GlyCombo Enables Rapid, Complete Glycan Composition Identification across Diverse Glycomic Sample Types.

Glycans are sugar-based polymers found to modify biomolecules, including lipids and proteins, as well as occur unconjugated as free polysaccharides. Due to their ubiquitous cellular presentation, glycans mediate crucial biological processes and are frequently sought after as biomarkers for a wide range of diseases. Identification of glycans present in samples acquired with mass spectrometry (MS) is a cornerstone of glycomics research; thus, the ability to rapidly identify glycans in each acquisition is integral to glycomics analysis pipelines. Here we introduce GlyCombo (https://github.com/Protea-Glycosciences/GlyCombo), an open-source, freely available software tool designed to rapidly assign monosaccharide combinations to glycan precursor masses including those subjected to MS2 in LC-MS/MS experiments. GlyCombo was evaluated across six diverse data sets, demonstrating MS vendor, derivatization, and glycan-type neutrality. Compositional assignments using GlyCombo are shown to be faster than the current predominant approach, GlycoMod, a closed-source web application. Two unique features of GlyCombo, multiple adduct search and off-by-one error anticipation, reduced unassigned MS2 scans in a benchmark data set by 40%. Finally, the comprehensiveness of glycan feature identification is exhibited in Skyline, a software that requires predefined transitions that are derived from GlyCombo output files.

Importance of evaluating protein glycosylation in pluripotent stem cell-derived cardiomyocytes for research and clinical applications.

Proper protein glycosylation is critical to normal cardiomyocyte physiology. Aberrant glycosylation can alter protein localization, structure, drug interactions, and cellular function. The in vitro differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CM) has become increasingly important to the study of protein function and to the fields of cardiac disease modeling, drug testing, drug discovery, and regenerative medicine. Here, we offer our perspective on the importance of protein glycosylation in hPSC-CM. Protein glycosylation is dynamic in hPSC-CM, but the timing and extent of glycosylation are still poorly defined. We provide new data highlighting how observed changes in hPSC-CM glycosylation may be caused by underlying differences in the protein or transcript abundance of enzymes involved in building and trimming the glycan structures or glycoprotein gene products. We also provide evidence that alternative splicing results in altered sites of glycosylation within the protein sequence. Our findings suggest the need to precisely define protein glycosylation events that may have a critical impact on the function and maturation state of hPSC-CM. Finally, we provide an overview of analytical strategies available for studying protein glycosylation and identify opportunities for the development of new bioinformatic approaches to integrate diverse protein glycosylation data types. We predict that these tools will promote the accurate assessment of protein glycosylation in future studies of hPSC-CM that will ultimately be of significant experimental and clinical benefit.

Facile Preparation of Peptides for Mass Spectrometry Analysis in Bottom-Up Proteomics Workflows.

Mass spectrometry (MS) is routinely used to identify, characterize, and quantify biological molecules. For protein analysis, MS-based workflows can be broadly categorized as top-down or bottom-up, depending on whether the proteins are analyzed as intact molecules or first digested into peptides. This article outlines steps for preparing peptide samples for MS as part of a bottom-up proteomics workflow, providing versatile methods suitable for discovery and targeted analyses in qualitative and quantitative workflows. Resulting samples contain peptides of suitable size for analysis by MS instrumentation generally available to modern research laboratories, including MS coupled to either liquid chromatography (LC) or matrix-assisted laser desorption/ionization (MALDI) interfaces. This article incorporates recent developments in methodologies and consumables to facilitate sample preparation. The protocols are well-suited to users without prior experience in proteomics and include methods for universally applicable suspension trap processing and for alternate in-solution processing to accommodate a range of sample types. Cleanup, quantification, and fractionation procedures are also described. © 2021 The Authors. Basic Protocol: Preparation of high-complexity peptide samples for mass spectrometry analysis using S-Trap™ processing Alternate Protocol 1: Preparation of low- to moderate-complexity peptide samples for mass spectrometry analysis using in-solution processing Alternate Protocol 2: Detergent, polymer, and salt removal from peptide samples before mass spectrometry analysis using SP2 processing Support Protocol 1: Protein quantification using Pierce 660 nm assay Support Protocol 2: Peptide quantification using Pierce quantitative fluorometric peptide assay Support Protocol 3: High-pH fractionation of complex peptide samples.

