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Gangliosides are glycosphingolipids composed of a sialylated glycan head group and a ceramide backbone. These anionic lipids form lipid rafts and play crucial roles in regulating various proteins involved in signal transduction, adhesion, and cell-cell recognition. Neuroblastoma, a pediatric cancer of the sympathetic nervous system, is treated with intensive chemotherapy, radiation, and an antibody targeting the GD2 ganglioside. Gangliosides are critical in neuroblastoma development and serve as therapeutic targets, making it essential to establish a reliable, rapid, and cost-effective method for profiling gangliosides, particularly one capable of isomeric separation of intact species. In this study, liquid chromatography-mass spectrometry (LC-MS) was optimized using standard gangliosides, followed by the optimization of sphingolipid extraction methods from cell lines by comparing Folch and absolute methanol extraction techniques. Percent recovery and the number of identified sphingolipids were used to evaluate the analytical merits of these methods. A standard gangliosides calibration curve demonstrated excellent linearity (R 2 = 0.9961-0.9975). The ZIC-HILIC column provided the best separation of ganglioside GD1 isomers with a 25 min runtime. GD1a elutes before GD1b on the ZIC-HILIC column. Absolute methanol yielded better percent recovery (96 ± 7) and identified 121 different sphingolipids, the highest number between the two extraction methods. The optimized method was applied to profile gangliosides in neuroblastoma (COG-N-683), pancreatic cancer (PSN1), breast cancer (MDA-MB-231BR), and brain tumor (CRL-1620) cell lines. The ganglioside profile of the neuroblastoma cell line COG-N-683 showed an inverse relationship between GD1 and GD2. Ceramide, Hex1Cer, GM1, and GM3 were highly abundant in CRL-1620, PSN1, and MDA-MB-231BR, respectively. These results suggest that our method provides a sensitive, reliable, and high-throughput workflow for ganglioside profiling across different cell types.

Mass spectrometry (MS) has revolutionized analytical chemistry, enabling precise identification and quantification of chemical species, which is pivotal for biomarker discovery and understanding complex biological systems. Despite its versatility, the presence of background ions in MS analysis hinders the sensitive detection of low-abundance analytes. Therefore, studies aimed at lowering background ion levels have become increasingly important. Here, we utilized the commercially available Active Background Ion Reduction Device (ABIRD) to suppress background ions and assess its effect on the liquid chromatography-electrospray ionization (LC-ESI)-MS analyses of N-glycans on the Q Exactive HF mass spectrometer. We also investigated the effect of different solvent vapors in the ESI source on N-glycan analysis by MS. ABIRD generally had no effect on high-mannose and neutral structures but reduced the intensity of some structures that contained sialic acid, fucose, or both when methanol vapor filled the ESI source. Based on our findings on the highest number of identified N-glycans from human serum, methanol vapor in the ion source compartment may enhance N-glycan LC-ESI-MS analyses by improving the desolvation of droplets formed during the ESI process due to its high volatility. This protocol may be further validated and extended to advanced bottom-up proteomic/glycoproteomic studies for the analysis of peptide/glycopeptide ions by MS., (© 2024 Wiley‐VCH GmbH.)

Glycosylation is an important post-translational modification that affects many critical cellular functions such as adhesion, signaling, protein stability, and function, among others. Abnormal glycosylation has been linked to many diseases. As such, the investigation of glycans and their roles in disease pathway and progression is important. Glycan analysis can be challenging, however, due to such factors as the heterogeneity of glycans and isomers as well as the poor ionization efficiency provided by mass spectrometry analyses. This chapter presents efficient methods that overcome these and other challenges for the analysis of native and permethylated N-glycan isomers in biological samples. Instructions regarding the packing of the MGC column, the N-glycan sample prep, and the LC-MS conditions are also provided., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The highly glycosylated S protein plays a vital role in host cell invasion, making it the principal target for vaccine development. Differences in mutations observed on the spike (S) protein of SARS-CoV-2 variants may result in distinct glycosylation patterns, thus influencing immunological evasion, infectivity, and transmissibility. The glycans can mask key epitopes on the S1 protein and alter its structural conformation, allowing the virus to escape the immune system. Therefore, we comprehensively characterize O -glycosylation in eleven variants of SARS-CoV-2 S1 subunits to understand the differences observed in the biology of the variants. In-depth characterization was performed with a double digestion strategy and an efficient LC-MS/MS approach. We observed that O -glycosylation is highly conserved across all variants in the region between the NTD and RBD, whereas other domains and regions exhibit variation in O -glycosylation. Notably, omicron has the highest number of O -glycosylation sites on the S1 subunit. Also, omicron has the highest level of sialylation in the RBD and RBM functional motifs. Our findings may shed light on how differences in O -glycosylation impact viral pathogenicity in variants of SARS-CoV-2 and facilitate the development of a robust vaccine with high protective efficacy against the variants of concern.

