High Protein Research Articles

Molecular Mechanism of lncRNAs in Regulation of Breast Cancer Metastasis; a Comprehensive Review.

Although the number of breast cancer deaths has decreased, and there have been developments in targeted therapies and combination treatments for the management of metastatic illness, metastatic breast cancer is still the second most common cause of cancer-related deaths in U.S. women. Numerous phases and a vast number of proteins and signaling molecules are involved in the invasion-metastasis cascade. The tumor cells penetrate and enter the blood or lymphatic vessels, and travel to distant organs via the lymphatic or blood vessels. Tumor cells enter cell cycle arrest, adhere to capillary beds in the target organ, and then disseminate throughout the organ's parenchyma, proliferating and enhancing angiogenesis. Each of these processes is regulated by changes in the expression of different genes, in which lncRNAs play a role in this regulation. Transcripts that are longer than 200 nucleotides and do not translate into proteins are called RNAs. LncRNA molecules, whose function depends on their unique molecular structure, play significant roles in controlling the expression of genes at various epigenetic levels, transcription, and so on. LncRNAs have essential functions in regulating the expression of genes linked to cell development in healthy and pathological processes, specialization, programmed cell death, cell division, invasion, DNA damage, and spread to other parts of the body. A number of cancer types have been shown to exhibit aberrant expression of lncRNAs. In this review, we describe the general characteristics, potential molecular mechanisms and targeted therapy of lncRNAs and discuss the emerging functions of lncRNAs in breast cancer.

Spray dried protein concentrates from white button and oyster mushrooms produced by ultrasound-assisted alkaline extraction and isoelectric precipitation.

In the present study, the optimization of ultrasound-assisted alkaline extraction (UAAE) and isoelectric precipitation (IEP) was applied to white button (WBM) and oyster (OYM) mushroom flours to produce functional spray dried mushroom protein concentrates. Solid-to-liquid ratio (5-15% w/v), ultrasound power (0-900 W) and type of acid [HCl or acetic acid (AcOH)] were evaluated for their effect on the extraction and protein yields from mushroom flours submitted to UAAE-IEP protein extraction. Prioritized conditions with maximized protein yield (5% w/v, 900 W, AcOH, for WBM; 5% w/v, 900 W, HCl for OYM) were used to produce spray dried protein concentrates from white button (WBM-PC) and oyster (OYM-PC) mushrooms with high solids recovery (62.3-65.8%). WBM-PC and OYM-PC had high protein content (5.19-5.81 g kg-1), in addition to remarkable foaming capacity (82.5-235.0%) and foam stability (7.0-162.5%), as well as antioxidant phenolics. Highly pH-dependent behavior was observed for solubility (> 90%, at pH 10) and emulsifying properties (emulsification activity index: > 50 m2 g-1, emulsion stability index: > 65%, at pH 10). UAAE-IEP followed by spray drying increased surface hydrophobicity and free sulfhydryl groups by up to 196.5% and 117.5%, respectively, which improved oil holding capacity (359.9-421.0%) and least gelation concentration (6.0-8.0%) of spray dried mushroom protein concentrates. Overall, the present study showed that optimized UAAE-IEP coupled with spray drying is an efficient strategy to produce novel mushroom protein concentrates with enhanced functional attributes for multiple food applications. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

N-terminal domain homologs of the orange carotenoid protein increase quenching of cyanobacterial phycobilisomes.

Stress exerted by excess captured light energy in cyanobacteria is prevented by the photoprotective activity of the orange carotenoid protein (OCP). Under high light, the OCP converts from an orange, inactive form (OCPO) into the red form (OCPR) that binds to and quenches the phycobilisome (PBS). Structurally, the OCP consists of two domains: the N-terminal effector domain and a C-terminal regulatory domain. Structural analysis of the OCP-PBS complex showed that the N-terminal domains of an OCP dimer interact with the PBS core. These N-terminal OCP domains have single domain protein paralogs known as Helical Carotenoid Proteins (HCPs). Using phycobilisome quenching assays, we show that the HCP4 and HCP5 homologs efficiently quench PBS fluorescence in vitro, surpassing the quenching ability of the OCP. This is consistent with computational quantum mechanics/molecular mechanics results. Interestingly, when using a maximum quenching concentration of OCP with phycobilisomes, HCP5 addition further increases phycobilisome quenching. Our results provide mechanistic insight into the quenching capacity and roles of HCP4 and HCP5 in cyanobacteria, suggesting that they are more than simply functionally redundant to the OCP.

What are the essential determinants of human papillomavirus carcinogenesis?

