Protein Kinetics Research Articles

C-reactive Protein Kinetics During In-Patient Treatment of COVID-19-Associated Rhino-Orbito-Cerebral Mucormycosis: A Retrospective Cohort Study in a Tertiary Hospital in Central India.

COVID-associated rhino-orbito-cerebral Mucormycosis (CA-ROCM), henceforth referred to as Covid-Associated Mucormycosis (CAM), is a serious and fatal condition unless treated promptly and completely. The main treatment of the CAM is complete surgical debridement and administration of systemic antifungals. The first line antifungal recommended for CAM is Amphotericin-B. Since Amphotericin-B has systemic side effects mainly on the renal system, a timely decision to start and end Amphotericin-B therapy is very essential. Besides the Computed Tomography (CT) scan, serum levels of C-reactive protein (CRP) levels are a good indicator of CAM-associated inflammation levels in the patient's body. By monitoring the CRP levels, we can titrate amphotericin treatment to cause minimal harm to the kidneys.Our study was done to analyze the kinetics of C-reactive protein in patients of CAM admitted in a tertiary-care hospital and compare it with the CRP levels in COVID-associated non-Mucormycosis Sinusitis patients. Aim and objective To study the kinetics of serum C-reactive protein (CRP) levels among patients undergoing in-patient care for COVID-associated rhino-orbito-cerebral mucormycosis and compare with serum CRPlevels in COVID-19 patients suffering from sinusitis without rhino-orbito-cerebral mucormycosis. Materials and methods This was a retrospective cohort study. The source of data was post-COVID sinusitis patients who were admitted during 2nd wave of COVID-19 in India in our hospital whose medical records were accessed by the Medical Records Department. The subjects were recruited into the two study groups namely the Mucormycosis group and the non-Mucormycosis group based on the histopathological report of the nasal biopsy specimen. The medical records of each member of the two groups were studied for the levels of serum C-reactive protein measured at the time of admission and every 5(+1) days thereafter till the time of discharge. The kinetics of serum C-reactive protein levels, which is a marker of inflammation is studied in each of the two groups and compared using statistical methods. Results There was a significant difference between Mucormycosis and Non-Mucormycosis groups in CRP-level kinetics. However, there was no significant trend of decrease or increase over time in Mucormycosis as well as non-Mucormycosis cases. Conclusion CRP is an important biomarker in assessing the septic response to COVID-associated rhino-orbito-cerebral mucormycosis. Detection of raised CRP levels helps in prompt early initiation of anti-fungal treatment. Also, monitoring the levels of serum CRP will guide in deciding the time to stop the antifungals at an appropriate time. CRP monitoring is commonly available and affordable. Hence, we recommend CRP monitoring of in-patients of CAM.

Droplet size dependency and spatial heterogeneity of lipid oxidation in whey protein isolate-stabilized emulsions

Spatiotemporal assessment of lipid and protein oxidation is key for understanding quality deterioration in emulsified food products containing polyunsaturated fatty acids. In this work, we first mechanistically validated the use of the lipid oxidation-sensitive fluorophore BODIPY 665/676 as a semi-quantitative marker for local peroxyl radical formation. Next, we assessed the impact of microfluidic and colloid mill emulsification (respectively producing mono- and polydisperse droplets) on local protein and lipid oxidation kinetics in whey protein isolate (WPI)-stabilized emulsions. We further used BODIPY 581/591 C11 and CAMPO-AFDye 647 as colocalisation markers for lipid and protein oxidation. The polydisperse emulsions showed an inverse relation between droplet size and lipid oxidation rate. Further, we observed less protein and lipid oxidation occurring in similar sized droplets in monodisperse emulsions. This observation was linked to more heterogeneous protein packing at the droplet surface during colloid mill emulsification, resulting in larger inter-droplet heterogeneity in both protein and lipid oxidation. Our findings indicate the critical roles of emulsification methods and droplet sizes in understanding and managing lipid oxidation.