The SARS-CoV-2 virus rapidly spread worldwide, threatening public health. Since it emerged, the scientific community has been engaged in the development of effective therapeutics and vaccines. The subunit S1 in the spike protein of SARS-CoV-2 mediates the viral entry into the host and is therefore one of the major research targets. The S1 protein is extensively glycosylated, and there is compelling evidence that glycans protect the virus' active site from the human defense system. Therefore, investigation of the S1 protein glycome alterations in the different virus variants will provide a view of the glycan evolution and its relationship with the virus pathogenesis. In this study, we explored the N -glycosylation expression of the S1 protein for eleven SARS-CoV-2 variants: five variants of concern (VOC), including alpha, beta, gamma, delta, and omicron, and six variants of interest (VOI), including epsilon, eta, iota, lambda, kappa, and mu. The results showed significant differences in the N -glycome abundance of all variants. The N -glycome of the VOC showed a large increase in the abundance of sialofucosylated glycans, with the greatest abundance in the omicron variant. In contrast, the results showed a large abundance of fucosylated glycans for most of the VOI. Two glycan compositions, GlcNAc 4 ,Hex 5 ,Fuc,NeuAc (4-5-1-1) and GlcNAc 6 ,Hex 8 ,Fuc,NeuAc (6-8-1-1), were the most abundant structures across all variants. We believe that our data will contribute to understanding the S1 protein's structural differences between SARS-CoV-2 mutations.

Engineered cardiac microtissues were fabricated using pluripotent stem cells with a hypertrophic cardiomyopathy associated c. 2827 C>T; p.R943x truncation variant in myosin binding protein C (MYBPC3 +/- ). Microtissues were mounted on iron-incorporated cantilevers, allowing manipulations of cantilever stiffness using magnets, enabling examination of how in vitro afterload affects contractility. MYPBC3 +/- microtissues developed augmented force, work, and power when cultured with increased in vitro afterload when compared with isogenic controls in which the MYBPC3 mutation had been corrected (MYPBC3 +/+ (ed)), but weaker contractility when cultured with lower in vitro afterload. After initial tissue maturation, MYPBC3 +/- CMTs exhibited increased force, work, and power in response to both acute and sustained increases of in vitro afterload. Together, these studies demonstrate that extrinsic biomechanical challenges potentiate genetically-driven intrinsic increases in contractility that may contribute to clinical disease progression in patients with HCM due to hypercontractile MYBPC3 variants., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mild cognitive impairment (MCI) is an early stage of memory loss that affects cognitive abilities, such as language or virtual/spatial comprehension. This cognitive decline is mostly observed with the aging of individuals. Recently, MCI has been considered as a prodromal phase of Alzheimer's disease (AD), with a 10-15% conversion rate. However, the existing diagnostic methods fail to provide precise and well-timed diagnoses, and the pathophysiology of MCI is not fully understood. Alterations of serum N -glycan expression could represent essential contributors to the overall pathophysiology of neurodegenerative diseases and be used as a potential marker to assess MCI diagnosis using non-invasive procedures. Herein, we undertook an LC-MS/MS glycomics approach to determine and characterize potential N -glycan markers in depleted blood serum samples from MCI patients. For the first time, we profiled the isomeric glycome of the low abundant serum glycoproteins extracted from serum samples of control and MCI patients using an LC-MS/MS analytical strategy. Additionally, the MRM validation of the identified data showed five isomeric N -glycans with the ability to discriminate between healthy and MCI patients: the sialylated N -glycans GlcNAc 5 ,Hex 6 ,Neu5Ac 3 and GlcNAc 6 ,Hex 7 ,Neu5Ac 4 with single AUCs of 0.92 and 0.87, respectively, and a combined AUC of 0.96; and the sialylated-fucosylated N -glycans GlcNAc 4 ,Hex 5 ,Fuc,Neu5Ac, GlcNAc 5 ,Hex 6 ,Fuc,Neu5Ac 2 , and GlcNAc 6 ,Hex 7 ,Fuc,Neu5Ac 3 with single AUCs of 0.94, 0.67, and 0.88, respectively, and a combined AUC of 0.98. According to the ingenuity pathway analysis (IPA) and in line with recent publications, the identified N -glycans may play an important role in neuroinflammation. It is a process that plays a fundamental role in neuroinflammation, an important process in the progression of neurodegenerative diseases.

New directions in material applications have allowed for the fresh insight into the coordination of biophysical cues and regulators. Although the role of the mechanical microenvironment on cell responses and mechanics is often studied, most analyses only consider static environments and behavior, however, cells and tissues are themselves dynamic materials that adapt in myriad ways to alterations in their environment. Here, we introduce an approach, through the addition of magnetic inclusions into a soft poly(dimethylsiloxane) elastomer, to fabricate a substrate that can be stiffened nearly instantaneously in the presence of cells through the use of a magnetic gradient to investigate short-term cellular responses to dynamic stiffening or softening. This substrate allows us to observe time-dependent changes, such as spreading, stress fiber formation, Yes-associated protein translocation, and sarcomere organization. The identification of temporal dynamic changes on a short time scale suggests that this technology can be more broadly applied to study targeted mechanisms of diverse biologic processes, including cell division, differentiation, tissue repair, pathological adaptations, and cell-death pathways. Our method provides a unique in vitro platform for studying the dynamic cell behavior by better mimicking more complex and realistic microenvironments. This platform will be amenable to future studies aimed at elucidating the mechanisms underlying mechanical sensing and signaling that influence cellular behaviors and interactions.