Dietary ethanol extract of mango increases antioxidant activity of pork.

Ethanol extract of mango seeds (EEMS) are composed of several polyphenolic compounds with considerable in vitro antioxidant activity that can be used in pig feed and may contribute positively to meat quality characteristics. The aim of this study was to evaluate the effect of EEMS as a source of antioxidants in growing-finishing pig diets on meat quality, lipid stability, sulfhydryl groups non-proteinaceous (SG-NP), total phenolic compounds, total antioxidant potential and total antioxidant activity of meat after 1 and 7 days of refrigeration storage. Thirty-two (60-day-old) barrows, weighing 20.20 ±â€¯1.34 kg, were used in a randomized block design consisting of eight animals with four treatment regimens. Treatments consisted of: Control = no dietary antioxidant; butylated hydroxytoluene (BHT) = diet with 200 ppm BHT; EEMS200 = diet with 200 ppm EEMS; EEMS400 = diet with 400 ppm EEMS. At 145 days of age and average weight of 95.47 ±â€¯6.19 kg, the animals were slaughtered and loin samples were collected and frozen before for qualitative analysis and evaluation of the effect of subsequent storage for 1 or 7 days at 8 °C on lipid stability, SG-NP, phenolic compounds, total antioxidant capacity and total antioxidant activity Meat from animals fed EEMS400 diet showed lower cooking loss (P < 0.0001) and higher non-protein sulfhydryl groups, phenolic compounds and total antioxidant activity at both 1 and 7 days of storage (P < 0.0001) compared to the other treatments. Greater antioxidant capacity was observed at 1 day storage in the meat of animals that consumed EEMS regardless of concentration when compared to the control group (P < 0.01). The dietary inclusion of EEMS to pig diets is more effective at 400 ppm in improving meat quality after cooking and antioxidant parameters of pork.

Parallel Determination of Polypeptide and Oligosaccharide Connectivities by Energy-Resolved Collison-Induced Dissociation of Protonated O-Glycopeptides Derived from Nonspecific Proteolysis.

Collision-induced dissociation (CID) is by far the most broadly applied dissociation method used for tandem mass spectrometry (MS/MS). This includes MS/MS-based structural interrogation of glycopeptides for applications in glycoproteomics. The end goal of such measurements is to determine the monosaccharide connectivity of the glycan, the amino acid sequence of the peptide, and the site of glycosylation for each glycopeptide of interest. In turn, this allows inferences with respect to the glycoprofile of the intact glycoprotein. For glycopeptide analysis, CID is best known for the ability to determine glycosidic topology of the oligosaccharide group; however, CID has also been shown to produce amide bond cleavage of the polypeptide group. Whether structural information is obtained for the glycan or the peptide has been found to depend on the applied collision energy. While these energy-resolved fragmentation pathways have been the subject of several studies on N-linked glycopeptides, there remains a dearth of similar work on O-linked glycopeptides. In this study, MS/MS via CID was shown to provide substantial peptide backbone fragmentation, in addition to glycosidic fragmentation, in an energy-dependent manner. While qualitatively similar to previous findings for N-glycopeptides, the energy-resolved CID (ER-CID) of O-glycopeptides was found to be substantially more sensitive to the collision energy setting. Thus, deliberately obtaining either glycan or peptide dissociation is a more delicate undertaking for O-glycopeptides. Establishing a more complete understanding of O-glycopeptide ER-CID is likely to have a substantive impact on how O-glycoproteomic analysis is approached in the future.

Two-point wavepacket modelling of jet noise.

