Pyruvate kinase M2 (PKM2) is preferentially expressed as a low-activity dimer over the active tetramer in proliferating tumor cells, resulting in metabolic reprogramming to achieve high energy requirements and nutrient uptake. This leads to a shift from the normal glycolytic pathway causing tumor cells to proliferate uncontrollably. This study utilizes knowledge-based drug discovery to determine the critical features from experimentally known PKM2 activators and design compounds that would significantly confer a stable structural and functional edge over the known compounds which are still at the preclinical stage.
Conscientious molecular modeling studies were carried out and critical structural features were identified and validated from the knowledge of experimentally known PKM2 activators to confer high-binding affinities. A virtual library of 200 palindromic and non-palindromic activators was designed based on these identified critical features to target a distinct activator binding-site. This binding would favor specific dimer-dimer association and subsequent protein tetramerization. The resultant compounds strongly correlated with identified structural features and binding affinities which further strengthened our findings. The designed activators were then subjected to pharmacokinetic profiling and toxicity prediction, followed by free-binding energy calculations and MD simulations.
All the virtually designed activators comprising the identified critical features were observed to confer high-binding affinities ranging from -9.1 to -15.0 kcal/mol to the receptor protein. The designed activators also demonstrated optimum pharmacokinetic and toxicity profiles.
The best activators selected for MD simulations studies were conclusively observed to stabilize the required tetrameric conformation suggesting that these activators could potentially target PKM2 tetramerization that might restore the normal glycolytic pathway and suppress tumor progression.

Telomere maintenance is a hallmark of cancer. Most tumors maintain telomere length via reactivation of telomerase reverse transcriptase (TERT) expression. Identifying clinically translatable imaging biomarkers of TERT can enable noninvasive assessment of tumor proliferation and response to therapy.
We used RNAi, doxycycline-inducible expression systems, and pharmacologic inhibitors to mechanistically delineate the association between TERT and metabolism in preclinical patient-derived tumor models. Deuterium magnetic resonance spectroscopy (2H-MRS), which is a novel, translational metabolic imaging modality, was used for imaging TERT in cells and tumor-bearing mice in vivo.
Our results indicate that TERT expression is associated with elevated NADH in multiple cancers, including glioblastoma, oligodendroglioma, melanoma, neuroblastoma, and hepatocellular carcinoma. Mechanistically, TERT acts via the metabolic regulator FOXO1 to upregulate nicotinamide phosphoribosyl transferase, which is the key enzyme for NAD+ biosynthesis, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which converts NAD+ to NADH. Because NADH is essential for pyruvate flux to lactate, we show that 2H-MRS-based assessment of lactate production from [U-2H]-pyruvate reports on TERT expression in preclinical tumor models in vivo, including at clinical field strength (3T). Importantly, [U-2H]-pyruvate reports on early response to therapy in mice bearing orthotopic patient-derived gliomas at early timepoints before radiographic alterations can be visualized by MRI.
Elevated NADH is a metabolic consequence of TERT expression in cancer. Importantly, [U-2H]-pyruvate reports on early response to therapy, prior to anatomic alterations, thereby providing clinicians with a novel tool for assessment of tumor burden and treatment response in cancer.

Cannabis is now legal in many countries and while numerous studies have reported on its impact on cognition and appetite regulation, none have examined fatty acid metabolism in young cannabis users. We conducted an exploratory analysis to evaluate cannabis impact on fatty acid metabolism in cannabis users (n = 21) and non-cannabis users (n = 16). Serum levels of some saturated and monounsaturated fatty acids, including palmitic, palmitoleic, and oleic acids were higher in cannabis users compared to nonusers. As palmitic acid can be derived from diet or lipogenesis from sugars, we evaluated lipogenesis using a de novo lipogenesis index (palmitate/linoleic acid) and carbon-specific isotope analysis, which allows for the determination of fatty acid 13 C signature. The significantly higher de novo lipogenesis index in the cannabis users group along with a more enriched 13 C signature of palmitic acid suggested an increase in lipogenesis. In addition, while serum glucose concentration did not differ between groups, pyruvate and lactate were lower in the cannabis user group, with pyruvate negatively correlating with palmitic acid. Furthermore, the endocannabinoid 2-arachidonoylglycerol was elevated in cannabis users and could contribute to lipogenesis by activating the cannabinoid receptor 1. Because palmitic acid has been suggested to increase inflammation, we measured peripheral cytokines and observed no changes in inflammatory cytokines. Finally, an anti-inflammatory metabolite of palmitic acid, palmitoylethanolamide was elevated in cannabis users. Our results suggest that lipogenic activity is increased in cannabis users; however, future studies, including prospective studies that control dietary intake are required.