Adhesion between silica glass or acrylic balls and silicone elastomers and various industrial rubbers is investigated. The work of adhesion during pull-off is found to strongly vary depending on the system, which we attribute to the two opposite effects: (1) viscoelastic energy dissipation close to an opening crack tip and (2) surface roughness. Introducing surface roughness on the glass ball is found to increase the work of adhesion for soft elastomers, while for the stiffer elastomers it results in a strong reduction in the work of adhesion. For the soft silicone elastomers a strong increase in the work of adhesion with increasing pull-off velocity is observed, which may result from the non-adiabatic processes associated with molecular chain pull-out. In general, the work of adhesion is decreased after repeated contacts due to the transfer of molecules from the elastomers to the glass ball. Thus, extracting the free chains (oligomers) from the silicone elastomers is shown to make the work of adhesion independent of the number of contacts. The viscoelastic properties (linear and nonlinear) of all of the rubber compounds are measured, and the velocity dependent crack opening propagation energy at the interface is calculated. Silicone elastomers show a good agreement between the measured work of adhesion and the predicted results, but carbon black filled hydrogenated nitrile butadiene rubber compounds reveal that strain softening at the crack tip may play an important role in determining the work of adhesion. Additionally, adhesion measurement under submerged conditions in distilled water and water + soap solutions are also performed: a strong reduction in the work of adhesion is measured for the silicone elastomers submerged in water, and a complete elimination of adhesion is found for the water + soap solution attributed to an osmotic repulsion between the negatively charged surface of the glass and the elastomer.

Fluid instabilities limit the ability of features to hold their shape in many types of 3D printing as liquid inks solidify into written structures. By 3D printing directly into a continuum of jammed granular microgels, these instabilities are circumvented by eliminating surface tension and body forces. However, this type of 3D printing process is potentially limited by inertial instabilities if performed at high speeds where turbulence may destroy features as they are written. Here, we design and test a high-speed 3D printing experimental system to identify the instabilities that arise when an injection nozzle translates at 1 m/s. We find that the viscosity of the injected material can control the Reynold's instability, and we discover an additional, unanticipated instability near the top surface of the granular microgel medium.

Background: Seafaring is known to be a demanding occupation but the implications of ageing in seafarers are poorly understood., Aims: To investigate task demands and work ability in merchant seamen at sea and to identify factors predicting work ability., Methods: This was a cross-sectional study carried out on a single vessel during a summer deployment. Instantaneous heart rate (HR) was recorded at 5-s intervals during representative 8-h shifts in 41 merchant seamen. Participants completed the work ability index and also rated their daily task demands using the National Aeronautics and Space Administration Task Load Index (NASA-TLX). Body mass index (BMI), waist circumference and demographic details were recorded., Results: Work demands were found to be moderate according to both HR data and TLX scores. The mean BMI was 27.5 kg/m(2) (standard deviation 3.3) and the mean age was 47 years. The majority of participants rated their work ability as 'good' to 'excellent'. The best predictor of work ability was the interaction between BMI and age, whereas there was a statistically significant negative correlation between HR and age., Conclusions: Work ability in this group of seafarers was found to be high and the work was shown to be moderately demanding, on average. Although work ability declined with age and with BMI independently, the best predictor of work ability was the interaction between BMI and age, with increased BMI having a deleterious effect on work ability in older employees doing moderately demanding work.

Objective: Increasing awareness of the high physical cost associated with lifting has led to the redesign of these tasks, incorporating manual handling devices and consequently pushing and pulling. Little research has focused on muscle activity responses to pushing and pulling, the current study therefore investigated these responses to further the understanding of risk of injury, informing ergonomics intervention strategies., Methods: A laboratory study was undertaken to determine the effect of three push/pull techniques and two loads (250 and 500 kg) on muscle activation in nine muscles, distributed through the upper and lower body. Unloaded forward and backward walking were used as control conditions for lower limb muscle activation., Participants: Thirty-six healthy male volunteers participated in the study. Subjects were required to manoeuvre a loaded pallet jack at a velocity of 0.45-0.55 statures., Results: The muscles of the shoulders and upper extremity were affected to a greater degree by technique and load changes than those of the lower limbs. Further, high levels of erector spinae activation were recorded across all six experimental conditions., Conclusions: Each technique displayed a unique muscle activation profile, indicating that alternating between techniques may reduce early onset of fatigue. Further understanding of muscle activation during pushing and pulling is necessary.