Isoenzyme characterization of Leishmania infantum toward checking the antioxidant activity of superoxide dismutase and glutathione peroxidase.

BACKGROUND: Leishmania infantum is the major causative agent of visceral leishmaniasis in Mediterranean regions. Isoenzyme electrophoresis (IE), as a biochemical technique, is applied in the characterization of Leishmania species. The current study attempted to investigate the isoenzyme patterns of logarithmic and stationary promastigotes and axenic amastigotes (amastigote-like) of L. infantum using IE. The antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX) was also checked in the aforementioned forms. METHOD: After L. infantum cultivation and obtaining logarithmic and stationary promastigotes, axenic amastigotes were achieved by incubation of stationary promastigotes at 37 °C for 48 h. The lysate samples were prepared and examined for six enzymatic systems including glucose-6-phosphate dehydrogenase (G6PD), nucleoside hydrolase 1 (NH1), malate dehydrogenase (MDH), glucose-phosphate isomerase (GPI), malic enzyme (ME), and phosphoglucomutase (PGM). Additionally, the antioxidant activity of SOD and GPX was measured. RESULTS: GPI, MDH, NH1, and G6PD enzymatic systems represented different patterns in logarithmic and stationary promastigotes and axenic amastigotes of L. infantum. PGM and ME showed similar patterns in the aforementioned forms of parasite. The highest level of SOD activity was determined in the axenic amastigote form and GPX activity was not detected in different forms of L. infantum. CONCLUSION: The characterization of leishmanial-isoenzyme patterns and the measurement of antioxidant activity of crucial antioxidant enzymes, including SOD and GPX, might reveal more information in the biology, pathogenicity, and metabolic pathways of Leishmania parasites and consequently drive to designing novel therapeutic strategies in leishmaniasis treatment.

Disposable stochastic sensors obtained by nanolayer deposition of copper, graphene, and copper-graphene composite on silk for the determination of isocitrate dehydrogenases 1 and 2.

Three disposable stochastic sensors designed using nanolayer deposition of copper (Cu), graphene (GR), and copper-graphene (Cu-GR) composite on the silk textile, as substrate, were modified with chitosan (n=371-744), for biomedical analysis. Isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) served as model analytes for molecular recognition and quantification in biological samples such as whole blood and brain tumor tissue samples. The best sensitivities (3.77×107s µg mL-1 for IDH1, and 1.88×107s µg mL-1 for IDH2) and the lowest limits of quantification (10-2fg mL-1 for IDH1, and 5×10-2fg mL-1 for IDH2) for both IDH1 and IDH2 were recorded for the disposable stochastic sensors based on chitosan/graphene nanolayer. Very good correlations between the screening method based on disposable stochastic sensors and enzyme-linked immunosorbent assay (ELISA) were obtained; this was also proved by the results obtained using the paired t-test.

Diagnostic utility of measuring lactate dehydrogenase levels and its isoenzyme activities for the evaluation of malignancy in feline pleural effusion and ascitic fluid.

Background: Lactate dehydrogenase (LDH) isoenzymes may be useful in the differential diagnosis of pleural effusion (PE) and ascitic fluid (AF) etiologies in cats since tissue damage induces their release, changing the pattern of their activity. Aim: This study aimed to determine the diagnostic utility of measuring LDH levels and isoenzyme activities in PE or AF in cats with malignancy. Methods: LDH levels and isoenzyme activities in the serum, PE, and AF were compared among cats in the malignant, infectious, and non-malignant, non-infectious groups. A receiver operating characteristic (ROC) analysis was performed to assess the accuracy in diagnosing feline malignancy. Results: Significant differences in LDH level and LDH isoenzyme activities in the PE and AF were observed among the three groups. The combination of LDH level and LDH-1 activity in PE or AF had the highest area under the ROC (AUC) values for discriminating malignant effusion from non-malignant effusion. The AUC of the combination of LDH level and LDH-1 activity in PE or AF was 0.874. The sensitivity and specificity of using the combination of LDH level (cut-off: <2,269 U/l) and LDH-1 activity (cut-off: <4.8%) in PE or AF for predicting malignancy with the highest AUC value were 94.4% and 72.7%, respectively. Conclusion: Our results suggest that the combination of LDH level and LDH-1 activity in PE or AF is a potential factor for diagnosing malignancy. Considering that LDH isoenzymes can be measured inexpensively and easily, LDH tests can be readily accommodated in veterinary clinical practice.