The current understanding of deviations of human microbiota caused by antibiotic treatment is poor. In an attempt to improve it, a proof-of-principle spectroscopic study of the breath of one volunteer affected by a course of antibiotics for Helicobacter pylori eradication was performed. Fourier transform spectroscopy enabled searching for the absorption spectral structures sensitive to the treatment in the entire mid-infrared region. Two spectral ranges were found where the corresponding structures strongly correlated with the beginning and end of the treatment. The structures were identified as methyl ester of butyric acid and ethyl ester of pyruvic acid. Both acids generated by bacteria in the gut are involved in fundamental processes of human metabolism. Being confirmed by other studies, measurement of the methyl butyrate deviation could be a promising way for monitoring acute gastritis and anti-Helicobacter pylori antibiotic treatment.

Pyruvate formate-lyase (PFL) catalyzes the reversible conversion of pyruvate and coenzyme A (CoA) into formate and acetyl-CoA in two half-reactions. For the second half-reaction to take place, the S-H group of CoA must enter the active site of the enzyme to retrieve a protein-bound acetyl group. However, CoA is bound at the protein surface, whereas the active site is buried in the protein interior, some 20-30 Å away. The PFL system was therefore subjected to a series of extensive molecular dynamics simulations (in the μs range) and a host of advanced analysis procedures. Models representing PFL before and after the first half-reaction were used to examine the possible effect of enzyme acetylation. All simulated structures were found to be relatively stable compared to the initial crystal structure. Although the adenine portion of CoA remained predominantly bound at the protein surface, the binding of the S-H group was significantly more labile. A potential entry channel for CoA, which would allow the S-H group to reach the active site, was identified and characterized. The channel was found to be associated with accentuated fluctuations and a higher probability of being in an open state in acetylated systems. This result suggests that the acetylation of the enzyme assumes a prominent functional role, whereby the formation of the acyl intermediate serves to initiate a subtle signaling cascade that influences the protein dynamics and facilitates the entry of the second substrate.

Chinese hamster ovary (CHO) cell lines are used to express a variety of therapeutic proteins. However, lactogenic behavior displayed by some CHO cell lines during manufacturing processes may result in a decline in viability, productivity, and possible alterations in product quality. In cultured cells, lactate is produced during glycolysis through irreversible conversion of phosphoenolpyruvate to pyruvate and then lactate via sequential function of pyruvate kinase and lactate dehydrogenase (LDH) enzymes. In the process of cell line development (CLD), two lactogenic cell lines expressing different antibody molecules are identified. The lactogenic behaviors of these cell lines can be differentially mitigated through optimization of either nutrient feeds or culture pH, depending on the cell line. Analysis of various proteins involved in the glycolysis pathway reveal a direct correlation between the pyruvate kinase muscle-1 (PKM-1) isoform levels and lactogenic behavior. CRISPR mediated knockout of the PKM-1 isoform abolishes lactate accumulation even under lactogenic conditions. Furthermore, a cell line lacking expression of both PKM-1 and PKM-2 enzymes capable of maintaining productivity, viability, and growth without displaying lactogenic behavior is identified. Targeted deletion of PKM in CHO cells may be tolerated due to expression of PKL (liver) and PKR (red blood cell) isoforms of pyruvate kinase. All together, these findings suggest that PKM-1 up-regulation during antibody production could trigger lactogenic behavior and that this enzyme is dispensable for CHO cell survival.

BACKGROUND: Fibromyalgia syndrome (FMS) is associated with central alterations, but controversies exist regarding the presence and role of peripheral factors. Microdialysis (MD) can be used in vivo to study muscle alterations in FMS. Furthermore for chronic pain conditions such as FMS, the mechanisms for the positive effects of exercise are unclear. This study investigates the interstitial concentrations of algesics and metabolites in the vastus lateralis muscle of 29 women with FMS and 28 healthy women before and after an exercise intervention.
METHODS: All the participants went through a clinical examination and completed a questionnaire. In addition, their pressure pain thresholds (PPTs) in their upper and lower extremities were determined. For both groups, MD was conducted in the vastus lateralis muscle before and after a 15-week exercise intervention of mainly resistance training of the lower limbs. Muscle blood flow and interstitial muscle concentrations of lactate, pyruvate, glutamate, glucose, and glycerol were determined.
RESULTS: FMS was associated with significantly increased interstitial concentrations of glutamate, pyruvate, and lactate. After the exercise intervention, the FMS group exhibited significant decreases in pain intensity and in mean interstitial concentrations of glutamate, pyruvate, and glucose. The decrease in pain intensity in FMS correlated significantly with the decreases in pyruvate and glucose. In addition, the FMS group increased their strength and endurance.
CONCLUSIONS: This study supports the suggestion that peripheral metabolic and algesic muscle alterations are present in FMS patients and that these alterations contribute to pain. After an exercise intervention, alterations normalized, pain intensity decreased (but not abolished), and strength and endurance improved, all findings that suggest the effects of exercise are partially peripheral.

