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Artificial insemination has proven to be an effective method for increasing population size and genetic quality of Kacang goats. However, innovation is required to maintain the quality of Kacang goat semen in storage. This study aimed to examine the effects of supplementing the 150 kDa protein assumed as IGF-I complex derived from bull seminal plasma in skim milk-egg yolk extender on the quality of Kacang goat sperm stored at 5°C. Twelve ejaculates collected from three Kacang goats were divided into three groups. In the control group (T0), the ejaculates were extended with skim milk-egg yolk only. In the treatment groups (T1 and T2), the ejaculates were extended with skim milk-egg yolk supplemented with the IGF-I complex protein at 12 μg and 24 μg/100 mL, respectively. The extended semen was stored at 5°C, and the viability, motility, intactness of the plasma membrane, malondialdehyde concentration, and apoptotic sperm percentage were evaluated daily for five days. The results showed that the T1 was the most effective treatment for maintaining Kacang goat semen at a quality acceptable for artificial insemination over five days of storage at 5°C. However, the T0 and T2 groups retained acceptable qualities for only three days at 5°C. It could be concluded that supplementation of 12 μg of the 150 kDa protein derived from bull seminal plasma per 100 mL extender successfully extended the life span of Kacang goat sperm for five days.

Bariatric surgery; Nutrition supplementation; Protein Cirugía bariátrica; Suplementación nutricional; Proteína Cirurgia bariàtrica; Suplementació nutricional; Proteïna Introduction: A significant reduction in fat-free mass (FFM) following bariatric surgery (BS) has been reported, and adequate protein intake is recommended for FFM preservation. Current guidelines of nutritional management after BS recommend complex protein (CP) compounds. However, Roux-en-Y-gastric bypass (RYGB) has a negative impact on CP digestion, leading to protein malabsorption. At present, there is no data regarding the impact of early supplementation with short peptide-based (SPB) or hydroxy methylbutyrate (HMB)-enriched formulas on the evolution of the FFM after the BS. Aim: The aim of this study is to evaluate the effect of nutritional products based on CP, HBM-enriched, or SPB formulas on the FFM of patients that undergo RYGB. Material and methods: This is a prospective interventional study, including three groups of patients (according to the type of protein product) as candidates for BS, recruited between December 2021 and April 2022, matched by age, gender, and BMI. All patients underwent evaluations at baseline and one month post-BS, including: medical history, physical and anthropometric evaluation, bioimpedance, and biochemical analysis. Results: A total of 60 patients were recruited: 63% women, mean age 43.13 ± 9.4 years, and BMI 43.57 ± 4.1 kg/m2. The % of FFM loss from total weight loss (TWL) was significantly lower in the SPB group than CP and HMB groups despite the major %TWL in this group (40.60 ± 17.27 in CP, 34.57 ± 13.15 in HMB, and 19.14 ± 9.38 in SPB, p < 0.001). TWL% was 9.98 ± 1.82 vs. 9.83 ± 2.71 vs. 13.56 ± 4.30, p < 0.001, respectively. Conclusion: In our study, the SPB supplementation prevented almost 50% FFM lost from the TWL than the CP- or HMB-enriched compounds at one month post-BS. These results are significant in the setting of muscle mass preservation after the BS, and have the potential to change the current guidelines for the management of nutritional supplementation after BS. This study was supported by grants from the Instituto de Salud Carlos III (Fondo de Investigacion Sanitaria, PI20/01086). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

O-GalNAc type glycosylation is an abundant and complex protein modification. Recent developments in mass spectrometry resulted in significant success in quantitative analysis of O-GalNAc glycosylation. The analysis of released O-GalNAc type glycans expanded our horizons of understanding the glycome of various biological models. The site-specific analysis of glycosylation micro-heterogeneity of purified proteins opened perspectives for the improved design of glycoprotein therapeutics. Advanced gene editing and chemical technologies applied to O-glycoproteomics enabled to identify O-GalNAc glycosylation at unprecedented depth. Progress in the analysis of intact glycoproteins under native and reduced conditions enabled the monitoring of glycosylation proteoform variants. Despite of the astonishing results in quantitative O-GalNAc glycoproteomics, site-specific mapping of the full O-GalNAc structural repertoire in complex samples is yet a long way off. Here, we summarize the most common quantitative strategies in O-GalNAc glycoproteomics, review recent progress and discuss benefits and limitations of the various approaches in the field.

Selective separation and enrichment of phosphoproteins possess the distinct clinical and biological importance in the diagnosis, treatment, and management of several fatal human diseases. In this study, a facile synthesis of titanium(IV) ion–immobilized arsenate-modified poly(glycidyl methacrylate) microparticles (denoted as Ti4+-arsenate-PGMA-MPs) was developed for the efficient enrichment of intact phosphoproteins found in biologically complex protein samples. By virtue of the strong interaction between the titanium ions immobilized on the surface of Ti4+-arsenate-PGMA-MPs and phosphate groups of phosphoproteins, Ti4+-arsenate-PGMA-MPs had a high saturated adsorption capacity for phosphoproteins (901 mg/g for β-casein), which was much higher than that of non-phosphoproteins (73.5 mg/g for BSA). Ti4+-arsenate-PGMA-MPs were characterized by SEM, TEM, and FT-IR, and the average particle diameter was about 2.5 μm with good dispersibility. Besides, the application of Ti4+-arsenate-PGMA-MPs in real biological samples was investigated by SDS-PAGE analysis, and the results showed that Ti4+-arsenate-PGMA-MPs were able to enrich phosphoproteins efficiently.