Analytical evaluation of the Nittobo Medical tartrate resistant acid phosphatase isoform 5b (TRACP-5b) EIA and comparison with IDS iSYS in different clinically defined populations.

OBJECTIVES: Tartrate-resistant acid phosphatase, isoform 5b (TRACP-5b) is a bone resorption marker not influenced by renal function or food intake. TRACP-5b can be measured with Nittobo Medical enzymatic-immunoassay and IDS-iSYS automated immunoassay. We evaluated the Nittobo assay and established reference ranges for a Western-European population. We compared Nittobo and IDS results in different well-defined clinical populations. METHODS: We established the limits of detection and quantification (LOD-LOQ), linearity, imprecision and the reference ranges in 119 males, 50 women (<45 years) and 120 women (>60 years) for TRACP-5b with the Nittobo assay. We compared both assays in 30 hemodialyzed (HD), and 40 stage 3-5 patients suffering from chronic kidney disease (CKD), 40 patients suffering from rheumatoid arthritis and osteoporosis and 80 post-menopausal women. We measured TRACP-5b, ß-crosslaps (ß-CTX), bone alkaline phosphatase (B-ALP) and PTH in 20 hemodialyzed (HD) and 40 CKD patients. RESULTS: LOD and LOQ were 0.02 and 0.35 U/L. CV ranged from 8.3 to 4.3% (2/5 samples presenting CV > desirable CV). Method was linear up to of 11.3 U/L. Upper and lower limits of normality were 0.8-7.6 U/L in men, 0.9-4.7 U/L in women <45 and 0.9-7.1 U/L in women >60. The regression equation between the 2 methods was Nittobo = 1.13 (95% CI: 1.09-1.16) × iSYS - 0.4 (95% CI: -0.5; -0.3). TRACP-5b and b-ALP were in their respective reference ranges for most of CKD and HD patients. That was not the case for ß-CTX, which increased with decreasing eGFR. CONCLUSIONS: Nittobo TRACP-5b presents interesting analytical features and a good concordance with IDS iSYS. These methods could thus potentially be harmonized.

Cancer Stem Cell Markers, CD44 and ALDH1, for Assessment of Cancer Risk in OPMDs and Lymph Node Metastasis in Oral Squamous Cell Carcinoma.

Tumour heterogeneity in oral cancer is attributed to the presence of cancer stem cells (CSCs). CSCs are the most migratory and metastatic cellular subpopulation within tumours. Assessment of CSC markers as significant predictors of lymph node metastasis may prove valuable in the clinical setting. Furthermore, analysis of this panel of putative stem cell markers in oral dysplasia may additionally inform of the likelihood for oral potentially malignant disorders (OPMDs) to progress to oral squamous cell carcinoma (OSCC). The present study aims to assess the significance of CSC markers in the progression of OPMDs to OSCC and assessment of lymph node metastasis in OSCC. CD44 and ALDH1 were assessed immunohistochemically in 25 normal, 30 OPMDs, and 24 OSCCs. CD44 is a membranous marker and ALDH1 is a cytoplasmic marker. The immunohistochemical expression of these markers were compared between OPMDs with and without dysplasia, as well as between low-risk and high-risk dysplasias. Similarly, expression was compared between OSCC with and without lymph node metastasis and among grades of OSCC. Positive CD44 expression was seen in all normal mucosal tissues. The expression decreased from normal epithelium to OPMDs but increased in OSCC. CD44 expression was positive in 21 cases of OSCC (87.5%) and reduced from well-differentiated to poorly differentiated OSCC. CD44 staining index was higher in OSCC without lymph node metastasis (3.59) when compared with OSCC with lymph node metastasis (1.33). There was a statistically significant difference observed in the ALDH1 staining index among three groups (p < 0.05), with highest expression seen in OSCC. Within OPMDs, the ALDH1 staining index was statistically higher in OPMDs with dysplasia as compared to OPMDs without dysplasia. Furthermore, the expression was higher in OPMDs with high-risk dysplasia when compared with low-risk dysplasia, but this was not statistically significant (p = 0.82). In conclusion, The CD44 positive population possesses properties of CSCs in head and neck carcinoma, and continuous shedding could be found after CD44 down-regulation. The present study reports differences in ALDH1 expression between OPMDs with and without dysplasia, dysplastic and non-dysplastic epithelia, and low-risk and high-risk dysplasia. These findings may suggest ALDH1 as a specific marker for dysplasia. CD44 demonstrated a difference in staining index in OSCC without lymph node metastasis versus OSCC with lymph node metastasis. These findings may suggest CD44 as a marker for lymph node metastasis. Both proteins may play key roles in the tumorigenicity of CSCs in OPMDs and OSCC.

