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Isothermal titration calorimetry allows the determination of thermodynamic parameters for the interaction between a protein and mono- or oligosaccharides in solution. For the study of protein-carbohydrate interactions, it is a robust way to determine the stoichiometry and affinity, as well as the enthalpic and entropic contributions to this interaction, without the use of labeled proteins or substrates. Here we describe a standard multiple-injection titration experiment for measuring the binding energetics between a carbohydrate-binding protein and an oligosaccharide...

Solution-state nuclear magnetic resonance (NMR) spectroscopy can be used to monitor protein-carbohydrate interactions. Two-dimensional 1 H-15 N heteronuclear single quantum coherence (HSQC)-based techniques described in this chapter can be used quickly and effectively to screen a set of possible carbohydrate-binding partners, to quantify the dissociation constant (Kd ) of any identified interactions, and to the map the carbohydrate-binding site on the structure of a protein. Here, we describe the titration of a family 32 carbohydrate-binding module from Clostridium perfringens (CpCBM32) with the monosaccharide N-acetylgalactosamine (GalNAc), in which we calculate the apparent dissociation of the interaction and map the GalNAc binding site onto the structure of CpCBM32...

Microscale thermophoresis (MST) is an emerging technology for studying a broad range of biomolecular interactions with a high sensitivity. The affinity constant can be obtained for a wide range of molecules within minutes based on reactions in microliters. Here we describe the application of MST in quantifying protein-carbohydrate interactions. A CBM3a and a CBM4 are titrated with insoluble substrate (cellulose nanocrystal) and soluble oligosaccharide (xylohexaose), respectively.

Affinity electrophoresis has long been used to study the interaction between proteins and large soluble ligands. The technique has been found to have great utility for the examination of polysaccharide binding by proteins, particularly carbohydrate-binding modules (CBMs). In recent years carbohydrate surface binding sites of proteins, mostly enzymes, have also been investigated by this method. Here we describe a protocol for identifying binding interactions between enzyme catalytic modules and a variety of carbohydrate ligands...

Expansins are proteins that loosen plant cell walls but lack enzymatic activity. Here we describe two protocols tailored to measure the biomechanical activity of bacterial expansin. The first assay relies on the weakening of filter paper by expansin. The second assay is based on induction of creep (long-term, irreversible extension) of plant cell wall samples.

Cellulosomes are multi-enzymatic nanomachines that have been fine-tuned through evolution to efficiently deconstruct plant biomass. Integration of cellulosomal components occurs via highly ordered protein-protein interactions between the various enzyme-borne dockerin modules and the multiple copies of the cohesin modules located on the scaffoldin subunit. Recently, designer cellulosome technology was established to provide insights into the architectural role of catalytic (enzymatic) and structural (scaffoldin) cellulosomal constituents for the efficient degradation of plant cell wall polysaccharides...

Lytic polysaccharide monooxygenases perform oxidative cleavage of glycosidic bonds in various polysaccharides. The majority of LMPOs studied so far possess activity on either cellulose or chitin and analysis of these activities is therefore the main focus of this review. Notably, however, the number of LPMOs that are active on other polysaccharides is increasing. The products generated by LPMOs from cellulose are either oxidized in the downstream end (at C1) or upstream end (at C4), or at both ends. These modifications only result in small structural changes, which makes both chromatographic separation and product identification by mass spectrometry challenging...

Use of the 3,5-dinitrosalicylic acid reagent allows the simple, rapid quantification of reducing sugars. The method can be used for analysis of biological samples or in characterization of enzyme reactions, as new reducing ends are generated when a polysaccharide substrate undergoes hydrolytic cleavage. Presented here is an application of the method in measuring the kinetics of a glycoside hydrolase reaction, including the optimization of the DNSA reagent, and the production of a standard curve of absorbance versus sugar concentration...

The quantitation of liberated reducing sugars by the copper-bicinchoninic acid (BCA) assay provides a highly sensitive method for the measurement of glycoside hydrolase (GH) activity, particularly on soluble polysaccharide substrates. Here we describe a straightforward method adapted to low-volume polymerase chain reaction (PCR) tubes that enables the rapid, parallel determination of GH kinetics in applications ranging from initial activity screening and assay optimization to precise Michaelis-Menten analysis...

Streptochlorin is a kind of indole alkaloid derived from marine microorganisms. It is a promising lead compound due to its potent bioactivity in preventing many phytopathogens, as shown in our previous study. To explore the potential applications of this natural product, a series of novel benzoxaborole-containing streptochlorin derivatives were designed and synthesized through a one-step and catalyst-free reaction in water at room temperature. All target compounds were first screened for their antifungal profiles in vitro against six common phytopathogenic fungi...