Gluten is a common dietary component with a complex protein structure. It forms incomplete products of digestion, which have the potential to mount an immune response in genetically predisposed individuals, resulting in celiac disease. It also has been linked with nonceliac gluten sensitivity and irritable bowel syndrome due to wheat allergy. A gluten-free diet is an effective treatment of these conditions; however, it can lead to micronutrient and mineral deficiencies and a macronutrient imbalance with higher sugar and lipid intake. Recent popularity has led to greater availability, but increasing cost, of commercially available gluten-free products.

I Ion channels are complex protein structures comprising the molecular systems of opening, closing, selectivity, inactivation and regulation. This review article aims to generalize and systematize literature data on the structural and functional characteristics of ion channels and methods for investigating their activity. Violations of the activity of ion channels may alter the functioning of both individual cells and the entire organism. Therefore, further research into the structural and physiological characteristics of ion channels can be considered promising and relevant.

The repeated failures of amyloid-targeting therapies have challenged our narrow understanding of Alzheimer’s disease (AD) pathogenesis and inspired wide-ranging investigations into the underlying mechanisms of disease. Increasing evidence indicates that AD develops from an intricate web of biochemical and cellular processes that extend far beyond amyloid and tau accumulation. This growing recognition surrounding the diversity of AD pathophysiology underscores the need for holistic systems-based approaches to explore AD pathogenesis. Here we describe how network-based proteomics has emerged as a powerful tool and how its application to the AD brain has provided an informative framework for the complex protein pathophysiology underlying the disease. Furthermore, we outline how the AD brain network proteome can be leveraged to advance additional scientific and translational efforts, including the discovery of novel protein biomarkers of disease.

Proteins in semen, either in spermatozoa (SPZ) or seminal plasma (SP), are directly involved in molecular processes and biological pathways regulating sperm function, including fertilizing ability. Therefore, semen proteins are candidates of choice for biomarkers discovery for fertility and for sperm (dys)function. Mass spectrometry (MS)-based proteomics has opened up a new era for characterizing and quantifying the protein profile of SP and SPZ, as well as for unveiling the complex protein interactions involved in the activation of sperm functionality. This article overviews existing literature on MS-based proteomics regarding porcine semen, with a specific focus on the potential practical application of the results achieved so far. The weaknesses of current studies and the perspectives for future research in MS-based pig semen proteomics are also addressed.

Discerning tyrosine phosphorylation (pTyr) catalyzed by Tyr kinase is central to the revelation of oncogenic mechanisms and the development of targeted anticancer drugs. Despite some techniques, this goal remains challenging, especially when faced with the interference of multiple phosphorylation events, including serine (pSer) and threonine phosphorylation (pThr). We describe here a functional polymer-modified artificial ion nanochannel, which enables the sensitive and selective recognition of phosphotyrosine (pY) peptide by the distinct ionic current change. Such a recognition effect allows for the nanochannel to work in a complex protein digest condition. Further, the implementation of nanofluidic logic functions with the addition of Ca2+ dramatically improves the selectivity of the nanochannel to pY peptide and thus can discern pTyr by the Tyr kinase from pSer by the Ser/Thr kinase through simultaneously monitoring multisite phosphorylation at the same or different peptide substrates in one-pot. This logic sensing platform displays the potential in differentiating Tyr kinase and Ser/Thr kinase and assessing multi-kinase activities in multi-targeted drug design.

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular processes in vitro. In recent years, encapsulation within GUVs has proven to be a helpful approach for reconstitution experiments in cell biology and related fields. It better mimics confinement conditions inside living cells, as opposed to conventional biochemical reconstitution. Methods for encapsulation inside GUVs are often not easy to implement, and success rates can differ significantly from lab to lab. One technique that has proven to be successful for encapsulating more complex protein systems is called continuous droplet interface crossing encapsulation (cDICE). Here, a cDICE-based method is presented for rapidly encapsulating cytoskeletal proteins in GUVs with high encapsulation efficiency. In this method, first, lipid-monolayer droplets are generated by emulsifying a protein solution of interest in a lipid/oil mixture. After being added into a rotating 3D-printed chamber, these lipid-monolayered droplets then pass through a second lipid monolayer at a water/oil interface inside the chamber to form GUVs that contain the protein system. This method simplifies the overall procedure of encapsulation within GUVs and speeds up the process, and thus allows us to confine and observe the dynamic evolution of network assembly inside lipid bilayer vesicles. This platform is handy for studying the mechanics of cytoskeleton-membrane interactions in confinement.