Human papillomavirus (HPV) infection is the leading viral cause of cancer. Over the past several decades, research on HPVs has provided remarkable insight into human cell biology and into the pathology of viral and non-viral cancers. The HPV E6 and E7 proteins engage host cellular proteins to establish an environment in infected cells that is conducive to virus replication. They rewire host cell signaling pathways to promote proliferation, inhibit differentiation, and limit cell death. The activity of the "high-risk" HPV E6 and E7 proteins is so potent that their dysregulated expression is sufficient to drive the initiation and maintenance of HPV-associated cancers. Consequently, intensive research efforts have aimed to identify the host cell targets of E6 and E7, in part with the idea that some or all of the virus-host interactions would be essential cancer drivers. These efforts have identified a large number of potential binding partners of each oncoprotein. However, over the same time period, parallel research has revealed that a relatively small number of genetic mutations drive carcinogenesis in most non-viral cancers. We therefore propose that a high-priority goal is to identify which of the many targets of E6 and E7 are critical drivers of HPV carcinogenesis. By identifying the cancer-driving targets of E6 and E7, it should be possible to better understand the distinct roles of other targets, perhaps in the viral life cycle, and to focus efforts to develop anti-cancer therapies on the subset of virus-host interactions for which therapeutic intervention would have the greatest impact.

Effects of spray drying and freeze drying on the protein profile of whey protein concentrate.

Whey protein concentrate (WPC) is consumed for its high protein content. The structure and biological functionality of whey proteins in WPC powders may be affected by the drying technique applied. However, the specific impact of spray drying and freeze drying on the overall protein profile of whey protein derived from sweet whey streams at scale is unknown. Herein, we examine the effects of commercial-scale freeze drying and spray drying on WPC to determine which method better preserves bioactive whey proteins, with the goal of helping the dairy industry create high-value products that meet the growing consumer demand for functional dairy products. WPCs were produced from pasteurized liquid whey using either a commercial spray dryer or freeze dryer. A variety of analytical techniques, including enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, and bottom-up proteomics using liquid chromatography-tandem mass spectroscopy were used to identify, quantify, and compare the retention of bioactive proteins in WPC before and after spray drying and freeze drying. In addition, the extent of denaturation was studied via solubility testing, differential scanning calorimetry, and hydrophobicity assessment. There was little to no difference in the retention or denaturation of key bioactive proteins between spray-dried and freeze-dried WPC powders. There was a higher percentage of select Maillard modifications in freeze-dried and spray-dried powders than in the control. The lack of significant differences between spray drying and freeze drying identified herein indicates that freeze drying does not meaningfully improve retention of bioactive proteins compared with spray drying when performed after multiple pasteurization steps. PRACTICAL APPLICATION: This study aimed to provide insight into the impacts of spray drying versus freeze drying on whey proteins. Overall, our results indicate that for commercial dairy processing that involves multiple rounds of pasteurization, freeze drying does not meaningfully improve the retention of bioactive proteins compared with spray drying. These findings may help the food and dairy industry make informed decisions regarding the processing of its whey protein products to optimize nutritional value.

Pair-EGRET: Enhancing the prediction of protein-protein interaction sites through graph attention networks and protein language models.

Proteins are responsible for most biological functions, many of which require the interaction of more than one protein molecule. However, accurately predicting protein-protein interaction (PPI) sites (the interfacial residues of a protein that interact with other protein molecules) remains a challenge. The growing demand and cost associated with the reliable identification of PPI sites using conventional experimental methods call for computational tools for automated prediction and understanding of PPIs. We present Pair-EGRET, an edge-aggregated graph attention network that leverages the features extracted from pre-trained transformer-like models to accurately predict PPI sites. Pair-EGRET works on a k-nearest neighbor graph, representing the three-dimensional structure of a protein, and utilizes the cross-attention mechanism for accurate identification of interfacial residues of a pair of proteins. Through an extensive evaluation study using a diverse array of experimental data, evaluation metrics, and case studies on representative protein sequences, we demonstrate that Pair-EGRET can achieve remarkable performance in predicting PPI sites. Moreover, Pair-EGRET can provide interpretable insights from the learned cross-attention matrix. Pair-EGRET is freely available in open source form at the GitHub Repository https://github.com/1705004/Pair-EGRET. Supplementary data are available at Bioinformatics online.

