Protein Loading Research Articles

Tubulointerstitial injury in proteinuric chronic kidney diseases

Proteinuria is an independent risk factor for chronic kidney disease progression and cardiovascular diseases. Apart from its prognostic role, the load of proteins that pass across the disrupted glomerular capillary wall trigger multiple pathophysiologic processes. These include, among others, intratubular complement activation and excessive proximal tubular reabsorption of filtered proteins, especially albumin and albumin-bound free fatty acids, which can set off several pathways of cellular damage. The activation of these pathways can cause apoptosis of proximal tubular cells and paracrine effects that incite the development of interstitial inflammation and fibrosis, ultimately leading to irreversible kidney injury. In this review, we provide a comprehensive overview of the current understanding on the mechanisms underlying the tubular toxicity of ultrafiltered proteins in the setting of proteinuric chronic kidney diseases. The acquired knowledge is expected to be instrumental for the development of novel therapeutic classes of medications to be tested on top of standard of care with optimized renin-angiotensin-aldosterone blockade and sodium-glucose cotransporter-2 inhibition, in order to further improve the clinical outcomes of patients with proteinuric chronic kidney diseases.

Evaluation of renal functional reserve in patients with preserved renal function and coronary microvascular disease

Abstract Background/introduction Renal Functional Reserve (RFR) stands as a subject of significant scientific discussion,holding promise as a diagnostic tool for early detection of subclinical renal disorders. Literature establishes a link between chronic kidney disease & cardiovascular disease (CVD). Purpose This study aims to assess RFR in patients with coronary microvascular dysfunction (CMD), while maintaining preserved renal function (eGFR≥60ml/min/1.73m2 by CKD-EPI criteria)&proteinuria<400mg/24hr. The investigation seeks to provide insights into the nuanced relationship between renal function&CMD, offering potential early indicators of CVD. Methods This is a single-center, prospective study enrolling patients with CMD. In the absence of significant coronary artery disease, functional coronary circulation assessment was performed. Coronary flow reserve (CFR) & index of microvascular resistance (IMR) were measured invasively with bolus thermodilution technique. In all participants, RFR was estimated by endogenous creatinine clearance after oral protein load (cooked meal, 1.2gr/kg). Normal RFR was defined as≥30 ml/min/1.73m2. Also, patients with CMD were offered 24-hour Ambulatory Blood Pressure Monitoring (ABPM). Results A total of 25 participants have been enrolled so far in study: 11 without CMD - control group [7 female, 64%, mean age: 52.9±8.8 years) & 14 with CMD – CMD group (11 female, 79%, mean age: 53.5±10,3 years). CMD patients were classified into 2 groups, structural & functional endotype (CFR<2.5 & IMR≥25 were considered abnormal). The RFR value for CMD & control group is 7.4±6.3 vs 36.3±5.8 ml/min/1.73m2 respectively (p<0.05). This difference between groups remained statistically significant after controlling for confounding factors. It was found that RFR value for functional & structural CMD endotype was 3.8±2.6 & 9.6±7,2 ml/min/1.73m2 respectively (p=0.06). Furthermore, no statistical significance was found between RFR and IMR, CFR indices. According to data from ABPM, there were no significant differences in ambulatory blood pressure (both systolic and diastolic) between the two endotypes of CMD, as well as no relationship with RFR was observed. However, the proportion of non-dipping BP pattern was significantly higher in functional CMD endotype (p<0.05). It was not found correlation between RFR & non-dipping phenotype. Conclusions Impaired RFR is evident across all individuals experiencing microvascular angina. Furthermore, those with functional CMD exhibit a diminished RFR when compared with their counterparts with structural CMD. RFR does not exhibit a correlation with blood pressure levels or the absence of a nocturnal dipping pattern. In light of the hypothesis asserting that appraising renal functional reserve serves as an early diagnostic measure for subclinical renal disorders, promptly recognizing individuals with distinct phenotypes could facilitate tailoring therapeutic interventions on an individual basis.Functional Coronary AngiogramCMD group baseline characteristics

How long do bacteria, fungi, protozoa, and viruses retain their replication capacity on inanimate surfaces? A systematic review examining environmental resilience versus healthcare-associated infection risk by "fomite-borne risk assessment".