We hypothesize that intrauterine hypoxia (HPX) alters the mitochondrial phenotype in fetal hearts contributing to developmental programming. Pregnant guinea pigs were exposed to normoxia (NMX) or hypoxia (HPX, 10.5% O2), starting at early [25 days (25d), 39d duration] or late gestation (50d, 14d duration). Near-term (64d) male and female fetuses were delivered by hysterotomy from anesthetized sows, and body/organ weights were measured. Left ventricles of fetal hearts were excised and frozen for measurement of expression of complex (I–V) subunits, fusion (Mfn2/OPA1) and fission (DRP1/Fis1) proteins, and enzymatic rates of I and IV from isolated mitochondrial proteins. Chronic HPX decreased fetal body weight and increased relative placenta weight regardless of timing. Early-onset HPX increased I, III, and V subunit levels, increased complex I but decreased IV activities in males but not females (all P < 0.05). Late-onset HPX decreased ( P < 0.05) I, III, and V levels in both sexes but increased I and decreased IV activities in males only. Both HPX conditions decreased cardiac mitochondrial DNA content in males only. Neither early- nor late-onset HPX had any effect on Mfn2 levels but increased OPA1 in both sexes. Both HPX treatments increased DRP1/Fis1 levels in males. In females, early-onset HPX increased DRP1 with no effect on Fis1, whereas late-onset HPX increased Fis1 with no effect on DRP1. We conclude that both early- and late-onset HPX disrupts the expression/activities of select complexes that could reduce respiratory efficiency and shifts dynamics toward fission in fetal hearts. Thus, intrauterine HPX disrupts the mitochondrial phenotype predominantly in male fetal hearts, potentially altering cardiac metabolism and predisposing the offspring to heart dysfunction.

The protein-bound uremic toxins (PBUT), evolving to avoid conventional hemodialysis, decreases the toxin-free dissemination by a higher degree of binding of proteins, in turn increasing the dialyzer redirected membranes from it. Therefore, PBUT kinetics mechanical understanding can open ways of improving dialytic removal. A robust PBUT kinetic model has been developed, which consists of the various levels of compartment and dialyzer. This model represents a dynamic balance between protein, toxin and complex protein toxins. Further, this model has been calibrated and validated through literature, clinical evidence and studies is presented in this paper with numerical results. This anticipates key aspects of PBUT kinetic, which includes free and binding PBUT concentration profiles, where the dialytic variance PBUT has the elimination of dialysis rate effect. A popular Deep learning (DL) integrated PBUT kinetic model has been used in the elimination of non-dialysis dose evaluation conditions. The new DL-PBUT algorithm removes interruptions, helps to estimate the dialysis quality parameter (MR/Y), online with considerable precision with minimized delay and more efficiently than the known algorithms. The test results have been computed for various datasets which has been analyzed from the patients at lab scale shows promising outcomes.

Assembly-line polyketide synthases (PKSs) are among the most complex protein machineries known in nature, responsible for the biosynthesis of numerous compounds used in the clinic. Their present-day diversity is the result of an evolutionary path that has involved the emergence of a multimodular architecture and further diversification of assembly-line PKSs. In this review, we provide an overview of previous studies that investigated PKS evolution and propose a model that challenges the currently prevailing view that gene duplication has played a major role in the emergence of multimodularity. We also analyze the ensemble of orphan PKS clusters sequenced so far to evaluate how large the entire diversity of assembly-line PKS clusters and their chemical products could be. Finally, we examine the existing techniques to access the natural PKS diversity in natural and heterologous hosts and describe approaches to further expand this diversity through engineering.

Significance Large proteins composed of multiple domains are abundant in all proteomes, but their folding and structural dynamics remain poorly understood. Using single-molecule force spectroscopy, we have defined how stabilizing interfaces among the domains of elongation factor G (EF-G) shape its folding pathway. Contrary to the expectation that multidomain proteins fold sequentially as they emerge from the ribosome, we find that folding cannot be completed until the full protein has been synthesized. This posttranslational folding mechanism results in a propensity for misfolding. It is dictated by an energetic coupling among domains that enables conformational flexibility crucial for EF-G function. EF-G thus provides an example of how distinct biological ends—robust folding and functionally important flexibility—come into conflict during protein biogenesis., Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.

Two complex protein defense systems—complement and coagulation—are based on amplifying enzyme cascades triggered by specific local stimuli. Excess systemic activation of either system is pathologic and is normally prevented by a family of regulatory proteins. The 2 systems are ancient biological processes which share a common origin that predates vertebrate evolution. Recent research has uncovered multiple opportunities for cross talk between complement and coagulation including proteins traditionally viewed as coagulation factors that activate and regulate complement, and proteins traditionally seen as part of the complement system that participate in coagulation. Ten examples of cross talk between the 2 systems are described. The mutual engagement of both systems is increasingly recognized to occur in human diseases. Three conditions—paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and the antiphospholipid syndrome—provide examples of the importance of interactions between complement and coagulation in human biology. A better understanding of the mutual engagement of these 2 ancient defense systems is expected to result in improved diagnostics and new treatments for systemic diseases.

The article describes specific aspects of biosimilars research and development. The aim of the study was to analyse the ways to conduct comparative studies of biotechnological medicinal products and the main approaches to the assessment of the obtained data. The paper highlights that biotechnological products are associated with a much higher potential variability of chemical and pharmacological characteristics than small molecules. The author analyses the reasons of this phenomenon, describes mechanisms underlying the microheterogeneity of protein molecules, primarily post-translational modification. The latter has an impact on the pharmacokinetic parameters, pharmacodynamics and immunogenicity of complex protein molecules, which increases the variability of test results and makes it difficult to conduct bioequivalence studies. In addition to bioequivalence studies, biosimilars research should include comparative studies of pharmacodynamics, evaluation of therapeutic equivalence and immunogenicity. Assessment of the medicines comparability should be based on the analysis of all data provided, which requires a more flexible and sometimes individual approach on the part of regulatory authorities.

