The use of isothermal titration calorimetry for the assay of enzyme activity: Application in higher education practical classes.

Determination of enzyme activity is crucial for discovery, research, and development in life sciences. The activity of enzymes is routinely determined using spectrophotometric assays that measure rates of substrate consumption or product formation. Though colorimetric-based detection systems are simple, rapid, and economical to perform, the majority of enzymes are unsuitable for this technique as their substrates/products do not absorb in the UV or visible range. This limitation can be addressed by the use of coupled-enzyme assays or artificial chromogenic substrates; however these approaches have their own drawbacks. Here, we describe a method based on the use of an isothermal titration calorimeter (ITC) to measure the heat produced or absorbed during any enzyme-catalyzed reaction. The concept of calorimetric enzyme assays was demonstrated for the determination of enzyme hexokinase activity, which cannot be monitored colorimetrically without first coupling it to another enzymatic reaction. The assay is suitable for incorporation into undergraduate laboratory classes, providing students with an appreciation for; the versatility and ease of use of ITC assays; ITC as a flexible generic method for exploring the functional characteristics of uncharacterized enzymes; an activity detection parameter suitable for enzymes that either have no straightforward colorimetric methods available or require the use of nonartificial chromogenic substrates.

APOBEC SBS13 Mutational Signature-A Novel Predictor of Radioactive Iodine Refractory Papillary Thyroid Carcinoma.

Standard surgery followed by radioactive iodine (131I, RAI) therapy are not curative for 5−20% of papillary thyroid carcinoma (PTC) patients with RAI refractory disease. Early predictors indicating therapeutic response to RAI therapy in PTC are yet to be elucidated. Whole-exome sequencing was performed (at median depth 198x) on 66 RAI-refractory and 92 RAI-avid PTCs with patient-matched germline. RAI-refractory tumors were significantly associated with distinct aggressive clinicopathological features, including positive surgical margins (p = 0.016) and the presence of lymph node metastases at primary diagnosis (p = 0.012); higher nonsilent tumor mutation burden (p = 0.011); TERT promoter (TERTp) mutation (p < 0.0001); and the enrichment of the APOBEC-related single-base substitution (SBS) COSMIC mutational signatures 2 (p = 0.030) and 13 (p < 0.001). Notably, SBS13 (odds ratio [OR] 30.4, 95% confidence intervals [CI] 1.43−647.22) and TERTp mutation (OR 41.3, 95% CI 4.35−391.60) were revealed to be independent predictors of RAI refractoriness in PTC (p = 0.029 and 0.001, respectively). Although SBS13 and TERTp mutations alone highly predicted RAI refractoriness, when combined, they significantly increased the likelihood of predicting RAI refractoriness in PTC. This study highlights the APOBEC SBS13 mutational signature as a novel independent predictor of RAI refractoriness in a distinct subgroup of PTC.

How to calculate thermostability of enzymes using a simple approach.

Determination of thermostability of enzymes is of prime importance for their successful industrial applications and, yet, the published data has often been incompletely analyzed to assess the suitability of enzymes. It is possible to determine meaningful thermostability parameters from the routinely acquired data through a straightforward method that is not only more informative but also provides a means to compare thermostability of enzymes from different sources. Here, we describe a simple, effective, and economical way to determine enzyme thermostability. In our opinion, including this method in Biochemistry and Molecular Biology curricula will encourage students to include thermostability analysis in their future work, leading to a more meaningful approach to evaluate and compare enzymes. Furthermore, as the method requires minimum specialized equipment, the analysis will be particularly suitable for labs that cannot afford expensive setup. © 2018 by The International Union of Biochemistry and Molecular Biology, 46:398-402, 2018.

Risk-adapted stratification for optimally intensive treatment assignment of pediatric patients with non-Hodgkin lymphoma is an effective strategy in developing countries.