This paper is focused on the study of a kinematic wavepacket model for jet noise based on two-point statistics. The model contains physical parameters that define its structure in terms of wavenumber, envelope shape and coherence decay. These parameters, which are necessary to estimate the sound pressure levels radiated by the source, were educed from a large-eddy simulation database of a Mach 0.4, fully turbulent jet. The sound pressure levels predicted by the model were compared with acoustic data and the results show that when the parameters are carefully educed from the data, the sound pressure levels generated are in good agreement with experimentally measured values for low Strouhal numbers and polar angles. Furthermore, here we show that a correct representation of both coherence decay and wavepacket envelope shape are key aspects to an accurate sound prediction. A Spectral Proper Orthogonal Decomposition (SPOD) of the model source was also performed motivated by the search for a low-rank model capable of capturing the acoustic efficiency of the full source. It is shown that only a few SPOD modes are necessary to recover acoustically important wavepacket traits.

Interpretation of Basic Clinical Images: How Are Surgical Residents Performing Compared to Other Trainees?

BACKGROUND: During medical training students, residents, and fellows learn how to accurately interpret basic radiographic images. This skill is mostly utilized by physicians in the acute and critical care settings. It is unclear whether surgical residents' interpretation skills differ from that of other trainees. METHODS: A 30-question online quiz was developed to evaluate trainees' skills in interpreting images using various radiologic modalities. The participating cohort included (1) medical students (MS), (2) general surgery residents (GST), internal medicine residents and fellows (IMT), and radiology trainees (RT). The impact of residency specialty and level of training on performance was evaluated. RESULTS: A total of 69 postgraduate trainees and 19 MS enrolled in the online quiz. The average score was 67.6% (±16.6). GST scored higher than IMT (74.2% ± 10.7% vs. 67.9% ± 11.3%, p = 0.038); however, they were equally proficient to RT. MS had the lowest interpretation accuracy rates compared to postgraduate trainees (57.4% ± 16.8%, p < 0.001). On different radiographic modalities, junior GST performance was comparable to MS, JR-IMT, and Junior Radiology Trainees (JR-RT). On computed tomography (CT) body, GST (83.1% ± 15.7%) scored higher than IMT (70.3% ± 17.7%, p = 0.026) and MS (61.7% ± 23.4%, p < 0.001). Similar findings were demonstrated on ultrasound modality. A difference in performance was not evident for X-rays, CT head, and tubes/lines localization images. CONCLUSIONS: GST were able to correctly interpret 74.2% of basic clinical images. Although superior in the evaluation of pathologies seen on CT body and ultrasound, GST have comparable performance to other trainees in X-rays, tube/line localization images, and CT head. Integration of radiology education in surgical training may enhance performance and potentially improve patient care.

Undernutrition risk at hospital admission and length of stay among pulmonology inpatients.

BACKGROUND: There is a lack of evidence regarding the association between the undernutrition risk at hospital admission with adverse clinical outcomes amongst pulmonology inpatients. The aim of this study was to quantify the association between undernutrition risk at hospital admission and time to discharge alive. METHODS: A retrospective cohort study including patients consecutively admitted to a pulmonology unit was conducted. Undernutrition risk at hospital admission was identified using the Malnutrition Universal Screening Tool. Survival analyses (Kaplan-Meier curves and Cox regression) were carried out. RESULTS: The sample was composed of 683 patients. Patients who presented high undernutrition risk on hospital admission had a longer length of hospital stay (approximately 50% were discharged to home after 14 days of hospitalization). In the multivariable Cox regression, high undernutrition risk was shown to be independently associated with a lower probability of discharge alive over time (adjusted hazard ratio=0.70; 95% confidence interval: 0.55-0.90). CONCLUSIONS: Pulmonology inpatients with high undernutrition risk have a longer length of hospital stay and had a lower probability of being discharged to home. In particular, lung cancer patients had a lower probability of being discharged to home, which corroborates a worse prognosis for these patients.

Cirugía a corazón abierto para el tratamiento de trombo auricular derecho relacionado con cateterización venosa central / Open heart surgery for management of right auricular thrombus related to central venous catheterization