In vitro protein folding is a complex process which often results in protein aggregation, low yields and low specific activity. We report the use of nanoscale exoshells (tES) to provide specific nanoenvironments for the folding and release of 12 highly diverse protein substrates ranging from small protein toxins to human albumin, a dimeric protein (alkaline phosphatase), a trimeric ion channel (Omp2a) and the tetrameric tumor suppressor, p53. These proteins represent a unique diversity in size, volume, disulfide linkages, isoelectric point and multi versus monomeric nature of their functional units. Crude soluble yield (3-fold to > 100-fold), functional yield (2-fold to > 100-fold) and specific activity (3-fold to > 100-fold) were increased for all the proteins tested. The average soluble yield of POI was 6.5 mg/100 mg of tES. Charge complementation between the tES internal cavity and the protein substrate was the primary determinant in functional folding. Our results confirm the importance of nanoscale electrostatic effects and provide a novel nanoparticle solution for folding proteins in vitro.

Enzymes are the complex protein moieties, catalyze the rate of chemical reactions by transforming various substrates to specific products and play an integral part in multiple biochemical cycles. Advancement in enzyme research and its integration with industries have reformed the biotech industries. It provides a superior monetary and ecological exchange to traditional material measures in an efficient and environmentally sustainable manner. The cost-effective production of pure and highly active enzymes is still a challenge for the biocatalyst industries. The use of high purity substrates further raises the cost of a typical biocatalyst. The use of low-cost plant-based biomasses as an enticing and sustainable substrate for enzyme production is the most cost-effective approach to these problems. Given the relevance of biomass as a substrate for enzyme development, this review article focuses on the key source, composition and major enzyme generated using various biomass residues. Furthermore, the difficulties associated with the use of biomass as a substrate and technical developments in this area, are also addressed. The use of waste biomass as a substrate lowers the ultimate cost for the production of biocatalysts while simultaneously reduces the waste burden from the environment.

Fluorescence lifetime microscopy (FLIM) and time-resolved cytometry (TRFC) are robust platforms that can resolve complex protein and cellular interactions. Flow cytometry has been a prominent staple in clinical and research for decades. In conventional form, flow cytometers can count cells and evaluate biophysical and biochemical attributes using fluorescence and inelastic scatter light. Cytometry has evolved beyond conventional paradigms are becoming polychromatic and mulitparametric apparatuses that can evaluate complex cellular interactions in real-time. A distancedependent technique known as Forster Resonance Energy Transfer, or FRET is a powerful quantitative tool that enables the ability to monitor binding interaction and morphological changes in the macro and microenvironment of cells. FRET measurements require sensitive instrumentation to capture and resolve subtle changes in biophysical and biochemical characteristics. TRFC captures a unique parameter known as fluorescence lifetime which is sensitive to microenvironmental changes. Past studies have demonstrated TRFC’s ability to resolve complex FRET interactions. Herein, we present the evolution of the TRFC modular platform that incorporates a microfluidic device. The microfluidic device in this contribution acoustically linearly focuses cells down the middle of the microcapillary, allowing for maximum optical excitation and optimizing optical geometries to maximize the capture of fluorescence.

International audience; Native mass spectrometry (nMS) is now widely applied to investigate non-covalently assembled biomolecule complexes. nMS requires the use of near-neutral pH and volatile buffers to preserve the native state of proteins. However, buffer exchange into nMS-compatible solvent is usually performed manually, which results in a time-consuming and tedious process, thus appearing as a major drawback for nMS analysis. Conversely, online coupling of size exclusion chromatography (SEC) to nMS affords a fast-automated and improved desalting, but also provides an additional dimension of separation for complex protein mixtures. We illustrate here the benefits of SEC-nMS compared to manual offline desalting for the characterization of a wide variety of biological systems, ranging from multiprotein assemblies, protein–ligand and protein–nucleic acid complexes, to proteins in a detergent environment. We then highlight the potential of the coupling to further integrate ion mobility while preserving the native conformations of proteins, allowing for rapid collision cross section measurement and even collision-induced unfolding experiments. Finally, we show that online SEC coupling can also serve as the basis for multidimensional non-denaturing liquid chromatography (LC) workflows, with the SEC acting as a fast desalting device, helping to achieve first dimension LC separation in optimal chromatographic conditions while being compatible with further nMS analysis.

Shells of pearl oysters are natural biominerals with remarkable properties that can be repaired after damage. The repair process can be regulated by biomacromolecules, especially shell matrix proteins (SMPs). Identifying SMPs is critical for further understanding the process. Although proteomic methods have been used to reveal the complex protein mixture in mature shells, the proteomics of repaired shells after shell damage have not been reported before. In the work described here, we studied the SMPs of the repaired shells (prismatic layers) 5-20 days after shell damage in the pearl oyster Pinctada fucata by integrating transcriptomics and proteomics and then compared the microstructure difference between repaired and mature shells. Although the repaired shells are calcite, similar to mature shells, the microstructures of repaired shells during the regeneration process were different, which could simulate the embryonic shell formation process. In total, we found 49 SMPs from the repaired shells, including some proteins that exist only in mature nacreous layers. Peroxidase-like protein and β-N-acetylhexosaminidase may be important players in shell repair. In addition, SMPs have the capability to affect the CaCO3 crystallization process in vitro, altering the packing and reducing the crystallinity of the crystals. This study could improve our understanding of the shell repair process and lay the foundation for studying SMPs-controlled biomineralization.