BACKGROUND: Pediatric patients with non-Hodgkin lymphoma (NHL) in developing countries (DCs) present with greater tumor load even at lower stages and with comorbidities that impact therapy delivery. This causes toxic mortality with "standard" intensive protocols or recurrences with "gentler" treatment. OBJECTIVES: We developed and evaluated a risk stratification schema that guides intensity of therapy. DESIGN/METHODS: Sixty-nine patients were prospectively assigned to five risk groups (A-E; n = 6, 15, 16, 15, and 17) following staging and treated with protocols of risk-stratified intensity. Risk stratification utilized St. Jude stage, disease bulk, and sites involved. RESULTS: Between 2006 and 2011, 69 patients with B-cell NHL were enrolled. Among these, 72.5% were boys with mean age of 6.9 years (±3.33 [SD]; range 2.4-14.2 years). Eighty-seven percent had Burkitt lymphoma, 82.6% had advanced stage (25 [36.2%] stage III; 32 [46.4%] stage IV), and 24.6% were central nervous system positive. Mean lactate dehydrogenase increased progressively across the risk strata. Among these, 0/6, 1/15, 3/16, 2/15, and 7/17 patients relapsed/progressed within each risk stratum. Fifteen patients died; three from treatment-related toxicity. At a median follow-up of 6.2 years, the overall and event-free survival (EFS) for all patients was 78.1 and 75.4%, respectively; EFS was related to risk assignment. The frequency of documented infectious and noninfectious toxicities increased with higher risk group assignment causing prolongation of admissions and potential treatment delays. CONCLUSIONS: Reduction in treatment intensity for an identified subset of patients with NHL is feasible, while high-intensity therapy is required for high-risk groups. This risk stratification system may be a first step toward improving the outcomes in some DCs.

Outcome of risk adapted therapy for relapsed/refractory acute lymphoblastic leukemia in children.

Results of second-line therapy for childhood acute lymphoblastic leukemia (ALL) remain suboptimal, particularly for high-risk groups identified using timing and site of relapse. We report results of prospectively collected data for pediatric patients with ALL who received risk adjusted second-line therapy. The 59 patients who failed first-line ALL therapy included 36 (61%) with bone marrow (BM), 13 (22.1%) with isolated extramedullary (EM) and 10 (16.9%) with BM + EM relapse. Some 51.8% patients were reinduced with high dose cytosine arabinoside (HDAraC)-based and 48.2% with standard four-drug regimens. In all, 38/56 (67.9%) achieved a complete remission (CR) with second-line therapy; the overall CR rate was 78.6% and was not associated with CR1 duration (p =0.8). Three-year overall survival (OS) was 45.3%, and was 61.4% for those achieving a CR. No risk group benefited from HSCT over chemotherapy. Patients with isolated EM relapse beyond 18 months of CR1 and BM relapse beyond 12 months off-therapy had an excellent outcome (OS 91.7%), identifying a particularly good-risk cohort. Patients not in this category continue with poor outcome even following hematopoietic stem cell transplant.

Identification of two residues essential for the stringent substrate specificity and active site stability of the prokaryotic l-arginine:glycine amidinotransferase CyrA.

A novel prokaryotic l-arginine:glycine amidinotransferase (CyrA; EC2.1.4.1) is involved in the biosynthesis of the polyketide-derived cytotoxin cylindrospermopsin in the cyanobacterium Cylindrospermopsis raciborskii AWT250, and was previously characterized with regard to kinetic mechanism and substrate specificity [Muenchhoff J et al. (2010) FEBS J277, 3844-3860]. In order to elucidate the structure-function-stability relationship of this enzyme, two residues in its active site were replaced with the residues that occur in the human l-arginine:glycine amidinotransferase (h-AGAT) at the corresponding positions (F245N and S247M), and a double variant carrying both substitutions was also created. In h-AGAT, both of these residues are critical for the function of this enzyme with regard to substrate binding, ligand-induced structural changes, and stability of the active site. In this study, we demonstrated that both single residue replacements resulted in a dramatic broadening of substrate specificity, but did not affect the kinetic mechanism. Experiments with substrate analogues indicate that donor substrates require a carboxylate group for binding. Evidence from initial velocity studies suggests that CyrA undergoes ligand-induced structural changes that involve Phe245. Stability parameters (T(opt) and T(max) ) of the CyrA variants differed from those of wild-type CyrA. Structural flexibilities of the wild type and all three variants were comparable on the basis of dynamic fluorescence quenching, indicating that changes in T(opt) are most likely attributable to localized effects within the active site. Overall, the results indicated that these two residues are essential for both stringent substrate specificity and the active site stability and flexibility of this unique cyanobacterial enzyme.

Chaperonins from an Antarctic archaeon are predominantly monomeric: crystal structure of an open state monomer.