Changes of adenosine deaminase activity in serum and saliva around parturition in sows with and without postpartum dysgalactia syndrome.

BACKGROUND: Postpartum dysgalactia syndrome (PDS) is associated with a significantly higher activation of the inflammatory and stress response at parturition than in the healthy sow. Therefore, reliable and possibly non-invasive biomarkers for substantial increases of inflammation are searched to support the PDS diagnosis. This report studies the possible changes of the inflammatory marker enzyme adenosine deaminase (ADA) in serum and saliva of 38 PDS positive sows (PDS+) and 38 healthy sows (PDS-). Sampling was performed every 24 h from 60 h before to 36 h after parturition. Isoenzyme 1 (ADA1) and isoenzyme 2 (ADA2), as well as total ADA (tADA), were measured and their statistical association with several serum and saliva biomarkers of inflammation and stress was investigated. RESULTS: Compared to a baseline (60 to 36h prepartum), salivary activities of ADA1, ADA2 and tADA increased significantly over time in both PDS+ and PDS- sows, reaching their peaks after parturition. In serum from PDS- sows, no changes were observed over time in either ADA1, ADA2 or tADA. In PDS+ sows, serum ADA2 activity decreased temporarily after parturition followed by a significant increase compared to baseline. ADA1, ADA2 and tADA were all significantly associated with several inflammatory biomarkers and ADA1 in serum was associated with serum cortisol. Although serum activity was higher in PDS+ than in PDS- sows, the differences were not statistically significant. Further, no difference was noted between the groups in the analyses of saliva. CONCLUSIONS: Salivary ADA1 and ADA2 increased in all sows after parturition, potentially as a response to the postpartum inflammation. However, no difference in the activity of ADA1, ADA2 and tADA were found between PDS+ and PDS- sows indicating inability to diagnose PDS under the conditions described in this report.

Hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC in conjunction with Lgl.

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here, we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell-cell contacts. In 3D Matrigel culture of human hepatocytic HepG2 cells, which mimics a process of liver development and regeneration, depletion of Par3, Cdc42, or aPKC results in an impaired establishment of apicobasolateral polarity and a loss of subsequent apical lumen formation. The aPKC activity is also required for bile canalicular (apical) elongation in mouse primary hepatocytes. The lateral membrane-associated proteins Lgl1 and Lgl2, major substrates of aPKC, seem to be dispensable for hepatocyte polarity establishment because Lgl-depleted HepG2 cells are able to form a single apical lumen in 3D culture. On the other hand, Lgl depletion leads to lateral invasion of aPKC, and overexpression of Lgl1 or Lgl2 prevents apical lumen formation, indicating that they maintain proper lateral integrity. Thus, hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC; Par3 cooperates with Cdc42 to recruit aPKC, which plays a crucial role in apical membrane development and regulation of the lateral maintainer Lgl.

