HK1 Gene Research Articles

SpRY-mediated screens facilitate functional dissection of non-coding sequences at single-base resolution

CRISPR mutagenesis screens conducted with SpCas9 and other nucleases have identified certain cis-regulatory elements and genetic variants but at a limited resolution due to the absence of protospacer adjacent motif (PAM) sequences. Here, leveraging the broad targeting scope of the near-PAMless SpRY variant, we have demonstrated that saturated SpRY mutagenesis and base editing screens can faithfully identify functional regulatory elements and essential genetic variants for target gene expression at single-base resolution. We further extended this methodology to investigate a genome-wide association study (GWAS) locus at 10q22.1 associated with a red blood cell trait, where we identified potential enhancers regulating HK1 gene expression, despite not all of these enhancers exhibiting typical chromatin signatures. More importantly, our saturated base editing screens pinpoint multiple causal variants within this locus that would otherwise be missed by Bayesian statistical fine-mapping. Our approach is generally applicable to functional interrogation of all non-coding genomic elements while complementing other high-coverage CRISPR screens.

Mild Intermittent Cold Stimulation Affects Cardiac Substance Metabolism via the Neuroendocrine Pathway in Broilers.

This study aimed to investigate the impact of cold adaptation on the neuroendocrine and cardiac substance metabolism pathways in broilers. The broilers were divided into the control group (CC), cold adaptation group (C3), and cold-stressed group (C9), and experimental period was divided into the training period (d 1-35), recovery period (d 36-43), and cold stress period (d 43-44). During the training period, the CC group was reared at ambient temperature, while C3 and C9 groups were reared at 3 °C and 9 °C lower than the ambient temperature, respectively, for 5 h/d at 1 d intervals. During the recovery period, all the groups were maintained at 20 °C. Lastly, during the cold stress period, the groups were divided into two sub-groups, and each sub-group was placed at 10 °C for 12 h (Y12) or 24 h (Y24) for acute cold stimulation. The blood, hypothalamic, and cardiac tissues samples were obtained from all the groups during the training, recovery, and acute stress periods. The results revealed that the transcription of calcium voltage-gated channel subunit alpha 1 C (CACNAIC) was increased in the hypothalamic tissues of the C3 group (p < 0.05). Moreover, compared to the CC group, the serum norepinephrine (NE) was increased in the C9 group (p < 0.05), but insulin (INS) was decreased in the C9 group (p < 0.05). In addition, the transcription of the phosphoinositide-3 kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), SREBP1c, FASN, ACC1, and SCD genes was down-regulated in the C3 and C9 groups (p < 0.05); however, their expression increased in the C3 and C9 groups after acute cold stimulation (p < 0.05). Compared to the CC group, the transcription of forkhead box O1 (FoxO1), PEPCK, G6Pase, GLUT1, HK1, PFK, and LDHB genes was up-regulated in the C3 and C9 groups (p < 0.05. Furthermore, compared to the CC and C9 groups, the protein and mRNA expressions of heat shock protein (HSP) 70 and HSP90 were significantly increased in the C3 group (p < 0.05). These results indicate that intermittent cold training can enhance cold stress tolerance in broilers by regulating their neuroendocrine and cardiac substance metabolism pathways.

Effects of Glucose Levels on Inflammation and Amino Acid Utilization in Lipopolysaccharide-Induced Bovine Mammary Epithelial Cells.