Archaea are abundant in permanently cold environments. The Antarctic methanogen, Methanococcoides burtonii, has proven an excellent model for studying molecular mechanisms of cold adaptation. Methanococcoides burtonii contains three group II chaperonins that diverged prior to its closest orthologues from mesophilic Methanosarcina spp. The relative abundance of the three chaperonins shows little dependence on organism growth temperature, except at the highest temperatures, where the most thermally stable chaperonin increases in abundance. In vitro and in vivo, the M. burtonii chaperonins are predominantly monomeric, with only 23-33% oligomeric, thereby differing from other archaea where an oligomeric ring form is dominant. The crystal structure of an N-terminally truncated chaperonin reveals a monomeric protein with a fully open nucleotide binding site. When compared with closed state group II chaperonin structures, a large-scale ≈ 30° rotation between the equatorial and intermediate domains is observed resulting in an open nucleotide binding site. This is analogous to the transition observed between open and closed states of group I chaperonins but contrasts with recent archaeal group II chaperonin open state ring structures. The predominance of monomeric form and the ability to adopt a fully open nucleotide site appear to be unique features of the M. burtonii group II chaperonins.

The RNA polymerase subunits E/F from the Antarctic archaeon Methanococcoides burtonii bind to specific species of mRNA.

RNA polymerase in Archaea is composed of 11 or 12 subunits - 9 or 10 that form the core, and a heterodimer formed from subunits E and F that associates with the core and can interact with general transcription factors and facilitate transcription. While the ability of the heterodimer to bind RNA has been demonstrated, it has not been determined whether it can recognize specific RNA targets. In this study we used a recombinant archaeal MbRpoE/F to capture cellular mRNA in vitro and a microarray to determine which transcripts it specifically binds. Only transcripts for 117 genes (4% of the total) representing 48 regions of the genome were bound by MbRpoE/F. The transcripts represented important genes in a number of functional classes: methanogenesis, cofactor biosynthesis, nucleotide metabolism, transcription, translation, import/export. The arrangement and characteristics (e.g. codon and amino acid usage) of genes relative to the putative origin of replication indicate that MbRpoE/F preferentially binds to mRNA of genes whose expression may be important for cellular fitness. We also compared the biophysical properties of RpoE/F from M. burtonii and Methanocaldococcus jannaschii, demonstrating a 50°C difference in their apparent melting temperatures. By using MbRpoE/F to capture and characterize cellular RNA we have identified a previously unknown functional property of the MbRpoE/F heterodimer.

A novel prokaryotic L-arginine:glycine amidinotransferase is involved in cylindrospermopsin biosynthesis.

We report the first characterization of an L-arginine:glycine amidinotransferase from a prokaryote. The enzyme, CyrA, is involved in the pathway for biosynthesis of the polyketide-derived hepatotoxin cylindrospermopsin from Cylindrospermopsis raciborskii AWT205. CyrA is phylogenetically distinct from other amidinotransferases, and structural alignment shows differences between the active site residues of CyrA and the well-characterized human L-arginine:glycine amidinotransferase (AGAT). Overexpression of recombinant CyrA in Escherichia coli enabled biochemical characterization of the enzyme, and we confirmed the predicted function of CyrA as an L-arginine:glycine amidinotransferase by (1) H NMR. As compared with AGAT, CyrA showed narrow substrate specificity when presented with substrate analogs, and deviated from regular Michaelis-Menten kinetics in the presence of the non-natural substrate hydroxylamine. Studies of initial reaction velocities and product inhibition, and identification of intermediate reaction products, were used to probe the kinetic mechanism of CyrA, which is best described as a hybrid of ping-pong and sequential mechanisms. Differences in the active site residues of CyrA and AGAT are discussed in relation to the different properties of both enzymes. The enzyme had maximum activity and maximum stability at pH 8.5 and 6.5, respectively, and an optimum temperature of 32 °C. Investigations into the stability of the enzyme revealed that an inactivated form of this enzyme retained an appreciable amount of secondary structure elements even on heating to 94 °C, but lost its tertiary structure at low temperature (T(max) of 44.5 °C), resulting in a state reminiscent of a molten globule. CyrA represents a novel group of prokaryotic amidinotransferases that utilize arginine and glycine as substrates with a complex kinetic mechanism and substrate specificity that differs from that of the eukaryotic L-arginine:glycine amidinotransferases.