SUMMARYIn healthcare settings, contaminated surfaces play an important role in the transmission of nosocomial pathogens potentially resulting in healthcare-associated infections (HAI). Pathogens can be transmitted directly from frequent hand-touch surfaces close to patients or indirectly by staff and visitors. HAI risk depends on exposure, extent of contamination, infectious dose (ID), virulence, hygiene practices, and patient vulnerability. This review attempts to close a gap in previous reviews on persistence/tenacity by only including articles (n = 171) providing quantitative data on re-cultivable pathogens from fomites for a better translation into clinical settings. We have therefore introduced the new term "replication capacity" (RC). The RC is affected by the degree of contamination, surface material, temperature, relative humidity, protein load, organic soil, UV-light (sunlight) exposure, and pH value. In general, investigations into surface RC are mainly performed in vitro using reference strains with high inocula. In vitro data from studies on 14 Gram-positive, 26 Gram-negative bacteria, 18 fungi, 4 protozoa, and 37 viruses. It should be regarded as a worst-case scenario indicating the upper bounds of risks when using such data for clinical decision-making. Information on RC after surface contamination could be seen as an opportunity to choose the most appropriate infection prevention and control (IPC) strategies. To help with decision-making, pathogens characterized by an increased nosocomial risk for transmission from inanimate surfaces ("fomite-borne") are presented and discussed in this systematic review. Thus, the review offers a theoretical basis to support local risk assessments and IPC recommendations.

Mesoporous Silica Microparticle-Protein Complexes: Effects of Protein Size and Solvent Properties on Diffusion and Loading Efficiency.

Oral administration of protein-based therapeutics is highly desirable due to lower cost, enhanced patient compliance, and convenience. However, the harsh pH environment of the gastrointestinal tract poses significant challenges. Silica-based carriers have emerged as potential candidates for the delivery of protein molecules, owing to their tuneable surface area and pore volume. We explored the use of a commercial mesoporous silica carrier, SYLOID, for the delivery of octreotide and bovine serum albumin (BSA) using a solvent evaporation method in three different solvents. The loading of proteins into SYLOID was driven by diffusion, as described by the Stokes-Einstein equation. Various parameters were investigated, such as protein size, diffusion, and solubility. Additionally, 3D fluorescence confocal imaging was employed to identify fluorescence intensity and protein diffusion within the carrier. Our results indicated that the loading process was influenced by the molecular size of the protein as octreotide exhibited a higher recovery rate (71%) compared to BSA (32%). The methanol-based loading of octreotide showed uniform diffusion into the silica carrier, whereas water and ethanol loading resulted in the drug being concentrated on the surface, as shown by confocal imaging, and further confirmed by scanning electron microscopy (SEM). Pore volume assessment supported these findings, showing that octreotide loaded with methanol had a low pore volume (1.2cc/g). On the other hand, BSA loading was affected by its solubility in the three solvents, its tendency to aggregate, and its low solubility in ethanol and methanol, which resulted in dispersed particle sizes of 223 and 231μm, respectively. This reduced diffusion into the carrier, as confirmed by fluorescence intensity and diffusivity values. This study underscores the importance of protein size, solvent properties, and diffusion characteristics when using porous carriers for protein delivery. Understanding these factors allows for the development of more effective oral protein-based therapeutics by enhancing loading efficiency. This, in turn, will lead to advances in targeted drug delivery and improved patient outcomes.