Cytotoxicity, metabolism, and isozyme mapping of the synthetic cannabinoids JWH-200, A-796260, and 5F-EMB-PINACA studied by means of in vitro systems.

Intake of synthetic cannabinoids (SC), one of the largest classes of new psychoactive substances, was reported to be associated with acute liver damage but information about their hepatotoxic potential is limited. The current study aimed to analyze the hepatotoxicity including the metabolism-related impact of JWH-200, A-796260, and 5F-EMB-PINACA in HepG2 cells allowing a tentative assessment of different SC subclasses. A formerly adopted high-content screening assay (HCSA) was optimized using a fully automated epifluorescence microscope. Metabolism-mediated effects in the HCSA were additionally investigated using the broad CYP inhibitor 1-aminobenzotriazole. Furthermore, phase I metabolites and isozymes involved were identified by in vitro assays and liquid chromatography-high-resolution tandem mass spectrometry. A strong cytotoxic potential was observed for the naphthoylindole SC JWH-200 and the tetramethylcyclopropanoylindole compound A-796260, whereas the indazole carboxamide SC 5F-EMB-PINACA showed moderate effects. Numerous metabolites, which can serve as analytical targets in urine screening procedures, were identified in pooled human liver microsomes. Most abundant metabolites of JWH-200 were formed by N-dealkylation, oxidative morpholine cleavage, and oxidative morpholine opening. In case of A-796260, most abundant metabolites included an oxidative morpholine cleavage, oxidative morpholine opening, hydroxylation, and dihydroxylation followed by dehydrogenation. Most abundant 5F-EMB-PINACA metabolites were generated by ester hydrolysis plus additional steps such as oxidative defluorination and hydroxylation. To conclude, the data showed that a hepatotoxicity of the investigated SC cannot be excluded, that metabolism seems to play a minor role in the observed effects, and that the extensive phase I metabolism is mediated by several isozymes making interaction unlikely.

Pattern of placental alkaline phosphatase (PLAP) expression in human tumors: a tissue microarray study on 12,381 tumors.

Placental alkaline phosphatase (PLAP) is commonly expressed at high levels in testicular germ cell tumors. PLAP immunohistochemistry (IHC) is thus often used to confirm this diagnosis, especially in cases of putative metastasis. However, other tumors can also express PLAP. To comprehensively determine PLAP expression in normal and tumor tissue, a tissue microarray containing 16,166 samples from 131 different tumor types and subtypes as well as 608 samples from 76 different normal tissue types was analyzed by IHC. Moderate to strong PLAP positivity was found in 27 (21%) of 131 different tumor types including seminoma (96%), embryonal carcinoma (85%), and yolk sac tumors of the testis (56%); endometrioid carcinoma of the endometrium (28%) and the ovary (20%); gastric adenocarcinoma (22%); serous carcinoma (not otherwise specified) of the ovary (17%) and the uterus (11%); adenocarcinoma of the ampulla of Vater (15%); carcinosarcoma of the ovary (11%) and the uterus (8%); esophageal adenocarcinoma (10%); invasive urothelial carcinoma (4%); cholangiocarcinoma (2%); and adenocarcinoma of the lung (1%). Low-level PLAP immunostaining, often involving only a small fraction of tumor cells, was seen in 21 additional tumor entities. The clinical significance of PLAP expression may vary between tumor types as high PLAP expression was linked to advanced pathological tumor stage (p = 0.0086), nodal metastasis (p = 0.0085), and lymphatic (p = 0.0007) and blood vessel invasion (p = 0.0222) in colorectal cancer, but to low pathological tumor stage in endometrial cancer (p = 0.0043). In conclusion, our data identify several tumor entities that can show PLAP expression at comparable levels to testicular germ cell tumors. These tumor entities need to be considered in cases of PLAP-positive metastasis. Low-level PLAP expression can be found in various other tumor entities and should generally not be viewed as a strong argument for germ cell neoplasia.