The genome sequence of the psychrophilic archaeon, Methanococcoides burtonii: the role of genome evolution in cold adaptation.

Psychrophilic archaea are abundant and perform critical roles throughout the Earth's expansive cold biosphere. Here we report the first complete genome sequence for a psychrophilic methanogenic archaeon, Methanococcoides burtonii. The genome sequence was manually annotated including the use of a five-tiered evidence rating (ER) system that ranked annotations from ER1 (gene product experimentally characterized from the parent organism) to ER5 (hypothetical gene product) to provide a rapid means of assessing the certainty of gene function predictions. The genome is characterized by a higher level of aberrant sequence composition (51%) than any other archaeon. In comparison to hyper/thermophilic archaea, which are subject to selection of synonymous codon usage, M. burtonii has evolved cold adaptation through a genomic capacity to accommodate highly skewed amino-acid content, while retaining codon usage in common with its mesophilic Methanosarcina cousins. Polysaccharide biosynthesis genes comprise at least 3.3% of protein coding genes in the genome, and Cell wall, membrane, envelope biogenesis COG genes are overrepresented. Likewise, signal transduction (COG category T) genes are overrepresented and M. burtonii has a high 'IQ' (a measure of adaptive potential) compared to many methanogens. Numerous genes in these two overrepresented COG categories appear to have been acquired from epsilon- and delta-Proteobacteria, as do specific genes involved in central metabolism such as a novel B form of aconitase. Transposases also distinguish M. burtonii from other archaea, and their genomic characteristics indicate they have an important role in evolving the M. burtonii genome. Our study reveals a capacity for this model psychrophile to evolve through genome plasticity (including nucleotide skew, horizontal gene transfer and transposase activity) that enables adaptation to the cold, and to the biological and physical changes that have occurred over the last several thousand years as it adapted from a marine to an Antarctic lake environment.

Clinical characteristics and outcome of pediatric patients with stage IV Hodgkin lymphoma.

BACKGROUND AND OBJECTIVES: While treatment outcomes for patients with Hodgkin lymphoma (HL) have improved remarkably, patients with disseminated disease still have a poorer outcome. Stage IV HL is often reported with other 'advanced stage' categories, confusing the specific contribution of disease dissemination to the outcome. This single-institution report looks at characteristics and outcomes of this specific category. PATIENTS AND METHODS: The medical records of pediatric HL patients (< 14 years) from 1975 through 2003 were retrospectively reviewed and the data analyzed. RESULTS: Stage IV patients (n = 67) had more poor-risk characteristics than patients in stages I-III (n = 300) (B symptoms 86.6% vs. 19.3%, bulky disease 57.6% vs. 45.5% and mediastinal mass 77.6% vs. 29.7%; P < .001 for all characteristics). The liver was the most common extralymphatic site (in 51.5% of patients with stage IV disease. Stage IV patients received chemotherapy (CT) alone (n = 55) or combined modality therapy (CMT) (n = 12). Fifty-four patients (80.6%) achieved complete remission, 2 (3%) partial remission, 10 (14.9%) had progressive disease and 1 was lost to follow up. Overall survival was 79.4% and event-free survival (EFS) was 63.9% at 5 years. There was a non-significant benefit for CMT (OS = 91.7% v. 77.1%, P = .3; EFS = 70.7% v. 62.7%, P = .3). Ten of 12 relapsed and only 1 of 10 progressive disease patients were salvaged. On multivariate analysis, failure to achieve complete remission with CT was associated with a poorer outcome. CONCLUSION: Stage IV disease is associated with poor risk features and confers a worse outcome than stage I-III disease. Achievement of complete remission with CT is an important prognostic feature. Slow responders may require novel and/or aggressive therapy to achieve complete remission.

Structure and function of cold shock proteins in archaea.