Biocatalytic Production of Solketal Esters from Used Oil Utilizing Treated Macauba Epicarp Particles as Lipase Immobilization Support: A Dual Valorization of Wastes for Sustainable Chemistry

This study describes the production of solketal esters from used soybean cooking oil (USCO) via enzymatic hydroesterification. This process consists of the complete hydrolysis of USCO into free fatty acids (FFAs) catalyzed by crude lipase extract from Candida rugosa (CRL). The resulting FFAs were recovered and utilized as the raw material for an esterification reaction with solketal, which was achieved via an open reaction. For this purpose, lipase Eversa® Transform 2.0 (ET2.0) was immobilized via physical adsorption on treated epicarp particles from Acrocomia aculeata (macauba), a lignocellulosic residue. A protein loading of 25.2 ± 1.3 mg g−1 with a support and immobilization yield of 64.8 ± 2.5% was achieved using an initial protein loading of 40 mg g−1 of support. The influence of certain parameters on the esterification reaction was evaluated using a central composite rotatable design (CCRD). Under optimal conditions, a FFAs conversion of 72.5 ± 0.8% was obtained after 150 min of reaction at 46 °C using a biocatalyst concentration of 20% wt. and a FFAs–solketal molar ratio of 1:1.6. The biocatalyst retained 70% of its original activity after ten esterification batches. This paper shows the conversion of two agro-industrial waste into valuable materials (enzyme immobilization support and solketal esters).

Synthesis of Dumbbell-Like Heteronanostructures Encapsulated in Ferritin Protein: Towards Multifunctional Protein Based Opto-Magnetic Nanomaterials for Biomedical Theranostic

Dumbbell-like hetero nanostructures based on gold and iron oxides is a promising material for biomedical applications, useful as versatile theranostic agents due the synergistic effect of their optical and magnetic properties. However, achieving precise control on their morphology, size dispersion, colloidal stability, biocompatibility and cell targeting remains as a current challenge. In this study, we address this challenge by employing biomimetic routes, using ferritin protein nanocages as template for these nanoparticles’ synthesis. We present the development of an opto-magnetic nanostructures using the ferritin protein, wherein gold and iron oxide nanostructures were produced within its cavity. Initially, we investigated the synthesis of gold nanostructures within the protein, generating clusters and plasmonic nanoparticles. Subsequently, we optimized the conditions for the superparamagnetic nanoparticles synthesis through controlled iron oxidation, thereby enhancing the magnetic properties of the resulting system. Finally, we produce magnetic nanoparticles in the protein with gold clusters, achieving the coexistence of both nanostructures within a single protein molecule, a novel material unprecedented to date. We observed that factors such as temperature, metal/protein ratios, pH, dialysis, and purification processes all have an impact on protein recovery, loading efficiency, morphology, and nanoparticle size. Our findings highlight the development of ferritin-based nanomaterials as versatile platforms for potential biomedical use as multifunctional theranostic agents.

Formulation of protein-loaded nanoparticles via freeze-drying.

Several nanotechnology-based formulation strategies have been reported for the oral administration of biological drugs. However, a prerequisite often overlooked in developing these formulations is their adaptation to a solid dosage form. This study aimed to incorporate a freeze-drying step, using either mannitol or sucrose laurate (SLAE), into the formulation of new insulin-zinc nanocomplexes to render them resistant to intestinal fluids while maintaining a high protein loading. The resulting freeze-dried insulin-zinc nanocomplexes exhibited physicochemical properties consistent with the target product profile, including a particle size of ∼ 100nm, a zeta potential close to neutrality (∼ -15 mV) and a high association efficiency (> 90%). Importantly, integrating the freeze-drying step in the formulation significantly improved the colloidal stability of the system and preserved the stability of the insulin molecules. Results from in vitro and in vivo studies indicated that the insulin activity remained fully retained throughout the entire formulation and freeze-drying processes. In brief, we present a novel protein formulation strategy that incorporates a critical freeze-drying step, resulting in a dry powder enabling efficient protein complexation with zinc and optimized for oral administration.

