BACKGROUND: Although great progress has been made in anti-cancer therapy, the prognosis of laryngeal squamous cell carcinoma (LSCC) patients remains unsatisfied. Quantities of studies demonstrate that glycolytic reprograming is essential for the progression of cancers, where triosephosphate isomerase 1 (TPI1) serves as a catalytic enzyme. However, the clinicopathological significance and potential biological functions of TPI1 underlying LSCC remains obscure.
METHODS: We collected in-house 82 LSCC tissue specimens and 56 non-tumor tissue specimens. Tissue microarrays (TMA) and immunohistochemical (IHC) experiments were performed. External LSCC microarrays and bulk RNA sequencing data were integrated to evaluate the expression of TPI1. We used a log-rank test and the CIBERSORT algorithm to assess the prognostic value of TPI1 and its association with the LSCC microenvironment. Malignant laryngeal epithelial cells and immune-stromal cells were identified using inferCNV and CellTypist. We conducted a comprehensive analysis to elucidate the molecular functions of TPI1 in LSCC tissue and single cells using Pearson correlation analysis, high dimensional weighted gene co-expression analysis, gene set enrichment analysis, and clustered regularly interspaced short palindromic repeats (CRISPR) screen. We explored intercellular communication patterns between LSCC single cells and immune-stromal cells and predicted several therapeutic agents targeting TPI1.
RESULTS: Based on the in-house TMA and IHC analysis, TPI1 protein was found to have a strong positive expression in the nucleus of LSCC cells but only weakly positive activity in the cytoplasm of normal laryngeal cells (p < 0.0001). Further confirmation of elevated TPI1 mRNA expression was obtained from external datasets, comparing 251 LSCC tissue samples to 136 non-LSCC tissue samples (standardized mean difference = 1.06). The upregulated TPI1 mRNA demonstrated a high discriminative ability between LSCC and non-LSCC tissue (area under the curve = 0.91; sensitivity = 0.87; specificity = 0.79), suggesting its potential as a predictive marker for poor prognosis (p = 0.037). Lower infiltration abundance was found for plasma cells, naïve B cells, monocytes, and neutrophils in TPI-high expression LSCC tissue. Glycolysis and cell cycle were significantly enriched pathways for both LSCC tissue and single cells, where heat shock protein family B member 1, TPI1, and enolase 1 occupied a central position. Four outgoing communication patterns and two incoming communication patterns were identified from the intercellular communication networks. TPI1 was predicted as an oncogene in LSCC, with CRISPR scores less than -1 across 71.43% of the LSCC cell lines. TPI1 was positively correlated with the half maximal inhibitory concentration of gemcitabine and cladribine.
CONCLUSIONS: TPI1 is dramatically overexpressed in LSCC than in normal tissue, and the high expression of TPI1 may promote LSCC deterioration through its metabolic and non-metabolic functions. This study contributes to advancing our knowledge of LSCC pathogenesis and may have implications for the development of targeted therapies in the future.

Homodimeric triosephosphate isomerases (TIMs) from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have markedly similar amino-acid sequences and three-dimensional structures. However, several of their biophysical parameters, such as their susceptibility to sulfhydryl agents and their reactivation speed after being denatured, have significant differences. The causes of these differences were explored with microsecond-scale molecular dynamics (MD) simulations of three different TIM proteins: TcTIM, TbTIM, and a chimeric protein, Mut1. We examined their electrostatic interactions and explored the impact of simulation length on them. The same salt bridge between catalytic residues Lys 14 and Glu 98 was observed in all three proteins, but key differences were found in other interactions that the catalytic amino acids form. In particular, a cation-π interaction between catalytic amino acids Lys 14 and His 96 and both a salt bridge and a hydrogen bond between catalytic Glu 168 and residue Arg 100 were only observed in TcTIM. Furthermore, although TcTIM forms less hydrogen bonds than TbTIM and Mut1, its hydrogen bond network spans almost the entire protein, connecting the residues in both monomers. This work provides new insight into the mechanisms that give rise to the different behavior of these proteins. The results also show the importance of long simulations.