Src kinases play fundamental roles in several crucial cell processes. Their activity is tightly regulated by conformational transitions between the active and the inactive forms, which are carried out by complex protein structural rearrangements. Here, we present an in-depth study of such structural transitions coupling extensive all-atoms molecular dynamic simulations coupled to an algorithm able to drive the system between defined conformational states. Our results, in line with the available experimental data, confirm the complexity of such a process indicating the main molecular determinants involved. Moreover, the role of an Src inhibitor—able to bind to the protein inactive state—is discussed and compared with available experimental data.

In recent decades, the increase in performance and versatility of mass spectrometric instrumentation has contributed to development of new analytical strategies for protein characterization. In particular, MS-based approaches have demonstrated their enormous potential in the characterization of the complex protein mixtures contained in food. Proteins are essential elements in the human diet. The investigation of the protein fraction of foodstuffs, including protein functionality (presence of bioactive peptides), origin (food traceability) and safety (e.g. absence of allergens, pathogens, etc.) is fundamental for understanding the relationship between protein content and the nutritional and technological properties of food, and, consequently, the influence of food proteins on the health of end-consumers. This chapter will give a brief description of MS-based techniques, proteomic approaches and bioinformatic tools for protein identification as well the most relevant aspects of the MS-based methodologies in food proteomics with their advantages and drawbacks.

Phosphopeptide enrichment is an essential step in large-scale, quantitative phosphoproteomics by mass spectrometry. Several phosphopeptide affinity enrichment techniques exist, such as immobilized metal-ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC). We compared zirconium(IV) IMAC (Zr-IMAC) magnetic microparticles to more commonly used titanium(IV) IMAC (Ti-IMAC) and TiO2 magnetic microparticles for phosphopeptide enrichment from simple and complex protein samples prior to phosphopeptide sequencing and characterization by mass spectrometry (liquid chromatography-tandem mass spectrometry, LC-MS/MS). We optimized sample-loading conditions to increase phosphopeptide recovery for Zr-IMAC-, Ti-IMAC-, and TiO2-based workflows by 22, 24, and 35%, respectively. The optimized protocol resulted in improved performance of Zr-IMAC over Ti-IMAC and TiO2 as well as high-performance liquid chromatography-based Fe(III)-IMAC with up to 23% more identified phosphopeptides. The different enrichment chemistries showed a high degree of overlap but also differences in phosphopeptide selectivity and complementarity. We conclude that Zr-IMAC improves phosphoproteome coverage and recommend that this complementary and scalable affinity enrichment method is more widely used in biological and biomedical studies of cell signaling and the search for biomarkers. Data are available via ProteomeXchange with identifier PXD018273.

Western blotting, followed by immunoblotting, provides a superb method to study antibody reactivity against several proteins separated according to their molecular weights. The difference between immune immunodominant and immunorecessive proteins is often problematic and is carried out by qualitative or experiential means. Dalit Talmi-Frank, Charles L. Jaffe, and Gad Baneth devised and described quantitative computerized western blotting (QCWB) to examine reactivity to specific antigens by giving a statistically measurable value for each band permitting distinction between immunodominant and immunorecessive determinants. QCWB is beneficial for both single time point studies and longitudinal investigation where several time points are assessed and the relativities against individual bands compared. This method can be used to investigate antibody responses to complex protein mixtures like allergens and infectious agents. This technique can also be used to detect serum markers for early diagnosis of cancer and autoimmune or infectious diseases or to monitor a patient’s disease condition.

ATP-independent chaperones are usually considered to be holdases that rapidly bind to non-native states of substrate proteins and prevent their aggregation. These chaperones are thought to release their substrate proteins prior to their folding. Spy is an ATP-independent chaperone that acts as an aggregation inhibiting holdase but does so by allowing its substrate proteins to fold while they remain continuously chaperone bound, thus acting as a foldase as well. The attributes that allow such dual chaperoning behavior are unclear. Here, we used the topologically complex protein apoflavodoxin to show that the outcome of Spy’s action is substrate specific and depends on its relative affinity for different folding states. Tighter binding of Spy to partially unfolded states of apoflavodoxin limits the possibility of folding while bound, converting Spy to a holdase chaperone. Our results highlight the central role of the substrate in determining the mechanism of chaperone action., Spy is an ATP independent chaperone that can act as both a holdase and a foldase towards topologically simple substrates. Assessing the interaction of Spy and apoflavodoxin, a complex client, the authors show that Spy’s activity is substrate specific. Spy binds partially unfolded states of apoflavodoxin tightly, which limits the possibility of folding and converts Spy to a pure holdase.

Recent advances in super-resolution imaging techniques, together with new fluorescent probes have enhanced our understanding of bacterial pathogenesis and their interplay within the host. In this review, we provide an overview of what these techniques have taught us about the bacterial lifestyle, the nucleoid organization, its complex protein secretion systems, as well as the secreted virulence factors.