A multi-epitope protein vaccine encapsulated in alginate nanoparticles as a candidate vaccine against Shigella sonnei

Shigellosis, caused by the Gram-negative bacterium Shigella, is a major global health challenge. Despite extensive research over the past two decades, no commercial vaccine is available to prevent Shigella infection. Developing multi-epitope vaccines offers a promising and innovative approach to tackling infectious diseases. In this study, we produced a multi-epitope vaccine candidate using E. coli BL21 (DE3) plysS bacteria and purified the vaccine protein with Ni-NTA affinity chromatography. We then prepared alginate nanoparticles containing the vaccine protein, with a particle size of 122 ± 6 nm, PDI 0.17, SPAN 0.83, and zeta potential of -27 ± 2 mV. Successful protein loading was confirmed through nanodrop and ATR-FTIR analyses. To evaluate the immunogenicity of the encapsulated vaccine, mice were orally vaccinated, and their serum was analyzed for IgG, IL-4, and IFN-γ levels cytokines. The results showed a significant increase in IgG level in the vaccinated group compared to controls. Additionally, the vaccinated group exhibited a notable increase in IL-4 and IFN-γ cytokines, indicating a robust Th-cell-mediated immune response essential for combating Shigella. Our nano-vaccine demonstrated high efficacy in activating both humoral and cellular immunity, effectively protecting against the bacteria. The alginate-based oral vaccine candidate thus emerges as a promising strategy for developing a multi-epitope vaccine candidate against Shigella.

Characterizing and Predicting Outcomes in Critically Ill Patients Receiving Low or High Protein Doses with Moderate Energy Support: A Retrospective Study.

Finding the best energy and protein dose and timing for critically ill patients remains challenging. Distinct populations may react differently to protein load. This study aimed to characterize and predict outcomes of critically ill patients who received moderate energy and high or low protein doses during their stay in the intensive care unit (ICU). The cohort included 646 adult patients (70% men and 30% women) hospitalized in Beilinson Hospital ICU (Petah Tikva, Israel) for over 5 days between 2011 and 2018. Patients received 10-20 kcal/kg/day and were classified into two groups: low (LP) and high (HP) protein support (≤1 g/kg/day vs. >1 g/kg/day), the LP group comprising 531 patients (82%) and the HP group 115 patients (18%). Multiple logistic regression was used to describe associations between patients' characteristics and 90-day survival in the LP and HP groups. Among LP, increased age, APACHE II, and receiving supplemental parenteral nutrition (SPN) were associated with decreased survival (OR = 0.986, 95% CI [0.973, 0.999]; OR = 0.915, 95% CI [0.886, 0.944], OR = 0.579, 95% CI [0.366, 0.917]). Trauma admission was associated with increased survival (OR = 1.826, 95% CI [1.001, 3.329]). Among HP, increased age was associated with decreased survival (OR = 0.956, 95% CI [0.924, 0.998]). Higher BMI was associated with improved survival (OR = 1.137, 95% CI [1.028, 1.258]). Likewise, in the HP group, the BMI of elderly survivors was higher compared to non-survivors (27.1 ± 6.2 vs. 24.7 ± 4.8, t (113) = 2.3, p < 0.05). Our results show that in patients with moderate energy support and low protein administration, survivors were younger, with a lower APACHE II score, mainly suffering from trauma and without renal failure. In the patients receiving high protein support, younger patients with a high BMI not suffering from sepsis were more likely to survive. We suggest confirming these findings with prospective RCTs.