The noctuid moth Spodoptera frugiperda (the fall armyworm) is endemic to the Western Hemisphere and appears to be undergoing sympatric speciation to produce two subpopulations that differ in their choice of host plants. The \'rice strain\' and \'corn strain\' are morphologically indistinguishable, requiring the use of genetic markers for identification. Because fall armyworm is a major pest of corn and several other agricultural crops, characterizing the strains has important economic consequences. In this study, comparisons were made of the intron sequences from the triose-phosphate isomerase (Tpi) gene isolated from 85 fall armyworm specimens collected from two host plants. Sixteen new strain-specific haplotypes based on intron polymorphisms are described that can facilitate the characterization of fall armyworm populations associated with different host plants. Comparisons of genetic diversity within and between the strains provides evidence that the corn strain is undergoing active selection and supports the proposal of directional interstrain mating occurring in the wild. Comparisons of the polymorphisms indicate that each intron undergoes different patterns of mutation that in some cases corresponds to host plant preferences. The results confirm that intron sequence comparisons are an effective approach to study fall armyworm population genetics.

Quantum mechanics/molecular mechanics (QM/MM) methods are excellent tools for the modeling of biomolecular reactions. Recently, we have implemented a new QM/MM method (Fireball/Amber), which combines an efficient density functional theory method (Fireball) and a well-recognized molecular dynamics package (Amber), offering an excellent balance between accuracy and sampling capabilities. Here, we present a detailed explanation of the Fireball method and Fireball/Amber implementation. We also discuss how this tool can be used to analyze reactions in biomolecules using steered molecular dynamics simulations. The potential of this approach is shown by the analysis of a reaction catalyzed by the enzyme triose-phosphate isomerase (TIM). The conformational space and energetic landscape for this reaction are analyzed without a priori assumptions about the protonation states of the different residues during the reaction. The results offer a detailed description of the reaction and reveal some new features of the catalytic mechanism. In particular, we find a new reaction mechanism that is characterized by the intramolecular proton transfer from O1 to O2 and the simultaneous proton transfer from Glu 165 to C2.

Recombinant triosephosphate isomerase from Plasmodium falciparum (PfTIM) and humans (hTIM) were expressed, purified and characterised. High specific activity (1207 U x mg(-1)) with a fold purification of -1.8 and a yield of 48% were obtained for hTIM after gel filtration while, in contrast PfTIM afforded a specific activity of 1387 U x mg(-1) with a fold purification of -6.8 and a yield of 57% after gel filtration and prior to dialysis. PfTIM had an optimal pH and temperature, K(m) and V(max) of 5.25, 25 degrees C, 12.8 mM and 1.13 μmol x mL(-1) min(-1) respectively while for hTIM the pH and temperature optima, K(m) and V(max) were 6.75, 30 degrees C; 8.2 mM and 1.35 μmol x ml(-1) min(-1). Polyvinylpyrrolidone stabilised silver nanoparticles (60 nM; 2-6 nm diameter) selectively inhibited PfTIM with a 7-fold decrease in enzyme catalytic efficiency (K(cat)/K(m)) over hTIM. Respective K(i) values were 283 nM [hTIM] and 85.7 nM [PfTIM]. Key structural differences between the two enzyme variants, especially with Cys13 at the dimer interface of PfTIM, were significant enough to suggest unique characteristics allowing for selective targeting of PfTIM by AgNPs.

BACKGROUND: A cross-sectional study was undertaken to determine the prevalence of Giardia and Cryptosporidium in calves of Palermo area (Sicily) and to evaluate the occupational risk associated with occurrence of zoonotic genotypes.
METHODS: A total of 217 faecal samples, from 149 calves (between 2 and 240 days of age) and 68 farmers, were collected in 19 cattle-farms of Palermo area. A questionnaire regarding demographic characteristics and personal hygienic measures was submitted to all farmers. All faecal samples were analyzed by Immunofluorescence assay and Polimerase Chain Reaction (PCR); genotypes were determined by DNA sequencing of Triose Phosphate Isomerase gene for Giardia and Small Subunit Ribosomal RNA gene for Cryptosporidium.
RESULTS: None farmer tested was positive for Giardia and Cryptosporidium, whereas these protozoa were respectively detected in 53 (including 5 with zoonotic G. duodenalis genotype A) and 17 (of which 1 with zoonotic C. ubiquitum) of the examined calves.
CONCLUSIONS: The results indicate that the risk of transmitting both protozoa to farmers in Palermo area is negligible although it cannot be considered null because of identification of human genotypes/species in calves.