Los catéteres venosos centrales se utilizan de manera generalizada en pacientes críticos; sin embargo, también se asocian a una elevada morbimortalidad. La literatura puede subestimar la incidencia de los trombos auriculares derechos asociados a catéter venoso, que son asintomáticos, pero potencialmente de riesgo. Los factores de riesgo reconocidos para su desarrollo incluyen infecciones relativas al catéter y lesiones endoteliales secundarias al daño mecánico y químico inducido por ciertos fármacos y líquidos infundidos. También desempeñan un papel añadido las características del paciente y del catéter, tales como tamaño, material, tipo, localización y facilidad de inserción, y duración de la misma. Reportamos el caso de un varón de 38 años que desarrolló trombos auriculares derechos asintomáticos asociados a catéter venoso y precisó cirugía a corazón abierto tras cateterización venosa central durante 35días. El presente caso destaca las limitaciones existentes a la hora de realizar un diagnóstico correcto y rápido, que debería anticiparse en pacientes con factores de riesgo múltiples de trombosis. Dadas las recomendaciones disponibles limitadas, consideramos que debería individualizarse la estrategia más adecuada

Central venous catheters are widely used in critically ill patients; however, they are also associated with increased morbidity and mortality. The literature may underestimate the incidence of catheter-inducible right atrial thrombi that are asymptomatic but potentially life threatening. The recognized risk factors for its development include infections related to the catheter, endothelial injury secondary to mechanical and chemical damage induced by certain medications and infused fluids. The characteristics of the patient and the catheter, such as size, material, type, location and ease of insertion, as well as the duration of placement play an additional role. We report the case of a 38-year-old man, who developed an asymptomatic catheter-inducible right atrial thrombi requiring open heart surgery, after taking a central venous catheter for thirty-five days. The present case highlights existing limitations in making a correct and fast diagnosis, which should be anticipated in patients with multiple risk factors for thrombosis. Given the limited recommendations available, we consider that the most appropriate strategy should be individualized

Open heart surgery for management of right auricular thrombus related to central venous catheterization. / Cirugía a corazón abierto para el tratamiento de trombo auricular derecho relacionado con cateterización venosa central.

Central venous catheters are widely used in critically ill patients; however, they are also associated with increased morbidity and mortality. The literature may underestimate the incidence of catheter-inducible right atrial thrombi that are asymptomatic but potentially life threatening. The recognized risk factors for its development include infections related to the catheter, endothelial injury secondary to mechanical and chemical damage induced by certain medications and infused fluids. The characteristics of the patient and the catheter, such as size, material, type, location and ease of insertion, as well as the duration of placement play an additional role. We report the case of a 38-year-old man, who developed an asymptomatic catheter-inducible right atrial thrombi requiring open heart surgery, after taking a central venous catheter for thirty-five days. The present case highlights existing limitations in making a correct and fast diagnosis, which should be anticipated in patients with multiple risk factors for thrombosis. Given the limited recommendations available, we consider that the most appropriate strategy should be individualized.

Electrotonic potentials in Aloe vera L.: Effects of intercellular and external electrodes arrangement.

Electrostimulation of plants can induce plant movements, activation of ion channels, ion transport, gene expression, enzymatic systems activation, electrical signaling, plant-cell damage, enhanced wound healing, and influence plant growth. Here we found that electrical networks in plant tissues have electrical differentiators. The amplitude of electrical responses decreases along a leaf and increases by decreasing the distance between polarizing Pt-electrodes. Intercellular Ag/AgCl electrodes inserted in a leaf and extracellular Ag/AgCl electrodes attached to the leaf surface were used to detect the electrotonic potential propagation along a leaf of Aloe vera. There is a difference in duration and amplitude of electrical potentials measured by electrodes inserted in a leaf and those attached to a leaf's surface. If the external reference electrode is located in the soil near the root, it changes the amplitude and duration of electrotonic potentials due to existence of additional resistance, capacitance, ion channels and ion pumps in the root. The information gained from this study can be used to elucidate extracellular and intercellular communication in the form of electrical signals within plants.

Matrix metalloproteinase 9 -1562C/T polymorphism increased protein levels in patients with colorectal cancer in a sample from southeastern Brazil.