Evaluation on potential application of lipase immobilized on rice husks in enzymatic glycerolysis reaction

Owing to high production cost and low reaction yield, immobilized lipase is rarely used in industrial glycerolysis. This research characterizes the performance of lipase immobilized on rice husk in glycerolysis reaction. By utilizing hexamethylenediamine (HMDA) and glutaraldehyde (GA) as coupling agents, lipase from Thermomyces lanuginosus was immobilized on oxidized rice husk (ORH). For comparison, another sample was prepared where the lipase was directly immobilized on ORH without the use of HMDA and GA. Then, monoglyceride production was performed via glycerolysis using the immobilized lipase. The FTIR analysis verify interactions on rice husk including rice husk oxidation, HMDA coupling, GA activation and lipase immobilization on rice husk. The study found that within the examined range of added lipase for immobilization (10–40 mg-protein/g-support), ORH–HMDA–GA–Lipase possessed superior outcomes in terms of protein loading, immobilization yield, and recovered glycerolysis activity compared to ORH–Lipase. Besides, ORH–HMDA–GA–Lipase exhibits better storage stability (60°C, 44.9 %) and higher reusability (90.0 % monoglyceride yield at the 8th cycle) against ORH–Lipase. The results confirm satisfying performance of the prepared immobilized lipase in glycerolysis and highlight its enhancements facilitated by coupling agents.

Engineering PEG10assembled endogenous virus-like particles with genetically encoded neoantigen peptides for cancer vaccination.

Tumor neoantigen peptide vaccines hold potential for boosting cancer immunotherapy, yet efficiently co-delivering peptides and adjuvants to antigen-presenting cells in vivo remains challenging. Virus-like particle (VLP), which is a kind of multiprotein structure organized as virus, can deliver therapeutic substances into cells and stimulate immune response. However, the weak targeted delivery of VLP in vivo and its susceptibility to neutralization by antibodies hinder their clinical applications. Here, we first designed a novel protein carrier using the mammalian-derived capsid protein PEG10, which can self-assemble into endogenous VLP (eVLP) with high protein loading and transfection efficiency. Then, an engineered tumor vaccine, named ePAC, was developed by packaging genetically encoded neoantigen into eVLP with further modification of CpG-ODN on its surface to serve as an adjuvant and targeting unit to dendritic cells (DCs). Significantly, ePAC can efficiently target and transport neoantigens to DCs, and promote DCs maturation to induce neoantigen-specific T cells. Moreover, in mouse orthotopic liver cancer and humanized mouse tumor models, ePAC combined with anti-TIM-3 exhibited remarkable antitumor efficacy. Overall, these results support that ePAC could be safely utilized as cancer vaccines for antitumor therapy, showing significant potential for clinical translation.

Engineering PEG10-assembled endogenous virus-like particles with genetically encoded neoantigen peptides for cancer vaccination

Tumor neoantigen peptide vaccines hold potential for boosting cancer immunotherapy, yet efficiently co-delivering peptides and adjuvants to antigen-presenting cells in vivo remains challenging. Virus-like particle (VLP), which is a kind of multiprotein structure organized as virus, can deliver therapeutic substances into cells and stimulate immune response. However, the weak targeted delivery of VLP in vivo and its susceptibility to neutralization by antibodies hinder their clinical applications. Here, we first designed a novel protein carrier using the mammalian-derived capsid protein PEG10, which can self-assemble into endogenous VLP (eVLP) with high protein loading and transfection efficiency. Then, an engineered tumor vaccine, named ePAC, was developed by packaging genetically encoded neoantigen into eVLP with further modification of CpG-ODN on its surface to serve as an adjuvant and targeting unit to dendritic cells (DCs). Significantly, ePAC can efficiently target and transport neoantigens to DCs, and promote DCs maturation to induce neoantigen-specific T cells. Moreover, in mouse orthotopic liver cancer and humanized mouse tumor models, ePAC combined with anti-TIM-3 exhibited remarkable antitumor efficacy. Overall, these results support that ePAC could be safely utilized as cancer vaccines for antitumor therapy, showing significant potential for clinical translation.

Functionalized α-Cyanostilbene Derivatives for Detection of Hypoxia or Proteostasis Imbalance in Live Cells.