‘Effectiveness of various sulbactam-based combination antibiotic therapy in the management of ventilator-associated pneumonia caused by carbapenem-resistant Acinetobacter baumannii in a tertiary care Health centre’

ObjectiveCarbapenem-resistant Acinetobacter baumannii (CRAB) is a common cause of ventilator-associated pneumonia (VAP). Some in vitro data favour various combination antibiotic therapy. However, there is a need for more in vivo studies for the management of VAP caused by CRAB. This retrospective study was done to evaluate the effectiveness of various combination antibiotic therapy including sulbactam on outcomes of VAP caused by CRAB. MethodsAdult patients (age ≥18 years) diagnosed with VAP caused by CRAB were included. Patients with polymicrobial infections were excluded from the study. Patients with CRAB associated VAP who were given sulbactam based antibiotic combinations were observed for outcomes. The primary outcome was 28-day mortality after diagnosis of VAP caused by CRAB. Reduction in serum HsCRP (High sensitivity C-reactive protein) during treatment and requirement of inotropes were the secondary outcomes. Outcomes were compared between various sulbactam based antibiotic combination therapies. ResultsA total of 103 patients were included. A total of 44 (42.7 %) patients received sulbactam and minocycline or sulbactam and polymyxin B dual antibiotic combination, and 59 (57.3 %) patients received sulbactam, polymyxin B and minocycline triple antibiotic combination. The percentage difference in 28 days mortality was 27.51 % (95 % CI 8.03 %–44.06 %; p = 0.005) in dual vs triple sulbactam based antibiotic combination therapy. The percentage difference in requirement of inotropes during therapy and HsCRP reduction after 7 days of therapy was 23.65 % (95 % CI 6.43 %–38.3 %; p = 0.007) and 25.1 % (95%CI 10.1 %–38.2 %; p < 0.001) respectively when compared between dual vs triple sulbactam based antibiotic combination therapy. ConclusionTreatment with sulbactam, polymyxin B and minocycline combination antibiotic therapy was associated with significantly lower 28-day mortality. Moreover, the lower requirement of inotropes during treatment and a significant reduction in HsCRP level favours this combination antibiotic therapy in VAP caused by CRAB.

Limitations of effective medium models for tissue phantoms in the THz frequency range.

The absorption of terahertz (THz) radiation by water molecules facilitates its application to several biomedical applications such as cancer detection. Therefore, it is critical for the THz technologies to be characterised with water content in a sample. In this paper, we analyse gelatine phantoms in the THz frequency range, with continuously varying hydration levels as they dry over time. Water molecules in close proximity to the protein molecule, termed 'bound water', feature properties different from the 'free water' molecules at larger distances. We find that a common model for predicting electromagnetic properties of phantoms and tissue samples, which assumes that only the free water varies with hydration while the bound water remains constant, does not agree well with measured results. To gain insight into this behaviour, we simultaneously measured the phantom in Raman spectroscopy, which shows a continuously varying concentration of bound water with hydration level. It follows from this investigation, that the permittivity contributions of neither the biomolecules nor water are expected to be linear with water density. This means that the often used, simple effective medium model will not be accurate for many biological tissues or phantoms.

Use of rice flour to produce plant-based yogurt alternatives.

Plant-based yogurt alternatives (YAs) are in demand due to the societal prevalence of milk sensitivities and allergies and some consumers abstaining from animal-derived products. Producing rice flour YAs has considerable potential because rice flour is hypoallergenic, more economical compared to plant milks, and there are no commercial rice-based YAs. A new higher protein variety of rice was developed, Frontière, which is sold as both brown and white rice. Therefore, the overall goals of this study were (1) to compare physicochemical properties of YAs from Frontière brown (Frontière brown rice flour [FBRF]) and white (Frontière white rice flour [FWRF]) high-protein rice flours to regular protein level rice flours and (2) to evaluate the sensory quality of Frontière YAs compared to commercial plant-based oat yogurt. Rice flours were fermented with Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lacticaseibacillus rhamnosus (a probiotic) to produce FBRF- and FWRF-YAs. A consumer study was conducted to compare FBRF- and FWRF-YAs to a commercially available oat-based YA. Consumers rated the YAs using 9-point hedonic and just-about-right (JAR) scales. Protein, fat, and ash levels were greater, whereas starch levels and peak viscosities were lower for BRFs than for WRFs. The use of BRF resulted in longer fermentation times but higher bacteria counts for YAs. FWRF-YA was preferred in terms of overall flavor and liking, sweetness, and tartness. Purchase intent (PI) for FWRF-YA increased 2.6 times after providing a health claim. This research showed that rice flour can be used to produce YAs with probiotic counts above the minimum recommended, which provides added health benefits for consumers. PRACTICAL APPLICATION: This research provides a possible new use of Frontière high-protein rice flour to produce plant-based yogurts. This will help the rice industry by adding value, and those who are vegan, allergic to casein, or lactose-intolerant will have another option for a plant-based yogurt. Moreover, the greater levels of probiotic bacteria found in the brown rice flour YAs can potentially provide greater health benefits, making brown rice flour a better choice for making rice-based YAs.

