Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP).

Ancestral sequence reconstruction is a technique that is gaining widespread use in molecular evolution studies and protein engineering. Accurate reconstruction requires the ability to handle appropriately large numbers of sequences, as well as insertion and deletion (indel) events, but available approaches exhibit limitations. To address these limitations, we developed Graphical Representation of Ancestral Sequence Predictions (GRASP), which efficiently implements maximum likelihood methods to enable the inference of ancestors of families with more than 10,000 members. GRASP implements partial order graphs (POGs) to represent and infer insertion and deletion events across ancestors, enabling the identification of building blocks for protein engineering. To validate the capacity to engineer novel proteins from realistic data, we predicted ancestor sequences across three distinct enzyme families: glucose-methanol-choline (GMC) oxidoreductases, cytochromes P450, and dihydroxy/sugar acid dehydratases (DHAD). All tested ancestors demonstrated enzymatic activity. Our study demonstrates the ability of GRASP (1) to support large data sets over 10,000 sequences and (2) to employ insertions and deletions to identify building blocks for engineering biologically active ancestors, by exploring variation over evolutionary time.

Hydroxyl substituted benzoic acid/cinnamic acid derivatives: Tyrosinase inhibitory kinetics, anti-melanogenic activity and molecular docking studies.

The inhibition of tyrosinase is an established strategy for treating hyperpigmentation. Our previous findings demonstrated that cinnamic acid and benzoic acid scaffolds can be effective tyrosinase inhibitors with low toxicity. The hydroxyl substituted benzoic and cinnamic acid moieties of these precursors were incorporated into new chemotypes that displayed in vitro inhibitory effect against mushroom tyrosinase. The most active compound, (2-(3-methoxyphenoxy)-2-oxoethyl (E)-3-(4-hydroxyphenyl) acrylate) 6c, inhibited tyrosinase with an IC50 of 5.7 µM, while (2-(3-methoxyphenoxy)-2-oxoethyl 2, 4-dihydroxybenzoate) 4d had an IC50 of 23.8 µM. In comparison, the positive control, kojic acid showed tyrosinase inhibition with an IC50 = 16.7 µM. Analysis of enzyme kinetics revealed that 6c and 4d displayed noncompetitive reversible inhibition of the second tyrosinase enzymatic reaction with Ki values of 11 µM and 130 µM respectively. In silico docking studies with mushroom tyrosinase (PDB ID 2Y9X) predicted possible binding modes in the catalytic site for these active compounds. The phenolic para-hydroxy group of the most active compound 6c is predicted to interact with the catalytic site Cu++ ion. The methoxy part of this compound is predicted to form a hydrogen bond with Arg 268. Compound 6c had no observable toxic effects on cell morphology or cell viability at the highest tested concentration of 91.4 µM. When dosed at 91.4 µM onto B16F10 melanoma cells in vitro6c showed anti-melanogenic effects equivalent to kojic acid at 880 µM. 6c displayed no PAINS (pan-assay interference compounds) alerts. Our results show that compound 6c is a more potent tyrosinase inhibitor than kojic acid and is a candidate for further development. Our exposition of the details of the interactions between 6c and the catalytic pocket of tyrosinase provides a basis for rational design of additional potent inhibitors of tyrosinase, built on the cinnamic acid scaffold.

Dysregulation of tyrosinase activity: a potential link between skin disorders and neurodegeneration.

OBJECTIVES: The co-occurrence of melanoma and Parkinson's disease (PD) is higher than expected. We review the relationship between melanoma and PD, then proffer a hypothesis of how dysregulated human tyrosinase could be involved in both diseases via the loss of dopamine and neuromelanin-positive neurons in PD and the genesis alterations in melanin content during melanoma. KEY FINDINGS: There are a surprising number of links between skin disorders and neurodegenerative diseases. Some risk factors related to the co-occurrence of PD and melanoma have been extensively investigated over the past 15 years. It has been proposed that human tyrosinase, an enzyme participating in the biosynthesis of neuromelanin in the brain and of melanin in the skin, plays a role. Abnormally dysregulated human tyrosinase impacts the genesis and progression of melanoma and PD. SUMMARY: The dual role of human tyrosinase places it as the potential critical link between these seemingly distinct conditions. Detecting and monitoring human tyrosinase activity in the progression of melanoma and PD opens new opportunities for early diagnosis and treatment of both diseases.

Liver and pancreatic-targeted interleukin-22 as a therapeutic for metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatohepatitis (MASH) is the most prevalent cause of liver disease worldwide, with a single approved therapeutic. Previous research has shown that interleukin-22 (IL-22) can suppress ß-cell stress, reduce local islet inflammation, restore appropriate insulin production, reverse hyperglycemia, and ameliorate insulin resistance in preclinical models of diabetes. In clinical trials long-acting forms of IL-22 have led to increased proliferation in the skin and intestine, where the IL-22RA1 receptor is highly expressed. To maximise beneficial effects whilst reducing the risk of epithelial proliferation and cancer, we designed short-acting IL-22-bispecific biologic drugs that successfully targeted the liver and pancreas. Here we show 10-fold lower doses of these bispecific biologics exceed the beneficial effects of native IL-22 in multiple preclinical models of MASH, without off-target effects. Treatment restores glycemic control, markedly reduces hepatic steatosis, inflammation, and fibrogenesis. These short-acting IL-22-bispecific targeted biologics are a promising new therapeutic approach for MASH.