α-Cyanostilbene represents one of the easily functionalized aggregation-induced emission (AIE) scaffolds. It has been widely adopted for the construction of fluorescent materials for broad applications. Here, we further expanded the utilization of α-cyanostilbene derivatives for the detection of hypoxia or proteostasis imbalance in live cells. Four different amine containing donors were introduced to construct α-cyanostilbene derivatives (R-ASC) with donor-acceptor scaffolds. Equipped with the cysteine (Cys) reactive group, maleimide (MI), R-ASC-MI shows fluorescence turn-on property upon binding with unfolded proteins in vitro and in live cells under proteostatic stress. By virtue of R-ASC-MI, the level of unfolded protein loads in cells can be quantified by flow cytometry, or visualized under microscope. Furthermore, we also characterized the performance of R-ASC-NO2, synthetic precursors of R-ASC-MI, in cellular hypoxia. R-ASC-NO2 revealed upregulated activities of nitroreductase, as well as increased hydrophobicity in live cells, under either chemical (NaN3) induced or atmospheric (1% O2) hypoxia. Together, the advantages of easy modification and high signal-to-noise ratio of new α-cyanostilbene derivatives reported in this work highlight the great potential of α-cyanostilbene in constructing functional biosensors and many other domains.

Postprandial glycemic response to isocaloric protein load in men with different types of fat distribution

Aim of the study was to quantify postprandial glucose levels in response to isocaloric protein load at main meals in men with different types of fat distribution. Material and methods. The study enrolled men aged 25 to 65 years. Group 1 (n = 17) consisted of obese men with subcutaneous fat distribution (SFD) type while group 2 (n = 16) was represented by obese men with abdominal type of fat distribution (AFD). Group 3 (comparators) consisted of 10 men with normal body weight (NBW). Glycemic response to standard isocaloric protein load was assessed by the results of glucose levels within 3 hours starting 5 minutes after end of food consumption on different days and mealtime. Standard protein lunch was introduced during the second day while standard protein dinner and breakfast were performed at the third and fourth days respectively. Results. It was found that protein intake leads to neither pronounced postprandial glycemic fluctuations nor decrease in glucose levels by the end of the 3rd hour of the test. Accordingly, it also doesn’t provoke hunger, unlike carbohydrate intake does. In men with NBW a more pronounced increase in glucose level after protein meal was found, both relative to the baseline levels and comparing with men from SFD and AFD groups. In NBW group at lunchtime and especially in the evening a double-humped glycemic curve was noted. Glycemic variability in men with different types of fat distribution was characterized by the fact that glycemic increment was more pronounced in men with AFD than in men with SFD whose glycemic curve was almost flat after all meals. Conclusions. Protein intake in men with NBW and different types of fat distribution does not lead to significant changes in postprandial glucose levels. Glycemic fluctuations don’t exceed 1 mmol/L within a 3-hour period after consuming of isocaloric breakfast, lunch or dinner. Protein intake results in greater postprandial glucose levels in men with AFD than in SFD men.