Archaea are abundant and drive critical microbial processes in the Earth's cold biosphere. Despite this, not enough is known about the molecular mechanisms of cold adaptation and no biochemical studies have been performed on stenopsychrophilic archaea (e.g., Methanogenium frigidum). This study examined the structural and functional properties of cold shock proteins (Csps) from archaea, including biochemical analysis of the Csp from M. frigidum. csp genes are present in most bacteria and some eucarya but absent from most archaeal genome sequences, most notably, those of all archaeal thermophiles and hyperthermophiles. In bacteria, Csps are small, nucleic acid binding proteins involved in a variety of cellular processes, such as transcription. In this study, archaeal Csp function was assessed by examining the ability of csp genes from psychrophilic and mesophilic Euryarchaeota and Crenarchaeota to complement a cold-sensitive growth defect in Escherichia coli. In addition, an archaeal gene with a cold shock domain (CSD) fold but little sequence identity to Csps was also examined. Genes encoding Csps or a CSD structural analog from three psychrophilic archaea rescued the E. coli growth defect. The three proteins were predicted to have a higher content of solvent-exposed basic residues than the noncomplementing proteins, and the basic residues were located on the nucleic acid binding surface, similar to their arrangement in E. coli CspA. The M. frigidum Csp was purified and found to be a single-domain protein that folds by a reversible two-state mechanism and to exhibit a low conformational stability typical of cold-adapted proteins. Moreover, M. frigidum Csp was characterized as binding E. coli single-stranded RNA, consistent with its ability to complement function in E. coli. The studies show that some Csp and CSD fold proteins have retained sufficient similarity throughout evolution in the Archaea to be able to function effectively in the Bacteria and that the function of the archaeal proteins relates to cold adaptation. The initial biochemical analysis of M. frigidum Csp has developed a platform for further characterization and demonstrates the potential for expanding molecular studies of proteins from this important archaeal stenopsychrophile.

The active site is the least stable structure in the unfolding pathway of a multidomain cold-adapted alpha-amylase.

The cold-active alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30 degrees C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was performed under various conditions of temperature (3 degrees C to 70 degrees C) and pH (7.5 to 10.4) in the absence or presence of Ca2+ and/or Tris (competitive inhibitor) to identify possible low-stability domains. Contrary to previous observations by strict thermal unfolding, two transitions were found at low temperature (12 degrees C). Within the duration of the TUG-GE, the structures undergoing the first transition showed slow interconversions between different conformations. By comparing the properties of the native enzyme and the N12R mutant, the active site was shown to be part of the least stable structure in the enzyme. The stability data supported a model of cooperative unfolding of structures forming the active site and independent unfolding of the other more stable protein domains. In light of these findings for AHA, it will be valuable to determine if active-site instability is a general feature of heat-labile enzymes from psychrophiles. Interestingly, the enzyme was also found to refold and rapidly regain activity after being heated at 70 degrees C for 1 h in 6.5 M urea. The study has identified fundamental new properties of AHA and extended our understanding of structure/stability relationships of cold-adapted enzymes.

Low-temperature extremophiles and their applications.

Psychrophilic (cold-adapted) organisms and their products have potential applications in a broad range of industrial, agricultural and medical processes. In order for growth to occur in low-temperature environments, all cellular components must adapt to the cold. This fact, in combination with the diversity of Archaea, Bacteria and Eucarya isolated from cold environments, highlights the breadth and type of biological products and processes that might be exploited for biotechnology. Relative to this undisputed potential, psychrophiles and their products are under-utilised in biotechnology; however, recent advances, particularly with cold-active enzymes, herald rapid growth for this burgeoning field.

Coupling of surface carboxyls of carboxymethylcellulase with aniline via chemical modification: extreme thermostabilization in aqueous and water-miscible organic mixtures.

We wish to report the attainment of the highest ever T(opt) by introducing approximately two aromatic rings through chemical modification of surface carboxyl groups in carboxymethylcellulase from Scopulariopsis sp. with concomitant decrease in V(max), K(m), and optimum pH! This extraordinary enhancement in thermophilicity of aniline-coupled CMCase (T(opt) = 122 degrees C) by a margin of 73 degrees C as compared with the native enzyme (T(opt) = 49 degrees C) is the highest reported for any mesophilic enzyme that has been modified either through chemical modification or site-directed mutagenesis. It is also reported for the first time that aniline coupled CMCase (ACC) is simultaneously thermostable in aqueous as well as water-miscible organic solvents. The T(opt) of native CMCase and ACC were 25 and 90 degrees C, respectively, in 40% (v/v) aqueous dioxan. The modified enzyme was also stabilized against irreversible thermal denaturation. Therefore, at 55 degrees C, ACC had a half-life of 136 min as compared with native CMCase whose half-life was only 5 min. We believe that the reasons for this elevated thermostability and thermophilicity are surface aromatic-aromatic interactions and aromatic interactions with the sugar backbone of the substrate, respectively.