A Numerical Integrator for Kinetostatic Folding of Protein Molecules Modeled as Robots with Hyper Degrees of Freedom

The kinetostatic compliance method (KCM) models protein molecules as nanomechanisms consisting of numerous rigid peptide plane linkages. These linkages articulate with respect to each other through changes in the molecule dihedral angles, resulting in a kinematic mechanism with hyper degrees of freedom. Within the KCM framework, nonlinear interatomic forces drive protein folding by guiding the molecule’s dihedral angle vector towards its lowest energy state in a kinetostatic manner. This paper proposes a numerical integrator that is well suited to KCM-based protein folding and overcomes the limitations of traditional explicit Euler methods with fixed step size. Our proposed integration scheme is based on pseudo-transient continuation with an adaptive step size updating rule that can efficiently compute protein folding pathways, namely, the transient three-dimensional configurations of protein molecules during folding. Numerical simulations utilizing the KCM approach on protein backbones confirm the effectiveness of the proposed integrator.

Effect of fermentation, malting and ultrasonication on sorghum, mopane worm and Moringa oleifera: improvement in their nutritional, techno-functional and health promoting properties

BackgroundFood processing offers various benefits that contribute to food nutrition, food security and convenience. This study investigated the effect of three different processes (fermentation, malting and ultrasonication) on the nutritional, techno-functional and health-promoting properties of sorghum, mopane worm and Moringa oleifera.MethodsThe fermented and malted flours were prepared at 35°C for 48 h, and for ultrasonication, samples were subjected to 10 min at 4°C with amplitudes of 40–70 Hz. The biochemical, nutritional quality and techno-functional properties of the obtained flours were analysed using standard procedures.ResultsFermentation resulted in significantly lower pH and higher titratable acidity in sorghum and mopane worm (4.32 and 4.76; 0.24 and 0.69% lactic acid, respectively), and malting resulted in higher total phenolic content and total flavonoid content in sorghum (3.23 mg GAE/g and 3.05 mg QE/g). Ultrasonication resulted in higher protein and fibre in raw sorghum flour (13.38 and 4.53%) and mopane worm (56.24 and 11.74%) while raw moringa had the highest protein (30.68%). Biomodification by fermentation in sorghum led to higher water and oil holding capacity and increased dispersibility in the ultrasonicated samples. Ultrasonication of mopane worms led to higher water holding capacity, oil holding capacity and dispersibility. Lightness was found to be significantly higher in the fermented samples in sorghum and mopane worm. Raw moringa had the greatest lightness compared to the ultrasonicated moringa. Moringa had the most redness and browning index among all samples.ConclusionIn this study, all the investigated processes were found to have caused variations in flours’ biochemical, nutritional and techno-functional properties. Ultrasonication process was noteworthy to be the most efficient to preserve the nutritional value in sorghum, mopane worm and M. oleifera flours.

A specific domain within the 3′ untranslated region of Usutu virus confers resistance to the exonuclease ISG20

Usutu virus (USUV) and West Nile virus (WNV) are two closely related emerging mosquito-borne flaviviruses. Their natural hosts are wild birds, but they can also cause severe neurological disorders in humans. Both viruses are efficiently suppressed by type I interferon (IFN), which interferes with viral replication, dissemination, pathogenesis and transmission. Here, we show that the replication of USUV and WNV are inhibited through a common set of IFN–induced genes (ISGs), with the notable exception of ISG20, which USUV is resistant to. Strikingly, USUV was the only virus among all the other tested mosquito-borne flaviviruses that demonstrated resistance to the 3′–5′ exonuclease activity of ISG20. Our findings highlight that the intrinsic resistance of the USUV genome, irrespective of the presence of cellular or viral proteins or protective post-transcriptional modifications, relies on a unique sequence present in its 3′ untranslated region. Importantly, this genomic region alone can confer ISG20 resistance to a susceptible flavivirus, without compromising its infectivity, suggesting that it could be acquired by other flaviviruses. This study provides new insights into the strategy employed by emerging flaviviruses to overcome host defense mechanisms.