Caraway (Carum carvi L.): Anther Culture and Production of DH Plants Caraway.

We describe the production of doubled haploids through anther culture in caraway. Induction conditions for the cultivation of donor plants, anther collection, composition of culture media, and physical induction conditions for embryogenesis have been described. As a result, responsive lines with numerous haploid embryo production were obtained, which after colchicine treatment became fertile. From a practical point of view, two doubled haploid populations are tested under field conditions.

Production of Homozygous Carrot (Daucus carota L.) Plants by Anther Culture.

Carrot is a vegetable of increasing economic importance. New hybrid cultivars are constantly required to meet the changing market needs. The application of anther culture significantly shortens the difficult and long-lasting breeding of carrot. We examined all the stages of the process of generating androgenic plants: induction of embryos in anther cultures, regeneration and acclimatization of produced plants, their evaluation, ploidy and homozygosity, and many other factors affecting their effectiveness. Every factor has been optimized by experimentally selecting the optimal level. As a result, a full protocol of producing homozygous plants using anther cultures was developed, which is presented in this chapter.

Core-Shell Nanozymes "Artificial Peroxidase": Stability with Superior Catalytic Properties.

We report on the nanoparticles composed of the catalytically synthesized Prussian Blue (PB) core stabilized with the nickel hexacyanoferrate (NiHCF) shell. Catalyzing hydrogen peroxide reduction, the resulting nanozymes (ø = 66 nm) display catalytic rate constants, which for pyrogallol or ferrocyanide are, respectively, 25 and 35 times higher than those for peroxidase enzyme. After more than half a year of storage at a room temperature, the core-shell PB-NiHCF nanozymes retain both their size and physicochemical properties; such stability is unreachable for the enzymes. Being immobilized, core-shell PB-NiHCF nanozymes (ø = 45 nm) result in a hydrogen peroxide sensor with a sensitivity similar to that of the sensor based on sole PB nanoparticles. However, whereas the latter response in hard inactivating conditions (25 min in 1 mM H2O2) drops down to 7.5%, the PB-NiHCF nanozymes-based sensor retains >75% of initial sensitivity. Application of the core-shell PB-NiHCF nanozymes "artificial peroxidase" would obviously open new horizons in elaboration of anti-inflammatory drugs and (bio)sensors.

NAT1 is a critical prognostic biomarker and inhibits proliferation of colorectal cancer through modulation of PI3K/Akt/mTOR.

The aim of this study was to analyze the correlations between NAT1 and clinicopathological features of and prognosis in colorectal cancer (CRC). RNA sequencing data and clinical information were retrieved from The Cancer Genome Atlas database. Wilcoxon test, logistic regression and Kaplan-Meier method were used to estimate the association between NAT1 and prognosis in CRC. In vitro experiments were conducted to confirm the role of NAT1. NAT1 is significantly less expressed in CRC and independently associated with poor prognosis in CRC patients. The authors further confirmed that expression of NAT1 was significantly lower in SW116 colon cancer cells than in NCM460 cells. Overexpressed NAT1 obviously inhibited the growth of CRC cells by downregulating phosphorylation of the PI3K/Akt/mTOR signaling pathway. NAT1 may be a potential therapeutic target for CRC.

Lay abstract Colorectal cancer (CRC) is a common malignancy worldwide. Because of the limited understanding of the pathogenesis and prognostic factors associated with CRC, the treatment effect in CRC remains poor. In the present study, the authors demonstrate that NAT1 is significantly less expressed in CRC and independently associated with poor prognosis in CRC patients. NAT1 may exert antitumor activity by inhibiting phosphorylation of the PI3K/Akt/mTOR signaling pathway. These results suggest that NAT1 may be a prognostic factor in and therapeutic target for CRC.

In-Depth Glycan Characterization of Therapeutic Glycoproteins by Stepwise PGC SPE and LC-MS/MS.