Purpose of Review This review a highlights that to use artificial intelligence (AI) tools effectively for hypertension research, a new foundation to further understand the biology of hypertension needs to occur by leveraging genome and RNA sequencing technology and derived tools on a broad scale in hypertension. Recent Findings For the last few years, progress in research and management of essential hypertension has been stagnating while at the same time, the sequencing of the human genome has been generating many new research tools and opportunities to investigate the biology of hypertension. Cancer research has applied modern tools derived from DNA and RNA sequencing on a large scale, enabling the improved understanding of cancer biology and leading to many clinical applications. Compared with cancer, studies in hypertension, using whole genome, exome, or RNA sequencing tools, total less than 2% of the number cancer studies. While true, sequencing the genome of cancer tissue has provided cancer research an advantage, DNA and RNA sequencing derived tools can also be used in hypertension to generate new understanding how complex protein network, in non-cancer tissue, adapts and learns to be effective when for example, somatic mutations or environmental inputs change the gene expression profiles at different network nodes. The amount of data and differences in clinical condition classification at the individual sample level might be of such magnitude to overwhelm and stretch comprehension. Here is the opportunity to use AI tools for the analysis of data streams derived from DNA and RNA sequencing tools combined with clinical data to generate new hypotheses leading to the discovery of mechanisms and potential target molecules from which drugs or treatments can be developed and tested. Summary Basic and clinical research taking advantage of new gene sequencing-based tools, to uncover mechanisms how complex protein networks regulate blood pressure in health and disease, will be critical to lift hypertension research and management from its stagnation. The use of AI analytic tools will help leverage such insights. However, applying AI tools to vast amounts of data that certainly exist in hypertension, without taking advantage of new gene sequencing-based research tools, will generate questionable results and will miss many new potential molecular targets and possibly treatments. Without such approaches, the vision of precision medicine for hypertension will be hard to accomplish and most likely not occur in the near future.

Screening for small-molecule fragments that can lead to potent inhibitors of protein&ndash, protein interactions (PPIs) is often a laborious step as the fragments cannot dissociate the targeted PPI due to their low &mu, M&ndash, mM affinities. Here, we describe an NMR competition assay called w-AIDA-NMR (weak-antagonist induced dissociation assay-NMR), which is sensitive to weak &mu, mM ligand&ndash, protein interactions and which can be used in initial fragment screening campaigns. By introducing point mutations in the complex&rsquo, s protein that is not targeted by the inhibitor, we lower the effective affinity of the complex, allowing for short fragments to dissociate the complex. We illustrate the method with the compounds that block the Mdm2/X-p53 and PD-1/PD-L1 oncogenic interactions. Targeting the PD-/PD-L1 PPI has profoundly advanced the treatment of different types of cancers.

Mimicking sensory principles encountered in animals, whereby numerous tastants and odorants are identified based on "pattern"-like sensory inputs that are generated by arrays of sensory cells, allows creating a unique technique that is distinct from conventional chemical sensing systems, as the latter usually require specific recognition of target analytes. Herein, we present a highly discriminative small fluorescent array of block copolymers that can recognize various bioanalytes in a biomimicry manner. These polyethylene glycol/poly-l-lysine block copolymers are functionalized with fluorescein as a fluorescent reporter unit and hydrophobic amino acids as cross-reactive recognition units, which provides the ability to generate fluorescent response patterns unique to proteins and cells. Multivariate analysis on the patterns obtained with an array consisting of solely 3 block copolymers allowed identifying not only 20 proteins and 10 mammalian cells individually but also complex protein mixtures with slightly different compositions. This design guideline for creating a versatile biomimicry sensing system, which is based on the bifunctionalization of polymeric materials, is expected to offer a powerful platform for simple and high-throughput sensing of a wide variety of bioanalytes.

In recent years, significant advancements have been made in the understanding of the population distributions and dynamic oligomeric states of the molecular chaperone αB-crystallin and its core domain variants. In this work, we provide solution-phase evidence of the polydispersity of αB- crystallin using microfluidic methods, used for separating the oligomeric species present in solution according to their different electrophoretic mobilities on-chip in a matter of seconds. We, in particular demonstrate that microfluidic high-field electrophoresis and diffusion can detect the oligomerisation of these highly dynamic molecular chaperones and characterise the dominant oligomeric species present. We thereby provide a robust microfluidic method for characterising the individual species within complex protein mixtures of biological relevance.