B-157 High Resolution Liquid Chromatography - Mass Spectrometry (LC-MS) For Rapid, High-Throughput Clinical Testing of β2-Transferrin in Human Nasal Secretions

Abstract Background Leakage of Cerebrospinal Fluid (CSF) can result from trauma, lacerations, and surgery, and if untreated can result in potentially life-threatening meningitis. CSF leaks can be diagnosed through the detection of β2-Transferrin (β2-Tf) from nasal secretions via gel electrophoresis. β2-Tf is a uniquely glycosylated proteoform of Transferrin found primarily in CSF, which can be distinguished from the most abundant Transferrin proteoform (β1-Tf) by its altered electrophoretic mobility. However, gel-based detection of β2-Tf requires hours of labor and has low specificity due to the numerous alternative glycoforms of Transferrin. Higher-throughput assays are thus desirable for improved patient care. Recently, our group applied High-Resolution Mass Spectrometry (HR-MS) coupled to Microprobe In-Emitter Elution (MPIE) to discriminate Transferrin isoforms. This method successfully resolved β2- from β1-Tf in nasal secretions derived from patient samples with suspected CSF leakage, but has relatively low sample-to-sample throughput. Herein, we describe a novel approach utilizing Liquid Chromatography Mass Spectrometry (LC-MS) to rapidly profile Transferrin glycoforms from patient nasal secretion samples. Furthermore, we explore solutions to address significant blood contamination in nasal secretions, which may otherwise interfere with this approach. Furthermore, we explore solutions to address significant blood contamination in some nasal secretions, which may otherwise interfere with this approach. Methods Our approach consists of two primary steps: 1) Sample processing and high abundance protein depletion via commercial filtration and depletion columns. 2) LC-MS analysis of the resulting extract to resolve Transferrin glycoforms by retention time and m/z. This approach retains the high specificity of the previously demonstrated MPIE-MS method, but takes advantage of existing high-throughput liquid chromatography platforms to reduce the sample-to-sample analysis time. Secretion samples were analyzed using a Vanquish HPLC system coupled to a Q-Exactive Plus Mass Spectrometer (ThermoFisher). Broadband mass spectra were collected (FWHM resolution of 17,500 at 200 m/z), and the deconvoluted average masses were used to identify Transferrin glycoforms. Results Our approach was benchmarked using remnant nasal secretion samples from patients with known CSF leakage. Examining 9 samples, the resulting ESI spectra revealed peaks at 79,554 Da and 78,008 Da, consistent with β1-Tf (Pred. MW (Average): 79,554.71 Da) and β2-Tf (Pred. MW (Average): 78,008.35 Da) within ∼10 ppm mass accuracy. The 79.5 kD peak was observed in all samples, while the 78 kD peak was observed in 7 of 9 samples. Samples with missing β2-Tf were marked by significant blood contamination. Genetic variants (2/9 samples) resulted in mass shifts for both β1- and β2-Tf variants, but Transferrin glycoforms identifiable due to the preserved mass shift between the β1- and β2-Tf peaks (∼1546.4 Da). An optimized LC-MS gradient enables rapid (∼10 minute) sample-to-sample injection times. Samples with blood contamination represent an additional challenge for identification of β2-Tf, owing to ion suppression from blood-derived β1-Tf. We have developed enrichment and depletion methods to increase the sensitivity for β2-Tf in blood contaminated samples, and anticipate that these approaches will reduce or eliminate this challenge. Conclusions Our approach enables high-throughput, MS-based measurement of Transferrin glycoforms (i.e. β1- and β2-Tf) from clinical nasal secretion samples for the first time.

Prevalence of and factors associated with Na + /K + imbalances in a population of children hospitalized with febrile urinary tract infection.

Hyponatremia was the most common Na + /K + imbalance in children hospitalized for fUTI, followed by hyperkalemia and concurrent hyponatremia and hyperkalemia. C-RP levels were most strongly associated with hyponatremia, age with hyperkalemia, and CAKUT with concurrent hyponatremia and hyperkalemia (suggestive of transient secondary pseudo-hypoaldosteronism). Therefore, in children who develop concurrent hyponatremia and hyperkalemia during the course of a fUTI, an underlying CAKUT could be suspected. • Na+ and K+ abnormalities can occur in patients hospitalized for febrile urinary tract infection (fUTI). • Concurrent hyponatremia and hyperkalemia during fUTI may suggest transient secondary pseudo-hypoaldosteronism (TPHA), for which limited data on prevalence are available. • The most common Na+/K+ imbalance in children hospitalized with fUTI was hyponatremia (23%), followed by hyperkalemia (6.4%), concurrent hyponatremia and hyperkalemia (2.9%), hypokalemia (0.7%), and hypernatremia (0.4%). • Concurrent hyponatremia and hyperkalemia were mainly associated with CAKUT, while hyponatremia alone correlated with high C-reactive protein and hyperkalemia alone with younger age.In cases of concurrent hyponatremia and hyperkalemia during fUTI, an underlying CAKUT should be suspected.