We examined whether the allelic and/or genotypic profile of locus -1562C/T of the matrix metalloproteinase (MMP-9) gene influences the protein expression levels of MMP-9 in patients with colorectal cancer (CRC) compared with controls. A total of 104 patients with CRC and 84 controls were evaluated. Peripheral blood was collected from both groups and DNA extraction was performed for -1562C/T genotyping; the plasma was used for MMP-9 quantification. The CT genotype was associated with increased MMP-9 expression (P = 0.0211). High levels of protein, independently of polymorphisms, were observed in the patient group (P < 0.0001) compared to controls. Mucinous tumors with signet ring cells were more frequent in females (P = 0.0177). Overall, patients older than 50 years showed a significant risk of developing CRC (P = 0.0001). MMP-9 plasma expression was increased in patients with CRC compared to controls, particularly in those with the heterozygous -1562CT genotype.

A real-time, fluorescence-based assay for Rho-associated protein kinase activity.

Inhibitors of Rho-associated protein kinase (ROCK) enzymatic activity have been shown to reduce the invasive phenotype observed in metastatic hepatocellular carcinoma (HCC). We describe the design, synthesis, and evaluation of a direct probe for ROCK activity utilizing a phosphorylation-sensitive sulfonamido-oxine fluorophore, termed Sox. The Sox fluorophore undergoes an increase in fluorescence upon phosphorylation of a proximal amino acid via chelation-enhanced fluorescence (CHEF, ex. = 360 nm and em. = 485 nm), allowing for the direct visualization of the rate of phosphate addition to a peptide substrate over time. Our optimal probe design, ROCK-S1, is capable of sensitively reporting ROCK activity with a limit of detection of 10 pM and a high degree of reproducibility (Z'-factor = 0.6 at 100 pM ROCK2). As a proof-of-principle for high-throughput screening (HTS) we demonstrate the ability to rapidly assess the efficacy of a 78 member, small molecule library against ROCK2 using a robotics platform. We identify two previously unreported ROCK2 inhibitor scaffolds, PHA665752 and IKK16, with IC50 values of 3.6 µM and 247 nM respectively. Lastly, we define conditions for selectively monitoring ROCK activity in the presence of potential off-target enzymes (PKCα, PKA, and PAK) with similar substrate specificities.

Comparison of fertility, regular returns-to-estrus, and calving interval between Ovsynch and CO-synch + CIDR protocols in dairy cows.

The main aims of the present study were to compare the pregnancy rate (PR), regular returns-to-estrus, and calving interval of a CO-Synch + controlled internal drug release (CIDR) device, commonly used to synchronize ovulations in beef cows, with the classical Ovsynch protocol in high-producing dairy cows. Holstein-Friesian cows (n = 128) from six commercial dairy herds, ≥40 days postpartum and not previously inseminated, were randomly assigned to one of two treatments. Cows submitted to Ovsynch protocol (group OS as control group; n = 66) received 10 µg of a GnRH analogue 7 days before and 48 hours after 25 mg PGF2α, followed by artificial insemination (AI) 16 hours after the second GnRH administration. Cows submitted to CO-Synch + CIDR (1.38 g of progesterone) inserted for 7 days beginning at the first GnRH administration (group CoS + CD; n = 62) had the second administration of GnRH concurrent with AI, 64 hours after CIDR removal/PGF2α administration. Nonpregnant cows with return-to-estrus between 18 and 24 days after first AI were reinseminated (second AI). Logistic regressions were used to analyze PR and returns-to-estrus. No effect of group or herd was observed in PR at first timed AI. However, the sum of cows pregnant at first AI and nonpregnant cows with regular returns-to-estrus and the total PR (first + second AI) were influenced by group treatment. Overall, cows of group CoS + CD (total PR = 56.5%) were 2.1 times more likely to became pregnant after AI and until first regular returns-to-estrus than cows of group OS. The calving interval was lower in group CoS + CD (425.9 ± 78.8 days; ±SD) than in group OS (475.3 ± 83.7 days). The CO-Synch + CIDR protocol was reliable to use in dairy herds and provided reproductive advantages when compared with Ovsynch protocol.

Morphing structures and signal transduction in Mimosa pudica L. induced by localized thermal stress.