Background and importance Rituximab (RTX) is a therapeutic monoclonal antibody used for the treatment of certain types of cancer. As a complex protein, routine handling or unintentional mishandling of its solutions may cause degradation that could remain unnoticed but could potentially compromise the clinical safety and efficacy of the drug product.1 Aim and objectives To assess the impact on the RTX (Mabthera) aggregation process promoted by slight modification in the concentration of the compound (NaCl 0.9% and glucose 5%) used to prepare the clinical diluted solution of RTX 1.0 mg/mL. Also, to assess the impact on the aggregation of RTX clinical diluted solutions (1.0 mg/mL in NaCl 0.9% and glucose 5%) promoted by manual shaking. Material and methods RTX (Mabthera 10 mg/mL) was diluted to 1 mg/mL using different NaCl (from 0.5% to 1.5%) and glucose (from 1% to 10%) concentrations. Manual gentle shaking was performed for 10 minutes. Particulate was tracked by dynamic light scattering (DLS) and readings were carried out in a protein solution DynaPro-99 system dynamic light scattering module equipped with a temperature control micro sampler (Wyatt, Santa Barbara, California, USA) for obtaining the hydrodynamic radius (HR) and polydispersity. Results Reference 1.0 mg/mL RTX samples diluted in NaCl 0.9% and glucose 5% showed a single particulate population with a HR of 10.51±2.210 nm and 10.72±2.694 nm, respectively, attributed to monomers of RTX. No significant changes were obtained for HR when the concentration of the diluents was changed. Also, no significant changes were observed when the samples were shaken, with the HR values always within the interval of the size of the monomers. Polydispersity remained unchanged in all of the samples analysed. Conclusion and relevance Variation in NaCl and glucose concentrations around clinical concentrations of 0.9% and 5% did not promote aggregation in a 1 mg/mL RTX solution detected by DLS. Also, shaking did not have any impact on aggregation in this clinical RTX solution. References and/or acknowledgements 1. Nejadnik MR, et al. J Pharm Sci 2018;107:2013–2019. Funded by Project FIS: PI-17/00547 (Instituto Carlos III, Spain), which means that it was also partially supported by the European Regional Development Funds. No conflict of interest.

One of the interesting focus points of research in protein science is the active site analysis and probe fixing Overall one can go on searching this interesting phenomenon of probe fixing but without proper explanation on how and why this happens at some site neglecting other places One of the probe fixing methods could be come along the way of newly discovered carbon role in protein stability that is taken here as starting point for due course of action Carbon role in protein domain formation and active portion are interestingly yielded good result Analysis includes ICOD of protein in native and domain state of active portion in complex protein ICOD analysis reveals that carbon does play an interesting role in probe of action Alternative method to detect active portion and probe development can be better exploited from this carbon domain of protein for life science to excel

Summary Comparing results from multiple MD simulations performed under different conditions is essential during the initial stages of analysis. We propose a tool called MD Contact Comparison (MDContactCom) that compares residue-residue contact fluctuations of two MD trajectories, quantifies the differences, identifies sites that exhibit large differences and visualizes those sites on the protein structure. Using this method, it is possible to identify sites affected by varying simulation conditions and reveal the path of propagation of the effect even when differences between the 3D structure of the molecule and the fluctuation RMSF of each residue is unclear. MDContactCom can monitor differences in complex protein dynamics between two MD trajectories and identify candidate sites to be analyzed in more detail. As such, MDContactCom is a versatile software package for analyzing most MD simulations. Availability and implementation MDContactCom is freely available for download on GitLab. The software is implemented in Python3. https://gitlab.com/chiemotono/mdcontactcom. Supplementary information Supplementary data are available at Bioinformatics online.

For successful protease inhibition, the reactive center loop (RCL) of the two-domain serine protease inhibitor, α1-antitrypsin (α1-AT), needs to remain exposed in a metastable active conformation. The α1-AT RCL is sequestered in a β-sheet in the stable latent conformation. Thus, to be functional, α1-AT must always fold to a metastable conformation while avoiding folding to a stable conformation. We explore the structural basis of this choice using folding simulations of coarse-grained structure-based models of the two α1-AT conformations. Our simulations capture the key features of folding experiments performed on both conformations. The simulations also show that the free energy barrier to fold to the latent conformation is much larger than the barrier to fold to the active conformation. An entropically stabilized on-pathway intermediate lowers the barrier for folding to the active conformation. In this intermediate, the RCL is in an exposed configuration, and only one of the two α1-AT domains is folded. In contrast, early conversion of the RCL into a β-strand increases the coupling between the two α1-AT domains in the transition state and creates a larger barrier for folding to the latent conformation. Thus, unlike what happens in several proteins, where separate regions promote folding and function, the structure of the RCL, formed early during folding, determines both the conformational and the functional fate of α1-AT. Further, the short 12-residue RCL modulates the free energy barrier and the folding cooperativity of the large 370-residue α1-AT. Finally, we suggest experiments to test the predicted folding mechanism for the latent state.

In this article, the trajectories of S-deoxyephedrine (SBD) along molecular channels within the complex protein structure of third dopamine receptor (D3R) are analyzed via molecular dynamic techniques, including potential mean force calculations of umbrella samplings from the 4.5 version of the GROMACS program. Changes in free energy due to the movement of SBD within D3R are determined, and the molecular dynamic mechanisms of SBD transmitting along molecular channels are probed. Molecular simulated results show that the change in free energy is calculated as 171.7 kJ·mol–1 for the transmission of SBD toward the outside of the cell along the y+ axis functional molecular channel and is 275.0 kJ·mol–1 for movement toward the intracellular structure along the y– axis. Within the internal structure of D3R, the changes in free energy are determined to be 103.6, 242.1, 459.7, and 127.8 kJ·mol–1 for transmission of SBD along the x+, x–, z+, and z– axes, respectively, toward the cell bilayer membrane, which indicates that SBD leaves much more easily along the x+ axis through the gap between the TM5 (the fifth transmembrane helix) and TM6 (the sixth transmembrane helix) from the internal structure of D3R. The values of free-energy changes indicate that SBD molecules can clear the protective channel within D3R, which helps dopamine molecules to leave the D3R internal structure along the x+ axis and to prevent them for exerting excessive neurotransmitter function. Therefore, our results suggest that SBD is effective for development as a drug for treating schizophrenia and its pharmacology is closely related to its dynamics and mechanisms within the molecular pathway of dopamine receptors.