Exploring the role of production and release of proteins for microbial interactions in kombucha

Driving kombucha fermentation through microbial consortia remains based on scarce scientific elements, especially regarding the consumption of nitrogenous nutrients. This study aimed at analyzing quantitatively proteins and free amino nitrogen (FAN) concentrations, and qualitatively the soluble proteins profiles during a 12-day production process. The effect of microbial interactions was evaluated by comparing the growth of previously isolated major microorganisms in monoculture and cocultures to original kombucha. Protein and FAN relative concentrations kinetics depended on microbial compositions. These parameters were associated to less variations and a positive effect on total microbial population in original kombucha, underlining a complex yet better management of the nutrients. Analysis of extracellular proteins by electrophoresis showed the presence of a microbial 15 kDa fraction common to all cultures. In contrast, a 21 kDa protein fraction was associated with the presence of Acetobacter indonesiensis. Exploratory proteomics analysis revealed the presence of characteristic extracellular moonlighting proteins, originating from this acetic bacteria. Production of extracellular vesicles was successfully confirmed with the isolation and observation of vesicles in A. indonesiensis culture, as well as in kombucha. The results characterize the central role of microbial interactions on FAN consumption and open new perspectives on the function of the bacterial vesicles observed.

C-reactive protein flare predicts response to checkpoint inhibitor treatment in melanoma.

The treatment of melanoma has been revolutionized by the use of immune checkpoint inhibition (ICI), but many patients do not benefit. Furthermore, immune-related adverse events may occur during therapy. A predictive biomarker is needed to reliably identify patients benefitting. In lung, renal cell and bladder cancer early C-reactive protein (CRP) kinetics were shown to be a predictive biomarker for ICI. Here, we investigate early CRP kinetics as predictive biomarker for ICI in melanoma patients. Two independent prospectively collected cohorts were analysed: Cohort 1 (n = 87) with advanced and Cohort 2 (n = 99) with completely resected melanoma. Patients were stratified by in the dynamics of CRP after ICI initiation: A doubling of baseline CRP within 30 days followed by at least a 30% drop within 3 months was classified as a CRP flare. If no doubling of CRP was reported, but a 30% drop within 3 months, patients were classified as CRP responders and all others as CRP non-responders. Analysed factors included clinical characteristics like S100B and LDH. Median follow-up was 1.5 and 1.7 years for Cohorts 1 and 2. In Cohort 1 CRP flare (n = 12), CRP responders (n = 43) and CRP non-responders (n = 32) with a progression-free survival (PFS) of 0.7, 0.6 and 0.2 years (p = 0.017) and an overall survival (OS) of 2.2, 1.5 and 1.0 years (p = 0.014), respectively. Multivariable Cox analysis showed an independent risk reduction of progression for CRP responders by 62% compared to CRP non-responders (p = 0.001). In Cohort 2 CRP flare (n = 13), CRP responders (n = 70) and CRP non-responders (n = 16) the log-rank analysis showed a significant difference between OS and recurrence-free survival (RFS) curves (p = 0.046 and p = 0.049). Early CRP kinetics could indicate a response to ICI with improved OS and RFS/PFS. CRP flare and CRP response indicating significantly improved outcomes compared to CRP non-responders.

The time revolution in macromolecular crystallography.

Macromolecular crystallography has historically provided the atomic structures of proteins fundamental to cellular functions. However, the advent of cryo-electron microscopy for structure determination of large and increasingly smaller and flexible proteins signaled a paradigm shift in structural biology. The extensive structural and sequence data from crystallography and advanced sequencing techniques have been pivotal for training computational models for accurate structure prediction, unveiling the general fold of most proteins. Here, we present a perspective on the rise of time-resolved crystallography as the new frontier of macromolecular structure determination. We trace the evolution from the pioneering time-resolved crystallography methods to modern serial crystallography, highlighting the synergy between rapid detection technologies and state-of-the-art x-ray sources. These innovations are redefining our exploration of protein dynamics, with high-resolution crystallography uniquely positioned to elucidate rapid dynamic processes at ambient temperatures, thus deepening our understanding of protein functionality. We propose that the integration of dynamic structural data with machine learning advancements will unlock predictive capabilities for protein kinetics, revolutionizing dynamics like macromolecular crystallography revolutionized structural biology.

CRP kinetics to predict long-term efficacy of first-line immune-checkpoint inhibition combination therapies in metastatic renal cell carcinoma: An updated multicenter real-world experience applying different CRP kinetics definitions.