Glycosylation of biologics, an important factor in pharmacological functions such as efficacy, safety, and biological activity, is easily affected by subtle changes in the cellular environment. Therefore, comprehensive and in-depth glycan characterization of therapeutic glycoproteins should be performed to ensure product quality and process consistency, but it is analytically challenging due to glycan microheterogeneity occurring in the glycan biosynthesis pathway. LC-based chromatographic separation combined with mass spectrometry (MS) has been widely used as a prominent tool for the qualitative and quantitative analysis of glycosylation of therapeutic glycoproteins. However, prior to LC/MS analysis, glycans are selectively captured and fractionated by solid-phase extraction (SPE) utilizing physicochemical characteristics for comprehensive characterization of a wide range of glycan heterogeneity on glycoengineered therapeutic proteins. In particular, porous graphitized carbon (PGC) SPE has been employed as a useful technique for the fractionation of native glycans having different sizes and polarities. Here, we describe a systematic method for comprehensive glycan characterization of therapeutic proteins using stepwise PGC SPE and LC/MS.

Cellular studies of the two main isoforms of human d-aspartate oxidase.

Human d-aspartate oxidase (hDASPO) is a FAD-dependent enzyme responsible for the degradation of d-aspartate (d-Asp). In the mammalian central nervous system, d-Asp behaves as a classical neurotransmitter, it is thought to be involved in neural development, brain morphology and behavior, and appears to be involved in several pathological states, such as schizophrenia and Alzheimer's disease. Apparently, the human DDO gene produces alternative transcripts encoding for three putative hDASPO isoforms, constituted by 341 (the 'canonical' form), 369, and 282 amino acids. Despite the increasing interest in hDASPO and its physiological role, little is known about these different isoforms. Here, the additional N-terminal peptide present in the hDASPO_369 isoform only has been identified in hippocampus of Alzheimer's disease female patients, while peptides corresponding to the remaining part of the protein were present in samples from male and female healthy controls and Alzheimer's disease patients. The hDASPO_369 isoform was largely expressed in E. coli as insoluble protein, hampering with its biochemical characterization. Furthermore, we generated U87 human glioblastoma cell clones stably expressing hDASPO_341 and, for the first time, hDASPO_369 isoforms; the latter protein showed a lower expression compared with the canonical isoform. Both protein isoforms are active (showing similar kinetic properties), localize to the peroxisomes, are very stable (a half-life of approximately 100 h has been estimated), and are primarily degraded through the ubiquitin-proteasome system. These studies shed light on the properties of hDASPO isoforms with the final aim to clarify the mechanisms controlling brain levels of the neuromodulator d-Asp.

Fluorogenic XY-69 in Lipid Vesicles for Measuring Activity of Phospholipase C Isozymes.

Mammalian phospholipase C (PLC) isozymes are major signaling nodes that regulate a wide range of cellular processes. Dysregulation of PLC activity has been associated with a growing list of human diseases such as cancer and Alzheimer's disease. However, methods to directly and continuously monitor PLC activity at membranes with high sensitivity and throughput are still lacking. We have developed XY-69, a fluorogenic PIP2 analog, which can be efficiently hydrolyzed by PLC isozymes either in solution or at membranes. Here, we describe the optimized assay conditions and protocol to measure the activity of PLC-γ1 (D1165H) with XY-69 in lipid vesicles. The described protocol also applies to other PLC isozymes.

Specificity of the lactate dehydrogenase isoenzyme index as a preoperative screen for uterine sarcoma before myomectomy.