Glucose and amino acids are important sources of nutrients in the synthetic milk of dairy cows, and understanding the fate of amino acids is essential to optimize the utilization of amino acids in milk protein synthesis, thereby reducing nutrient inefficiencies during lactation. The purpose of this study was to investigate the effects of LPS and different concentrations of glucose on (1) the expression of inflammatory factors and genes, (2) the glucose metabolism, and (3) amino acid utilization in BMECs. The results showed that there was an interaction (LPS × glucose, p < 0.05) between LPS and glucose content in the inflammatory cytokine genes (IL-6 and TNF-α) and the inflammatory regulatory genes (CXCL2, CXCL8, and CCL5). With the addition of LPS, the HG + LPS group caused downregulated (p < 0.05) expression of IL-6 and TNF-α, compared with the LG + LPS group. Interestingly, compared with the LG + LPS group, the HG + LPS group upregulated (p < 0.05) the expression of CXCL2, CXCL8, and CCL5. LPS supplementation increased (p = 0.056) the consumption of glucose and GLUT1 gene expression (p < 0.05) and tended to increase (p = 0.084) the LDHA gene expression of BMECs under conditions of different concentrations of glucose culture. High glucose content increased (p < 0.001) the consumption of glucose and enhanced (p < 0.05) the GLUT1, HK1, HK2, and LDHA gene expression of BMECs with or without LPS incubation, and there was an interaction (LPS × glucose, p < 0.05) between LPS and glucose concentrations in GLUT1 gene expression. In this study, LPS enhanced (p < 0.05) the consumption of amino acids such as tryptophan, leucine, isoleucine, methionine, valine, histidine, and glutamate, while high levels of glucose decreased (p < 0.01) consumption, except in the case of tyrosine. For histidine, leucine, isoleucine, and valine consumption, there was an interaction (LPS × glucose, p < 0.05) between LPS and glucose levels. Overall, these findings suggest that relatively high glucose concentrations may lessen the LPS-induced BMEC inflammatory response and reduce amino acid consumption, while low glucose concentrations may increase the demand for most amino acids through proinflammatory responses.

Induced Expression of CYP51a and HK1 Genes Associated with Penconazole and Fludioxonil Resistance in the Potato Pathogen Fusarium oxysporum.

Preventing antifungal resistance development and identifying pathogens with high, medium, and low risk of resistance development to a particular fungicide or fungicide class is crucial in the fight against phytopathogens. We characterized the sensitivity of potato wilt-associated Fusarium oxysporum isolates to fludioxonil and penconazole and assessed the effect of these fungicides on the expression of fungal sterol-14-α-demethylase (CYP51a) and histidine kinase (HK1) genes. Penconazole stunted the growth of F. oxysporum strains at all concentrations used. While all isolates were susceptible to this fungicide, concentrations of up to 1.0 μg/mL were insufficient to cause a 50% inhibition. At low concentrations (0.63 and 1.25 μg/mL), fludioxonil stimulated growth in F. oxysporum. With an increase in the concentration of fludioxonil, only one strain (F. oxysporum S95) exhibited moderate sensitivity to the fungicide. Interaction of F. oxysporum with penconazole and fludioxonil leads to respective elevated expressions of the CYP51a and HK1 genes, which upsurge with increasing concentration of the fungicides. The data obtained indicate that fludioxonil may no longer be suitable for potato protection and its continuous use could only lead to an increased resistance with time.

Expanding the Molecular Spectrum of HK1-Related Charcot-Marie-Tooth Disease, Type 4G; the First Report in Iran.

Charcot-Marie-Tooth disease type 4G (CMT4G) was first reported in Balkan Gypsies as a myelinopathy starting with progressive distal lower limb weakness, followed by upper limb involvement and prominent distal sensory impairment later in the patient's life. So far, CMT4G has been only reported in European Roma communities with two founder homozygous variants; g.9712G>C and g.11027G>A, located in the 5'-UTR of the HK1 gene. Here, we present the first Iranian CMT4G patient manifesting progressive distal lower limb weakness from 11 years of age and diagnosed with chronic demyelinating sensorimotor polyneuropathy. Whole-exome sequencing for this patient revealed a homozygous c.19C>T (p. Arg7*) variant in the HK1 gene. This report expands the mutational spectrum of the HK1-related CMT disorder and provides supporting evidence for the observation of CMT4G outside the Roma population. Interestingly, the same Arg7* variant is recently observed in another unrelated Pakistani CMT patient, proposing a possible prevalence of this variant in the Middle Eastern populations.

The deletion of HK-1 gene affects the bacterial virulence of Pseudomonas stutzeri LH-42.

Two-component systems (TCSs) are widespread regulatory systems in bacteria, which control cellular functions and play an important role in sensing various external stimuli and regulating gene expression in response to environmental changes. Among the nineteen genes for the two-component system found in the whole genome of Pseudomonas stutzeri LH-42, one of the TCS coded by the HK-1 gene, has a structural domain similar to the HAMP domain, which plays an important role in regulating bacterial virulence in other bacteria. In this study, the deletion mutant LH-42△HK-1 was successfully constructed using the lambda Red recombinase system. Compared with the wild-type strain, the mutant strain LH-42△HK-1 showed a significantly slower growth time and a longer stationary phase time. In addition, in the plate bacteriostatic experiment with Escherichia coli DH5α as an indicator strain, the inhibition zone size of the mutant strain showed significantly less than the wild-type strain(P<0.05), indicating that the virulence of the mutant strain was significantly reduced compared with the wild-type strain. Overall, the results indicate that the deletion of the gene HK-1 decreased bacterial virulence in Pseudomonas stutzeri LH-42.