BACKGROUND: Racial/ethnic (R/E) minorities with head and neck squamous cell carcinoma (HNSCC) have worse survival outcomes compared to White patients. While disparities in patient outcomes for R/E minorities have been well documented, the specific drivers of the inferior outcomes remain poorly understood. PATIENTS AND METHODS: This was a population-based retrospective cohort study that analyzed HNSCC patients using the National Cancer Database (NCDB) from 2000-2016...

BACKGROUND: Evaluating the burden of ischemic heart disease (IHD) as the first cause of mortality worldwide is necessary to develop healthcare policies. This study aimed to report the national and subnational IHD burden and risk factors in Iran according to the Global Burden of Disease (GBD) study 2019. METHODS: We extracted, processed, and presented the results of the GBD 2019 study regarding incidence, prevalence, deaths, years lived with disability (YLDs), years of life lost (YLLs), disability-adjusted life years (DALYs), and attributable burden to risk factors of IHD in Iran during 1990-2019...

A survey on field pea (Pisum sativum) root rot was carried out in Prince Edward Island (PEI) and New Brunswick (NB), Canada, between July and August, 2018. The average disease incidence was 75% and 78%, and severity was 3.5 and 2.8 on a 1 to 7 scale for NB and PEI, respectively (Schneider and Kelly 2000). Symptoms included seedling stunting, root rot, and wilting. Surface sterilized diseased root segments were incubated on water agar at 25°C for 5 days. Pure isolates were obtained by single spore culturing...

In this study, a series of structurally novel N-(benzene sulfonyl) acetamide derivatives were designed, synthesized, and biologically evaluated as COX-2/5-LOX/TRPV1 multitarget inhibitors for anti-inflammatory and analgesic therapy. Among them, 9a and 9b displayed favorable COX-2 (9a IC50 =0.011 μM, 9b IC50 =0.023 μM), 5-LOX (9a IC50 =0.046 μM, 9b IC50 =0.31 μM) and TRPV1 (9a IC50 =0.008 μM, 9b IC50 =0.14 μM) inhibitory activities. The pharmacokinetic (PK) study of 9a in SD rats at the dosage of 10 mg/kg demonstrated a high oral exposure, an acceptable clearance and a favorable bioavailability (Cmax=5807...

MirDIP is a well-established database that aggregates microRNA-gene human interactions from multiple databases to increase coverage, reduce bias, and improve usability by providing an integrated score proportional to the probability of the interaction occurring. In version 5.2, we removed eight outdated resources, added a new resource (miRNATIP), and ran five prediction algorithms for miRBase and mirGeneDB. In total, mirDIP 5.2 includes 46 364 047 predictions for 27 936 genes and 2734 microRNAs, making it the first database to provide interactions using data from mirGeneDB...

BACKGROUND: In the City and County of San Francisco, frequent users of emergent and urgent services across different settings (i.e., medical, mental health (MH), substance use disorder (SUD) services) are referred to as high users of multiple systems (HUMS). While often grouped together, frequent users of the health care system are likely a heterogenous population composed of subgroups with differential management needs. OBJECTIVE: To identify subgroups within this HUMS population using a cluster analysis...

Torasemide is a loop diuretic with a molecule that is chemically similar to the sulphonamides described as eosinophilic granulomatosis with polyangiitis (EGPA) triggering drugs. The presented case is probably the first description of torasemide-induced vascular purpura in the course of EGPA. Any diagnosis of vasculitis should be followed by an identification of drugs that may aggravate the disease. A 74-year-old patient was admitted to the Department of Dermatology with purpura-like skin lesions on the upper, and lower extremities, including the buttocks...

OBJECTIVES: Understanding changes in moderate to vigorous aerobic physical activity (MVPA) and muscle-strengthening activity (MSA) at the start of the COVID-19 pandemic and their correlates (socio-demographics, health characteristics, living and exercise conditions and pre-pandemic MVPA/MSA) can inform interventions. DESIGN: A cross-sectional analysis of retrospective and concurrent data on MVPA/MSA. SETTING: An online survey in the UK. PARTICIPANTS: 2657 adults (weighted n=2442, 53...

Importance: Functional abdominal pain disorders (FAPDs) can severely affect the life of children and their families, with symptoms carrying into adulthood. Management of FADP symptoms is also a financial and time burden to clinicians and health care systems. Objective: To systematically review various randomized clinical trials (RCTs) on the outcomes of cognitive behavioral therapy (CBT), educational support, yoga, hypnotherapy, gut-directed hypnotherapy, guided imagery, and relaxation in the management of FAPDs...

INTRODUCTION: The aims of this observational study were to: 1) Determine if Endodontists' practice in early 2021 experienced changes in patients' characteristics compared to a comparable pre-pandemic period; 2) Determine whether the changes reported during the initial outbreak of COVID-19 in 2020 were reversed one year later. METHODS: Demographic, diagnostic, and procedural data of 2657 patient visits from two endodontist private offices, from March-16 to May-31 in 2019, 2020, and 2021 were included...