OBJECTIVE: To evaluate the applicability of the Uterine mass Magna Graecia (UMG) risk index (elevation defined by a lactate dehydrogenase isoenzyme index >29) in women undergoing surgery for benign fibroids and to determine whether other factors were associated with an elevated index. An elevated UMG index has been reported to be associated with an increased risk of uterine sarcoma in Italian women. DESIGN: Retrospective cohort study. SETTING: University fibroid center. PATIENTS: All women presenting from July 1, 2013, through June 30, 2019, with fibroids who had lactate dehydrogenase isoenzymes collected and surgery performed. INTERVENTIONS: Calculation of UMG index. MAIN OUTCOME MEASURE: Applicability of UMG index. RESULTS: Of 272 patients initially identified, 179 met inclusion criteria, 163 with UMG index ≤29 and 16 with UMG index >29. There were no cases of uterine sarcoma. Race, age, and presence of endometriosis, adenomyosis, or degenerating fibroids were not predictors of elevated UMG index. Body mass index (BMI) was positively associated with elevated UMG index. Specificity of UMG index to exclude uterine sarcoma was 91.1% (163/179) and higher in non-obese (BMI<30; 95.1%) than obese women (85.5%). CONCLUSION: A previously reported UMG index cutoff of 29 had a specificity of 91.1% (higher with normal BMI and lower when obese) in our patient population. Although lower than previously reported, the index could be a useful initial method of preoperative screening of women with symptomatic fibroids. Higher BMI appears to be associated with elevated UMG indices, increasing the false-positive rate in obese women.

Optimization of a Colorimetric Assay to Determine Lactate Dehydrogenase B Activity Using Design of Experiments.

Lactate dehydrogenase B (LDH-B) is overexpressed in lung and breast cancer, and it has been considered as a potential target to treat these types of cancer. Herein, we propose a straightforward incomplete factorial (IF) design composed of 12 combinations of two reaction buffers, three pH values, three salt (NaCl) concentrations, and three incubation times, which we called IF-BPST (Buffer/pH/Salt/Time), for the optimization of a colorimetric LDH-B assay in a final volume of 100 µL using 96-well plates. The assay is based on the absorbance change at ~570 nm and the color change of the reaction mixture due to the release of NADH that reacts with nitroblue tetrazolium (NBT) and phenazine methosulfate (PMS), resulting in the formation of a blue-purple formazan. The results obtained using the IF-BPST were comparable with those obtained by response surface methodology. Our work revealed that the NBT/PMS assay with some modifications can be used to measure the activity of LDH-B and other dehydrogenases in a high-throughput screening format at the early stages of drug discovery. LDH-B containing lysates cannot be assayed directly, however, due to the sensitivity of the method toward detergents. Thus, we suggest precipitating the proteins in the lysates to remove the interfering detergents, and then to dissolve the protein pellet in a suitable buffer and carry out the assay.

Advances in Activity-Based Sensing Probes for Isoform-Selective Imaging of Enzymatic Activity.

Until recently, there were no generalizable methods for assessing the effects of post-translational regulation on enzymatic activity. Activity-based sensing (ABS) has emerged as a powerful approach for monitoring small-molecule and enzyme activities within living systems. Initial examples of ABS were applied for measuring general enzymatic activity; however, a recent focus has been placed on increasing the selectivity to monitor a single enzyme or isoform. The highest degree of selectivity is required for differentiating between isoforms, where the targets display significant structural similarities as a result of a gene duplication or alternative splicing. This Minireview highlights key examples of small-molecule isoform-selective probes with a focus on the relevance of isoform differentiation, design strategies to achieve selectivity, and applications in basic biology or in the clinic.

Insights of tankyrases: A novel target for drug discovery.

Tankyrases are the group of enzymes belonging to a class of Poly (ADP-ribose) polymerase (PARP) recently named ADP-ribosyltransferase (ARTD). The two isoforms of tankyrase i.e. tankyrase1 (TNKS1) and tankyrase2 (TNKS2) were abundantly expressed in various biological functions in telomere regulation, Wnt/ß-catenin signaling pathway, viral replication, endogenous hormone regulation, glucose transport, cherubism disease, erectile dysfunction, and apoptosis. The structural analysis, mechanistic information, in vitro and in vivo studies led identification and development of several classes of tankyrase inhibitors under clinical phases. In the nutshell, this review will drive future research on tankyrase as it enlighten the structural and functional features of TNKS 1 and TNKS 2, different classes of inhibitors with their structure-activity relationship studies, molecular modeling studies, as well as past, current and future perspective of the different class of tankyrase inhibitors.