinase 2 (HK2), catalyzing the critical step in glycolysis and influencing several oncogenic pathways. Previous preclinical work has suggested a role for repurposed posaconazole (PCZ) in downregulating HK2 activity, reducing lactate and pyruvate production, interfering with tumor cell metabolism, and increasing mouse survival.
To establish brain tumor penetrance, neuropharmacokinetic profile, and mechanistic effect on tumor cell metabolism of PCZ in adults with GBM.
This is an open label, nonrandomized, parallel arm trial involving patients with GBM. Cohorts will receive PCZ (intervention, n = 5) or will not receive PCZ (control, n = 5), followed by tumor resection and microdialysis catheter placement. Dialysate, plasma, and tumor samples will be analyzed for lactate and pyruvate concentrations. Tumor samples will also be assessed for PCZ concentration, HK2 expression, angiogenesis, and apoptosis. PCZ\'s neuropharmacokinetics will be determined based on the concentration vs time profile and area under the curve 0 to 24 hours of PCZ concentration in the brain interstitium.
(1) Increased PCZ concentration in contrast-enhancing brain regions compared with nonenhancing regions; (2) inverse correlation between lactate/pyruvate and PCZ concentrations in dialysate samples from treated patients, over time; and (3) decreased HK2 activity in PCZ-treated tumor samples.
A successful trial will support the decision to proceed to advanced phase trials. Any tumor penetration by PCZ, with concomitant effect on glycolysis, warrants further in-depth analysis, as therapeutic options for these deadly tumors are currently limited.

Breast cancer remains a leading cause of female mortality worldwide. Therefore, novel complementary treatments have been sought. Recently, there has been a growing interest in investigating the possible complementary effects of polyphenolic compounds against various malignancies. In the present study, using MCF-7 and MDA-MB-231 human breast adenocarcinoma cells, the anticancer efficacy of a polyphenolic mixture (PFM) was investigated. PFM is composed of curcumin, resveratrol, epigallocatechin gallate, and quercetin. PFM treatment led to a dose-dependent inhibition of cell proliferation, with IC50 values of 25.9 ± 3 µg/ml and 29.4 ± 0.9 µg/ml for MCF-7 and MDA-MB-231 cells, respectively. In addition, PFM induced apoptosis in MDA-MB-231 cells and cell cycle arrest at the S phase in MCF-7 cells. Using RT-qPCR, PFM treatment was observed to result in significant downregulation of the oncogenic miR-155 (P < 0.05), as well as significant downregulation of the rate-limiting glycolytic enzyme, hexokinase 2 (HK2) (P < 0.05), while upregulating the expression of the zinc finger E-box binding homeobox 2 gene (P < 0.01). PFM was also found to exert an anti-migration effect in breast cancer cells using the wound healing assay, as well as significantly (P < 0.05) increasing the median survival of Ehrlich ascites carcinoma (EAC) tumor-bearing mice. These results suggest that PFM possesses potential antitumor effects against breast cancer. A possible mechanism of action could be due to PFM\'s effect in modulating the expression of the glycolytic enzyme HK2 through suppression of miR-155 in MCF-7 cells. Combining polyphenolic compounds that interact with one another could result in synergistic effects that potentially target various tumour hallmarks.

Pesticides are extensively employed worldwide, especially in agriculture to control weeds, insect infestation and diseases. Besides their targets, pesticides can also affect the health of non-target organisms, including humans The present study was conducted to study the effect of oral exposure of thiram, a dithiocarbamate fungicide, on the intestine of rats. Male rats were administered thiram at doses of 100, 250, 500 and 750 mg/kg body weight for 4 days. This treatment reduced cellular glutathione, total sulfhydryl groups but enhanced protein carbonyl content and hydrogen peroxide levels. In addition, the activities of all major antioxidant enzymes (catalase, thioredoxin reductase, glutathione peroxidase and glutathione-S-transferase) except superoxide dismutase were decreased. The antioxidant power of the intestine was impaired lowering the metal-reducing and free radical quenching ability. Administration of thiram also led to inhibition of intestinal brush border membrane enzymes, alkaline phosphatase, γ-glutamyl transferase, leucine aminopeptidase and sucrase. Activities of enzymes of pentose phosphate pathway, citric acid cycle, glycolysis and gluconeogenesis were also inhibited. Histopathology showed extensive damage in the intestine of thiram-treated rats at higher doses. All the observed effects were in a thiram dose-dependent manner. The results of this study show that thiram causes significant oxidative damage in the rat intestine which is associated with the marked impairment in the antioxidant defense system.