Variants influencing age at diagnosis of HNF1A-MODY

BackgroundHNF1A-MODY is a monogenic form of diabetes caused by variants in the HNF1A gene. Different HNF1A variants are associated with differences in age of disease onset, but other factors are postulated to influence this trait. Here, we searched for genetic variants influencing age of HNF1A-MODY onset.MethodsBlood samples from 843 HNF1A-MODY patients from Czech Republic, France, Poland, Slovakia, the UK and the US were collected. A validation set consisted of 121 patients from the US. We conducted a genome-wide association study in 843 HNF1A-MODY patients. Samples were genotyped using Illumina Human Core arrays. The core analysis was performed using the GENESIS package in R statistical software. Kinship coefficients were estimated with the KING and PC-Relate algorithms. In the linear mixed model, we accounted for year of birth, sex, and location of the HNF1A causative variant.ResultsA suggestive association with age of disease onset was observed for rs2305198 (p = 2.09E−07) and rs7079157 (p = 3.96E−06) in the HK1 gene, rs2637248 in the LRMDA gene (p = 2.44E−05), and intergenic variant rs2825115 (p = 2.04E−05). Variant rs2637248 reached nominal significance (p = 0.019), while rs7079157 (p = 0.058) and rs2825115 (p = 0.068) showed suggestive association with age at diabetes onset in the validation set.Conclusionsrs2637248 in the LRMDA gene is associated with age at diabetes onset in HNF1A-MODY patients.

Charcot-Marie-Tooth Disease 4G Caused by Homozygous Deletion of Exon 4 in HK1 Gene due to Inbreeding: A Case Report and Literature Review

Objectives: The objective is to investigate the clinical and genotypic characteristics of Charcot-Marie-Tooth disease, caused by HK1 gene mutation. Methods: The detailed medical history of the child was collected and the clinical symptoms were summarized. The genomic DNA was extracted from the 2ml of peripheral blood of child and their parents, and the whole exome sequencing was performed, and the related literatures were reviewed. Results: A 7-year-old girl with unstable walking for 7 months, left claudication, obvious valgus of left foot, unable to squate , unable to jump on one foot, grade IV of muscle strength of lower extremity and slight limitation of dorsal extension of left foot. Electromyography showed multiple peripheral neurogenic lesions (motor and sensory nerve demyelination with axonal damage, more severe in lower limbs than in upper limbs). Whole exome sequencing and PCR verification indicated that the patient had a homozygous deletion in exon 4 of HK1 gene, and the variation site was located in the range of chr10:71048499-71048526, which had not been reported before, and the associated disease was peroneal muscular atrophy type 4G. Conclusion: This study expands the number of reported cases of CMT4G and the mutation spectrum of HK1 gene, and provides reference for prenatal diagnosis and genetic counseling.

Sensitivity of Colletotrichum nymphaeae to Six Fungicides and Characterization of Fludioxonil-Resistant Isolates in China.

Colletotrichum nymphaeae is the dominant species causing anthracnose disease of peach in China. In this study, 140 isolates of C. nymphaeae were assessed for their sensitivity to six fungicides. It was found that C. nymphaeae was highly resistant to carbendazim, procymidone, and boscalid but sensitive to pyraclostrobin and prochloraz. For fludioxonil, the fungus exhibited differential sensitivities (i.e., approximately 14% of isolates were resistant to fludioxonil and the resistance was stable). Fludioxonil-resistant isolates had a mean EC50 value of 2.2380 µg/ml, whereas the mean EC50 value was 0.0194 µg/ml in fludioxonil-sensitive isolates. The mean EC50 values of C. nymphaeae for pyraclostrobin and prochloraz were 0.0083 µg/ml and 0.002 µg/ml, respectively. No cross-resistance was observed between fungicides from different groups. Mycelial growth rate, control efficacy, and osmotic stress responses were significantly different (P < 0.05) between fludioxonil-sensitive (FluS) and -resistant (FluR) isolates, but no significant difference was observed (P > 0.05) in virulence and sporulation between FluS and FluR isolates. No mutation was detected in coding regions of the CnOs-1, Cal, Hk1, Hog1, TPI, and Mrr1 genes. Interestingly, with fludioxonil treatment, the expression of ABC transporter gene atrB was significantly overexpressed in some resistant isolates. However, overexpression of the atrB gene was not detected in one moderately and one highly resistant isolate, indicating that other unknown mechanisms may be involved. Current findings uncovered several effective chemicals and provided the foundation for designing management strategies to practically control peach anthracnose with the most effective demethylation inhibitor fungicides and quinone outside inhibitor fungicides.