405 Background: Although biomarkers predicting therapy response in 1st line metastatic renal carcinoma (mRCC) therapy remain to be defined, C-reactive protein (CRP) kinetics have recently been associated with immunotherapy (IO) response. To assess the predictive and prognostic power of two contemporary CRP kinetics definitions in a large, real-world 1st line mRCC cohort. Methods: mRCC patients treated with IO-based 1st line therapy within 5 years were retrospectively included in this multi-center study. According to Fukuda et al., patients were defined as ‘CRP flare-responder’, ’CRP responder’ and ‘non-CRP responder’; according to Ishihara et al., as ‘normal’, ‘normalized’ and ‘non-normalized’ based on their early CRP kinetics. Patient and tumor characteristics were compared, and treatment outcome was measured by overall (OS) and progression-free survival (PFS), including multivariable Cox regression analyses. Results: Out of 316 mRCC patients, 227 (72%) were assigned to CRP groups according to Fukuda. Both CRP flare- (HR [Hazard ratio]: 0.59) and CRP responders (HR: 0.52) had a longer PFS, but not OS, than non-CRP responders. According to Ishihara, 276 (87%) patients were assigned to the respective groups, and both normal and normalized patients had a significantly longer PFS and OS, compared to non-normalized group. Conclusions: Different early CRP kinetics may predict therapy response in 1st line mRCC therapy in a large real-world cohort. However, further research regarding the optimal timing and frequency of measurement is needed.

Spatiotemporally Controlled Photolabeling of Genetically Unmodified Proteins in Live Cells.

Selective labeling of the protein of interest (POI) in genetically unmodified live cells is crucial for understanding protein functions and kinetics in their natural habitat. In particular, spatiotemporally controlled installation of the labels on a POI under light control without affecting their original activity is in high demand but is a tremendous challenge. Here, we describe a novel ligand-directed photoclick strategy for spatiotemporally controlled labeling of endogenous proteins in live cells. It was realized with a designer labeling reagent skillfully integrating the photochemistries of 2-nitrophenylpropyloxycarbonyl and 3-hydroxymethyl-2-naphthol with an affinity ligand. Highly electrophilic ortho-naphthoquinone methide was photochemically released and underwent a proximity coupling reaction with nucleophilic amino acid residues on the POI in live cells. With fluorescein as a marker, this photoclick strategy enables time-resolved labeling of carbonic anhydrase subtypes localized either on the cell membrane or in the cytoplasm and a discriminable visualization of their metabolic kinetics. Given the versatility underlined by facilely tethering other functional entities (e.g., biotin, a peptide short chain) via acylation or (in cell) Huisgen cycloaddition, this affinity-driven photoclick chemistry opens up enormous opportunities for discovering dynamic functions and mechanistic interrogation of endogenous proteins in live cells.

Kinetics of IL-6, C-reactive Protein and Fibrinogen Levels in COVID-19 Outpatients Who Evolved to Hypoxemia.

Despite the efficacy of the COVID-19, the search for improvements in the management of severe/critical cases continues to be important. The aim is to demonstrate the kinetics of 4 serological markers in patients with COVID-19 who evolved in hypoxemia. From June to December 2020, the Health Secretariat of Rondônia State, Brazil, established a home medical care service team (HMCS) that provided clinical follow-up for health professionals and military personnel with COVID-19. The clinical and laboratory monitoring was individualized at home by a nursing and medical team. In addition to laboratory parameters, C-reactive protein (CRP), interleukin-6 (IL-6), fibrinogen, and D-dimer levels were periodically taken to monitor the evolution of treatment. Of 218 patients telemonitored, 48 patients needed special care by the HMCS team due to shortness of breath. Chest tomography showed multiple ground-glass shadows and lung parenchymal condensations that was compatible with secondary bacterial infection associated with leukocytosis, for which antibiotics were prescribed. The symptoms were accompanied by increases of CRP and IL-6 levels followed by fibrinogen after a few days, for which an anticoagulant therapy was included. Thirty-three patients evolved to improvements in clinical signs and laboratory results. Between the sixth and eighth day of illness, 15 patients presented signs of hypoxemia with low O2 saturation accompanied with an increase in the respiratory rate, with some of them requiring oxygen therapy. As they did not present signs of clinical severity, but their laboratory markers showed an abrupt IL-6 peak that was higher than the increase in CRP and a new alteration in fibrinogen levels, they received a supplemental dose of anticoagulant and a high dose of corticosteroids, which resulted in clinical improvement. Our study demonstrates that monitoring of IL-6 and CRP may identify precocious hypoxemia in COVID-19 patients and prevented the progressive deterioration of the lung injury.