Objective: To study the cytotoxicity and malignant transformation ability of chrysotile on MeT-5A cells. Methods: In June 2016, lactate dehydrogenase (LDH) method was used to detect the cytotoxicity of chrysotile to MeT-5A cells. MeT-5A cells were treated with 5 μg/cm(2) chrysotile intermittently for 24 h a time, once a week and a total of 28 times. After the cells showed anchorage independent growth, the cell features of malignant transformation were identified by colony forming frequency in soft agar, and the soft agar colony formation rates were calculated. The activities of key speed limiting enzymes of glycolysis metabolism including hexokinase (HK) , phosphofructokinase (PFK) and pyruvate kinase (PK) were determined by UV colorimetry. Results: Chrysotile was cytotoxic to MeT-5A cells in a concentration-dependent decline. Compared with the control group, the relative survival rates of MeT-5A cells were significantly decreased after exposed to chrysotile at 10, 20, 40 and 80 μg/cm(2) (P<0.05) . After 28 times of exposure, the growth rate of the cells in chrysotile transformed MeT-5A cells was accelerated, the arrangement was disordered, the contact inhibition was lost, and the double layer growth appeared, which could grow on soft agar. The colony forming rate of the chrysotile transformed MeT-5A cells was 18.33‰±2.49‰. Compared with the control group (0) , the difference was statistically significant (P<0.01) . The activities of glycolysis related kinase including PK [ (19.51±1.52) U/L], PFK[ (0.12±0.02) U/10(4) cell] and HK[ (0.26±0.01) U/10(4) cell] were increased in the chrysotile transformed MeT-5A cells compared with control group [ (25.00±1.04) U/L、(0.15±0.01) U/10(4) cell and (0.33±0.01) U/10(4) cell] (P<0.01) . Conclusion: Chrysotile can induce malignant transformation of MeT-5A cells and increase the activities of glycolysis related kinases including PK, PFK and HK.
目的： 研究温石棉对人胸膜间皮细胞MeT-5A细胞毒性与诱导细胞恶性转化的能力。 方法： 于2016年6月，采用乳酸脱氢酶（LDH）法检测温石棉对MeT-5A细胞的细胞毒性；以5 μg/cm(2)温石棉间断染毒MeT-5A细胞，24 h/次，每周一次，共28次，待细胞呈现锚着不依赖性生长后，用软琼脂集落形成试验判断细胞恶性转化，并计算软琼脂集落形成率，紫外比色法测定糖代谢过程中的关键限速酶活性，包括丙酮酸激酶（PK）、磷酸果糖激酶（PFK）和己糖激酶（HK）。 结果： 温石棉对MeT-5A细胞具有细胞毒性，细胞相对存活率呈浓度依赖性下降。与未染毒时相比，10、20、40、80 μg/cm(2)温石棉染毒后MeT-5A细胞相对存活率显著下降（P<0.05）。染毒28次后，温石棉转化组细胞生长速度加快、排列紊乱，失去接触抑制，出现叠层生长，并可在软琼脂上生长，细胞集落形成率为18.33‰±2.49‰，与对照组（0）比较，差异有统计学意义（P<0.01）；温石棉转化组细胞PK[（19.51±1.52）U/L]、PFK[（0.12±0.02）U/10(4)个细胞]和HK[（0.26±0.01）U/10(4)个细胞]酶活性水平高于对照组细胞[（25.00±1.04）U/L、（0.15±0.01）U/10(4)个细胞和（0.33±0.01）U/10(4)个细胞]（P<0.01）。 结论： 温石棉可诱导MeT-5A细胞发生恶性转化，并升高细胞糖酵解相关激酶PK、PFK、HK酶活性水平。.

Increased apoptotic lymphocytes have been correlated to a high incidence of infection in poorly controlled diabetes. This study aimed to determine whether altered voltage-dependent anion channel (VDAC)-hexokinase (HK) association contributes to the increase in apoptosis. Mouse peripheral blood lymphocytes (PBL) exposed to high glucose (Glc)/palmitic acid (PA) were used as the in vitro model, which was compared with PBL isolated from alloxan-induced diabetic mice (in vivo model). Our results showed a significant increase in apoptosis as indicated by the apoptotic index, caspase-3 activity, mitochondrial membrane potential and ultrastructural study. HK and glucose-6-phosphate dehydrogenase (G6PDH) activities were markedly reduced with a profound increase in glucose-6-phosphate level. Co-immunoprecipitation confirms HK interaction with VDAC, an outer mitochondrial membrane protein. Inhibited glycolytic enzyme, i.e. HK and reduced HK-VDAC interaction in our study could contribute to increased apoptosis in lymphocytes exposed to high Glc/PA. Targeting HK-VDAC interaction may therefore provide therapeutic potential for the treatment of diabetes-associated infection.