RetSat stabilizes mitotic chromosome segregation in pluripotent stem cells

BackgroundChromosome stability is crucial for homeostasis of pluripotent stem cells (PSCs) and early-stage embryonic development. Chromosomal defects may raise carcinogenic risks in regenerative medicine when using PSCs as original materials. However, the detailed mechanism regarding PSCs chromosome stability maintenance is not fully understood.MethodsMouse embryonic stem cells (line D3) and human embryonic stem cells (line H9) were cultured under standard conditions. To confirm the loading of RetSat protein on mitotic chromosomes of PSCs, immunostaining was performed in PSCs spontaneous differentiation assay and iPSC reprogramming assay from mouse embryonic fibroblasts (MEFs), respectively. In addition, qPCR, immunoprecipitation, LC-MS/MS and immunoblotting were used to study the expression of RetSat, and interactions of RetSat with cohesin/condensin components. RNA sequencing and teratoma formation assay was conducted to evaluate the carcinogenic risk of mouse embryonic stem cells with RetSat deletion.ResultsWe reported a PSC high-expressing gene, RetSat, plays key roles in chromosome stabilization. We identified RetSat protein localizing onto mitotic chromosomes specifically in stemness positive cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We found dramatic chromosome instability, e.g. chromosome bridging, lagging and interphase micronuclei in mouse and human ESCs when down regulating RetSat. RetSat knock-out mouse ESCs upregulated cancer associated gene pathways, and displayed higher tumorigenic capacities in teratoma formation assay. Mechanistically, we confirmed that RetSat interacts with cohesin/condensin components Smc1a and Nudcd2. RetSat deletion impaired the chromosome loading dosage of Smc1a, Smc3 and Nudcd2.ConclusionsIn summary, we reported RetSat to be a key stabilizer of chromosome condensation in pluripotent stem cells. This highlights the crucial roles of RetSat in early-stage embryonic development, and potential value of RetSat as an effective biomarker for assessing the quality of pluripotent stem cells.

Exploring Mutation-Driven Changes in the ATP-ADP Conformational Cycle of Human Hsp70 by All-Atom MD Adaptive Sampling.

Hsp70 belongs to a family of molecular chaperones ubiquitous through organisms that assist client protein folding and prevent aggregation. It works through a tightly ATP-regulated allosteric cycle mechanism, which organizes its two NBD and SBD into alternate open and closed arrangements that facilitate loading and unloading of client proteins. The two cytosolic human isoforms Hsc70 and HspA1 are relevant targets for neurodegenerative diseases and cancer. Illuminating the molecular details of Hsp70 functional dynamics is essential to rationalize differences among the well-characterized bacterial homologue DnaK and the less explored human forms and develop subtype- or species-selective allosteric drugs. We present here a molecular dynamics-based analysis of the conformational dynamics of HspA1. By using an "allosterically impaired" mutant for comparison, we can reconstruct the impact of the ADP-ATP swap on interdomain contacts and dynamic coordination in full-length HspA1, supporting previous predictions that were, however, limited to the NBD. We model the initial onset of the conformational cycle by proposing a sequence of structural steps, which reveal the role of a specific human sequence insertion at the linker, and a modulation of the angle formed by the two NBD lobes during the progression of docking. Our findings pinpoint functionally relevant conformations and set the basis for a selective structure-based drug discovery approach targeting allosteric sites in human Hsp70.

Engineered exosomes with a photoinducible protein delivery system enable CRISPR-Cas-based epigenome editing in Alzheimer's disease.

Effective intracellular delivery of therapeutic proteins can potentially treat a wide array of diseases. However, efficient delivery of functional proteins across the cell membrane remains challenging. Exosomes are nanosized vesicles naturally secreted by various types of cells and may serve as promising nanocarriers for therapeutic biomolecules. Here, we engineered exosomes equipped with a photoinducible cargo protein release system, termed mMaple3-mediated protein loading into and release from exosome (MAPLEX), in which cargo proteins can be loaded into the exosomes by fusing them with photocleavable protein (mMaple3)-conjugated exosomal membrane markers and subsequently released from the exosomal membrane by inducing photocleavage with blue light illumination. Using this system, we first induced transcriptional regulation by delivering octamer-binding transcription factor 4 and SRY-box transcription factor 2 to fibroblasts in vitro. Second, we induced in vivo gene recombination in Cre reporter mice by delivering Cre recombinase. Last, we achieved targeted epigenome editing in the brains of 5xFAD and 3xTg-AD mice, two models of Alzheimer's disease. Administration of MAPLEXs loaded with β-site amyloid precursor protein cleaving enzyme 1 (Bace1)-targeting single guide RNA-incorporated dCas9 ribonucleoprotein complexes, coupled with the catalytic domain of DNA methyltransferase 3A, resulted in successful methylation of the targeted CpG sites within the Bace1 promoter. This approach led to a significant reduction in Bace1 expression, improved recognition memory impairment, and reduced amyloid pathology in 5xFAD and 3xTg-AD mice. These results suggest that MAPLEX is an efficient intracellular protein delivery system that can deliver diverse therapeutic proteins for multiple diseases.