A-282 Purpura fulminans caused by Haemophilus influenzae: case report of unusual etiology

Abstract Background Fulminant purpura is a clinical syndrome characterized by a purplish, ischemic-looking skin rash, commonly accompanied by systemic symptoms ranging from acute febrile syndrome to septic shock. Due to high lethality of possible infectious etiologies, immediate recognition and early initiation of empirical antimicrobial therapy are crucial to ensure better outcomes. Interdisciplinary collaboration among healthcare teams, infectious disease specialists and microbiologists is essential to ensure accurate and early microbiological diagnosis. Methods This work aims to report a case of Haemophilus influenzae-induced purpura fulminans that occurred in Fortaleza-CE, a coastal city in northeastern Brazil, highlighting clinical recognition and microbiological identification, based on review of medical records. Results Case report A 59-year-old man, originally from Germany, was on vacation in Brazil. He presented to emergency medical service with an acute onset of fever, abdominal pain and diarrhea, progressing to decreased level of consciousness and purplish ischemic skin lesions with hemorrhagic bullae on trunk and extremities (Figure 1), with no history of trauma. Upon admission, the blood exams showed bicytopenia - 1900 leukocytes/µL (86% segmented) and 51,000 platelets/µL - and very high C-reactive protein (41.58 mg/dL). Although empirical antimicrobial therapy with vancomycin and piperacillin-tazobactam was initiated, patient progressed to septic shock, hepatic dysfunction, requiring mechanical ventilation, vasopressor support, and renal replacement therapy. Blood cultures from hospital admission were positive after about 14 hours and allowed isolation of small and translucent colonies in Chocolate PVX plate Agar (BioMerieux™). Maldi tof analysis by Vitek MS identified H. influenzae (99.9 % accuracy). Despite prolonged hospitalization due to multiple organ dysfunctions, patient had a positive outcome. Conclusions This case illustrates the importance of immediate recognition of the syndrome coupled with assertive empirical treatment and early microbiological diagnosis for a positive clinical outcome, even in a severe presentation. Multiprofessional approach is essential for accurate and early diagnosis.

Modification of an AIE Fluorescent Probe for Monitoring the Polarity of Lipid Droplets Based on a Series of Synthesized Aryl Naphthalizes.

Lipid droplets (LDs) are subcellular organelles that are dynamic and play a central role in energy homeostasis and lipid metabolism. They also contribute to the transport and maturation of cellular proteins and are closely associated with several diseases. The important role of the cellular microenvironment in maintaining cellular homeostasis. Changes in cell polarity, particularly in organelles, have been found to be strongly linked to inflammation, Alzheimer's disease, cancer, and other illnesses. It is essential to check the polarity of the LDs. A series of arylated naphthalimide derivatives were synthesized using the Suzuki reaction. Modification of synthesized aryl naphthalimides using oligomeric PEG based on intramolecular charge transfer (ICT) mechanism. A series of fluorescent probes were designed to target LDs and detect their polarity. Nap-TPA-PEG3 probe exhibited high sensitivity to polarity. The addition of oligomeric polyethylene glycol (PEG) to the probe not only significantly improved its solubility in water, but also effectively reduced its cytotoxicity. In addition, the probe exhibited excellent aggregation-induced luminescence (AIE) properties and solvent discolouration effects. Nap-TPA-PEG3 probe exhibited high Pearson correlation coefficient (0.957163) in lipid droplet co-localization in cells. Nap-TPA-PEG3 could be used as an effective hand tool to monitor cell polarity.