Leaf movements in Mimosa pudica, are in response to thermal stress, touch, and light or darkness, appear to be regulated by electrical, hydrodynamical, and chemical signal transduction. The pulvinus of the M. pudica shows elastic properties. We have found that the movements of the petiole, or pinnules, are accompanied by a change of the pulvinus morphing structures. After brief flaming of a pinna, the volume of the lower part of the pulvinus decreases and the volume of the upper part increases due to the redistribution of electrolytes between these parts of the pulvinus; as a result of these changes the petiole falls. During the relaxation of the petiole, the process goes in the opposite direction. Ion and water channel blockers, uncouplers as well as anesthetic agents diethyl ether or chloroform decrease the speed of alert wave propagation along the plant. Brief flaming of a pinna induces bidirectional propagation of electrical signal in pulvini. Transduction of electrical signals along a pulvinus induces generation of an action potential in perpendicular direction between extensor and flexor sides of a pulvinus. Inhibition of signal transduction and mechanical responses in M. pudica by volatile anesthetic agents chloroform or by blockers of voltage gated ion channels shows that the generation and propagation of electrical signals is a primary effect responsible for turgor change and propagation of an excitation. There is an electrical coupling in a pulvinus similar to the electrical synapse in the animal nerves.

Electrotonic and action potentials in the Venus flytrap.

The electrical phenomena and morphing structures in the Venus flytrap have attracted researchers since the nineteenth century. We have observed that mechanical stimulation of trigger hairs on the lobes of the Venus flytrap induces electrotonic potentials in the lower leaf. Electrostimulation of electrical circuits in the Venus flytrap can induce electrotonic potentials propagating along the upper and lower leaves. The instantaneous increase or decrease in voltage of stimulating potential generates a nonlinear electrical response in plant tissues. Any electrostimulation that is not instantaneous, such as sinusoidal or triangular functions, results in linear responses in the form of small electrotonic potentials. The amplitude and sign of electrotonic potentials depend on the polarity and the amplitude of the applied voltage. Electrical stimulation of the lower leaf induces electrical signals, which resemble action potentials, in the trap between the lobes and the midrib. The trap closes if the stimulating voltage is above the threshold level of 4.4V. Electrical responses in the Venus flytrap were analyzed and reproduced in the discrete electrical circuit. The information gained from this study can be used to elucidate the coupling of intracellular and intercellular communications in the form of electrical signals within plants.

Venus flytrap biomechanics: forces in the Dionaea muscipula trap.

Biomechanics of morphing structures in the Venus flytrap has attracted the attention of scientists during the last 140 years. The trap closes in a tenth of a second if a prey touches a trigger hair twice. The driving force of the closing process is most likely due to the elastic curvature energy stored and locked in the leaves, which is caused by a pressure differential between the upper and lower layers of the leaf. The trap strikes, holds and compresses the prey. We have developed new methods for measuring all these forces involved in the hunting cycle. We made precise calibration of the piezoelectric sensor and performed direct measurements of the average impact force of the trap closing using a high speed video camera for the determination of time constants. The new equation for the average impact force was derived. The impact average force between rims of two lobes in the Venus flytrap was found equal to 149 mN and the corresponding pressure between the rims was about 41 kPa. Direct measurements of the constriction force in the trap of Dionaea muscipula was performed during gelatin digestion. This force increases in the process of digestion from zero to 450 mN with maximal constriction pressure created by the lobes reaching to 9 kPa. The insects and different small prey have little chance to escape after the snap of the trap. The prey would need to overpower the "escaping" force which is very strong and can reach up to 4N.

Schistosoma mansoni: evaluation of an RNAi-based treatment targeting HGPRTase gene.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential gene of the parasite Schistosoma mansoni and it is well conserved in its hosts (mouse and human) at the protein but not at the RNA level. This feature prompted us to assess RNA interference (RNAi) to combat schistosomiasis. Small interfering RNAs (siRNAs) were produced against HGPRTase, injected in infected mice and the number of worms was counted six days after injection. The total number of parasites was reduced by approximately 27% after treatment. RT-PCR analyzes showed a significant reduction in parasite target mRNA but not in host's homologue. The use of low doses of molecules did not oversaturate si- or miRNA pathways as mice survival rates were not affected by siRNAs. This is the first successful in vivo demonstration of a RNAi-based treatment against schistosomiasis. We believe that improvements in molecule delivery and an increase on siRNA dose could rapidly eliminate parasite.