Correlation of 18F-Fluorodeoxyglucose Glucose Uptake by Liver Cancer and Transcriptional Regulation of the Warburg Effects in ATT-MYC Mouse Model of Liver Cancer

Background It was previously reported that diethylnitrosamine (DEN) enhanced liver cancer progression in ATT-MYC mouse model of liver cancer. Radiogenomics is a new tool in advanced science technology that gives information on tumor biology, non-tumor surrounding tissue, the degree of tumor size and presence of necrosis of cells especially with joined micro computed tomography – positron emission tomographys (CT/PETs). Aim To evaluate the correlation of gene expression and non-invasive microPET information of the liver tumors at different points of the stage of growth. Methods Exon array expression of the liver of ATT-MYC mice treated with DEN or butylated hydroxytoluene (BHT) compared to control non-transgenic mice were analyzed by array track and the current data were also compared to microarray expression of liver tumor of ATT-MYC mice. Results The expression of genes responsible for glucose transport such as glut1, 3, 4, hk1, slc1a5, slc1a1, slc1a4, slc1a2, gp6c and gpc-1-3-4 were up-regulated significantly in DEN-treated transgenic mice immediately after end of treatment (p≤0.05), while glut2 (fold change 0.9503, p-value 0.4385) and hk2 (fold change 3.0589, p-value 0.0565) genes were increased not significantly immediately after end of treatment. Additionally, at 4.5-months of observation after the end of treatment slc1a5, slc38a2, glut1, glut4 and gpc3-4 genes had a significant fold change in liver tumor tissue in DEN treated mice when compared to BHT or control transgenic or non-transgenic one. While hk1, 2, slc5a1, slc1a4, glut2, glut3, g6pc and gpc-1 genes were increased non-significantly in the liver of treated mice when compared to control group at 4.5-months of observation after the end of treatment. Notably, c-myc, hif-1 and aldoa glycolytic genes were expressed significantly both time points of 4 and 8.5-months while ldhb, hk-2 and PKM2 were increased non-significantly in DEN treatment when compared to BHT/control non-transgenic animals. Conclusion There is a definitive correlation between genes responsible for glucose transport and 18F-Fluorodeoxyglucose (FDG) uptake in the early and advanced degree of liver carcinogenesis. This study of glucose pathway in Hepatocellular carcinoma (HCC) at different stages of early and advanced one is the potential for therapeutic anticancer therapy.

YTHDC1-mediated augmentation of miR-30d in repressing pancreatic tumorigenesis via attenuation of RUNX1-induced transcriptional activation of Warburg effect

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers. It thrives in a malnourished environment; however, little is known about the mechanisms by which PDAC cells actively promote aerobic glycolysis to maintain their metabolic needs. Gene Expression Omnibus (GEO) was used to identify differentially expressed miRNAs. The expression pattern of miR-30d in normal and PDAC tissues was studied by in situ hybridization. The role of miR-30d/RUNX1 in vitro and in vivo was evaluated by CCK8 assay and clonogenic formation as well as transwell experiment, subcutaneous xenograft model and liver metastasis model, respectively. Glucose uptake, ATP and lactate production were tested to study the regulatory effect of miR-30d/RUNX1 on aerobic glycolysis in PDAC cells. Quantitative real-time PCR, western blot, Chip assay, promoter luciferase activity, RIP, MeRIP, and RNA stability assay were used to explore the molecular mechanism of YTHDC1/miR-30d/RUNX1 in PDAC. Here, we discover that miR-30d expression was remarkably decreased in PDAC tissues and associated with good prognosis, contributed to the suppression of tumor growth and metastasis, and attenuation of Warburg effect. Mechanistically, the m6A reader YTHDC1 facilitated the biogenesis of mature miR-30d via m6A-mediated regulation of mRNA stability. Then, miR-30d inhibited aerobic glycolysis through regulating SLC2A1 and HK1 expression by directly targeting the transcription factor RUNX1, which bound to the promoters of the SLC2A1 and HK1 genes. Moreover, miR-30d was clinically inversely correlated with RUNX1, SLC2A1 and HK1, which function as adverse prognosis factors for overall survival in PDAC tissues. Overall, we demonstrated that miR-30d is a functional and clinical tumor-suppressive gene in PDAC. Our findings further uncover that miR-30d is a novel target for YTHDC1 through m6A modification, and miR-30d represses pancreatic tumorigenesis via suppressing aerobic glycolysis.