B-247 A Missing Peptide Link: A Case Study Involving Detection of an Unusual IGF-1 Protein Form Using High-Resolution Mass Spectrometry

Abstract Background Previously, we described a method for monitoring insulin-like growth factor-1 (IGF-1) variants using liquid chromatography coupled with high-resolution, accurate-mass, mass spectrometry (LC-HRAM-MS). The method split s variants in 4 Variant Groups (VG) and characterizes them by the Isotopic Peak index (IPi) and relative retention time (rRT). Uncommon variants are analyzed by tandem mass-spectrometry (MS/MS) and specimens suspected to have a novel variant are deidentified and routed for DNA sequencing. A potentially novel species was detected that did not match characteristics of any previously identified IGF-1 variant. Methods LC-HRAM-MS was used for the detection of wild type (WT) IGF-1 protein and its variants in a serum specimen. Results The patient’s IGF-1 WT concentration was 210 ng/mL and z-score was -1.5, which were within normal reference ranges values for their sex and age. LC-HRAM-MS also revealed the isotopic distribution and charge for the variant, but with new characteristics (VG2:IP2, rRT= 0.01 min). The area under curve for the variant was 36% of that of the WT, which is a stark contrast to the usual 100% found in a typical heterozygous patient. MS/MS of the suspected variant showed 2 sets of b- and y-ions, which has not been seen in our laboratory. Sequencing of the fragments revealed that one set of C- and N-terminal amino acids were from the WT (C-terminus GPETL sequence and N-terminus PLKPAKSA sequence). The intact molecular weight (MW) of the species (monoisotopic m/z 1095.5250 at charge 7+) was 18 Da heavier than that of the WT (monoisotopic m/z 1092.9482 at charge 7+), so further investigation was warranted. The other set of b- and y-ions sequenced were manually identified as GL(or I)VD and PTGYG, respectively. BLAST search did not identify relevant proteins with these N- and C-termini. However, closer inspection revealed that identified sequences were found in the WT protein, albeit not at its termini. The rest of the amino acids were found that complete termini: C-terminal b-ions sequence RAPQTGIVD corresponded to positions 37-45 of the WT sequence, while N-terminal y-ions sequence KPTGYGSSSR corresponded to positions 27-36. All evidence indicates that the species is the WT protein, but with loss of a peptide bond between 2 arginines at positions 36-37 resulting in a gain of a water molecule to “cap” the newly formed termini, which is confirmed by its observed intact m/z. Conclusions We identified an extremely rare IGF-1 species formed through loss of a peptide bond between arginines at positions 36 and 37 resulting in a “missing link” in IGF-1 amino acid sequence. Despite the cleavage, the variant was a single protein molecule with intact disulfide bonds and 2 sets of C- and N-termini. The processes involved in generating this species (e.g., hydrolysis or enzymatic cleavage) and potential consequences for the patient are unknown. Because binding to receptors and binding proteins are potentially affected, and the total level of active WT IGF-1 is reduced, we are working with patient’s physician to establish whether this unusual form of IGF-1 protein may contribute to the etiology of the patient’s condition.

High-affinity antibodies specific to the core region of the tau protein exhibit diagnostic and therapeutic potential for Alzheimer’s disease

BackgroundRecent advances in blood-based biomarker discovery are paving the way for simpler, more accessible diagnostic tools that can detect early signs of Alzheimer’s disease (AD). Recent successes in the development of amyloid-targeting immunotherapy approaches mark an important advancement in providing new options for the treatment of AD. We have developed a set of high-affinity monoclonal antibodies (mAbs) to tau protein that have the potential as tools for diagnosis and treatment of AD.MethodsSheep were immunised with either full-length tau (1-441) or truncated paired helical filament (PHF)-core tau (297–391). A stringent bio-panning and epitope selection strategy, with a particular focus directed to epitopes within the disease-relevant PHF-core tau, was used to identify single-chain antibodies (scAbs). These scAbs were ranked by affinity for each epitope class, with leads converted to high-affinity mAbs. These antibodies and their potential utility were assessed by their performance in tau immunoassays, as well as their ability to prevent tau aggregation and propagation. Further characterisation of these antibodies was performed by immunohistochemical staining of brain sections and immuno-gold electronmicroscopy of isolated PHFs.ResultsOur work resulted in a set of high-affinity antibodies reacting with multiple epitopes spanning the entire tau protein molecule. The tau antibodies directed against the core tau unit of the PHF inhibited pathological aggregation and seeding using several biochemical and cell assay systems. Through staining of brain sections and PHFs, the panel of antibodies revealed which tau epitopes were available, truncated, or occluded. In addition, highly sensitive immunoassays were developed with the ability to distinguish between and quantify various tau fragments.ConclusionThis article introduces an alternative immunodiagnostic approach based on the concept of a “tauosome” – the diverse set of tau fragments present within biological fluids. The development of an antibody panel that can distinguish a range of different tau fragments provides the basis for a novel approach to potential diagnosis and monitoring of disease progression. Our results further support the notion that tau immunotherapy targeting the PHF-core needs to combine appropriate selection of both the target epitope and antibody affinity to optimise therapeutic potential.