The disturbances of β-hydroxybutyrate (β-HB) and pyruvate are linked with impaired brain energy utilization which involves in the psychopathology of schizophrenia. This study investigates the difference in levels of β-HB and pyruvate between patients with schizophrenia and healthy controls, and explores their relationship with metabolic profiles and disease characteristics. We recruited 54 physically-health schizophrenic patients and 54 age- and gender-matched healthy control subjects. Blood samples were gathered to determine the serum levels of β-HB and pyruvate and plasma levels of metabolic profiles, including fasting glucose, triglycerides, total cholesterol, high- and low-density lipoprotein-cholesterol and adiponectin. The disease characteristics and psychopathology of patients with schizophrenia were assessed by using the Positive and Negative Syndrome Scale. Of patients with schizophrenia, serum levels of β-HB were significantly correlated with fasting glucose (p=0.007) and triglycerides (p=0.021). Pyruvate was significantly correlated with fasting glucose (p=0.018), total cholesterol (p=0.005), triglycerides (p=0.014) and LDL-C (p=0.006). After controlling the metabolic profiles, β-HB was still significantly higher in schizophrenia patients than in controls (p<0.001), but no difference in pyruvate was observed. Neither β-HB nor pyruvate was significantly correlated with disease characteristics. However, pyruvate was higher in patients treated with olanzapine or clozapine than in those treated with other antipsychotics (p=0.048). Findings suggest that schizophrenic patients had significantly higher serum levels of β-HB than control subjects, possibly reflecting higher demands in energy utilization. Serum levels of β-HB, rather than pyruvate, may act as a potential indicator of energy utilization impairment for schizophrenia.

The structures and relative stabilities as well as the rotational and vibrational spectra of the three low-energy conformers of pyruvic acid (PA) have been characterized using a state-of-the-art quantum-mechanical approach designed for flexible molecules. By making use of the available experimental rotational constants for several isotopologues of the most stable PA conformer, Tc-PA, the semiexperimental equilibrium structure has been derived. The latter provides a reference for the pure theoretical determination of the equilibrium geometries for all conformers, thus confirming for these structures an accuracy of 0.001 Å and 0.1 deg for bond lengths and angles, respectively. Highly accurate relative energies of all conformers (Tc-, Tt-, and Ct-PA) and of the transition states connecting them are provided along with the thermodynamic properties at low and high temperatures, thus leading to conformational enthalpies accurate to 1 kJ mol(-1). Concerning microwave spectroscopy, rotational constants accurate to about 20 MHz are provided for the Tt- and Ct-PA conformers, together with the computed centrifugal-distortion constants and dipole moments required to simulate their rotational spectra. For Ct-PA, vibrational frequencies in the mid-infrared region accurate to 10 cm(-1) are reported along with theoretical estimates for the transitions in the near-infrared range, and the corresponding infrared spectrum including fundamental transitions, overtones, and combination bands has been simulated. In addition to the new data described above, theoretical results for the Tc- and Tt-PA conformers are compared with all available experimental data to further confirm the accuracy of the hybrid coupled-cluster/density functional theory (CC/DFT) protocol applied in the present study. Finally, we discuss in detail the accuracy of computational models fully based on double-hybrid DFT functionals (mainly at the B2PLYP/aug-cc-pVTZ level) that avoid the use of very expensive CC calculations.

OBJECTIVE: To analyze the clinical characteristics and genetype of one children who had been diagnosed with pyruvate dehydrogenase complex deficiency.
METHODS: Comprehensive analyses of this case were performed, including clinical symptoms, signs, biochemical examinations and therapeutic effects. The eleven exons and splicing areas of PDHA1 were amplified with genomic DNA from whole blood. And variations were investigated by sequencing the PCR product. The patient was diagnosed with pyruvate dehydrogenase complex deficiency by sequence analysis of PDHA1 gene.
RESULTS: The patient was a 2 years and 4 monthes old boy. He presented with muscle hypotonia and weakness for one year, and experienced recurrent episodes of unstable head control, unable to sit by himself or stand without support, with persistently hyperlactacidemia. Metabolic testing revealed blood lactate 5.37 mmol/L, pyruvate 0.44 mmol/L, and lactate/pyruvate ratio was 12.23. MRI of the brain showed hyperintense signals on the T2 and T2 Flair weighted images in the basal ganglia bilaterally. Sequence analysis of PDHA1 gene showed a G>A point mutation at nucleotide 778, resulting in a substitution of glutarnine for arginine at position 263 (R263Q). And the diagnosis of pyruvate dehydrogenase complex deficiency was identified. By giving the therapy with ketogenic diet, vitamin B(1), coenzyme Q(10) and L-carnitine , the boy was in a stable condition.
CONCLUSIONS: The severity and the clinical phenotypes of pyruvate dehydrogenase complex deficiency varied. Sequence analysis of PDHA1 gene revealed a 788G>A (R263Q) mutation. Patients who presented with unexplained muscle hypotonia, weakness and hyperlactacidemia could be diveded by gene analysis. And appropriate treatment can improve the quality of life.