Chronic exposure to PPCPs mixture at environmentally relevant concentrations (ERCs) altered carbohydrate and lipid metabolism through gut and liver toxicity in zebrafish.

Pharmaceuticals and personal care products (PPCPs) have been widely distributed and posed ecotoxicological risks in the aquatic environment. This study aims to evaluate the toxic effects after chronic exposure to PPCPs mixture at the environment relevant concentrations (ERCs). Our results indicated that PPCPs induced serious metabolic effects by disturbing the carbohydrate and lipid metabolism pathways. Chronic exposure caused a significant reduction in the hepatosomatic index (HSI), the gut weight ratios, and histological alterations in liver and gut tissues. Further, exposure to the combined PPCPs disrupted the carbohydrate metabolism via significant upregulation of hk1, gk, pck1, and insr genes. The lipid metabolism was affected with higher ppars expression levels that increased the fatty acid β-oxidation and ultimately decreased the lipidogenesis. Moreover, the altered responses of the insulin growth factor (IGF) pathway more in male gut tissue than that of female revealed sex-dependent disturbance in the gut homeostasis induced by PPCPs mixture. In conclusion, chronic exposure to PPCPs mixtures at ERCs can induce developmental effects and metabolic dysfunction in both male and female fish. The consumption and environmental disposal of these PPCPs should be regulated to ensure ecological health and environmental safety.

Novel pathogenic variant c.2714C>A (p. Thr905Lys) in the HK1 gene causing severe haemolytic anaemia with developmental delay in an Indian family

Hexokinase (EC 2.7.1.1, Adenosine Tri Phosphate (ATP): D-hexose-6-phosphotransferase) is a crucial regulatory enzyme of the glycolytic pathway (Embden-Meyerhof pathway). Hexokinase deficiency is associated with chronic non-spherocytic haemolytic anaemia (HA) with...

Non-Immune Hemolytic Anemia of Pregnancy: Heterozygous RBC Variants Elude Diagnosis

A 25 year old G3P2002 El Salvadorean female, with a prior history of pregnancy related anemia of unknown etiology, presented at 24 weeks gestation with symptomatic anemia (hemoglobin 2.9 g/dL) including dizziness, weakness and fatigue and no active signs of bleeding. Blood work included normal range results for LDH, haptoglobin, indirect and direct Coombs indicating no intravascular or immune driven hemolysis. Peripheral smear showed spherocytosis and stomatocytosis, concerning for an intrinsic RBC defect. Other workup included hemoglobin electrophoresis with slight increase in Hb A2 of 3.3%, flow cytometry negative for a lymphoproliferative disorder or paroxysmal nocturnal hemoglobinuria, no evidence of G6PD deficiency, and a bone marrow biopsy negative for marrow dysplasia, aplasia or HLH. Abdominal ultrasound revealed hypersplenism. The anemia was attributed to a non-immune hemolytic anemia with extra corpuscular RBC destruction in the spleen but without evidence of RBC destruction in the bone marrow or peripheral blood. After a prolonged 10-week hospitalization, the patient received a trial of steroids, 8 IV immunoglobulin infusions with minimal benefit, and total of 22 units of packed red blood cells. She underwent an elective induction and delivery at 34 weeks of pregnancy. During the postpartum period, she continued to have persistent anemia. A partial splenic embolization was attempted, complicated by splenic abscesses resulting in a splenectomy. Post splenectomy, the patient's hemoglobin and hematocrit stabilized to 11.7/37.8 at her three week outpatient visit. Molecular testing for Next Generation Sequencing (NGS) with Laboratory Hereditary Hemolytic Anemia Comprehensive Panel was also performed, revealing four different heterozygous variants. While these mutations individually have not been proven to cause hemolysis, the four alterations together, with the stressor of pregnancy, likely induced a non-immune hemolytic anemia. Non-immune hemolytic anemia is caused by intracorpuscular defects within the red blood cells or extracorpscular by environmental factors. The patient was found with four heterozygous variants in HK1, RPS19, SPTA1 and HBB, implicated in intracorpuscular defects. The HK1 gene, expressed in erythrocytes, encodes hexokinase, and provides red blood cells ATP. HK deficiency is a rare hereditary disorder associated with mild to severe non-spherocytic hemolytic anemia. The RPS19 gene encodes for a ribosomal protein involved in erythropoiesis. Clinically significant mutations in this gene cause Diamond Blackfan anemia. The SPTA1 gene encodes alpha spectrin subunits, which are a part of red cell membrane cytoskeleton and maintains its shape. Mutations in this gene have been implicated in hereditary spherocytosis. One case report described severe non-immune hemolytic anemia in a neonate with hereditary spherocytosis secondary a heterozygous mutation of the SPTA1 gene. Lastly, the HBB gene encodes for hemoglobin beta globin chains where alterations have been associated with hemolytic anemia, sickle cell anemia, and beta thalassemia. Several case reports described heterozygous variants of HBB and association with hemoglobin instability and extravascular hemolysis. Heterozygous mutations in the above genes have been rarely reported in literature to cause non-immune hemolytic anemia. Although unclear, pregnancy appeared to be the inciting factor in our patient with these mutational variants that have a potential role in extravascular hemolysis. While there have been few case reports describing autoimmune hemolytic anemia caused by pregnancy, a non-immune hemolytic anemia from 4 heterozygous variants in RBC genes, as seen in our patient, has not been previously described. Disclosures No relevant conflicts of interest to declare.