Mathematical modelling of Alzheimer's disease biomarkers: Targeting Amyloid beta, Tau protein, Apolipoprotein E and Apoptotic pathways.

Introduction: The kinetics of brain cell death in Alzheimer's disease (AD) is being studied using mathematical models. These mathematical models utilize techniques like differential equations, stochastic processes, and network theory to explore crucial signalling pathways and interactions between different cell types. One crucial area of research is the intentional cell death known as apoptosis, which is crucial for the nervous system. The main purpose behind the mathematical modelling of this is for identification of which biomarkers and pathways are most influential in the progression of AD. In addition, we can also predict the natural history of the disease, by which we can make early diagnosis. Mathematical modelling of AD: Current mathematical models include the Apolipoprotein E (APOE) Gene Model, the Tau Protein Kinetics Model, and the Amyloid Beta Peptide Kinetic Model. The Bcl-2 and Bax apoptosis theories postulate that the balance of pro- and anti-apoptotic proteins in cells determines whether a cell experiences apoptosis, where the Bcl-2 model, depicts the interaction of pro- and anti-apoptotic proteins, it is also being used in research on cell death in a range of cell types, including neurons and glial cells. How peptides are produced and eliminated in the brain is explained by the Amyloid beta Peptide (Aβ) Kinetics Model. The tau protein kinetics model focuses on production, aggregation, and clearance of tau protein processes, which are hypothesized to be involved in AD. The APOE gene model investigates the connection between the risk of Alzheimer's disease and the APOE gene. These models have been used to predict how Alzheimer's disease would develop and to evaluate how different inhibitors will affect the illness's course. Conclusion: These mathematical models reflect physiological meaningful characteristics and demonstrates robust fits to training data. Incorporating biomarkers like Aβ, Tau, APOE and markers of neuronal loss and cognitive impairment can generate sound predictions of biomarker trajectories over time in Alzheimer's disease.

Protein Compartments Modulate Fibrillar Self-Assembly.

A notable feature of complex cellular environments is protein-rich compartments that are formed via liquid-liquid phase separation. Recent studies have shown that these biomolecular condensates can play both promoting and inhibitory roles in fibrillar protein self-assembly, a process that is linked to Alzheimer's, Parkinson's, Huntington's, and various prion diseases. Yet, the exact regulatory role of these condensates in protein aggregation remains unknown. By employing microfluidics to create artificial protein compartments, the self-assembly behavior of the fibrillar protein lysozyme within them can be characterized. It is observed that the volumetric parameters of protein-rich compartments can change the kinetics of protein self-assembly. Depending on the change in compartment parameters, the lysozyme fibrillation process either accelerated or decelerated. Furthermore, the results confirm that the volumetric parameters govern not only the nucleation and growth phases of the fibrillar aggregates but also affect the crosstalk between the protein-rich and protein-poor phases. The appearance of phase-separated compartments in the vicinity of natively folded protein complexes triggers their abrupt percolation into the compartments' core and further accelerates protein aggregation. Overall, the results of the study shed more light on the complex behavior and functions of protein-rich phases and, importantly, on their interaction with the surrounding environment.

Early kinetics of C reactive protein for cancer-agnostic prediction of therapy response and mortality in patients treated with immune checkpoint inhibitors: a multicenter cohort study

BackgroundC reactive protein (CRP) kinetics have recently been suggested as predictive biomarkers for the efficacy of immune checkpoint inhibitor (ICI) therapy in selected cancer types. The aim of this study...

Choroid plexus protein turnover in human choroid plexus organoids recapitulates turnover in humans measured using stable isotope labeling kinetics (SILK)