Effect of an intensive nutrition intervention of a high protein and low glycemic load diet on weight of kidney transplant recipients: a randomized clinical trial.

The purpose of this study is to evaluate the effect of a high protein and low glycemic load diet in preventing weight gain after kidney transplantation. We designed a prospective, single-center, open-label, randomized controlled study to compare the efficacy of a high protein (1.3-1.4 g/kg/day) and low glycemic load diet versus a conventional diet (0.8-1.0 g/kg/day of protein and no recommendations on glycemic load) in preventing weight gain (ClinicalTrials.gov identifier: NCT02883777). A total of 120 patients were evaluated. Patients were followed for 12 months, and the primary outcome was weight maintenance or weight gain lower than 5%. There were no differences in total energy intake, carbohydrates, and total fats between groups. Intervention group (IG) increased protein intake to 1.38 ± 0.56 g/kg/day and decreased the glycemic load to 87.27 ± 4.54 g/day, while control group (CG) had a dietary protein intake of 1.19 ± 0.43 g/kg/day and a glycemic load of 115.60 ± 7.01 g/day. Total fiber intake was greater and trans-fat was lower in IG. Dietetic cholesterol increased in IG over time and was significantly different between groups. Overall, patients had an increase in body weight over time, with a mean increment of 4.1 ± 5.5 kg (5.75%). The percentage of patients who achieved the primary outcome was 50% of sample size, without differences between groups. The glomerular filtration rate improved over time in both groups. Considering 24-h proteinuria and albuminuria, a similar rise was observed in both groups. The present dietary intervention was safe, but had no effect on weight gain in kidney transplant subjects. Our findings suggest that other strategies, including alternative dietary and/or pharmacological and psychological interventions might be tested in randomized control trials in order to improve patients' body weight outcomes after transplant.

Laponite nanoclays for the sustained delivery of therapeutic proteins

Protein therapeutics hold immense promise for treating a wide array of diseases. However, their efficacy is often compromised by rapid degradation and clearance. The synthetic smectite clay Laponite emerges as a promising candidate for their sustained delivery. Despite its unique properties allow to load and release proteins mitigating burst release and extending their effects, precise control over Laponite-protein interactions remains challenging since it depends on a complex interplay of factors whose implication is not fully understood yet. The aim of this review article is to shed light on this issue, providing a comprehensive discussion of the factors influencing protein loading and release, including the physicochemical properties of the nanoclay and proteins, pH, dispersion buffer, clay/protein concentration and Laponite degradation. Furthermore, we thoroughly revise the array of bioactive proteins that have been delivered from formulations containing the nanoclay, highlighting Laponite-polymer nanocomposite hydrogels, a promising avenue currently under extensive investigation.

Tailor-Made Bioactive Papers by Site-Specific and Orthogonal Covalent Immobilization of Proteins.

A strategy for the bioorthogonal immobilization of proteins onto commercially available filter paper is presented. Recently, a two-step approach has been described that relies on covalent immobilization of a linker molecule to paper, followed by enzyme-mediated conjugation of a protein of interest containing an enzyme-recognition tag. Here, this strategy was expanded by evaluating four different chemical and chemoenzymatic reactions and investigating paper loading efficiency and orthogonality. Enhanced green fluorescent protein (EGFP) was used as a model protein to allow quantification of protein loading via fluorescence imaging. Two approaches were identified that showed significantly increased loading efficiencies compared with the previously applied conjugation strategy. Additionally, all four methods were proven orthogonal to each other, allowing simultaneous immobilization of a mixture of proteins to a premodified assembly of two paper sheets.