Genotype-phenotype correlations of Berardinelli-Seip congenital lipodystrophy and novel candidate genes prediction

BackgroundBerardinelli-Seip congenital lipodystrophy (BSCL) is a heterogeneous autosomal recessive disorder characterized by an almost total lack of adipose tissue in the body. Mutations in the AGPAT2, BSCL2, CAV1 and PTRF genes define I-IV subtype of BSLC respectively and clinical data indicate that new causative genes remain to be discovered. Here, we retrieved 341 cases from 60 BSCL-related studies worldwide and aimed to explore genotype-phenotype correlations based on mutations of AGPAT2 and BSCL2 genes from 251 cases. We also inferred new candidate genes for BSCL through protein-protein interaction and phenotype-similarity.ResultsAnalysis results show that BSCL type II with earlier age of onset of diabetes mellitus, higher risk to suffer from premature death and mental retardation, is a more severe disorder than BSCL type I, but BSCL type I patients are more likely to have bone cysts. In BSCL type I, females are at higher risk of developing diabetes mellitus and acanthosis nigricans than males, while in BSCL type II, males suffer from diabetes mellitus earlier than females. In addition, some significant correlations among BSCL-related phenotypes were identified. New candidate genes prediction through protein-protein interaction and phenotype-similarity was conducted and we found that CAV3, EBP, SNAP29, HK1, CHRM3, OBSL1 and DNAJC13 genes could be the pathogenic factors for BSCL. Particularly, CAV3 and EBP could be high-priority candidate genes contributing to pathogenesis of BSCL.ConclusionsOur study largely enhances the current knowledge of phenotypic and genotypic heterogeneity of BSCL and promotes the more comprehensive understanding of pathogenic mechanisms for BSCL.