AbstractBackgroundThe choroid plexus secretes protein‐rich cerebrospinal fluid (CSF). CSF turnover is a key mechanism for protein clearance. Neurodegenerative diseases such as AD are characterised by accumulation of excess proteins that form pathological inclusions. CSF flow dysregulation could therefore be involved in the pathophysiology of neurodegenerative diseases (ND) such as Alzheimer’s Disease. A method for measuring CSF turnover in vivo may be achieved by quantitating CSF derived protein kinetics, providing a biomarker tool with to interrogate the relationship between CSF flow disequilibrium and neurodegeneration.MethodUsing untargeted proteomics we identify the most abundant proteins in a) stem‐cell derived choroid plexus organoids and b) human CSF. We then develop targeted mass spectrometry methods to quantitate protein specific peptides in both matrices. Using a stable isotope (13C6 leucine) we label a) choroid plexus organoids and b) humans with suspected NPH undergoing a 72 hour lumbar drainage protocol. We collect serial samples of the CSF‐like organoid fluid and CSF. We then measure the ratio of labelled to unlabelled peptides to quantitate the rates of protein labelling (fractional synthesis rate) and label loss (fractional clearance rate), defining the protein turnover rate.ResultHighly‐expressed proteins in human CSF from nine individuals were also present in CSF‐like fluid from CP organoids. Of these common proteins, we were able to quantitate the turnover rates of transthyretin, apolipoprotein E, cystatin C and pigment epithelium‐derived factor, and the synthesis rates of serotransferrin and albumin. We will correlate these findings with clinical measures of CSF production.ConclusionChoroid plexus protein synthesis and clearance can be measured using SILK; choroid plexus organoid fluid protein turnover recapitulates that seen in human CSF.

Treatment Patterns and Real-World Outcomes for Locally Advanced or Metastatic Urothelial Cancer in the Era of Immunotherapy

Background and objectiveThere are limited data on real-world outcomes for patients with advanced or metastatic urothelial cancer (mUC) since immune checkpoint inhibitors (ICIs) became available. Our objective was to analyze outcomes for patients with mUC since ICIs became available. MethodsWe performed a retrospective analysis of 131 patients with mUC attending the outpatient clinic of a single tertiary care center who received systemic therapy between June 2017 and July 2021 with follow-up up to December 2022. Summary and descriptive statistics were calculated for categorical and continuous variables. The Kaplan-Meier method was applied to calculate survival, and a Cox proportional-hazards model was used to explore associations between clinical variables and outcomes. Key findings and limitationsThe median patient age was 68 yr (range 35–90). The first systemic therapy administered was platinum-based in 79% of cases and ICI-based in 21%. Some 61% of the cohort received a second systemic treatment, with 75% of these an ICI. Median overall survival for the entire cohort was 24 mo (interquartile range 9–35). Patients on ICI therapy for ≥6 mo had median overall survival of 59 mo (95% confidence interval 39 mo–not reached). Metastatic sites on initiation of ICI therapy and C-reactive protein kinetics were prognostic in patients receiving ICIs. Limitations include the retrospective design and inherent selection bias. Conclusions and clinical implicationsMore than 60% of patients with mUC received second-line treatment, and 75% of these received an ICI. Patients staying on immunotherapy for more than 6 mo have substantially better outcomes in comparison to patients with less time on immunotherapy and historical cohorts. Patient summaryWe looked at the lines of therapy and outcomes for patients with advanced or metastatic cancer of the urinary tract, starting from when immunotherapy drugs called immune checkpoint inhibitors (ICIs) became available. We found that 60% of patients have received second-line therapy, which is a double the rate in comparison to historical groups of patients. Patients with long-term ICI therapy (>6 months) had significantly better outcomes, with a median survival of more than 3 years.

Understanding HDL Metabolism and Biology Through In Vivo Tracer Kinetics.

HDL (high-density lipoprotein), owing to its high protein content and small size, is the densest circulating lipoprotein. In contrast to lipid-laden VLDL (very-low-density lipoprotein) and LDL (low-density lipoprotein) that promote atherosclerosis, HDL is hypothesized to mitigate atherosclerosis via reverse cholesterol transport, a process that entails the uptake and clearance of excess cholesterol from peripheral tissues. This process is mediated by APOA1 (apolipoprotein A-I), the primary structural protein of HDL, as well as by the activities of additional HDL proteins. Tracer-dependent kinetic studies are an invaluable tool to study HDL-mediated reverse cholesterol transport and overall HDL metabolism in humans when a cardiovascular disease therapy is investigated. Unfortunately, HDL cholesterol-raising therapies have not been successful at reducing cardiovascular events suggesting an incomplete picture of HDL biology. However, as HDL tracer studies have evolved from radioactive isotope- to stable isotope-based strategies that in turn are reliant on mass spectrometry technologies, the complexity of the HDL proteome and its metabolism can be more readily addressed. In this review, we outline the motivations, timelines, advantages, and disadvantages of the various tracer kinetics strategies. We also feature the metabolic properties of select HDL proteins known to regulate reverse cholesterol transport, which in turn underscore that HDL lipoproteins comprise a heterogeneous particle population whose distinct protein constituents and kinetics likely determine its function and potential contribution to cholesterol clearance.