A new efficient method for the parallel and sequential stepwise generation of single-domain antibodies to various high-abundance human-plasma proteins has been described. Single-domain antibodies have a number of features that favorably distinguish them from classical antibodies. In particular, they are able to recognize unusual unique conformational epitopes of native target proteins, small in size, and relatively easily produced and modified; have enhanced stability; and rapidly renature after denaturation. As a consequence, the immunosorbents that utilize these antibodies can be reused without any significant loss of activity. The principal novelty and universality of the described method is that it enables the sequential generation of antibodies to a number of high-abundance and yet unknown antigens of a complex protein mixture without the need for purified antigens. The effectiveness of the method is demonstrated by the example of generation of single-domain antibodies to a number of high-abundance proteins of the human blood plasma. The produced antibodies are promising biotechnological tools that can be used to develop prototypes for new diagnostic and therapeutic agents, as well as appropriate immunoaffinity-based methods for removal, enrichment, analysis, and/or targeting of specified proteins and their complexes from (in) the human blood. As we show, the generated single-domain antibodies can be efficiently used in designing new immunosorbents. As a rule, commercially available analogous immunosorbents that utilize classical antibodies remove many major proteins from the blood plasma immediately, while immunosorbents for many individual proteins are difficult to find and rather expensive. Single-domain antibodies generated by our method are unique new materials that allow for the development of more efficient and delicate approaches to pretreatment of plasma and the analysis of various blood plasma biomarkers.

The PubMed, MEDLINE databases and China National Knowledge Infrastructure (CNKI) were searched for information regarding the etiology and pathogenesis of conjunctivochalasis (CCh) and the synthesis and degradation of elastic fibers. After analysis of the literature, we found elastic fibers was a complex protein molecule from the structure and composition; the degradation of elastic fibers was one of the histopathological features of the disease; the vast majority of the factors related to the pathogenesis of CCh ultimately pointed to abnormal elastic fibers. By reasonably speculating, we considered that abnormal elastic fibers cause the conjunctival relaxation. In conclusion, we hypothesize that elastic fibers play an important role in the pathogenesis of CCh. Studies on the mechanism of synthesis, degradation of elastic fibers are helpful to clarify the pathogenesis of the disease and to find effective treatment methods.

Fluorous solid-phase extraction (FSPE) has been employed to isolate target compounds from complex chemical or biological samples in many research areas. However, the lack of efficient and economical available perfluorinated materials impeded its development. In this study, we present a novel nanographite fluoride-based fluorous solid-phase extraction (GF-FSPE) as a replacement of commercially available cartridge-based FSPE, which showed remarkable selectivity, low LOD, high post enrichment recovery, and large enrichment capacity. To demonstrate the potency of GF-FSPE, it was designed and successfully applied to isolate cysteine-containing peptide subsets from complex protein samples to improve the proteome coverage. Additionally, since graphite fluoride was inexpensive and highly commercialized, this study was expected to promote the popularization of FSPE in both chemical and biological separations as well as encourage the synthesis and broaden the application of highly fluorinated carbon fluoride in material science.

Protein subcellular localization prediction is important for studying the function of proteins. Recently, as significant progress has been witnessed in the field of microscopic imaging, automatically determining the subcellular localization of proteins from bio-images is becoming a new research hotspot. One of the central themes in this field is to determine what features are suitable for describing the protein images. Existing feature extraction methods are usually hand-crafted designed, by which only one layer of features will be extracted, which may not be sufficient to represent the complex protein images. To this end, we propose a deep model based descriptor (DMD) to extract the high-level features from protein images. Specifically, in order to make the extracted features more generic, we firstly trained a convolution neural network (i.e., AlexNet) by using a natural image set with millions of labels, and then used the partial parameter transfer strategy to fine-tune the parameters from natural images to protein images. After that, we applied the Lasso model to select the most distinguishing features from the last fully connected layer of the CNN (Convolution Neural Network), and used these selected features for final classifications. Experimental results on a protein image dataset validate the efficacy of our method.

For decades, chemists have strived to mimic the intricate design and diverse functions of naturally occurring systems through the bioinspired synthesis of programmable inorganic nanomaterials. The development of thiol-capped gold nanoparticles (AuNPs) has driven advancement in this area; however, although versatile and readily accessible, hybrid AuNPs are rarely atomically precise, which limits control over their surface topology and therefore the study of complex structure-function relationships. Here, we present a bottom-up approach to the systematic assembly of atomically precise hybrid nanoclusters employing a strategy that mimics the synthetic ease with which thiol-capped AuNPs are normally constructed, while producing welldefined covalent nanoscale assemblies with diverse surface topologies. For the first time, using a structurally characterized cluster-based organometallic building block, we demonstrate the systematic synthesis of nanoclusters with multivalent binding capabilities to complex protein targets.

Fritz-Laylin studies the evolution of complex protein networks associated with cell movement.