PS1014 GLYCOLYSIS VERSUS OXIDATIVE PHOSPHORYLATION – POTENTIAL THERAPEUTIC TARGETS IN AML

Background:Cancer cells alter their metabolism to sustain high levels of growth and proliferation. One of the most common alterations is the metabolic shift of cancer cells from oxidative phosphorylation (OXPHOS) to glycolysis, regardless of oxygen presence (aerobic glycolysis), resulting in lower ATP production. This alteration is associated with up‐regulation of glucose transporters (GLUTs) and glycolytic enzymes, such as hexokinases, and enhanced lactate fermentation. This shift is mainly regulated by HIF‐1, p53 and MYC. Hence, metabolism constitute a new target in acute myeloid leukemia (AML), where mutations in metabolism [isocitrate dehydrogenase 1/2 (IDH1/2)] were already describe. AML is a heterogeneous hematologic neoplasia characterized by myeloid differentiation arrest and uncontrolled proliferation.Aims:This study aimed to metabolically characterize AML cell lines and assess the potential of glycolysis and OXPHOS metabolism as a therapeutic target in AML using in vitro models.Methods:Six AML cell lines of different subtypes and with different genetic backgrounds were used (HEL, HL‐60, K‐562, KG‐1, NB‐4 and THP‐1). The presence of IDH1/2 exon 4 mutations was assessed using Sanger sequencing. Intracellular peroxides, superoxide anion and mitochondrial membrane potential levels were measured by fluorimetry using DCFH2‐DA, DHE and JC‐1 probes, respectively. Cells were incubated in presence or absence of 2‐deoxy‐D‐glucose (2‐DG; glycolysis inhibitor) or oligomycin complex (OXPHOS inhibitor). Metabolic activity was assessed by resazurin assay and cell death by flow cytometry (FC) using Annexin V and 7‐AAD double staining. Cell cycle was also assessed by FC, using propidium iodide/RNase. Glucose intake was measured by 18F‐FDG uptake and expression levels of 8 SLC2A, 2 hexokinases and HIF‐1α were assessed using RT‐PCR.Results:NB‐4 cell line has the highest 18F‐FDG uptake, whereas HL‐60 and THP‐1 cells showed the lowest. HEL, K‐562 and KG‐1 had similar 18F‐FDG uptake. THP‐1 cells expressed the 8 tested SLC2A genes. NB‐4 cell line also had the highest peroxide and superoxide anion levels. K‐562 and KG‐1 cells had similar ROS levels and IDH1 genotype (single nucleotide variant rs11554137 in heterozygosity). Glycolysis inhibition reduced metabolic activity in a dose‐ and cell line dependent manner and HEL and KG‐1 were the most sensitive cell lines to 2‐DG. OXPHOS inhibition led to a decrease in metabolic activity in a cell line‐dependent manner. K‐562 cells were the most sensitive to this inhibition and THP‐1 cells were the most resistant to both compounds (2‐DG and oligomycim). These inhibitors induce cell death by apoptosis and cell cycle arrest in G0/G1 phase. 2‐DG induced a more evident cytotoxic effect on K‐562 and NB‐4 and a cytostatic effect on KG‐1 and THP‐1 cells. Conversely, oligomycin had a cytotoxic effect on K‐562 and KG‐1 cells and a cytostatic effect on NB‐4, THP‐1 and K 562. Moreover, 2‐DG decreased 18F‐FDG uptake, mainly in KG‐1 and NB‐4 cell lines, inducing up‐regulation of SLC2A11 and down‐regulation of HK1 gene expression. Lastly, oligomycin increased 18F‐FDG uptake, particularly in K‐562 cells, with up‐regulation of SLC2A1 and HK2 gene expression.Summary/Conclusion:AML cell lines have different metabolic preferences, with HEL, KG‐1 and NB‐4 cells appearing to be more glycolytic, whereas K‐562 cells more dependent on OXPHOS. Contrary, THP‐1 cells seems to be OXPHOS independent. These cell lines seem to reprogram their metabolism in order to overcome OXPHOS inhibition, suggesting that glycolysis could be a better therapeutic target in AML.

Diagnosis of red blood cell hexokinase deficiency and literature review

Objective To investigate the value of gene sequencing and protein conformation in the diagnosis process by reporting a case of erythrocyte hexokinase deficiency and reviewing relevant literature. Methods One patient with erythrocyte hexokinase deficiency and his families in the pediatrics of Sun Yat-sen Memorial Hospital of Sun Yat-sen University on May 23, 2016 were selected as the research objects. Clinical data was collected from the initial records by retrospective analysis. Pathogenic gene was detected by target sequence capture and next generation high-throughput sequencing, and the effects of mutant genes on hexokinase function were studied by observing changes in the 3-dimensional structure of proteins. Results The patient exhibited chronic hemolytic anemia after birth. The high-throughput second-generation gene sequencing result of the DNA sample of the child showed that a de novo mutation in the HK1 gene located on chromosome 10, the substitution of cytidine at site 34 of exon1 by thymine : NM_033496: exon1: c. C34T: p. Arg12X, which was a nonsense mutation. Bioinformatics analysis and protein function simulation confirmed that the mutation of the gene can lead to the premature termination of the synthesis of the peptide chain. The short peptide synthesized after the mutation does not contain the binding group and catalytic group of the erythrocyte hexokinase, resulting in the loss of enzyme activity. The patient was eventually diagnosed with erythrocyte hexokinase-deficient hemolytic anemia caused by the HK1 gene mutation. Conclusions In clinical practice, patients with chronic non-spherical erythrocyte hemolytic anemia should be alert to erythrocyte hexosaminidase deficiency disease. Those with conditions should be diagnosed by gene sequencing and protein configuration analysis. Key words: Hexokinase deficiency; Hemolytic anemia; Sequence analysis; Protein structure, quaternary; HK1 gene

Functional Divergence of Poplar Histidine-Aspartate Kinase HK1 Paralogs in Response to Osmotic Stress.