Multiplex-microsphere-quantitative polymerase chain reaction: nucleic acid amplification and detection on microspheres.

We report the development of a new system to monitor the amplification of nucleic acids on microspheres. This was realized by the design of (i) a "universal" oligonucleotide "tagged" polymerase chain reaction (PCR) forward primer, (ii) a sensor sequence complementary to the universal sequence on the forward PCR primer modified with a fluorescent dye, and (iii) a universal oligonucleotide coupled to Luminex microspheres. The PCR takes place with the microspheres present in the reaction tube. With the consumption of the universal oligonucleotide tagged forward primer, the fluorescently labeled sequences can bind to the universal oligonucleotide on the microspheres. We tested the microsphere quantitative PCR system with up to three different target genes (Neisseria meningitides porA and ctrA and influenza A M gene segment) as templates in a single PCR tube. The analytical sensitivity of this quantitative PCR system was tested and compared with the TaqMan system. The multiplex-microsphere-quantitative PCR system does not require design of unique internal probes for each target and has potential for a high degree of multiplexing, greater than the limited multiplexing achievable with current real-time protocols.

PrimRglo: a multiplexable quantitative real-time polymerase chain reaction system for nucleic acid detection.

We report the development of a new real-time polymerase chain reaction (PCR) detection system that uses oligonucleotide "tagged" PCR primers, a fluorophore-labeled "universal" detection oligonucleotides, and a complementary quenching oligonucleotide. The fluorescence signal decreases as PCR product accumulates due to the increase in detection/quencher hybrid formation as the tagged primer is consumed. We use plasmids containing the influenza A matrix gene and the porA and ctrA genes of Neisseria meningitidis as targets for developing the system. Cycle threshold (Ct) values were generated, and the sensitivity of the new system (dubbed "PrimRglo") compared favorably with the commonly used SYBR green and Taqman detection systems and, unlike the latter system, does not require the design of a new dual-labeled detection oligonucleotide for each new target sequence.

Risk Factors for Heart Disease in Working Railwaymen.

Heart diseases are the most common non-communicable diseases worldwide. We examined the prevalence of risk factors for heart disease among a sub-population of working men. In total, 11,059 railway crew workers of the Russian Railways Company were included in the study. We also asked participants to answer several questions based on the WHO STEPwise approach to surveillance (STEPS) translated into Russian. Only 30% of drivers had normal body mass index (BMI), whereas 70% were overweight or obese. In 12% of subjects, total cholesterol was higher than 5 mmol/L. In 15% of participants, glucose level was higher than 5.5 mmol/L. 38% of drivers reported smoking. Physical inactivity was registered in 54% of persons. Only 29% ate according to the key principles of good diet quality. 24% of respondents had a family history of heart disease. MANOVA demonstrated that BMI was determined by age, profession, smoking, physical inactivity, and diet quality. As age increased, the number of people with normal cholesterol levels decreased. It was demonstrated that a correlation existed between glucose levels and BMI. In the total group, the correlation was 0.46 (p < .05). The correlation between those parameters increased due to age, from 0.33 in the <30 years of age group up to 0.52 in the >50 years of age group. This study demonstrated that there is a high prevalence of risk factors for heart disease in train drivers in the Russian Federation.

Antiinflammatory and Anticancer Properties of Grewia asiatica Crude Extracts and Fractions: A Bioassay-Guided Approach.

The study was an extension of our earlier work on antiinflammatory and anticancer properties of G. asiatica fruit. We aimed to develop a bioassay guided multistep purification technique for producing bioactive fractions of G. asiatica crude extracts. Dried fruit powder was sequentially fractionated with 100% dichloromethane, 100% methanol (MeOH), and 50% MeOH. Active extracts were subjected to liquid-liquid partitioning followed by subfractionation using RP-HPLC. Antioxidant, antiinflammatory, and anticancer activities of the fruit extracts, and their potent fractions were evaluated in vitro, while identification of compounds from the bioactive fractions was performed by ESI-MS/MS analysis. The amount of the identified compounds present was confirmed using external standards adopting a simple, accurate, and rapid analytical HPLC method. The results showed that 100% and 50% MeOH extracts possessed bioactivity; one of which (the 50% MeOH extract) displayed potent activity in all in vitro bioassays. MeOH extract (50%) derived fraction C and hydroalcoholic fraction 5 (GAHAF5) were observed to possess higher antioxidant, antiinflammatory, and in vitro anticancer activity. IC50 of GAHAF5 against MCF-7, HEp-2, and NCI-H522 cancer cells was recorded as 26.2, 51.4, and 63 µg/mL, respectively. ESI-MS/MS and HPLC analysis identified catechin, chlorogenic acid, caffeic acid, and morin as potential bioactive compounds in the GAHAF5 fraction with concentrations of 1230, 491, 957, and 130 µg/g, respectively. The findings indicated that G. asiatica bioactive fractions possessed antiinflammatory activity in vitro and were cytotoxic against breast cancer, lung cancer, and laryngeal cancer cell lines.