B-150 Chromatographic Separation and Quantitation of Human Insulin and Lispro Analog in Serum Using Liquid Chromatography High-Resolution Mass Spectrometry

Abstract Background Factitious hypoglycemia, a deliberate attempt to induce low blood glucose levels occurring secondary to the surreptitious administration of exogenous insulin, can be difficult to diagnose due to a variety of factors including the increased use of synthetic insulin analogs in clinical practice. Traditional insulin immunoassays demonstrate variable cross-reactivity with commonly prescribed insulin analogues and their metabolites, making it difficult to measure specific analogs. The development of liquid chromatography high resolution accurate mass (LC-HRAM)-mass spectrometry (MS) assays has largely circumvented the limitations of traditional immunoassays for detection and quantitation of insulin analogs. Analytical challenges remain, however, specifically in differentiating lispro, an isomeric human insulin analog formed by the transposition of proline and lysine near the C-terminal end of the B chain, from human insulin in patient samples due to the equivalent mass-to-charge ratios of their precursor ions. Methods Immunoaffinity purification of insulin from calibrators and quality control material prepared by spiking insulin-depleted serum with pharmaceutical insulin preparations, as well as patient samples, was performed using tips coupled with anti-insulin monoclonal antibody. An automated liquid handler was programmed to perform aspiration and dispense cycles. Eluate from the purification protocol was submitted for analysis using a TLX-2 multiplex LC system paired with a Thermo Scientific Q Exactive Plus mass spectrometer. Various HPLC column chemistries were evaluated, a column passivation procedure was established, and column heater temperature was optimized to identify a combination of parameters yielding the best chromatographic separation of human insulin and lispro. Additionally, parallel reaction monitoring (PRM) targeted tandem mass spectrometry (MS/MS) analysis was utilized in combination with the full scan data to increase specificity through the monitoring of fragment ions produced from human insulin and each insulin analog. Results We found that a 1000 Å Diphenyl, 2.7 µm, 2.1 x 100 mm analytical column in a column heater maintained at a temperature of 80°C allowed for human insulin and lispro to be differentiated as two chromatographically separated extracted ion chromatographic peaks (XICs) and quantified down to 2.5 mcIU/mL with near-baseline separation. Before use, the column required passivation with a high protein buffer (0.05% bovine serum albumin in 20% acetonitrile [ACN] + 0.2% acetic acid) followed by increasingly organic buffers (20% ACN + 0.2% acetic acid and 50% ACN + 0.2% acetic acid). MS/MS was used to confirm the presence of analogs detected. Conclusions The LC-HRAM-MS method described here, coupled with optimized column chemistry, column passivation, and proper column temperature control allows for chromatographic separation of human insulin and lispro. Incorporating a PRM scan into the mass spectrometry full scan method provides confirmation of the insulin detected in the XICs. The results obtained from preliminary studies suggest this method is appropriate for use to facilitate the clinical investigation of suspected factitious hypoglycemia with the ability to differentiate isomeric compounds, such as human insulin and lispro.

Enhancing Resistance to Corrosion and Fouling Using Epoxy Coatings with Superhydrophobic Cells

AbstractAs maritime transport becomes increasingly important, metal corrosion results in significant economic losses. Superhydrophobic materials have significant potential for corrosion prevention. However, poor compatibility between superhydrophobic materials and resin reduces the long‐term corrosion resistance. Interfacial strengthening cells (IS‐Cells) are designed to maintain superhydrophobicity and retain active hydroxyl groups participating in resin‐curing. IS‐Cells also contribute to achieving the maze effect. A novel anti‐corrosion IS coating is fabricated by compatible IS‐Cells and epoxy. IS coatings feature smooth surfaces and vertical compactness, protecting against liquid and gaseous corrosive medium. Strong diffusion resistance and extended diffusion distance ensure long‐term corrosion resistance of IS coatings. Approximately 85 µm of IS coatings remain corrosion‐free after 112 days of immersion in the NaCl solution. The low‐frequency impedance, self‐corrosion potential, and self‐corrosion current density are 1.68 × 109 Ω cm2, −0.0708 V, and 5.38 × 10−11 A cm−2, respectively. IS coatings can also withstand 2400 h of neutral salt spray testing without corrosion in the artificial scratches. Due to the electrostatic repulsion between the hydrophobic C–F chains onto IS‐Cells and protein molecules, IS coatings maintain anti‐fouling properties after dry‐wet immersion tests in the natural marine environment for 90 days. IS coatings show great potential for marine anti‐corrosion applications.