Previous works have shown the existence of protein partnerships belonging to a MultiStep Phosphorelay (MSP) in Populus putatively involved in osmosensing. This study is focused on the identification of a histidine-aspartate kinase, HK1b, paralog of HK1a. The characterization of HK1b showed its ability to homo- and hetero-dimerize and to interact with a few Histidine-containing Phosphotransfer (HPt) proteins, suggesting a preferential partnership in poplar MSP linked to drought perception. Furthermore, determinants for interaction specificity between HK1a/1b and HPts were studied by mutagenesis analysis, identifying amino acids involved in this specificity. The HK1b expression analysis in different poplar organs revealed its co-expression with three HPts, reinforcing the hypothesis of partnership participation in the MSP in planta. Moreover, HK1b was shown to act as an osmosensor with kinase activity in a functional complementation assay of an osmosensor deficient yeast strain. These results revealed that HK1b showed a different behaviour for canonical phosphorylation of histidine and aspartate residues. These phosphorylation modularities of canonical amino acids could explain the improved osmosensor performances observed in yeast. As conserved duplicates reflect the selective pressures imposed by the environmental requirements on the species, our results emphasize the importance of HK1 gene duplication in poplar adaptation to drought stress.

Effect of lentivirus-mediated shRNA inactivation of HK1, HK2, and HK3 genes in colorectal cancer and melanoma cells.

BackgroundThe switch from oxidative phosphorylation to glycolysis in proliferating cancer cells, even under aerobic conditions, has been shown first in 1926 by Otto Warburg. Today this phenomenon is known as the “Warburg effect” and recognized as a hallmark of cancer. The metabolic shift to glycolysis is associated with the alterations in signaling pathways involved in energy metabolism, including glucose uptake and fermentation, and regulation of mitochondrial functions. Hexokinases (HKs), which catalyze the first step of glycolysis, have been identified to play a role in tumorigenesis of human colorectal cancer (CRC) and melanoma. However, the mechanism of action of HKs in the promotion of tumor growth remains unclear.ResultsThe purpose of the present study was to investigate the effect of silencing of hexokinase genes (HK1, HK2, and HK3) in colorectal cancer (HT-29, SW 480, HCT-15, RKO, and HCT 116) and melanoma (MDA-MB-435S and SK-MEL-28) cell lines using short hairpin RNA (shRNA) lentiviral vectors. shRNA lentiviral plasmid vectors pLSLP-HK1, pLSLP-HK2, and pLSLP-HK3 were constructed and then transfected separately or co-transfected into the cells. HK2 inactivation was associated with increased expression of HK1 in colorectal cancer cell lines pointing to the compensation effect. Simultaneous attenuation of HK1 and HK2 levels led to decreased cell viability. Co-transfection with shRNA vectors against HK1, HK2, and HK3 mRNAs resulted in a rapid cell death via apoptosis.ConclusionsWe have demonstrated that simultaneous inactivation of HK1 and HK2 was sufficient to decrease proliferation and viability of melanoma and colorectal cancer cells. Our results suggest that HK1 and HK2 could be the key therapeutic targets for reducing aerobic glycolysis in examined cancers.

HSMNR belongs to the most frequent types of hereditary neuropathy in the Czech Republic and is twice more frequent than HMSNL.

Hereditary motor and sensory neuropathy type Russe (HMSNR), also called CMT4G, is an autosomal recessive inherited peripheral neuropathy (IPN) caused by a founder mutation in the HK1 gene. HMSNR affects only patients with Roma origin, similar to the better known HMSN type Lom clarified earlier. By testing IPN patients with Roma origin, we realized that HMSNR affects surprisingly many patients in the Czech Republic. HMSNR is one of the most frequent types of IPN in this country and appears to be twice more frequent than HMSNL. Pronounced lower limb atrophies and severe deformities often lead to walking inability in even young patients, but hands are usually only mildly affected even after many years of disease duration. The group of 20 patients with HMSNR presented here is the first report about the prevalence of HMSNR from central Europe.