A Marcus-Type Inverted Region in the Translocation Kinetics of a Knotted Protein.

Knotted proteins are rare but important species, yet how their complex topologies affect their physical properties is not fully understood. Here we combine single molecule nanopore experiments and all-atom MD simulations to study the electric-field-driven unfolding during the translocation through a model pore of individual protein knots important for methylating tRNA. One of these knots shows an unusual behavior that resembles the behavior of electrons hopping between two potential surfaces: as the electric potential driving the translocation reaction is increased, the rate eventually plateaus or slows back down in the "Marcus inverted regime". Our results shed light on the influence of topology in knotted proteins on their forced translocation through a pore connecting two electrostatic potential wells.

Effects of slit dual-frequency ultrasound-assisted pulping on the structure, functional properties and antioxidant activity of Lycium barbarum proteins and in situ real-time monitoring process

To improve the protein dissolution rate and the quality of fresh Lycium barbarum pulp (LBP), we optimized the slit dual-frequency ultrasound-assisted pulping process, explored the dissolution kinetics of Lycium barbarum protein (LBPr), and established a near-infrared spectroscopy in situ real-time monitoring model for LBPr dissolution through spectral information analysis and chemometric methods. The results showed that under optimal conditions (dual-frequency 28–33 kHz, 300 W, 31 min, 40 °C, interval ratio 5:2 s/s), ultrasonic treatment not only significantly increased LBPr dissolution rate (increased by 71.48 %, p < 0.05), improved other nutrient contents and color, but also reduced the protein particle size, changed the amino acid composition ratio and protein structure, and increased the surface hydrophobicity, zeta potential, and free sulfhydryl content of protein, as well as the antioxidant activity of LBPr. In addition, ultrasonication significantly improved the functional properties of the protein, including thermal stability, foaming, emulsification and oil absorption capacity. Furthermore, the real-time monitoring model of the dissolution process was able to quantitatively predict the dissolution rate of LBPr with good calibration and prediction performance (Rc = 0.9835, RMSECV = 2.174, Rp = 0.9841, RMSEP = 1.206). These findings indicated that dual-frequency ultrasound has great potential to improve the quality of LBP and may provide a theoretical basis for the establishment of an intelligent control system in the industrialized production of LBP and the functional development of LBPr.

Folding kinetics of an entangled protein.

The possibility of the protein backbone adopting lasso-like entangled motifs has attracted increasing attention. After discovering the surprising abundance of natively entangled protein domain structures, it was shown that misfolded entangled subpopulations might become thermosensitive or escape the homeostasis network just after translation. To investigate the role of entanglement in shaping folding kinetics, we introduce a novel indicator and analyze simulations of a coarse-grained, structure-based model for two small single-domain proteins. The model recapitulates the well-known two-state folding mechanism of a non-entangled SH3 domain. However, despite its small size, a natively entangled antifreeze RD1 protein displays a rich refolding behavior, populating two distinct kinetic intermediates: a short-lived, entangled, near-unfolded state and a longer-lived, non-entangled, near-native state. The former directs refolding along a fast pathway, whereas the latter is a kinetic trap, consistently with known experimental evidence of two different characteristic times. Upon trapping, the natively entangled loop folds without being threaded by the N-terminal residues. After trapping, the native entangled structure emerges by either backtracking to the unfolded state or threading through the already formed but not yet entangled loop. Along the fast pathway, trapping does not occur because the native contacts at the closure of the lasso-like loop fold after those involved in the N-terminal thread, confirming previous predictions. Despite this, entanglement may appear already in unfolded configurations. Remarkably, a longer-lived, near-native intermediate, with non-native entanglement properties, recalls what was observed in cotranslational folding.