Recent molecular dynamics simulations have suggested important roles for nanoscale dewetting in the stability, function, and folding dynamics of proteins. Using a synergistic simulation-experimental approach on the αTS TIM barrel protein, we validated this hypothesis by revealing the occurrence of drying inside hydrophobic amino acid clusters and its manifestation in experimental measures of protein stability and structure. Cavities created within three clusters of branched aliphatic amino acids [isoleucine, leucine, and valine (ILV) clusters] were found to experience strong water density fluctuations or intermittent dewetting transitions in simulations. Individually substituting 10 residues in the large ILV cluster at the N-terminus with less hydrophobic alanines showed a weakening or diminishing effect on dewetting that depended on the site of the mutation. Our simulations also demonstrated that replacement of buried leucines with isosteric, polar asparagines enhanced the wetting of the N- and C-terminal clusters. The experimental results on the stability, secondary structure, and compactness of the native and intermediate states for the asparagine variants are consistent with the preferential drying of the large N-terminal cluster in the intermediate. By contrast, the region encompassing the small C-terminal cluster experiences only partial drying in the intermediate, and its structure and stability are unaffected by the asparagine substitution. Surprisingly, the structural distortions required to accommodate the replacement of leucine by asparagine in the N-terminal cluster revealed the existence of alternative stable folds in the native basin. This combined simulation-experimental study demonstrates the critical role of drying within hydrophobic ILV clusters in the folding and stability of the αTS TIM barrel.

BACKGROUND: Triosephosphate isomerase (TIM) is a ubiquitous enzyme that has been targeted for the discovery of small molecular weight compounds with potential use against Trypanosoma cruzi, the causative agent of Chagas disease. We have identified a new selective inhibitor chemotype of TIM from T. cruzi (TcTIM), 1,2,4-thiadiazol-5(4H)-one.
OBJECTIVE: Study the mechanism of TcTIM inhibition by a 1,2,4-thiadiazol derivative.
METHODS: We performed the biochemical characterization of the interaction of the 1,2,4-thiadiazol derivative with the wild-type and mutant TcTIMs, using DOSY-NMR and MS experiments. Studies of T. cruzi growth inhibition were additionally carried out.
CONCLUSIONS: At low micromolar concentrations, the compound induces highly selective irreversible inactivation of TcTIM through non-covalent binding. Our studies indicate that it interferes with the association of the two monomers of the dimeric enzyme. We also show that it inhibits T. cruzi growth in culture.

Giardiasis, the most prevalent intestinal parasitosis in humans, is caused by Giardia lamblia. Current drug therapies have adverse effects on the host, and resistant strains against these drugs have been reported, demonstrating an urgent need to design more specific antigiardiasic drugs. ATP production in G. lamblia depends mainly on glycolysis; therefore, all enzymes of this pathway have been proposed as potential drug targets. We previously demonstrated that the glycolytic enzyme triosephosphate isomerase from G. lamblia (GlTIM), could be completely inactivated by low micromolar concentrations of thiol-reactive compounds, whereas, in the same conditions, the activity of human TIM (HuTIM) was almost unaltered. We found that the chemical modification (derivatization) of at least one Cys, of the five Cys residues per monomer in GlTIM, causes this inactivation. In this study, structural and functional studies were performed to describe the molecular mechanism of GlTIM inactivation by thiol-reactive compounds. We found that the Cys222 derivatization is responsible for GlTIM inactivation; this information is relevant because HuTIM has a Cys residue in an equivalent position (Cys217). GlTIM inactivation is associated with a decrease in ligand affinity, which affects the entropic component of ligand binding. In summary, this work describes a mechanism of inactivation that has not been previously reported for TIMs from other parasites and furthermore, we show that the difference in reactivity between the Cys222 in GlTIM and the Cys217 in HuTIM, indicates that the surrounding environment of each Cys residue has unique structural differences that can be exploited to design specific antigiardiasic drugs.

Predicting how point mutations in genes alter the tertiary and quarternary structure of proteins is central to a number of areas of molecular biology and has implications in relation to the function and evolution of molecules. In the present study, we theoretically assessed the effects of 20 point mutations detected previously in a region of the triose-phosphate isomerase gene (tpi) of the protozoan Giardia duodenalis on the three-dimensional structure of the \'wild-type\' protein (TPI). Amino acid substitutions arising from codon variations were mainly located at surface-accessible sites or in hydrophobic pockets of TPI. None of the substitutions was predicted to exert a significant change to the fold or functionality of the enzyme, with the exception of one alteration (Arg100). Almost all substitutions were either conservative or semi-conservative, and retained or even improved the expected stability of the fold. Overall, the findings provide support for the \"neutral theory\", which contends that evolution at the molecular level is not solely shaped by \"Darwinian selection but also by random drift of selectively neutral or nearly neutral mutants\".