PSXIV-23 In Vitro Evaluation of Protein Kinetics of Soy-Based Ingredients

Abstract Protein kinetics is the evaluation of how quickly a protein can be digested, absorbed, and entered into the portal system. It is more indicative of growth, feed efficiency, and protein deposition than the extent of digestibility per se. Feed ingredients may have the same digestibility (SID), but will differ in the speed of protein digestion. It was therefore decided to evaluate differences in speed of in vitro protein hydrolysis between 9 different soy-based protein ingredients: soybean meal (SBM), enzyme-treated soy protein (ESP), 3 types of soy protein concentrate (SPC), 2 types of extruded soy (ExS), and 2 types of fermented soybean meal (FSBM). Protein hydrolyzation rate was determined in vitro for the soy-based samples (SBM, ESP, SPC1, SPC2, SPC3, ExS1, ExS2, FSBM1, and FSBM2) using a pH-stat method that quantifies progress of protein hydrolysis by indirect measurement of released acid during hydrolysis. For each sample a 10 mL aqueous suspension containing 1 mg N/mL was prepared and adjusted to pH 8.0 at 39 oC using 0.1 M NaOH. One mL of a proteolytic enzyme-solution (trypsin, chymotrypsin and intestinal peptidase) was added to each sample to start protein hydrolysis followed by auto-titration (Mettler Toledo Titrator T9) with 0.1 M NaOH for 120 minutes. The volume of 0.1 M NaOH added to maintain pH at 8 throughout the incubation was recorded and used to calculate degree of hydrolysis (DH). The curve representing DH followed a second-order reaction kinetics and regression was used to estimate the rate constant (k) for each sample. The control sample (SBM) and ESP were analyzed in quadruplicates, the rest of the samples were in duplicates. A pairwise t-test was conducted in the statistical software R (version 4.0.0) to separate means. Significant difference was declared at P &amp;lt; 0.05. Degree of protein hydrolysis increased exponentially for all tested protein ingredients. The speed of protein hydrolysis (k-value) varied with soy-based protein ingredient (Table 1). The greater the k-value is representative of faster protein hydrolysis. There was a numerical 40% increase (P &amp;gt; 0.05) of ESP k-value when compared with the control, which was SBM. The SPC and FSBM samples were significantly less (P &amp;lt; 0.05) when compared with ESP, SBM and ExS2, with ExS1 sample being intermediate. In conclusion, soy processing technique (enzymatic, fermented, extrusion, or alcohol extraction) affects speed of in vitro protein hydrolysis. Therefore, more research is needed to see if these results coincide with protein utilization in vivo.

High-protein diet with excess leucine prevents inactivity-induced insulin resistance in women

Background and aimsMuscle inactivity leads to muscle atrophy and insulin resistance. The branched-chain amino acid (BCAA) leucine interacts with the insulin signaling pathway to modulate glucose metabolism. We have tested the ability of a high-protein BCAA-enriched diet to prevent insulin resistance during long-term bed rest (BR). MethodsStable isotopes were infused to determine glucose and protein kinetics in the postabsorptive state and during a hyperinsulinemic-euglycemic clamp in combination with amino acid infusion (Clamp+AA) before and at the end of 60 days of BR in two groups of healthy, young women receiving eucaloric diets containing 1 g of protein/kg per day (n=8) or 1.45 g of protein/kg per day enriched with 0.15 g/kg per day of BCAAs (leucine/valine/isoleucine=2/1/1) (n=8). Body composition was determined by Dual X-ray Absorptiometry. ResultsBR decreased lean body mass by 7.6±0.3% and 7.2±0.8% in the groups receiving conventional or high protein-BCAA diets, respectively. Fat mass was unchanged in both groups.At the end of BR, percent changes of insulin-mediated glucose uptake significantly (p=0.01) decreased in the conventional diet group from 155±23% to 84±10% while did not change significantly in the high protein-BCAA diet group from 126±20% to 141±27% (BR effect, p=0.32; BR/diet interaction, p=0.01; Repeated Measures ANCOVA). In contrast, there were no BR/diet interactions on proteolysis and protein synthesis Clamp+AA changes in the conventional diet and the high protein-BCAA diet groups. ConclusionA high protein-BCAA enriched diet prevented inactivity-induced insulin resistance in healthy women.

Early C‐reactive Protein Kinetics Predict Response to Immune Checkpoint Blockade in Unresectable Hepatocellular Carcinoma

Early C‐reactive Protein Kinetics Predict Response to Immune Checkpoint Blockade in Unresectable Hepatocellular Carcinoma